JOURNAL OF PURE AND APPLIED MICROBIOLOGY, March 2015. Vol. 9(1), p. 349-355

Phenotypic and Genotypic Haemolysin Properties ofStaphylococcus aureus Obtained from Milk of

Cattle and Buffalo with Clinical Mastitis

Rahul Yadav' *, Sandeep Kumar Sharma",Jyotika Yadav", Taruna Bhati' and Anil Kumar Kataria'

'Department of Veterinary Microbiology and Biotechnology, College of Veterinary and AnimalSciences, Bikaner. Rajasthan University of Veterinary and Animal Science,

Bikaner-334001 (Rajasthan), India.2Department of Veterinary Microbiology and Biotechnology, Post graduate institute of veterinary

Education and Research, Jaipur. Rajasthan University of Veterinary and Animal science,Bikaner-334001 (Rajasthan), India.

3College of Veterinary and Animal Sciences, Hisar. Lala Lajpat Rai University of Veterinary & AnimalSciences, Hisar-125001 (Haryana), India.

(Received: 06 April 2014; accepted: 19 August 2014)

The qualitative and quantitative phenotypic expression of haemolysins alongwith presence of genes encoding (1- and Ji-haemolysin were determined in 32Staphylococcus aureus isolates from milk of cattle and buffalo with clinical mastitis.Overall haemolytic reactions on sheep blood agar revealed five (15.62%) isolates toshow complete haemolysis, 20 (62.50%) isolates to show partial haemolysis, four(12.50%) isolates to show both complete and partial and three (9.37%) did not show anyhaemolysis. All the 32 (100%) isolates from both cattle (16) and buffalo (16) produced (1-

toxin, the maximum titre of which was 1: 5120. Whereas beta-toxin was produced by 11(68.75%) cattle isolates and by seven (43.75%) buffalo isolates with maximum titre of1:1280 and 1:240, respectively. Delta toxin was detected to be produced by only five(15.62%) isolates, two from cattle and three from buffalo. The genotypic characterizationrevealed an overall hla gene prevalence in 96.8% isolates from both cattle and buffalowherein a single amplicons of 534 bp was produced while hlb gene was amplified by84.3% (13 cattle and 14 buffalo isolates) producing single amplicon of 833 bp.

Key word: Staphylococcus aureus, Cattle, Buffalo, Mastitis, Haemolysis, hla and hlb gene.

Staphylococcus aureus is recognizedworldwide as a major pathogen causing clinicalintramammary infections in dairy cattle and buffalo.The disease is associated with reduced milk qualityand high economic loss (Salasia et al., 2004; Graberet al., 2013), and is therefore a key problem fordairy industry. Three types ofhaemolysins namelya, ~ and d designated in the order of their discovery

* Towhom all correspondence should be addressed.Mob.: +91-9466930987; 9694065689;E-mail: drrahul16889@gmail.com

have been reported to be produced by S. aureusand are considered true virulent factors incausation of mastitis. Alpha-hemolysin (a-toxin)is considered a main pathogenicity factor becauseof its hemolytic, cytotoxic, dermonecrotic andneurotoxic effects on rabbit erythrocytes (Dinges,2000; Aryanti et al., 2011). Beta- hemolysin is asphingo-myelinase that is highly active againstsheep and bovine erythrocytes (Larsen et aI., 2002)while d-hemolysin as well as a-hemolysin inducespore formation perturbing the cell membranepermeability (Butt et al., 1998). Though it has theability to lyse erythrocytes and other cells of

350 YADAVet al.: S. aureus HAEMOLYSIS HLA & HLB GENES FROM MASTITIS MILK

different animals species, it is more active againsthorse red blood cells (Quinn et aI., 1994).

The typing and titration of thesehaemolysins may well be an indicator ofpathogenicity ofthese organisms in bovine clinicalmastitis (Sanjiv and Kataria, 2007; Yang et al.,2012). Several studies have been carried out todemonstrate hemolysin production by S. aureusobtained from bovine mastitis (Fitzgerald et al.,1997; Dinges, 2000 and Larsen et al., 2002). Thegene hla responsible for n-haemolysin is able todissolve many types of the cells in human andanimals including monocytes, Iymohocytes, redblood cells, platelets and endothelial cells.Whereas, cytotoxic effect of ~-haemolysin(governed by hlb gene) suggesting that its primaryvirulent activity is to modulate host processes thateffects pathogenesis rather than to directly kill thehost cells. The gene hlb can promote themultiplication of S. aureus and increase the harmto bovine (Wang et al., 2011). Many workers havecarried out typing of hla and hlb genes (EI-Sayedet al., 2005; Haveri et al., 2007; Sudagidan et aI.,2008; Coelho et aI., 2011; Salasia et aI., 2011). Thepresent work elucidated various haemolysinsproduced by S. aureus in terms of qualitative andquantitative assays along with characterization ofisolates for hla and hlb genes.

