Research ArticlePrevalence of Haemoplasma Infections in Stray Cats inNorthern Italy

Eva Spada1 Daniela Proverbio1 Paola Galluzzo2 Alessandra Della Pepa1

Giada Bagnagatti De Giorgi1 Roberta Perego1 and Elisabetta Ferro1

1 Reparto di Medicina Emotrasfusionale Veterinaria (REV) Dipartimento di Scienze Veterinarie per la Salutela Produzione Animale e la Sicurezza Alimentare (VESPA) Universita degli Studi di Milano Via G Celoria 10-20133 Milano Italy

2 Centro di Referenza Nazionale per Anaplasma Babesia Rickettsia e Theileria (CRABaRT)Istituto Zooprofilattico Sperimentale della Sicilia Via G Marinuzzi 3-90129 Palermo Italy

Correspondence should be addressed to Eva Spada evaspadaunimiit

Received 8 December 2013 Accepted 16 January 2014 Published 23 February 2014

Academic Editors R E Levin and D Rodrıguez-Lazaro

Copyright copy 2014 Eva Spada et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study investigated the prevalence of feline haemoplasma infections in a number of stray cat colonies in Milan Northern ItalyBlood samples from 260 stray cats were evaluated with conventional PCR for the presence of DNA associated with Mycoplasmahaemofelis (Mhf) and ldquoCandidatus Mycoplasma haemominutumrdquo (CMhm) Odd ratios (OR) were calculated to identify risk factorsfor haemoplasma infections PCRwas positive in 86 out of 260 subjects (331) with a prevalence of 108 (28260 cats) forMhf and223 (58260 cats) for CMhm No coinfections were registeredThere were significant associations between infections and seasonof sampling that is a negative association between winter sampling and a haemoplasma positive status (OR = 029 119875 = 0001) orCMhm positive status (OR = 029 119875 = 001) Haemoplasma infections are common in stray cats inMilanThus domestic cats withoutdoor access should be routinely monitored and treated for ectoparasites to minimize risks of disease acquisition Moreover asthese infections are transmitted via blood feline blood donors from this area should be screened by PCR and preferably be drawnfrom a population of indoor cats regularly treated for fleas

1 Introduction

Haemotropic mycoplasmas (haemoplasmas) are bacterialorganisms without cell walls that attach to and grow on thesurface of red blood cells Three feline haemoplasma speciesare described Mycoplasma haemofelis (Mhf) ldquoCandidatusMycoplasma haemominutumrdquo (CMhm) and ldquoCandidatusMycoplasma turicensisrdquo (CMt) [1] These feline haemoplas-mas are the causative agents of infectious anemia in catsand in several mammalian species There is also potentialinterspecies transmission of some of these agents as recordedfrom cats to immunocompromised dogs [2] Their zoonoticpotential has recently been substantiated by the molecularidentification of a feline haemoplasma isolate (Mhf) in anHIV-positive immunocompromised human patient [3] Thethree feline haemoplasma species have different pathogenici-ties Mhf often resulting in haemolysis and severe anaemia incontrast to CMhm and CMt which are less pathogenic [4]

Although several studies worldwide have reported onthe epidemiology of feline hemoplasmosis in sick or healthyclient-owned pet cats with prevalences ranging from 72 to458 [5ndash19] and few studies have focused on stray cat (withprevalences from 115 to 60 [20ndash24]) there have been nostudies investigating stray cats in Northern Italy Informationon regional prevalence of haemoplasmas could be used tolimit the spread of diseases in feline populations and forpredicting the likelihood of infection in cats presented withanemia

The transmission of haemoplasmas is still poorly under-stood The vector potential of Ctenocephalides felis has beendemonstrated in experimental Mhf infections [25] and straycats may play a role in multiplying the organism in fleasthat then infest pet cats and dogs and human beings Directtransmission by aggressive interaction of cats or interspeciestransmission might play a role in the epidemiology ofthese organisms In addition haemoplasmas can be directly

Hindawi Publishing CorporationISRN MicrobiologyVolume 2014 Article ID 298352 8 pageshttpdxdoiorg1011552014298352

2 ISRNMicrobiology

transmitted through intravenous infusion of infected blood[4 26] and have been shown to survive for up to 1 weekin stored blood products [27] As administration of freshor stored whole blood becoming more common in felinemedicine the knowledge of the regional prevalence of bloodtransmitted pathogens in owned and stray cats that sharethe same environment and parasites is increasingly impor-tant This would provide useful information for evaluatingthe risks of transmission of blood-borne infections frompotential blood donors and in the development of optimalscreening protocols in blood donors

The aimof this studywas to evaluate using a conventionalPCR assay the prevalence of Mhf and CMhm infections instray cats from colonies in Milan and to identify possible riskfactors for these infections

2 Materials and Methods

21 Sample Population and Data Collection During a 2-year collection period (January 2008 to January 2010) bloodsamples were taken from 260 stray cats from urban coloniesinMilan (Northern Italy) under a trap-neuter-release (TNR)program approved by the local authority of the city councilThe program was conducted as previously described [28 29]

Age (estimated based on dentition animals lt6 monthsof age were considered juvenile whereas all others wereconsidered adult) gender (male or female) origin (prove-nance area of colonies) and body condition score (BCS 4ndash6 indicating normal weight 1ndash3 underweight) were recordedtogether with data obtained from physical examination of thecats (healthy or unhealthy) Unhealthy cats were defined ascats with one or more of the following clinical abnormalitieslymph node enlargement pale mucous membranes stomati-tis or signs of ocular and respiratory infections

The seasonal analysis based on the date of sample col-lection was grouped as follows winter (January Februaryand March) spring (April May and June) summer (JulyAugust and September) autumn (October November andDecember)The catswere not systematically examined for thepresence of ticks or fleas and so rates of ectoparasitism werenot recorded

22 Haematological and Serological Analyses Blood sampleswere collected aseptically from the jugular vein while catswere anaesthetized for neutering and placed in EDTA-treatedtubes and in serum separator tubes

Within 24 h of sample collection a complete blood count(CBC) was performed on whole blood using an ADVIA 120System (Siemens Healthcare Diagnostics Milan Italy) Catswere categorized in terms of presence or absence of anaemia(HCT lt 24) leukopenia (WBCs count lt 10570120583L) leuko-cytosis (WBC gt 14390120583L) and thrombocytopenia (PLT lt200670120583L) [30] Surplus blood was stored at minus20∘C to uselater in PCR assay

Following separation serum samples were tested forantibodies to FIV (relative to gp40 and p24 FIV antigens)and for FeLV p27 antigen with a commercial enzyme-linkedimmunosorbent assay (ELISA) kit (Snap FeLVFIV Combo

Plus Test Idexx Laboratories Hoofddorp The Netherlands)Toxoplasma gondii sera IgG antibodies were detected usingindirect fluorescent antibody tests (IFAT) performed witha commercial kit (Fuller-laboratories Fullerton CA USA)Titres ge1 64 were considered seroreactive and thereforeindicative of T gondii exposure The results of these sero-logical tests have been already published [28] and werereanalyzed with the present results

23 PCR Assay Conventional PCR was performed on bloodsamples to amplify DNA associated with haemoplasmas(Mhf and CMhm) The reaction mixture included 2120583L oftemplate DNA 025mM dNTPs 04mM of each primer1x reaction buffer and 25U Tap DNA polymerase (GoTaqDNAPolymerase PromegaMadisonWIUSA)The volumeof this mixture was adjusted to 25 120583L with sterile waterPrimers were used that target part of the 16S rRNA geneproducing a 170 base pair (bp) product from Mhf and 193 bpamplicon from MChm (forward primer 51015840-ACG AAA GTCTGA TGG AGC AAT A-31015840 and reverse primer 51015840-ACG CCCAAT AAA TCC GRA TAA T-31015840) [6 31] PCR reactions wereperformed using an automated thermocycler PCR productswere resolved using 2 agarose gels and fragment size wasestimated using aDNAmolecular weightmarker (50 bpDNALadder Promega Madison WI USA) Control reactionswere done in the absence of template DNA to rule outcontaminations during PCR

24 Statistical Analysis Association between the threegroups of haemoplasma status (haemoplasma positive Mhfpositive only and CMhm positive only) and categoricalvariables (age gender colony of origin BCS season of sam-pling health status presence or absence of selected clinicaland CBC abnormalities FeLVFIV status and T gondii testresults) were analysed by univariate analysis using the chi-squared test (cell frequencies of gt5) or Fisherrsquos exact test(cell frequencies of le5) Any parameters statistically linked topositive PCR results were used in a logistic regression modelto test for independent risk factors associated with infection

Descriptive statistics (including minimum maximummean median and standard deviation (SD)) were obtainedfor the continuous variables RBCs count HCT HB WBCcount and PLT count values Distribution of the data fornormality was assessed with Kolmogorov Smirnov test anda 119905-test or a Mann-Whitney 119880 test respectively was used totest the differences between the feline haemoplasma positiveand negative cats depending on whether data was normallydistributed or not

Associations were considered statistically significantwhen 119875 lt 005 Both the 119875 value and odds ratio (OR) with95 confidence interval (CI) are reported Data were ana-lyzed using MedCalc Software (version 1230 MariakerkeBelgium)

3 Results

Haemoplasmic DNA was detected in 331 (86260 95 CI265ndash409) of blood samples Of the positive samples 28

ISRNMicrobiology 3

0

5

10

15

20

25

30

35

40

Neg Mhf CMhm

HCT

()

Figure 1 HCT values of Italian stray cats grouped by haemoplasmainfectious status Boxes represent the 25th 50th (median) and 75thquartiles withwhiskers extending to the greatest and smallest valuesBlue circles indicate outliers (cases with values greater than 15 boxlengths from the upper or lower edge of the box) CMhm ldquoCan-didatus M haemominutumrdquo positive Mhf M haemofelis positiveThe HCT values of CMhm group and the Mhf group were notsignificantly lower than the negative group The HCT values of theMhf group were also not significantly lower than the CMhm group

cats (108 95CI 72ndash156) were infected withMhf and 58cats (223 95 CI 169ndash288) were infected with CMhmNo comorbid infections were registered

Characteristics of the population and association betweenrisk factors and haemoplasma PCR status (negative or pos-itive) are reported in Table 1 and between Mhf alone andCMhm alone positive results in Table 2

None of the risk factors were associated with the PCRresults for haemoplasmas (Tables 1 and 2) with the excep-tion of negative associations at univariate and multivariateanalysis between winter season of sampling and haemo-plasma positive status (119875 = 001 OR = 029 95 CI = 014ndash061 119875 = 0001) and CMhm positive status (119875 = 001 OR =029 95 CI = 012ndash070 119875 = 001)

CBC results from 150260 cats are reported in Table 3Data was normally distributed according to KolmogorovSmirnov test No significant associations were found withCBC abnormalities and haemoplasma PCR-positive results(Figure 1) or in the number of anaemic and nonanaemic catsusing a 119905-test

4 Discussion

We present the first study investigating the prevalence ofhaemoplasmas in urban stray colony cats from the city ofMilan in Northern Italy

The prevalence of haemoplasma infection in our sampleshowed a similar pattern to that reportedworldwide in client-owned [5ndash7 9ndash15 17ndash19] and stray cats [19ndash23] CMhminfection is reported to be the most common infectionranging from 8 in Arizona [21] to 416 in Portugal[19] Mhf infection was less common ranging from 05 in

Switzerland [9] to 128 in Portugal [19] and dual infectionwas absent or rare (no more than 54 as found in Korea)[22]

The prevalence of haemoplasmas in stray cats in ourstudy (331) was higher than that reported in other studieson stray cats performed worldwide (Table 4) Differences inprevalence among countries can be explained by geographicalvariation such as climate vector distribution and the catpopulation surveyedMoreover direct comparisons of preva-lence results are of limited value because of the characteristicsof sample of cat populations investigated (number of catsincluded in the study healthy versus sick cats) inclusioncriteria diagnostic techniques (molecular tools versusmicro-scopical detection) different statistical methods stage ofinfection (acute versus chronic) or a combination of all themresulting in differences between studies

Stray cats in this study had higher rates of infections thanthose reported for pet cats in Northern Italy [12] in which189 of PCR tested cats were reported to be haemoplasmapositive A high incidence of haemoplasmas in stray cats isnot surprising as outdoor access is a recognized risk factor forinfection For example in a study on haemoplasma in client-owned cats fromBarcelona (Spain) outdoor accesswas foundto be a risk factor for infection (OR = 38) [15] Additionallyin a study involving feline blood donors from the USA theprevalence of haemoplasmas was 197 in domestic cats withoutdoor access and only 36 in domestic cats not allowedoutdoors [8] Free-ranging cats may have more exposureto bloodsucking arthropods and exhibit a higher fightingactivity than owned indoor cats thus they might experiencea higher infection risk for haemoplasmas

In our study there was a negative statistical associationbetween sampling in the winter season and haemoplasmaPCR-positive status and CMhm positive status This may bedue to reduced outdoor activity of fleas in winter seasonsalthough this hypothesis is purely speculative There wereno statistically significant differences or any relationshipbetween the presence of haemoplasma infection and anaemiastatus or HCT levels between positive and negative haemo-plasma group This finding was somewhat surprising andwas in agreement with studies undertaken in client-ownedcats in Switzerland [9] and in Italy [12] which also foundno association between haemoplasma infection and anaemiaor HCT variations In addition other studies have failed todemonstrate a significant difference in prevalence rates ofhaemoplasma between healthy and anaemic cats [10 32]These results could be explained by the higher prevalenceof CMhm (a less pathogenic species than Mhf) in this andprevious studies or by the fact that the stage of infectionis not known in our cats with a positive PCR result sincechronically infected cats that recover from acute illnessmay be asymptomatic Another explanation for the lack ofassociation between infection status and the presence ofanaemia in the present study could be that the HCT wasknown only for 150 of the 260 cats tested

Epidemiological data on haemoplasma are particularlyimportant in areas where feline blood donor programmesare active as in Milan where a donor programme has beenrunning since 2010 at University Veterinary Transfusion Unit

4 ISRNMicrobiology

Table 1 Sample characteristics of 260 stray cats testing positive and negative for haemoplasma infection by conventional PCR in NorthernItaly

Factor Category Total number PCR119875 value

() Negative () Positive ()

Origin of the cats(119899 = 260)

Zone 1 3 (12) 3174 (17) 086 (00) 119875 = 054

Zone 2 11 (42) 6174 (34) 586 (58) 119875 = 057

Zone 4 108 (415) 68174 (391) 4086 (465) 119875 = 031

Zone 5 12 (46) 8174 (46) 486 (47) 119875 = 077

Zone 6 27 (104) 15174 (86) 1286 (140) 119875 = 027

Zone 7 55 (212) 41174 (236) 1486 (163) 119875 = 023

Zone 8 22 (85) 14174 (80) 886 (93) 119875 = 092

Zone 9 22 (85) 19174 (109) 386 (35) 119875 = 007

Age (119899 = 260) Juvenile (le6 months) 118 (454) 81174 (466) 3786 (430) 119875 = 069

Adult (gt6 months) 142 (546) 93174 (534) 4986 (570)

Gender (119899 = 260) Male 90 (346) 30174 (172) 6086 (698) 119875 = 037

Female 170 (654) 144174 (827) 2686 (302)

BCS (119899 = 243) Poor (1ndash39) 18 (74) 10165 (61) 878 (103) 119875 = 037

Good (4ndash69) 225 (926) 155165 (939) 7078 (897) 119875 = 054

Seasons (119899 = 260)

Winter 64 (246) 5464 (944) 1064 (156)

P = 001OR = 029

CI = 014ndash061P = 0001lowast

Spring 69 (265) 3869 (551) 3169 (449) 119875 = 009

Summer 31 (119) 2331 (742) 831 (258) 119875 = 054

Autumn 96 (369) 5996 (615) 3796 (385) 119875 = 040

Health status (119899 = 260) Healthy 72 (277) 47174 (270) 2586 (291) 119875 = 084

Unhealthy 188 (723) 127174 (730) 6186 (709)

Clinical abnormalities inunhealthy cats (119899 = 188)

Lymph node enlargement 133 (707) 87102 (853) 4686 (535) 119875 = 069

Pale mucous membranes 14 (74) 11102 (108) 386 (35) 119875 = 051

Stomatitis 101 (537) 71102 (696) 3086 (349) 119875 = 043

Signs of respiratory tractinfection 22 (117) 12102 (118) 1086 (116) 119875 = 029

Signs of ocular infection 40 (213) 26102 (255) 1486 (163) 119875 = 092

CBC abnormalities(119899 = 150)

Anaemia 62 (413) 3391 (363) 2959 (492) 119875 = 069

Leukopenia 15 (100) 1191 (121) 459 (68) 119875 = 056

Leukocytosis 5 (33) 391 (33) 259 (34) 119875 = 066

Thrombocytopenia 10 (67) 491 (44) 659 (102) 119875 = 030

FIV test results (119899 = 166) Positive 13 (78) 8102 (78) 564 (78) 119875 = 082

Negative 153 (922) 94102 (922) 5964 (922)FeLV test results(119899 = 166)

Positive 6 (36) 4102 (39) 264 (31) 119875 = 090

Negative 160 (964) 98102 (961) 6264 (969)T gondii test results(119899 = 113)

Positive 31 (274) 1887 (207) 1346 (283) 119875 = 092

Negative 82 (274) 4987 (563) 3346 (717)PCR polymerase chain reaction BCS body condition score CBC complete blood count CI 95 confidence interval OR odds ratio FeLV feline leukemiavirus and FIV feline immunodeficiency virus119875 values in bold are statistically significant (119875 lt 005)lowastResults from multivariate logistic regression analysis

ISRNMicrobiology 5

Table 2 Characteristics of 28 Mhf PCR positive stray cats and 58 CMhm PCR positive stray cats in Northern Italy

Factor Category PCR positive resultsMhf 119899 () 119875 value CMhm 119899 () 119875 value

Origin of the cats

Zone 1 03 (00)

119875 = 063

03 (00)

119875 = 047

Zone 2 111 (91) 411 (364)Zone 4 12108 (111) 28108 (259)Zone 5 112 (83) 312 (250)Zone 6 627 (222) 627 (222)Zone 7 555 (91) 955 (164)Zone 8 222 (91) 622 (273)Zone 9 122 (45) 222 (91)

