15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt

MCE4A GENE IS A SPECIFIC GENE FOR DETECTION OF M.BOVIS IN EGYPT

(With 3 Tab&1Fig)

By

Saad El-din, Nasr.; Nagah, M. Saad.; Nasr, E. A.; Nahed, M. Wahba and Walaa, M. Elsherif

فى بوفز الميكوباكتيريم عن للكشف متخصص جينMce4A االلبان منتجات وبعض الخام اللبن

بواسطة

, , , محمود وولاء وهبه محمد ناهد نصر امين عصام سعد محمد نجاح نصر الشريف سعدالدينالعربى الملخص

رة تفاعل أثبت طريقة انه المتسلسل البلم اكتيريم مرض عن للكشف ومحددة وحساسة دقيقة سريعة، وفز الميكوب ب الطرق مقارنة ة. وفى التقليدية ب ذه والكيميائي تم الدراسة ه

وائية عينه ثالثمائة جمع اطق من عش ة في مختلف ة من أس يوط. منها مدين ام حليب عينه 150 ات بعض من150و خ ان منتج منها األلب

ادي نعة الزب ا المص دة محلي ريش والجبن والزب لكل عينه 50) الق ان منهم(. بة وك زل نس و 2 ،4 الميكوباكتيريا ع

تخدام 0٪ تاينلوفين باس ن ش و 0 ،2 و (LJ) Pyruvated جنس تخدام 0٪ تاين باس ريناتد لوفينش ات من اللبن من كلل جليس الحيوان

لبية االيجابية ار والس تيوبركلين الختب اع واللبن ال المحالت المب على ب ان القريش الجبنو الزبدةو محليا مصنعةال الزبادى عينات في التوالي. تك ات لوفينشتاين باستخدام2 ،.2 ،4 النسبة تاين ٪2 ،2 ،0 و بيروف لوفينش

ريناتد والي على جليس ارت .الت د وأش زالت تحدي ان الع اكتيريم ب الميكوب ات من ٪2و ٪4 بنسبةMOTT وبوفز ات من اللبن عين ال الحيوان و هإيجابي

MOTT (2٪) لميكوباكتيريما عن الكشف نامك كما السلبية. الحيوانات من وفز رى الميكوبكتريا و ب بةMOTT االخ )1  بنس دة من ٪(2 واللبن الزب

المحالت المباع اللبن من يعزل ولم القريش الجبن الزبادي، عمل تم وقد بPCR باستخدام العزالت هذه لتأكيد بوفز الميكوباكتيريم من عترات 6 على )  استخدام نقترح للعترات. لذا ومؤكدة ايجابية النتيجة وكانت Mce4A جين

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15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt

Mce4A الالت( ا. المعزولة للس اك محلي ود من مزيد إلى حاجة وهن الجه.المرض انتشار لتجنب الحليب بيع ونقاط المزارع وتشجيع لتعزيز

SUMMARY

Polymerase Chain Reaction (PCR) proved to be a rapid, accurate, sensitive and specific method for detection of M. bovis in regard to conventional, biochemical and serological methods. Three hundred random samples were collected from different localities in Assiut city including 150 raw milk samples and some dairy products as locally manufactured yogurt, Kareish cheese and cooking butter (50 samples for each). The incidence of Mycobacteria was 4, 2 and 0% using Lowen stein Jensen (L-J) medium Pyruvated and 2, 0 and 0% using L-J medium Glycerinated for tuberculin positive reactors, tuberculin negative reactors dairy animals and marketable milk samples, respectively. In locally manufactured yogurt, Kareish cheese and cooking butter samples the incidence was 4, 2, 2% for pyruvated media and 0, 2, 2% for glycerinated media, respectively. Identification of isolates indicated M. bovis (4%) and Mycobacteria other than tuberculosis (MOTT) (2%) from tuberculin positive reactors and (2%) from tuberculin negative reactors animals. M. bovis and MOTT could be detected in 1 (2%) of locally manufactured yogurt, Kareish cheese and cooking butter and failed detection in marketable milk samples. PCR was done to 6 M. bovis strains previously identified by biochemical tests and positive results were obtained. To confirm these isolates we suggest using specific primer (Mce4A) as a specific primer for locally isolated strains. More efforts are needed to enhance and promote farms and sale points of milk.

