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Open Access
STUDIES ON PESTICIDE RESIDUES IN WATER, SOIL, FODDER AND MILK ALONG MUST RIVER BELT
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2013-10) KOTINAGU, KORRAPATI; KRISHNAIAH, N(MAJOR); MADAHAVA RAO, T; KONDAL REDDY, K; SASHI BHUSHAN, V
ABSTRACT: A study was conducted to estimate residues of certain pesticides of organochlorines viz., DDT (o,p'- DDE, o,p' - DDD, p,p'- DDT and o,p'- DDT and Dicofol), HCH (Alpha, Beta, Gamma, Delta), cyclodiene compounds (Aldrin, Endosulfan Sulphate and Heptachlor) and organophosphates (Triazophos, Dimetheoate, Chlorpyrifos and Methyl-chlorpyrifos) in soil, water, fodder and milk samples collected from six zones of Musi river belt area. To evaluate the pollution level of Musi river, the river belt was divided in to six zones viz., Zone 1 (Attapur to High court), Zone 2 (Chadhar ghat to Uppal), Zone 3 (Peerzadiguda to Chinna viralla), Zone 4 (Pillai Palli to Alinagar), Zone 5 (Indriyala to Manimadde), Zone 6 (Musi reservoir to Wazirabad). Only soil samples collected hm Zone 1 showed residual levels (in ppm) of 0.06 + 0.005 (0.035 to 0.083), 0.73 * 0.01 (0.675 to 0.791), 1.27 * 0.09 (1.023 to l.893), 0.14 =k 0.015 (0.098 to 0.243) and 0.55 * 0.02 (0.481 to 0.685) for p,p'- DDE, o,p'- DDD, p,p'- DDT, o,p' - DDT and Total DDT respectively. Dicofol was present only in fodder samples of zone 5 at concentration of 0.07 + 0.0007 (0.071 to 0.077). Among HCH compounds only delta HCH was found in soil samples of Zone 1 at a concentration of 0.08 *0.003 (0.065 to 0.098). Water, fodder and milk samples from zone 2 to 6 did not contain any residues of DDT and HCH. None of the samples water, soil, fodder and Milk from all the 6 zones contain the residues of Cyclodiene compounds. Among organophosphorus compounds Triazophos was present in soil samples of zone 1 at a level of 0.03 * 0.001 (0.032 to 0.045) and Dimetheoate was present in milk samples collected from Zone 6 at a level of 0.13 & 0.006 (0.1 11 to 0.167). From this study, it can be concluded that all the pesticide residues in soil were well below the MRL values, whereas Dicofol in fodder and Dimethoate in milk were slightly above the MRL values specified by EU and CODEX.
Open Access
IN VITRO EVALUATION OF DIFFERENT LACTIC ACID BACTERIAL STRAINS FOR THEIR PROBIOTIC CHARACTERISTICS
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2012-11) SRINU, B; MADHAVA RAO, T(MAJOR); SHASHI KUMAR, M; KONDAL REDDY, K
ABSTRACT : Identifying probiotic characteristics of bacterial strains by in vitro studies forms basis for selection of functional probiotics for commercial use. The objective of study was to collect different cultures and screen to study their functional probiotic characteristics such as acid tolerance, bile tolerance, antibacterial activity, antibiotic sensitivity, temperature tolerance and acidifying activity. Among the eighteen Lactic acid bacterial strains studied, 15 showed good survivability at high bile salt concentration (0.3 to 2 % oxgall) and good growth at a pH of 1.5 to 3.5. These probiotic species showed good survival abilities in acidic pH 2.0 to 3.5 except Lactobacillus delbruckii bulgaricus 281, Bifidobacterium bifidum 232 and Bifidobacterium bifidum 235, which did not grow at pH 2.0. Lactobacillus fermentum 141 and Pediococcus acidolactici 252 was able to grow even at pH 1.5 also. All the 18 lactic acid bacterial strains showed a count (CFU/ml) in the range of 0.23x107 to 2.7x107 at pH 2.0 except Lactobacillus delbruckii bulgaricus 281, Bifidobacterium bifidum 232 and Bifidobacterium bifidum 235, which did not form detectable CFU/ml, whereas Lactobacillus helviticus 194 showed highest count of 2.7x107 CFU/ml at pH 2.0. Among all the Lactobacillus spp. Lactobacillus paraplantarum 321 and Lactobacillus paracasei 22 showed maximum growth at 0.3% oxgall concentrations similarly Bifidobacterium bifidum 233 and Bifidobacterium bifidum 236 showed maximum growth when compared to other bifidobacterial strains. Among eighteen Lactic acid bacterial strains Lactobacillus casei 17, Pediococcus acidolactici 252 (except Clindamycin), Lactobacillus delbruckii bulgaricus 281 (except for Nitrofurantoin, Clindamycin and Cefpodoxime) and Lactobacillus helviticus 194 (except for Gentamycin, Nitrofurantoin and Streptomycin) were resistant to all the antibiotics tested. Bifidobacterium bifidum 231 (except for Streptomycin and Kanamycin) and Bifidobacterium bifidum 236 (except Norfloxicin) showed resistance to all antibiotics tested. All these Lactic acid bacterial strains were screened for in vitro inhibition of pathogenic microorganisms namely Proteus vulgaris, E.coli ATCC, E.coli 448, Pseudomonas aeruginosa, Klebsialla pneumoneae, Salmonella typhimurium, Bacillus cereus, Salmonella para-B, Staphylococci aureus. All the eighteen strains showed good antibacterial activity against the tested pathogenic bacteria. The strains of Lactobacillus paraplantarum 321, Pedicocci acidolactici 252 and Lactobacillus bulgaricus 281 inhibited the growth of all pathogenic bacteria. Bifidobacterium bifidum 232 and Bifidobacterium bifidum 233 (except Bacillus cereus and Salmonella typhimurium) also inhibited the growth of all pathogens tested. All the eighteen Lactic acid bacterial strains showed growth at all the tested temperatures (15oC, 40oC, and 45oC) except Bifidobacterium bifidum 229, Lactobacillus fermentum 141 and Lactobacillus rhamnosus 18 which have shown growth only at the temperatures of 15oC and 40oC, except at 45oC Among twelve Lactobacillus spp. Lactobacillus helviticus 194 has shown the highest acidifying activity, whereas Lactobacillus casei 17 shown lowest acidifying activity. Similarly Bifidobacterium bifidum 231 shown highest acidifying activity, whereas Bifidobacterium bifidum 236 shown lowest acidification values when compared with the other Bifidobacterial strains. The present study identified functional probiotics for future in vivo studies to commercialize probiotic strains of lactic acid bacteria to promote public health.
