iraqi journal of veterinary sciences

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010

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Page 1: Iraqi Journal of Veterinary Sciences

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Diagnosis of bovine brucellosis in Mosul city by indirect ELISA and conventional

serological tests

M. S. Rhaymah, K. A. AL-Saad,and O. KH. AL-Hankawe

Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Mosul, Mosul, Iraq

Abstract

The study was conducted on 126 cattle (94 females and 32 males) of different ages (1->5 years) randomly selected from

July 2007 to August 2008 in Mosul. Indirect ELISA test and other traditional tests (rose Bengal test, tube agglutination test and

2- mercapto-ethanol test) were used to determine the incidence of bovine brucellosis. The highest incidence of disease was

recorded by Indirect ELISA, 23.01%, whereas it was 18.25%, 11.90% and 4.76% by rose Bengal, tube agglutination and 2-

Mercapto-ethanol tests, respectively. The highest incidence was in females in all serological tests and the highest incidence

was in females at the age between 1-3 years whereas in males more than 3 years of age it was 23.07%. The results of tube

agglutination test revealed the titer 1/40 occurred mostly compared with other titers. Six chronic cases were determined by 2-mercapto-ethanol test. The degree of agreement of negative samples with rose Bengal test and indirect ELISA, tube

agglutination, and 2- mercapto-ethanol tests was 94.17%, 100% and 100%, respectively, and by indirect ELISA with rose

Bengal, tube agglutination and 2-mercapto-ethanol tests was 79.31%, 51.72% and 20.68%, respectively.

Available online at http://www.vetmedmosul.org/ijvs

Page 9: Iraqi Journal of Veterinary Sciences

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de Nagues M P, Cau C. Efficacy of different rose Bengal and

complement of fixation antigen for the diagnosis of Brucella

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12. Beh KJ. Quantitative distribution of Brucella antibody amongst

immunoglobulin classes in vaccinated and infected cattle. Res Vet

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13. Cruickshank R, Duguid JP, Marmion BP, Swain RH A. Medical

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198p.

16. Wright PF, Kelly W, Gall D E. Application of a timing protocol to the

reduction of inter plate variability of anti-Brucella antigen. J

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of enzyme immunoassay for detection of antibody to Brucella abortus

in cattle. Vet Immunol Immunopath. 1988;18:331-347.

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brucellosis laboratory. Paris: INRA; 1988.

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health organization. Geneve. 1975. 20. Hadad JJ, AL-Azawy ZS. Incidence of Brucellosis in Sheep and goats

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in Ninevah province. Iraqi J Vet Sci. 1990;5:159-164.

22. Hussain K A, Saleem AN, Fatoohi FAM. prevalence brucellosis in

buffaloes, cattle and sheep in Mosul region. Iraqi J Vet Sci. 1994;7:

233-238.

�0. ����"����) �� . $ ')�� �� F��� �?3(� !� � )���M�"" R$�;�) .�()��� ��)� ( ���� ���� �� ���� ��

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���� ���� ��������: ���*. 25. Kadohira M, Dermott MCJJ, Shouki MM, Kyule MN. variations in

the prevalence of antibody to brucella infection in cattle by farm, area

and district in Kenya. Epidemiol Infect. 1997;118:53-41. 26. Omer MK, Skjerva E, Woldehiwet Z, Holstad G. Risk factors for

Brucella Spp. Infection in dairy cattle farms in Asmara, state of

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27. AL-Thwyni A, AL-Bayatti S, Abass A, Abdulhussin A. A study in the

epidemiology of brucellosis in some production animals in the

province of Baghdad. The Veterinarian. 2000;10:168-174.

�C. ��" � 5 ,);� �,�V����. $ ���)�� F�Q�&� F��� !� � e)� "����� "�� $ ��. &� . ��� �� ���� ����� )��.�� ������*;

� :�0��A. 29. Darwesh M, Benkirane A. Field investigations of brucellosis in cattle

and small ruminants in Syria, 1990-1996. Rev Sci tech Int Epiz. 2001;

20:769-775.

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among livestock in himachal Pradesh. Indian J comp Microbiol

Immunol Infect Dis. 2004;25:15-18.

0�. ��) �51? ��5 �����";��. F�� T?3(� "��.� )��� �� (?� ���?()� M�"" R$�;� $ 1����� ,<V�� $ 1)��)�� ��

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32. Kolar J. Diagnosis and control of brucellosis in small ruminants. Prev

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Microbiology. 1st ed. London: Elservier; 1999.78-79p.

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between brucella abortus and yersinia enterocoliticao: 9 Int. J

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1.55pdf.

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Epiz. 2004;23:989-1002.

Page 14: Iraqi Journal of Veterinary Sciences

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M���K�>75� L��� ,� (� �6D = ���� I:N ,���, �� �5���� 4������ ��)>5� �/ 6������ 3��) A3 ,����� $�2(7� )5@�G 3�%� %83.8 6������ �O A1.5 $�2(7� )5 � %81.3� ��D6������ B3 $�2(7*� )5 35�# %78.2 6������ P��7D�

B1.5 )5�$�2(7 %74.6.

Effect of immunization with protoscolices antigens of hydatid cysts on growth of

Echinococcus granulosus in dogs

M. A. Aljawady and M. N. Al-Shahery

Department of Microbiology, College of Veterinary Medicine, University of Mousl, Mosul, Iraq

Abstract

The study was designed to evaluate the immune response of puppies, injected by different protein fractions extracted from

protoscolieces, obtained from ovine hydatid cyst. Indirect heamagglutination revealed a remarkable increase in the antibody

titers for the immunized groups (A1.5, A3, B1.5, and B3) before and after challenge when compared with the control.

Biological variations showed decline in numbers of adult Echinococcus granulosus in the immunized groups when compared

with the control. Other variations proved dropping in numbers of the worms within the same immunized groups. Subsequent

reductions of cestodes were reported which were 83.8%, 81.3%, 78.2% and 74.6% for the groups A3, A1.5, B3, and B1.5,

respectively.

Available online at http://www.vetmedmosul.org/ijvs *���� L�� �� I���(#��� ����D ,� $() $

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Page 15: Iraqi Journal of Veterinary Sciences

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Page 16: Iraqi Journal of Veterinary Sciences

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Page 17: Iraqi Journal of Veterinary Sciences

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1. Andersen FL, Ouhelli H, Kachani M. Compendium on cystic

echinococcosis in Africa and in Middle Eastern countries with Special

Reference to Morocco. Brigham Young University print services,

provo, Utah 84604,1997:54-85.

2. Al-Khalidi NW. Investigation of the Immunity of Dogs to

Echinococcus granulosus during the prepatent infection. [dissertation],

Ohio State University; 1982:1-35p.

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Diagnosis of Hydatid Disease. Clin Microbiol Rev. 2003;16(1):18-36.

4. Zhang WB, Zhang ZZ, Chi PS. Vaccination of dogs against

Echinococcus granulosus using soluble antigens of protoscolieces.

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Hydat Madrid 1985:84-95p.

6. Smyth JD, Barett NJ. Procedures for testing the viability of Human

Hydatid cysts following surgical removal especially after

chemotherapy. Trans Roy Soc Trop Med Hyg. 1980;74:649-652.

7. Taherkhani H, Rogan MT. General characterization of laminated layer

of Echinococcus granulosus. Irn J Med Sci. 2000;25:3-4.

.. !������� ��()�� � 6 �����. ��� 3�#�-��� =�>�� 6�5� )��� ��5AD $%D ,� ���� 3�)<��� 3��H()� 5�/���� "'#�� = .

I���(#� ����D�!�� �� "��� �# �$%���� ���� � &����� ���[.

9. Robyte JF, White BJ. Gel permeation chromatography. II. Organic

solvent protein precipitation. in: Biochemical Techniques, Theory and

practice. Wadsworth Inc., Belmont, California, USA. 1987;88-268p.

10. Hudson L, Hay FC. Application of gel chromatography. In: practical

immunology, 1st edition, p156. Blackwell Scientific publication Osney

Mead, Oxford, London Edinburgh, U.K. North Balwyn, Victoria,

Australia. 1976.

11. Eckert J, Schantz PM, Gasser RB, Torgerson PR, Bessonov AS,

Movses-sian SO, Thakur A, Grimm F, Nikogossian MA. Geographic

distribution and prevalence, p. 100-142. In: Eckert, J. Gemmell, M. A.

Meslin, F.-X. and Pawlowski Z. S. (ed.), WHO/OIE manual on

echinococcosis in humans and animals: a public health problem of

global concern. World Organisation for Animal Health, Paris, France.

2001.

12. Alton GG. Recent development in vaccination against bovine

brucellosis. Aust Vet J. 1978;54:551-557.

13. Dalsgaard K. Adjuvants. Vet Immunol Immunopathol. 1987;17:145-

152.

14. Jones WO, Emery DL, McClure SJ, Wagland BM. Changes in

inflammatory mediators and larval inhibitory activity in intestinal

contents and mucus during primary and challenge infections of sheep

with Trichostrongylus colubriformis. Int J Parasitol. 1994;24:519-525.

Page 18: Iraqi Journal of Veterinary Sciences

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Clinical study of foot and mouth disease in feedlot calves in Mosul region

M. A. Abd- Alhameed and M. SH. Rhaymah

Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Mosul, Mosul, Iraq

Abstract

The affected calves showed clinical signs including presence of vesicles at the bucal cavity and hoof (90.7%), fever (36%), salivation (32.9%), depression (20.5%), Anorexia (19.6%), loss of body weight (7.94%), lamness (15.9%), lesions at the

muzzle (15.3%), presence of the lesions at the feet (6.0%), prostration (4.7%), diarrhea (3.3%), presence of the lesion at the

mouth cavity (3.0%), paralysis of hind quarters (2.2%) and sudden death (1.6%). There was significant increase in the rectal

temperature, respiration rates, and heart rates in the infected animals compared with control group. Recovered animals

appeared some diseases including theileriosis (40.4%), tympany (22.8%), pneumonia (19.9%), foot abscess (14.7%), and

babesiosis (2.2 %). The mortality rate varied between 3.4% to 27.9% in different herds and the mean was 3.3 % in all herds.

There was significant relationship between age of animals and the incidence of the disease.

Available online at http://www.vetmedmosul.org/ijvs * #�)� R:� �� �'(����� :���� ,� #'(� R: ��

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Page 23: Iraqi Journal of Veterinary Sciences

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1. Donaldson AI. Foot and mouth disease: the principl features. Irish Vet J. 1987;41:325–327.

2. Kitching RP. Clinical variation in foot and mouth disease: Cattles. Rev

Sci Tech Int Epiz. 2002;21:499–504.

3. Gibson CF, Donaldson AI. Exposure of sheep to natural aerosols of

foot and mouth disease virus. Res Vet Sci. 1986;41:45–49.

4. Radostitis OM, Gay CC, Bood DC, Hinchcliff KW. Veterinary

Medicine: A textbook of the disease of cattle, sheep, goat, pigs, and

horses. 9th ed. London: W.B. Saunders company; 2000.1059–1066p.

5. Azab AS, Shahawy MI, Hassos EI. Detection of antibody to foot and

mouth disease virus –infection associated (VIA) antigen in cattle. Sera

in Nineveah Iraq Zanco J.1987;5:2.

6. Al-Bana AS, Shony MO. Foot and mouth disease in Iraqi Native

Gazella: Virus Isolation, serology and characterization.(master’s

thesis) Baghadad University; 1988.

?. ������(��� ��:� ��'(��� 8 ,(�()�2-2.( 3��;��� <$�'� #18A���:�� (�(:�� �;� 8&.�� 9�:�� L�� ��$��� �����»o«. ��(�

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������H���� 8� ��� %����� �#$���� �#$����. 9. Gailiunas P, Cottral GE. Presence and persistence of foot and mouth

disease virus in bovine skin. J Bacteriol. 1966;91:2333–238.

10. Kitching RP, Alexanderson S. Clinical variation in foot and mouth

disease: Pigs Rev Sci Tech Int Epiz. 2002;21:513–518.

11. Kitching RP, Hughes GJ. Clinical variation in foot and mouth disease:

Sheep and goats. Rev Sci Tech Int Epiz. 2002;21:505–512.

��. A�:'��� ��k� 8��1��� OW+� �5��)����(. ,��� 7�� ���C� 9��+� %�H�� ���B 9�8 ��Z��=� � 8&.�� 9�:�� 7��� �. �� .

9��+� %�H�� ,��H���� L��(�� ����G� ��'X���,,��4� . 2 – 0 ��OE����.

Page 24: Iraqi Journal of Veterinary Sciences

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K�� �M =�" I������/*� ������ 4���� OF�� ���/* A� D .

Biotyping of Clostridium Perfringens strains isolated from enterotoxemia cases in

sheep using ELISA technique

M. A. Hamad1, N. Habra

2 and A. Kalb Allouz

3

1 Department of Microbiology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq,

2 Department of Microbiology, 3 Department of Animal Diseases, College of Veterinary Medicine, AL-Baath University, Syria

Abstract

The study was designed to determine the types of Clostridium perfringens and their toxins in sheep with suspected

enterotoxemia in Hama province, Syria. For this purpose, 89 bacterial isolates were isolated from 132 samples collected from

dead and some slaughtered sheep with suspected enterotoxemia and diagnosed with classical diagnostic tests and by using enzyme-linked immunosorbent assay (ELISA) technique to determine the types and toxins of C. perfringens. These isolates

appeared as G+ bulged rods with curved ends and were as positive to lecithinase, gelatin hydrolysis and sugar fermented, as

where negative to catalase, oxidase, and indole. Based on the ELISA results all isolates were C. perfringens types A 84 isolate

(94.38 %), D 3 (3.37 %), and C 2(2.25 %). Clostridium perfringens type A was the dominant type in cases of enterotoxemia in

sheep in Hama province detected by ELISA test. The enterotoxaemia causes considerable economic loss to the sheep industry

particularly in Hama province and generally in Syria. Therefore, it is recommended that a proper vaccination schedule against

enterotoxemia should be implemented for sheep flocks in Hama province. These vaccines should provide adequate protective

immunity against all C. perfringens types specially types A and D.

Available online at http://www.vetmedmosul.org/ijvs

Page 25: Iraqi Journal of Veterinary Sciences

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���.��

1. Quinn PJ, Carter ME, Markey B, Carter GR. Clinical Veterinary

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M, Stackebrandt E and Titball R.EUROPEAN COMMISSION-

European Conce- rted Action QLK2-CT2001-01267: Clostridia in

medical, veterinary and food microbiology Diagnosis and typing. Key

Action 2 – Control of infectious diseases EUR 21463 EN. 2006.

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dairy cows with hemo-rrhagic bowel syndrome or left –displaced

abomasums.JAVMA 2005;227(1):132-138. 21. Quinn PJ, Markey BK, Carter ME, Donnelly WJC, Leonard FC.

Veterinary Microbiology and Microbial Diseases. USA: Blackwell

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Page 30: Iraqi Journal of Veterinary Sciences

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) '()*� �� $+� ,��" ���-$�.�� / �� 0�( ���1 (

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,2 34"�� �)����� 5!�6()� ���( �)���78� �9��� �)����' :����� ,'�;�� �! ���(<� $�"���� ���� #���(<� ��<()�� �4����� ��= �0��*� �>� ( ?�� ��@ ��2�$%� ) 1@ ,'�;�� ��:����� (��( 5��" �(�� A������� ,��� . 5�6C8 D�;

����) E��(� ����+� ,���.��*���(<$�"���� ���� ������ �F% ��)�� � ���)� 5��" ���(���78 '�) ���� �9��� ���(<� ��<()���4����� ��= �0��*� ,'�;�� �"> D��� �!) % 29.34 10 % (���(�� A�2 5H�� ���" ��)�� �@,�% � �! �6�)� 5H�� ,�; �!:����� �"> D���) % 55 0.0% ( ���(�� A�2 5H�� ���" ��)�� �J,@% .5��� A�2 ,����(<*� ,�� &!�( �� �2 E��(���

��� kappa. ,� E(�()� � ��= �0��*� ���(<� A�2 ���(2*� ,"�� K�� �)����� L>M ,2 34"�� �! �4���� �9��� ���7� ���('�)����$�"���� ���� ���(<� ,� *�� �%�<4( �;)� ���(<�" :����� ,'�;�� �! .

Detection of anti brucella antibodies in lambs and goat kids using rose bengal test

and indirect ELISA in Gugjeli - Ninavah province, Iraq

S. A. Esmaeel, B. A. Mohmmed, S. D. Hassan and Kh. J. Hassain

Department of Internal and Preventive Medicine, Collage of Veterinary Medicine, University of Mosul, Mosul, Iraq

Abstract

The object of the study was to detection of anti-brucella antibodies in lambs and goat kids by using Rose Bengal and

indirect ELISA tests. Sera samples were collected from four flocks, including 135 samples (95 from lambs and 40 from goat

kids). The sera examined by rose Bengal test showed negative result in all of flocks, in percentage was zero %. While the

percentage of anti-brucella antibodies using indirect ELISA test in female and male lambs was (29.34 %, 10 %) respectively,

with total percentage 25.3% and in female and male kids was (55 %, 0.0 %) respectively, with total percentage 27. 5%. The

results showed incompatibility between rose Bengal test and indirect ELISA test with kappa value. The study concluded that

indirect ELISA test can be considerable as a screening and diagnostic test instead of rose Bengal test for detection of anti-

brucella antibodies in lambs and kids.

Available online at http://www.vetmedmosul.org/ijvs

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Vet Microbial. 2002;20:81-110.

2. Radostits OM, Gay CC, Hincheliff KW, Constable PD. Veterinary

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3. Fich TA. Intracellular survival of Brucella defining the link with

persistence. Vet Microbial. 2003;92:213-223. 4. Adams LG. The pathology of brucellosis reflects the outcome of the

battle between the host genome and the brucella genome. Vet

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5. Alton GG. In: Nielson K, Duncan JR,eds. Animal brucellosis. Boca

Raton, FL, CRC press; 1990. 379p.

6. Grillo MJ. Transmission of Brucella melitensis from sheep lambs. Vet

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8. Martin SW. The evaluation of tests. Can J Com Med. 1997;41:19-25.

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45-47.

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2000 . 12. Saleem AN, Rhaymah MS, Shamoon GN. Isoltion and Seroprevalence

of ovine brucellosis. Iraqi J Vet Sci. 2004;18(1):31-38.

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14. Quinn PJ, Carter ME, Markey B, Carter GR. Clinical Veterinary

Microbiology. 1 st ed Elsevier LTD. London. 1999;78-79.

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on human brucellosis in Assiut Governorate. Egypt J Immumol

2005;12:49-56.

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A review. Pakistan Vet J. 2007;27(3):145-151.

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Page 35: Iraqi Journal of Veterinary Sciences

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Reproductive efficiency of rats whose mothers treated with lead acetate during

lactation: role of vitamin E

W. H. Yousif and S. T. Abdullah

Department of Physiology Biochemistry and Pharmacology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq

Abstract

Reproductive efficiency of young male and female rats yielded from mothers treated with lead acetate (10 mg/kg B.W) and

vitamin E as an antioxidant (500 mg/kg) during lactation period was studied. 27 pregnant albino rats aged 4-5 months divided

into 3 groups, the first group given 0.2 ml distilled water through oral intubations (control). The second group given lead

acetate (10 mg/kg B.W), while the 3rd group given lead acetate (10 mg/kg B.W) with vitamin E (500 mg/kg B.Wt). Lead

acetate treatment did not cause significant difference (P>0.05) in young male rats body, testes, epididymal (head, tail), prostate

gland, seminal vesicle weights, number of epidiymal sperms count, percentage of live/dead sperms and sperms abnormalities

accompanied with a significant increase (P<0.05) in seminiferous tubules diameter. On the other hand, lead acetate treatment caused a significant decrease (P<0.05) in the duration of vaginal opening with no significant changes (P>0.05) in ovary and

uterus weights. Lead acetate with vit. E cause significant decrease (P<0.05) in body, testes, epididymal (head, body) weights,

number of epididymal sperm count, percentage of live/dead and sperm abnormalities. Concerning young female rats treated

with lead acetate and vit E showed a significant decrease (P<0.05) in body, uterus weights with a significant increase (P<0.05)

in the duration of vaginal opening. Histopathological study revealed changes in the testes such as desquemination in some

epithelial lining of seminiferous tubules and sertoli cells. Undevelopment of localized area of some seminiferous tubules with

blood vessels congestion in both testes and ovaries with hyperemia in ovarian stroma, increase in follicular cells, decrease in

corpus luteum formation, degeneration of ovarian follicles and increase in ovarian adipose tissue. This study concluded that

lead acetate can be transmitted through mother milk to their pups. The dose of lead acetate (10 mg/kg B.W) was enough to

make bad effects on both male and female reproductive systems. Vitamin E (as an antioxidant) found to have no improving

effect in treatment of lead acetate disturbances on reproductive systems of both sexes.

Available online at http://www.vetmedmosul.org/ijvs

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������� �0g0 20 :0�. 3. El-Sayed EA, Ebrahim AZ. Effect of atmospheric lead pollution on

rachitic infants. First scientific conference on Environment and

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�. %�!C������" ' - r�. =!(���� /� �!$�� ��$�� '�O �.5 � -�!1� �� ,� ����!$���4!.����� \78 K� R!� /��!��� .\��� $� "���O�

������ �$�'(���� ��!� �)����� �����.

5. Al-Wakil BNA. Effect of lead exposure on the erythrocyte delta –

amino levulinic acid dehydratase activity. (master's thesis), College of

Medicine, University of Mosul; 1986.

6. Shaper AG, Pocock SJ, Walker M, Thompson AG. British regional

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Environ Med. 1980;6:104-117.

0. %������,�F /�" ���!.� ���!� �8�� W �)���� ���"� /�" . &�"� L ,� �1.�� /�!���� H� ��9� ,���� �.5 ��Scenedesms

quadricanda���� ���� �- ��� L �� 6!" O ��� . ����2 �)�( :�0�A�.

10. Hulse M, John SM, Gene DS, Carl SH, Stanley MP. Environmentally

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Extensor and flexor digit synovial sheath, sac and synovial capsule in the distal part

of the limbs in buffalos and camels and its relation of surgical interference

S. AL-sadi1, G. Alosh

2 and A. AL-Omari

3

1 Department of Anatomy, College of Veterinary Medicine, University of Mosul, Mosul, Iraq, 2 Department of Anatomy, 3 Department of Surgery, College of Veterinary Medicine, University of Al-baath, Hama, Syria

Abstract

Sixty one samples of the distal parts of limbs were obtained from different ages of buffalo and camels of both sex to study

the synovial structures to determine the suitable sites for injection of surgical interference. The result showed that extensor

digit synovial sheath was extend between middle or distal part of metacarpal (metatarsal) to the extensor processes and this

formed with synovial capsule dorsal pouches which serve in surgical interference. The flexor digit synovial sheath extended to palmar (planter) between distal extremity of metacarpal (metatarsal) to the middle of second phalanx in buffalo while in camel

it extended to the proximal extremity of second phalanx, that sheath was formed with suspensory ligament and sessamoid bone

palmar or planter pouches which were serve the surgical interference. Fourth synovial bursa observed situated dorsally

Page 43: Iraqi Journal of Veterinary Sciences

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between the extensor digit laterals tendon and capsule of fetlock joint, forms site of injection during surgical interference,

while the other two synovial bursa were located to palmer (planter) between deep flexor tendon and distal sessamoid bone in

buffalo while in camel these bursa were located between deep flexor tendon and cartilage of the second phalanx, these bursa

were served for surgical interference. The synovial capsule which serve the surgical interference through digit cushion these

were shown extended from the claw capsule. The result show that surgical interference was form six pouches in buffalo and

eight pouches in camel, which formed by synovial structures and the tissue associated with them.

Available online at http://www.vetmedmosul.org/ijvs

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Page 45: Iraqi Journal of Veterinary Sciences

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������

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Page 56: Iraqi Journal of Veterinary Sciences

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Diagnosis of foot and mouth disease by indirect competitive ELISA test in feedlot

calves - in Mosul province

M. A. Abd-Alhameed and M. Sh. Rhaymah

Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Mosul, Mosul, Iraq

Abstract

Indirect competitive ELISA test used, for detection of non-structural protein (3ABC) in 460 serum samples of infected

animals revealed that 421 samples were positive (91.5%) and 39 samples were negative (8.5%), with 91.5% sensitivity and

28.4% specifity. Blood picture revealed significant (P<0.05) increase in total leukocytic count, mean corpuscular volume, mean corpuscular hemoglobin and the percentage of granulocytes, and significant decrease in the total erythrocytic count and

packed cell volume.

Available online at http://www.vetmedmosul.org/ijvs * ����� ���� � ������� ������ �� ���� ���

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Page 60: Iraqi Journal of Veterinary Sciences

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Evaluation the suitable level of standard concentrated ration for fattening growing

Sharabi calves

N. M. Abdullah1, A. K. Nasser

2 and N. Y. Abou

2

1 Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul,

2 State of Board of Agriculture Research, Department of Agriculture Research, Nineveh, Mosul, Iraq

Abstract

Three groups of growing Sharabi calves were used in this study to evaluate the best suitable level of concentrate ration.

Each group consisted of four calves between 8-12 months of age, with an average live body weight around 145 kg. They were

fed for 90 days on concentrated standard ration according to NRC, 1984, at three different levels of 2, 3, 4 % of live body

weight, respectively, consisted of 80:20 concentrate to roughage contained 14% crude protein, 72% TDN and 2.4 Mkcal/kg.