Age Juvenile (le6 months) 13118 (110)119875 = 093

24118 (203)119875 = 059

Adult (gt6 months) 15142 (106) 34142 (239)

Gender Male 1190 (122)119875 = 015

1590 (167)119875 = 015

Female 17170 (100) 43170 (253)

BCS Poor (1ndash39) 318 (167)119875 = 046

518 (278)119875 = 084

Good (4ndash69) 191225 (849) 51225 (227)

Seasons

Winter 464 (63) 119875 = 027 664 (94)

P = 001OR = 029lowast

CI = 012ndash070lowast

P = 001lowast

Spring 1069 (145) 119875 = 035 2169 (304) 119875 = 008

Summer 231 (65) 119875 = 060 631 (194) 119875 = 085

Autumn 1296 (125) 119875 = 063 2596 (260) 119875 = 034

Health status Healthy 972 (125)119875 = 074

1672 (222)119875 = 088

Unhealthy 19188 (101) 42188 (223)

Clinical abnormalities inunhealthy cats

Lymph node enlargement 13133 (98) 119875 = 074 33133 (248) 119875 = 040

Pale mucous membranes 114 (71) 119875 = 099 214 (143) 119875 = 068

Stomatitis 11101 (109) 119875 = 088 19101 (188) 119875 = 035

Signs of respiratory tract infection 322 (141) 119875 = 093 722 (318) 119875 = 039

Signs of ocular infection 340 (75) 119875 = 065 1140 (275) 119875 = 052

CBC abnormalities

Anaemia 769 (101) 119875 = 081 2269 (319) 119875 = 069

Leukopenia 014 (00) 119875 = 040 414 (286) 119875 = 081

Leukocytosis 05 (00) 119875 = 100 25 (400) 119875 = 097

Thrombocytopenia 110 (100) 119875 = 059 510 (500) 119875 = 027

FIV test results Positive 113 (77)119875 = 083

413 (308)119875 = 084

Negative 15150 (100) 44150 (293)

FeLV test results Positive 16 (167)119875 = 090

16 (167)119875 = 081

Negative 15157 (96) 47157 (299)

T gondii test results Positive 631 (194)119875 = 014

731 (226)119875 = 038

Negative 681 (74) 2781 (333)PCR polymerase chain reaction BCS body condition score CBC complete blood count CI 95 confidence interval OR odds ratio FeLV feline leukemiavirus FIV feline immunodeficiency virus 119875 values in bold are statistically significant (119875 lt 005)lowastResults from multivariate logistic regression analysis

(REV Reparto di Medicina Emotrasfusionale Veterinaria)Feline haemoplasmas can be directly transmitted by intra-venous infusion of fresh EDTA-anticoagulated blood [26]heparinized blood [4] and by infusion of blood storedin CPDA-1 solution for 1 h (Mhf) and 1 week (CMhm)

[27] Cats do not reliably eliminate the organism followinginfection [26] and most infections with CMhm are chronicand asymptomatic [1] A significant number of asymptomaticcats are positive for haemoplasma infection [16] and mayplay a role along with infected cats in the maintenance

6 ISRNMicrobiology

Table 3 Selected haematological findings in subgroups of haemoplasma PCR-positive and PCR-negative cats

Variable reference range Number of cats Mean SD Median Lowest value Highest valuePCR-positive for all haemoplasma

WBC 59 11019 4427 10720 1558 23240RBC 59 6406 1059 6300 2100 8750HB 59 91 37 89 47 124HCT 59 247 14 247 132 322PLT 59 369 145 380 90 693

PCR-negative for all haemoplasmaWBC 91 10815 3957 10440 1516 22870RBC 91 6555 1113 6550 3780 9190HB 91 255 17 92 146 132HCT 91 94 44 252 42 384PLT 91 378 141 367 119 800

PCR-positive MhfWBC 15 10547 3062 9990 6400 15460RBC 15 6541 800 6310 5610 8460HB 15 94 12 94 76 124HCT 15 248 31 252 204 309PLT 15 371 122 376 135 693

PCR-positive CMhmWBC 43 11181 4825 10790 1558 23240RBC 43 6360 1138 6285 2100 8750HB 43 90 15 89 47 121HCT 43 246 4 245 132 322PLT 43 369 154 386 90 662WBC white blood cell RBC red blood cells HB haemoglobin HCT haematocrit PLT platelet

Table 4 Prevalence of haemoplasma infection in stray cats inworldwide studies

Sample source(number of tested cats) [Ref]

Positive samplesTotal prevalence Mhf CMhm

Italy current study (260) 331 108 223USA (Florida) (484) [20] 205 83 122Korea (331) [22] 145 42 103USA (Arizona) (112) [21] 277 45 80Ireland (75) [23] 173 13 133Canada (96) [24] 115 31 84MhfMycoplasma haemofelis CMhm ldquoCandidatus Mycoplasma haemomin-utumrdquo

of haemoplasma infection within a population All thesecharacteristics of feline haemotropic mycoplasma infectionneed to be consideredwhen choosing potential blood donors

The limitations of this study include the lack of infor-mation on ldquoCandidatus Mycoplasma turicensisrdquo in our studypopulation Risk factors analyzed were not available forall 260 cats Lastly statistical analysis was limited in somegroups because of the sample size and so some conclusionsor associations may be affected by type I errors that isno difference between haemoplasma positive and negative

groups was observed due to insufficient sample size forexample no association was recorded between anaemia anda positive haemoplasma result because of the low number ofMhf positive cats (28260) Regardless of these limitations webelieve that this study provides new and useful informationon feline haemoplasma infections in stray cats in Italy

5 Conclusion

From this study it can be concluded that feline haemotropicmycoplasma Mhf and CMhm are common in the stray catpopulation of Milan Indeed pet cats with outdoor accessin this region should be regularly monitored and treated forectoparasites to minimize health risks Importantly felineblood donors in this area should undergo PCR screeningfor these infections before donations and preferably donorsshould be drawn from exclusively indoor cats that receiveregular flea control

Conflict of Interests

All the authors (Eva Spada Daniela Proverbio Paola Gal-luzzo Alessandra Della Pepa Roberta Perego Giada Bag-nagatti De Giorgi and Elisabetta Ferro) declare to have noconflict of interests

ISRNMicrobiology 7

Acknowledgment

This research received no specific grant from any fundingagency in the public commercial or not-for-profit sectors

References

[1] J B Messick and J W Harvey ldquoHemotropic mycoplasmosis(Hemobartonellosis)rdquo in Infectious Diseases of the Dog and CatC E Greene Ed pp 310ndash319 Elsevier-Saunders St Louis MoUSA 4th edition 2012

[2] H Obara M Fujihara Y Watanabe H K Ono and RHarasawa ldquoA feline hemoplasma ldquoCandidatus Mycoplasmahaemominutumrdquo detected in dog in Japanrdquo Journal of Veteri-nary Medical Science vol 73 no 6 pp 841ndash843 2011

[3] A P dos Santos R P dos Santos A W Biondo et al ldquoHemo-plasma infection in HIV-positive patient Brazilrdquo EmergingInfectious Diseases vol 14 no 12 pp 1922ndash1924 2008

[4] S Tasker I R Peters K Papasouliotis et al ldquoDescription ofoutcomes of experimental infection with feline haemoplasmascopy numbers haematology Coombsrsquo testing and blood glu-cose concentrationsrdquo Veterinary Microbiology vol 139 no 3-4pp 323ndash332 2009

[5] S Tasker J A Braddock R Baral et al ldquoDiagnosis of felinehaemoplasma infection inAustralian cats using a real-timePCRassayrdquo Journal of Feline Medicine and Surgery vol 6 no 6 pp345ndash354 2004

[6] K E Kewish G D Appleyard S L Myers B A Kidneyand M L Jackson ldquoMycoplasma haemofelis and Mycoplasmahaemominutum detection by polymerase chain reaction in catsfrom Saskatchewan and Albertardquo Canadian Veterinary Journalvol 45 no 9 pp 749ndash752 2004

[7] M R Lappin B Griffin J Brunt et al ldquoPrevalence of Bartonellaspecies Haemoplasma species Ehrlichia species Anaplasmaphagocytophilum and Neorickettsia risticii DNA in the bloodof cats and their fleas in the United Statesrdquo Journal of FelineMedicine and Surgery vol 8 no 2 pp 85ndash90 2006

[8] T B Hackett W A Jensen T L Lehman et al ldquoPrevalenceof DNA of Mycoplasma haemofelis ldquoCandidatus Mycoplasmahaemominutumrdquo Ananplasma phagocytophilum and species ofBartonella Neorickettsia and Ehrlichia in cats used as blooddonors in the United Statesrdquo Journal of the American VeterinaryMedical Association vol 229 no 5 pp 700ndash705 2006

[9] B Willi F S Boretti C Baumgartner et al ldquoPrevalence riskfactor analysis and follow-up of infections caused by threefeline hemoplasma species in cats in Switzerlandrdquo Journal ofClinical Microbiology vol 44 no 3 pp 961ndash969 2006

[10] N Bauer H-J Balzer S Thure and A Moritz ldquoPrevalence offeline haemotropicmycoplasmas in convenience samples of catsin Germanyrdquo Journal of Feline Medicine and Surgery vol 10 no3 pp 252ndash258 2008

[11] I R Peters C R Helps B Willi R Hofmann-Lehmann and STasker ldquoThe prevalence of three species of feline haemoplasmasin samples submitted to a diagnostics service as determined bythree novel real-time duplex PCR assaysrdquo Veterinary Microbiol-ogy vol 126 no 1ndash3 pp 142ndash150 2008

[12] F Gentilini M Novacco M E Turba B Willi M L Bacciand R Hofmann-Lehmann ldquoUse of combined conventionaland real-time PCR to determine the epidemiology of felinehaemoplasma infections in northern Italyrdquo Journal of FelineMedicine and Surgery vol 11 no 4 pp 277ndash285 2009

[13] M Tanahara S Miyamoto T Nishio et al ldquoAn epidemiologicalsurvey of feline hemoplasma infection in Japanrdquo Journal ofVeterinary Medical Science vol 72 no 12 pp 1575ndash1581 2010

[14] V R Barrs J A Beatty B J Wilson et al ldquoPrevalence of Bar-tonella species Rickettsia felisHaemoplasmas and the Ehrlichiagroup in the blood of cats and fleas in eastern AustraliardquoAustralian Veterinary Journal vol 88 no 5 pp 160ndash165 2010

[15] X Roura I R Peters L Altet et al ldquoPrevalence of hemotropicmycoplasmas in healthy and unhealthy cats and dogs in SpainrdquoJournal of Veterinary Diagnostic Investigation vol 22 no 2 pp270ndash274 2010

[16] AD Bennett DAGunn-MooreM Brewer andMR LappinldquoPrevalence of Bartonella species Haemoplasmas and Toxo-plasma gondii in cats in Scotlandrdquo Journal of FelineMedicine andSurgery vol 13 no 8 pp 553ndash557 2011

[17] R Lobetti and M R Lappin ldquoPrevalence of Toxoplasma gondiiBartonella species and haemoplasma infection in cats in SouthAfricardquo Journal of Feline Medicine and Surgery vol 14 pp 857ndash862 2012

[18] S Assarasakorn J K Veir J R Hawley et al ldquoPrevalenceof Bartonella species hemoplasmas and Rickettsia felis DNAin blood and fleas of cats in Bangkok Thailandrdquo Research inVeterinary Science vol 93 pp 1213ndash1216 2012

[19] V L Martınez-Dıaz A C Silvestre-Ferreira H Vilhena etal ldquoPrevalence and co-infection of haemotropic mycoplasmasin Portuguese cats by real-time polymerase chain reactionrdquoJournal of Feline Medicine and Surgery vol 15 no 10 pp 879ndash885 2013

[20] B J Luria J K Levy M R Lappin et al ldquoPrevalence ofinfectious diseases in feral cats in Northern Floridardquo Journal ofFeline Medicine and Surgery vol 6 no 5 pp 287ndash296 2004

[21] J M Eberhardt K Neal T Shackelford and M R LappinldquoPrevalence of selected infectious disease agents in cats fromArizonardquo Journal of Feline Medicine and Surgery vol 8 no 3pp 164ndash168 2006

[22] D-H Yu H-W Kim A R Desai et al ldquoMolecular detection offeline hemoplasmas in feral cats in Koreardquo Journal of VeterinaryMedical Science vol 69 no 12 pp 1299ndash1301 2007

[23] F Juvet M R Lappin S Brennan and C T Mooney ldquoPreva-lence of selected infectious agents in cats in Irelandrdquo Journal ofFeline Medicine and Surgery vol 12 no 6 pp 476ndash482 2010

[24] V Stojanovic and P Foley ldquoInfectious disease prevalence ina feral cat population on Prince Edward Island CanadardquoCanadian Veterinary Journal vol 52 no 9 pp 979ndash982 2011

[25] J E Woods M M Brewer J R Hawley N Wisnewskiand M R Lappin ldquoEvaluation of experimental transmissionof Candidatus Mycoplasma haemominutum and Mycoplasmahaemofelis by Ctenocephalides felis to catsrdquo The AmericanJournal of Veterinary Research vol 66 no 6 pp 1008ndash10122005

[26] D S Westfall W A Jensen W J Reagan S V Radecki andM R Lappin ldquoInoculation of two genotypes ofHemobartonellafelis (California and Ohio variants) to induce infection incats and the response to treatment with azithromycinrdquo TheAmerican Journal of Veterinary Research vol 62 no 5 pp 687ndash691 2001

[27] A T Gary H L Richmond S Tasker T B Hackett and M RLappin ldquoSurvival of Mycoplasma haemofelis and ldquoCandidatusMycoplasma haemominutumrdquo in blood of cats used for transfu-sionsrdquo Journal of Feline Medicine and Surgery vol 8 no 5 pp321ndash326 2006

8 ISRNMicrobiology

[28] E Spada D Proverbio A Della Pepa et al ldquoSeroprevalenceof feline immunodeficiency virus feline leukaemia virus andToxoplasma gondii in stray cat colonies in northern Italy andcorrelation with clinical and laboratory datardquo Journal of FelineMedicine and Surgery vol 14 pp 369ndash377 2012

[29] E Spada D Proverbio A Della Pepa et al ldquoPrevalence offaecal-borne parasites in colony stray cats in northern ItalyrdquoJournal of Feline Medicine and Surgery vol 15 no 8 pp 672ndash677 2013

[30] A Moritz Y Fickenscher K Meyer K Failing and D J WeissldquoCanine and feline hematology reference values for the ADVIA120 hematology systemrdquo Veterinary Clinical Pathology vol 33no 1 pp 32ndash38 2004

[31] W A Jensen M R Lappin S Kamkar and W J Reagan ldquoUseof a polymerase chain reaction assay to detect and differentiatetwo strains of Haemobartonella felis in naturally infected catsrdquoThe American Journal of Veterinary Research vol 62 no 4 pp604ndash608 2001

[32] A M Ishak S Radecki and M R Lappin ldquoPrevalence ofMycoplasma haemofelis ldquoCandidatus Mycoplasma haemomin-utumrdquo Bartonella species Ehrlichia species and Anaplasmaphagocytophilum DNA in the blood of cats with anemiardquoJournal of FelineMedicine and Surgery vol 9 no 1 pp 1ndash7 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 ISRNMicrobiology

transmitted through intravenous infusion of infected blood[4 26] and have been shown to survive for up to 1 weekin stored blood products [27] As administration of freshor stored whole blood becoming more common in felinemedicine the knowledge of the regional prevalence of bloodtransmitted pathogens in owned and stray cats that sharethe same environment and parasites is increasingly impor-tant This would provide useful information for evaluatingthe risks of transmission of blood-borne infections frompotential blood donors and in the development of optimalscreening protocols in blood donors

The aimof this studywas to evaluate using a conventionalPCR assay the prevalence of Mhf and CMhm infections instray cats from colonies in Milan and to identify possible riskfactors for these infections

2 Materials and Methods

21 Sample Population and Data Collection During a 2-year collection period (January 2008 to January 2010) bloodsamples were taken from 260 stray cats from urban coloniesinMilan (Northern Italy) under a trap-neuter-release (TNR)program approved by the local authority of the city councilThe program was conducted as previously described [28 29]

Age (estimated based on dentition animals lt6 monthsof age were considered juvenile whereas all others wereconsidered adult) gender (male or female) origin (prove-nance area of colonies) and body condition score (BCS 4ndash6 indicating normal weight 1ndash3 underweight) were recordedtogether with data obtained from physical examination of thecats (healthy or unhealthy) Unhealthy cats were defined ascats with one or more of the following clinical abnormalitieslymph node enlargement pale mucous membranes stomati-tis or signs of ocular and respiratory infections

The seasonal analysis based on the date of sample col-lection was grouped as follows winter (January Februaryand March) spring (April May and June) summer (JulyAugust and September) autumn (October November andDecember)The catswere not systematically examined for thepresence of ticks or fleas and so rates of ectoparasitism werenot recorded

22 Haematological and Serological Analyses Blood sampleswere collected aseptically from the jugular vein while catswere anaesthetized for neutering and placed in EDTA-treatedtubes and in serum separator tubes

Within 24 h of sample collection a complete blood count(CBC) was performed on whole blood using an ADVIA 120System (Siemens Healthcare Diagnostics Milan Italy) Catswere categorized in terms of presence or absence of anaemia(HCT lt 24) leukopenia (WBCs count lt 10570120583L) leuko-cytosis (WBC gt 14390120583L) and thrombocytopenia (PLT lt200670120583L) [30] Surplus blood was stored at minus20∘C to uselater in PCR assay

Following separation serum samples were tested forantibodies to FIV (relative to gp40 and p24 FIV antigens)and for FeLV p27 antigen with a commercial enzyme-linkedimmunosorbent assay (ELISA) kit (Snap FeLVFIV Combo

Plus Test Idexx Laboratories Hoofddorp The Netherlands)Toxoplasma gondii sera IgG antibodies were detected usingindirect fluorescent antibody tests (IFAT) performed witha commercial kit (Fuller-laboratories Fullerton CA USA)Titres ge1 64 were considered seroreactive and thereforeindicative of T gondii exposure The results of these sero-logical tests have been already published [28] and werereanalyzed with the present results