MATERIALS AND METHODS

Sample collection, Isolation and identificationThe milk samples were collected in 5-10

ml amounts from cattle and buffalo affected withclinical mastitis. The sample phenotypicallyidentified as per standard procedures (Quinn etal., 1994). All phenotypically identifiedStaphylococci isolates were further confirmed tobe S. aureus based on 23S rRNA gene ribotyping(Straub et aI., 1999).Haemolytic properties and Haemolysin assays

The hemolytic activity was evaluated byplating staphylococci isolates on triplicate platesof blood agar base supplemented with 5% sheep,bovine and horse blood for alpha, beta and delta-hemolysin assays, respectively (Quinn et aI., 1994).Isolates were inoculated in the form of streaks onthe surface of plates and incubated at 37°C for 24and 48 h. The criteria for hemolysin identificationwere: complete lytic zone (transparent) with blurred

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edges for a-hemolysin on ovine and incomplete(non-transparent) lytic zone, which becamecomplete with sharp edges after overnightincubation at 4°C on bovine blood agar, for beta-hemolysin. The delta-hemolysin production wasdetermined as complete hemolytic zones on horseblood agar (Quinn et aI., 1994; Bedidi-Madani etal., 1998; da Silva et al., 2005). Qualitative andquantitative assays for haemolysins were doneusing rabbit, cattle and horse erythrocytes for a-,~- and d-haemolysin, respectively (Sanjiv andKataria, 2007).Toxin Production

The test culture suspension (about 1-2ml, 24h old) was poured, spread well onto surfaceof semisolid nutrient agar plate and then plateswere incubated at 37°C in an atmosphere of 20%carbon dioxide tension for 48 h. Followingincubation, the agar medium was sliced into smallpieces and the plates were then transferred to deepfreezer at -20°C for 30 min. Alternate freezing andthawing was carried out to obtain the fluid fromculture. It was then centifuged at 4000 rpm inrefrigerated centrifuge machine for 45 min. Thesupernatant having toxin was collected in screwcapped plastic test tubes and was stored at -20°Cin deep freezer till use for titration ofhaemolysins.Titration of haemolysins

The preparation of erythrocytes andtitration ofhaemolysins were done as per methoddescribed by (Sanjiv and Kataria, 2007).Amplification hla and hlb gene

Amplification of these gene was carriedout as described by Booth et al. (2001) usingforward primer 5'GGTTTAGCCTGGCCTIC3' andreverse primer 5'CATCACGAACTCGTTCG3' forhla gene and forward primer 5'GCCAAAGCCGAATCTAAG3' and reverse primer 5'CGCATATACATCCCATGGC3' for hlb gene. Briefly, thereaction mixture of30 IIIwas prepared by mixing19.4111deionised water, 2.5 1111OxBuffer, 1.8111MgC~1.5 IIIPrimer-I (10 PM/ill), 1.5 IIIPrimer-2 (10 pMiIll), 0.6 III dNTP-mix (I OmM), 0.2 III Taq DNApolymerase (5U/IlI) and 2.5111 template DNA (25ngiIll). Amplification was carried out in aVeriti thermalcycler (Applied biosystem) as follows: initial 30cycle of amplification (denaturation at 94°C for 30see, primer annealing at 53°C for 60 see and primerextension at noc for 30 sec) and final extension at10°C for 2 min. The PCR products, after addition of

YADAVetal.: S. aureus HAEMOLYSTSHLA& HLB GENES FROM MASTITIS MILK 351

2 IIIof trekking dye were resolved in 1.2% agarosegels prepared in 1.0 x TBE buffer containing 0.5Ilg/ml of ethidium bromide and 500 bp DNA ladderwas used as molecular marker. The amplificationproducts were electrophoresed for 50-60 min at100 V. The gel was then visualized under geldocumentation system (ENDURO GDS).

RESULTS AND DISCUSSION

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The ribotyping produced an amplicon of1250 bp in all the 32 isolates confirming them to beStaphylococcus aureus. Of the 16 cattle isolatesfive (31.20%) isolates exhibited completehaemolysis, nine (56.20%) isolates exhibitedincomplete/partial haemolysis of which sevenisolate showed phenomenon of hot-cold lysis, one(6.25%) isolate showed both complete and partialhaemolysis and one (6.25%) isolate was recordednot to produce any haemolysis and was consideredas ahaemolytic on sheep blood agar. Of the 16buffalo isolates 11 (68.75%) showed partialhaemolysis of which one showed hot-cold lysis,three (18.75%) showed both complete and partialhaemolysis and two (12.5%) did not showhaemolysis on sheep blood agar. In the presentstudy the overall haemolytic reactions on sheepblood agar revealed five (15.62%) isolates to showcomplete haemolysis, 20 (62.50%) isolates to showpartial haemolysis, four (12.50%) isolates to showboth complete and partial and three (9.37%) didnot show any haemolysis.