Open Access
STUDY ON CHARACTERIZATION OF SLAUGHTERHOUSE WASTEWATER IN AND AROUND HYDERABAD CITY
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2011-02) ENUMULA KUMAR; MADHAVA RAO, T(MAJOR); KRISHNAIAH, N; GOPALA REDDY, A; PRABU PRASADINI, P
ABSTRACT : The present study was undertaken to characterize the physical, chemical and microbiological properties of raw wastewater collected from three slaughterer houses located in and around Hyderabad city and to assess the efficiency of the treatment available at the plant and the effect of such wastewater on the environment. In raw wastewater from three slaughterhouses, the physical characteristics such as temperature of 31oC, pH of 6.8, turbidity in the range of 1847-4287 NTU, alkalinity varied between 433-903 mg/L, electrical conductivity in the range of 3.31-3.57 mS/cm, TDS varied between 1705-1896 mg/L, TSS ranged between 3001-5479 mg/L. The biochemical characteristics like DO varied between 0.41-1.45 mg/L, BOD ranged between 3559-4169 mg/L, COD varied between 4812-5308 mg/L, TOC varied between 2046-3345 mg/L, calcium ranged from 369 to 595 mg/L, NH4-N varied between 66-152 mg/L, nitrates in the range of 89-111 mg/L and phosphates of 76 mg/L were recorded. Major heavy metals such as lead, nickel and cadmium detected in wastewater from three slaughterhouses lead content ranged between 5.73 and 10.88 mg/L, nickel content of 1.03 mg/L and cadmium content varied from 0.59 to 1.47 mg/L detected. Except lead, cadmium and nickel values were below the effluent discharges limits recommended by pollution control board. In raw wastewater from three slaughterhouses, the microbiological characteristics such as TVC ranged from 68.21x106 - 105.14x 106 cfu/ml, total coliform count varied between 10.41 x106 - 35.16x106 cfu/ml recorded. The pathogens were isolated and the total count of Salmonella spp. ranged between 11.3-14.1x10 4 cfu/ml, Shigella count varied between 2.67x10 4-5.3x10 4 cfu/ml, S. faecalis count ranged between 8.58 x104 - 10.91x104 cfu/ml, S. aureus varied between 18.96x104 - 21.19x104 cfu/ml, B. cereus varied between 9.17 x104 - 10.19 x104 cfu/ml, and L. monocytogenes varied between 1900-6300 cfu/ml. The two slaughterhouses (i.e. II and III) have no treatment facilities and slaughterhouse I only having the UASB and DAF treatment facilities. The treated wastewater from slaughterhouse I has shown insufficient reduction in wastewater strength which was above the recommended limits for effluent discharges. The UASB reactor significantly reduced the wastewater strength. It has shown the removal rates of turbidity, TSS, BOD, COD, TOC, NH4-N, nitrates and phosphates as 71%, 80%, 79%, 71%, 63%, 70%, 53% and 42%, respectively. Whereas the DAF unit has shown the removal rates as 53%, 47%,42%, 55%, 48%, 36%, 49% and 77% respectively. The treatment of wastewater by UASB and DAF units has not been shown any effect on the levels of heavy metals. During UASB and DAF treatments the heavy metal followed an irregular trend with slight variation in levels. Further, the efficiency of UASB reactor has shown efficient removal rates for microbiological properties like TVC, TCC, total count of Salmonella spp., Shigella spp. S. faecalis, S. aureus, B .cereus and L. monocytogenes as 78%, 87.4%, 92%, 87%, 85%, 76%, 78% and 72% respectively, whereas the DAF unit has shown these removal rates as 82.6%, 85%, 64%, 70%, 72%, 58% and 72% respectively. The effect of the treated and raw wastewater from slaughterhouses on some physical and chemical properties of the soils was investigated. The results obtained revealed that both raw and treated slaughterhouse wastewater decreased the soil pH to 6.95 and to 6.88 from pH of 7.34 (control soil) and increased the electrical conductivity of soils to 1.59 mS/cm and to 1.48 mS/cm from 0.62 mS/cm (control soil). The raw and treated wastewaters also increased the organic carbon of control soils (1.75%) to 5.29% and to 6.24% and the available nitrogen of control soils (339.04 kg/ha) to 752.64 kg/ha and to 815.36 kg/ha. The heavy metal (Pb, Ni, and Cd) levels in soils polluted with raw and treated wastewaters are significantly increased to 4.11 mg/L, 0.38 mg/L and 0.54 mg/L when compared to that of control soils such as 11.4 mg/L, 0.05 mg/L and 0.13 mg/L, respectively.