23 PCR Assay Conventional PCR was performed on bloodsamples to amplify DNA associated with haemoplasmas(Mhf and CMhm) The reaction mixture included 2120583L oftemplate DNA 025mM dNTPs 04mM of each primer1x reaction buffer and 25U Tap DNA polymerase (GoTaqDNAPolymerase PromegaMadisonWIUSA)The volumeof this mixture was adjusted to 25 120583L with sterile waterPrimers were used that target part of the 16S rRNA geneproducing a 170 base pair (bp) product from Mhf and 193 bpamplicon from MChm (forward primer 51015840-ACG AAA GTCTGA TGG AGC AAT A-31015840 and reverse primer 51015840-ACG CCCAAT AAA TCC GRA TAA T-31015840) [6 31] PCR reactions wereperformed using an automated thermocycler PCR productswere resolved using 2 agarose gels and fragment size wasestimated using aDNAmolecular weightmarker (50 bpDNALadder Promega Madison WI USA) Control reactionswere done in the absence of template DNA to rule outcontaminations during PCR

24 Statistical Analysis Association between the threegroups of haemoplasma status (haemoplasma positive Mhfpositive only and CMhm positive only) and categoricalvariables (age gender colony of origin BCS season of sam-pling health status presence or absence of selected clinicaland CBC abnormalities FeLVFIV status and T gondii testresults) were analysed by univariate analysis using the chi-squared test (cell frequencies of gt5) or Fisherrsquos exact test(cell frequencies of le5) Any parameters statistically linked topositive PCR results were used in a logistic regression modelto test for independent risk factors associated with infection

Descriptive statistics (including minimum maximummean median and standard deviation (SD)) were obtainedfor the continuous variables RBCs count HCT HB WBCcount and PLT count values Distribution of the data fornormality was assessed with Kolmogorov Smirnov test anda 119905-test or a Mann-Whitney 119880 test respectively was used totest the differences between the feline haemoplasma positiveand negative cats depending on whether data was normallydistributed or not

Associations were considered statistically significantwhen 119875 lt 005 Both the 119875 value and odds ratio (OR) with95 confidence interval (CI) are reported Data were ana-lyzed using MedCalc Software (version 1230 MariakerkeBelgium)

3 Results

Haemoplasmic DNA was detected in 331 (86260 95 CI265ndash409) of blood samples Of the positive samples 28

ISRNMicrobiology 3

0

5

10

15

20

25

30

35

40

Neg Mhf CMhm

HCT

()

Figure 1 HCT values of Italian stray cats grouped by haemoplasmainfectious status Boxes represent the 25th 50th (median) and 75thquartiles withwhiskers extending to the greatest and smallest valuesBlue circles indicate outliers (cases with values greater than 15 boxlengths from the upper or lower edge of the box) CMhm ldquoCan-didatus M haemominutumrdquo positive Mhf M haemofelis positiveThe HCT values of CMhm group and the Mhf group were notsignificantly lower than the negative group The HCT values of theMhf group were also not significantly lower than the CMhm group

cats (108 95CI 72ndash156) were infected withMhf and 58cats (223 95 CI 169ndash288) were infected with CMhmNo comorbid infections were registered

Characteristics of the population and association betweenrisk factors and haemoplasma PCR status (negative or pos-itive) are reported in Table 1 and between Mhf alone andCMhm alone positive results in Table 2

None of the risk factors were associated with the PCRresults for haemoplasmas (Tables 1 and 2) with the excep-tion of negative associations at univariate and multivariateanalysis between winter season of sampling and haemo-plasma positive status (119875 = 001 OR = 029 95 CI = 014ndash061 119875 = 0001) and CMhm positive status (119875 = 001 OR =029 95 CI = 012ndash070 119875 = 001)

CBC results from 150260 cats are reported in Table 3Data was normally distributed according to KolmogorovSmirnov test No significant associations were found withCBC abnormalities and haemoplasma PCR-positive results(Figure 1) or in the number of anaemic and nonanaemic catsusing a 119905-test

4 Discussion

We present the first study investigating the prevalence ofhaemoplasmas in urban stray colony cats from the city ofMilan in Northern Italy

The prevalence of haemoplasma infection in our sampleshowed a similar pattern to that reportedworldwide in client-owned [5ndash7 9ndash15 17ndash19] and stray cats [19ndash23] CMhminfection is reported to be the most common infectionranging from 8 in Arizona [21] to 416 in Portugal[19] Mhf infection was less common ranging from 05 in

Switzerland [9] to 128 in Portugal [19] and dual infectionwas absent or rare (no more than 54 as found in Korea)[22]

The prevalence of haemoplasmas in stray cats in ourstudy (331) was higher than that reported in other studieson stray cats performed worldwide (Table 4) Differences inprevalence among countries can be explained by geographicalvariation such as climate vector distribution and the catpopulation surveyedMoreover direct comparisons of preva-lence results are of limited value because of the characteristicsof sample of cat populations investigated (number of catsincluded in the study healthy versus sick cats) inclusioncriteria diagnostic techniques (molecular tools versusmicro-scopical detection) different statistical methods stage ofinfection (acute versus chronic) or a combination of all themresulting in differences between studies

Stray cats in this study had higher rates of infections thanthose reported for pet cats in Northern Italy [12] in which189 of PCR tested cats were reported to be haemoplasmapositive A high incidence of haemoplasmas in stray cats isnot surprising as outdoor access is a recognized risk factor forinfection For example in a study on haemoplasma in client-owned cats fromBarcelona (Spain) outdoor accesswas foundto be a risk factor for infection (OR = 38) [15] Additionallyin a study involving feline blood donors from the USA theprevalence of haemoplasmas was 197 in domestic cats withoutdoor access and only 36 in domestic cats not allowedoutdoors [8] Free-ranging cats may have more exposureto bloodsucking arthropods and exhibit a higher fightingactivity than owned indoor cats thus they might experiencea higher infection risk for haemoplasmas

In our study there was a negative statistical associationbetween sampling in the winter season and haemoplasmaPCR-positive status and CMhm positive status This may bedue to reduced outdoor activity of fleas in winter seasonsalthough this hypothesis is purely speculative There wereno statistically significant differences or any relationshipbetween the presence of haemoplasma infection and anaemiastatus or HCT levels between positive and negative haemo-plasma group This finding was somewhat surprising andwas in agreement with studies undertaken in client-ownedcats in Switzerland [9] and in Italy [12] which also foundno association between haemoplasma infection and anaemiaor HCT variations In addition other studies have failed todemonstrate a significant difference in prevalence rates ofhaemoplasma between healthy and anaemic cats [10 32]These results could be explained by the higher prevalenceof CMhm (a less pathogenic species than Mhf) in this andprevious studies or by the fact that the stage of infectionis not known in our cats with a positive PCR result sincechronically infected cats that recover from acute illnessmay be asymptomatic Another explanation for the lack ofassociation between infection status and the presence ofanaemia in the present study could be that the HCT wasknown only for 150 of the 260 cats tested

Epidemiological data on haemoplasma are particularlyimportant in areas where feline blood donor programmesare active as in Milan where a donor programme has beenrunning since 2010 at University Veterinary Transfusion Unit

4 ISRNMicrobiology

Table 1 Sample characteristics of 260 stray cats testing positive and negative for haemoplasma infection by conventional PCR in NorthernItaly

Factor Category Total number PCR119875 value

() Negative () Positive ()

Origin of the cats(119899 = 260)

Zone 1 3 (12) 3174 (17) 086 (00) 119875 = 054

Zone 2 11 (42) 6174 (34) 586 (58) 119875 = 057

Zone 4 108 (415) 68174 (391) 4086 (465) 119875 = 031

Zone 5 12 (46) 8174 (46) 486 (47) 119875 = 077

Zone 6 27 (104) 15174 (86) 1286 (140) 119875 = 027

Zone 7 55 (212) 41174 (236) 1486 (163) 119875 = 023

Zone 8 22 (85) 14174 (80) 886 (93) 119875 = 092

Zone 9 22 (85) 19174 (109) 386 (35) 119875 = 007

Age (119899 = 260) Juvenile (le6 months) 118 (454) 81174 (466) 3786 (430) 119875 = 069

Adult (gt6 months) 142 (546) 93174 (534) 4986 (570)

Gender (119899 = 260) Male 90 (346) 30174 (172) 6086 (698) 119875 = 037

Female 170 (654) 144174 (827) 2686 (302)

BCS (119899 = 243) Poor (1ndash39) 18 (74) 10165 (61) 878 (103) 119875 = 037

Good (4ndash69) 225 (926) 155165 (939) 7078 (897) 119875 = 054

Seasons (119899 = 260)

Winter 64 (246) 5464 (944) 1064 (156)

P = 001OR = 029

CI = 014ndash061P = 0001lowast

Spring 69 (265) 3869 (551) 3169 (449) 119875 = 009

Summer 31 (119) 2331 (742) 831 (258) 119875 = 054

Autumn 96 (369) 5996 (615) 3796 (385) 119875 = 040

Health status (119899 = 260) Healthy 72 (277) 47174 (270) 2586 (291) 119875 = 084

Unhealthy 188 (723) 127174 (730) 6186 (709)

Clinical abnormalities inunhealthy cats (119899 = 188)

Lymph node enlargement 133 (707) 87102 (853) 4686 (535) 119875 = 069

Pale mucous membranes 14 (74) 11102 (108) 386 (35) 119875 = 051

Stomatitis 101 (537) 71102 (696) 3086 (349) 119875 = 043

Signs of respiratory tractinfection 22 (117) 12102 (118) 1086 (116) 119875 = 029

Signs of ocular infection 40 (213) 26102 (255) 1486 (163) 119875 = 092

CBC abnormalities(119899 = 150)

Anaemia 62 (413) 3391 (363) 2959 (492) 119875 = 069

Leukopenia 15 (100) 1191 (121) 459 (68) 119875 = 056

Leukocytosis 5 (33) 391 (33) 259 (34) 119875 = 066

Thrombocytopenia 10 (67) 491 (44) 659 (102) 119875 = 030

FIV test results (119899 = 166) Positive 13 (78) 8102 (78) 564 (78) 119875 = 082

Negative 153 (922) 94102 (922) 5964 (922)FeLV test results(119899 = 166)

Positive 6 (36) 4102 (39) 264 (31) 119875 = 090

Negative 160 (964) 98102 (961) 6264 (969)T gondii test results(119899 = 113)

Positive 31 (274) 1887 (207) 1346 (283) 119875 = 092

Negative 82 (274) 4987 (563) 3346 (717)PCR polymerase chain reaction BCS body condition score CBC complete blood count CI 95 confidence interval OR odds ratio FeLV feline leukemiavirus and FIV feline immunodeficiency virus119875 values in bold are statistically significant (119875 lt 005)lowastResults from multivariate logistic regression analysis

ISRNMicrobiology 5

Table 2 Characteristics of 28 Mhf PCR positive stray cats and 58 CMhm PCR positive stray cats in Northern Italy

Factor Category PCR positive resultsMhf 119899 () 119875 value CMhm 119899 () 119875 value

Origin of the cats

Zone 1 03 (00)

119875 = 063

03 (00)

119875 = 047

Zone 2 111 (91) 411 (364)Zone 4 12108 (111) 28108 (259)Zone 5 112 (83) 312 (250)Zone 6 627 (222) 627 (222)Zone 7 555 (91) 955 (164)Zone 8 222 (91) 622 (273)Zone 9 122 (45) 222 (91)

Age Juvenile (le6 months) 13118 (110)119875 = 093

24118 (203)119875 = 059

Adult (gt6 months) 15142 (106) 34142 (239)

Gender Male 1190 (122)119875 = 015

1590 (167)119875 = 015

Female 17170 (100) 43170 (253)

BCS Poor (1ndash39) 318 (167)119875 = 046

518 (278)119875 = 084

Good (4ndash69) 191225 (849) 51225 (227)

Seasons

Winter 464 (63) 119875 = 027 664 (94)

P = 001OR = 029lowast

CI = 012ndash070lowast

P = 001lowast

Spring 1069 (145) 119875 = 035 2169 (304) 119875 = 008

Summer 231 (65) 119875 = 060 631 (194) 119875 = 085

Autumn 1296 (125) 119875 = 063 2596 (260) 119875 = 034

Health status Healthy 972 (125)119875 = 074

1672 (222)119875 = 088

Unhealthy 19188 (101) 42188 (223)

Clinical abnormalities inunhealthy cats

Lymph node enlargement 13133 (98) 119875 = 074 33133 (248) 119875 = 040

Pale mucous membranes 114 (71) 119875 = 099 214 (143) 119875 = 068

Stomatitis 11101 (109) 119875 = 088 19101 (188) 119875 = 035

Signs of respiratory tract infection 322 (141) 119875 = 093 722 (318) 119875 = 039

Signs of ocular infection 340 (75) 119875 = 065 1140 (275) 119875 = 052

CBC abnormalities

Anaemia 769 (101) 119875 = 081 2269 (319) 119875 = 069

Leukopenia 014 (00) 119875 = 040 414 (286) 119875 = 081

Leukocytosis 05 (00) 119875 = 100 25 (400) 119875 = 097

Thrombocytopenia 110 (100) 119875 = 059 510 (500) 119875 = 027

FIV test results Positive 113 (77)119875 = 083

413 (308)119875 = 084

Negative 15150 (100) 44150 (293)

FeLV test results Positive 16 (167)119875 = 090

16 (167)119875 = 081

Negative 15157 (96) 47157 (299)

T gondii test results Positive 631 (194)119875 = 014

731 (226)119875 = 038

Negative 681 (74) 2781 (333)PCR polymerase chain reaction BCS body condition score CBC complete blood count CI 95 confidence interval OR odds ratio FeLV feline leukemiavirus FIV feline immunodeficiency virus 119875 values in bold are statistically significant (119875 lt 005)lowastResults from multivariate logistic regression analysis

(REV Reparto di Medicina Emotrasfusionale Veterinaria)Feline haemoplasmas can be directly transmitted by intra-venous infusion of fresh EDTA-anticoagulated blood [26]heparinized blood [4] and by infusion of blood storedin CPDA-1 solution for 1 h (Mhf) and 1 week (CMhm)

[27] Cats do not reliably eliminate the organism followinginfection [26] and most infections with CMhm are chronicand asymptomatic [1] A significant number of asymptomaticcats are positive for haemoplasma infection [16] and mayplay a role along with infected cats in the maintenance

6 ISRNMicrobiology

Table 3 Selected haematological findings in subgroups of haemoplasma PCR-positive and PCR-negative cats

Variable reference range Number of cats Mean SD Median Lowest value Highest valuePCR-positive for all haemoplasma

WBC 59 11019 4427 10720 1558 23240RBC 59 6406 1059 6300 2100 8750HB 59 91 37 89 47 124HCT 59 247 14 247 132 322PLT 59 369 145 380 90 693

PCR-negative for all haemoplasmaWBC 91 10815 3957 10440 1516 22870RBC 91 6555 1113 6550 3780 9190HB 91 255 17 92 146 132HCT 91 94 44 252 42 384PLT 91 378 141 367 119 800

PCR-positive MhfWBC 15 10547 3062 9990 6400 15460RBC 15 6541 800 6310 5610 8460HB 15 94 12 94 76 124HCT 15 248 31 252 204 309PLT 15 371 122 376 135 693

PCR-positive CMhmWBC 43 11181 4825 10790 1558 23240RBC 43 6360 1138 6285 2100 8750HB 43 90 15 89 47 121HCT 43 246 4 245 132 322PLT 43 369 154 386 90 662WBC white blood cell RBC red blood cells HB haemoglobin HCT haematocrit PLT platelet

Table 4 Prevalence of haemoplasma infection in stray cats inworldwide studies

Sample source(number of tested cats) [Ref]

Positive samplesTotal prevalence Mhf CMhm

Italy current study (260) 331 108 223USA (Florida) (484) [20] 205 83 122Korea (331) [22] 145 42 103USA (Arizona) (112) [21] 277 45 80Ireland (75) [23] 173 13 133Canada (96) [24] 115 31 84MhfMycoplasma haemofelis CMhm ldquoCandidatus Mycoplasma haemomin-utumrdquo

of haemoplasma infection within a population All thesecharacteristics of feline haemotropic mycoplasma infectionneed to be consideredwhen choosing potential blood donors

The limitations of this study include the lack of infor-mation on ldquoCandidatus Mycoplasma turicensisrdquo in our studypopulation Risk factors analyzed were not available forall 260 cats Lastly statistical analysis was limited in somegroups because of the sample size and so some conclusionsor associations may be affected by type I errors that isno difference between haemoplasma positive and negative

groups was observed due to insufficient sample size forexample no association was recorded between anaemia anda positive haemoplasma result because of the low number ofMhf positive cats (28260) Regardless of these limitations webelieve that this study provides new and useful informationon feline haemoplasma infections in stray cats in Italy

5 Conclusion

From this study it can be concluded that feline haemotropicmycoplasma Mhf and CMhm are common in the stray catpopulation of Milan Indeed pet cats with outdoor accessin this region should be regularly monitored and treated forectoparasites to minimize health risks Importantly felineblood donors in this area should undergo PCR screeningfor these infections before donations and preferably donorsshould be drawn from exclusively indoor cats that receiveregular flea control

Conflict of Interests

All the authors (Eva Spada Daniela Proverbio Paola Gal-luzzo Alessandra Della Pepa Roberta Perego Giada Bag-nagatti De Giorgi and Elisabetta Ferro) declare to have noconflict of interests

ISRNMicrobiology 7

Acknowledgment

This research received no specific grant from any fundingagency in the public commercial or not-for-profit sectors

References

[1] J B Messick and J W Harvey ldquoHemotropic mycoplasmosis(Hemobartonellosis)rdquo in Infectious Diseases of the Dog and CatC E Greene Ed pp 310ndash319 Elsevier-Saunders St Louis MoUSA 4th edition 2012

[2] H Obara M Fujihara Y Watanabe H K Ono and RHarasawa ldquoA feline hemoplasma ldquoCandidatus Mycoplasmahaemominutumrdquo detected in dog in Japanrdquo Journal of Veteri-nary Medical Science vol 73 no 6 pp 841ndash843 2011