ME. The total gain and amount of consumed ration were measured. At the end of the trial, samples of rumen fluid were

Page 61: Iraqi Journal of Veterinary Sciences

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collected from all calves in order to estimate the activity of bacteria and micro organism. Results showed that the average

amount of the daily ration consumed by the three groups of calves were 3.68, 6.91 and 8.12 kg/calves, respectively. There

were significant differences (P<0.05) in average daily gain among the three groups of calves (311, 869 and 1000 gm, for the

three respective treatments). Also the feed conversion ratio was improved for the second and third groups compared with the

first group and they were 7.96, 8.12 and 11.8 kg. ration/kg live body weight gain, respectively. Ration levels showed no effect

on rumen fluid pH but increased the time of methylene blue reduction (more than 15 mints) and sedimentation activity test

(more than 20 minuets) than their normal standard levels. The cost to produce one kg live body weight gain was about the

same for the second and third groups and it was 50% less than first group and they were 3184, 3248 and 4746 I.D.,

respectively. It was concluded that the fattening regime for growing Sharabi calves on concentrated ration given at 4% of live

body weight for three months had more economic profit.

Available online at http://www.vetmedmosul.org/ijvs

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�$����� ���� �!�� �� "������J.

Page 65: Iraqi Journal of Veterinary Sciences

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��

7. Rosenberger G. Clinical Examination of Cattle. 2nd

ed. Verlage Paul

Parey, Berlin and Hamburg, Germany. 1977. 197-212p.

�. f� I�9 ���� &&��� � %� ����� <:�9 �!����� . $��0� ���0"���0�� %��&��. �� ��� ,,Z� ���� ��:*��� %� ��� "0#

�$����� �$����� �5��. 5. +0� ��1�� % ��, � +0� �� f�� % ��)�� ��Q�# +�% ����9�� .

"#�0�� !���#�� ������ I���� ����� (�7C�� �3��� . %��&�� ?��&�?��=�� ��� �+%��&�� \/�b�� �7C0�� �,� ������ *�����

�5J�. 10. Association of Official Analytical (AOAC). Official Methods of

Analysis, 11th ed., Washington DC. 1970.

11. Arafat EA. A comparative study of the effect of different level of urea

on performance of IRAQI local breed bull calves. [master's thesis],

College of Agriculture and Forestry, University of Mosul; 1977. 12. Murphy TA, Loerch SC. Effects of restricted feeding growing steers

on performance and carcass characteristics. Ohio Beef Cattle Research

and Industry Report. 1993:93-1; 99-110p. 13. Hicks RB, Owens FN, Gill RD, Martin JJ, Strasia CA. Effects of

controlled feed intake on performance and carcass characteristics of

feedlot steers and heifers. J Anim Sci. 1993;68:233. 14. Plegge SD. Restricting intake of feedlot cattle, Oklohoma State Univ.

Stillwater. 1987. 15. Zinn RA, Owens FN. Influence of feed inake level on site of digestion

in steers fed a high concentrate diet. J Anim Sci. 1983;56:471-475.

16. Brown MS, Ponce CH, Pulikanti R. Adaptation of beef cattle on high–

concentrate diets: Performance and ruminal metabolism. J Anim Sci.

2006;84:E25- E33.

Page 66: Iraqi Journal of Veterinary Sciences

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(� �� ���� �� �� ��� � ������ ���� �!)��� �� ���

������ ������� ������� �� � �!�� �� "��� �# ��� �� � ��� $��� %�&

)�'()*� +,�- # .- /�� ������ 0�� 1 �� 2��( ����(

���*��

�( 32�( �45 )�6 �$��� "-��*� ��#7 ,�, 8� �59 � – + 3 )9 3� �� /'/ ;�<� ����� � )(� =*��� .& � 0(�;��>� 5������ =7? �$�� @-�2�9 ;�5 �� 0�� .& A � �# ) 0��NRC� ���� 5������ =7?� ;�5 /� /��� - /�� BC-

) 0�� 0���� ��� () �� �D� % A���)�� $�� )# F���� ,(�� ���� ��& )# ,(�� ,�)Nigella Sativa Meal( )- ) 0�� 0���� ;�< =CG9 H$� ��� % ;�5 � @-�.���(��. =��()�7J(�� $�(C��� K���'6 = )$ 3 )9 - �/ @�

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�-�� .& /'/�� 3� ���� , =- # H$ .O�7J�� ��$(�� KA C#� �#�� -2��� K� 2�� =*����D�� ��� � �D+ � �? �,�� �,� � �,+0�5 �J# /-2� K� 2 �J#� .���(�� ;�5 . K� 2 �$�� ���?��# T (-< C�# .& U C6-� )$�� NO (-�� =�@P9 �#

)- -2��� %��� "-��V� ;�5 �($��� 0���� �� -(�� % 3� -� 0� A���)�� $�� )#"-��>�) 0�� 0���� �� -(��� - /�� 0����� @(�� =- # H$ ����� 3� -� 0� �D�D ����� .���(�� ;�5 �.���5 � -�. NO (-�� =�@P9O �$Q� ��5

.& /'/�� 3� ���� , �-�� = ���& ������$ 7�� = ) � =*� .)����� F78 ,� N(-() �'$9 - #�9D� % ,(�� ,� �O'5 .& A���)�� $�� )# F���� ,(�� ���� ��& )#"-��>� � (-Q� @( C� 3�� ;�5 . �) �/W( !9 ,�� �$���

$ 7�� = C�� .

The effect of substituting Nigella Sativa Meal as a source of protein in the rations of

local rabbits on their productive performance and carcass traits

N. M. Abdullah and A. A. AL-Kuhla

Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, mosul, Iraq

Abstract

Fifteen local male rabbits (6-8 weeks old) were allocated into three nutritional groups. The first group fed standard ration, 5

and 10% of the Nigella Sativa Meal (NSM) were added to the concentrated rations of the 2nd and 3rd groups replacing 36 and

72% of the soybean meal (SBM) protein respectively. The feeding period lasted for eight weeks. Feed consumption and body

weight gain were recorded weekly. At the end of feeding period, all rabbits were slaughtered and carcass traits were studied.

No significant differences were found in total body weight gain and feed conversion rate (475, 502, 478 gm) and (4.8, 4.8, 4.9

kg ration/1 kg wt. gain). Feed cost per 1 kg body gain declined 16% in the 3rd group, which cost 2294 ID, compared with the

1st group (2717) and the 2nd group (2561 ID). No significant differences in all carcass traits were found. Substituting 72% of SBM protein by NSM protein in rabbit ration showed no negative effects on all productive parameters and carcass traits.

Available online at http://www.vetmedmosul.org/ijvs

Page 67: Iraqi Journal of Veterinary Sciences

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.&NRC� ���� )�(. � (�( )# ������ ���� ��& �����,(�� � �0�� �7 � 6�� ���� ��� ��� [ G( .(��� .&

�O'5 = -��$�� 5� 2��� H$ @-9 3�� �&�( U �$>� ����G�� -�* @� X�< � 49 �#)�(. "���� ���29 Y�7�

5 ���� ��& )# ;� X�5 �L �� 5 �C(�� 8� �)9 =$ �W& � �/$ �� ,� = -��$�� 7J( � �� .&5� 2��� ,5 H$ �

\�69 -(�� �� ���� � K�/# K�&�(�� C�# ��� �$( � ���� ��& )# �$�� �&�(� ,� ��0� =)� = �#)# "

� ��6()( .(�� (2�� ��7 �� U� O����� = 5 -��� .&� �7� A���)�� $�� ��7 @-�� �0&=�&�(,� = �# @( )#

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L �-��)D( )��� =� 49 �)+ (;�< - #�< )- @(& G9 �� % 0�5 .&"-��>� .(�� � -�� 8� �59 3 )9 - �/

@(7J(� !9 ,�� ,��@4 K����/W( = C�� ��-�� ;�5 . �) ���� .

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�$�� K�(C�� @- .&� $�(C� K��� � =-2� 3��"-��>� ��(9 P$'�& ��= �-��.& =*��� ,�2�>� , ��� 3� �

=- # H$ �/'/�� @����D ±��� ��� ±�� ���� ± �� ��? .���(�� ;�5 =� (5�� F78,�2�>� =*��� .8

,�2�*�O��( *� "-��V�/'/�� 3� ���� .& .=9�( � K�(& .& ��C�� ��(���/�/���+ 7J( Y�7� "-��>� .&

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A���)�� $�� Nigella Sativa Meal (NSM) F78 =CG9 H$ )#��;�<���� )- ) 0�� 0� � �� % ;�5 � @-�.���(��.& MG�� �# ��� ���� )�.( =��()� 7J(�� K�(&

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Page 68: Iraqi Journal of Veterinary Sciences

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=769� K� ��� )- @& = )$� /'/�� �O'��� ,� T7 �- ")$� & ���)��(. .&� �69 3�� =-2� ��(�� ,� ��

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��#W���� #�� =��4 .(�� �,(�#��� "�0�� � .& �(�� )- )) � $�� $ 7�� ,2� + ,2�A�2�>� ��#W��� / �-5 ,2���

M 7��( × ���( )-�A�2�>� �? ��#W��� =��4�B9���� ������ ���>� A 4$>���6���� . ���?��# T (-9 C�# = )$

,� �$�� ,2�� $�� $ 7��.

��� ����)� :( @ #�(� ��(�� �O'��� = -�#�!� �#�� )& ��� K� ��� B )9 ;�5.(

0���� = -�#�

0����;��>�

Control

ration

0����- /��

� % NSM

0����/� /��

��% NSM

=��� B�� �� �� �� �� "� $��4 �� �� ��

A��C� K�7 �� �� �D �-$�� � 6- �� �� ��

���� ��& )# �� � � �)�� $�� )#A�� � � ��

B*�� � � � M��� ���� �,� �,� �,�

B�#�� ��$ � � � ,� ��� = -� (& �,� �,� �,�

!� �#�� "#�(�� % & ��� K� ��� ��,� ��,D ��,+

� 6�� ,(�� �� ��,� ��,� ��,� �/Q� ]�6()� �,� �,D �,+

[ �>�� 6�� ��,� ��,� ��,� O�7�� =���8� � #�� ��,D ��,� ��,+

� ���� � � � ��G@��� � ���

��#K��)/0�5 �J# ��+� ���� ���+

�� #�� .O��4��� ���(�� ��6()9)CRD ( = - ��$(� .G ��� T7��-�� �� �� ")$� )����� �.(d� !��9�� (6�

��$(� ,#-� ���C��= = C��� 3�� =*��� , �-���� .& )������ .& A � �#� ��(�� F78)��.(

,9 H$ :yij = K�8 4���)j ( �� ���� ,�)i) ( !9 ��K�8 4�( �f = 3�(���� � ��� �)�(��� �ti = �� ���� �/W()i (

K�8 4��� F7@ � 6�� �eij = K�8 4��� . ��(�� W�6��)j ( ,� �� ����)i(.

.�!���

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=*����� �� -(��� = �# ��(�� �O'��� ,� �#�� "� 0( 3� ��� @(�� .&"-��>� @��$( ,� ,#�(- �� .(��� /'/��

O �$< 5 ���� 7J(�� " ) "-��V�H$ =- # ���D� ���� � �����? /"-�9 ����.���(�� ;�5 �/'/�� 3� .

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� 3� ���� .$�� K��#7��F'59 .���(�� ;�5 �) ��� �����.( NO (-�� =� 49;�< & ��� K� ��� �G8 �� �� P�$�� ,)$(

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Page 69: Iraqi Journal of Veterinary Sciences

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= ) 0��� =*���� � $�� $ 7�� ,2� =*��� �� =- # H$ .& �(�� )-.8 ���� ���� ��� � �?��,D� �D,�

���,� %'/�� 3� ����;�5 / � ��� ���� .���(��)� .( �#

C�# 0��(��� NO (-�� ,9T (-9 � $�� $ 7�� ,2� ,� �$�� �J# 8 -�9 =- #"-��V� /� /�� 5������ .& � ;�5 K�7J��� 0���

;�5 �($����� %NSM @(�("-��>� � ;�5 K�7J��� 0��� ;�5 �($���� %NSM =- #� 8'59 "-��V� J��� ;�5 K�7

� @�� =J� H$ ) 0�� 0�����+�� ���� � ���� � -� .���5/ K��#7��� 3� ���� � $�� $ 7�� ,2� ,� �J#F'59.���(�� ;�5 �.

��� ����)�(: �/W( # & G9 )A���)�� $�� � (-Q� = C��� ;�5 �"-��V.

3� ��"-��>�

)������ = C���* � 5������ ;��>Control ration

- /�� 5������ � % NSM

/� /�� 5��������% NSM

.O��( *� ,2��� �����) �?( ��D ± �� ��� ± �� ��� ± �� .O @-�� ,2��� �����) �?( ���� ± �� ���D ± +� ���� ± ��

�#�� -2��� K� 2�� �����) �?( �D� ± �� ��� ± �� �D+ ± �� ����� �#0����� -(��� �) �?( ���D ���� ����

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Page 70: Iraqi Journal of Veterinary Sciences

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� !����

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2. National Research Council. Nutrient requirements of poultry. 9th

revised , National academy press , Washington. D.C. 1994.

�. X��T $�� ��$9 �, $�& .�5 ��$� �# 4 .��7J(��� A�7J. "(#�� ��� 5 ��� �4-���� ������ �� �. ��+�.

4. Khalifa MM. Nigella seeds oil meal as a protein supplement in broiler

diets.(master's thesis), Face. Agric. Alexandria Univ. 1995.

5. EL-Nattat WS, EL-Kady RI. Effect of different medical plant seeds

residues on the nutritional and reproductive performance of adult male

rabbits. Int J Agric Biol. 2007;9:(3).

6. Zeweil HZ. Evaluation of substituting Nigella seed oil meal for soybean meal on the performance of growing and laying Japanese

quail. Egypt. Poultry Sic. 1996;16:451-477.

7. Nasr AS, Attia AI, Rashwan AA, Abdine AMM. Growth performance

of New Zealand White rabbits as affected by partial replacement of

diet with Nigella sativa or soyaben meals. Egyptian J. Rabbits

Sciences. 1996;6:129-141. 8. Zeweil HS, Ahmed MH, EL-Adawy MM, Zaki B. Evaluation of

substituting Nigella seed meal as a source of protein for soybean meal

in diets of New Zealand white rabbits, 9th World Rabbit congress, June

10-13, 2008, Verona–Italy. �. �P # .�5 ����6�� ..( �� i�� 5 � @�� ..(� �$*�� 5 ��) .

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��D+. 10. AOAC. Official methods of analysis. 16

th ed., Association of Official

Analytical Chemists, Washington DC, 1996.

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Page 71: Iraqi Journal of Veterinary Sciences

Vol. 24, No. 1, 2010

Editor-in-Chief Prof. Dr. Fouad K. Mohammad (BVMS, MS, PhD) Editoria Secretary Dr. Nabeel A. Al-Hussary (BVMS, MSc, PhD) Executive Editorial Board Prof. Dr. Mohammed N. Al-Shahery (BVMS, DSc) Prof. Dr. Samih H. Arslan (BVMS, PhD) Prof. Dr. Fadwa K. Tawfeek (BVMS, MSc, PhD) Dr. Talal H. Hussein (BSc, MSc, PhD) Dr. Tariq S. Qubih (BVMS, MSc, PhD)

Page 72: Iraqi Journal of Veterinary Sciences

Iraqi Journal of Veterinary Sciences is printed and organized by:

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Iraqi Journal of Veterinary Sciences (Iraqi J. Vet. Sci.)

ISSN 1607-3894 (Print), ISSN 2071-1255 (Online)

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Introduction

Describes briefly the background of the investigation with updated information and states the aim of the study.

Materials and Methods

Provide sufficient details to enable the experiments to be reproduced. Support the techniques and methods used with references. Investigations on animals must comply with institutional and/or equivalent guides for the care and use of animals. Metric and standard international units should be used in this section and throughout the manuscript. Specify the computer software used for statistical analysis and define statistical terms, abbreviations, and symbols applied. Results Present the results and their significance clearly. Graphs and tables should be self explanatory. Do not repeat in figures or in the text the data presented in tables. Tables and figures should be numbered by Arabic numbers in the order of their mention in the text.

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Master's thesis: Roguskie JM. The role of Pseudomonas aeruginosa 1244 pilin glycan in virulence [master's thesis]. Pittsburgh (PA): Duquesne University; 2005. 111 p.

Journal article on the Internet: Polgreen PM, Diekema DJ, Vandeberg J, et al. Risk factors for groin wound infection after femoral artery catheterization: a case-control study. Infect Control Hosp Epidemiol [Internet]. 2006 Jan [cited 2007 Jan 5];27(1):34-7. Available from: http://www.journals.uchicago.edu/ICHE/journal/issues/v27n1/2004069/2004069.web.pdf

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Page 76: Iraqi Journal of Veterinary Sciences

Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010

Contents

Research articles Page

Detection of Brucella antigen in the aborted ovine fetal stomach contents using a modified

ELISA test

M. I. Al-Farwachi, B. A. Al- Badrani and Th. M. Al-Nima

1

Effect of bone marrow and low power lasers on fracture healing with destruction of both

periosteum and endosteum in rabbits

M. G. Thanoon and S. M. Ibrahim

5

Concurrent aflatoxicosis and caecal coccidiosis in broilers

A. M. Shareef

11

Molds and mycotoxins in poultry feeds from farms of potential mycotoxicosis

A. M. Shareef

17

Prevalence of intestinal ciliate Buxtonella sulcata in cattle in Mosul

T. M. Al-Saffar, E. G. Suliman, H. S. Al-Bakri

27

Clinical, haematological and biochemical studies of babesiosis in native goats in Mosul

E. G. Sulaiman, S. H. Arslan, Q. T. Al-Obaidi, E. Daham

31

Effect of industrial product IMBO® on immunosuppressed broilers vaccinated with Newcastle

disease vaccine

O. G. Mohammadamin and T. S. Qubih

37

Serodiagnosis of Johne's disease by indirect ELISA in ovine

I. M. Ahmed

41

Effect of treating lactating rats with lead acetate and its interaction with vitamin E or C on

neurobehavior, development and some biochemical parameters in their pups

A. A. Hassan and H. M. Jassim

45

Use of saturated sodium chloride solution as a tissue fixative

A. Al-Saraj

53

Page 77: Iraqi Journal of Veterinary Sciences

Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (1-4)

1

Detection of Brucella antigen in the aborted ovine fetal stomach contents

using a modified ELISA test

M. I. Al-Farwachi*, B. A. Al- Badrani* and Th. M. Al-Nima**

*Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Mosul,

** Veterinary Hospital in Mosul, Mosul, Iraq

(Received November 17, 2008; Accepted July 13, 2009)

Abstract

This study was conducted on two flocks of sheep suffering from abortion in Mosul city, Iraq. The clinical findings in ewes

were abortion during the 3-4 months of gestation period in the both flocks. The total percentage of abortion was 11.7 %,

whereas stillbirth percentage was 4 %. Brucella spp. was isolated from four (33.3 %) of the 12 samples (stomach contents of

the aborted fetuses). All culture – positive samples had also positive with direct smears. By a modified enzyme-linked

immunosorbent assay (ELISA), Brucella antigens were detected in the fetal stomach contents of 5 samples. The sensitivity and

specificity of the modified ELISA were 100 % and 87.5 % respectively. The test had a good negative predictive value but only

a moderate positive predictive value. Therefore, the test would be useful for confirming the existence of suspect disease.

Comparison of modified ELISA with bacterial isolation demonstrated a close agreement (Kappa value = 0.92). Of the 12

serum samples from aborted ewes, eight samples were positive with Rose Bengle test (66.7 %), more than 10 samples (83.3 %)

were detected by indirect ELISA test. Keywords: Ovine brucellosis, ELISA, Fetus, Stomach contents.

Available online at http://www.vetmedmosul.org/ijvs

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Page 78: Iraqi Journal of Veterinary Sciences

Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (1-4)

2

Introduction

Ovine brucellosis is usually caused by Brucella

melitensis, less frequently by B. abortus, and rarely by B.

ovis (1,2). It is usually manifested by abortion, with

excretion of the organisms in uterine discharges and in milk

(2,3). Diagnosis depends on the isolation of Brucella spp.

from aborted fetuses, milk or from tissues removed at

postmortem examination (1). Alternatively, specific cell-

mediated or serological responses to Brucella antigens can

be demonstrated (3,4).

The demonstration by modified acid-fast or

immunospecific staining of organisms of Brucella

morphology in abortion material or vaginal discharges

provides presumptive evidence of brucellosis, especially if

supported by serological tests (5). Whenever possible, the

species and biovar should be isolated, and identified by

phage lysis or oxidative metabolic tests, or both, and by

cultural, biochemical and serological criteria (5-7). The recently developed polymerase chain reaction (PCR) and

DNA-probe methods provide additional means of detection

(8,9).

No serological test is appropriate for all epidemiological

situations (6). The buffered Brucella antigen tests (Rose

Bengal plate agglutination test and buffered plate

agglutination test) are suitable for screening herds and

individual animals (10). The reactivity of positive samples

should be confirmed by the complement fixation test or by

enzyme-linked immunosorbent assay (ELISA), both of

which can also be used for both screening and confirmation (11,12). The serum agglutination test is inferior to other

tests in specificity and sensitivity, and is not recommended

if other procedures are available (12). Another

immunological test was developed for detection of brucella

antigen in the bovine fetal stomach contents as agar gel

precipitation and counter – immunoelectrophoresis (13).

Counter – immunoelectrophoresis also was used for

detection of brucella antigen in the stomach contents of

aborted buffalo fetuses (14), while coagglutination test was

used for detection of Brucella antigens in aborted ovine

fetal stomach contents (15). The aim of the study to describe a simple and rapid method (Modified ELISA) for

detection of Brucella antigen in aborted ovine fetal stomach

contents.

Materials and methods

The study was conducted in April 2008 on two flocks of

sheep in the Mosul city (Badoosh and Zumar) covering 120

and 52 sheep for each respectively. Complete examination

was performed to all aborted animals and 12 were subjected

to thorough postmortem examination. The two flocks were not vaccinated against brucellosis.

The following specimens were collected for diagnosis

of the disease:

1. Bacterial isolation trials from stomach contents of the

aborted fetuses were made on the modified Brucella agar

medium supplemented with antibiotic (Himedia, India)

and incubated at 37 oC in an atmosphere of 10 % CO2

and in air for 6 days and then Brucella colonies were identified and typed according to conventional methods

(4). Suspected colonies were further identified and

subcultured on Brucella spp. agar slants. We identified

Brucella spp. isolates according to morphologic

characteristics, microscopic appearance. Brucella spp.

isolates were typed according to their CO2 requirement,

H2S production, and growth in the presence of basic

fuchsin and thionin at final concentrations of 20 µg/ml,

as described (4, 5).

2. Direct smear were prepared from aborted fetal stomach

contents, then stained with Koster stain and examined

under light microscope (16). 3. Detection of the brucella antigen from aborted fetal

stomach contents by modified ELISA: Apart of the

samples was heated for 1 hr at 70 oC and centrifuged.

The precipitates were washed twice with phosphate

buffered saline (PBS) and centrifuged. The precipitates

were then used for ELISA. A hyperimmune antibrucella

serum for using in the modified ELISA was prepared in

sheep by four biweekly injections subcutaneously with

live Brucella vaccine (Rev.1 strain of Brucella

melitensis) produced in (CZ Veterinaria, Spain) with the

standard dose of 1×109 colony forming units (CFU) (17). Modified ELISA was done according to the general

principals of immunoblotting (18). Brifly, 20µl of each

of the samples and controls (BPS) were applied to

nitrocellulose membrane (Amersham). The membrane

was blocked in 5 % skim milk in PBS for 45 minutes.

Membrane was washed three times, 5 min each with

PBS- T (PBS containing 0.05 % Tween 20), was placed

in 1: 100 dilution of anti brucella serum in PBS- T and

incubated at room temperature for 1 hr. The membranes

were then incubated in conjugate goat antimouse IgG

horse – radish peroxidase (Svanovir ®, Sweden) diluted 1: 1000 in PBS – T for 1 hr. The same washing

procedure was repeated after incubation with the anti –

sheep peroxidase conjugate (Svanovir ®, Sweden),

followed by reaction with a chromogen – substrate

solution consisting of 30 µl of 30 % H2O2 in 50 ml of

PBS mixed with 30 mg of 4 – Chloro – 1 – naphtol in 10

ml of cold methanol. The results were determined by

observation of violet – stained spots on the membrane.

4. Serum samples: 12 blood samples from all aborted ewes

(12) were collected by venipuncture at 15 days after

abortion. Rose Bengal (RBT), and indirect ELISA were

used to detect antibodies against brucellosis. The Rose Bengal test was performed as described by (19). A

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3

commercially available indirect ELISA (Brucella – Ab I-

ELISA kit Svanovir ®, Sweden) was used to detect

specific antibodies for Brucella spp. according to

manufacturer´s instruction.

Statistical analysis: The sensitivity, specificity, positive

and negative predictive values were calculated. The Kappa

value was used to evaluate the correlation between modified ELISA and bacterial isolation (20,21)

Results

Clinical examination showed abortion occurred during

the 3-4 months of gestation period in the both flocks. Table

1 illustrate the number of aborted ewes and average of their

gestation period in months. The total percentage of abortion

was 11.7 %, while stillbirth percentage was 4 %.Brucella

spp. was isolated from 4 (33.3 %) of the 12 samples (stomach contents of the aborted fetuses). All culture –

positive samples had also positive results with direct smears

(stained with Koster stain). By modified ELISA, Brucella

antigens were detected in the fetal stomach contents of 5

samples (42.0 %). Other culture – negative samples were

also negative with modified ELISA test (Table 2).