The observation about haemolytic S.aureus in the present study was similar to thatreported by Jasper et al. (1985) who recorded 99%of the isolates to produce haemolysins. Our resultsin regards to partial haemolysis by 62.5% S. aureusisolates support earlier observations of Matsunagaet al. (1993) who recorded 65.5% S. aureus frombovine mastitic milk and Morandi et at. (2009) whorecorded 62% of the isolates from various cow dairyproducts showing incomplete haemolysis.Likewise, Aarestrup et al. (1999) recorded 72% S.aureus of bovine mastitic origin to produceincomplete haemolysis. However, our results arein contrast to observation ofBoerlin et al. (2003)who did not detect incomplete haemolysis onblood agar plate by S. aureus isolates. Similar toour observations Islam et at. (2007) also recordedmore 86.3% S. aureus from cattle showing

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incomplete haemolysis. Our results on haemolysisare also similar to observation of Annemuller et at.(1999) who recorded production of completehaemolysis by eight isolates and partial haernolysis

Fig. 1. Agarose gel electrophoresis of amplicons of hlagene of S. aureus isolates obtained from cattle withclinical mastitis

" .' e~.:f" B;4·,··;'"el~~~'B~ ',~8~~' ,611 817 ail 618"_'~·"""""_i :';.1::';"';" .. ...;...".:_ .. -.~ .----- •..--~--..-..•.i- M: MOlltUlAR MARJR f!tOObp)

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Fig. 2. Agarose gel electrophoresis of amplicons of hlagene of S. aureus isolates obtained from buffalo withclinical mastitis

Fig. 3. Agarose gel electrophoresis of amplicons of hlbgene of S. aureus isolates obtained from cattle withclinical mastitis

Fig. 4. Agarosc gel electrophoresis of amplicons of hlbgene of S. aureus isolates obtained from buffalo withclinical mastitis

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352 YADAVet al.: S. aureus HAEMOLYSIS HLA & HLB GENES FROM MASTITIS MILK

by 13 out of 25 S. aureus isolates of bovinemastitis. Similar to our observation Sharma et al.(2013) also reported that 12 out of 15 isolatesshowed partial haemolysis of which four isolateslater showed hot-cold lysis whereas three isolatedshowed complete haemolysis.

In the present study 9.37% of the isolateswere ahaemolytic. Graber et al. (2013) also recordedvery low percentage (0-2%) ofnon-haemolytic S.aureus in their study. Likewise, Sanjiv and Kataria(2007) and Upadhyay and Kataria (2010) did notrecord presence of ahaemolytic S. aureus isolatesfrom the present study area. Similar to ourobservations of obtaining ahaemolytic isolates,Salasia et al. (2004) also reported 10non-haemolyticisolates out of 35 S. aureus isolates from bovinesubclinical mastitis. The study of Ariyanti et at.(2011), the types of haemolysins of S. aureus onthe sheep blood agar plate, revealed completehaemolysis for two isolates (18.18%), partialhaemolysis for three isolates (27.27%) and nohaemolysis for six isolates (54.55%). Production ofdelta (d) haemolysin is also an important propertyof S. aureus recorded as complete haemolysis onhorse blood agar (Quinn et al., 1994). In the presentinvestigation, production of delta haemolysin wasshown by nine (28.12%) isolates only. Ourobservations are contrary to findings of Garcia etat. (1980) who found delta-haemolysin productionby 47 (82.45%) out of the 57 strains. Likewise, daSilva et at. (2005) reported 83.3% isolates of clinicaland subclinical caprine mastitic origin to producedelta (d) haemolysin in combination with otherhaemolysins but not alone. Similarly, Ebrahimi andTaheri (2009) reported production of delta toxin by62.5% of S. aureus isolates from clinical andsubclinical mastitis of cow in combination withalpha, beta toxins. Chu et al. (2013) also reported100% isolates to produce complete haemolysis onsheep and horse blood agar.Toxin assay

Toxin production is considered related topathogenicity of S. aureus. To study the qualitativeand quantitative production of toxins, all theisolates were subjected to haemolytic assays usingerythrocytes from rabbit, cattle and horse for alpha-, beta- and delta-toxins, respectively.Qualitative Assay

In the present investigation, all the 32(100%) isolates from both cattle and buffalo

] PURE APPL MICROBIO, 9(1), MARCH 2015.

haemolysed rabbit erythrocytes indicating presenceof alpha-toxin whereas beta-toxin was producedby II (68.75%) cattle isolates and by seven (43.75%)buffalo isolates whereas five (15.62%) isolates, twofrom cattle and three from buffalo were detected asdelta-toxin producer. Similar to present observationUpadhyay and Kataria (20 I 0) also reportedproduction of ±-toxin by all the isolates from bovineand goat mastitic mille Likewise, Jasper et al. (\ 985)also observed alpha and beta toxin by 99% of theisolates and Kenny et at. (1992) detected 94.3% ofS. aureus from bovine mammary glands to producealpha-haemolysin and suggested alpha-haemolysin production is a feature of bovinemammary isolates.