Open Access
STUDIES ON THE INCIDENCE OF SHIGELLA IN LIVESTOCK PRODUCTS AND ENVIRONMENTAL SAMPLES
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2010-07) DURGA RAMA DEVI, N; KRISHNAIAH, N(MAJOR); MADHAVA RAO, T; ANJANEYULU, Y
ABSTRACT: Out of 60 samples each of stools, drinking water, farm water, beef, chicken, egg and fish, Shigella spp. were found in 12 ,4, 15, 4, 3, 4, 3 and 5 respectively. Out of 12 positive stool samples for Shigella, 8 were S. flexneri, 3 were S. sonnei and 1 was S. dysenteriae. Out of 7 and 15 positive samples of drinking and farm water, S. flexneri was found in 4 and 10, S. sonnei 2 and 3 respectively and S. dysenteriae was one in each. Out of 4 positive samples each of beef and milk. S. flexneri was found in 3 and 2 S. sonnei one each and S. dysenteriae was 1 in milk and not found in beef. Out of 3, 3 and 5 positive samples of chicken, eggs and fish, S. flexneri was found in 2, 2 and 4 respectively. S. sonnei was found in one each and S. dysenteriae was not found. Out of 480 total different samples 53 were positive for Shigella spp., out of which 35 were S. flexeneri, 13 were S. sonnei and 4 were S. dysenteriae. For isolation and identification of Shigella spp., BHI broth enrichment was good for S. flexneri and SB broth was good for S. sonnei and S. dysenteriae. The isolation with or without enrichment and plating has shown that XLD and HEA media are superior over DCA, MAC and SS agar for S. flexneri and S. sonnei, where as for S. dysenteriae XLD, DCA, HEA and MAC has given moderate growth, with no growth on SS medium. In general, all the three enrichment broths have given better growth compared to direct plating. S. flexneri was highly sensitive to Kanamycin (94.28%) and Nalidixic acid (84.7%) and highly resistant to Gentamycin (97%) and Ampicillin (91.4%), where as S.sonnei was highly sensitive to Kanamycin (92.3%), Ampicillin (84.6%), Chloramphenical (69.23%) and highly resistant to Gentamycin (92.3%), Nalidixic acid (76.9%) and Cotrimaxozole (69.23%). S.dysenteriae was sensitive only to Kanamycin (75%) and Ciprofloxacin (25%) and resistant to Ampicillin, Gentamycin, Tetracycline , Nalidixic acid , Erythromycin, Chloramphenical and Cotrimoxazole (75%).
Open Access
DETECTION OF VIBRIO CHOLERAE IN FOODS BY PCR TECHNIQUE
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2010-06) MAHESHWARI, MANI; KRISHNAIAH, N(MAJOR); MADHAVA RAO, T; ANJANEYULU, Y
ABSTRACT: The present study was undertaken to standardize PCR assay for detection of Vibrio cholerae, its serogroups (O1 and O139) and cholera toxin from foods and compare its efficacy with conventional cultural methods. Primers derived from ompW gene, rfbO1 and rfbO139 genes and ctxA gene were used which gave specific amplification products of 304, 192, 449, 302 bp for V.cholerae, its serogroups (O1 and O139) and cholera toxin respectively. To determine their suitability to PCR, four different template preparation methods viz. genomic DNA extraction, heat lysis, lysis buffers-1 and 2 were compared, of which heat lysis was found to be efficient and convenient. The specificity for V.cholerae, its serogroups (O1 and O139) and cholera toxin was tested using primers from ompW, rfbO1, rfbO139 and ctxA genes with V.cholerae and nine other non-V.cholerae strains, which gave specific products at 304, 192, 449 and 302 bp for V.cholerae, its serogroups and cholera toxin respectively. The minimum detection level was 2.5 cfu for V.cholerae, its serogroups and cholera toxin. Evaluation of two non-selective broths, i.e Luria Broth (LB) and Tryptic Soy Broth (TSB) and two selective broths, i.e Alkaline Peptone Water Broth (APW) and Salt Polymyxin Broth (SPB) for PCR compatibility, it was revealed that APW was superior followed by SPB, LB and TSB. Spiking studies were carried out by inoculating with pure culture of V.cholerae in selective enrichments broths (8h and 18h), which revealed that earliest detection time for V.cholerae by PCR was 18h and APW was the most suited selective broth for PCR assay. Screening of 245 naturally contaminated samples (35 each of water, fish, crab, shrimp, meat, milk and clinical stool samples) revealed 80 positive for V.cholerae (water-19, fish-16, crab-20, shrimp-6, meat-4, milk-3, and clinical stool samplres-12) out of which 45 belonged to O1 serogroup (water 11, fish-9, crab-12, shrimp-3, meat-2, milk-1 and clinical stool samples-7), 25 belonged to O139 (water-6, fish-5, crab-6, shrimp-2, meat-1, milk-1 and clinical stool samples-4). Out of 45 and 25 positive samples for O1 and O139, 35 and 20 were positive for cholera toxin respectively.