INTRODUCTION

Tuberculosis (TB) is still a major public health problem in many parts of the world despite multiple efforts to combat it (Isdore et al., 2010). Every minute (somewhere in the world) four people die from tuberculosis (Khansobis et al., 2002).W H O (2009) estimated 13.7 million people had active TB disease, with 9.3 million new cases and 1.8 million deaths; the annual incidence rate varied from 363 per 100,000 in Africa to 32 per 100,000 in the Americas. The high prevalence of

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15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt

Bovine Tuberculosis among the human population in Egypt is due to the way of living of more than 20 million Egyptians at rural areas, where those human and farm animals live side by side in close contact and the insufficient extension service in the rural areas (Siam, 1992).

The polymerase chain reaction (PCR) showed more promising for Mycobacterial spp. detection in clinical samples. This technique seemed to have sensitivity equal or greater than that of the culture method. Thus PCR increases the rate of detection and will be a useful tool for control programs especially when considering the lower sensitivity of diagnosis by bacteriological culture, the occupational risk, technical difficulties with the protocol and the prolonged time of incubation (Bermudez et al., 2010). Mammalian cell entry (mce) was able to confer the ability to invade HeLa cells and macrophages to E. coli cells transformed with plasmids containing it. They found that the mce gene sequence contains two open reading frames (ORF). The first (ORF1) has been shown to contain the element responsible for attachment and entry into mammalian cells; the second was shown to confer the ability to survive inside cells. Kumar et al. (2008) demonstrated that recombinant mce4A protein facilitates the invasion of non-pathogenic strain of E. coli into non-phagocytic HeLa cells. They also observe that mce4A gene has a role comparable to mce1A in the survival of recombinant E. coli in human macrophages. This study aimed to detect specific gene in Egypt that help in rapid diagnosis of tuberculosis.

MATERIAL AND METHODS

1- Tuberculin skin test: was done according to Ovdiennkop et al., (1987)2- Isolation and identification of M. bovis from milk and some milk products samples:A- Collection of samples: A total of 300 random samples of raw milk from (tuberculin positive, negative reactors and marketable milk) and some milk products (yoghurt, kareish cheese and cooking butter) were collected from different localities in Assiut Governorate. B- Preparation of samples according to Neill et al., (1988) and A.P.H.A, (1992):-C- Identification of isolates: using Ziehl-Neelsen stain for acid-fast bacilli and conventional methods (rate of growth, colonial morphology, and pigmentation and biochemical properties) according to Brasil (1994).

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15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt

D- PCR analysis: (Sambrook et al., 1989).1- Extraction of genomic DNA from field isolates: One hundred mg (wet weight) of the cell pellet of each 9 isolate was re-suspended in 0.5ml TE buffer, allowed for 2 cycles freezing and thawing. The cells were then homogenized in glass homogenizer and then incubated 4 hours at 37oC with 5ul lysozymes (final concentration 100μg/ml). Proteinase-K was added 25μl/0.5 ml (final concentration 100μg/ml) and incubated for further 3 hours at 56 oC with shaking. DNA was then extracted using Trizol reagent.

The supernatant was completely discarded and the DNA pellet was washed twice with 0.1ml sodium citrate in 10% ethanol. At each time, the DNA pellet was kept in the washing solution for 30 minutes at RT with periodical mixing and centrifuged at 4000 rpm/5 minutes at 4oC. Following the 2 washes, the DNA was re-suspended in 2ml of 75% ethanol, kept at RT for 20 minutes with periodical mixing and then centrifuged. The DNA pellet was finally dried briefly for 5 minutes under vacuum and re-dissolved in 50ul of 8 mM NaOH. The pH was adjusted at 8. Two µl of RNAase were then added and incubated at 37oC for 1 hour.2- Purification of DNA: Purification of DNA was done according to manufacturer instruction. 3- Measuring DNA concentration and purity: One µl of DNA was diluted with 49 µl dist. water and the optical density (OD) was measured in a quarter cuvette at 260 and 280 nm.