Table 1: Numbers of aborted animals and average of their

gestation period in months.

Gestation

period (Months)

Stillbirth

No. (%)

Abortion

No (%)

Total

lambing Regions

3 - 4 3 (3.6 %) 8 (9.6 %) 120 Badoosh

3 - 3.5 4 (2.0 %) 12 (6.3 %) 52 Zumar

3 - 4 7 (4.0 %) 20 (11.7%) 172 Total

Table 2: Results of the tests used for detection of brucella

antigens in stomach contents of the aborted fetuses.

Modified

ELISA

Direct

smears

Bacterial

culture Samples

- - - 1

+ + + 2

- - - 3

+ + + 4

- - - 5

+ + + 6

- - - 7 - - - 8

- - - 9

+ + + 10

- - - 11

+ - - 12

5 (42.0 %) 4 (33.0 %) 4 (33.0 %) Total

(-) negative results, (+) positive results.

From the results, it appeared that the modified ELISA

has a high sensitivity (100 %), but only moderate

specificity (87.5 %). The test also has a good negative

predictive value(100 %) and moderate positive predictive

value (80 %). Therefore, the test is would be useful for

confirming the existence of suspected disease. Comparison

of modified ELISA with bacterial isolation demonstrated a close agreement (Kappa value = 0.92). Of the 12 serum

samples from aborted ewes, eight samples were positive

with Rose Bengle test (66.7 %), more than 10 samples

(83.3%) were detected by indirect ELISA test.

Discussion Brucellosis is widely regarded as an extremely insidious

disease, demanding the most exhaustive care in diagnosis

and many cases are more difficult to diagnosis than others,

and a few are extremely difficult to pick up, and could

easily be missed altogether (1,2). In this study, Brucella

spp. was isolated from 4 (33.3 %) of the 12 samples (stomach contents of the aborted fetuses). All culture –

positive samples were also positive with direct smears

(stained with Koster stain). By modified ELISA, Brucella

antigens were detected in the fetal stomach contents of 5

samples (42.0 %). Other culture – negative samples were

also negative with modified ELISA test. According to the

results of this study modified ELISA is able to detect

Brucella antigens in fetal stomach content because the

modified ELISA has a high sensitivity (100 % = false

negative rate of 0%), but only moderate specificity (87.5 %

= false positive rate of 12.5%). The test also has a good negative predictive value (proportion of modified ELISA

negative animals which do not have disease = 100 %) and

moderate positive predictive value (proportion of modified

ELISA positive animals which have the disease = 80 %)

(20). Therefore, the test would be useful for confirming the

existence of suspected disease because the test has a few

false positive rate and moderate positive predictive value

(21). Comparsion of modified ELISA with bacterial isolation

demonstrated a close agreement (Kappa value = 0.92).The

Kappa test can be used to measure the level of agreement beyond that which may be obtained by chance. The Kappa

lies within a range between -1 and +1 (20). (20), (21) were

noted that the Kappa value gives no which of the tests is

better and that a good agreement may indicate that both test

are equally good or equally bad.

Acknowledgment

This study was supported by the College of Veterinary

Medicine, University of Mosul, Mosul, Iraq.

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4

References

1. Radostits OM, Gay CC, Hinchcliff KW, Constable PD. Veterinary

Medicine: A textbook of the diseases of cattle, horses, sheep, pigs and

goats. 10th ed. London: Saunders Elsevier; 2007. 966-994p.

2. Cutler SJ, Whatmore AM, Commander NJ. Brucellosis- new aspects

of an old disease. J Applied Microbiol. 2005;98:1270-1281.

3. Garin-Bastuji B, Blasco JM, Marin C. The diagnosis of brucellosis in

sheep and goats, old and new tools. Small Rum Res. 2006;62:63-70.

4. Forbes BA, Sahm DF, Weissfeld AS. Diagnostic Microbiology. 11th

ed. USA: Elsevier; 2002. 196-201p.

5. Songer JG, Post KW. Veterinary microbiology. Bacterial and fungal

agents of Animal Disease. Missouri: Saunders Elsevier; 2005. 200-

206p.

6. Nielsen K. Diagnosis of Brucellosis by Serology. Vet Microbiol. 2002;

90:447-459.

7. Aggad H. Serological studies of animal brucellosis in Algeria. Assiut

Vet Med J. 2003;49(98):121-130.

8. Fekete A, Bantle A, Halling SM. Detection of Brucella by polymerase

chain reaction in bovine fetal and maternal tissues. J Vet Diag Invest.

1992;11:79-83.

9. Gallien P, Dorn C, Alban G, Staak C, Protz D. Brucella species in

organs of naturally infected cattle by polymerase chain reaction. Vet

Rec. 1998;142:512-514.

10. Baum M, Zamir O, Bergman-Rios R, Katz E, Beider Z, Cohen A,

Banai, M. Comparative evaluation of microagglutination test and

serum agglutination test as supplementary diagnostic methods for

brucellosis. J Clinic Microbiol. 1995;33(8):2166-2170.

11. Blasco JM, Garin-Bastuji B, Marin CM, Gerbier G, Fanlo J, Jimenez

de Bagues MP, Cau C. Efficacy of different rosebengal and

complement fixation antigens for the diagnosis of Brucella melitensis

in sheep and goats. Vet Rec. 1994;134:415-420

12. Minas A, Stournara A, Christodoulopoulos G, Katsoulos PD.

Validation of a competitive ELISA for diagnosis of Brucella

melitensis infection in sheep and goats. The Vet J. 2007;111:102-105.

13. Chand P, Gupta RK, Khanna RN, Sadana IR. Detection of Brucella

antigen in bovine fetal stomach contents by agar gel precipitation and

counter immunoelectrophoresis. Res Vet Sci. 1987;43(1):132-136.

14. Chand P, Khanna RN, Sadana JR. Counter – immunoelectrophoresis

for the detection of Brucella antigen and antibodies in the diagnosis of

brucellosis in buffaloes. J Appl Bacteriol. 1988;64(5):445-449.

15. Erganis O, Kaya O, Hadimli H, Guler L. Rapid diagnosis of ovine

Brucella,Campylobacter and Salmonella infections from fetal stomach

contents by coagglutination test. Small Rum Res. 2002;45:123-127.

16. Coles EH. Veterinary Clinical Pathology. 4th ed. Philadelphia: W.B.

Saunders Company; 1986. 43-79p.

17. Little SF, Ivins BE, Fellows PF, Friedlander AM. Passive protection

by polyclonal antibodies against Bacillus anthracis infection in guinea

pigs. Infect Immunol.1997;65:5171–5175.

18. Golds RA, Kinde TJ, Osborne BA, Kaby J. Enzyme linked

immunosorbent assay: In immunology 5th ed. Philadelphia: W.B.

Saunders Company; 2003. 43-79p.

19. Alton GG, Jones LM Angus RD, Verger JM. Techniques for the

brucellosis laboratory. Paris: INRA;1988.

20. Thrusfield M. Veterinary Epidemiology 2nd

ed. Oxford: Blackwell

Science Ltd; 1995. 95- 112p.

21. Pfeffer D. Veterinary Epidemiology. An Introduction. NewZealand:

Institute of Veterinary, Animal and Biomedical Sciences.Massey

University; 2000. 32-34p.

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5

Effect of bone marrow and low power lasers on fracture healing with

destruction of both periosteum and endosteum in rabbits

M. G. Thanoon and S. M. Ibrahim

Department of Veterinary Surgery and Theriogenology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq

(Received December 18, 2008; Accepted July 13, 2009)

Abstract

Ten mature rabbits of local breed were used in this study; weighing between 1.5 to 1.75 kg and aged about 1–2 years.

These animals were divided into two equal groups; in group A destruction of both periosteum and endosteum was done one

centimeter from each side of mid-shaft femoral bone fracture, then sufficient amount of autogenously bone marrow was

injected directly at the fracture site after immobilization by intramedullary pin. In group B a similar procedure was achieved as

in group A, but in additional to that He-Ne infrared laser therapy was used for several sessions. The result of radiological

findings indicated that, the fracture healing occurred within group B at fifteen weeks, whereas in group A the healing occurred

at eighteen weeks after operation. The implantation of autologous bone marrow enhanced the fracture healing, whereas using

of combinations of autologous bone marrow and He-Ne infrared laser therapy hastened the healing.

Keywords: Fracture, Bone marrow, Laser, Rabbit. Available online at http://www.vetmedmosul.org/ijvs

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Introduction

Studies suggested that, the periosteum was more

essential than endosteum in callus formation. If there is

destruction of both periosteum and endosteum the results

will be absence of bone formation around the destructed

area (1,2). Bone marrow stem cells residing in the bone

marrow are the progenitors for osteoblasts (3-5).

Autologous bone marrow is a safe, simple and reliable

method for treating delayed and nonunion (6-8). Low

energy laser irradiation has positive effects on bone fracture

healing. The mechanisms by which low-energy laser

irradiation affect on bone healing still not clear (9). He-Ne

low-level energy treatment accelerated the deposition of

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (5-9)

6

bone matrix and increases vascularization after seven days

of irradiation (10). Direct irradiation of the whole injury

with He-Ne laser on days five, six post-injury altered the

osteoblast and osteoclast cell population (11,12). Studies on

animals were performed on the effect of low-level laser of

fracture healing indicated that, the laser enhanced healing

(13-15). The aim of this study was to investigate the effect

of bone marrow and low-level laser energy on healing of

femoral fracture after destruction of both periosteum and

endosteum in rabbits.

Materials and methods

Ten mature rabbits of both sex of local breed aged 1-2

years and weighing between 1.5-1.75 kg. All rabbits

tolerated on the same manner of condition and housing

along the periods of study. The experimental design divided

into two equal series; Group A: five animals underwent

transverse fracture at the mid-shaft of femur bone then

about one centimeter of both periosteum and endosteum has

been destroyed and treated with sufficient amount of bone

marrow, Group B: five animals underwent the same

conditions of group A in additional to that irradiated with

He-Ne infrared laser for several sessions.

Surgical procedure was performed under general

anesthesia by using protocol of anesthesia include atropine

sulphate 1mg/kg b.w. intramuscularly as a premedication

after fifteen minutes later a mixture of Xylazine and

Ketamine hydrochloride given intramuscularly at a dose of

5 and 20 mg/kg b.w., respectively. The surgical site was

prepared under aseptic technique. Skin incised directly at

the mid-shaft of femoral bone to expose the bone and

osteotomy was performed, about one centimeter of

periosteum and endosteum were destroyed from both sides

of fractured end with bistoury scalpel. The fractured ends

fixed by stainless steel 2.4φ×120 mm intramedullary

pinning. Muscles sutured by simple continuous using catgut

NO. 2/0, then sufficient amount of bone marrow which

aspirated from femoral head of other side (using needle

gauge 18) was injected at the site of fracture in-between the

stitches, after that, suture the fascia and skin. While in

group B in additional to that, animals were exposed to

radiation with He-Ne infrared lasers (the He-Ne wave

length: 632.8 nanometers, frequency: 50-60 Hz were the

wave length and frequency of infrared are 904 nanometers,

700-1200 Hz respectively) applied at the fractured site, in a

series of eight sessions (1-3 , 5, 7, 9, 11 and 13 days) after

operation, the total dose of He-Ne infrared lasers for each

animal was 3.6 joule/cm2.

Post-operative care by using penicillin-streptomycin at a

dose 10000 I.U, 10 mg/kg b.w. injected intramuscularly for

four days. Clinical examination and weekly radiological

studies were performed for the fracture site to determine the

stage of fracture healing.

Results

The results of this study showed that mild inflammation

at the site of operation during the first few days after

operation, theses signs represented by; pain, red, hot,

swelling, but subsided quickly during fifth days in group A

and fourth days in group B. The callus formation was tested

clinically by stress of the fracture site by finger palpation, at

the end of 2nd

week in group A and at the end of 3rd

week

on B. It has noticed that the callus formation around the

fractured area in group A larger than in group B. The callus

disappeared clinically at eight and six weeks in group A

and B respectively. The radiographical finding revealed

that, the healing occurred at the eighteenth and fifteenth

week in group A and B respectively, which characterized

by the invisibility of fracture line and the bone taken about

the normal shape. The periods of fracture healing are

summarized in (Table 1).

Discussion

The periosteum, endosteum and bone marrow provided

cells that proliferate and differentiate into osteoblasts,

chondroblasts and fibroblasts, which contribute to new

bone formation (2,3). It was demonstrated that autologous

bone marrow contains mesenchymal stem cells that are able

to form bone, cartilage and enhancement of the

osteogenesis in fracture healing (5,6).

In our study the destruction of both periosteum and

endosteum at the fracture site and injection of sufficient

amount of autologous bone marrow leaded to healing at

about eighteenth week. This observation was confirmed by

other authors (5,7). Who said that, the autologous bone

marrow enhanced of bone healing. On the other hand this

result agreed with other authors (1,2). Who recorded that,

the periosteum and endosteum are important in fracture

healing, which when both removed at the fracture area

resulting in absence of callus formation.

Low level laser therapy can accelerate the bone

regeneration. This was appeared by altered osteoblast

activity at the fracture site as reflected by alkaline

phosphatase activity (11). Laser irradiation also caused a

significant increase in calcium accumulation at the fracture

site (13). In this study, the results confirm these facts,

which revealed that, the laser therapy enhanced the fracture

healing in-group B when compared with group A. The

destruction of both periosteum and endosteum at the

fracture site then treated with a combination of laser and

autologous bone marrow as in group B, which exhibited

better results than using autologous bone marrow alone by

other authors (6-8). We believed that this combination of

autologous bone marrow and He-Ne infrared laser therapy

may be not mentioned in literature.

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7

Table 1: Radiographic findings of group A and group B.

Week Group A Group B

1 Slight proliferation of periosteum at a distance from

fracture site. Clear fracture line.

Slight proliferation of periosteum at a distance from

fracture site. Clear fracture line.

2 External callus become active and migrated in to the

fracture site. Clear fracture line.

Slight increase of external callus around the fracture site.

Clear fracture line.

3 Increase internal callus and partially bridged fracture

site. Clear fracture line.

External callus around the fracture site. Visible fracture

line.

5 Thick external callus bridged the fracture site. Still

clear fracture line (fig. 1).

Thin external callus bridged fracture site. Still visible

fracture line.

8 External callus began to absorb. Still visible fracture

line (fig. 2).

Good alignment of the cortex at the fracture site. Bone

almost taken about the normal shape. Disappearance of

fracture line (fig. 3).

10 Thin external callus around the fracture site. Invisible

fracture line.

Thin external callus around the fracture site. The bone

taken about the normal shape (fig. 4).

12 The bone began to taken about the normal shape. Thin

external callus still around the fracture site (fig. 5).

The bone taken the normal shape. Still appear very thin

external callus around the fracture site (fig. 6).

15 Still visible external callus. Bone almost taken the

normal shape.

The bone is normal in shape. Still very thin external

callus around the fractured area (fig. 7).

18 Slight invisible callus. Bone may be taken about the

normal shape.

Fig. 1: Radiographic picture shows thick external callus

bridged the fracture line. Still clear fracture line, five weeks

after operation in group A.

Fig. 2: Radiographic picture shows external callus began to

absorb. Still visible fracture line, eight weeks after

operation in group A.

Fig. 3: Radiographic picture shows bone almost taken the

normal shape. Invisible fracture line, eight weeks after

operation in group B.

Fig. 4: Radiographic picture shows thin external callus

around the fracture site. The bone taken about normal

shape, ten weeks after operation in group B.

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8

Fig. 5: Radiographic picture shows the bone began to taken

about the normal shape. Thin external callus around the

fracture site, twelve weeks after operation in group A.

Fig. 6: Radiographic picture shows the bone taken the

normal shape. Still appear very thin external callus around

the fracture site, twelve weeks after operation in group B.

Fig. 7: Radiographic picture shows normal shape of bone.

Very thin external callus around the fracture area, fifteen

weeks after operation in group B.

The radiographic findings revealed that, the healing of

the fractured bone is noticed in eighteenth, fifteenth week

in group A and B respectively. These findings may be due

to the He-Ne infrared laser therapy enhancement of

osteogenesis and hasten the healing. This result was agreed

with other authors (11,13). Who said that, the low level

laser therapy can accelerate the osteogenesis. The external

callus was larger in animals of group A than in group B,

this may be due to the He-Ne infrared lasers causes high

activity of the source of osteoblasts and mesenchymal stem

cells of autologous bone marrow for direct formation of

bony material to fill the gap of fracture line instead of

cartilage or fibrocartilage tissue formation as seen in

common stages of fracture healing. The bone return to the

normal feature approximately in eighteenth week in group

A and fifteenth week in group B, this variation of period

between these both groups may be due to the action of

combination of autologous bone marrow and He-Ne

infrared laser which accelerate the osteoblasts and

osteoclasts activity to deposition and resorption of bone

matrix.

In conclusion of this study which revealed that, the

using of autologous bone marrow was enhanced the

fracture healing. While using of combinations of

autologous bone marrow and He-Ne infrared laser therapy

caused more hastened healing.

Acknowledgement

This study was supported by the College of Veterinary

Medicine, University of Mosul.

References

1. Kojimoto H, Yasui N, Goto T, Matsuda S, Shimomura Y. Bone

lengthening in rabbits by callus distraction. The role of periosteum and

endosteum. J Bone Joint Surg Br. 1988;70(4):543-549.

2. Guichet JM, Braillon P, Bodenreider O, Lascombes P. Periosteum and

bone marrow in bone lengthening: a DEXA quantitative evaluation in

rabbits. Acta Orthop Scand. 1998;69(5):527-531.

3. Reynders P. Intra-osseous injection of concentrated autologous bone

marrow in 62 cases of delayed union. Folia Traumat Lovaniensia.

2003;108-116.

4. Porter SE, Hanley EN. The musculoskeletal effect of smoking. J AM

Acad Orthop Surg. 2001;9(1):9-17.

5. Srouji S, Livne E. Bone marrow stem cells and biological scaffold for

bone repair in aging and disease. Mech Ageing Dev. 2005;126(2):281-

287.

6. Sim R, Liang TS, Tay BK. Autologous marrow injection in the

treatment of delayed and non-union in long bones. Singapor Med J.

1993;34(5):412-417.

7. Liang YT, Zhang BX, Lu SB. Experimental study and clinical

application on osteogenesis of percutaneous autogenous bone marrow

grafting in bone defects. Zhongguo Xiu Fu Chong Jian Wai Ke Za

Zhi. 1999;13(3):148-151.

8. Connolly JF, Guse R, Tiedeman J, Dehne R. Autologous marrow

injection as a substitute for operative grafting of tibial nonunions. Clin

Orthop Relat Res. 1991;(266):259-270.

9. Ninomiya T, Miyamoto Y, Ito T, Yamashita A, Wakita M, Nishisaka

T. High-intensity pulsed laser irradiation accelerates bone formation in

metaphyseal trabecular bone in rat femur. J Bone Miner Metab.

2003;21(2):67-73.

10. Garavello I, Baranauskas V, da Cruz-Hofling MA. The effects of low

laser irradiation on angiogenesis in injured rat tibiae. Histol

Histopathol. 2004;19(1):43-48.

11. Yaakobi T, Maltz L, Oron U. Promotion of bone repair in the cortical

bone of the tibia in rats by low energy laser (He-Ne) irradiation. Calcif

Tissue Int. 1996;59(4):297-300.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (5-9)

9

12. Barushka O, Yaakobi T, Oron U. Effect of low-energy laser (He-Ne)

irradiation on the process of bone repair in the rat tibia. Bone J.

1995;16(1):47-55.

13. Luger EJ, Rochkind S, Wollman Y, Kogan G, Dekel S. Effect of low-

power laser irradiation on the mechanical properties of bone fracture

healing in rats. Lasers Surg Med. 1998;22(2):97-102.

14. Guzzardella GA, Fini M, Torricelli P, Giavaresi G, Giardino R. Laser

stimulation on bone defect healing: an in vitro study. Lasers Med Sci.

2002;17(3):216-220.

15. Khadra M, Kasem N, Haanaes HR, Ellingsen JE, Lyngstadaas SP.

Enhancement of bone formation in rat calvarial bone defects using

low-level laser therapy. Oral Surg Oral Med Oral Pathol Oral Radijol

Endod. 2004;97(6):693-700.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (11-16)

11

Concurrent aflatoxicosis and caecal coccidiosis in broilers

A. M. Shareef

Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, Mosul, Iraq

(Received January 27, 2008; Accepted July 13, 2009)

Abstract

A farm of two broiler flocks fed from one day old to 56 days of age a diet naturally contaminated with high level of

Aflatoxin (AF), were suffered from caecal coccidiosis at 35 days of age Aflatoxin levels in feed commodities and mixed feed

were determined using ELISA test. Results of ELISA showed that the average level of corn, soybean and mixed feeds contamination with aflatoxin were 1915, ppb, 229 ppb and 860 ppb respectively. Diagnosis of caecal coccidiosis was

confirmed by clinical signs, post-mortem findings, fecal oocyst excretion per gram, oocyt size, morphological characteristic of

eggs, lesion score and schizonts size. The significance of AF as predisposing factor to coccidiosis infection was discussed.

Keywords: Coccidiosis, Aflatoxin, Broilers.

Available online at http://www.vetmedmosul.org/ijvs

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Introduction

Poultry aflatoxicosis was first reported when 100000

turkey poults in the United Kingdom in 1960 died

following the ingestion of poultry feed containing Brazilian

groundnut cake which led to the discovery of a group of

compounds now called the aflatoxins (1). Soon afterwards,

however, aflatoxicosis became a worldwide problem (2-4).

Mycotoxins are secondary metabolites produced by fungi of various genera when they grow on agricultural products

before or after harvest or during transportation or storage.

Mycotoxins are regularly found in feed ingredients such as

maize, sorghum grain, barley, wheat, rice meal, cottonseed

meal, groundnuts and other legumes. Most are relatively

stable compounds and are not destroyed by processing of

feed and may even be concentrated in screenings. Naturally

occurring toxins present a different problems (5,6).

Mycotoxins cannot be avoided entirely or eliminated from

animal feeds because the molds that create them occur

naturally in grains and other food commodities Aflatoxins

B1, B2, G1, and G2 are mycotoxins that may be produced by three moulds of the Aspergillus species: A. flavus,

parasiticus and A. nomius (7) Aflatoxin B1 is an

immunosuppressive mycotoxin for cell-mediated immunity

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12

(CMI), humoral immunity, suppression of peritoneal

macrophage phagocytosis and the primary splenic antibody

response (8-11). Aflatoxin has been reported to increase the

severity of avian infectious diseases such as salmonellosis

(12), crop mycosis (13), aspergillosis (14), and coccidiosis

(15).

Avian coccidiosis is the major parasitic disease of poultry with a substantial economic burden on the cost the

industry (16). In-feed medication for prevention and

treatment contributes a major portion of these losses in

addition to mortality, malabsorption, inefficient feed

utilization and impaired growth rate in broilers, and a

temporary reduction of egg production in layers. The

pathogenicity of coccidia depends largely on the successful

replication of developing parasites inside the host.

Theoretical estimates indicate that a single oocyst of a

virulent species such as E. tenella could yield 2,520,000

invasive parasites after the 2nd merogony stage (17). The

profuse bleeding in the ceca is a characteristic feature of E.

tenella infection due to its extensive destruction of the

mucosa with histological lesions (18). In general, young

animals are more susceptible to coccidiosis and more

readily display signs of disease, whereas older chickens are

relatively resistant to infection (19). Young animals which

recover from coccidiosis may later be able to partly

compensate for the loss of body growth, but their growth

potential remains severely compromised. Because the life

cycle of Eimeria parasites is complex and comprised of

intracellular, extracellular, asexual, and sexual stages, host

immune responses are quite diverse and complex. After invasion of the host intestine, Eimeria elicit both

nonspecific and specific immune responses which involve

many facets of cellular and humoral immunity (20,21)

Caecal coccidiosis of broilers has been extensively studied

(22), and as the worlds poultry industry has developed the

disease has continued to be of major economic importance

(23). Interactions occur between chicken caecal coccidiosis

and other diseases, caused by various pathogens (24).

Among those other diseases, aflatoxin intercurrent with

coccidiosis are an increasing health risk to poultry and

poultry coccidiosis due to primary exposure to aflatoxin had been reported previously (25-30). A natural broiler

aflatoxicosis or broiler caecal coccidiosis had been reported

in mosul province (4, 31).

The natural concurrence of these both cases in broilers

is what we are tried to describe in a farm of two broiler

flocks, in Mosul province, Iraq.

Materials and methods

Chicks and diet:

A broiler farm, in Al-Hamdanyea / Mosul province,

reared on letter 8600 and 9650 broilers in two separate house, had been claimed at 35 days of age from mortality

rate of 26% with signs of reduction in feed consumption,

depression, ruffled feathers, closed eyes, stunted growth,

and profuse bloody diarrhea. These birds were fed a

commercial diet, with 22% protein and 3060 kcal/kg

metabolizable energy, without coccidiostatic or Aflatoxin

binder additives.

Pathological examination:

Forty chicks from the two flocks were necropsed for

pathological examination. These birds were scored for cecal

lesions as devised by (32) as follows; Score 0: no gross

lesions; Score1: very few scattered petichiae on the caecal

wall with normal caecal contents; Score 2: lesions are more

numerous, blood in content, wall is somewhat thickened,

normal caecal content; Score 3: large amounts of blood or

caecal cores, caecal wall greatly thickened, little if any fecal

content in ceca; Score 4: cecum is greatly distended, blood

or caseous core, no fecal debris, dead birds; +4.