Open Access
STUDIES ON ISOLATION AND MOLECULAR CHARACTERIZATION OF LISTERIA MONOCYTOGENES OF ANIMAL ORIGIN AND THEIR PUBLIC HEALTH SIGNIFICANCE
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2017-01) PRATAPA REDDY, VENNA; SRINIVASA RAO, T(MAJOR); BINDU KIRANMAYI, Ch.; RAMANI PUSHPA, R.N
ABSTRACT: The present study was undertaken for isolation, identification and molecular characterization of L. monocytogenes of animal origin and to assess their extent of public health significance. Out of 502 samples collected from foods of animal origin, clinical and human diarrheal samples, 97 (19.32%) were found positive for Listeria spp. of which 39 (7.76%, 39/502) isolates were identified as L. monocytogenes after morphological and biochemical confirmation. Among 39 isolates, 24 (7.27%, 24/330) were from foods of animal origin and 15 (13.15%, 15/114) were from clinical samples and none of the human diarrheic samples revealed L. monocytogenes. Among foods of animal origin, highest prevalence was noticed in badam milk with 20% followed by ice cream 13.3%, 10% in both butter and chicken and 8% in both raw milk and cheese and among clinical samples highest prevalence was found in buffalo abortions (20%, 9/45) followed by 10% (1/10 and 5/50) each from sheep abortions and vaginal swabs of infertility cases of buffaloes. All L. monocytogenes isolates were subjected to mPCR detection of virulence genes hlyA, iapA, actA, prfA and plcA using specific primers. Results revealed that hlyA and iapA found in all tested isolates, actA in 33 (84.61%) isolates and no isolate was found to contain prfA and plcA genes. Serotyping of L. monocytogenes revealed majority of isolates belonged to serogroup 4b, 4d, 4e (79.48%, 31/39) and 8 (20.51%, 8/39) isolates (2 each from raw milk and butter and 4 from cheese) belonged to serogroup 1/2b and 3b. L. monocytogenes isolates from various sources showed 100% resistance towards nalidixic acid followed by erythromycin (94%), fosfomycin (15%) and chloramphenicol (7%). All the isolates showed 100% sensitivity towards ampicillin, tetracycline, vancomycin, norfloxacin and a high number showed sensitivity to cefotaxime, cefotaxime/clavulanic acid, ceftriaxone, ceftriaxone+sulbactam and ceftazidime (97%) followed by ceftazidime+clavulanic acid (94%) and streptomycin (74%). Multidrug resistant strains of L. monocytogenes along with predominant serogroup 4b, 4d, 4e in both foods and clinical samples may pose a threat to public health indicating possibility of transfer of the pathogen from animals to foods of animal origin and thereafter to the food chain.