4- PCR amplification of mce4A gene: In a 0.2 ml PCR tubes the following reaction mixture was added: DNA tamplet (100 ng/µl) 10 μl, Taq polymerase 5u/μl. (1 µl), 10x enzyme buffer 5 μl, dNTPs 2 μl then added Q-Solution 10 μl, Primer 1 (1 μl), Primer 2 (1 µl): ~ Primer 1 (Sense): 5´-CAC-CTT-CCT-CAT-CCC-CTC-3´, ~ Primer 2 (Antisense): 5´-GAT-GAG-GGA-TTG-GAA-CAA-C -3´ and Bidist. water to 50 µl. The mixture was placed in the thermal cycler (T- gradient, Biometra Inc, Germany), which was programmed as follow: Initial denaturing applied at (95oC/3 minute), number of Cycles (40 cycles), denaturing at (95oC/1 minute), annealing at (56 oC/45 seconds), extension at (72oC/1 minute) and final extension at (72oC/10 minutes).5- Agarose gel electrophoresis: Electrophoresis was done at 80 v/15 minutes and examined using UV Trans-illuminator.

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15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt

RESULTTable 1: Incidence of Acid Fast Bacilli in milk and some milk products samples examined microscopically using Ziehl-Neelsen stain

Positive samples Negativesamples

No. % No. %

Milk

from

tuberculin positive reactors

50 7 14 43 86

tuberculin negative reactors

Marketable milk 50 1 2 49 98

locally manufactured yoghurt

50 4 8 46 92

Kareish cheese

Cooking butter 50 2 4 48 96

Table 2: Incidence of Mycobacteria isolated from milk and some milk products samples using conventional culture method.

Lowenstein- Jensen media pyruvated glycerinated

+ve % +ve %

Milk

from

tuberculin positive reactors

50 2 4 1 2 3

tuberculin negative reactors

Marketable milk 50 0 0 0 0 0

locally manufactured yoghurt

50 2 4 0 0 2

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15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt

Kareish cheese 1 2 2 1

Cooking butter 50 1 2 1 2 1

Table 3: Identification of Mycobacteria isolated from milk and some milk products samples.

Type of samples No. ofsamples

Positive culture for AFB

Types of MycobacteriaM. bovis MOTT

No. % No. % No. %

Milk from

tuberculinpositivereactors

50 3 6 2 4 1 2

tuberculinnegativereactors

50 1 2 1 2 0 0

Marketable milk 50 0 0 0 0 0 0

locally manufactured yoghurt 50 2 4 1 2 1 2

Kareish cheese 50 2 4 1 2 1 2

Cooking butter 50 2 4 1 2 1 2

Total 300 10 3.3 6 2 4 1.3

Photo 1:- 1% agarose gel staining by 0.5μg/ml ethidium bromides showing:--ve referred to a negative control (unamplified PCR products), +ve referred to positive control, M referred to 100 base pair ladder marker.

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15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt

Lanes: 1-6 positive for M. bovis by using mce4A gene (700bp) and Lanes: 7-10 Mycobacteria other Than M. bovis.

DISCUSSION

The results of AFB in milk samples examined by direct smear using ZN stain was shown in Table (1). It is clear that the total positive samples were 7 (14%), 3 (6%) and 1 (2%) in milk from tuberculin positive reactors, tuberculin negative reactors and marketable milk samples, respectively. While, the results of AFB in some milk products samples examined by direct smear in locally manufactured yoghurt was 4 (8%), Kareish cheese 5 (10%) and Cooking butter 2 (4%). Nearly the same detected by Shaimaa Shalapy (2009). The reason for such results may be due to obtaining some milk samples from infected farms in Assiut. Although the direct microscopical examination is easy, rapid and cheap, it will be positive only in the presence of large number of bacilli and it can't differentiate between tubercle bacilli and saprophytic AFB. El-Guindi et al. (1980) and Dunn and Hodgson (1982) proved that milk samples with enough viable tubercle bacilli excreted can infect the milk of 100 clean cow to be of infectious level and confirmed by Bastawrous (1992).