Ten of these dead chicks were subjected to caecal mucosal scraping for schizont determination. Oocyst size

and shape were determined from about 50 predominant

oocysts. A concentration McMaster technique has been

used for calculation of average fecal oocyst excretion per

gram (33).

Feed sampling: Twenty five feed samples (each 1 kg) were taken from

the broiler farm feed store, five samples of corn, barley,

wheat, soybean and mixed feed according to (34).

Aflatoxin assay:

The levels of aflatoxin contamination of feed samples

were determined by the method of direct competitive

enzyme-linked immunoassay using Neogen extraction kit

(Neogen Corporation). Results of the yield optical densities

of the controls and samples were obtained by using

computerized Neogen Verotex Software program version

2.0.16 (Neogen Corporation).

Results

The most prominent lesions seen in 80 necropsid birds

in both flocks were summarized in (Table 1), in which

pathological changes were mainly seen in the liver and two

caecai. Those in the liver were characterized by creasy,

yellow, ocher discoloration, with scattered areas of sub

capsular hemorrhage. The caecal lesions were characterized

by accumulation with varying quantities of blood and

caseous necrotic materials (Figure 1). From table 1, it is

evident that 5% of necropsed broilers in both flocks show

(+) intensity of liver discoloration. More liver discoloration

intensity (++) were recognized in both flocks with 22.5% in

flock No.1, and 24% in flock No.2. Similar percentage of liver discoloration changes intensity (+++) were noticed in

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13

both flocks. Higher percentage (37.5%) of liver intensity

(++++) was recorded in flock No. 1, than that of 35% in

flock No. 2. The intensity of sub capsular hemorrhage in

livers of necropsed broiler chicks in both flocks, showed

that only 2.5% of the necropsed birds in flock No. 1 with

sub capsular hemorrhage of (+) was recorded, in

comparison of 10% in flock No. 2. More sub capsular hemorrhage intensity (++) was noticed in flock No. 1

(27.5%), compared with 25% in flock No. 2. Still more

intensity of (+++) in flock No. 1 of 35% was recorded,

compared with 30% in the other flock. The same picture

was continued with the highest sub capsular hemorrhage

intensity of (++++) was also recorded in flock No. 1 with

37.5%, while it was 35% in flock No. 2.

Table 1: Number of necropsed birds, their percentages and

intensity of pathological changes seen in livers of two

broiler flocks.

Broiler flocks

Flock No. 1 Flock No. 2

Liver

lesions

No

. of

nec

rop

sed

bir

ds

%

Inte

nsi

ty

No

. of

nec

rop

sed

bir

ds

%

Inte

nsi

ty

2/40 5 + 2/40 5 +

9/40 22.5 ++ 10/40 5 +

14/40 35 +++ 14/40 35 +++

Dis

colo

rati

on

(Yel

low

och

er)

15/40 37.5 ++++ 14/40 35 ++++

1/40 2.5 + 4/40 10 +

11/40 27.5 ++ 10/40 25 ++

13/40 35 +++ 12/40 30 +++

Su

b c

apsu

lar

hem

orr

hag

e

15/40 37.5 ++++ 14/40 35 ++++

Individual and mean caecal lesion scores (Mean lesion

score, MLS) (MLS = Sum of scores/number of birds) are

presented in Table 2. From table it is clear that the total

mean score lesions in the first flock was 3.5, i.e., higher

than that MLS in the flock N0. 2 (3.05). The distributional

percentages of MLS in both broiler flocks are illustrated in Figure 2. From this table it is clear that score 4 represents

73% in flock No.1, compared with 52% in flock N0.2.

Score 3 in flock No. 2 was about double that in flock

N0.1was about, they were 28 and 16% respectively. Two

percent of score 2 was recorded in both broiler flocks.

Relatively similar percentage for both broiler flocks 1 and

2, and they were 10 and 8% respectively. Nor score lesion

of (0) was reported in flock No. 1, while the score was

higher ten times in flock No.2.

Figure 1: Necropsy liver and caecal lesions of broiler 35

days of age, showing creasy, yellow-ocher discolored liver,

with ballooned two caeci, filled with free blood.

Parasitological findings of both flocks are summarized

in Table 3. The MLS in both flocks was 3.26, with fecal

oocyst excretion per gram X1000 = 636. The morphological

features of examined caecal oocysts were identical for

Eimeria tenella.

Aflotoxin assay of feed commodities taken from both broiler flocks are presented in Table 4. Of these

commodities corn samples were heavily contaminated with

AF. The range of contamination was between 831-3485

ppb, with average concentration of 1915ppb.Soybean

samples were also reported to be contaminated with AF but

at a lower rate than that of corn. Similar rate of

contamination was noted, with range occurred between

220-238ppb, and an average of 229 ppb. No AF

contamination was detected in both wheat and barley

commodities. Mixed feed was consequently contaminated

from the contaminated ingredients made from them, corn

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14

and soybean. They were contaminated in a rate of 720-1006

ppb, with an average of 860 ppb.

Table 2: Individual and mean total caecal score lesion of

broiler chicks in both flocks.

Broiler flocks

Caecal score

lesion

No. of birds in

flock No. 1

No. of birds in

flock No. 2

0 4

1 4 3

2 1 1

3 6 10

4 29 22

Mean lesion

score(MLS) 140/40 = 3.5 122/40=3.05

Figure 2: Percentages of caecal score lesions in both broiler

flocks.

Table 3: Summary of the parasitological examination of

necropsed birds (Average findings of broilers in both

broiler flocks).

Parameters Results

Mortality 26%

Fecal oocyst excretion per

gram X1000 636

Oocyt size width X length

(µm) 26 X 23

Mophological characteristic

of eggs Broadly ovoid

Mean lesion score for 80

dead birds 262/80 = 3.27

Schizonts maximum in µm 55.0

Table4: Aflatoxin contamination of feed commodities (ppb)

in of both broiler flocks.

Feed commodities

Corn Soybean Wheat Barley Mixed

feed

Sample

No.

Concentrations of AF ppb

1 3485 238 ND � ND 1006

2 3485 233 ND ND 931

3 931 228 ND ND 831

4 831 224 ND ND 753

5 866 220 ND ND 720

Mean 1915 229 ND ND 860

�ND= not detected

Discussion

Aflatoxin was reported to increase susceptibility to

coccidiosis under experimentally prior exposure poultry to

Aflatoxin (25-30,35). To our knowledge, no report is reported here in Mosul province referring to a natural

concurrent secondary caecal coccidiosis due to

contamination of broiler feed commodities with Aflatoxin.

In this study, although anticoccidial agent was not

included in the diet of broiler flocks as prophylactic

measure, but the presence of high natural Aflatoxin

contamination levels (860 ppb) of the mixed feed used in

the feeding of these flocks, could largely impose a great

stress on the birds immune system against coccidial

infection.. This relation is supported by (22), who stated

that 380 ppb AF or more is responsible for a significant reduction in cell mediated immunity (CMI). This effect on

CMI include (a) macrophages, (b) natural killer (NK) cell

lymphocytes and (c) two types of T-derived lymphocyte

population (CD4+and CD8+) (35,36). Aflatoxin causes a

substantial diminution in phagocytic activity by

macrophages (37). Macrophages, CD4+ and NK cell

lymphocytes dominate the CMI reaction upon primary

contact/infection of coccidiosis, while CD8+ cells multiply

particularly upon second contact (reinfection) of coccidiosis

(38). CD4+ and CD8+ cells are cytotoxic and cytolytic, this

means they can kill and lyses parasitic stages during

coccidiosis cycle (39). However, it was reported that specific serum antibodies might also play a part in

protecting chickens against coccidiosis (40) Antibodies,

especially local immunoglobulin A (IgA) in the intestinal

lumen can bind and damage sporozoites, causing loss in the

ability of extra cellular differentiation and thereby

preventing parasite invasion and intracellular development

(41). So, antibodies, although play a minor role as a

protective component against coccidiosis compared with

the role of cell mediated immunity, but it is still negatively

0 1 2 3 4 1 2 3 4

Caecal score lesions Flock 1 Flock 2

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15

affected by Aflatoxin, since their production was reported

to be decreased by exposing birds to high toxin levels from

300 to 6000 ppb (42). The other possible roles of AF in

predisposing broilers here to coccidial infection is the toxin

reduction effect of vitamin A level in chicken liver and

serum (43). Here, although vitamin A level was not

measured in liver or serum, but it is highly possible that vitamin A deficiency is likely to occur due to feeding

highly AF contaminated diet. Since, it is well known that

disease resistance is a function of cell differentiation, and

one of the primary function of vitamin A is to maintain

proper epithelial tissue differentiation and prevent epithelial

keratinization which occurs in vitamin A deficiency and it

is important for enhancing both cellular and humoral

immunity and enhance phagocytosis activity, and for

maintaining cellularity of lymphoid organs (44,45). The

possible reduction of vitamin A may be related to the high

mortality rate reported here, since vitamin A has been

reported to reduce mortality in chicks infected with coccidial oocyst from E- tenella and E- acervulina (46).

The high coccidial pathogenicity noticed in our study,

expressed by high lesion score of 3.27, could be related, to

high AF diet contamination (15), and the high fecal oocyst

excretion per gram (636000) oocyst/gram could also be

attributed to this contamination. This effect was supported

by (2,28), who confirmed that Aflatoxicosis was

responsible for increased oocyst production and

reproduction potential during coccidiosis. It is evident from

the parasitic parameters examined in this study, that lesion

score was found to be directly proportional to the mortality and oocyst count per gram of faeces/ dropping. Any way, it

should be stressed that other mycotoxins like ochratoxin, T-

2 toxin, and fumonisins may be present or contaminate feed

commodities or mixed feed samples in our study, but the

estimation was tried only with the most effective

immunosuppressant mycotoxins, Aflatoxin. Moreover, no

attempt was carried out to isolate immunosuppressive

viruses like reo virus and Merk disease virus to elucidate

their possible role if they were present in exacerbating

caecal coccidiosis beside aflatoxin on broiler chickens.

From all above, and under field conditions, it seems that mycotoxins and here AF are highly stressful, when fed

for extended period of time to birds on their immune

system, which may be weakened to the point where it

cannot control the organism and set the stage for the

development of coccidiosis. So to prevent Aflatoxicosis and

coccidiosis in poultry, sorbents like sodium bentonite and

hydrated sodium calcium aluminosilicates should be added

to their diet in order to decrease Aflatoxin severity (47), and

by using coccidiostats/coccidiocidal drugs, or by Appling

an alternative system of vaccination against coccidiosis..

Acknowledgement

The author thanks the College of Veterinary Medicine,

Mosul, Iraq, for providing all supports for this study.

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17

Molds and mycotoxins in poultry feeds from farms of potential

mycotoxicosis

A. M. Shareef

Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, Mosul, Iraq

(Received June 19, 2008; Accepted July 13, 2009)

Abstract

Forty five finished poultry feed samples, collected from different broilers, broiler breeders and layers farms were divided

into two parts, for mycological and mycotoxins examination. In counting of molds, dilute plate technique was used, whereas

feed parts were used for mycotoxin estimation, they were subjected to four standard kits of Aflatoxin, Ochratoxin, T-2 toxin

and Fumonisins. Mold counts were around 105 cfu.g-1 sample. Fourteen mold genera were recovered. From the systematic point of view, 2 genera belonged to Zygomycetes (i.e. Mucor, Rhizopus,), 1 genus belong to Ascomycetes (i.e. Eurotium); the

majority, within so-called mitotic fungi (formerly Deuteromycetes), encompassed 11 genera (i.e. Acremonium, Alternaria,

Aspergillus, Fusarium, Paecilomyces, Penicillium, Scopulariopsis,, Trichothecium, Ulocladium and Aerobasidium). The most

frequent fungi were those from the genus Aspergillus. The concentrations of the four analyzed mycotoxins in the poultry

finished feeds, and the percentages of the recovered mycotoxins, revealed that aflatoxins was recovered in 91.1% of the

examined samples, with a mean value of 179.1µg/kg. The same percentage was found with Ochratoxins, but with lower mean

concentration of 159.4µg/kg. In the third order were Fumonisins mycotoxins were in the third order, and they were recovered

in 51.1% of the tested samples with a mean value of 127µg/kg. In the fourth order was T-2 toxin, with a percentage of 2.2%

and a value of 50.0µg/kg.

Key words: Molds, Mycotoxins, Mycotoxicosis, Poultry feed.

Available online at http://www.vetmedmosul.org/ijvs

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18

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Introduction

Mold occurrence and growth on poultry feeds is one of

the major threats to poultry economic and health. Besides

their negative impacts on nutritional and organoleptic

properties, moulds can also synthesize different

mycotoxins. More than 100.000 fungal species are considered as natural contaminants of agricultural and food

products. However, due to genetical and ecological factors,

relatively few can actually generate mycotoxins (1).

According to Leibetseder (2), 30 to 40 % of existing

moulds can elaborate toxic substances under favorable

conditions. The majority of the toxic species belong to the

genera Aspergillus, Penicillium, Fusarium and Alternaria

(3). The effects of mycotoxins on higher animals include

hepatotoxicity, nephrotoxicity, immunotoxicity,

oncogenesis and genotoxicity (4-6). Despite the great

attention that has been paid to the study of toxigenic moulds and their mycotoxins in various foods and feeds,

little is known about fungal and mycotoxin contamination

of poultry feed in Mosul governorate yet. During the last

two or three decades, the production of mixed poultry feeds

were significantly increased in parallel with the evolution

of poultry industry in the country. Furthermore, it is well

established that contamination of poultry feeds with

mycotoxins may induce sanitary disturbances and mortality

among the birds and secondary contamination of the human

consumer via eggs, poultry meat and giblets (7,8). Variety

of complex and divers clinical signs of potential

mycotoxicosis were always observed in different broiler, broiler breeder and layer farms with potential

mycotoxicosis. Affected flocks showed one or more of the

following symptoms; decreased weight gain; anorexia;

reduced feed conversion efficiency; decreased egg

production; poor egg shell quality ;increased egg blood

spots; spiking mortalities; immunosuppression and failure

of vaccination programs; increased susceptibility to

diseases especially E-coli infection; reduced fertility and

hatchability; visceral hemorrhages; leg weakness and high

percentages of leg deformities; pale bird syndrome; fatty

liver with pale, muddy to yellowish discoloration; increased bruising; enlarged pale kidneys; wet litter; urate deposition

in the body cavities; increased incidence of viral diseases

like Newcastle disease, infectious bursal disease and

inclusion body hepatitis; oral lesions; tibial

dyschondroplasia; gizzard erosions; paralysis; extension of

leg and neck.

The purpose in the present work was to initiate a study

on the toxigenic mycoflora of poultry feed in Ninevah

governorate/ Iraq that It includes: enumeration and

identification of moulds genera naturally contaminating

different kinds of poultry feeds and detection and

quantification of the 4 major mycotoxins in the feed

samples, aflatoxin, ochratoxin A, fumonisins and T-2 toxin.

Materials and methods

Samples The samples of finished poultry feed were delivered to

the College of Veterinary Medicine, University of Mosul

from different broilers, broiler breeders and layers farms.

These farms were located in Nineveh governorate and were

claimed from potential mycotoxicosis, feed samples were

collected during a 2-years-long period from Apr 2005 to

Sep 2007. Random 10 to 30 representative samples of I kg

were collected from several locations within a batch of feed

and combined thoroughly to provide a composite sample of 1 Kg for submission. They were then divided to two parts,

one for mycological examination, and the other for

mycotoxins detection. Feed samples intended for

mycological examination were usually analyzed

immediately upon arrival or, if necessary, they were stored

for 2-3 days in paper bags at room temperature (22-25ºC).

The other parts of feed samples intended for mycotoxins

analysis were stored at -20Co.

Isolation of poultry feed fungi Dilute plate technique was used for isolation of fungi

from the samples (9). General molds count was carried out by weighing 20 g of the poultry mixed feed samples and

their mixing with 180 ml of saline solution (0.85% sodium

chloride) with 0.05% Tween 80 on a horizontal shaker for

ca. 30 minutes. Then, 0.1 ml of appropriate dilutions made

up to 10-5 was applied on Dichloran Rose Bengal

Chloramphenicol agar (DRBC (10, 11). Plates were

incubated at 25oC for 5-7 days. The mold genera were

identified according to (11-15).

Mycotoxins analyses Feed parts intended for mycotoxin estimation were

subjected to four standard kits for aflatoxin, ochratoxin, T-2

toxin and fumonisins using Neogen mycotoxin extraction

kits (Neogen corporation) as follows:

Samples preparation and extraction: Twenty five gram-

samples were collected for analysis. These samples were

finely ground,so that at least 75% of them pass through a 20

mesh. Five gram samples were blended with 25 ml of 70%

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19

v/v methanol/ water solution for 3 minutes. Extracts were

filtered through a Whattman No.1 filter paper.

Test procedure: All Neogen extraction reagents were

allowed to warm at room temperature (18-30 ºC) before

use. Red marked mixing wells were prepared, one for each

sample plus four red wells for controls. All red- marked

wells prepared were placed in the well holder. An equal number of antibody coated (AB) white wells to those red-

marked wells were also prepared. Hundred µl of conjugate

were transferred to each red-marked mixing well. To those

red wells containing the conjugate another (with new

pipette tips) 100 µl of controls and samples were added by

using a 12-cannel pipettor liquid in wells were mixed and

pipetting it up and down for 3 minutes. After mixing 100 µl

of the (conjugate + samples, or conjugate + controls) were

transferred to AB – coated wells. These wells were moved

back and froth for well mixing the contents in each well for

10-20 seconds without splashing reagents from the wells.

Antibody-coated wells were then incubated at room temperature (18-30 ºC). The contents in AB- coated wells

were shacked out, by filling the wells with deionized water

and dumping them out. This step was repeated 5 times.

Turning the wells upside down and tamping them out on a

paper was carried out until the remaining water has been

removed. Substrate was then added to AB- coated wells, by

using the 12-channel pipettor through pipetting 100 µl of

substrate to these wells. Mixing was done by sliding the

well holder back and froth for 10-20 seconds, followed by

incubation for 3 minutes. Stop solution was poured to these

wells (100 µl) to each, mixing was done by sliding well holder back and froth on a flat surface. Within 20 minutes

after the addition of stop solution. Results were read, using

a micro well reader (ELx00) with a 650 nm filter. More

blue color means less toxins. Results of the yield optical

densities of the controls and samples were obtained by

using computerized Neogen Verotex Softwere Program

version 2.9.16 (Neogen Corporation).

Results

The total number and percentages of enumerated mold

genera in Nineveh finished poultry feed are presented in

Table 1, and the ability to produce appropriate mycotoxins

is shown in Table 2. Total fungal counts were ranged from

0.1 × 101 to 6.5 X 106 cfu.g-1 of feed sample, with an

average 7.2 X 105 cfu.g-1 sample (Table 1. Fourteen mold

genera were recovered during this study.

From the systematic point of view, 2 genera belong to

Zygomycetes (i.e. Mucor, Rhizopus,), 1 genus belong to

Ascomycetes (i.e. Eurotium); the majority, within so-called

mitotic fungi (formerly Deuteromycetes), encompassed 11

genera (i.e. Acremonium, Alternaria, Aspergillus,

Fusarium,, Paecilomyces, Penicillium, Scopulariopsis,,

Trichothecium, Ulocladium and Aerobasidium). The most

frequent fungi were those from the genus Aspergillus,

recovered from 40 samples (88%) with a range of 0.1 X 10 5- 5.3 X 106 and a mean value of 2.6 X 106. In the second

order were the molds from the genus Penicillium and

Mucor, both recovered from 28 samples (64%) with a range

of 0.2 X 105 - 4.4 X 106 and3 X 104 - 2.6 X 105 and a mean

of 2.2 X 106 and 1.4 X 105 respectively. To the third and fourth orders were the molds from the genera Rhizopus, and

Scopulariopsis, recovered from 23 and 22 samples (50%

and 48%) respectively with a range of 1.5 X 104- 2.1 X 105

and 0.2 X 104- 1.8 X 105, and a mean of 1.1 X 105 and 9.1X

105 respectively. The following recovered genera were the

molds of Alternaria and Eurotium, from 20 and 19 samples

with percentages of 45% and 42% and a range of 8 X 10 3 -

1.8 X 1053 and X 10 3 - 1.7 X 105, with a mean of 9.4 X 104

and 8.5 X 104 respectively. In the descending pattern

occurred the molds of the genera Cladosporium and

Fusarium, recovered from 17 and 14 samples (37% and

31%), representing a range between0.2 X 10 3- 9.8 X 104 and 0.4 X 10 3 - 9.2 X 104, and a mean of 4.9X 104 and 4.6

X 104 respectively. From less than 10 samples recovered

the genera of the mitotic molds from Acremonium,,

Paecilomyces, Ulocladium, Aureobasidium, and

Trichothecium, with 11%,7%,7%,2% and 2% respectively,

with an average of 4300,110,70,50 and 25 CFU g-

1respectively.

Table 2 illustrate the frequency of recovered mold

genera out of 45 finished poultry feeds tested through the

study period, of these 41 samples were positives for

aflatoxins and ochratoxins (91.1%).To a lower extent were the positive samples of Fumonisins (51.1%). The lowest

positive sample was that of T-2 Toxin (2.2%). The

mycotoxins concentrations were in general positive

proportion to the number of respective molds producing

them, being highest in aflatoxins and ochratoxins. These

mycotoxins are produced mainly by aspergillus and

penicillium mold genera, the most prominanat mold

enumerated in this study. In a descenging manner were also

the average levels of aflatoxins, ochratoxin A, fumonisins

and T-2 toxin. The highest levels of these mycotoxins were

as follows; aflatoxins: < 475 µg/kg ; ochratoxin A: < 460 µg/kg ; fumonisins: < 350 µg/kg and T-2 toxin: 50 µg/kg.

Table 3 shows the distribution concentration of

different mycotoxins in the tested finished poultry feeds.

Only aflatoxins and ochratoxin A were ranged from 0-500

µg/kg. The highest number of samples contaminated with

aflatoxin were within levels of 200-300 µg/kg. Although

ochratoxin level was also ranged from 0-500 µg/kg, but the

highest levels were occurred in concentrations of 50 and

250 µg/kg. Fumonisins highest levels were occurred in

concentrations between 50-150 µg/kg, and the highest

levels did not exceeds 350 µg/kg. The only one sample

contaminated with T-2 toxin had level of 50 µg/kg.

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Table 1: Frequency and average count of recovered mold genera from 45 Nineveh poultry finished feed samples.

Average

counts cfu g-1 Range

Percentage of mold

genera frequencies

Frequency of Positive

samples Molds

2.6 X 106 0.1 X 10 5- 5.3 X 106 88.8 40 Aspergillus spp.

2.2 X 106 0.2 X 105 - 4.4 X 106 62.2 28 Penicillium spp.

1.4 X 105 3 X 104 - 2.6 X 105 62.2 29 Mucor spp.

1.1 X 105 1.5 X 104- 2.1 X 105 51.1 23 Rhizopus spp.

9.1X 105 0.2 X 104- 1.8 X 105 48.8 22 Scopulariopsis spp.

9.4 X 104 8 X 10 3 - 1.8 X 105 44.4 20 Alternaria spp.

8.5 X 104 3 X 10 3 - 1.7 X 105 42.2 19 Eurotium spp.

4.9X 104 0.2 X 10 3- 9.8 X 104 37.7 17 Cladosporium spp. 4.6 X 104 0.4 X 10 3 - 9.2 X 104 33.3 14 Fusarium spp.

4.3 X 103 0.6 X 10 2 - 8.7 X 103 11.1 5 Acremonium spp.

1.1 X 102 1 X 10 1 - 2.1 X 102 6.6 3 Paecilomyces spp.

7 X 101 2 X 10 1 - 1.2 X 102 6.6 3 Ulocladium spp.

5 X 101 0.2 X 10 1 - 0.8 X 101 2.2 1 Trichothecium spp.

2.5 X 101 0.2 X 10 1 - 0.8 X 101 2.2 1 Aureobasidium spp.

Table 2: Number of tested, positive, percentage and levels of detected mycotoxins in PMFS tested.

Mycotoxins Parameters

Aflatoxins Ochratoxin A Fumonisins T-2 Toxin

No. of samples tested 45 45 45 45

No. of positive samples 41 41 23 1

Percentage positive (%) 91.1 91.1 51.1 2.2 Average level (µg/kg) 179.1 159.4 127 50

Highest level (µg/kg) 475 460 350 50

Figure 4 shows the different AF concentrations (ppb)

in positive AF-PFF samples. It is evident that 24% of the

samples had AF concentration between 201-250 ppb,

followed by 16.6% between 151-200ppb and 11.1%

between 351-400 ppb. The remaining concentrations were

recovered in less than 10% of the samples.

Figure 5 shows the different Ochratoxin concentrations

(ppb) in positive Ochratoxin -PFF samples. From figure it

is clear that 20% of the samples had Ochratoxin up to 50 ppb, followed by 17.7% between 201-250 ppb, 13.3%

between 51-199 and 351-400, 11.1% between 101-150,

while the remaining concentration were occurred in

percentages less than 10%.

Figure 6 shows the different fumonisins concentrations

(ppb) in positive fumonisins -PFF samples. It is evident that

48.8% were negative samples. The positive samples

showed that 22% of them had fumonisins concentrations up

to 50 ppb, and 13.3% between 101-150 ppb, while the

concentrations up to 350 ppb had percentages of less than

10%. No more than 350 ppb of fumonisins mycotoxin was detected.

Figure 7 shows that 97.75 of the tested feed samples

were negative for T-2 toxin, and only one sample with 50

ppb was detected.