Open Access
STUDIES ON Arcobacter SPECIES OF ANIMAL AND HUMAN ORIGIN
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-12) SOMA SEKHAR, M; SRINIVASA RAO, T(MAJOR); BINDU KIRANMAYI, Ch.; SUBRAMANYAM, K.V
ABSTRACT: Arcobacter is an emerging foodborne pathogen having worldwide public health concern. The present study was undertaken to characterize Arcobacter species of animal and human origin based on cultural isolation, PCR detection, antibiogram, virulence profiles and genetic diversity. A total of 510 samples comprising faecal swabs of livestock (245), raw foods of animal origin (180) and human stools (85) were examined. Overall prevalence of Arcobacter species was found to be 11.7% (60/510) by genus-specific PCR. Among faecal samples of healthy livestock, pigs (23.3%) revealed the highest prevalence, followed by chicken (16.6%), turkey (15.0%), cattle (10.0%), duck (10.0%) and sheep (6.6%). Among foods of animal origin, pork samples (15.0%) revealed the highest prevalence, followed by chicken meat (12.5%), milk (10.0%) and mutton (7.5%). Among human stool samples, farm workers revealed highest prevalence (13.3%, 4/30) followed by veterinary students (8.0%, 2/25) and diarrhoeic humans (6.66%, 2/30). Out of 60 genus-specific PCR positive samples, multiplex-PCR assay enabled detection of A. butzleri (16/60), A. cryaerophilus (10/60), A. skirrowii (10/60), both A. cryaerophilus and A. skirrowii (9/60), both A. butzleri and A. skirrowii (5/60), both A. butzleri and A. cryaerophilus (4/60) and all the three Arcobacter species (6/60). Using cultural methods, a total of 41 (8.03%) Arcobacter isolates were recovered. Multiplex-PCR assay of 41 Arcobacter isolates enabled detection of A. butzleri (16/41), A. cryaerophilus (13/41) and A. skirrowii (12/41). All the A. butzleri isolates carried all six putative virulence genes. For A. cryaerophilus and A. skirrowii, the cadF gene was detected in 61.5% and 50%, ciaB in 84.6% and 91.6%, cj1349 in 76.9% and 83.3%, mviN in 76.9% and 66.6%, pldA in 61.5% and 50% and tlyA in 61.5% and 50% of isolates, respectively. Antibiogram of Arcobacter isolates revealed sensitivity to tetracycline (100%), ciprofloxacin (95.1%) and gentamicin (82.9%). Higher resistance was observed for vancomycin (100%), co-trimoxazole (87.8%), chloramphenicol (78%) and erythromycin (51.2%) with remarkable intermediate resistance against kanamycin (68.2%), nalidixic acid (53.6%) and cefoxitin (43.9%). Resistance to β-lactam antibiotics like aztreonam (65.8%), cefotaxime (63.4%), ceftazidime (58.5%) and ceftriaxone (53.6%) was detected. An ESBL phenotype was confirmed in a total of 15 Arcobacter isolates. β-lactamase genes were detected in 63.4% of Arcobacter isolates, with blaTEM being the predominant gene detected (51.2%, 21/41) followed by blaCTX-M group 1 (36.5%, 15/41), blaAmpC (29.2%, 12/41), blaOXA (29.2%, 12/41), blaSHV (14.6%, 6/41) and blaCTX-M group 2 (14.6%, 6/41). CTX-M beta-lactamase was found to be the most frequent mechanism of ESBL resistance in Arcobacter isolates. ERIC PCR and rep-PCR analysis revealed a greater degree of heterogeneity among Arcobacter isolates from different sources. ERIC-PCR genotyping distinguished 14, 13 and 12 genotypes among 16, 13 and 12 isolates of A. butzleri, A. Cryaerophilus and A. skirrowii, respectively. Rep-PCR genotyping distinguished 15, 12 and 11 genotypes among 16, 13 and 12 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively. The discriminatory power ERIC-PCR and rep-PCR for Arcobacter isolates was found to be 0.997 and 0.996, respectively. Genetic diversity of A. butzleri, A. cryaerophilus and A. skirrowii recovered from animals, foods of animal origin and humans in India adds to the heterogeneity reports among Arcobacter species world-wide, supporting diversity among same species. Close clustering between Arcobacters of animal and human origin are indicative of probable zoonotic significance.