Our findings are contrary for delta toxinto those ofSanjiv and Kataria (2007) who did notrecord production of delta-toxin by S. aureusisolates obtained from H-F crossbred and Rathicattle with clinical mastitis. Our results are in partialagreement to those ofUpadhyay and Kataria (2010)who observed the production of d-toxin by all ofthe isolates of S. aureus from mastitic milk.Quantitative Assay

In the present investigation all the 32isolates produced alpha-toxin of which eight cattleisolates and seven buffalo isolates produced titreof I :2560 and eight cattle and nine buffalo isolatesproduced the titre of I: 5120. The production ofbeta-toxin was shown by lesser number of isolateswhere 11 cattle and seven buffalo isolates producedbeta-toxin. The titres of beta-toxin were also muchless than that for alpha-toxin ranging between 1:5and 1:1280. Most of the isolates produced lowertitres. In this study buffalo isolates produced lowerbeta-toxin titres than the cattle isolates. In ourinvestigation delta toxin was detected to beproduced by only five isolates, two from cattleand three from buffalo. The overall analysis of thehaemolysis assay revealed that there was nodifference in the qualitative and quantitativeproduction of toxins by the isolates from cattleand from buffalo. Our results were in completeagreement to those of Sanjiv and Kataria (2007)and Upadhyay and Kataria (2010) who alsoreported production of alpha-haemolysin by all theisolates and with reported high alpha-toxin titres.The lower titres of 2-toxin than that of ±-toxin inthe present investigation is in complete agreementto the findings of Sanjiv and Kataria (2007);

YADAVet a!.: S. aureus HAEMOLYSIS HLA & HLB GENES FROM MASTITIS MILK 353

Upadhyay and Kataria (20 I0) who also recordedcomparatively lower titres of2-toxin in their studies.hla and hlb genotyping

The pathogenicity of Saureus is relatedto the production of a wide variety of exoproteinsincluding alpha and beta haemolysins whichcontribute to its ability to cause diseases in manymammalian species (da Silva et al., 2005). Alpha-haemolysin or alpha-toxin is considered a mainpathogenicity factor because of its haemolytic,dermonecrotic and neurotoxic effects and it isgoverned by hla gene. Beta-haemolysin containssphingomyelinase that is more active against sheepand bovine erythrocytes (da Silva et al., 2005;Dinges et al., 2000; Larsen et al., 2002) and isgoverned by hlb gene. In the present study all theisolates from cattle except one (C26) amplified hlagene producing amplicons of 534 bp (Figure I).Similar amplicons were produced by all buffaloisolates (Figure 2). The overall hla gene prevalencewas recorded as 96.8%. The absence of hla genein C26 isolates was well correlated with absence ofhaemolysis on sheep blood agar.

The hlb gene was amplified by 13 cattleand 14 buffalo isolates producing single ampliconof 833 bp in each (Figure 3 & 4). The overallprevalence of hlb gene was recorded as 84.3%which was lower than that of hla gene. Theprevalence of hla and hlb recorded in the presentstudy was almost similar to observations ofHaveriet al., (2007) who recorded the prevalence of97.4%and 76.7% of the 116 strains for the hla and hlbgenes, respectively. Salasia et al., (20 II) alsorecorded the prevalence of 81.81 % isolates for hlagene with amplicons size of 534 bp. Likewise, Yanget al., (2012) recorded the prevalence of hla andhlb gene as 85% and 82%, respectively. However,Booth et al., (2001) observed only 38% (77/200) ofthe isolates to possess hlb gene. Wang et al.,(20 II) also recorded that 47 (34.88%) of the Saureus isolates possessed hla gene. Similarly,Coelho et al., (2011) also reported that only 24 and16% of the isolates were positive for the hla andhlb genes, respectively.

The present investigation was incomplete agreement with the findings of theSudagidan et al., (2008), Salasia et al., (2011),Ariyanti et al., (2011) and Memon et al., (20l3)who also found similar amplified product size ofthe hla and hlb gene. However, El-Sayed et al.,

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(2005) detected hlb gene with a size ofapproximately 840 bp in all 24 S aureus isolates(100%) obtained from clinical mastitis and in l3isolates (81.3%) from subclinical mastitis whereasgene hla of 550 bp was found in all the S aureusisolates.

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