[3] A P dos Santos R P dos Santos A W Biondo et al ldquoHemo-plasma infection in HIV-positive patient Brazilrdquo EmergingInfectious Diseases vol 14 no 12 pp 1922ndash1924 2008

[4] S Tasker I R Peters K Papasouliotis et al ldquoDescription ofoutcomes of experimental infection with feline haemoplasmascopy numbers haematology Coombsrsquo testing and blood glu-cose concentrationsrdquo Veterinary Microbiology vol 139 no 3-4pp 323ndash332 2009

[5] S Tasker J A Braddock R Baral et al ldquoDiagnosis of felinehaemoplasma infection inAustralian cats using a real-timePCRassayrdquo Journal of Feline Medicine and Surgery vol 6 no 6 pp345ndash354 2004

[6] K E Kewish G D Appleyard S L Myers B A Kidneyand M L Jackson ldquoMycoplasma haemofelis and Mycoplasmahaemominutum detection by polymerase chain reaction in catsfrom Saskatchewan and Albertardquo Canadian Veterinary Journalvol 45 no 9 pp 749ndash752 2004

[7] M R Lappin B Griffin J Brunt et al ldquoPrevalence of Bartonellaspecies Haemoplasma species Ehrlichia species Anaplasmaphagocytophilum and Neorickettsia risticii DNA in the bloodof cats and their fleas in the United Statesrdquo Journal of FelineMedicine and Surgery vol 8 no 2 pp 85ndash90 2006

[8] T B Hackett W A Jensen T L Lehman et al ldquoPrevalenceof DNA of Mycoplasma haemofelis ldquoCandidatus Mycoplasmahaemominutumrdquo Ananplasma phagocytophilum and species ofBartonella Neorickettsia and Ehrlichia in cats used as blooddonors in the United Statesrdquo Journal of the American VeterinaryMedical Association vol 229 no 5 pp 700ndash705 2006

[9] B Willi F S Boretti C Baumgartner et al ldquoPrevalence riskfactor analysis and follow-up of infections caused by threefeline hemoplasma species in cats in Switzerlandrdquo Journal ofClinical Microbiology vol 44 no 3 pp 961ndash969 2006

[10] N Bauer H-J Balzer S Thure and A Moritz ldquoPrevalence offeline haemotropicmycoplasmas in convenience samples of catsin Germanyrdquo Journal of Feline Medicine and Surgery vol 10 no3 pp 252ndash258 2008

[11] I R Peters C R Helps B Willi R Hofmann-Lehmann and STasker ldquoThe prevalence of three species of feline haemoplasmasin samples submitted to a diagnostics service as determined bythree novel real-time duplex PCR assaysrdquo Veterinary Microbiol-ogy vol 126 no 1ndash3 pp 142ndash150 2008

[12] F Gentilini M Novacco M E Turba B Willi M L Bacciand R Hofmann-Lehmann ldquoUse of combined conventionaland real-time PCR to determine the epidemiology of felinehaemoplasma infections in northern Italyrdquo Journal of FelineMedicine and Surgery vol 11 no 4 pp 277ndash285 2009

[13] M Tanahara S Miyamoto T Nishio et al ldquoAn epidemiologicalsurvey of feline hemoplasma infection in Japanrdquo Journal ofVeterinary Medical Science vol 72 no 12 pp 1575ndash1581 2010

[14] V R Barrs J A Beatty B J Wilson et al ldquoPrevalence of Bar-tonella species Rickettsia felisHaemoplasmas and the Ehrlichiagroup in the blood of cats and fleas in eastern AustraliardquoAustralian Veterinary Journal vol 88 no 5 pp 160ndash165 2010

[15] X Roura I R Peters L Altet et al ldquoPrevalence of hemotropicmycoplasmas in healthy and unhealthy cats and dogs in SpainrdquoJournal of Veterinary Diagnostic Investigation vol 22 no 2 pp270ndash274 2010

[16] AD Bennett DAGunn-MooreM Brewer andMR LappinldquoPrevalence of Bartonella species Haemoplasmas and Toxo-plasma gondii in cats in Scotlandrdquo Journal of FelineMedicine andSurgery vol 13 no 8 pp 553ndash557 2011

[17] R Lobetti and M R Lappin ldquoPrevalence of Toxoplasma gondiiBartonella species and haemoplasma infection in cats in SouthAfricardquo Journal of Feline Medicine and Surgery vol 14 pp 857ndash862 2012

[18] S Assarasakorn J K Veir J R Hawley et al ldquoPrevalenceof Bartonella species hemoplasmas and Rickettsia felis DNAin blood and fleas of cats in Bangkok Thailandrdquo Research inVeterinary Science vol 93 pp 1213ndash1216 2012

[19] V L Martınez-Dıaz A C Silvestre-Ferreira H Vilhena etal ldquoPrevalence and co-infection of haemotropic mycoplasmasin Portuguese cats by real-time polymerase chain reactionrdquoJournal of Feline Medicine and Surgery vol 15 no 10 pp 879ndash885 2013

[20] B J Luria J K Levy M R Lappin et al ldquoPrevalence ofinfectious diseases in feral cats in Northern Floridardquo Journal ofFeline Medicine and Surgery vol 6 no 5 pp 287ndash296 2004

[21] J M Eberhardt K Neal T Shackelford and M R LappinldquoPrevalence of selected infectious disease agents in cats fromArizonardquo Journal of Feline Medicine and Surgery vol 8 no 3pp 164ndash168 2006

[22] D-H Yu H-W Kim A R Desai et al ldquoMolecular detection offeline hemoplasmas in feral cats in Koreardquo Journal of VeterinaryMedical Science vol 69 no 12 pp 1299ndash1301 2007

[23] F Juvet M R Lappin S Brennan and C T Mooney ldquoPreva-lence of selected infectious agents in cats in Irelandrdquo Journal ofFeline Medicine and Surgery vol 12 no 6 pp 476ndash482 2010

[24] V Stojanovic and P Foley ldquoInfectious disease prevalence ina feral cat population on Prince Edward Island CanadardquoCanadian Veterinary Journal vol 52 no 9 pp 979ndash982 2011

[25] J E Woods M M Brewer J R Hawley N Wisnewskiand M R Lappin ldquoEvaluation of experimental transmissionof Candidatus Mycoplasma haemominutum and Mycoplasmahaemofelis by Ctenocephalides felis to catsrdquo The AmericanJournal of Veterinary Research vol 66 no 6 pp 1008ndash10122005

[26] D S Westfall W A Jensen W J Reagan S V Radecki andM R Lappin ldquoInoculation of two genotypes ofHemobartonellafelis (California and Ohio variants) to induce infection incats and the response to treatment with azithromycinrdquo TheAmerican Journal of Veterinary Research vol 62 no 5 pp 687ndash691 2001

[27] A T Gary H L Richmond S Tasker T B Hackett and M RLappin ldquoSurvival of Mycoplasma haemofelis and ldquoCandidatusMycoplasma haemominutumrdquo in blood of cats used for transfu-sionsrdquo Journal of Feline Medicine and Surgery vol 8 no 5 pp321ndash326 2006

8 ISRNMicrobiology

[28] E Spada D Proverbio A Della Pepa et al ldquoSeroprevalenceof feline immunodeficiency virus feline leukaemia virus andToxoplasma gondii in stray cat colonies in northern Italy andcorrelation with clinical and laboratory datardquo Journal of FelineMedicine and Surgery vol 14 pp 369ndash377 2012

[29] E Spada D Proverbio A Della Pepa et al ldquoPrevalence offaecal-borne parasites in colony stray cats in northern ItalyrdquoJournal of Feline Medicine and Surgery vol 15 no 8 pp 672ndash677 2013

[30] A Moritz Y Fickenscher K Meyer K Failing and D J WeissldquoCanine and feline hematology reference values for the ADVIA120 hematology systemrdquo Veterinary Clinical Pathology vol 33no 1 pp 32ndash38 2004

[31] W A Jensen M R Lappin S Kamkar and W J Reagan ldquoUseof a polymerase chain reaction assay to detect and differentiatetwo strains of Haemobartonella felis in naturally infected catsrdquoThe American Journal of Veterinary Research vol 62 no 4 pp604ndash608 2001

[32] A M Ishak S Radecki and M R Lappin ldquoPrevalence ofMycoplasma haemofelis ldquoCandidatus Mycoplasma haemomin-utumrdquo Bartonella species Ehrlichia species and Anaplasmaphagocytophilum DNA in the blood of cats with anemiardquoJournal of FelineMedicine and Surgery vol 9 no 1 pp 1ndash7 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

ISRNMicrobiology 3

0

5

10

15

20

25

30

35

40

Neg Mhf CMhm

HCT

()

Figure 1 HCT values of Italian stray cats grouped by haemoplasmainfectious status Boxes represent the 25th 50th (median) and 75thquartiles withwhiskers extending to the greatest and smallest valuesBlue circles indicate outliers (cases with values greater than 15 boxlengths from the upper or lower edge of the box) CMhm ldquoCan-didatus M haemominutumrdquo positive Mhf M haemofelis positiveThe HCT values of CMhm group and the Mhf group were notsignificantly lower than the negative group The HCT values of theMhf group were also not significantly lower than the CMhm group

cats (108 95CI 72ndash156) were infected withMhf and 58cats (223 95 CI 169ndash288) were infected with CMhmNo comorbid infections were registered

Characteristics of the population and association betweenrisk factors and haemoplasma PCR status (negative or pos-itive) are reported in Table 1 and between Mhf alone andCMhm alone positive results in Table 2

None of the risk factors were associated with the PCRresults for haemoplasmas (Tables 1 and 2) with the excep-tion of negative associations at univariate and multivariateanalysis between winter season of sampling and haemo-plasma positive status (119875 = 001 OR = 029 95 CI = 014ndash061 119875 = 0001) and CMhm positive status (119875 = 001 OR =029 95 CI = 012ndash070 119875 = 001)

CBC results from 150260 cats are reported in Table 3Data was normally distributed according to KolmogorovSmirnov test No significant associations were found withCBC abnormalities and haemoplasma PCR-positive results(Figure 1) or in the number of anaemic and nonanaemic catsusing a 119905-test

4 Discussion

We present the first study investigating the prevalence ofhaemoplasmas in urban stray colony cats from the city ofMilan in Northern Italy

The prevalence of haemoplasma infection in our sampleshowed a similar pattern to that reportedworldwide in client-owned [5ndash7 9ndash15 17ndash19] and stray cats [19ndash23] CMhminfection is reported to be the most common infectionranging from 8 in Arizona [21] to 416 in Portugal[19] Mhf infection was less common ranging from 05 in

Switzerland [9] to 128 in Portugal [19] and dual infectionwas absent or rare (no more than 54 as found in Korea)[22]

The prevalence of haemoplasmas in stray cats in ourstudy (331) was higher than that reported in other studieson stray cats performed worldwide (Table 4) Differences inprevalence among countries can be explained by geographicalvariation such as climate vector distribution and the catpopulation surveyedMoreover direct comparisons of preva-lence results are of limited value because of the characteristicsof sample of cat populations investigated (number of catsincluded in the study healthy versus sick cats) inclusioncriteria diagnostic techniques (molecular tools versusmicro-scopical detection) different statistical methods stage ofinfection (acute versus chronic) or a combination of all themresulting in differences between studies

Stray cats in this study had higher rates of infections thanthose reported for pet cats in Northern Italy [12] in which189 of PCR tested cats were reported to be haemoplasmapositive A high incidence of haemoplasmas in stray cats isnot surprising as outdoor access is a recognized risk factor forinfection For example in a study on haemoplasma in client-owned cats fromBarcelona (Spain) outdoor accesswas foundto be a risk factor for infection (OR = 38) [15] Additionallyin a study involving feline blood donors from the USA theprevalence of haemoplasmas was 197 in domestic cats withoutdoor access and only 36 in domestic cats not allowedoutdoors [8] Free-ranging cats may have more exposureto bloodsucking arthropods and exhibit a higher fightingactivity than owned indoor cats thus they might experiencea higher infection risk for haemoplasmas

In our study there was a negative statistical associationbetween sampling in the winter season and haemoplasmaPCR-positive status and CMhm positive status This may bedue to reduced outdoor activity of fleas in winter seasonsalthough this hypothesis is purely speculative There wereno statistically significant differences or any relationshipbetween the presence of haemoplasma infection and anaemiastatus or HCT levels between positive and negative haemo-plasma group This finding was somewhat surprising andwas in agreement with studies undertaken in client-ownedcats in Switzerland [9] and in Italy [12] which also foundno association between haemoplasma infection and anaemiaor HCT variations In addition other studies have failed todemonstrate a significant difference in prevalence rates ofhaemoplasma between healthy and anaemic cats [10 32]These results could be explained by the higher prevalenceof CMhm (a less pathogenic species than Mhf) in this andprevious studies or by the fact that the stage of infectionis not known in our cats with a positive PCR result sincechronically infected cats that recover from acute illnessmay be asymptomatic Another explanation for the lack ofassociation between infection status and the presence ofanaemia in the present study could be that the HCT wasknown only for 150 of the 260 cats tested

Epidemiological data on haemoplasma are particularlyimportant in areas where feline blood donor programmesare active as in Milan where a donor programme has beenrunning since 2010 at University Veterinary Transfusion Unit

4 ISRNMicrobiology

Table 1 Sample characteristics of 260 stray cats testing positive and negative for haemoplasma infection by conventional PCR in NorthernItaly

Factor Category Total number PCR119875 value

() Negative () Positive ()

Origin of the cats(119899 = 260)

Zone 1 3 (12) 3174 (17) 086 (00) 119875 = 054

Zone 2 11 (42) 6174 (34) 586 (58) 119875 = 057

Zone 4 108 (415) 68174 (391) 4086 (465) 119875 = 031

Zone 5 12 (46) 8174 (46) 486 (47) 119875 = 077

Zone 6 27 (104) 15174 (86) 1286 (140) 119875 = 027

Zone 7 55 (212) 41174 (236) 1486 (163) 119875 = 023

Zone 8 22 (85) 14174 (80) 886 (93) 119875 = 092

Zone 9 22 (85) 19174 (109) 386 (35) 119875 = 007

Age (119899 = 260) Juvenile (le6 months) 118 (454) 81174 (466) 3786 (430) 119875 = 069

Adult (gt6 months) 142 (546) 93174 (534) 4986 (570)

Gender (119899 = 260) Male 90 (346) 30174 (172) 6086 (698) 119875 = 037

Female 170 (654) 144174 (827) 2686 (302)

BCS (119899 = 243) Poor (1ndash39) 18 (74) 10165 (61) 878 (103) 119875 = 037

Good (4ndash69) 225 (926) 155165 (939) 7078 (897) 119875 = 054

Seasons (119899 = 260)

Winter 64 (246) 5464 (944) 1064 (156)

P = 001OR = 029

CI = 014ndash061P = 0001lowast

Spring 69 (265) 3869 (551) 3169 (449) 119875 = 009

Summer 31 (119) 2331 (742) 831 (258) 119875 = 054

Autumn 96 (369) 5996 (615) 3796 (385) 119875 = 040

Health status (119899 = 260) Healthy 72 (277) 47174 (270) 2586 (291) 119875 = 084

Unhealthy 188 (723) 127174 (730) 6186 (709)

Clinical abnormalities inunhealthy cats (119899 = 188)

Lymph node enlargement 133 (707) 87102 (853) 4686 (535) 119875 = 069

Pale mucous membranes 14 (74) 11102 (108) 386 (35) 119875 = 051

Stomatitis 101 (537) 71102 (696) 3086 (349) 119875 = 043

Signs of respiratory tractinfection 22 (117) 12102 (118) 1086 (116) 119875 = 029

Signs of ocular infection 40 (213) 26102 (255) 1486 (163) 119875 = 092

CBC abnormalities(119899 = 150)

Anaemia 62 (413) 3391 (363) 2959 (492) 119875 = 069

Leukopenia 15 (100) 1191 (121) 459 (68) 119875 = 056

Leukocytosis 5 (33) 391 (33) 259 (34) 119875 = 066

Thrombocytopenia 10 (67) 491 (44) 659 (102) 119875 = 030

FIV test results (119899 = 166) Positive 13 (78) 8102 (78) 564 (78) 119875 = 082

Negative 153 (922) 94102 (922) 5964 (922)FeLV test results(119899 = 166)

Positive 6 (36) 4102 (39) 264 (31) 119875 = 090

Negative 160 (964) 98102 (961) 6264 (969)T gondii test results(119899 = 113)

Positive 31 (274) 1887 (207) 1346 (283) 119875 = 092

Negative 82 (274) 4987 (563) 3346 (717)PCR polymerase chain reaction BCS body condition score CBC complete blood count CI 95 confidence interval OR odds ratio FeLV feline leukemiavirus and FIV feline immunodeficiency virus119875 values in bold are statistically significant (119875 lt 005)lowastResults from multivariate logistic regression analysis

ISRNMicrobiology 5

Table 2 Characteristics of 28 Mhf PCR positive stray cats and 58 CMhm PCR positive stray cats in Northern Italy

Factor Category PCR positive resultsMhf 119899 () 119875 value CMhm 119899 () 119875 value

Origin of the cats

Zone 1 03 (00)

119875 = 063

03 (00)

119875 = 047

Zone 2 111 (91) 411 (364)Zone 4 12108 (111) 28108 (259)Zone 5 112 (83) 312 (250)Zone 6 627 (222) 627 (222)Zone 7 555 (91) 955 (164)Zone 8 222 (91) 622 (273)Zone 9 122 (45) 222 (91)

Age Juvenile (le6 months) 13118 (110)119875 = 093

24118 (203)119875 = 059

Adult (gt6 months) 15142 (106) 34142 (239)

Gender Male 1190 (122)119875 = 015

1590 (167)119875 = 015

Female 17170 (100) 43170 (253)

BCS Poor (1ndash39) 318 (167)119875 = 046

518 (278)119875 = 084

Good (4ndash69) 191225 (849) 51225 (227)

Seasons

Winter 464 (63) 119875 = 027 664 (94)