Discussion

Both field and storage fungi were recovered in this study

through the examination of poultry finished feed samples

collected from different poultry farms with potential

mycotoxicosis. These molds include members of the genera

Aspergillus, Penicillum, Fusarium, Alternaria and

Cladosporium which could contaminate many agricultural

commodities used in the formulation of poultry finished feed samples like corn, wheat, soybean, barley and others

commodities. These molds are of great importance because

of potential mycotoxin production (16). The total fungal

loads in the analyzed finished feed samples in our study

were around 105 cfug-1 which is higher than that reported in

Slovakia of 103 cfu.g-1 (17), and 104 cfu.g-1 (18). Similar

results were found to those reported in Turky (19), Spain

(20) and from Argentina (21,22). The predominance of

mold genera were to large extent resembles those found in

Saudi Arabia (23), which could be due to some

resemblance in the ecological and climatologically conditions. The main contaminating molds appeared to be

from the genus Aspergillus with a percentage recovery of

91.11%.

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21

Table 3: Mycotoxins recovered from Nineveh poultry

finished feed.

Sample

No.

Aflatoxins

ppb

Ochratoxins

ppb

Fumonisins

ppm

T-2

toxin

ppb

PFF 1* 57 30 ND ND** PFF 2 189 290 100 ND

PFF 3 254 230 ND ND PFF 4 370 61 ND ND PFF 5 69 291 ND ND

PFF 6 165 242 200 ND PFF 7 ND. 159 250 ND PF F8 250 ND. ND ND

PF F9 350 42 ND ND PF F10 452 460 200 ND PFF 11 54 91 ND ND PFF 12 152 420 ND ND

PF F13 250 223 150 ND PFF 14 340 133 150 ND PF F15 ND. 173 30 ND

PF F16 458 ND. ND ND PFF 17 146 228 ND ND PFF 18 ND 191 150 ND PF F19 240 ND. ND ND

PF F20 36 133 200 ND PFF 12 362 23 350 ND PFF 22 475 214 ND ND

PFF 23 76 38 ND ND PF F24 158 28 150 ND PF F25 370 212 30 ND PF F26 356 273 ND ND

PF F27 235 ND. 20 ND PFF 28 230 19 50 ND PF F29 235 12 130 ND

PFF 30 453 73 20 ND PF F31 ND. 260 ND ND PFF 32 293 142 20 ND

PF F33 250 212 ND ND PF F34 323 57 ND ND PF F35 230 72 ND ND PF F36 196 12 250 ND

PF F37 226 68 ND ND PF F38 180 50 20 ND PFF 39 400 252 250 ND

PFF 40 196 222 10 ND PF F41 188 142 ND ND PFF 42 250 123 20 ND PF F43 219 183 ND ND

PF F44 90 280 150 ND PF F45 22 320 150 50

*PFF=Poultry finished feed from farm 1 ND**=Not detect.

This finding in consistent with (22) and (24) and (25) who

found that the most dominant species isolated of poultry

feed samples belonged to the genus Aspergillus, but not in

consistent with (17), (22) and (25), who found that the most

frequent fungi were those from the genus Penicillium.

Although molds of Aspergillus species are more often soil

fungi or saprophytes but several are important because they produce mycotoxins. Penecilli in our results were in the

second order in counting and percentages among recovered

molds, as they are one of the main contaminants of stored

cereals as well as feeds with worldwide occurrence (26-28)

being producers of toxicologically significant mycotoxins

(29-31). In order, our results were in consistent with (17)

that the third mostly encountered fungi were representatives

of the genus Mucor, who found this genus with frequency

of 44%, followed by the genus Rhizopus. Our results also

revealed that molds from the genus Eurotium were

recovered in 19%, and Fusarium in 14% of the samples.

These results were less than that reported by (17), who found that Eurotium and Fusarium genera were widespread

through the samples they examined and were occurred with

the same frequency of 42%. In this study, the mean of

aflatoxins were recovered at a rate of 91.1%, with an

average of 179.1µg/kg of finished poultry feed samples,

confirming our previous results of aflatoxin contamination

to poultry feed samples (32). Comparing with other Asian

countries, our results were higher than that reported in north

Asia (China, Taiwan and Korea) of 3%, and higher than

south East Asia (Malaysia, Philippines, Thailand and

Vietnam) of 36%, and higher than south Asia (India, Pakistan and Bangladesh) of 53% (33). The second

mycotoxin recovered was Ochratoxin (91.1%), in an

average of 159.4 µg/kg, confirming our previous results of

ochratoxin contamination of poultry finished feeds (34).

Our results were also more than those reported in north

Asia (China, Taiwan and Korea) of 15%, and higher than

south East Asia (Malaysia, Philippines, Thailand and

Vietnam) of 9%, and higher than south Asia (India,

Pakistan and Bangladesh) of 50% (33). Again our high

results here were due to sampling feeds from mycotoxicosis

suffering poultry farms. Ochratoxin has been implicated in significant field outbreaks of mycotoxicosis in poultry (35).

Fumonisins mycotoxins were the third type of mycotoxins

recovered in this study, in a percentage of 53.3% and an

average of 127µg/kg of finished poultry feed samples.

These mycotoxins recovered here in Mosul governorate in

corn samples intended for use in finished poultry feeds

(36). Our results agreed with the percentages reported in

north Asia (China, Taiwan and Korea) of 52%, and with

those of south East Asia (Malaysia, Philippines, Thailand

and Vietnam) of 52%, but higher than hose reported in

south Asia (India, Pakistan and Bangladesh) of 32% (33).

The fourth mycotoxin recovered in this study was T-2 toxin in a percentage of 2% at a rate of 50 µg/kg. This toxin was

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22

Figure 4:Distribution of different AF

concentrations(ppb) in positive AF- PFF samples

0

5

10

15

20

25

30

1234567891011

Concentrations

%

1=ND 2=0-50ppb 3=51-100ppb 4=101-150ppb 5=151-200ppb 6=201-250ppb

7=351-300ppb 8=301-350ppb 9=351-400ppb 10=401-450 ppb 11=451-500ppb

11.1%

16.6%

24.4%

Figure5: Distribution of different Ochratoxin

concentrations (ppb) in positive Ochratoxin-PFF

samples

0

5

10

15

20

25

1234567891011

Concentrations

%

1=ND 2=0-50ppb 3=51-100ppb 4=101-150ppb 5=151=200ppb 6=201-250ppb

7=351-300ppb 8=301-350ppb 9=351-400ppb 10=401-450 ppb 11=451-500ppb

20%17.7%

13.3% 13.3%

11.1%

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23

Figure 6:Distribution of different Fumonisins (ppb)

concentrations in positive Fumonisins-PFF in PFF

samples

0

10

20

30

40

50

60

1234567891011

Concentrations

%

1=ND 2=0-50ppb 3=51-100ppb 4=101-150ppb 5=151=200ppb 6=201-250ppb

7=351-300ppb 8=301-350ppb 9=351-400ppb 10=401-450 ppb 11=451-500ppb

48.8%

22.2%

13.3%

Figure 7:Distribution of different T-2 toxin (ppb)

in positive T-2 toxin - PFF samples

0

20

40

60

80

100

120

1234567891011

Concentrations

%

1=ND 2=0-50ppb 3=51-100ppb 4=101-150ppb 5=151=200ppb 6=201-250ppb

7=351-300ppb 8=301-350ppb 9=351-400ppb 10=401-450 ppb 11=451-500ppb

97.7%

2.2%

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24

also recovered by us through the study performed during

2004-2005 in Mosul governorate (37). Our results were

close to those reported in north Asia (China, Taiwan and

Korea) of 0%, and those found in south East Asia

(Malaysia, Philippines, Thailand and Vietnam) of 1%. In

general our results were agreed with those reported by

Pacin et al., (38) who found that the most recovered mycotoxins from feed associated fungi were ochratoxin A,

aflatoxin and fumonisin. It may be stated that Aspergillus

(including Eurotium), Penicillium and Fusarium are the

typical fungal genera inhabiting poultry feed mixtures. In

fact, they are very important contaminants being renowned

for their ability to form a huge number of various types of

toxic extrolites-mycotoxins (39,40), and that the outcomes

of this study clearly show that finished poultry feeds in

Nineveh governorate represent a rich source of significant

mycotoxin producers, especially those from the

Penicillium, Fusarium and Aspergillus genera. It is possible

that these co-contaminant of estimated mycotoxins in our study, of two carcinogenic mycotoxins, aflatoxin and

fumonisins, and two cancer promoting mycotoxins,

ochratoxin and T-2 toxin, could exert great negative effects

on farms health and productivity than do each of them

singly and of public health concern to the health of

consumer through the food chain by ingestion residual

levels of these toxins in poultry meat and poultry products

(meat and eggs) (41,42). These toxic substances are known

to be either carcinogenic (e.g. aflatoxin B1, fumonisin B1,

Ochratoxin A), neurotoxic (fumonisin B1), nephrotoxic

(ochratoxin A), dermatotoxic (trichothecenes), or immunosuppressive (aflatoxin B1, ochratoxinA and T-2

toxin) (33). the senerginestic effects of these mycotoxins on

poultry productivity and health were well documented.(43-

45).

Acknowledgements

The author thanks the College of Veterinary Medicine,

University of Mosul for providing all supports for this

experiment.

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Prevalence of intestinal ciliate Buxtonella sulcata in cattle in Mosul

T. M. Al-Saffar*, E. G. Suliman**, H. S. Al-Bakri**

*Department of Internal and Preventive Medicine, **Department of Microbiology, College of Veterinary Medicine,

University of Mosul, Mosul, Iraq

(Received June 11, 2008; Accepted July 13, 2009)

Abstract

The current study was conducted to detect the presence of Buxtonella sulcata (an intestinal ciliate) in faecal samples of

cattle suffering from diarrhea in Mosul city. One hundred and twenty faecal samples were examined, and collected from calves

(44), beef cattle (34) and dairy cattle (42) these animals were divided into two groups those showed diarrhea (86) and (34) had

no symptomatic diarrhea. Direct smear and formalin-ether sedimentation methods were used for detection of this parasite. The

total percentage of infection with Buxtonella sulcata was 24.16%. There was no significant differences in the percentage of

infection and intensity of infection between calves, beef and dairy cattle where as there were significant differences between

diarrheic and non-diarrheic animals.

Keywords: Buxtonella sulcata, Diarrhea, Cattle.

Available online at http://www.vetmedmosul.org/ijvs

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Introduction

Buxtonella sulcata is one of the parasitic protozoa

(Ciliophora) type which inhabited in the colon of the

ruminants and the original role for diarrhea or for

classification has not been fully explained (1-5). Buxtonella

sulcata is similar to Balantidium coli found in the swine

and man, some authors included them into the same genus

(6). However, the classification of Buxtonella sulcata is:

Kingdom: Protozoa, phylum: Ciliophora, Class:

Kinetofragminophora, order: Trichostromatida, Family:

Pyenotrichidae, Genus: Buxtonella (1).

Henrisken (7) reported that percentage of infection

with Buxtonella sulcata in Danish cattle was (71.8%), Fox

(2) recovered Buxtonella sulcata cysts in faeces of adult

British cow from nine commercial dairy farms with a percentage of 44.6% and in Kynoggi-don the prevalence of

Buxtonella sulcata during 1984 and 1994 were 33.6%,

34.5% respectively (8). A higher percentage 38.0%, 21.6%

was reported in a dairy and beef cattle in Costa Rica (9). In

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (27-30)

28

Germany a more controversial prevalence was reported

which has been range from Zero-73% without any obvious

association with clinical symptoms (10). The first recorded

of infection with Buxtonella sulcata from cattle was in Al

Qadissiyh city (Iraq) and the percentage of infection was

47% (11). The problem of Buxtonellosis in ruminants is not

considered of importance yet, therefore the aim of this study was to diagnose this parasite in faeces of cattle in

Mosul city and to examine the role of this parasite as

etiological agent of diarrhea in calves, dairy and beef cattle.

Materials and Methods

Faecal samples were collected from 120 cattle (44

calves, 42 dairy frezinn cattle, and 34 beef cattle) from

teaching veterinary Medicine College, farms of Agriculture

College and Kogyali village and cattle market in Mosul city

during October 2007 – April 2008, faecal samples were

taken from animals with diarrheal disease (86) and apparently healthy animals (34), samples kept in a clean

plastic container. Direct smear method (12), sedimentation

methods by using ether-formalin (13,14) were used for

detection Buxtonella sulcata. Differentiation of Buxtonella

cyst or trophozoites was based on morphological specific

feature and by microscopic measurement by using ocular

micrometer (2,3,6,8,11). Coproscopical examination for

detection the degree of intensity of infection according to

(15,16) and examination of the 20 microscopical fields. The

data were analyzed statistically by using Z-test, Fisher- test

and χ2 – test (17).

Results

During coproscopical examination of 120 faecal

samples of cattle, infection with Buxtonella sulcata was

appeared in 29 animals with a total percentage of 24.16%

(Table 1), all of these animals infected with Buxtonella

sulcata cysts. These cysts are round or oval in shape,

yellow in color reach 68.6-107.8 µm in diameter with a

mean of 74.58 µm, these cysts surrounded by a two layered capsule (Fig.1). Four positive samples which infected with trophozoites (vegetative forms) with a percentage of

13.79% (Table 1). The vegetative forms were oval, with

diameters of 107.8-137.2X 49-102.9, with a mean of

121.25X94.06 µm. The surface of the cilliate's cell was evenly covered with short cilia, deep syncystoma was at the

anterior pole and the nucleus lies in the centre of the

vegetative form (Fig.2). From Table (1) statistical analysis

showed significant differences between calves and beef

cattle and between calves and dairy cattle while no

significant differences between beef and dairy cattle. From

Table (2) it is evident that there were three degree of

infection, low 51.72%, moderate 24.13% and high 24.13%

according to the number of parasites under high power field (40x hpf) there was no significant differences between

calves, beef and dairy cattle. Buxtonella sulcata cysts were

appeared in 27 animals with diarrhea symptom in a

percentage of 31.39% while those show no diarrhea, the

percentage were 5.88% (Table 3). Statistical analysis

showed significant differences between animals with and

without diarrhea.

Table (1): Incidence of Buxtonella sulcata of cattle in Mosul.

Animals No. of samples No. Of positive

samples Rate of

infection Infection with cyst No.(%)

Infection with Trophozoite No.(%)

Calves (3-8 months) 44 5 11.36 a 5(100.0) 0(0.0) Beef cattle (2-7 years) 34 12 35.29 b 12(100.0) 3(25.0) Dairy cattle (4-8 years) 42 12 28.57 b 12(100.0) 1(8.3) Total 120 29 24.16 29(100.0) 4(13.79)

Rates with different letters have significant difference at P<0.05 according to Z-test between two proportion. Table (2): Distribution and intensity of infection with Buxtonella sulcata of cattle.

Infection Animals No. of +ve samples Low degree* Moderate degree** High degree** Statistical group Calves 5 4(80.0) 1(20.0) 0(0.0) A Beef cattle 12 5(41.6) 3(25.0) 4(33.33) A Dairy cattle 12 6(50.0) 3(25.0) 3(25.0) A Total 29 15(51.72) 7(24.13) 7(24.13)

Groups with same letters have non-significant difference at p<0.05 using Fisher Freeman Halton test. Low degree: 1 cyst/hpf , Moderate degree: 2-4 cyst/hpf , High degree: 5 and more than 5 cyst/hpf

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (27-30)

29

Figure (1): Cysts of Buxtonella sulcata (10X,40X) by using digital camera.

Table (3): Relationship of Buxtonella sulcata infection with

diarrheal symptoms in cattle by using χ2 – test.

Symptoms No. of

animals

No.of +ve

B.sulcata

% of infection

Animals with diarrhea 86 27 31.39a Animals with normal faeces

34 2 5.88b

Total 120 29 24.16

Groups with different letters have significant differences at P<0.016.

Figure (2): Trophozoites of Buxtonella sulcata (10X,40X)

by using digital camera.

Discussion

In this study, the total percentage of infection with

Buxtonella sulcata was 24.16% and the infection with cysts

appeared in all positive cases while the trophozoites

appeared only in four positive cases from total of all

positive number, while other study accomplished in Iraq by

Ayaiz (11) reported that the percentage of infection was 47%. Other studies which performed in different part of

world such as England, Poland, Costa Rica, Bangalore and

Thailand (2,6,9,18,19) showed a wide differences in

percentage and ranged between 2-87%.The differences in

the percentages of infection could be due to many different

factors, such as environmental conditions, animal, farm

management practices and stress factors. Fox and Jacobs

(2) itself showed that seasonal fluctuations in the

prevalence of the infection and cyst excretion rates were

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (27-30)

30

related to changes in the diet and opportunities for

transmission, furthermore, the delivery rate may be lead to

an increase in prevalence of infection. The shape and size

of the cysts and trophozoites which observed in this study

are in agreement with those described by (3, 8,11).

The statistical analysis of infection with Buxtonella

sulcata in our study showed a significant differences between the infection of calves, beef and dairy cattle, while

no significant differences was noticed between beef and

dairy cattle, moreover, no significant differences were

observed in the intensity of the infection between calves,

beef and dairy cattle. High rate of infection with Buxtonala

sulcata cysts appeared in animals affected with diarrhea

and which are found to have large number of cysts and

trophozoites (Table 3), the result was in agreement with

(2,6,11,19,20) who reported that B. sulcata can be one of

the causative agent of diarrhea in ruminants. The

pathogenic effect of this parasite has not been found to be

of great interest and it is suggested that more comprehensive studies should be done in order to explain

the cause of diarrhea. In an observations of Tomczuk et al.,

(6) they reported that the pathogensis of B. sulcata may be

compared with the incidence of similar ciliate Balantidium

coli living in the large intestine of pig, man and many other

mammals. In a different studies of Skotarczak and

Zielinski, Urman and Kelky, and Skotarczak (21-24) was

proved that especially significant effect of Balantidium coli

in change in the pH of large intestine content on the

intensively of invasion and the damaging effect on the

mucosa of large intestine results in a secondary bacterial infection and increase in the pathological changes.

In this study, our observation was based on detection of

the B. sulcata cysts in animals which have signs of diarrhea

and those with normal faeces. All examined positive cases

was affected with B. sulcata only, furthermore, no clinical

signs observed other than diarrhea on the affected animals

and no other parasitic agents were diagnosed, this indicates

that B. sulcata can be considered as one of the agents of

unexplained etiology of diarrhea in cattle.

Acknowledgement

This study was supported by the College of Veterinary

Medicine, University of Mosul.

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31

Clinical, haematological and biochemical studies of babesiosis in native goats in Mosul

E. G. Sulaiman1, S. H. Arslan

2, Q. T. Al-Obaidi

2, E. Daham

2

1Department of Microbiology, 2Department of Internal and Preventive Medicine,

College of Veterinary Medicine, University of Mosul, Mosul, Iraq

(Received March 26, 2008; Accepted July 13, 2009)

Abstract

The study included examination of 175 native goats, 27 were infected with Babesia ovis, B. motasi, B. foliata and B. taylori, (recorded in Mosul for the first time) and 25 were clinically normal and served as control. Results indicated that the percentage of the infection with Babesiosis was 15.42% and the percentage of parasitemia ranged between 3.5-10.4% with a mean 6.95%, infected goats showed signs of loss of appetite, weakness, pale mucous membranes, jaundice, fever, coughing, nasal discharge, recumbency, diarrhea and haemoglobinuria. A statistically significant decrease were recorded in total red blood cells (RBC), haemoglobin concentration (Hb), packed cell volume (PCV) and platelets counts. Anemia was of microcytic hypochromic type. A statistically significant increase in erythrocyte sedimentation rate and significant increase in total white blood cells was recorded due to significant increase in lymphocyte and neutrophile count. Results of the biochemical testes indicated an increase in activity of alanin amino transferase (AST), aspartate amino transferase (ALT), total bilirubin, blood urea nitrogen and icterus index, with a significant decrease in total serum protein, albumin and globulin levels. Results also indicated the presence of Rhipecephalus ticks which were: Rh. sanguineus and Rh. turanicus. Keywords: Babesia, Goats, Hematology.

Available online at http://www.vetmedmosul.org/ijvs

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (31-35)

32

Introduction

Babesiosis is caused by intraerythrocytic protozoan

parasites of the genus Babesia, the disease which is

transmitted by hard ticks (family: Ixodidae), affect a wide

range of domestic and wild animals and occasionally

humans (1). Babesia occurs seasonally and the peaks of infection were observed in rainy season (2). Babesia forms

can vary as pear-shaped, round and elongated (3) Clinically

Babesiosis is characterized by fever, inappetence, increased

respiratory rate, muscle tremors, anemia, jaundice, body

weight loss, and hemoglobinuria in the final stages (4,5).

Anemia is very common for all infected animals,

hemoglobinuria may not observed in animals infected with

B.ovis (6) Four species of Babesia have been reported from

sheep and goats mainly consisting of one large form (B.

motasi) and three small forms (B. ovis, B. foliata and B.

tyalori) (7), while Friedhoff referred that the Babesiosis in

domestic small ruminants is due to at least three species, namely : B. ovis, B. motasi and B. crassa (8). B. ovis is less

pathogenic than B. motasi for sheep infection and cause

relatively moderate haemolytic anemia (9,10), whereas

Friedhoff considered the B. ovis is the most important

causative agent which transmitted by Rhipicephalus bursa,

R. turanicus, Hyalomma anatolicum excavatum and

probably by R. evertsi evertsi (11), whereas the known

vector of B. motasi are Haemophysalis punctata and R.

bursa and the B. motasi is more pathogenic than B. ovis in

India and northern Africa. In animls affected with Babesia

spp. The studies revealed that decrease in the total erythrocytes counts, haemoglobin concentration,packed cell

volume and platelets counts, and in biochemicals studies

showed increase in activity of alanine amino transferase,

aspartate amino transferase, total bilirubin, blood urea

nitrogen and icterus index, with decrease in total serum

protein. (12-14)

The percentage of infection with B. ovis in goats in Al-

Arich city and El-Hassanah center were 7.0% (15) whereas

the prevalence of B. ovis infection in Awassi sheep in Urfa,

Turkey was 41.02% (16). Little work has been done on

Babesia spp in Mosul, Iraq on goats, hence the present study was taken to determine the occurrence of Babesia spp

together with clinical, haematological and some

biochemical changes.

Materials and methods

A total of 175 goats, 2-5 years of age and from both

sexes were examined in the Teaching Veterinary Hospital

(College of Veterinary Medicine, University of Mosul) and

from other regions in Mosul city, for the possibility of

infection with babesiosis. Clinically normal goats (n=25)

served as control. Careful clinical examination of all suspected animal were carried out.

Blood was collected from jugular vein for

haematological examination by using (Automatic full

digital cell counter, Beckman USA) to get of total red blood

cells (RBC), haemoglobin concentration (Hb), packed cell

volume (PCV), mean corpuscular volume (MCV), mean

corpuscular hemoglobin concentration (MCHC), platelets

counts and total and differential leuckoytes count (TLC & DLC), and westergren method used for get erythrocyte

sedimentation rate (ESR) (17). Thin blood smears were

taken from the vein of the ear and stained with Giemsa

were used to identify the Babesia and the percentage of

parasitemia (18). Blood serum samples were tested spectro-

photometrically for the biochemical changes of alanine

amino transferase, aspartate amino transferase, blood urea

nitrogen and total serum protein, albumin and globulin by

using available kits (Randox, U.K.), total bilirubine using

available kits (Biomerex, France), and Icterus Index by

using potassium dichromate according to (18). Statistical

analysis were done by using t-test (19).

Results

Clinically infected goats showed different signs

graduated from loss of appetite, emaciation, pale mucous

membranes, jaundice, fever, coughing, nasal discharge,

recumbency, diarrhea and haemoglobinuria as well as

presence of ticks (Rhipicephalus sanguineus and Rh.

turanicus) were detected on different parts of the body

(Table 1).

Table (1): Clinical signs of infected goats (n=27) with

babesiosis.

Clinical signs

No. of

infected

goats

%

Pale mucous membranes 24 88.9

Loss of appetite 21 77.8

Haemoglobinuria 21 77.8

Diarrhea 16 59.3

Emaciation 15 55.6

Jaundice 13 48.1

Nasal discharge 10 37 Coughing 9 33.3

Recumbency 7 25.9

Ticks (Rhipicephalus

sanguineus and Rh. turanicus) 15 55.6

Significant increase (P<0.01) were encountered in

body temperature, respiratory and heart rates While,

ruminal contractions were reduced significantly (Table 2).

Examination of blood smear revealed four types of

Babesia in infected goats, which were: B.ovis,, B.taylori,

B.foliata and B. motasi. (Fig1). The number of goats which

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33

were infected with Babesia spp was (27) with percentage

(15.42%) and the parasitemia ranged between (3.5-10.4%)

in a mean of (6.95%).

Figure (1): four types of babesia in infected goats, which

were: B.ovis (1), B.taylori

(2), B.foliata (3) and B. motas i

(4) in

blood smear.

Table (2): Body temperature, respiratory rates, heart rates

and ruminal contractions of infected goats with babesiosis.

Control Infected

Parameters Mean±S.E. Mean±S.E.

Body temperature oC 39.10±0.51 40.8±0.82**

Respiratory rate/min 26.21±3.82 53.32±7.95**

Heart rate/min 79.81±8.62 112.4±10.90**

Ruminal contraction/5 min 4.09±0.20 2.52±1.5**

** P<0.01, * P<0.05

There was a significant reduction (P<0.01) in the mean

values of TRBC, Hb, PCV, platelets count, anemia was of

Microcytic hypochromic type due to significant reduction

(P<0.01) in the Mean Corpuscular Volume (MCV) and

Mean Corpuscular Haemoglubin Concentration (MCHC).