Mycobacteria was detected by culturing in 3 (6%) and 1 (2%) milk samples from tuberculin positive and negative reactors while, failed detection in marketable milk (Table 2). Higher results where detected by Vekemans et al. (1999) who detected Mycobacteria in 26% of retailed milk samples collected in markets in Burkina Faso, Munreo et al. (2000) 9.3% in Canadian cattle and Ameni et al. (2003) (13.3%). Clarice Fujimura Leite et al. (2003) isolated Mycobacteria from 15 of 22 (68.2%) caseous lesions from livestock and from 23 of 128 (18%) milk samples. Hamid et al. (2003) recorded 28.07% from milk of tuberculin positive buffaloes and from milk of 25% tuberculin positive cows. Richardson (1970) who reported that atypical mycobacteria were probably present in the environment and may get entrance to udder by contamination during animal lay down or during non-proper milking with contaminated equipment, these environmental atypical mycobacteria may succeeded to induce ascending infection and even lead to positive tuberculin results.

The results of Mycobacteria by culturing was 2 (4%), 1 (2%) and 1 (2%) for locally manufactured yoghurt, Kareish cheese and Cooking butter, respectively (Table 2). Higher values were recorded by Munreo

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15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt

et al. (2000) and Clarice Fujimura Leite et al. (2003), but nearly similar to Cousins and Dawson (1999), Hamid et al. (2003), Shirima et al. (2003) and Faye et al. (2005).

In this study the isolated Mycobacteria from collected milk samples were 4, 2 and 0% of M. bovis and 2, 0 and 0% of MOTT for tuberculin positive, negative reactors and marketable milk, respectively while the results of locally manufactured yoghurt, Kareish cheese and cooking butter were 2% of both M. bovis and MOTT for each (Table 3). While, M. tuberculosis could not be detected from all samples. L-J medium with Glycerol was used for the cultivation and differentiation of human and bovine types of the tubercle bacillus. Maureen (1981) stated that the growth of M. bovis could be stimulated by the addition of sodium pyruvate to the medium.

The mce proteins are a family of invasion-like proteins located at the Mycobacterial cell surface (Chitale et al., 2001 and Ahmad et al., 2005). The mce gene sequence contains two open reading frames. The first (ORF1) has been shown to contain the element responsible for attachment and entry into mammalian cells; the second was shown to confer the ability to survive inside cells (Ereny Markos, 2011).

Photo (1) showed (-ve) a negative control (unamplified PCR products); (+ve) positive control; (M) 100 base pair ladder marker; lanes (1-6) revealed to all M. bovis at 700bp (100%) while the lanes (7-10) indicated MOTT and they were isolated from milk of tuberculin positive reactors, locally manufactured yoghurt, Kareish cheese and Cooking butter. These results were consistent with (Nassar et al., 2007 and Ereny Markos, 2011). Higher results obtained by Sechi et al., (2000) (86.7%) and Cristina et al., (2005) (76.5%).

PCR is a sensitive and fast diagnostic tool that can be used to detect the agent in clinical samples in 48 h, but the presence of inhibitors in samples can interfere with its performance (Haddad et al., 2004; Singh et al., 2004 and Brasil, 2005) although, the sensibility of PCR from culture is more accurate (Sakamoto, 1997) and more confirmatory for isolates. Genetic elements specific to virulence of the target species would constitute attractive probe targets. The presence of virulence factors can help to establish the disease potential of different samples (Lang et al., 1994). The genetic elements responsible for the virulence of Mycobacteria are not yet well established. Many virulence genes of various levels of complexity are probably found in the MTBC. Also, Stuart et al. (1993) recorded that the PCR of samples prepared by the chaotrope-silica method had a sensitivity of 75% and a specificity of 100%, whereas, PCR of samples prepared by the chloroform method had

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15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt

a sensitivity of 92% and a specificity of 100% when compared with the sensitivities and specificities of the combined classical microbiological methods for the diagnosis of tuberculosis. It is concluded that the PCR was at least as sensitive as microscopy, but had greater specificity because samples with atypical mycobacteria were not detected by PCR.

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