P = 001OR = 029lowast

CI = 012ndash070lowast

P = 001lowast

Spring 1069 (145) 119875 = 035 2169 (304) 119875 = 008

Summer 231 (65) 119875 = 060 631 (194) 119875 = 085

Autumn 1296 (125) 119875 = 063 2596 (260) 119875 = 034

Health status Healthy 972 (125)119875 = 074

1672 (222)119875 = 088

Unhealthy 19188 (101) 42188 (223)

Clinical abnormalities inunhealthy cats

Lymph node enlargement 13133 (98) 119875 = 074 33133 (248) 119875 = 040

Pale mucous membranes 114 (71) 119875 = 099 214 (143) 119875 = 068

Stomatitis 11101 (109) 119875 = 088 19101 (188) 119875 = 035

Signs of respiratory tract infection 322 (141) 119875 = 093 722 (318) 119875 = 039

Signs of ocular infection 340 (75) 119875 = 065 1140 (275) 119875 = 052

CBC abnormalities

Anaemia 769 (101) 119875 = 081 2269 (319) 119875 = 069

Leukopenia 014 (00) 119875 = 040 414 (286) 119875 = 081

Leukocytosis 05 (00) 119875 = 100 25 (400) 119875 = 097

Thrombocytopenia 110 (100) 119875 = 059 510 (500) 119875 = 027

FIV test results Positive 113 (77)119875 = 083

413 (308)119875 = 084

Negative 15150 (100) 44150 (293)

FeLV test results Positive 16 (167)119875 = 090

16 (167)119875 = 081

Negative 15157 (96) 47157 (299)

T gondii test results Positive 631 (194)119875 = 014

731 (226)119875 = 038

Negative 681 (74) 2781 (333)PCR polymerase chain reaction BCS body condition score CBC complete blood count CI 95 confidence interval OR odds ratio FeLV feline leukemiavirus FIV feline immunodeficiency virus 119875 values in bold are statistically significant (119875 lt 005)lowastResults from multivariate logistic regression analysis

(REV Reparto di Medicina Emotrasfusionale Veterinaria)Feline haemoplasmas can be directly transmitted by intra-venous infusion of fresh EDTA-anticoagulated blood [26]heparinized blood [4] and by infusion of blood storedin CPDA-1 solution for 1 h (Mhf) and 1 week (CMhm)

[27] Cats do not reliably eliminate the organism followinginfection [26] and most infections with CMhm are chronicand asymptomatic [1] A significant number of asymptomaticcats are positive for haemoplasma infection [16] and mayplay a role along with infected cats in the maintenance

6 ISRNMicrobiology

Table 3 Selected haematological findings in subgroups of haemoplasma PCR-positive and PCR-negative cats

Variable reference range Number of cats Mean SD Median Lowest value Highest valuePCR-positive for all haemoplasma

WBC 59 11019 4427 10720 1558 23240RBC 59 6406 1059 6300 2100 8750HB 59 91 37 89 47 124HCT 59 247 14 247 132 322PLT 59 369 145 380 90 693

PCR-negative for all haemoplasmaWBC 91 10815 3957 10440 1516 22870RBC 91 6555 1113 6550 3780 9190HB 91 255 17 92 146 132HCT 91 94 44 252 42 384PLT 91 378 141 367 119 800

PCR-positive MhfWBC 15 10547 3062 9990 6400 15460RBC 15 6541 800 6310 5610 8460HB 15 94 12 94 76 124HCT 15 248 31 252 204 309PLT 15 371 122 376 135 693

PCR-positive CMhmWBC 43 11181 4825 10790 1558 23240RBC 43 6360 1138 6285 2100 8750HB 43 90 15 89 47 121HCT 43 246 4 245 132 322PLT 43 369 154 386 90 662WBC white blood cell RBC red blood cells HB haemoglobin HCT haematocrit PLT platelet

Table 4 Prevalence of haemoplasma infection in stray cats inworldwide studies

Sample source(number of tested cats) [Ref]

Positive samplesTotal prevalence Mhf CMhm

Italy current study (260) 331 108 223USA (Florida) (484) [20] 205 83 122Korea (331) [22] 145 42 103USA (Arizona) (112) [21] 277 45 80Ireland (75) [23] 173 13 133Canada (96) [24] 115 31 84MhfMycoplasma haemofelis CMhm ldquoCandidatus Mycoplasma haemomin-utumrdquo

of haemoplasma infection within a population All thesecharacteristics of feline haemotropic mycoplasma infectionneed to be consideredwhen choosing potential blood donors

The limitations of this study include the lack of infor-mation on ldquoCandidatus Mycoplasma turicensisrdquo in our studypopulation Risk factors analyzed were not available forall 260 cats Lastly statistical analysis was limited in somegroups because of the sample size and so some conclusionsor associations may be affected by type I errors that isno difference between haemoplasma positive and negative

groups was observed due to insufficient sample size forexample no association was recorded between anaemia anda positive haemoplasma result because of the low number ofMhf positive cats (28260) Regardless of these limitations webelieve that this study provides new and useful informationon feline haemoplasma infections in stray cats in Italy

5 Conclusion

From this study it can be concluded that feline haemotropicmycoplasma Mhf and CMhm are common in the stray catpopulation of Milan Indeed pet cats with outdoor accessin this region should be regularly monitored and treated forectoparasites to minimize health risks Importantly felineblood donors in this area should undergo PCR screeningfor these infections before donations and preferably donorsshould be drawn from exclusively indoor cats that receiveregular flea control

Conflict of Interests

All the authors (Eva Spada Daniela Proverbio Paola Gal-luzzo Alessandra Della Pepa Roberta Perego Giada Bag-nagatti De Giorgi and Elisabetta Ferro) declare to have noconflict of interests

ISRNMicrobiology 7

Acknowledgment

This research received no specific grant from any fundingagency in the public commercial or not-for-profit sectors

References

[1] J B Messick and J W Harvey ldquoHemotropic mycoplasmosis(Hemobartonellosis)rdquo in Infectious Diseases of the Dog and CatC E Greene Ed pp 310ndash319 Elsevier-Saunders St Louis MoUSA 4th edition 2012

[2] H Obara M Fujihara Y Watanabe H K Ono and RHarasawa ldquoA feline hemoplasma ldquoCandidatus Mycoplasmahaemominutumrdquo detected in dog in Japanrdquo Journal of Veteri-nary Medical Science vol 73 no 6 pp 841ndash843 2011

[3] A P dos Santos R P dos Santos A W Biondo et al ldquoHemo-plasma infection in HIV-positive patient Brazilrdquo EmergingInfectious Diseases vol 14 no 12 pp 1922ndash1924 2008

[4] S Tasker I R Peters K Papasouliotis et al ldquoDescription ofoutcomes of experimental infection with feline haemoplasmascopy numbers haematology Coombsrsquo testing and blood glu-cose concentrationsrdquo Veterinary Microbiology vol 139 no 3-4pp 323ndash332 2009

[5] S Tasker J A Braddock R Baral et al ldquoDiagnosis of felinehaemoplasma infection inAustralian cats using a real-timePCRassayrdquo Journal of Feline Medicine and Surgery vol 6 no 6 pp345ndash354 2004

[6] K E Kewish G D Appleyard S L Myers B A Kidneyand M L Jackson ldquoMycoplasma haemofelis and Mycoplasmahaemominutum detection by polymerase chain reaction in catsfrom Saskatchewan and Albertardquo Canadian Veterinary Journalvol 45 no 9 pp 749ndash752 2004

[7] M R Lappin B Griffin J Brunt et al ldquoPrevalence of Bartonellaspecies Haemoplasma species Ehrlichia species Anaplasmaphagocytophilum and Neorickettsia risticii DNA in the bloodof cats and their fleas in the United Statesrdquo Journal of FelineMedicine and Surgery vol 8 no 2 pp 85ndash90 2006

[8] T B Hackett W A Jensen T L Lehman et al ldquoPrevalenceof DNA of Mycoplasma haemofelis ldquoCandidatus Mycoplasmahaemominutumrdquo Ananplasma phagocytophilum and species ofBartonella Neorickettsia and Ehrlichia in cats used as blooddonors in the United Statesrdquo Journal of the American VeterinaryMedical Association vol 229 no 5 pp 700ndash705 2006

[9] B Willi F S Boretti C Baumgartner et al ldquoPrevalence riskfactor analysis and follow-up of infections caused by threefeline hemoplasma species in cats in Switzerlandrdquo Journal ofClinical Microbiology vol 44 no 3 pp 961ndash969 2006

[10] N Bauer H-J Balzer S Thure and A Moritz ldquoPrevalence offeline haemotropicmycoplasmas in convenience samples of catsin Germanyrdquo Journal of Feline Medicine and Surgery vol 10 no3 pp 252ndash258 2008

[11] I R Peters C R Helps B Willi R Hofmann-Lehmann and STasker ldquoThe prevalence of three species of feline haemoplasmasin samples submitted to a diagnostics service as determined bythree novel real-time duplex PCR assaysrdquo Veterinary Microbiol-ogy vol 126 no 1ndash3 pp 142ndash150 2008

[12] F Gentilini M Novacco M E Turba B Willi M L Bacciand R Hofmann-Lehmann ldquoUse of combined conventionaland real-time PCR to determine the epidemiology of felinehaemoplasma infections in northern Italyrdquo Journal of FelineMedicine and Surgery vol 11 no 4 pp 277ndash285 2009

[13] M Tanahara S Miyamoto T Nishio et al ldquoAn epidemiologicalsurvey of feline hemoplasma infection in Japanrdquo Journal ofVeterinary Medical Science vol 72 no 12 pp 1575ndash1581 2010

[14] V R Barrs J A Beatty B J Wilson et al ldquoPrevalence of Bar-tonella species Rickettsia felisHaemoplasmas and the Ehrlichiagroup in the blood of cats and fleas in eastern AustraliardquoAustralian Veterinary Journal vol 88 no 5 pp 160ndash165 2010

[15] X Roura I R Peters L Altet et al ldquoPrevalence of hemotropicmycoplasmas in healthy and unhealthy cats and dogs in SpainrdquoJournal of Veterinary Diagnostic Investigation vol 22 no 2 pp270ndash274 2010

[16] AD Bennett DAGunn-MooreM Brewer andMR LappinldquoPrevalence of Bartonella species Haemoplasmas and Toxo-plasma gondii in cats in Scotlandrdquo Journal of FelineMedicine andSurgery vol 13 no 8 pp 553ndash557 2011

[17] R Lobetti and M R Lappin ldquoPrevalence of Toxoplasma gondiiBartonella species and haemoplasma infection in cats in SouthAfricardquo Journal of Feline Medicine and Surgery vol 14 pp 857ndash862 2012

[18] S Assarasakorn J K Veir J R Hawley et al ldquoPrevalenceof Bartonella species hemoplasmas and Rickettsia felis DNAin blood and fleas of cats in Bangkok Thailandrdquo Research inVeterinary Science vol 93 pp 1213ndash1216 2012

[19] V L Martınez-Dıaz A C Silvestre-Ferreira H Vilhena etal ldquoPrevalence and co-infection of haemotropic mycoplasmasin Portuguese cats by real-time polymerase chain reactionrdquoJournal of Feline Medicine and Surgery vol 15 no 10 pp 879ndash885 2013

[20] B J Luria J K Levy M R Lappin et al ldquoPrevalence ofinfectious diseases in feral cats in Northern Floridardquo Journal ofFeline Medicine and Surgery vol 6 no 5 pp 287ndash296 2004

[21] J M Eberhardt K Neal T Shackelford and M R LappinldquoPrevalence of selected infectious disease agents in cats fromArizonardquo Journal of Feline Medicine and Surgery vol 8 no 3pp 164ndash168 2006

[22] D-H Yu H-W Kim A R Desai et al ldquoMolecular detection offeline hemoplasmas in feral cats in Koreardquo Journal of VeterinaryMedical Science vol 69 no 12 pp 1299ndash1301 2007

[23] F Juvet M R Lappin S Brennan and C T Mooney ldquoPreva-lence of selected infectious agents in cats in Irelandrdquo Journal ofFeline Medicine and Surgery vol 12 no 6 pp 476ndash482 2010

[24] V Stojanovic and P Foley ldquoInfectious disease prevalence ina feral cat population on Prince Edward Island CanadardquoCanadian Veterinary Journal vol 52 no 9 pp 979ndash982 2011

[25] J E Woods M M Brewer J R Hawley N Wisnewskiand M R Lappin ldquoEvaluation of experimental transmissionof Candidatus Mycoplasma haemominutum and Mycoplasmahaemofelis by Ctenocephalides felis to catsrdquo The AmericanJournal of Veterinary Research vol 66 no 6 pp 1008ndash10122005

[26] D S Westfall W A Jensen W J Reagan S V Radecki andM R Lappin ldquoInoculation of two genotypes ofHemobartonellafelis (California and Ohio variants) to induce infection incats and the response to treatment with azithromycinrdquo TheAmerican Journal of Veterinary Research vol 62 no 5 pp 687ndash691 2001

[27] A T Gary H L Richmond S Tasker T B Hackett and M RLappin ldquoSurvival of Mycoplasma haemofelis and ldquoCandidatusMycoplasma haemominutumrdquo in blood of cats used for transfu-sionsrdquo Journal of Feline Medicine and Surgery vol 8 no 5 pp321ndash326 2006

8 ISRNMicrobiology

[28] E Spada D Proverbio A Della Pepa et al ldquoSeroprevalenceof feline immunodeficiency virus feline leukaemia virus andToxoplasma gondii in stray cat colonies in northern Italy andcorrelation with clinical and laboratory datardquo Journal of FelineMedicine and Surgery vol 14 pp 369ndash377 2012

[29] E Spada D Proverbio A Della Pepa et al ldquoPrevalence offaecal-borne parasites in colony stray cats in northern ItalyrdquoJournal of Feline Medicine and Surgery vol 15 no 8 pp 672ndash677 2013

[30] A Moritz Y Fickenscher K Meyer K Failing and D J WeissldquoCanine and feline hematology reference values for the ADVIA120 hematology systemrdquo Veterinary Clinical Pathology vol 33no 1 pp 32ndash38 2004

[31] W A Jensen M R Lappin S Kamkar and W J Reagan ldquoUseof a polymerase chain reaction assay to detect and differentiatetwo strains of Haemobartonella felis in naturally infected catsrdquoThe American Journal of Veterinary Research vol 62 no 4 pp604ndash608 2001

[32] A M Ishak S Radecki and M R Lappin ldquoPrevalence ofMycoplasma haemofelis ldquoCandidatus Mycoplasma haemomin-utumrdquo Bartonella species Ehrlichia species and Anaplasmaphagocytophilum DNA in the blood of cats with anemiardquoJournal of FelineMedicine and Surgery vol 9 no 1 pp 1ndash7 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 ISRNMicrobiology

Table 1 Sample characteristics of 260 stray cats testing positive and negative for haemoplasma infection by conventional PCR in NorthernItaly

Factor Category Total number PCR119875 value

() Negative () Positive ()

Origin of the cats(119899 = 260)

Zone 1 3 (12) 3174 (17) 086 (00) 119875 = 054

Zone 2 11 (42) 6174 (34) 586 (58) 119875 = 057

Zone 4 108 (415) 68174 (391) 4086 (465) 119875 = 031

Zone 5 12 (46) 8174 (46) 486 (47) 119875 = 077

Zone 6 27 (104) 15174 (86) 1286 (140) 119875 = 027

Zone 7 55 (212) 41174 (236) 1486 (163) 119875 = 023

Zone 8 22 (85) 14174 (80) 886 (93) 119875 = 092

Zone 9 22 (85) 19174 (109) 386 (35) 119875 = 007

Age (119899 = 260) Juvenile (le6 months) 118 (454) 81174 (466) 3786 (430) 119875 = 069

Adult (gt6 months) 142 (546) 93174 (534) 4986 (570)

Gender (119899 = 260) Male 90 (346) 30174 (172) 6086 (698) 119875 = 037

Female 170 (654) 144174 (827) 2686 (302)

BCS (119899 = 243) Poor (1ndash39) 18 (74) 10165 (61) 878 (103) 119875 = 037

Good (4ndash69) 225 (926) 155165 (939) 7078 (897) 119875 = 054

Seasons (119899 = 260)

Winter 64 (246) 5464 (944) 1064 (156)

P = 001OR = 029

CI = 014ndash061P = 0001lowast

Spring 69 (265) 3869 (551) 3169 (449) 119875 = 009

Summer 31 (119) 2331 (742) 831 (258) 119875 = 054

Autumn 96 (369) 5996 (615) 3796 (385) 119875 = 040

Health status (119899 = 260) Healthy 72 (277) 47174 (270) 2586 (291) 119875 = 084

Unhealthy 188 (723) 127174 (730) 6186 (709)

Clinical abnormalities inunhealthy cats (119899 = 188)

Lymph node enlargement 133 (707) 87102 (853) 4686 (535) 119875 = 069

Pale mucous membranes 14 (74) 11102 (108) 386 (35) 119875 = 051

Stomatitis 101 (537) 71102 (696) 3086 (349) 119875 = 043

Signs of respiratory tractinfection 22 (117) 12102 (118) 1086 (116) 119875 = 029

Signs of ocular infection 40 (213) 26102 (255) 1486 (163) 119875 = 092

CBC abnormalities(119899 = 150)

Anaemia 62 (413) 3391 (363) 2959 (492) 119875 = 069

Leukopenia 15 (100) 1191 (121) 459 (68) 119875 = 056

Leukocytosis 5 (33) 391 (33) 259 (34) 119875 = 066

Thrombocytopenia 10 (67) 491 (44) 659 (102) 119875 = 030

FIV test results (119899 = 166) Positive 13 (78) 8102 (78) 564 (78) 119875 = 082

Negative 153 (922) 94102 (922) 5964 (922)FeLV test results(119899 = 166)

Positive 6 (36) 4102 (39) 264 (31) 119875 = 090

Negative 160 (964) 98102 (961) 6264 (969)T gondii test results(119899 = 113)

Positive 31 (274) 1887 (207) 1346 (283) 119875 = 092

Negative 82 (274) 4987 (563) 3346 (717)PCR polymerase chain reaction BCS body condition score CBC complete blood count CI 95 confidence interval OR odds ratio FeLV feline leukemiavirus and FIV feline immunodeficiency virus119875 values in bold are statistically significant (119875 lt 005)lowastResults from multivariate logistic regression analysis