A statistically significant increase of ESR encountered and

results also indicated a significant increase (P<0.01) in total

leukocytes count due to significant increase in neutrophils

and lymphocytes count (Table 3).

Table (3): Blood parameters of infected goats with babesiosis and control group.

Control Infected Parameters

Mean ± S.E. Mean ± S.E.

RBC x 106 microliter 6.10 ± 0.54 4.21 ± 1.15**

Hb g/100 ml 10.35 ± 2.43 6.93 ± 2.52**

PCV % 32.40 ± 3.22 26.13 ± 4.33**

MCV % 64.37 ± 5.68 61.40 ± 5.46**

MCHC g/100 ml 32.35 ± 4.33 28.31 ± 2.20**

Platelets x 103 microliter 769 ± 157 349 ± 103**

ESR mm/24 h 0.62 ± 0.43 3.94 ± 1.34**

WBC x 103 microliter 7.68 ± 2.35 10.95 ± 3.73**

Neutrophils % 61.33 ± 1.46 61.87 ± 1.02*

Lymphocytes % 37.50 ± 0.93 39.20 ± 1.31**

Monocytes % 0.20 ± 0.03 0.15 ± 0.10 Eosinophils % 0.15 ± 0.03 0.00 ± 0.00

Basophils % 0.04 ± 0.01 0.06 ± 0.01

Parasitemia --- 3.5 – 10.4% ** P<0.01, * P<0.05.

Results of biochemical examination showed a

significant increase (P<0.01) in activity of AST, ALT, as

well as in levels of total bilirubin, BUN and Icterus index,

however significant decrease were recorded in total protein,

albumin and globuline values of infected goats (Table 4).

B. ovis (round

form) at the

margin of the

red cells

(1.66-2.32)

micron with

mean 1.88

micron (1).

B.taylori

(round

appear) under

go several

fission (1.66-

1.19) micron with mean

1.74 micron

(2).

B. foliata.

round lies more

centrally in

red cells

(1.66-1.99)

micron with

mean 1.83

micron (3).

B.motasi

(pyriform stages) (2.9-

4.15) micron

with mean

3.27 micron

(4).

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (31-35)

34

Table (4): Biochemical parameters of infected goats with

babesiosis and control group.

Control Infected Parameters

Mean ± S.E. Mean ± S.E.

AST U/L 27.13 ± 9.52 77.37 ± 6.94**

ALT U/L 23.65 ± 5.12 29.63 ± 4.59**

Total bilirubin mg/100 ml 0.26 ± 0.10 0.69 ± 0.17**

Total protein g/100 ml 6.70 ± 0.79 3.63 ± 0.53**

albumin g/100 ml 4.07 ± 0.22 2.10 ± 0.21**

Globulin g/100 ml 2.64 ± 0.57 1.27 ± 0.12*

BUN mg/100 ml 32.78 ± 7.64 68.46 ± 12.89**

Icterus index units 2.67 ± 0.39 6.31 ± 1.16**

** P<0.01, * P<0.05.

Discussion

The results of this study showed that the clinical signs

observed in infected goats were in agreement with the

results of other studies carried out by different researchers

concerning ruminants infected with babesiosis (2,3,7). The

cause of pale mucous membrane was development of anemia and decrease in erythrocyte count and haemoglobin

(12), while jaundice due to increase in the total bilirubine

(direct and indirect) and icterus index (18), the

haemoglobinuria may be due to intravascular haemolysis

and high rate of destruction of erythrocytes and

haemoglobinemia (20,21).

Two types of ticks were diagnosed in this study, they

were Rhipicephalus sanguninus and Rh. turanicus and

Friedhoff added that Babesia ovis is transmitted by Rh.

bursa, Rh. turanicus Hyalomma anatolicum excavatun and

probably R. evertsi evertsi (11), while Mazyed and Khalaf

identified Hyalomma anatolicum excavatum and Haemophysalis sulcata in infected goats with B. ovis and

Theileria ovis (15). In general the distribution of the

parasite is correlated with the distribution of tick vector

species (16). Four species of babesia (B. ovis, B. motasi, B.

foliata, B. taylori) were diagnosed in this study, which were

also described by (7,12).

The percentage of infection with Babesia spp was

(15.42%) whereas Mazyed and Khalaf recorded (7%) of

infection with B. ovis in goats (15) and in another study on

goats in a village in south west Nigeria showed (20.4%)

infection with B. motasi (2). Haematological parameters showed relatively

significant decrease in values total Red blood cells,

haemoglobin concentration, packed cell volume and

platelets counts, compared to control group, these results

were in agreement with studies carried out on sheep and

goats by others (2,12,22) and this might be due to

intravascular haemolysis of erythrocyte, increase

erythrocyte phagocytosis by reticloendothelial system and

restricted erythropoitic activity in bone marrow (20,21).

Anemia was microcytic hypochromic type due to

significant reduction in the mean corpuscular volume and

mean corpuscular haemoglubin concentration this was in

agreement with the study of (23) in foals. The increase in

ESR values refers to the correlation between the

sedimentation of RBCs and the intensity of anemia (18),

and Allen added that increase in ESR values due to decrease in Packed cell volume (24). The significant

increase in WBCs was due to increase in lymphocytes, this

was in agreement with (3) in sheep.

Biochemical parameters showed relatively significant

increase in the AST and ALT may be due to indirect

damage of liver, kidney tissue and myocardium, changes

indicated to a possible damage to the liver, kidney tissue

(25), Wright added that increase in the AST due to

distraction of RBCs (26). `

The increase in total bilirubin due to damage of liver

and increase in the indirect bilirubin was due to erythrocyte

haemolysis (18), Babesia also reported to causes nephrosis, renal ischemia, dehydration and some heart diseases that

causes increase in BUN, (26,27). The increase in the icterus

index was due to increase in Total bilirubin in serum and

dehydration (26).

The reduction in total protein, albumin and glubulin

values is might be due to decrease production from the liver

due to direct and indirect effect of parasite, digestive

distribance (diarrhea), loss of appetite and high fever

(28,29).

Acknowledgement

This study was supported by the College of Veterinary

Medicine, University of Mosul.

References

1. Merck Co. The Merck veterinary manual. Babesiosis: Overview,

published in educational partner ship, USA, 2006. Available from:

http://www.merk.vetmanual.com/mvm/htm/bc/10402.htm.

2. Opasine BA. Blood parasites of village goats in South West Nigeria.

Animal Genetic Res. 1984.

3. Kozat S, Yuksek N, Altug N, Agaoglu ZT, Ercin F. Studies on the

effect of Iron (Fe) Preparations in addition to Babesiosis treatment on

the haematological and some mineral levels in sheep naturally infected

with Babesia ovis, YYU Vet. Fak Derg. 2003;14(2):18-21.

4. Urquhart GM, Armour J, Duncan JL, Dunn AM, Jennings FW.

Veterinary parasitology. 2nd

ed. Blackwell Co. Great Briatain; 1996.

242-245p.

5. Ferrer D, Castella J, Guticrrez JF, Lavin S, Marco I. Seroprevalence of

Babesia ovis in Mouflon sheep in Spain. J Wildlife diseases. 1998;

34(3):637-639.

6. Popa E. Ixodid ticks vectors of Babesiosis in animals in Romania.

Revista-Romana-de-Medicina-Veterinara. 1998;8(2):61-67.

7. Soulsby EJL. Helminths, Arthopods and Protozoa of domesticated

animals. 7th

ed. Philadelphia, London, Toronto. 1986.718-719p.

8. Friedhoff KT. Transmission of Babesia, In: Babesiosis of domestic

animals and man. M. Ristic(ed.) CRC. Press, Boca Raton. Florida.

1988.23-52p.

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9. Aytug CN, Alacam E, Gorgul S, Koyun Ve. Keci Hastaliklarive

Yetistiri cillige Istanbul, Tum-Vet Hayvancilik Yayini. 1990;201-202.

10. Imren HY, Sahal M. Veteriner ic Hastaliklari. Ankara. Feryal

Matbacilik San. ve Tic. Ltd. Sti. 1991;207-208p.

11. Friedhoff KT. Tick-borne diseases of sheep and goats caused by

Babesia, Theileria or Anaplasma spp. Parasitologia. 1997;39(2):99-

109.

12. Radostitis OM, Gay CC, Blood DC, Hinchcliff. Veterinary medicine,

A text of the diseases of cattle, sheep, pigs, goats and horse. 9th ed.

Philadelphia: W.B. Saunders Company; 2000.1289-1296p.

13. Arsalan SH. Clinical, haematological and biochemical studies of some

blood protozoa in dogs in ninavah. Iraqi J Vet Sci. 2005;1(19):63-77.

14. Al-Mula GM. Clinical, pathological and therapeutical studies of

Equine Babesiosis in draught horses in mosul. [master's thesis].

Collage of Veterinary Medicine: University of Mosul; 2004.

15. Mazyed SA, Khalaf SA. Studies on Theileria and Babesia infecting

live and slaughtered animals in Al-Arish and El-Hassnah, North Sinai

Governorate, Egypt. J Egypt Soc Parasitol. 2002;32(2):601-610.

16. Emre Z, Duzgun A, Iriadam M, Sert H. Seroprevalence of Babesia

ovis in Awassi sheep in Urfa, Turkey. Turk J Vet Anim Sci. 2001;25:

759-762.

17. Meyer DJ, Harvy JW. Veterinary laboratory medicine. 2nd

ed. W.B.

Saunders Co; 1998.157-199p.

18. Jain NC. Schalm's Veterinary hematology. 4th ed. Lea and Febiger,

Philadelphia; 1986.610-612p.

19. Steel RG, Torrie JH. Principles and Procedures of Statistics. 2nd

ed.

McGraw, Hill Inc.; 1985.120p.

20. Lewis D, Holman MR, Purnell RE, Young ER, Herbert IV, Bevar WJ.

Investigation on Babesia motasi isolated from wales. Res Vet Sci.

1981;31(2):239-243.

21. Voyvoda H, Sekin S, Kaya A, Bildik A. Koyunlarin Dogal Babesia

ovis Enfeksiyonunda Serum Demir. Bakir Konsantrasyona (Fe, Cu)

total Ve Latent Demir Baglama Kapasitesi (TDBK : LDBK) Ve

Transferrin Doynma (TD) Modifikasyonlari Tr. J Vet Ani Sci.

1997;21(1):31-37.

22. Alani AJ, Herbert IV. The pathogenesis of Babesia motasi (Wales)

Infection in sheep. Vet Parasitol. 1998;27:202-220.

23. Guimaraes AM, Lima JD, Tafuri WL, Ribeiro MF, Sciavicco CJ,

Botelho AC. Clinical and histopathological aspects of splenectomized

foals infected by Babesia equi. JEVS.1997;17(4):211-216.

24. Allen BV. Relationships between the erythrocyte sedimentation rate,

plasma proteins and viscosity, and leukocytes counts in thoroughbred

racehorses. Vet Rec. 1988;122:329-332.

25. Yfruham I, Hadani A, Galker F, Avidar Y, Bogin E. Some

epizootiological and clinical aspects of ovine babesiosis caused by

Babesia ovis. Vet Parasitol. 1998;4(2-4):153-63.

26. Wright IG. Biochemical characteristics of Babesia and physio-

chemical reactions in host. In: Babesiosis, ed. By: Ristic M, Kreier JP.

Academic press, New York & London; 171-205p.

27. Coles EH. Veterinary clinical pathology. 4th

ed. Philadelphia: W.B.

Saunders Co; 1984.25-29p.

28. Al-Aboud AY, Al-Deoun MA, Maroun EA. Haematological and

histopathological in sheep and goats naturally infected with some

single blood protozoa. Bas J Vet Res. 2005;4(1):10-14.

29. Hailat NQ, Lafi SQ, Al-Darraj AM, Al-Ani FK. Equine Babesiosis

associated with strenuous execise: clinical and pathological studies in

Jordon. Vet Parasitol. 1997;69:1-8.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (37-40)

37

Effect of industrial product IMBO® on immunosuppressed broilers

vaccinated with Newcastle disease vaccine

O. G. Mohammadamin* and T. S. Qubih

Department of Pathology and Poultry Diseases, College of Veterinary Medicine, University of Mosul, Mosul, Iraq

(Received May 13, 2009; Accepted October 5, 2009)

Abstract

The effect of IMBO was investigated on humoral immune response to Newcastle disease vaccines in broiler chickens.

Haemagglutination inhibition test and enzyme-linked immunosorbent assay were used to assess the immune response. Results

showed that although IMBO significantly enhanced humoral immune response to live Newcastle disease vaccine, it did not

decrease post virulent NDV challenge mortality.

Keywords: Humoral immunity; Newcastle disease,Vaccine, IMBO.

Available online at http://www.vetmedmosul.org/ijvs

������� ����� �������� ®�� ����� ������� ����� ������ ����� ���� ������� ���� ��� !�"�" ���� #�$ ���� %���

��� ������ ����� ���� ������ �� ��������� ���� ����� ����

&�'(��

� ! "���#�� �� $%�� &&�� ��'()*� ���%+) ���� #, -�� "�%�.� ����/ ����*� ���)#����*� 0� 1��� �%� .��') ���)/ ��/)#� � �&3) , &�)#, ���.) ����*� ��� .�#% �4 ���� +)*� � �#�� "�56���)#, ����*� �/ ��� -% �#����* ��� 7�.� � �� 6�%�� ��8��. ��� "��35 �� 9����:� ;�%) ;��< �#����*.

Introduction

Severe outbreaks of Newcastle disease often occur in

areas of° intensive poultry production, which is reasoned mainly to break down in immunity. Although poor vaccine

quality is one of several possible factors that could lead to vaccination failures (1).The failure of protection usually

results from: (i) mycotoxin and/or drug induced

immunomodulation (ii) cold or heat stress (iii) infectious

agents (iv) malfunctioning of the host defense mechanism

and (v) presence of high titers of maternal antibodies (1-7).

Immunostimulation of a bird may lead to increased

antibody production, increased cellular immune responses,

* Part of MSc thesis submitted by the first auther to the College of veterinary Medicine, Mosul University.

and increased macrophage phagocytic ability which

positively correlates with enhanced resistance to various

viral and bacterial infections (8,9). Probiotics are defined as

direct feed microbials or microbial cell preparations with a beneficial effect on the health and well-being of the host

(10). Probiotic represent one of the most recent examples of

natural substances that influence adaptive immune

responses by activating the innate immune system (11), and

enhancing the systemic antibody.response to some antigens

in chickens (12). Recently, the beneficail effect of Biomin®

C-X (Enterotococcus faecium + prebiotic+cell wall extract)

on humoral immunity to Newcastle disease vaccine of

commercial broilers was studied (13). This experiment was

conductd to investigate the effect of Biomin®IMBO

(Biomin G.T.I. GmbH., Ember AG-Austria; containing Enterotococcus faecium 5×1011 cfu /kg, prebiotic, cell wall

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (37-40)

38

and algae extracts) as a potential immunostimulator to

enhance humoral immune response to live and killed

Newcastle disease vaccines in broiler chickens.

Materials and methods

A total of 210 day-old Hubbard-Flex broiler chicks were procured from a local supplier. They were reared in cages

in a separate rooms of the animal house, College of

Veterinary Medicine, University of Mosul and fed ad

libitum with a Hubbard-Flex recommended diet. Ambient

temperature, lighting, ventilation and other environmental

conditions fully met the requirements for management of

Hubbard-Flex birds.

Biomin®IMBO

(Biomin G.T.I. GmbH., Ember AG-Austria, it contains

Enterotococcus faecium 5×1011 cfu / kg, prebiotic, cell wall

and algae extracts. IMBO was added to the feed free from antibiotics and administered throughout the study as

recommended by the manufacturer 1.5g/kg.

Drugs Cyclophosphamide (CPA) (Cycloxan® manufaured in

India, Biochem Pharmaceutical Industries LTD) was

procured from a local pharmacy. Day-old chickens of

groups G2 and G5 were given 3 mg per chicken per day for

4 consecutive days intramuscularly into leg muscle (14).

Challenge virus One day before challenge, birds in G1 splited

randomely to two halves; negative control group (G1); left

wthout challeneg and positive control group (G8) which

submitted to challenge. At 39 days of age chickens were

intramuscularly inoculated with virulent field NDV strain

(obtained from the Microbiology Department, College of

Veterinary Medicine, Mosul University). The virus titer

was determined to be 1×106.5 EID50 / 0.1ml.

Sampling On day 7(before vaccination 0, blood samples were

taken from each group to assess the maternal immunity.

Blood samples were taken at weekly intervals after

vaccination and challenge.

Serological Test Antibodies to NDV were quantitified by

hemagglunation inhibition test (HI) using the diluted

serum-constant virus procedure (15) and by indirect

Enzyme-linked Immunosorbent Assay (ELISA).

Experimental design Chickens were randomly divided to 7 groups with 30

birds each. These groups consisted of: 1) Non IMBO + Non

CPA + Non challenge referred to as negative control (G1),

2) IMBO + CPA&L-K (G2), 3) IMBO + Non CPA&L

(G3), 4) IMBO + Non CPA&L-K (G4), 5) Non IMBO +

CPA&L-K (G5), 6) Non IMBO + Non CPA&L (G6), 7)

Non IMBO + Non CPA&L-K (G7). Birds of all groups

except negative and positive controls were vaccinated with

live NDV (Cevac®Vitapest L; CEVA) at seven days old individually by oral route using 1 ml syringe. In addition,

each bird in groups G2,G4, G5, and G7 was

intramuscularly injected with 0.1 ml of killed NDV vaccine

(Cevac®Broiler NDK) at seven days of age. Revaccination

with live ND vaccine LaSota strain (Cevac® NEW

L;CEVA) was done at 21 days of age by spraying.

Results

HI titer serum antibody response According to figure (1), chicks contained maternal

antibody level before vaccination and gradually declined to low levels with time. At 28 days of age, production of

antibody detected in all groups except G1 group. The G2

(vaccine + IMBO+CPA) and G5 (vaccine + CPA) groups

produced significantly (P<0.05) lower levels of antibody in

comparison to other treatment groups indicating that

immunosuppression occurred. Furthermore, no significant

differences were found when the two groups were

compared at different time points post booster vaccination.

The data also revealed that GMT of G3 ( live vaccine +

IMBO) group was significantly (P<0.05) higher than G6

(live vaccine alone) group while GMT of G4 (live& killed vaccine + IMBO) group was statistically (P>0.05) not

different compared with G7 (live& killed vaccines).

Furthermore, when GMTs of G3 and G4 groups were

compared no significant (P>0.05) difference were found.

ELISA titer immune response The results of ELISA test are presented in figure (2). On

day 28, only birds in G3 and G7 groups showed

seroconversions, however their mean titers were not

significantly (P>0.05) different. The data also demonstrated

that the antibody titer of G3 was significantly higher (P<0.05) when compared with titer of G6, meanwhile the

titer of G4 was not significantly (P>0.05) different when

compared with G7. The titers of G2 and G5 groups

remained low and did not differ signficantly.

Mortality The post-challenge test results are shown in Table (1).

The table shows that the protection rate in G2, G3,G4, G5,

G6, G7 and G8 were 28.6, 85.71%, 96.29%, 31.8%, 83.87,

96.66%, and 0% respectively. No significant differences in

mortality were found between probiotic fed and their

corresponding control groups.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (37-40)

39

Fig. 1: Geometric mean HI antibody titer (log2) in chickens

with or without IMBO supplementation.

Fig. 2: Mean ELISA antibody titer in chickens with or

without IMBO supplementation.

Table 1: Response to challenge with virulent Newcastle

disease virus in chickens

Groups

No

dead/No

challenged

No

survived/No

challenged

Negative control group

(G1) 0/14

14/14 a

(100)B

IMBO,CPA &L-K vaccine

(G2) 20/28

8/28 c

(28.57)

IMBO&live vaccine

(G3) 4/28

24/28 a

(85.71)

IMBO&L-K vaccine (G4)

1/27 26/27 a

(96.29)

CPA &L-K vaccine

(G5) 15/22

7/22 c

(31.81)

Live vaccine

(G6) 5/31

26/31 a

(83.87)

L-K vaccine

(G7) 1/30

29/30 a

(96.66)

Positive control group

G8) 13/13 0b

Discussion

In this experiment, significantly higher HI and ELISA

titers were seen in birds received live NDV +IMBO(G3).

This is in agreement with finding of (13), however, IMBO

had no effect in birds immunized with live and killed

vaccine G4. The data on the effect of probiotics on immunity are extremely controversial due to the variety of

variables reported (16). More over(17) reported treating

with just one bacterial type may not be as effective and

increasing the types of bacteria in the mix could enhance

the efficacy of probiotic functions. The post-challenge

mortality rates observed in immunosuppressed (G2 and G5

groups) were higher compared with immune-competent

birds (G3, G4, G6 and G7 groups). The protection rate did

not differ significantly in G3 compared with G6 despite

enhancement of humoral immune response which

contradicts preivious report (18). The different results

might be due to twofold increase in titer observed in latter study and in addition, they challenged birds orally with

virulent NDV compared with IM challenge used in our

study, in addition Leghorn male chickens were used

compared to broilers in the present experiment. Under the

conditions of this study, IMBO significantly enhaced

humoral immune response to only live vaccine,and this is in

agreement with (19), but did not restore immunity in

immunosuppressed chickens and did not decrease post

challenge mortality in immunosuppressed and

immunocompetent broiler chickens.

References

1. Chaudhry K M, Chaudhry RA. Causes of vaccine failure. Vet Int.

1996;12: 13-15

2. Li Y C, Ledoux D R, Bermudez A J, Fritsche K L, Rottinghaus G E.

Effects of fumonisin B1 on selected immune responses in broiler

chicks. Poult Sci. 1999;78:1275-1282.

3. Sharma J M, Kim I J, Rautenschlein S, Yeh H Y. Infectious bursal

disease virus of chickens: pathogenesis and immunosuppression. Dev

Comp Immunol. 2000;24:223-235.

4. El-Lethey H, Huber-Eicher B, Jungi T W. Exploration of stress-

induced immunosuppression in chickens reveals both stress- resistant

and stress-susceptible antigen responses. Vet Immunol Immunopathol.

2003;95:91-101

5. Shivachandra SB, Sah RL, Singh SD, Kataria J M, Manimaran K.

Immunosuppression in broiler chicks fed aflatoxin and inoculated with

fowl adenovirus serotype-4 (FAV-4) associated with

hydropericardium syndrome. Vet Res Com. 2003;27:39-51

6. Kim Y, Brown TP, Pantin-Jackwood MJ. The effects of

cyclophosphamide treatment on the pathogenesis of subgroup J avian

leukosis virus (ALV-J) infection in broiler chickens with Marek’s

disease virus exposure. JVet Sci. 2004;5:49-58.

7. Muruganandan S, Lai J, Gupta PK. Immunotherapeutic effects of

mangiferin mediated by the inhibition of oxidative stress to activated

lymphocytes, neutrophils and macrophages. Toxicology. 2005; 215:

57-68.

8. Dugas B, Mercenier A, Lenoir-Wijnkoop I, Arnaud C. Dugas N and

Postaire E. Immunity and probiotics. Immunol Today. 1999;20:387-

390.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (37-40)

40

9. Maassen C B J D Laman W J Boersma and E Claassen. Modulation of

cytokine expression by lactobacilli and its possible therapeutic use. In

R. Fuller and G. Perdigon (ed.), Probiotics2001; 3:

immunomodulation

10. Nemcová R. Criteria for selection of lactobacilli for probiotic use. Vet.

Med. 1997; 42: 19-27.

11. Maldonado Galdeano C and Perdigo G. The probiotic bacterium

Lactobacillus casei induces activation of the gut mucosal immune

system through innate immunity. Clin. vaccine Immunol. 2006; 13:

219-226.

12. Haghighi H R Gong J Gyles C L Hayes M A Sanei B Parvizi P Gisavi

H Chambers J R and Sharif S H. Modulation of antibody-mediated

immune response by probiotics in chickens. Clin. Diagn. Lab.

Immunol. 12:1387-1392. by the gut microflora and probiotics. Kluwer

Academic, Dordrecht, the Netherlands. 2005; PP: 176-192.

13. Al-Dalo N R A A. Effects of probiotics on some aspects of pathology

and productivity of chicken broilers. A Ph.D. Thesis. College of

Veterinary Medicine, University of Mosul. Mosul. Iraq. 2007

14. Sharma J M and Lee L F. Suppressive effect of cyclophosphamide on

the T-cell system in chickens. Infect. Immun. 1977; 17:227-230.

15. Young M Alders R Grimes S Spradbrow P Dias P da Silva A and

LoboQ. Controlling Newcastle disease in Village Chickens: A

laboratory manual.PP:72-85.2002

16. De Roos N M and Katan M B. Effects of probiotic bacteria on

diarrhea lipid metabolism and carcinogenesis: A review of papers

published between 1988 and 1998. Am. J. Clin. Nutr. 2000 71:405-

411.

17. Sharif S. Probiotics Help Produce Safer, Healthier Chickens.

University of Guelph Ontario Veterinary College. College News

Published January 19. 2007; 338 Views

18. Zhang X Zhang X and Yang Q. Effect of compound mucosal immune

adjuvant on mucosal and systemic immune responses in chicken orally

vaccinated with attenuated Newcastle-disease. Vaccine.

2007;25:3254-3262.