Open Access
MOLECULAR CHARACTERIZATION OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS FROM ANIMALS AND ITS PUBLIC HEALTH SIGNIFICANCE
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2014-06) SIVA SANKARA REDDY, M; JAGADEESH BABU, A(MAJOR); SREENIVASULU, D; SRINIVASA RAO, T
ABSTRACT: The present study was undertaken to isolate the Staphylococcus aureus from domestic animals viz: goat, pigs, dogs and from humans who were in close contact with animals, to study their antibiotic sensitivity/resistant patterns, the molecular characterization of species specific Staphylococcus aureus and their methicillin resistance by using the primers derived from nuc and mecA genes which gave specific amplification product of 270 bp and 533 bp for Staphylococcus aureus and for its methicillin resistance respectively. The nasal swabs collected from the domestic animals viz: goat, pigs, dogs and from humans who were in close association with those animals were used for isolation of Staphylococcus aureus. Out of 455 samples collected from different sources, 398 samples were positive for staphylococcus on Gram’s staining and developed purple coloured cocci in clusters. Positive methyl red, voges-proskauer, catalase, urease, nitrate reduction test and negative indole, citrate utilization and oxidase tests confirmed the presence of S. aureus. Among the 398 isolates 122(goats-61, pigs-23, dogs-21, humans-17) isolates were confirmed as pathogenic Staphylococcus aureus by a positive coagulase test. DNase test revealed the presence of blue to purple coloured colonies with clear zones around the colonies which are the characteristic colonies on DNase agar. On blood agar plates, the isolates were produced β haemolysis. A pannel of 11 antibiotic discs were tested by using the standard disc diffusion method. Among the 398 isolates all of them were not resistant to vancomycin (0%). Maximum resistance was observed for ciprofloxacin (56.78%), followed by ampicillin (48.24%), penicillin (43.96%), gentamycin (42.21%), streptomycin (37.68%), tetracycline (28.89%), erythromycin (23.86%), cephoxitin (23.11%), oxacillin (17.83%) and cephalothin (14.82%). For the detection of MRSA all the isolates were streaked on Hi-crome Me Re Sa agar plates and the results revealed that 5 isolates were grown as bluish-green coloured colonies. Two sets of primers derived from nuc gene and mecA genes were used for the identification of Staphylococcus aureus and its methicillin resistance respectively for the PCR assay. Initial denaturation at 940C for 3 minutes followed by 35 cycles each of denaturation at 940C for 30 sec, annealing at 550C for 35 sec and extension at 720C for 1 min with a final extension period of 10 minutes at 720C was found to be optimum for obtaining the desired PCR amplification of 270bp from nuc gene of Staphylococcus aureus and 533 bp from mecA gene of Methicillin resistant Staphylococcus aureus. Electrophoretic analysis of the PCR product revealed the specific amplification of a 270 bp and 533 bp fragmens without the presence of any spurious product. Out of 151 goat samples 115 (76.15%) were positive for Staphylococcus aureus by cultural methods and 112 (74.1%) were positive by PCR method. Out of 112 PCR positives none were found positive for methicillin resistant Staphylococcus aureus by PCR. Out of 102 pig samples 96 (94.1%) were positive for Staphylococcus aureus by both cultural as well as PCR method and one isolate was found positive for mecA by PCR which accounts to 0.98% over the total number of samples and 1.04% over the positive samples for Staphylococcus aureus by PCR. Out of 140 human samples 129 (92.1%) were positive for Staphylococcus aureus by cultural methods and 128 (91.4%) were positive by PCR method and two isolates were found positive for mecA by PCR which accounts to 1.42% over the total number of samples and 1.56% over the positive samples for Staphylococcus aureus by PCR. Out of 62 dog samples 58 (93.5%) were positive for Staphylococcus aureus by both cultural as well as PCR methods and two isolates were found positive for mecA by PCR which accounts to 3.22% over the total number of samples and 3.44% over the positive samples for Staphylococcus aureus by PCR.
Open Access
STUDIES ON Arcobacter SPECIES OF ANIMAL AND HUMAN ORIGIN
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI – 517 502. (A.P) INDIA, 2016-12) SOMA SEKHAR M, M; SRINIVASA RAO, T(Major); BINDU KIRANMAYI, Ch; SUBRAMANYAM, K.V
ABSTRACT: Arcobacter is an emerging foodborne pathogen having worldwide public health concern. The present study was undertaken to characterize Arcobacter species of animal and human origin based on cultural isolation, PCR detection, antibiogram, virulence profiles and genetic diversity. A total of 510 samples comprising faecal swabs of livestock (245), raw foods of animal origin (180) and human stools (85) were examined. Overall prevalence of Arcobacter species was found to be 11.7% (60/510) by genus-specific PCR. Among faecal samples of healthy livestock, pigs (23.3%) revealed the highest prevalence, followed by chicken (16.6%), turkey (15.0%), cattle (10.0%), duck (10.0%) and sheep (6.6%). Among foods of animal origin, pork samples (15.0%) revealed the highest prevalence, followed by chicken meat (12.5%), milk (10.0%) and mutton (7.5%). Among human stool samples, farm workers revealed highest prevalence (13.3%, 4/30) followed by veterinary students (8.0%, 2/25) and diarrhoeic humans (6.66%, 2/30). Out of 60 genus-specific PCR positive samples, multiplex-PCR assay enabled detection of A. butzleri (16/60), A. cryaerophilus (10/60), A. skirrowii (10/60), both A. cryaerophilus and A. skirrowii (9/60), both A. butzleri and A. skirrowii (5/60), both A. butzleri and A. cryaerophilus (4/60) and all the three Arcobacter species (6/60). Using cultural methods, a total of 41 (8.03%) Arcobacter isolates were recovered. Multiplex-PCR assay of 41 Arcobacter isolates enabled detection of A. butzleri (16/41), A. cryaerophilus (13/41) and A. skirrowii (12/41). All the A. butzleri isolates carried all six putative virulence genes. For A. cryaerophilus and A. skirrowii, the cadF gene was detected in 61.5% and 50%, ciaB in 84.6% and 91.6%, cj1349 in 76.9% and 83.3%, mviN in 76.9% and 66.6%, pldA in 61.5% and 50% and tlyA in 61.5% and 50% of isolates, respectively. Antibiogram of Arcobacter isolates revealed sensitivity to tetracycline (100%), ciprofloxacin (95.1%) and gentamicin (82.9%). Higher resistance was observed for vancomycin (100%), co-trimoxazole (87.8%), chloramphenicol (78%) and erythromycin (51.2%) with remarkable intermediate resistance against kanamycin (68.2%), nalidixic acid (53.6%) and cefoxitin (43.9%). Resistance to β-lactam antibiotics like aztreonam (65.8%), cefotaxime (63.4%), ceftazidime (58.5%) and ceftriaxone (53.6%) was detected. An ESBL phenotype was confirmed in a total of 15 Arcobacter isolates. β-lactamase genes were detected in 63.4% of Arcobacter isolates, with blaTEM being the predominant gene detected (51.2%, 21/41) followed by blaCTX-M group 1 (36.5%, 15/41), blaAmpC (29.2%, 12/41), blaOXA (29.2%, 12/41), blaSHV (14.6%, 6/41) and blaCTX-M group 2 (14.6%, 6/41). CTX-M beta-lactamase was found to be the most frequent mechanism of ESBL resistance in Arcobacter isolates. ERIC PCR and rep-PCR analysis revealed a greater degree of heterogeneity among Arcobacter isolates from different sources. ERIC-PCR genotyping distinguished 14, 13 and 12 genotypes among 16, 13 and 12 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively. Rep-PCR genotyping distinguished 15, 12 and 11 genotypes among 16, 13 and 12 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively. The discriminatory power ERIC-PCR and rep-PCR for Arcobacter isolates was found to be 0.997 and 0.996, respectively. Genetic diversity of A. butzleri, A. cryaerophilus and A. skirrowii recovered from animals, foods of animal origin and humans in India adds to the heterogeneity reports among Arcobacter species world-wide, supporting diversity among same species. Close clustering between Arcobacters of animal and human origin are indicative of probable zoonotic significance.

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