ISRNMicrobiology 5

Table 2 Characteristics of 28 Mhf PCR positive stray cats and 58 CMhm PCR positive stray cats in Northern Italy

Factor Category PCR positive resultsMhf 119899 () 119875 value CMhm 119899 () 119875 value

Origin of the cats

Zone 1 03 (00)

119875 = 063

03 (00)

119875 = 047

Zone 2 111 (91) 411 (364)Zone 4 12108 (111) 28108 (259)Zone 5 112 (83) 312 (250)Zone 6 627 (222) 627 (222)Zone 7 555 (91) 955 (164)Zone 8 222 (91) 622 (273)Zone 9 122 (45) 222 (91)

Age Juvenile (le6 months) 13118 (110)119875 = 093

24118 (203)119875 = 059

Adult (gt6 months) 15142 (106) 34142 (239)

Gender Male 1190 (122)119875 = 015

1590 (167)119875 = 015

Female 17170 (100) 43170 (253)

BCS Poor (1ndash39) 318 (167)119875 = 046

518 (278)119875 = 084

Good (4ndash69) 191225 (849) 51225 (227)

Seasons

Winter 464 (63) 119875 = 027 664 (94)

P = 001OR = 029lowast

CI = 012ndash070lowast

P = 001lowast

Spring 1069 (145) 119875 = 035 2169 (304) 119875 = 008

Summer 231 (65) 119875 = 060 631 (194) 119875 = 085

Autumn 1296 (125) 119875 = 063 2596 (260) 119875 = 034

Health status Healthy 972 (125)119875 = 074

1672 (222)119875 = 088

Unhealthy 19188 (101) 42188 (223)

Clinical abnormalities inunhealthy cats

Lymph node enlargement 13133 (98) 119875 = 074 33133 (248) 119875 = 040

Pale mucous membranes 114 (71) 119875 = 099 214 (143) 119875 = 068

Stomatitis 11101 (109) 119875 = 088 19101 (188) 119875 = 035

Signs of respiratory tract infection 322 (141) 119875 = 093 722 (318) 119875 = 039

Signs of ocular infection 340 (75) 119875 = 065 1140 (275) 119875 = 052

CBC abnormalities

Anaemia 769 (101) 119875 = 081 2269 (319) 119875 = 069

Leukopenia 014 (00) 119875 = 040 414 (286) 119875 = 081

Leukocytosis 05 (00) 119875 = 100 25 (400) 119875 = 097

Thrombocytopenia 110 (100) 119875 = 059 510 (500) 119875 = 027

FIV test results Positive 113 (77)119875 = 083

413 (308)119875 = 084

Negative 15150 (100) 44150 (293)

FeLV test results Positive 16 (167)119875 = 090

16 (167)119875 = 081

Negative 15157 (96) 47157 (299)

T gondii test results Positive 631 (194)119875 = 014

731 (226)119875 = 038

Negative 681 (74) 2781 (333)PCR polymerase chain reaction BCS body condition score CBC complete blood count CI 95 confidence interval OR odds ratio FeLV feline leukemiavirus FIV feline immunodeficiency virus 119875 values in bold are statistically significant (119875 lt 005)lowastResults from multivariate logistic regression analysis

(REV Reparto di Medicina Emotrasfusionale Veterinaria)Feline haemoplasmas can be directly transmitted by intra-venous infusion of fresh EDTA-anticoagulated blood [26]heparinized blood [4] and by infusion of blood storedin CPDA-1 solution for 1 h (Mhf) and 1 week (CMhm)

[27] Cats do not reliably eliminate the organism followinginfection [26] and most infections with CMhm are chronicand asymptomatic [1] A significant number of asymptomaticcats are positive for haemoplasma infection [16] and mayplay a role along with infected cats in the maintenance

6 ISRNMicrobiology

Table 3 Selected haematological findings in subgroups of haemoplasma PCR-positive and PCR-negative cats

Variable reference range Number of cats Mean SD Median Lowest value Highest valuePCR-positive for all haemoplasma

WBC 59 11019 4427 10720 1558 23240RBC 59 6406 1059 6300 2100 8750HB 59 91 37 89 47 124HCT 59 247 14 247 132 322PLT 59 369 145 380 90 693

PCR-negative for all haemoplasmaWBC 91 10815 3957 10440 1516 22870RBC 91 6555 1113 6550 3780 9190HB 91 255 17 92 146 132HCT 91 94 44 252 42 384PLT 91 378 141 367 119 800

PCR-positive MhfWBC 15 10547 3062 9990 6400 15460RBC 15 6541 800 6310 5610 8460HB 15 94 12 94 76 124HCT 15 248 31 252 204 309PLT 15 371 122 376 135 693

PCR-positive CMhmWBC 43 11181 4825 10790 1558 23240RBC 43 6360 1138 6285 2100 8750HB 43 90 15 89 47 121HCT 43 246 4 245 132 322PLT 43 369 154 386 90 662WBC white blood cell RBC red blood cells HB haemoglobin HCT haematocrit PLT platelet

Table 4 Prevalence of haemoplasma infection in stray cats inworldwide studies

Sample source(number of tested cats) [Ref]

Positive samplesTotal prevalence Mhf CMhm

Italy current study (260) 331 108 223USA (Florida) (484) [20] 205 83 122Korea (331) [22] 145 42 103USA (Arizona) (112) [21] 277 45 80Ireland (75) [23] 173 13 133Canada (96) [24] 115 31 84MhfMycoplasma haemofelis CMhm ldquoCandidatus Mycoplasma haemomin-utumrdquo

of haemoplasma infection within a population All thesecharacteristics of feline haemotropic mycoplasma infectionneed to be consideredwhen choosing potential blood donors

The limitations of this study include the lack of infor-mation on ldquoCandidatus Mycoplasma turicensisrdquo in our studypopulation Risk factors analyzed were not available forall 260 cats Lastly statistical analysis was limited in somegroups because of the sample size and so some conclusionsor associations may be affected by type I errors that isno difference between haemoplasma positive and negative

groups was observed due to insufficient sample size forexample no association was recorded between anaemia anda positive haemoplasma result because of the low number ofMhf positive cats (28260) Regardless of these limitations webelieve that this study provides new and useful informationon feline haemoplasma infections in stray cats in Italy

5 Conclusion

From this study it can be concluded that feline haemotropicmycoplasma Mhf and CMhm are common in the stray catpopulation of Milan Indeed pet cats with outdoor accessin this region should be regularly monitored and treated forectoparasites to minimize health risks Importantly felineblood donors in this area should undergo PCR screeningfor these infections before donations and preferably donorsshould be drawn from exclusively indoor cats that receiveregular flea control

Conflict of Interests

All the authors (Eva Spada Daniela Proverbio Paola Gal-luzzo Alessandra Della Pepa Roberta Perego Giada Bag-nagatti De Giorgi and Elisabetta Ferro) declare to have noconflict of interests

ISRNMicrobiology 7

Acknowledgment

This research received no specific grant from any fundingagency in the public commercial or not-for-profit sectors

References

[1] J B Messick and J W Harvey ldquoHemotropic mycoplasmosis(Hemobartonellosis)rdquo in Infectious Diseases of the Dog and CatC E Greene Ed pp 310ndash319 Elsevier-Saunders St Louis MoUSA 4th edition 2012

[2] H Obara M Fujihara Y Watanabe H K Ono and RHarasawa ldquoA feline hemoplasma ldquoCandidatus Mycoplasmahaemominutumrdquo detected in dog in Japanrdquo Journal of Veteri-nary Medical Science vol 73 no 6 pp 841ndash843 2011

[3] A P dos Santos R P dos Santos A W Biondo et al ldquoHemo-plasma infection in HIV-positive patient Brazilrdquo EmergingInfectious Diseases vol 14 no 12 pp 1922ndash1924 2008

[4] S Tasker I R Peters K Papasouliotis et al ldquoDescription ofoutcomes of experimental infection with feline haemoplasmascopy numbers haematology Coombsrsquo testing and blood glu-cose concentrationsrdquo Veterinary Microbiology vol 139 no 3-4pp 323ndash332 2009

[5] S Tasker J A Braddock R Baral et al ldquoDiagnosis of felinehaemoplasma infection inAustralian cats using a real-timePCRassayrdquo Journal of Feline Medicine and Surgery vol 6 no 6 pp345ndash354 2004

[6] K E Kewish G D Appleyard S L Myers B A Kidneyand M L Jackson ldquoMycoplasma haemofelis and Mycoplasmahaemominutum detection by polymerase chain reaction in catsfrom Saskatchewan and Albertardquo Canadian Veterinary Journalvol 45 no 9 pp 749ndash752 2004

[7] M R Lappin B Griffin J Brunt et al ldquoPrevalence of Bartonellaspecies Haemoplasma species Ehrlichia species Anaplasmaphagocytophilum and Neorickettsia risticii DNA in the bloodof cats and their fleas in the United Statesrdquo Journal of FelineMedicine and Surgery vol 8 no 2 pp 85ndash90 2006

[8] T B Hackett W A Jensen T L Lehman et al ldquoPrevalenceof DNA of Mycoplasma haemofelis ldquoCandidatus Mycoplasmahaemominutumrdquo Ananplasma phagocytophilum and species ofBartonella Neorickettsia and Ehrlichia in cats used as blooddonors in the United Statesrdquo Journal of the American VeterinaryMedical Association vol 229 no 5 pp 700ndash705 2006

[9] B Willi F S Boretti C Baumgartner et al ldquoPrevalence riskfactor analysis and follow-up of infections caused by threefeline hemoplasma species in cats in Switzerlandrdquo Journal ofClinical Microbiology vol 44 no 3 pp 961ndash969 2006

[10] N Bauer H-J Balzer S Thure and A Moritz ldquoPrevalence offeline haemotropicmycoplasmas in convenience samples of catsin Germanyrdquo Journal of Feline Medicine and Surgery vol 10 no3 pp 252ndash258 2008

[11] I R Peters C R Helps B Willi R Hofmann-Lehmann and STasker ldquoThe prevalence of three species of feline haemoplasmasin samples submitted to a diagnostics service as determined bythree novel real-time duplex PCR assaysrdquo Veterinary Microbiol-ogy vol 126 no 1ndash3 pp 142ndash150 2008

[12] F Gentilini M Novacco M E Turba B Willi M L Bacciand R Hofmann-Lehmann ldquoUse of combined conventionaland real-time PCR to determine the epidemiology of felinehaemoplasma infections in northern Italyrdquo Journal of FelineMedicine and Surgery vol 11 no 4 pp 277ndash285 2009

[13] M Tanahara S Miyamoto T Nishio et al ldquoAn epidemiologicalsurvey of feline hemoplasma infection in Japanrdquo Journal ofVeterinary Medical Science vol 72 no 12 pp 1575ndash1581 2010

[14] V R Barrs J A Beatty B J Wilson et al ldquoPrevalence of Bar-tonella species Rickettsia felisHaemoplasmas and the Ehrlichiagroup in the blood of cats and fleas in eastern AustraliardquoAustralian Veterinary Journal vol 88 no 5 pp 160ndash165 2010

[15] X Roura I R Peters L Altet et al ldquoPrevalence of hemotropicmycoplasmas in healthy and unhealthy cats and dogs in SpainrdquoJournal of Veterinary Diagnostic Investigation vol 22 no 2 pp270ndash274 2010

[16] AD Bennett DAGunn-MooreM Brewer andMR LappinldquoPrevalence of Bartonella species Haemoplasmas and Toxo-plasma gondii in cats in Scotlandrdquo Journal of FelineMedicine andSurgery vol 13 no 8 pp 553ndash557 2011

[17] R Lobetti and M R Lappin ldquoPrevalence of Toxoplasma gondiiBartonella species and haemoplasma infection in cats in SouthAfricardquo Journal of Feline Medicine and Surgery vol 14 pp 857ndash862 2012

[18] S Assarasakorn J K Veir J R Hawley et al ldquoPrevalenceof Bartonella species hemoplasmas and Rickettsia felis DNAin blood and fleas of cats in Bangkok Thailandrdquo Research inVeterinary Science vol 93 pp 1213ndash1216 2012

[19] V L Martınez-Dıaz A C Silvestre-Ferreira H Vilhena etal ldquoPrevalence and co-infection of haemotropic mycoplasmasin Portuguese cats by real-time polymerase chain reactionrdquoJournal of Feline Medicine and Surgery vol 15 no 10 pp 879ndash885 2013

[20] B J Luria J K Levy M R Lappin et al ldquoPrevalence ofinfectious diseases in feral cats in Northern Floridardquo Journal ofFeline Medicine and Surgery vol 6 no 5 pp 287ndash296 2004

[21] J M Eberhardt K Neal T Shackelford and M R LappinldquoPrevalence of selected infectious disease agents in cats fromArizonardquo Journal of Feline Medicine and Surgery vol 8 no 3pp 164ndash168 2006

[22] D-H Yu H-W Kim A R Desai et al ldquoMolecular detection offeline hemoplasmas in feral cats in Koreardquo Journal of VeterinaryMedical Science vol 69 no 12 pp 1299ndash1301 2007

[23] F Juvet M R Lappin S Brennan and C T Mooney ldquoPreva-lence of selected infectious agents in cats in Irelandrdquo Journal ofFeline Medicine and Surgery vol 12 no 6 pp 476ndash482 2010

[24] V Stojanovic and P Foley ldquoInfectious disease prevalence ina feral cat population on Prince Edward Island CanadardquoCanadian Veterinary Journal vol 52 no 9 pp 979ndash982 2011

[25] J E Woods M M Brewer J R Hawley N Wisnewskiand M R Lappin ldquoEvaluation of experimental transmissionof Candidatus Mycoplasma haemominutum and Mycoplasmahaemofelis by Ctenocephalides felis to catsrdquo The AmericanJournal of Veterinary Research vol 66 no 6 pp 1008ndash10122005

[26] D S Westfall W A Jensen W J Reagan S V Radecki andM R Lappin ldquoInoculation of two genotypes ofHemobartonellafelis (California and Ohio variants) to induce infection incats and the response to treatment with azithromycinrdquo TheAmerican Journal of Veterinary Research vol 62 no 5 pp 687ndash691 2001

[27] A T Gary H L Richmond S Tasker T B Hackett and M RLappin ldquoSurvival of Mycoplasma haemofelis and ldquoCandidatusMycoplasma haemominutumrdquo in blood of cats used for transfu-sionsrdquo Journal of Feline Medicine and Surgery vol 8 no 5 pp321ndash326 2006

8 ISRNMicrobiology

[28] E Spada D Proverbio A Della Pepa et al ldquoSeroprevalenceof feline immunodeficiency virus feline leukaemia virus andToxoplasma gondii in stray cat colonies in northern Italy andcorrelation with clinical and laboratory datardquo Journal of FelineMedicine and Surgery vol 14 pp 369ndash377 2012

[29] E Spada D Proverbio A Della Pepa et al ldquoPrevalence offaecal-borne parasites in colony stray cats in northern ItalyrdquoJournal of Feline Medicine and Surgery vol 15 no 8 pp 672ndash677 2013

[30] A Moritz Y Fickenscher K Meyer K Failing and D J WeissldquoCanine and feline hematology reference values for the ADVIA120 hematology systemrdquo Veterinary Clinical Pathology vol 33no 1 pp 32ndash38 2004

[31] W A Jensen M R Lappin S Kamkar and W J Reagan ldquoUseof a polymerase chain reaction assay to detect and differentiatetwo strains of Haemobartonella felis in naturally infected catsrdquoThe American Journal of Veterinary Research vol 62 no 4 pp604ndash608 2001

[32] A M Ishak S Radecki and M R Lappin ldquoPrevalence ofMycoplasma haemofelis ldquoCandidatus Mycoplasma haemomin-utumrdquo Bartonella species Ehrlichia species and Anaplasmaphagocytophilum DNA in the blood of cats with anemiardquoJournal of FelineMedicine and Surgery vol 9 no 1 pp 1ndash7 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

ISRNMicrobiology 5

Table 2 Characteristics of 28 Mhf PCR positive stray cats and 58 CMhm PCR positive stray cats in Northern Italy

Factor Category PCR positive resultsMhf 119899 () 119875 value CMhm 119899 () 119875 value

Origin of the cats

Zone 1 03 (00)

119875 = 063

03 (00)

119875 = 047

Zone 2 111 (91) 411 (364)Zone 4 12108 (111) 28108 (259)Zone 5 112 (83) 312 (250)Zone 6 627 (222) 627 (222)Zone 7 555 (91) 955 (164)Zone 8 222 (91) 622 (273)Zone 9 122 (45) 222 (91)

Age Juvenile (le6 months) 13118 (110)119875 = 093

24118 (203)119875 = 059

Adult (gt6 months) 15142 (106) 34142 (239)

Gender Male 1190 (122)119875 = 015

1590 (167)119875 = 015

Female 17170 (100) 43170 (253)

BCS Poor (1ndash39) 318 (167)119875 = 046

518 (278)119875 = 084

Good (4ndash69) 191225 (849) 51225 (227)

Seasons

Winter 464 (63) 119875 = 027 664 (94)

P = 001OR = 029lowast

CI = 012ndash070lowast

P = 001lowast

Spring 1069 (145) 119875 = 035 2169 (304) 119875 = 008

Summer 231 (65) 119875 = 060 631 (194) 119875 = 085

Autumn 1296 (125) 119875 = 063 2596 (260) 119875 = 034

Health status Healthy 972 (125)119875 = 074

1672 (222)119875 = 088

Unhealthy 19188 (101) 42188 (223)

Clinical abnormalities inunhealthy cats

Lymph node enlargement 13133 (98) 119875 = 074 33133 (248) 119875 = 040

Pale mucous membranes 114 (71) 119875 = 099 214 (143) 119875 = 068

Stomatitis 11101 (109) 119875 = 088 19101 (188) 119875 = 035

Signs of respiratory tract infection 322 (141) 119875 = 093 722 (318) 119875 = 039

Signs of ocular infection 340 (75) 119875 = 065 1140 (275) 119875 = 052

CBC abnormalities

Anaemia 769 (101) 119875 = 081 2269 (319) 119875 = 069

Leukopenia 014 (00) 119875 = 040 414 (286) 119875 = 081

Leukocytosis 05 (00) 119875 = 100 25 (400) 119875 = 097

Thrombocytopenia 110 (100) 119875 = 059 510 (500) 119875 = 027

FIV test results Positive 113 (77)119875 = 083

413 (308)119875 = 084

Negative 15150 (100) 44150 (293)