19. Emad J K and Amjad H E.Study of some Impact of Enterococcus

faecium As Probiotic on chick 2-NewCastle Disease Antibody and

White blood cells.Iraqi J Vet Med 2007;31(1):78-86.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (41-43)

41

Serodiagnosis of Johne's disease by indirect ELISA in ovine

I. M. Ahmed

Department of Microbiology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq

(Received August 11, 2008; Accepted October 5, 2009)

Abstract

The study included collection of 92 serum samples from local Awasii breed in Mosul and Karakosh regions, some of them

show clinical signs for John's disease, all samples were assayed using indirect Enzyme-Linked Immunosorbent Assay (ELISA)

to detect antibodies against Mycobacterium avium subsp. paratuberculosis (Map). The results showed that 7/92 (7.6%)

samples were positive for antibodies against (Map), and 7/89 (7.9%) were positive in female and 0/3 (0%) in male.

Keywords: Mycobacterium, Paratuberculosis, Ovine Johne’s disease, Indirect ELISA, Serodiagnosis.

Available online at http://www.vetmedmosul.org/ijvs

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��0��( ��!�� 1�2 ���"3 %�3"��� �� �� ���� 4 � %"� 5 �� 6��� ���3 ������ ���7� 5 ��& �� "� �8���� '(� %��� 9��! ���:���� ���3Mycobacterium avium subsp. paratuberculosis (Map) . <=�" ���0> '>?/#$ �( � � � ������� %���A 9��! ��� �%7.6 . '> ��� ����� �2� B� C *� � ��89/7 ��� � %7.9���D *� � �� '� *� E/F ��� �E.%

Introduction

Johne's disease is also known as "paratuberculosis"

because it is caused by Mycobacterium avium subspecies

paratuberculosis (Map). It is a contagious disease in dairy

cattle, sheep, goats and other ruminants (1). The clinical

manifestation of paratuberculosis in sheep tends to prevail

at younger age than in cattle. Chronic weight loss is the

primary clinical sign of paratuberculosis in sheep and goats.

Affected sheep will experience progressive weight loss over a period of weeks to months and eventually die (2). Only

10-20% of clinical cases present with diarrhoea or

clumping of faeces in the advanced stage of the disease (3).

Johne’s disease is commonly found in dairy cattle herds and

sheep flocks but identification of individual sheep with

subclinical infections is difficult. Animals apparently are

infected when young but, while shedding the organism via

faeces, these animals may not show clinical symptoms for

several years. Infected animals may have reduced feed

efficiency without obvious clinical signs of disease. (4,5).

In sheep flocks, the fecal culture detects less than 12 % of

clinical cases and requires up to 12 months of incubation,

making it an impractical diagnostic test (6,7). Three

different tests are currently available for measuring

antibodies against Mycobacterium avium subsp. paratuberculosis in the serum of infected animals. These are

the complement fixation test (CFT), the agar gel

immunodiffusion (AGID) test and enzyme-linked

immunosorbent assay (ELISA). ELISA or AGID are still

the main options in live animals (8). Among various

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42

serological tests for Johne’s disease, ELISA-based tests are

widely used and can be conducted rapidly and require

limited expertise (9).

Materials and methods

A total of 92 blood samples, 3 samples from males and

the others from females were collected from local Awassi

breed sheep that some of them showed clinical signs for

John's disease including emaciation, unresponsive to

dewormers and antibiotics. Appetite was often good, in

spite of weight loss, all animals wer not previously

vaccinated against Johne's disease and they were >2 years

old. The samples were collected through April 2008 from

different regions, 69 samples represent 3 flocks containing

580 sheep from Mosul and 23 samples represent 2 flocks

containing 264 sheep from Karakosh. Samples were submitted to the department of microbiology (College of

Veterinary Medicine, Mosul, Iraq); within 24 h after

bleeding, serum samples were separated and stored at –

20°C until they were assayed (2,10).

A commercial ELISA kit (ID SCREEN®,

Paratuberculosis Indirect) for detection of antibodies

against Mycobacterium avium subsp. paratuberculosis in

ovine serum samples was used, the kit has been supplied

from (ID Vet (innovative diagnostics)-France). The

principle of the test depends on indirect ELISA. Sera were

tested according to the manufacturer's instructions for

ovine, the absorbance reading O.D in all ELISA plate wells were measured at 450 nm using an automated ELISA

reader. ELISA optical density (OD) readings were

transformed to Serum/Positive percentage (S/P%)

according to a specific equation cited by manufacturer. The

sample considered positive if it gives S/P % ≥ 70%, 60% <

S/P % < 70% considered doubtful, S/P % ≤ 60% considered

negative. S/P%=(OD sample-ODNC)/(ODPC-ODNC).

Results

The results showed that 7/92 samples 7.6% were positive for antibodies against Mycobacterium avium

subsp. paratuberculosis, P<0.001. The results are detailed in

table 1. Also the results showed no positive results in males

0/3 (0.0%) when compared with females 7/89 (7.9%). The

distribution of S/P % values for samples, positive control

and negative control are given in figure 1.

Table 1: Positive samples according to Mosul regions

Region No. of samples Positive %

Mosul 69 5 7.3

Karakosh 23 2 8.7

Total 92 7 7.6

Figure 1: Distribution of ELISA S/P % values for (n=92),

positive controls (PC) and negative controls (NC). ID

SCREEN®, Paratuberculosis Indirect ELISA kit, ID Vet

(innovative diagnostics).

Discussion

The aim of this study was to investigate Johne's disease

in sheep by serodiagnosis using ELISA as there are

indications for M. avium subsp. paratuberculosis infection

and unresponsive to dewormers and antibiotics. All sheep

were selected >2 years old as the clinical signs are

commonly not evident until at least 18 months of age (6).

As the cultivation of sheep strains of M. avium subsp.

paratuberculosis using culture media (Herrold's egg yolk

medium) has been extremely difficult to perform, the selection of ELISA in this study was based on the studies

by (6,12,13). Among the antibody tests, ELISA is more

sensitive than AGID and CFT test (14,15). It's performance

is similar in cattle, sheep, and goats (16,17) and can be used

with comparable sensitivity for either milk or serum

samples (18). AGID test is considered less sensitive than

ELISA and CFT (19). Since a strong humoral response

does not occur until the later stage of Johne's disease, the

sensitivity of these 3 tests is the highest for animals with

lepromatous lesions (8,20,21), those with clinical symptoms

(20,22,23), or those that shed large numbers of bacteria

(18,24). Therefore, the main limitation of these antibody tests is their inability to identify animals in early infection

(25,26). Conversely, all of these tests are highly specific,

with false-positive results occurring at low frequency (25).

The ELISA couldn't detect all animals with clinical signs.

Comparative studies of the CFT, AGID test and ELISA

repeatedly show discrepancies in the ability to identify all

infected animals (27,28). This may be due to genetic

variation of the individual animal or the lack of

representation of the entire range of immunodominant

antigens for Mycobacterium avium subsp. paratuberculosis

within a given test (29). There are few studies about ovine Johne's disease in the Arab countries, so there is no

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (41-43)

43

sufficient data to compare with the results of this study. The

total positivity % (7.6%) is considered high as the disease

now has a virtually worldwide distribution in farmed

ruminants as well as many species of wild ruminants (1,

11), also the high S/P% for the all 7 positive sera indicate

the high levels of antibodies against (Map) in the affected

sheep (20, 22, 23), this mean that we need more investigations to confirm Johne's disease in sheep and also

in other susceptible ruminants. In this study few numbers of

male samples were included, only 3 samples were tested

and all gives negative results 0.0%, because of the breading

style in keeping few numbers of rams for each flock. This

study has resulted in detection of antibodies against

Mycobacterium avium subsp. paratuberculosis in sheep.

The study was done for the first time in Iraq to provide

information about this disease for subsequent studies.

Acknowledgments

This study was supported by College of Veterinary

Medicine, University of Mosul. Mosul, Iraq.

References

1. Kennedy DJ and Benedictus G. Control of Mycobacterium avium

subsp. paratuberculosis infection in agricultural species. Rev. sci.

Tech. Off. Int. Epiz 2001; 20, 151-179.

2. Navneet K D, Jeff E, Whittington RJ, Jenny-Ann LML. Risk factors

for ovine Johne’s disease in infected sheep flocks in Aust. Preve Vet

Med. 2007; 82:51–71.

3. Stehman SM. Paratuberculosis in small ruminants, deer, and South

American camelides. Vet Clin North Am. Food Anim Pract. 1996;12,

441–455.

4. Reddacliff LA, McClure SJ and Whittington RJ Immunoperoxidase

studies of cell mediated immune effector cell populations in early

Mycobacterium avium subsp. paratuberculosis infection in sheep. Vet

Immunol Immunopathol. 2004;97:149-162.

5. Sigurethardottir OG, Valheim M, PressCM. Establishment of

Mycobacterium avium subsp. paratuberculosis infection in the

intestine of ruminants. Adv. Drug Deliv. Rev. 2004;56:819-834.

6. Collins DM, Hilbink F, West DM, Investigation of Mycobacterium

paratuberculosis in sheep by faecal culture, DNA characterization and

the polymerase chain reaction. Vet Rec. 1993;133(24):599.

7. Satoko K, Deborah LT, Yasuyuki M, Whittington RJ. Detection of

Mycobacterium avium subsp. paratuberculosis in ovine faeces by

direct quantitative PCR has similar or greater sensitivity compared to

radiometric culture. Vet Microb. 2007;125:36–48.

8. Clarke CJ, Patterson IA, Armstrong KE, Low JC. Comparison of the

absorbed ELISA and agar gel immunodiffusion test with

clinicopathological findings in ovine clinical paratuberculosis. Vet

Rec.1996;139:618-621.

9. McKenna SL, Keefe GP, Barkema HW and Sockett DC., Evaluation

of three ELISAs for Mycobacterium avium subsp. paratuberculosis

using tissue and fecal culture as comparison standards. Vet. Microbiol

2005;110:105–111.

10. Quinn PJ, Carter ME, Markey B and Carter GR. Clinical Veterinary

Microbiology. USA: Mosby, an imprint of Elsevier limited 2004;157.

11. Sergeant, ESG. Ovine Johne’s disease in Australia – the first 20 years.

Aust Vet J. 2001;79:484-491.

12. De Lisle GW, Collins DM and Huchzermeyer HF. Characterization of

ovine strains of Mycobacterium paratuberculosis by restriction

endonuclease analysis and DNA hybridization. Onderstepoort J Vet

Res.1992;59(2):163.

13. Shulaw WP, Bech-Nielsen S, Rings DM, Getzy DM and Woodruff

TS. Serodiagnosis of paratuberculosis in sheep by use of agar gel

immunodiffusion. Am J Vet Res 1993;54:13-19.

14. Cocito CP Gilot, Coene M, De Kesel M, Poupart P,Vannuffel P.

Paratuberculosis. Clin Microbiol Rev 1994;7:328-345.

15. Collins MT. Diagnosis of paratuberculosis. Vet Clin North Am. Food

Anim Pract. 1996;12:357-371.

16. Burnside DM, Rowley BO. Evaluation of an enzyme-linked

immunosorbent assay for diagnosis of paratuberculosis in goats. Am J

Vet Res. 1994; 55:465-466.

17. Dubash K, Shulaw WP, Bech-Nielsen S, Stills HF and Slemons RD.

Evaluation of an agar gel immunodiffusion test kit for detection of

antibodies to Mycobacterium paratuberculosis in sheep. J Am Vet

Med Assoc. 1996;208:401-403.

18. Sweeney RW, Whitlock RH, Buckley CL, Spencer PA. Evaluation of

a commercial enzyme-linked immunosorbent assay for the diagnosis

of paratuberculosis in dairy cattle. J Vet Diagn Investig. 1995;7:488-

493.

19. Nielsen SS, Nielsen KK, Huda A, Condron R, Collins MT. Diagnostic

techniques for paratuberculosis. In: Bulletin of the international dairy

federation. IDF 2001;362:5–17.

20. Perez VJ, Tellechea JJ, Badiola MG, Garcia-Marin JF. Relation

between serologic response and pathologic findings in sheep with

naturally acquired paratuberculosis. Am J Vet Res 1997;58:799-803.

21. Sven B, John PB, Frank J, Griffin T. Autoreactive antibodies are

present in sheep with Johne’s disease and cross-react with

Mycobacterium avium subsp. paratuberculosis antigens. Microbes

Infec. 2007;9:963-970.

22. Dubash K, Shulaw WP, Bech-Nielsen S, Stills HF, Slemons RD.

Evaluation of an enzyme-linked immunosorbent assay licensed by the

USDA for use in cattle for diagnosis of ovine paratuberculosis. J Vet

Diagn Investig. 1995;7:347-351.

23. Hilbink F, West DM, DeLisle GW, Kittelberger R, Hosie BD, Hutton

J, Cooke MM, Penrose M. Comparison of a complement fixation test,

a gel diffusion test and two absorbed and unabsorbed ELISAs for the

diagnosis of paratuberculosis in sheep. Vet Microbiol. 1994;41:107-

116.

24. Sockett DC, Conrad TA, Thomas CB and Collins M. T. Evaluation of

four serological tests for bovine paratuberculosis. J Clin Microbiol.

1992;30:1134-1139.

25. Beth HN, Raúl GB. Mycobacterium avium subsp. paratuberculosis in

Veterinary Medicine. Clin Microb Reviews 2001;14: 489-512.

26. Stewarta DJ, Vaughana JA, Stilesa PL, Noskea PJ, Tizarda MLV,

Prowsea SJ, Michalskia WP, Butlerb KL and Jonesc SL. A long-term

study in Merino sheep experimentally infected with Mycobacterium

avium subsp. paratuberculosis: clinical disease, faecal culture and

immunological studies. Vet Microb. 2004;104:165–178.

27. John PB, Valentina R, Stefania Z, Stefano R, Niyaz A and Leonardo

AS. Antigenic profiles of recombinant proteins from Mycobacterium

avium subsp. paratuberculosis in sheep with Johne’s disease. Vet

Immunol Immunopatho 2008;122:116–125.

28. Darcel C and Logan-Handsaeme B. ELISA testing for antibody to

Mycobacterium paratuberculosis. Can Vet J. 1998;39:335-336.

29. Sugden EA, Stilwell K and Michaelides AA. comparison of

lipoarabinomannan with other antigens used in absorbed enzyme

immunoassays for the serological detection of cattle infected with

mycobacterium paratuberculosis. J Vet Diagn Investig. 1997;9:413-

417.

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45

Effect of treating lactating rats with lead acetate and its interaction with

vitamin E or C on neurobehavior, development and some biochemical

parameters in their pups

A. A. Hassan and H. M. Jassim

Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Mosul, Iraq

(Received February 17, 2009; Accepted October 5, 2009)

Abstract

The current study investigated the effect of administration of vitamin E (600mg/ kg diet) concomitantly with lead acetate

(10mg/kg, orally) and vitamin C (100mg/kg, orally) concomitantly with lead acetate (10mg/kg, orally) to the female lactating

rats on the neurobehavioral, landmarks development and some biochemical tests in their pups. Administration of lead acetate

to the female lactating rats caused a significant increase in open field activity test including (the number of squares crossed and

rearing test within 3 minutes), olfactory discrimination test, triglycerides and malondialdehyde brain tissue, with a significant

decrease in glutathione brain tissue and high density lipoproteins in their pups. The present study demonstrated that treatment

of female lactating rats with vitamin C and lead acetate produced a significant decrease in righting reflex test in their pups.

Administration of vitamin E concomitantly with lead acetate to the female lactating rats caused a significant increase in

glutathione level accompanied with a significant decrease in malondialdehyde and triglycerides levels in their pups. The present study showed that treatment of female lactating rats with vitamin E or C with lead acetate produced a significant

decrease in rearing test, whereas a significant increase in high density lipoproteins in their pups. It is concluded that

administration vitamin E or C to the female lactating rats reverse the adverse effects produced by lead acetate on

neurobehavioral. Vitamin E had positive effect on the levels of glutathione, malondialdehyde brain tissue, triglyceride and high

density lipoproteins in their lactating pups.

Keywords: Vitamin E, Vitamin C, Lead acetate, Neurobehavior, Glutathione, Malondialdehyde, Rat pups.

Available online at http://www.vetmedmosul.org/ijvs

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Page 118: Iraqi Journal of Veterinary Sciences

Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (45-52)

46

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Introduction

Lead is an ubiquitous element in the environment, it is

used in many industrial activities including mining, refining

and producing lead – acid batteries (1). Although this heavy

metal is less widely used today, it remains a significant

public health problem. Animals may be exposed to lead via contaminated food or water and fuel additives (2). The

alimentary and respiratory tract are the major routes of lead

entry into the body (3). Once the lead is in the bloodstream,

it is distributed into soft and hard tissues (4). Milk is the

most important food source for newborn, however, also be

a pathway of maternal excretion of toxic elements such as

lead, and these toxins impact most severely on the newborn

at a time of rapid development of the central nervous

system (5). In the nervous system, all neurons and glial

cells form a very large network, integrate all external and

internal stimuli and contributes to the elaboration of

adequate responses (6). Lead enters the brain and selectively deposited in the hippocampus and cortex, as

well as in non-neuronal elements that are important in the

maintenance of the blood brain barrier function. Lead

exposure causes distractibility inability to maintain physical

balance and it affects some complex functions including

learning (7). The neuropathlogical effects of lead include

nervousness, anxiety and symptomatic encephalopathy at

the end (8). Pervious study has shown that the level of lead

in milk are thus similar to those in plasma (5). In rodents

lead mobilized from the skeleton is transferred to the

suckling offspring during lactation (9), and that lactational transfer after current or recent exposure to lead in dams was

considerably higher than placental transfer (10). The main

targets organ of lead toxicity are the red blood cell, central

nervous system, peripheral nerves and the kidney (11).

Recent study reported that lead acetate can be

transmitted through mother milk to their offspring. Lead

acetate make bad effects on the reproductive systems of

both males and females rats. While giving vitamin E as

antioxidant found to have no improving effect in the

treatment of lead acetate disturbances on the reproductive

system of both sexes (12). Lead-induced oxidative stress

contributes to the pathogenesis of lead poisoning for disrupting the delicate prooxidant/antioxidant balance that

exists within mammalian cells. Production of reactive

oxygen species (ROS) is increased after lead treatment in

vitro studies, moreover other studies in vivo suggest that

lead exposure cause generation of ROS and alteration of

antioxidants defense system in animals (13). The aim of present study is to assess the role of

administration of vitamin E & C concomitantly with lead

acetate to the dams during lactation period (21days) on the neurobehavioral, landmarks development and some

biochemical changes in their pups.

Materials and methods

Adult healthy albino rats were obtained from the

animals house of the Veterinary Medical College –

University of Mosul at 3-4 months of age weighing 150-

200g. The rats were mated (3:1 females to male). Pregnant

rats were removed and kept in separated polyproplyne

cages under condition of temperature (22-26 C°) and

lighting (12hours light /12hours dark). The rats were

supplied a standard pellet diet and tap water ad libitum. At

birth dams were separated with their pups from the first day

of parturition.

In this experiment, animals divided into four groups:

Group 1: dams (rats during lactation period) received

normal saline orally as control group. Group2: dams

received lead acetate at (10mg/kg B. Wt.) orally during

lactation period, lead acetate dissolved in distalled water

and given at 1 ml/kg (12). Group3: dams received lead

acetate (10mg/kg B. Wt.) orally with concomitant

administration of vitamin E at (600 mg /kg diet) during lactation period (14) (Shang Hang, China). Group 4: dams

received lead acetate (10mg/kg B. Wt.) orally with

simultaneous administration of vitamin C at (100 mg /kg B.

Wt.) orally during lactation period (15) (Chemical Suppl,

South Australia).

Twenty pups from each groups were selected randomly

for examination. Pups were examined at the 7 day of age

for neural behaviour which included righting reflex, cliff

avoidance and olfactory discrimination tests (16). Central

nervous system activity test were examined at the 21 day

which include onset of movement test, open field activity

test (17). This test measures the general locomotor activity, exploration which include (the number of squares crossed

and rearing test within 3 minutes) and negative geotaxis test

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (45-52)

47

(16). Sensimotor system activity were examined which

include approach response, touch response, click response

and tail pinch response (18). Landmarks development tests

included opening of ear, appearance of hair and teeth,

opening of eyes, descending time of testis and appearance

time of vaginal opening (16).

Blood was collected from pups at age 21 days for biochemical examination. Total cholesterol, high density

lipoproteins, glucose, alanine aminotransfererase, aspartate

aminotransfererase, and albumin, were measured using

colorimetric assay kit (Syrbio, Syria) and triglyceride was

measured using kit (bioMerieux, France), Pups weights

monitored at birth and at weaning time. Pups were

sacrificed at the end of experiment and the brain was placed

in ice normal saline for glutathione (Moron method) (19)

and malondialdehyde estimation (Gilbert method) (20).

Data were analyzed statistically using one way analysis

of variance. Group differences were determined using

Duncan multiple range test. Data of approach response, touch response, click response and tail pinch response

analyzed statistically using Mann _ whitny _ U _ test.

Statistical significance was considered at (P < 0.05) (21).

Results

In the current study Table 1- revealed that

administration of lead acetate to the dams during lactation

period did not effect the onset of movement and negative

geotaxis tests in their pups compared the pups of control

group. Treatment with vitamin E to the rats receiving lead acetate during lactation period did not effect significantly

the onset of movement and negative geotaxis tests in their

pups, but the treatment with vitamin C to the rats receiving

lead acetate during lactation period produced a significant

increase (P≤0.05) in the onset of movement test. Treatment

with each of vitamin E & C to the rats receiving lead

acetate during lactation period did not effect significantly

the negative geotaxis test in their pups compared with pups

of lead acetate group. Table 1- showed that rats receiving

lead acetate during lactation period cause a significant

increase (P≤0.05) in rearing and the number of squares

crossed tests within 3 minutes in their pups compared with

the pups of the control group. Treatment with vitamin E & C to the rats receiving lead acetate during lactation period

caused no significant changes in rearing test in their pups.

On the other hand, the number of square crossed test was

significantly decrease by vitamin E & C in pups compared

with the lead acetate group.

Table 2 shows that administration of lead acetate to the

rats during lactation period caused a significant increase in

olfactory discrimination (P≤0.05) in their pups compared

with the pups of control group.

Treatment with vitamin E & C to the rats receiving lead

acetate during lactation period did not affect significantly in

olfactory discrimination test in their pups compared with the pups of lead acetate group. No significant changes

between groups in cliff avoidance test. Same Table

demonstrated that administration of lead acetate to the rats

during lactation period did not effect significantly in

righting reflex test in their pups compared with the pups of

control values. Treatment with vitamin E of rats receiving

lead acetate during lactation period did not affect

significantly in righting reflex in their pups compared with

the pups of lead acetate group, but treatment with vitamin C

of rats receiving lead acetate during lactation period cause a

significant decrease (P≤0.05) in righting reflex test in their pups compared with the pups of lead acetate group.

The data of vitamin E & C revealed no significant

differences on sensimotor include (approach, touch, click

and tail pinch responses),landmarks development and

weighing of pups (at 21 days) from rats receiving lead

acetate during lactation period are shown in Table 3,4,5.

Table 1. The onset of movement, open field activity tests and negative geotaxis in suckling pups at(21 days) from dams treated

with lead acetate and their interaction with vitamin E or C for 21 consecutive days (lactation period).

Open field Negative geotaxis/sec square crossed with 3 mins Rearing with 3 min

Onset of movement/ sec

Treatment of dams

a

4.65±0.41 a

11.20±0.51 a

56.75±2.42 ab

2.15±0.13 control

ab

5.80±0.51 b

14.50±0.48 b

65.25±3.48 a

2.05±0.13 Lead acetate (10mg/ kg) orally

b 6.7±0.64

a 11.90±0.46

b 65.20±2.52

ab 2.30±0.14

Lead acetate (10mg/ kg) orally

+ vitamin E (600mg/ kg diet)

ab

6.05±0.29 a

12.05±0.65 ab

58.35±1.96 b

2.55±0.18 Lead acetate (10mg/ kg) orally

+ vitamin C (100mg/kg)

Values were expressed as means ± SE from 20pups per treatment.

Values with different letters are significantly different at (P≤0.05).

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (45-52)

48

Table 2. The righting reflex,cliff avoidance and olfactory discrimination tests in suckling pups at (7 days) from dams treated

with lead acetate and their interaction with vitamin E or C for 21 consecutive days (lactation period).

Treatment of dams Righting reflex(sec) Cliff avoidance

(sec)

Olfactory discrimination

(sec)

Control b

2.95±0.13

a

9.35±0.57

a

11.75±0.42

Lead acetate (10mg/ kg) orally b

2.90±0.17

a

8.75±0.89

b

23.85±1.33

Lead acetate (10mg/ kg) orally

+ vitamin E(600mg/ kg diet)

ab

2.65±0.18

a

10.25±0.95

b

20.70±1.93

Lead acetate (10mg/ kg) orally

+vitamin C(100mg/kg)

a

2.30±0.11

a

10.30±0.82

b

20.90±1.48

Values were expressed as means ± SE from 20pups per treatment.

Values with different letters are significantly different at (P≤0.05).

Table 3. The approach, touch, click, tail pinch responses in suckling pups at (21 days) from dams treated with lead acetate and

their interaction with vitamin E or C for 21 consecutive days (lactation period).

Treatment of dams Approach response/

score

Touch response/ score

Click response/ score

Tail pinch

response/ score

Control a

1.90±0.02

a

2±0

a

2.60±0.16

a

3±0.1

Lead acetate (10mg/ kg) orally a

1.90±0.02

a

2.05±0.02

a

2.70±0.14

a

2.90±0.12

Lead acetate (10mg/ kg) orally

+ vitamin E (600mg/ kg diet)

a

1.90±0.02

a

2.05±0.02

a

2.70±0.14

a

2.95±0.13

Lead acetate (10mg/ kg) orally

+ vitamin C (100mg/kg)

a

1.95±0.02

a

2±0

a

2.85±0.1

a

3±0.02

Values were expressed as means ± SE from 20 pups per treatment.

Table 4. The landmarks development in suckling pups from dams treated with lead acetate and their interaction with vitamin E

or C.

Treatment of dams

Appearance

of

ear opening

Appearan

ce of hair

Appearan

ce of teeth

Appearance

of eye

Descending

time of testis

Appearance

of vaginal

opening

Control a

2.1±0.12

a

6.4±0.21

a

5.9±0.17

a

16.16±0.2

a

33.5±0.29

a

47.2±0.40

Lead acetate (10mg/ kg) a

2.15±0.13

a

6.7±0.16

a

5.9±0.17

a

17.15±0.26

a

32.8±0.46

a

47.5±0.36

Lead acetate (10mg/ kg)

+ vitamin E (600mg/ kg diet)

a

2.15±0.13

a

6.4±.19

a

6±0.16

a

17.3±0.17 a 33.15±0.37

a

47.0±0.24

Lead acetate (10mg/ kg)

+ vitamin C (100mg/kg)

a

2.1±0.12

a

6.6±0. 2

a

5.9±0.18

a

17.10±0.2

a 33.25±0.33

a

47.4±0.32

Values were expressed as means ± SE from 5 pups per treatment.

In the present study, Table 6 demonstrated that

administration of lead acetate to the rats during lactation

period cause a significant decrease in glutathione level with

increase in malondialdehyde in brain tissue of their pups

compared with the control group. Administration of vitamin

E to the rats receiving lead acetate during lactation period

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (45-52)

49

produce a significant increased in glutathione and decrease

(P≤0.05)in malondialdehyde levels in their pups compared

with the pups of lead acetate group. No significant

differences in the level of glutathione and malodialdehyde

of the pups when their dams treated with vitamin C and

lead acetate concomitantly during lactation period

compared with the pups of lead acetate group. In same Table the data shows no significant differences in the levels

of alanine aminotransferase, aspartate aminotransferase and

albumin in the pups of all group.

Table 7 demonstrated that administration of lead acetate

alone or concomitantly with vitamin E & C to the rats

during lactation period did not effect significantly glucose

and cholesterol levels in their pups. Administration of lead

acetate to rats during lactation period caused a significant

increase (P≤0.05) in triglyceride level and significant

decrease in high density lipoproteins in their pups

compared with the pups of control value. Treatment with

vitamin E to the rats receiving lead acetate during lactation

period produced a significant decrease (P≤0.05) in

triglyceride and significant increase in high density lipoproteins level in their pups compared with the pups of

lead acetate group. Rats administered lead acetate with

vitamin C during lactation period did not affect triglyceride

level significantly in their pups compared with that of lead

acetate group, on the other hand vitamin C caused a

significant increase (P≤0.05) in high density lipoproteins in

pups compared with the pups of lead acetate group.

Table 5. The body weight in suckling pups from dams treated with lead acetate and their interaction with vitamin E or C for 21

consecutive days (lactation period).

Treatment of dams Weight(g) at 1 day Weight (g)at 21 day

Control a

5.46±0.02 a

25.27±0.19

Lead acetate (10mg/ kg) orally a

5.44±0.02

a

25.31±0.45

Lead acetate (10mg/ kg) orally

+ vitamin E (600mg/ kg diet)

a

5.33±0.10

a

25.06±0.15

Lead acetate (10mg/ kg) orally

+ vitamin C (100mg/kg)

a

5.43±0.02

a

25.13±0.16

Values were expressed as means ± SE from 20 pups per treatment.

Table 6. The glutathione, malondialdehyde brain tissue, alanine amimotraferase, aspartate amimotraferase and albumin in

suckling pups from dams treated with lead acetate and their interaction with vitamin E or C for 21 consecutive days (lactation

period).

Treatment of dams Glutathione

µmlg

Malodialdehy

de nm/g

Alanine

Amimotraferase

Unit/ L

aspartate

amimotraferase

Unit/ L

Albumin

g/dl

Control a

2.73±0.2

b

107±4.19

a

11.54±0.62

a

23.34±1.61

a

3.06±0.17

Lead acetate (10mg/ kg) orally c

1.19±0.17 a

181.8±8.39 a

11.16±0.99 a

22.12±0.99 a 2.97±0.11

Lead acetate (10mg/ kg) orally

+ vitamin E (600mg/ kg diet)

b

1.8±0.15

b

112±6.71

a

10.56±0.54

a

24.92±0.8

a

3.2±0.38

Lead acetate (10mg/ kg) orally

+ vitamin C (100mg/kg)

c

0.99±0.002

a

167.4±9.9

a

11.08±0.47

a

22.6±1.28

a

3.42±0.48

Values were expressed as means ± SE from 5pups per treatment.

Values with different letters are significantly different at (P≤0.05).

Discussion

The present study showed that administration of lead

acetate to rats during lactation period caused a significant

increase in the open field activity test including (rearing and

the number of squares crossed tests within 3 minutes) and

olfactory discrimination test in their pups compared to pups

of the control group.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (45-52)

50

The nervous system is the most sensitive target of lead

exposure. Fetuses and young animals are especially

vulnerable to the neurologic effects of lead because their

brains and nervous system are still developing and blood

brain barrier is incomplete (22). Lead neurotoxicity results

in behavioral and neurochemical alteration in neurons as a

result of changes and disruption of main structural components of the blood brain barrier, through primary

injury to astrocytes and to secondary damage of the

endothelial microvasculature (3). There are numerous

studies utilizing experimental animal models on the central

nervous system, These studies have mainly been concerned

with possible effects of lead on certain performance tasks

that might reflects a cognitive function (learning and

memory) or sensorimotor function in the infant animal

exposed to lead very early in life or in utero (23). Some

investigators studied effects of lead on the action of

neurotransmitters using isolated peripheral nerve

preparation, both cholinergic and adrenergic synaptic

evoked transmitter released is inhibited by lead, and this effect is prevented by calcium (23). Lead affects primarily

the metabolism of calcium (24), and inhibits the action of

calcium as a result lead can affect calcium-dependent

processes and interact with proteins including sulfhydryl,

amine, phosphate, and carboxyl groups (21).

Table 7. The glucose, cholesterol, triglyceride and high density lipoproteins in suckling pups from dams treated with lead

acetate and their interaction with vitamin E or C for 21 consecutive days (lactation period).

Treatment of dams glucose

mg/dl

Cholesterol

mg/dl

Triglyceride

mg/dl

High density Lipoprotein

mg/dl

Control a

102±6.5

a

71.3±4.8

b

121±4.8

a

44.1±3.9

Lead acetate (10mg/ kg) orally a

106±3.7

a

91.9±10.4

a

186.8±5.8

b

29.2±3.4

Lead acetate (10mg/ kg) orally

+ vitamin E (600mg/ kg diet)

a

104±4.3

a

71.6±5.9

b

118.2±6.3 a

42.1±2.3

Lead acetate (10mg/ kg) orally

+ vitamin C (100mg/kg)

a

106±3

a

80.5±7.8

a

196±4.8 a

40.9±4.7

Values were expressed as means ± SE from 5pups per treatment.

Values with different letters are significantly different at (P≤0.05).

The nervous system is the most sensitive target of lead

exposure. Fetuses and young animals are especially

vulnerable to the neurologic effects of lead because their

brains and nervous system are still developing and blood

brain barrier is incomplete (22). Lead neurotoxicity results

in behavioral and neurochemical alteration in neurons as a

result of changes and disruption of main structural components of the blood brain barrier, through primary

injury to astrocytes and to secondary damage of the

endothelial microvasculature (3). There are numerous

studies utilizing experimental animal models on the central

nervous system, These studies have mainly been concerned

with possible effects of lead on certain performance tasks

that might reflects a cognitive function (learning and

memory) or sensorimotor function in the infant animal

exposed to lead very early in life or in utero (23). Some

investigators studied effects of lead on the action of

neurotransmitters using isolated peripheral nerve

preparation, both cholinergic and adrenergic synaptic evoked transmitter released is inhibited by lead, and this

effect is prevented by calcium (23). Lead affects primarily

the metabolism of calcium (24), and inhibits the action of

calcium as a result lead can affect calcium-dependent

processes and interact with proteins including sulfhydryl,

amine, phosphate, and carboxyl groups (21).

Neurotoxicity may be a consequence of alterations in

cholinergic function mediated by the enzyme

acetylcholinesterase (AchE) (25).

Maged recorded that lead caused a progressive decrease in the activity of acetylcholinesterase in different brain

regions and spinal cord (26).

The enzyme inhibition is generally reached its

significance after 10 to 20 days of lead acetate intake orally

to the rabbits, such alteration in cholinergic transmission

suggests that lead is able to reach the CNS and exerts its

neurotoxic effect (26). It was supposed that oxidative stress

was one possible mechanism for lead neurotoxicity. lead–

induced oxidative stress might result from accumulation of

5-aminolevulinic acid (ALA), a potential endogenous

source of free radical, induced by inhibition of lead to ALA

dehydratase, overload of ALA seemed to be involved in the neurological disturbances, which leads to inhibition γ- aminobutyric acid (GABA) release from synaptosomes and

blocking GABA receptor (27). Nihei et al (28) have

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (45-52)

51

reported ALA can cause oxidative stress to rats brain.

Additionally, direct interaction of lead to biological

membranes was to induce lipid peroxidation. Lead-

exposure might also induce decrease in activities of free

radical scavenging enzymes. This mainly attributed to high

affinity of lead to sulfhydryl-groups in these enzymes (29).

The result of the current study showed that administration of lead acetate to the rats during lactation

period caused a significant increase in malondialdehyde and

triglyceride with a significant decrease in glutathione in

their pups compared with the control group. Numerous

reports have documented increased lipid peroxidation

(LPO) and decreased glutathione (GsH) and superoxide

dismutase (SOD) activity in the brain homogenates of lead

treated rats (30). Furthermore, lead exposure led to

depletion of brain glutathione content, superoxide

dismutase activity, and increase in thiobarbituric acid

reactive substances (TBARS), and the activity of

glutathione S-transferase bound enzyme (31). Oxidative damage associated with lead in the brain has been proposed

as a possible mechanism of lead toxicity (31). Some

investigators revealed that lead –treated cultured aortic

endothelial cells caused increase in the production of the

lipid peroxidation products malondialdehyde and enhanced

generation of hydroxyl radical compared with control cells

which is considered as a direct cause of oxidative stress

(33). It should be noted that hydroxyl radicals are primarily

produced from sequential reduction of superoxide radical

and hydrogen peroxide radical (33). Lead-induced oxidative

stress associated with hyperglycemia suggested to contribute in the overproduction of very low density

lipoprotein (VLDL), increasing the burden of triglyceride-

rich lipoproteins on the common lipolytic pathway at the

level of lipoprotein in lipase (34).

In the current study administration vitamin E &C to the

rats receiving lead acetate during the lactation period

ameliorating the effect of lead acetate in their lactating

pups. Recent study carried out effects of lead actate at 600

p.p.m. in drinking water during pregnancy and lactation

caused significant decrease in activities of superoxide

dismutase, glutathione peroxidaes and glutathione reductase in hypothalamus, corpora quadrigemina and corpus striatum

in weaned pups (mouse) (27).

Vitamin E is necessary for the maintenance of normal

neurological structures and function, and play a role in

protecting lipid rich structures such as the brain from free

radical (35). Antioxidant /chelating action represented by

vitamin E improved the enzyme activity in the central

nervous system (25). A major contributor to non-enzymatic

protection of polyunsaturated fatty acid and low density

lipoprotein from oxidation by free radicals against lipid

peroxidation is vitamin E (35). Vitamin E as a lipid soluble,

chain breaking antioxidant (37), plays a major protective role against oxidative stress (38), and prevents the

production of lipid peroxide by scavenging free radicals in

biological membrane (39).

Previous studies have revealed that vitamin E possesses

an antioxidant activity in protecting cells from damage by

highly reactive superoxide free radicals production (35). In

the tissue of vitamin E deficient animals, it is reported that

lipid peroxidation is enhanced suggesting that vitamin E plays a role as physiological antioxidant on its chemical

properties, and prevent oxidation of low density lipoprotein

(40). While vitamin C the most abundant water- soluble

antioxidant in the body acts primarily in cellular fluid of

particular in combating free radical for caused by pollution

furthermore vitamin C help vitamin E to return to its active

form (40). Moreover, Frei (41) found that only ascorbic

acid is protectively enough to intercept oxidant in the

aqueous phase before they can attack and cause detectable

oxidative damage to lipids, as compared to many other

lipophilic and hydrophilic antioxidants. (42) revealed that

administration of vitamin E to rats during lactation period caused a positive effects on lipid profile, glutathione and

malondialdehyde in brain tissue, of their offspring.

Acknowledgements

This study was supported by the College of Veterinary

Medicine, University of Mosul.

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31. Saxena G, Flora S. Lead –induced oxidative stress and hematological

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32. Kang JK, Sul D, Nam SY, Kim HJ, Lee E. Effect of lead exposure on

the expression of phosphlipidhydroperoxidase glutathione peroxidase

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33. Ding Y, Gonick HC, Vaziri ND. Lead promotes hydroxyl radical

generation and lipid perxidation in cultured aortic endothelial cells.

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34. De-Man FH, Gabezas MC, Van-Barlingen HH, Erkelens DW, Bruin

TW. Triglyceride-rich lipoproteins in non-insulin-dependent diabetes

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35. Yoshioka T, Motoyama H, Yamasaki F, Ando M, Takehara Y,

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53

Use of saturated sodium chloride solution as a tissue fixative

A. Al-Saraj

Department of Dental Basic Sciences, College of Dentistry, University of Mosul, Mosul, Iraq

E.mail : [email protected]

(Received March 4, 2009; Accepted October 5, 2009)

Abstract

The present study was carried out to examine the capability of saturated sodium chloride solution as a fixative agent instead

of formalin which is regarded as a carcinogenic material. For this purpose 3 rabbits were used and their livers, kidneys and

spleens were exposed and removed. Neutral buffered formalin solution, saturated sodium chloride solution and distilled water

were used as fixatives for specimens obtained from the first, second and third rabbits respectively. Routine histological

technique was performed to prepare a stained histological sections for light microscopic examination. The result showed that

the tissue sections which were obtained by using sodium chloride have the same histological features and without any artifacts

when they compared with the results obtained using formalin fixation method. We conclude that the saturated sodium chloride solution can be used as a fixative agent in some circumstances when no any fixative agent is available.

Keywords: Sodium chloride, Formalin, Tissue fixative.

Available online at http://www.vetmedmosul.org/ijvs

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Introduction

As early as 400 BC Hippocrates discussed the biological

effects of mercury and alcohol as fixatives (1). The

objective of fixation is to preserve cells and tissue

constituents in as close a life-like state as possible and to

allow them to undergo further preparative procedures

without change. Fixation arrests autolysis and bacterial

decomposition and stabilises the cellular and tissue

constituents so that they withstand the subsequent stages of

tissue processing. Fixation should also provide for the

preservation of tissue substances and proteins. Therefore, it

is regarded the first step and the foundation in a sequence of

events that culminates in the final examination of a tissue

section (2).

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54

A large variety of fixatives is now available but no

single substance or known combination of substances has

the ability to preserve and allow the demonstration of every

tissue component. It is for this reason that some fixatives

have only special and limited applications, and in other

instances, a mixture of two or more reagents is necessary to

employ the special properties of each. The selection of an appropriate fixative is based on considerations such as the

structures and entities to be demonstrated and the effects of

short-term and long-term storage (3). Each fixative has

advantages and disadvantages, some are restrictive while

others are multipurpose. Ferdinard Blum has been credited

as the first person to use formaldehyde as a tissue fixative.

Formaldehyde, as 4% buffered formaldehyde (10%

buffered formalin), is the most widely employed universal

fixative particularly for routine paraffin embedded sections

(4).

The aim of the present work is to test the possibility of

using saturated sodium chloride solution as fixative and preserving agent during histological and pathological

procedures and compared with the results obtained by using

conventional formalin fixation.

Materials and methods Three male rabbits were randomly assigned irrespective

of age and weight into the following: First rabbit:

specimens fixed in neutral buffered formalin (positive

control rabbit). Second rabbit: specimens fixed in saturated

sodium chloride solution. Third rabbit: specimens immersed in distilled water (negative control rabbit).

Each rabbit was anaesthetized with chloroform in an air-

tighted jar, then the animal was laid down on dissecting

board. The liver, kidneys and spleen were exposed,

removed and washed by water. Specimens of 5mm

thickness of these organs were excised.

The fixation was made immediately after the removal of

the above mentioned organs for 24 hours by using the

following:

First rabbit: Neutral buffered formalin fixative solution which

composed of: Formaldehyde 37%, 100 ml. Distilled water

900 ml. Sodium phosphate monobasic (NaH2PO3), 4 gm.

Sodium phosphate bibasic (Na2HPO3), 6.5 gm.

Second rabbit: Saturated sodium chloride solution.

Third rabbit: Distilled water.

Then procedure of preparing the paraffin section slides

and staining by Harrie`s Haematoxylin and Eosin (H&E)

was performed to prepare a stained histological sections for

light microscopic examination (5).

Results

Liver: fixed in neutral buffered formalin There were no tissue artifacts such as shrinkage,

precipitate and swelling, therefore the liver appears to be

divided clearly into many lobules, each one is hexagonal in

shape with central vein located at its center. The portal canal which consist of a bile duct, a branch of hepatic artery

and a tributary of the portal vein, all appeared clearly and

enclosed in a common investment of connective tissue (take

pink color). The paranchymal tissue of the liver appeared to

be arranged in one or two cell thickness plates converging

from the periphery to the center of lobule and the sinusoids

are intermingled in between these plates. The cytoplasm

appeared to be acidophilic and the nucleus basophilic and

located at the center of the cells. Some hepatocytes

appeared to contain more than one nucleus, and each

nucleus contains a prominent nucleolus (Fig. 1 and 2).

Liver: fixed in saturated sodium chloride solution The sodium chloride solution was preserve the tissue

and cells without any shrinking or swelling and without

distorting or dissolving cellular constituents. Therefore the

liver lobule architecture is similar to those of the control

group. The central vein being located at the center of the

hexagonal liver lobule. The hepatocytes appeared normal

and arranged in form of plates. The cytoplasm appeared to

be acidophilic while the nuclei basoplilic and some of

hepatocytes were binucleated, this indicates that the tissue

sections were taken the stained in good manner and without any precipitate (Fig. 3 and 4).

Liver: immersed in distilled water There was no section appear under microscope.

Kidney: fixed in neutral buffered formalin The cortex of the kidney showed clear cellular details of

its all structures (renal corpuscles, convoluted and straight

tubules) and stain well by (H &E) stain. There was no

shrinkage or swelling of cells. While the medulla of the

kidney also showed clear cellular details of it all structures (straight portions of tubules, thin segments of Henel`s loop

and collecting tubules). There were no tissue artifacts

(shrinkage, precipitate and swelling) and all of sections

appeared to be stained well (Fig. 5 and 6).

Kidney: fixed in saturated sodium chloride solution Sections taken from kidneys of this rabbit revealed that

the general structure of the kidney is nearly similar to that

of the control group and seemed to be normal i.e. preserved

cortex and medulla architecture without any morphological

changes of the cells such as swelling and shrinkage or any

artifacts. Also the cellular details appeared clearly and stained well (Fig. 7 and 8).

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Figure 1: Photomicrograph of the liver of rabbit fixed in

neutral buffered formalin showing hepatocytes (H) and the

sinusoids (S) (H&E X400).

Figure 2: Photomicrograph of the liver of rabbit fixed in

neutral buffered formalin showing the small bile duct (B),

the terminal branch of the portal vein (V) and the terminal

branch of hepatic artery (A) in the portal tract (H&E X400).

Figure 3: Photomicrograph of the liver of rabbit fixed in

sodium chloride solution showing hepatocytes (H) and the

sinusoids (S) (H&E X400).

Figure 4: Photomicrograph of the liver of rabbit fixed in

sodium chloride solution showing the small bile duct (B)

and the terminal branch of the portal vein (V) in the portal

tract (H&E X400).

Figure 5: photomicrograph of the kidney (cortex) of rabbit

fixed in neutral buffered formalin showing the glomerulus

(G), proximal (P) and distal (D) convoluted tubules (H&E

X400).

Figure 6: photomicrograph of the kidney (medulla) of

rabbit fixed in neutral buffered formalin showing the

straight segment of the proximal tubule (SP) and the

straight segment of the distal tubule (SD) (H&E X400).

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Figure 7: photomicrograph of the kidney (cortex) of rabbit

fixed in sodium chloride solution showing the glomerulus

(G), proximal (P) and distal (D) convoluted tubules (H&E

X400).

Figure 8: photomicrograph of the kidney (medulla) of

rabbit fixed in sodium chloride solution showing the

straight segment of the proximal tubule (SP) and the

straight segment of the distal (SD) tubule (H&E X400).

Figure 9: photomicrograph of spleen of rabbit fixed in

neutral buffered formalin showing the white (w) and the red (R) pulps (H&E X100).

Figure 10: photomicrograph of spleen of rabbit fixed in

neutral buffered formalin showing the central artery (A) of

the white pulp and the sinusoids (S) of the red pulp. (H&E

X400).

Figure 11: photomicrograph of spleen of rabbit fixed in

sodium chloride solution showing the white (W) and the red

(R) pulps (H&E X100).

Figure 12: photomicrograph of spleen of rabbit fixed in

sodium chloride solution showing the central artery (A) of

the white pulp and the sinusoids (S) of the red pulp. (H&E

X400)

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Kidney: immersed in distilled water There was no section appear under microscope

Spleen: fixed in neutral buffered formalin Histological section throughout the spleen showed a

well defined cellular structures arranged into white (splenic

nodules and central artery) and red (splenic cords and sinuses) pulps without any artifacts (Fig. 9 and 10).

Spleen: fixed in saturated sodium chloride solution Microscopically, there was a preserved normal

histological sections architecture nearly similar to those of

control group without any morphological (shrinkage or

swelling) changes. The well defined histological structures

indicates well stained and preserved sections (Fig. 11 and

12).

Spleen: immersed in distilled water There was no section appear under microscope.

Discussion

A systematic study of the fixatives began in the latter

half of the 19th century, however, it must be noted that

fixation by itself introduces a major artifact. Much attention

was focused on developing fixatives that would preserve

cells and tissue constituents in as close a life-like state as

possible while allowing them to undergo further preparative

procedures without change (6).

The mechanisms by which fixatives act may be broadly categorized as dehydrants, heat effects, cross-linkers, and

effects of acids and combinations of these. Agents that

combine with proteins are called additives and those that

precipitate proteins are called coagulants. At this time, it is

accepted that no one fixative fulfils all of the aims of cell or

tissue preservation: namely prevention of autolysis and

preservation of physical and chemical properties of the

tissue (7).

In aldehydes include formaldehyde (formalin) the tissue

is fixed by cross-linkages formed in the proteins,

particularly between lysine residues. This cross-linkage does not harm the structure of proteins greatly, so that

antigenicity is not lost. Formalin penetrates tissue well, but

is relatively slow and the standard solution is 10% neutral

buffered formalin. A buffer prevents acidity that would

promote autolysis and cause precipitation of formol-heme

pigment in the tissues (8).

Formalin is used for all routine surgical pathology and

autopsy tissues when (H and E) slide is to be produced. It is

the most forgiving of all fixatives when conditions are not

ideal, and there is no tissue that it will harm significantly.

Most clinicians and nurses can understand what formalin is

and it smells bad enough that they are careful handling it.

However previous workers have indicated that exposure

to formaldehyde has a serious effects and is highly

suspected to be a human carcinogenic (9,10).

A method to overcome the problems of formaldehyde is

to use an alternative fixative that is better suited for the

preservation of tissues.

A study conclude that the cheap saturated table salt solution can be used as an alternative to the formaldehyde

in preserving the bodies of animals (11).

Fixation of skin and lymph node fragments in anhydric

sodium chloride at room temperature for periods of weeks

or months was found to preserve morphological structures

and immunoreactivity (12).

Segments of rat's aorta were harvested, immersed in

pulverized dehydrated sodium chloride, and stored for 1

week to 3 months. Thereafter, they were desalinated and

transplanted. The tensile strength and maximum

intraluminal pressures did not significantly differ from

freshly harvested, transplanted aortic segments (13). Human skin fragments can be preserved in anhydric

sodium chloride at room temperature for periods of weeks

or months and successfully transplanted, retaining normal

morphological structure (14).

The result of the present study showed that the saturated

sodium chloride solution can be used as a fixative for tissue

in pathological or histological procurers. The tissue sections

which obtained have the same histological features when

they compared with the results obtained using conventional

formaldehyde fixation methods. It penetrates tissue but is

relatively slow and prevents autolysis as well as it cause no precipitation of pigment in the tissues nor morphological

changes (swelling or shrinkage) of the cells.

The exact mechanism by which the sodium chloride act

on the tissue is unclear and no one mentioned its precise

action on the tissue. Therefore it need more investigations

and can be classified as unknown mechanism fixative, such

as mercuric chloride, picric acid.

We suggest that this new (cheap and safe) method of

fixation can be considered as an alternative fixative

procedure which can be used in emergency cases when no

any fixative is available (for pathologist, veterinarian, biologist…etc) in such cases for tissue preservation.

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