FeLV test results Positive 16 (167)119875 = 090

16 (167)119875 = 081

Negative 15157 (96) 47157 (299)

T gondii test results Positive 631 (194)119875 = 014

731 (226)119875 = 038

Negative 681 (74) 2781 (333)PCR polymerase chain reaction BCS body condition score CBC complete blood count CI 95 confidence interval OR odds ratio FeLV feline leukemiavirus FIV feline immunodeficiency virus 119875 values in bold are statistically significant (119875 lt 005)lowastResults from multivariate logistic regression analysis

(REV Reparto di Medicina Emotrasfusionale Veterinaria)Feline haemoplasmas can be directly transmitted by intra-venous infusion of fresh EDTA-anticoagulated blood [26]heparinized blood [4] and by infusion of blood storedin CPDA-1 solution for 1 h (Mhf) and 1 week (CMhm)

[27] Cats do not reliably eliminate the organism followinginfection [26] and most infections with CMhm are chronicand asymptomatic [1] A significant number of asymptomaticcats are positive for haemoplasma infection [16] and mayplay a role along with infected cats in the maintenance

6 ISRNMicrobiology

Table 3 Selected haematological findings in subgroups of haemoplasma PCR-positive and PCR-negative cats

Variable reference range Number of cats Mean SD Median Lowest value Highest valuePCR-positive for all haemoplasma

WBC 59 11019 4427 10720 1558 23240RBC 59 6406 1059 6300 2100 8750HB 59 91 37 89 47 124HCT 59 247 14 247 132 322PLT 59 369 145 380 90 693

PCR-negative for all haemoplasmaWBC 91 10815 3957 10440 1516 22870RBC 91 6555 1113 6550 3780 9190HB 91 255 17 92 146 132HCT 91 94 44 252 42 384PLT 91 378 141 367 119 800

PCR-positive MhfWBC 15 10547 3062 9990 6400 15460RBC 15 6541 800 6310 5610 8460HB 15 94 12 94 76 124HCT 15 248 31 252 204 309PLT 15 371 122 376 135 693

PCR-positive CMhmWBC 43 11181 4825 10790 1558 23240RBC 43 6360 1138 6285 2100 8750HB 43 90 15 89 47 121HCT 43 246 4 245 132 322PLT 43 369 154 386 90 662WBC white blood cell RBC red blood cells HB haemoglobin HCT haematocrit PLT platelet

Table 4 Prevalence of haemoplasma infection in stray cats inworldwide studies

Sample source(number of tested cats) [Ref]

Positive samplesTotal prevalence Mhf CMhm

Italy current study (260) 331 108 223USA (Florida) (484) [20] 205 83 122Korea (331) [22] 145 42 103USA (Arizona) (112) [21] 277 45 80Ireland (75) [23] 173 13 133Canada (96) [24] 115 31 84MhfMycoplasma haemofelis CMhm ldquoCandidatus Mycoplasma haemomin-utumrdquo

of haemoplasma infection within a population All thesecharacteristics of feline haemotropic mycoplasma infectionneed to be consideredwhen choosing potential blood donors

The limitations of this study include the lack of infor-mation on ldquoCandidatus Mycoplasma turicensisrdquo in our studypopulation Risk factors analyzed were not available forall 260 cats Lastly statistical analysis was limited in somegroups because of the sample size and so some conclusionsor associations may be affected by type I errors that isno difference between haemoplasma positive and negative

groups was observed due to insufficient sample size forexample no association was recorded between anaemia anda positive haemoplasma result because of the low number ofMhf positive cats (28260) Regardless of these limitations webelieve that this study provides new and useful informationon feline haemoplasma infections in stray cats in Italy

5 Conclusion

From this study it can be concluded that feline haemotropicmycoplasma Mhf and CMhm are common in the stray catpopulation of Milan Indeed pet cats with outdoor accessin this region should be regularly monitored and treated forectoparasites to minimize health risks Importantly felineblood donors in this area should undergo PCR screeningfor these infections before donations and preferably donorsshould be drawn from exclusively indoor cats that receiveregular flea control

Conflict of Interests

All the authors (Eva Spada Daniela Proverbio Paola Gal-luzzo Alessandra Della Pepa Roberta Perego Giada Bag-nagatti De Giorgi and Elisabetta Ferro) declare to have noconflict of interests

ISRNMicrobiology 7

Acknowledgment

This research received no specific grant from any fundingagency in the public commercial or not-for-profit sectors

References

[1] J B Messick and J W Harvey ldquoHemotropic mycoplasmosis(Hemobartonellosis)rdquo in Infectious Diseases of the Dog and CatC E Greene Ed pp 310ndash319 Elsevier-Saunders St Louis MoUSA 4th edition 2012

[2] H Obara M Fujihara Y Watanabe H K Ono and RHarasawa ldquoA feline hemoplasma ldquoCandidatus Mycoplasmahaemominutumrdquo detected in dog in Japanrdquo Journal of Veteri-nary Medical Science vol 73 no 6 pp 841ndash843 2011

[3] A P dos Santos R P dos Santos A W Biondo et al ldquoHemo-plasma infection in HIV-positive patient Brazilrdquo EmergingInfectious Diseases vol 14 no 12 pp 1922ndash1924 2008

[4] S Tasker I R Peters K Papasouliotis et al ldquoDescription ofoutcomes of experimental infection with feline haemoplasmascopy numbers haematology Coombsrsquo testing and blood glu-cose concentrationsrdquo Veterinary Microbiology vol 139 no 3-4pp 323ndash332 2009

[5] S Tasker J A Braddock R Baral et al ldquoDiagnosis of felinehaemoplasma infection inAustralian cats using a real-timePCRassayrdquo Journal of Feline Medicine and Surgery vol 6 no 6 pp345ndash354 2004

[6] K E Kewish G D Appleyard S L Myers B A Kidneyand M L Jackson ldquoMycoplasma haemofelis and Mycoplasmahaemominutum detection by polymerase chain reaction in catsfrom Saskatchewan and Albertardquo Canadian Veterinary Journalvol 45 no 9 pp 749ndash752 2004

[7] M R Lappin B Griffin J Brunt et al ldquoPrevalence of Bartonellaspecies Haemoplasma species Ehrlichia species Anaplasmaphagocytophilum and Neorickettsia risticii DNA in the bloodof cats and their fleas in the United Statesrdquo Journal of FelineMedicine and Surgery vol 8 no 2 pp 85ndash90 2006

[8] T B Hackett W A Jensen T L Lehman et al ldquoPrevalenceof DNA of Mycoplasma haemofelis ldquoCandidatus Mycoplasmahaemominutumrdquo Ananplasma phagocytophilum and species ofBartonella Neorickettsia and Ehrlichia in cats used as blooddonors in the United Statesrdquo Journal of the American VeterinaryMedical Association vol 229 no 5 pp 700ndash705 2006

[9] B Willi F S Boretti C Baumgartner et al ldquoPrevalence riskfactor analysis and follow-up of infections caused by threefeline hemoplasma species in cats in Switzerlandrdquo Journal ofClinical Microbiology vol 44 no 3 pp 961ndash969 2006

[10] N Bauer H-J Balzer S Thure and A Moritz ldquoPrevalence offeline haemotropicmycoplasmas in convenience samples of catsin Germanyrdquo Journal of Feline Medicine and Surgery vol 10 no3 pp 252ndash258 2008

[11] I R Peters C R Helps B Willi R Hofmann-Lehmann and STasker ldquoThe prevalence of three species of feline haemoplasmasin samples submitted to a diagnostics service as determined bythree novel real-time duplex PCR assaysrdquo Veterinary Microbiol-ogy vol 126 no 1ndash3 pp 142ndash150 2008

[12] F Gentilini M Novacco M E Turba B Willi M L Bacciand R Hofmann-Lehmann ldquoUse of combined conventionaland real-time PCR to determine the epidemiology of felinehaemoplasma infections in northern Italyrdquo Journal of FelineMedicine and Surgery vol 11 no 4 pp 277ndash285 2009

[13] M Tanahara S Miyamoto T Nishio et al ldquoAn epidemiologicalsurvey of feline hemoplasma infection in Japanrdquo Journal ofVeterinary Medical Science vol 72 no 12 pp 1575ndash1581 2010

[14] V R Barrs J A Beatty B J Wilson et al ldquoPrevalence of Bar-tonella species Rickettsia felisHaemoplasmas and the Ehrlichiagroup in the blood of cats and fleas in eastern AustraliardquoAustralian Veterinary Journal vol 88 no 5 pp 160ndash165 2010

[15] X Roura I R Peters L Altet et al ldquoPrevalence of hemotropicmycoplasmas in healthy and unhealthy cats and dogs in SpainrdquoJournal of Veterinary Diagnostic Investigation vol 22 no 2 pp270ndash274 2010

[16] AD Bennett DAGunn-MooreM Brewer andMR LappinldquoPrevalence of Bartonella species Haemoplasmas and Toxo-plasma gondii in cats in Scotlandrdquo Journal of FelineMedicine andSurgery vol 13 no 8 pp 553ndash557 2011

[17] R Lobetti and M R Lappin ldquoPrevalence of Toxoplasma gondiiBartonella species and haemoplasma infection in cats in SouthAfricardquo Journal of Feline Medicine and Surgery vol 14 pp 857ndash862 2012

[18] S Assarasakorn J K Veir J R Hawley et al ldquoPrevalenceof Bartonella species hemoplasmas and Rickettsia felis DNAin blood and fleas of cats in Bangkok Thailandrdquo Research inVeterinary Science vol 93 pp 1213ndash1216 2012

[19] V L Martınez-Dıaz A C Silvestre-Ferreira H Vilhena etal ldquoPrevalence and co-infection of haemotropic mycoplasmasin Portuguese cats by real-time polymerase chain reactionrdquoJournal of Feline Medicine and Surgery vol 15 no 10 pp 879ndash885 2013

[20] B J Luria J K Levy M R Lappin et al ldquoPrevalence ofinfectious diseases in feral cats in Northern Floridardquo Journal ofFeline Medicine and Surgery vol 6 no 5 pp 287ndash296 2004

[21] J M Eberhardt K Neal T Shackelford and M R LappinldquoPrevalence of selected infectious disease agents in cats fromArizonardquo Journal of Feline Medicine and Surgery vol 8 no 3pp 164ndash168 2006

[22] D-H Yu H-W Kim A R Desai et al ldquoMolecular detection offeline hemoplasmas in feral cats in Koreardquo Journal of VeterinaryMedical Science vol 69 no 12 pp 1299ndash1301 2007

[23] F Juvet M R Lappin S Brennan and C T Mooney ldquoPreva-lence of selected infectious agents in cats in Irelandrdquo Journal ofFeline Medicine and Surgery vol 12 no 6 pp 476ndash482 2010

[24] V Stojanovic and P Foley ldquoInfectious disease prevalence ina feral cat population on Prince Edward Island CanadardquoCanadian Veterinary Journal vol 52 no 9 pp 979ndash982 2011

[25] J E Woods M M Brewer J R Hawley N Wisnewskiand M R Lappin ldquoEvaluation of experimental transmissionof Candidatus Mycoplasma haemominutum and Mycoplasmahaemofelis by Ctenocephalides felis to catsrdquo The AmericanJournal of Veterinary Research vol 66 no 6 pp 1008ndash10122005

[26] D S Westfall W A Jensen W J Reagan S V Radecki andM R Lappin ldquoInoculation of two genotypes ofHemobartonellafelis (California and Ohio variants) to induce infection incats and the response to treatment with azithromycinrdquo TheAmerican Journal of Veterinary Research vol 62 no 5 pp 687ndash691 2001

[27] A T Gary H L Richmond S Tasker T B Hackett and M RLappin ldquoSurvival of Mycoplasma haemofelis and ldquoCandidatusMycoplasma haemominutumrdquo in blood of cats used for transfu-sionsrdquo Journal of Feline Medicine and Surgery vol 8 no 5 pp321ndash326 2006

8 ISRNMicrobiology

[28] E Spada D Proverbio A Della Pepa et al ldquoSeroprevalenceof feline immunodeficiency virus feline leukaemia virus andToxoplasma gondii in stray cat colonies in northern Italy andcorrelation with clinical and laboratory datardquo Journal of FelineMedicine and Surgery vol 14 pp 369ndash377 2012

[29] E Spada D Proverbio A Della Pepa et al ldquoPrevalence offaecal-borne parasites in colony stray cats in northern ItalyrdquoJournal of Feline Medicine and Surgery vol 15 no 8 pp 672ndash677 2013

[30] A Moritz Y Fickenscher K Meyer K Failing and D J WeissldquoCanine and feline hematology reference values for the ADVIA120 hematology systemrdquo Veterinary Clinical Pathology vol 33no 1 pp 32ndash38 2004

[31] W A Jensen M R Lappin S Kamkar and W J Reagan ldquoUseof a polymerase chain reaction assay to detect and differentiatetwo strains of Haemobartonella felis in naturally infected catsrdquoThe American Journal of Veterinary Research vol 62 no 4 pp604ndash608 2001

[32] A M Ishak S Radecki and M R Lappin ldquoPrevalence ofMycoplasma haemofelis ldquoCandidatus Mycoplasma haemomin-utumrdquo Bartonella species Ehrlichia species and Anaplasmaphagocytophilum DNA in the blood of cats with anemiardquoJournal of FelineMedicine and Surgery vol 9 no 1 pp 1ndash7 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 ISRNMicrobiology

Table 3 Selected haematological findings in subgroups of haemoplasma PCR-positive and PCR-negative cats

Variable reference range Number of cats Mean SD Median Lowest value Highest valuePCR-positive for all haemoplasma

WBC 59 11019 4427 10720 1558 23240RBC 59 6406 1059 6300 2100 8750HB 59 91 37 89 47 124HCT 59 247 14 247 132 322PLT 59 369 145 380 90 693

PCR-negative for all haemoplasmaWBC 91 10815 3957 10440 1516 22870RBC 91 6555 1113 6550 3780 9190HB 91 255 17 92 146 132HCT 91 94 44 252 42 384PLT 91 378 141 367 119 800

PCR-positive MhfWBC 15 10547 3062 9990 6400 15460RBC 15 6541 800 6310 5610 8460HB 15 94 12 94 76 124HCT 15 248 31 252 204 309PLT 15 371 122 376 135 693

PCR-positive CMhmWBC 43 11181 4825 10790 1558 23240RBC 43 6360 1138 6285 2100 8750HB 43 90 15 89 47 121HCT 43 246 4 245 132 322PLT 43 369 154 386 90 662WBC white blood cell RBC red blood cells HB haemoglobin HCT haematocrit PLT platelet

Table 4 Prevalence of haemoplasma infection in stray cats inworldwide studies

Sample source(number of tested cats) [Ref]

Positive samplesTotal prevalence Mhf CMhm

Italy current study (260) 331 108 223USA (Florida) (484) [20] 205 83 122Korea (331) [22] 145 42 103USA (Arizona) (112) [21] 277 45 80Ireland (75) [23] 173 13 133Canada (96) [24] 115 31 84MhfMycoplasma haemofelis CMhm ldquoCandidatus Mycoplasma haemomin-utumrdquo

of haemoplasma infection within a population All thesecharacteristics of feline haemotropic mycoplasma infectionneed to be consideredwhen choosing potential blood donors

The limitations of this study include the lack of infor-mation on ldquoCandidatus Mycoplasma turicensisrdquo in our studypopulation Risk factors analyzed were not available forall 260 cats Lastly statistical analysis was limited in somegroups because of the sample size and so some conclusionsor associations may be affected by type I errors that isno difference between haemoplasma positive and negative

groups was observed due to insufficient sample size forexample no association was recorded between anaemia anda positive haemoplasma result because of the low number ofMhf positive cats (28260) Regardless of these limitations webelieve that this study provides new and useful informationon feline haemoplasma infections in stray cats in Italy

5 Conclusion

From this study it can be concluded that feline haemotropicmycoplasma Mhf and CMhm are common in the stray catpopulation of Milan Indeed pet cats with outdoor accessin this region should be regularly monitored and treated forectoparasites to minimize health risks Importantly felineblood donors in this area should undergo PCR screeningfor these infections before donations and preferably donorsshould be drawn from exclusively indoor cats that receiveregular flea control

Conflict of Interests

All the authors (Eva Spada Daniela Proverbio Paola Gal-luzzo Alessandra Della Pepa Roberta Perego Giada Bag-nagatti De Giorgi and Elisabetta Ferro) declare to have noconflict of interests

ISRNMicrobiology 7

Acknowledgment

This research received no specific grant from any fundingagency in the public commercial or not-for-profit sectors

References

[1] J B Messick and J W Harvey ldquoHemotropic mycoplasmosis(Hemobartonellosis)rdquo in Infectious Diseases of the Dog and CatC E Greene Ed pp 310ndash319 Elsevier-Saunders St Louis MoUSA 4th edition 2012

[2] H Obara M Fujihara Y Watanabe H K Ono and RHarasawa ldquoA feline hemoplasma ldquoCandidatus Mycoplasmahaemominutumrdquo detected in dog in Japanrdquo Journal of Veteri-nary Medical Science vol 73 no 6 pp 841ndash843 2011

[3] A P dos Santos R P dos Santos A W Biondo et al ldquoHemo-plasma infection in HIV-positive patient Brazilrdquo EmergingInfectious Diseases vol 14 no 12 pp 1922ndash1924 2008

[4] S Tasker I R Peters K Papasouliotis et al ldquoDescription ofoutcomes of experimental infection with feline haemoplasmascopy numbers haematology Coombsrsquo testing and blood glu-cose concentrationsrdquo Veterinary Microbiology vol 139 no 3-4pp 323ndash332 2009

[5] S Tasker J A Braddock R Baral et al ldquoDiagnosis of felinehaemoplasma infection inAustralian cats using a real-timePCRassayrdquo Journal of Feline Medicine and Surgery vol 6 no 6 pp345ndash354 2004

[6] K E Kewish G D Appleyard S L Myers B A Kidneyand M L Jackson ldquoMycoplasma haemofelis and Mycoplasmahaemominutum detection by polymerase chain reaction in catsfrom Saskatchewan and Albertardquo Canadian Veterinary Journalvol 45 no 9 pp 749ndash752 2004

[7] M R Lappin B Griffin J Brunt et al ldquoPrevalence of Bartonellaspecies Haemoplasma species Ehrlichia species Anaplasmaphagocytophilum and Neorickettsia risticii DNA in the bloodof cats and their fleas in the United Statesrdquo Journal of FelineMedicine and Surgery vol 8 no 2 pp 85ndash90 2006

[8] T B Hackett W A Jensen T L Lehman et al ldquoPrevalenceof DNA of Mycoplasma haemofelis ldquoCandidatus Mycoplasmahaemominutumrdquo Ananplasma phagocytophilum and species ofBartonella Neorickettsia and Ehrlichia in cats used as blooddonors in the United Statesrdquo Journal of the American VeterinaryMedical Association vol 229 no 5 pp 700ndash705 2006

[9] B Willi F S Boretti C Baumgartner et al ldquoPrevalence riskfactor analysis and follow-up of infections caused by threefeline hemoplasma species in cats in Switzerlandrdquo Journal ofClinical Microbiology vol 44 no 3 pp 961ndash969 2006

[10] N Bauer H-J Balzer S Thure and A Moritz ldquoPrevalence offeline haemotropicmycoplasmas in convenience samples of catsin Germanyrdquo Journal of Feline Medicine and Surgery vol 10 no3 pp 252ndash258 2008

[11] I R Peters C R Helps B Willi R Hofmann-Lehmann and STasker ldquoThe prevalence of three species of feline haemoplasmasin samples submitted to a diagnostics service as determined bythree novel real-time duplex PCR assaysrdquo Veterinary Microbiol-ogy vol 126 no 1ndash3 pp 142ndash150 2008

[12] F Gentilini M Novacco M E Turba B Willi M L Bacciand R Hofmann-Lehmann ldquoUse of combined conventionaland real-time PCR to determine the epidemiology of felinehaemoplasma infections in northern Italyrdquo Journal of FelineMedicine and Surgery vol 11 no 4 pp 277ndash285 2009

[13] M Tanahara S Miyamoto T Nishio et al ldquoAn epidemiologicalsurvey of feline hemoplasma infection in Japanrdquo Journal ofVeterinary Medical Science vol 72 no 12 pp 1575ndash1581 2010

[14] V R Barrs J A Beatty B J Wilson et al ldquoPrevalence of Bar-tonella species Rickettsia felisHaemoplasmas and the Ehrlichiagroup in the blood of cats and fleas in eastern AustraliardquoAustralian Veterinary Journal vol 88 no 5 pp 160ndash165 2010

[15] X Roura I R Peters L Altet et al ldquoPrevalence of hemotropicmycoplasmas in healthy and unhealthy cats and dogs in SpainrdquoJournal of Veterinary Diagnostic Investigation vol 22 no 2 pp270ndash274 2010

[16] AD Bennett DAGunn-MooreM Brewer andMR LappinldquoPrevalence of Bartonella species Haemoplasmas and Toxo-plasma gondii in cats in Scotlandrdquo Journal of FelineMedicine andSurgery vol 13 no 8 pp 553ndash557 2011

[17] R Lobetti and M R Lappin ldquoPrevalence of Toxoplasma gondiiBartonella species and haemoplasma infection in cats in SouthAfricardquo Journal of Feline Medicine and Surgery vol 14 pp 857ndash862 2012

[18] S Assarasakorn J K Veir J R Hawley et al ldquoPrevalenceof Bartonella species hemoplasmas and Rickettsia felis DNAin blood and fleas of cats in Bangkok Thailandrdquo Research inVeterinary Science vol 93 pp 1213ndash1216 2012

[19] V L Martınez-Dıaz A C Silvestre-Ferreira H Vilhena etal ldquoPrevalence and co-infection of haemotropic mycoplasmasin Portuguese cats by real-time polymerase chain reactionrdquoJournal of Feline Medicine and Surgery vol 15 no 10 pp 879ndash885 2013

[20] B J Luria J K Levy M R Lappin et al ldquoPrevalence ofinfectious diseases in feral cats in Northern Floridardquo Journal ofFeline Medicine and Surgery vol 6 no 5 pp 287ndash296 2004

[21] J M Eberhardt K Neal T Shackelford and M R LappinldquoPrevalence of selected infectious disease agents in cats fromArizonardquo Journal of Feline Medicine and Surgery vol 8 no 3pp 164ndash168 2006

[22] D-H Yu H-W Kim A R Desai et al ldquoMolecular detection offeline hemoplasmas in feral cats in Koreardquo Journal of VeterinaryMedical Science vol 69 no 12 pp 1299ndash1301 2007

[23] F Juvet M R Lappin S Brennan and C T Mooney ldquoPreva-lence of selected infectious agents in cats in Irelandrdquo Journal ofFeline Medicine and Surgery vol 12 no 6 pp 476ndash482 2010

[24] V Stojanovic and P Foley ldquoInfectious disease prevalence ina feral cat population on Prince Edward Island CanadardquoCanadian Veterinary Journal vol 52 no 9 pp 979ndash982 2011

[25] J E Woods M M Brewer J R Hawley N Wisnewskiand M R Lappin ldquoEvaluation of experimental transmissionof Candidatus Mycoplasma haemominutum and Mycoplasmahaemofelis by Ctenocephalides felis to catsrdquo The AmericanJournal of Veterinary Research vol 66 no 6 pp 1008ndash10122005

[26] D S Westfall W A Jensen W J Reagan S V Radecki andM R Lappin ldquoInoculation of two genotypes ofHemobartonellafelis (California and Ohio variants) to induce infection incats and the response to treatment with azithromycinrdquo TheAmerican Journal of Veterinary Research vol 62 no 5 pp 687ndash691 2001

[27] A T Gary H L Richmond S Tasker T B Hackett and M RLappin ldquoSurvival of Mycoplasma haemofelis and ldquoCandidatusMycoplasma haemominutumrdquo in blood of cats used for transfu-sionsrdquo Journal of Feline Medicine and Surgery vol 8 no 5 pp321ndash326 2006

8 ISRNMicrobiology

[28] E Spada D Proverbio A Della Pepa et al ldquoSeroprevalenceof feline immunodeficiency virus feline leukaemia virus andToxoplasma gondii in stray cat colonies in northern Italy andcorrelation with clinical and laboratory datardquo Journal of FelineMedicine and Surgery vol 14 pp 369ndash377 2012

[29] E Spada D Proverbio A Della Pepa et al ldquoPrevalence offaecal-borne parasites in colony stray cats in northern ItalyrdquoJournal of Feline Medicine and Surgery vol 15 no 8 pp 672ndash677 2013

[30] A Moritz Y Fickenscher K Meyer K Failing and D J WeissldquoCanine and feline hematology reference values for the ADVIA120 hematology systemrdquo Veterinary Clinical Pathology vol 33no 1 pp 32ndash38 2004

[31] W A Jensen M R Lappin S Kamkar and W J Reagan ldquoUseof a polymerase chain reaction assay to detect and differentiatetwo strains of Haemobartonella felis in naturally infected catsrdquoThe American Journal of Veterinary Research vol 62 no 4 pp604ndash608 2001

[32] A M Ishak S Radecki and M R Lappin ldquoPrevalence ofMycoplasma haemofelis ldquoCandidatus Mycoplasma haemomin-utumrdquo Bartonella species Ehrlichia species and Anaplasmaphagocytophilum DNA in the blood of cats with anemiardquoJournal of FelineMedicine and Surgery vol 9 no 1 pp 1ndash7 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

ISRNMicrobiology 7

Acknowledgment

This research received no specific grant from any fundingagency in the public commercial or not-for-profit sectors

References

[1] J B Messick and J W Harvey ldquoHemotropic mycoplasmosis(Hemobartonellosis)rdquo in Infectious Diseases of the Dog and CatC E Greene Ed pp 310ndash319 Elsevier-Saunders St Louis MoUSA 4th edition 2012

[2] H Obara M Fujihara Y Watanabe H K Ono and RHarasawa ldquoA feline hemoplasma ldquoCandidatus Mycoplasmahaemominutumrdquo detected in dog in Japanrdquo Journal of Veteri-nary Medical Science vol 73 no 6 pp 841ndash843 2011

[3] A P dos Santos R P dos Santos A W Biondo et al ldquoHemo-plasma infection in HIV-positive patient Brazilrdquo EmergingInfectious Diseases vol 14 no 12 pp 1922ndash1924 2008

[4] S Tasker I R Peters K Papasouliotis et al ldquoDescription ofoutcomes of experimental infection with feline haemoplasmascopy numbers haematology Coombsrsquo testing and blood glu-cose concentrationsrdquo Veterinary Microbiology vol 139 no 3-4pp 323ndash332 2009

[5] S Tasker J A Braddock R Baral et al ldquoDiagnosis of felinehaemoplasma infection inAustralian cats using a real-timePCRassayrdquo Journal of Feline Medicine and Surgery vol 6 no 6 pp345ndash354 2004

[6] K E Kewish G D Appleyard S L Myers B A Kidneyand M L Jackson ldquoMycoplasma haemofelis and Mycoplasmahaemominutum detection by polymerase chain reaction in catsfrom Saskatchewan and Albertardquo Canadian Veterinary Journalvol 45 no 9 pp 749ndash752 2004

[7] M R Lappin B Griffin J Brunt et al ldquoPrevalence of Bartonellaspecies Haemoplasma species Ehrlichia species Anaplasmaphagocytophilum and Neorickettsia risticii DNA in the bloodof cats and their fleas in the United Statesrdquo Journal of FelineMedicine and Surgery vol 8 no 2 pp 85ndash90 2006

[8] T B Hackett W A Jensen T L Lehman et al ldquoPrevalenceof DNA of Mycoplasma haemofelis ldquoCandidatus Mycoplasmahaemominutumrdquo Ananplasma phagocytophilum and species ofBartonella Neorickettsia and Ehrlichia in cats used as blooddonors in the United Statesrdquo Journal of the American VeterinaryMedical Association vol 229 no 5 pp 700ndash705 2006

[9] B Willi F S Boretti C Baumgartner et al ldquoPrevalence riskfactor analysis and follow-up of infections caused by threefeline hemoplasma species in cats in Switzerlandrdquo Journal ofClinical Microbiology vol 44 no 3 pp 961ndash969 2006

[10] N Bauer H-J Balzer S Thure and A Moritz ldquoPrevalence offeline haemotropicmycoplasmas in convenience samples of catsin Germanyrdquo Journal of Feline Medicine and Surgery vol 10 no3 pp 252ndash258 2008

[11] I R Peters C R Helps B Willi R Hofmann-Lehmann and STasker ldquoThe prevalence of three species of feline haemoplasmasin samples submitted to a diagnostics service as determined bythree novel real-time duplex PCR assaysrdquo Veterinary Microbiol-ogy vol 126 no 1ndash3 pp 142ndash150 2008

[12] F Gentilini M Novacco M E Turba B Willi M L Bacciand R Hofmann-Lehmann ldquoUse of combined conventionaland real-time PCR to determine the epidemiology of felinehaemoplasma infections in northern Italyrdquo Journal of FelineMedicine and Surgery vol 11 no 4 pp 277ndash285 2009

[13] M Tanahara S Miyamoto T Nishio et al ldquoAn epidemiologicalsurvey of feline hemoplasma infection in Japanrdquo Journal ofVeterinary Medical Science vol 72 no 12 pp 1575ndash1581 2010

[14] V R Barrs J A Beatty B J Wilson et al ldquoPrevalence of Bar-tonella species Rickettsia felisHaemoplasmas and the Ehrlichiagroup in the blood of cats and fleas in eastern AustraliardquoAustralian Veterinary Journal vol 88 no 5 pp 160ndash165 2010

[15] X Roura I R Peters L Altet et al ldquoPrevalence of hemotropicmycoplasmas in healthy and unhealthy cats and dogs in SpainrdquoJournal of Veterinary Diagnostic Investigation vol 22 no 2 pp270ndash274 2010

[16] AD Bennett DAGunn-MooreM Brewer andMR LappinldquoPrevalence of Bartonella species Haemoplasmas and Toxo-plasma gondii in cats in Scotlandrdquo Journal of FelineMedicine andSurgery vol 13 no 8 pp 553ndash557 2011

[17] R Lobetti and M R Lappin ldquoPrevalence of Toxoplasma gondiiBartonella species and haemoplasma infection in cats in SouthAfricardquo Journal of Feline Medicine and Surgery vol 14 pp 857ndash862 2012

[18] S Assarasakorn J K Veir J R Hawley et al ldquoPrevalenceof Bartonella species hemoplasmas and Rickettsia felis DNAin blood and fleas of cats in Bangkok Thailandrdquo Research inVeterinary Science vol 93 pp 1213ndash1216 2012

[19] V L Martınez-Dıaz A C Silvestre-Ferreira H Vilhena etal ldquoPrevalence and co-infection of haemotropic mycoplasmasin Portuguese cats by real-time polymerase chain reactionrdquoJournal of Feline Medicine and Surgery vol 15 no 10 pp 879ndash885 2013

[20] B J Luria J K Levy M R Lappin et al ldquoPrevalence ofinfectious diseases in feral cats in Northern Floridardquo Journal ofFeline Medicine and Surgery vol 6 no 5 pp 287ndash296 2004

[21] J M Eberhardt K Neal T Shackelford and M R LappinldquoPrevalence of selected infectious disease agents in cats fromArizonardquo Journal of Feline Medicine and Surgery vol 8 no 3pp 164ndash168 2006

[22] D-H Yu H-W Kim A R Desai et al ldquoMolecular detection offeline hemoplasmas in feral cats in Koreardquo Journal of VeterinaryMedical Science vol 69 no 12 pp 1299ndash1301 2007

[23] F Juvet M R Lappin S Brennan and C T Mooney ldquoPreva-lence of selected infectious agents in cats in Irelandrdquo Journal ofFeline Medicine and Surgery vol 12 no 6 pp 476ndash482 2010

[24] V Stojanovic and P Foley ldquoInfectious disease prevalence ina feral cat population on Prince Edward Island CanadardquoCanadian Veterinary Journal vol 52 no 9 pp 979ndash982 2011

[25] J E Woods M M Brewer J R Hawley N Wisnewskiand M R Lappin ldquoEvaluation of experimental transmissionof Candidatus Mycoplasma haemominutum and Mycoplasmahaemofelis by Ctenocephalides felis to catsrdquo The AmericanJournal of Veterinary Research vol 66 no 6 pp 1008ndash10122005

[26] D S Westfall W A Jensen W J Reagan S V Radecki andM R Lappin ldquoInoculation of two genotypes ofHemobartonellafelis (California and Ohio variants) to induce infection incats and the response to treatment with azithromycinrdquo TheAmerican Journal of Veterinary Research vol 62 no 5 pp 687ndash691 2001

[27] A T Gary H L Richmond S Tasker T B Hackett and M RLappin ldquoSurvival of Mycoplasma haemofelis and ldquoCandidatusMycoplasma haemominutumrdquo in blood of cats used for transfu-sionsrdquo Journal of Feline Medicine and Surgery vol 8 no 5 pp321ndash326 2006

8 ISRNMicrobiology

[28] E Spada D Proverbio A Della Pepa et al ldquoSeroprevalenceof feline immunodeficiency virus feline leukaemia virus andToxoplasma gondii in stray cat colonies in northern Italy andcorrelation with clinical and laboratory datardquo Journal of FelineMedicine and Surgery vol 14 pp 369ndash377 2012

[29] E Spada D Proverbio A Della Pepa et al ldquoPrevalence offaecal-borne parasites in colony stray cats in northern ItalyrdquoJournal of Feline Medicine and Surgery vol 15 no 8 pp 672ndash677 2013

[30] A Moritz Y Fickenscher K Meyer K Failing and D J WeissldquoCanine and feline hematology reference values for the ADVIA120 hematology systemrdquo Veterinary Clinical Pathology vol 33no 1 pp 32ndash38 2004

[31] W A Jensen M R Lappin S Kamkar and W J Reagan ldquoUseof a polymerase chain reaction assay to detect and differentiatetwo strains of Haemobartonella felis in naturally infected catsrdquoThe American Journal of Veterinary Research vol 62 no 4 pp604ndash608 2001

[32] A M Ishak S Radecki and M R Lappin ldquoPrevalence ofMycoplasma haemofelis ldquoCandidatus Mycoplasma haemomin-utumrdquo Bartonella species Ehrlichia species and Anaplasmaphagocytophilum DNA in the blood of cats with anemiardquoJournal of FelineMedicine and Surgery vol 9 no 1 pp 1ndash7 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 ISRNMicrobiology

[28] E Spada D Proverbio A Della Pepa et al ldquoSeroprevalenceof feline immunodeficiency virus feline leukaemia virus andToxoplasma gondii in stray cat colonies in northern Italy andcorrelation with clinical and laboratory datardquo Journal of FelineMedicine and Surgery vol 14 pp 369ndash377 2012

[29] E Spada D Proverbio A Della Pepa et al ldquoPrevalence offaecal-borne parasites in colony stray cats in northern ItalyrdquoJournal of Feline Medicine and Surgery vol 15 no 8 pp 672ndash677 2013

[30] A Moritz Y Fickenscher K Meyer K Failing and D J WeissldquoCanine and feline hematology reference values for the ADVIA120 hematology systemrdquo Veterinary Clinical Pathology vol 33no 1 pp 32ndash38 2004

[31] W A Jensen M R Lappin S Kamkar and W J Reagan ldquoUseof a polymerase chain reaction assay to detect and differentiatetwo strains of Haemobartonella felis in naturally infected catsrdquoThe American Journal of Veterinary Research vol 62 no 4 pp604ndash608 2001

[32] A M Ishak S Radecki and M R Lappin ldquoPrevalence ofMycoplasma haemofelis ldquoCandidatus Mycoplasma haemomin-utumrdquo Bartonella species Ehrlichia species and Anaplasmaphagocytophilum DNA in the blood of cats with anemiardquoJournal of FelineMedicine and Surgery vol 9 no 1 pp 1ndash7 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology