Rapid detection of Mycobacterium bovis in bovine cytological smears and tissue sections by peptide nucleic acid fluorescence in-situ hybridization.

BACKGROUND: Bovine tuberculosis is the leading cause of death in cattle and other species worldwide. Quick and precise identification of mycobacteria is critical to control the occurrence of tuberculosis in cattle. METHODS: We developed a fluorescent peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) approach to detect Mycobacterium bovis and Mycobacterium avium in cytological smears and tissue sections of bovines suspected of having tuberculosis. PNA-FISH was conducted on smears of lung and lymph node tissues. Standard bovine mycobacterial cultures were used to standardize the probes using 50 % formamide for M. bovis and 30 % formamide for M. avium. M. bovis probe (MTBCcy3), which was standardized at hybridization conditions of (55 °C and 40 % formamide) concentrations, was positive in all cytological smears. RESULTS: Four out of twenty five samples tested positive in tissue sections observed as a bright red fluorescence with a cy3 filter (MTBC probe). No results were observed with (MAVTAMRA) probe for M. avium which was standardized at hybridization conditions of (55 °C and 30 % formamide). No fluorescence was observed in the control tissue sections. Additionally, the results were juxtaposed with those of other commonly used detection methods such as immunohistochemistry and Polymerase Chain Reaction (PCR) by targeting the esxA gene. None of the samples tested positive for M. avium infection. CONCLUSION: PNA-FISH can be used to obtain cytological impression smears and tissue sections. When compared to PCR it consumes less time in the diagnosis of bovine tuberculosis in post mortem cases.

Prevalence and antimicrobial resistance of food safety related Vibrio species in inland saline water shrimp culture farms / Prevalencia y resistencia a los antimicrobianos de especies de Vibrio relacionadas con la inocuidad de los alimentos en granjas de cultivo de camarones de aguas saladas continentales

This study evaluated the potential pathogenicity and antimicrobial resistance (AMR) of Vibrio species isolated from inland saline shrimp culture farms. Out of 200 Vibrio isolates obtained from 166 shrimp/water samples, 105 isolates were identified as V. parahaemolyticus and 31 isolates were identified as V. alginolyticus and V. cholerae, respectively. During PCR screening of virulence-associated genes, the presence of the tlh gene was confirmed in 70 and 19 isolates of V. parahaemolyticus and V. alginolyticus, respectively. Besides, 10 isolates of V. parahaemolyticus were also found positive for trh gene. During antibiotic susceptibility testing (AST), very high resistance to cefotaxime (93.0%), amoxiclav (90.3%), ampicillin (88.2%), and ceftazidime (73.7%) was observed in all Vibrio species. Multiple antibiotic resistance (MAR) index values of Vibrio isolates ranged from 0.00 to 0.75, with 90.1% of isolates showing resistance to ≥ 3 antibiotics. The AST and MAR patterns did not significantly vary sample-wise or Vibrio species-wise. During the minimum inhibitory concentration (MIC) testing of various antibiotics against Vibrio isolates, the highest MIC values were recorded for amoxiclav followed by kanamycin. These results indicated that multi-drug resistant Vibrio species could act as the reservoirs of antibiotic resistance genes in the shrimp culture environment. The limited host range of 12 previously isolated V. parahaemolyticus phages against V. parahaemolyticus isolates from this study indicated that multiple strains of V. parahaemolyticus were prevalent in inland saline shrimp culture farms. The findings of the current study emphasize that routine monitoring of emerging aquaculture areas is critical for AMR pathogen risk assessment.(AU)

A study on the occurrence of Burkholderia cepacia complex in ultrasound gels used in different veterinary clinical settings in India.

Burkholderia cepacia complex (Bcc) organisms are emerging multidrug-resistant pathogens. They are opportunistic and cause severe diseases in humans that may result in fatal outcomes. They are mainly reported as nosocomial pathogens, and transmission often occurs through contaminated pharmaceutical products. From 1993 to 2019, 14 Bcc outbreaks caused by contaminated ultrasound gels (USGs) have been reported in several countries, including India. We screened a total of 63 samples of USGs from various veterinary and human clinical care centers across 17 states of India and isolated 32 Bcc strains of Burkholderia cenocepacia (46.8%), B. cepacia (31.3%), B. pseudomultivorans (18.8%) and B. contaminans (3.1%) species. Some isolates were co-existent in a single ultrasound gel sample. The isolation from unopened gel bottles revealed the intrinsic contamination from manufacturing sites. The MALDI-TOF analysis to identify the Bcc at the species level was supported by the partial sequencing of the recA gene for accurate species identification. The phylogenetic analysis revealed that isolates shared clades with human clinical isolates, which is an important situation because of the possible infections of Bcc by USGs both in humans and animals. The pulsed field gel electrophoresis (PFGE) typing identified the genetic variation among the Bcc isolates present in the USGs. The findings indicated USGs as the potential source of Bcc species.

Prevalence and antimicrobial resistance of food safety related Vibrio species in inland saline water shrimp culture farms.

This study evaluated the potential pathogenicity and antimicrobial resistance (AMR) of Vibrio species isolated from inland saline shrimp culture farms. Out of 200 Vibrio isolates obtained from 166 shrimp/water samples, 105 isolates were identified as V. parahaemolyticus and 31 isolates were identified as V. alginolyticus and V. cholerae, respectively. During PCR screening of virulence-associated genes, the presence of the tlh gene was confirmed in 70 and 19 isolates of V. parahaemolyticus and V. alginolyticus, respectively. Besides, 10 isolates of V. parahaemolyticus were also found positive for trh gene. During antibiotic susceptibility testing (AST), very high resistance to cefotaxime (93.0%), amoxiclav (90.3%), ampicillin (88.2%), and ceftazidime (73.7%) was observed in all Vibrio species. Multiple antibiotic resistance (MAR) index values of Vibrio isolates ranged from 0.00 to 0.75, with 90.1% of isolates showing resistance to ≥ 3 antibiotics. The AST and MAR patterns did not significantly vary sample-wise or Vibrio species-wise. During the minimum inhibitory concentration (MIC) testing of various antibiotics against Vibrio isolates, the highest MIC values were recorded for amoxiclav followed by kanamycin. These results indicated that multi-drug resistant Vibrio species could act as the reservoirs of antibiotic resistance genes in the shrimp culture environment. The limited host range of 12 previously isolated V. parahaemolyticus phages against V. parahaemolyticus isolates from this study indicated that multiple strains of V. parahaemolyticus were prevalent in inland saline shrimp culture farms. The findings of the current study emphasize that routine monitoring of emerging aquaculture areas is critical for AMR pathogen risk assessment.

Complete genome sequencing and characterization of single-stranded DNA Vibrio parahaemolyticus phage from inland saline aquaculture environment.

Despite their evolutionary, molecular biology and biotechnological significance, relatively fewer numbers of single-stranded DNA (ssDNA) filamentous phages belonging to the family Inoviridae have been discovered and characterized to date. The present study focused on genome sequencing and characterization of an ssDNA Vibrio parahaemolyticus phage V5 previously isolated from an inland saline shrimp culture farm. The complete circular genome of phage V5 consisted of 6658 bp with GC content of 43.7%. During BLASTn analysis, only 36% of phage V5 genome matched with other Vibrio phage genomes in the NCBI database with a sequence identity value of 79%. During the phylogenetic analysis, phage V5 formed a separate branch in the minor clade. These features indicate the novel nature of the phage V5 genome. Among 10 predicted open reading frames (ORFs) in the phage V5 genome, 6 encoded for the proteins of known biological functions, whereas the rest were classified as hypotheticals. Proteins involved in replication and structural assembly were encoded by the phage genome. However, the absence of genes encoding for DNA/RNA polymerases and tRNAs signified that phage V5 is dependent on the host`s molecular machinery for its propagation. As per our knowledge, this is the first study describing the novel genome sequence of an ssDNA V. parahaemolyticus phage from the inland saline environment.

Prevalence and molecular characterization of canine parvovirus.

BACKGROUND AND AIM: Canine parvovirus (CPV) belonging to family Parvoviridae causes hemorrhagic gastroenteritis in dogs and heavy mortality in young dogs. The virus has three structural (VP1, VP2 and VP3) and two non-structural proteins (NS1 and NS2), VP2 being highly immunogenic. This study aims to study molecular epidemiology of CPV by sequence analysis of VP2 gene to determine the prevailing antigenic type(s) in the northern regions of India. MATERIALS AND METHODS: A total of 118 rectal swabs collected from dogs exhibiting clinical signs of CPV infection were processed for the isolation of DNA and subjected to polymerase chain reaction (PCR) and nested PCR (NPCR). A total of 13 NPCR products selected randomly were subjected to sequence analysis of VP2 gene. RESULTS: The percent positivity of CPV was found 28% and 70% by PCR and NPCR, respectively. Dogs with vaccination history against CPV too were found positive with a percent positivity of 24.10%. Gene sequencing and phylogenetic analysis of VP2 gene from these isolates revealed that most samples formed a clade with CPV-2a isolates. CONCLUSION: Sequence analysis and phylogenetic analysis of VP2 gene in the studied regions of northern India revealed that CPV-2a was the most prevalent antigenic type.

Evaluation of adenosine deaminase activity in serum of cattle and buffaloes in the diagnosis of bovine tuberculosis.

BACKGROUND AND AIM: Bovine tuberculosis (bTB) is a chronic bacterial disease of cattle caused by Mycobacterium bovis. bTB causes severe economic losses resulting from livestock deaths, chronic disease, and trade restrictions. Determination of serum levels of adenosine deaminase (ADA), an enzyme produced by monocytes/macrophages and lymphocytes, has been used in the diagnosis of human TB. This study aimed to evaluate the role of ADA enzyme activity in the diagnosis of bTB. MATERIALS AND METHODS: In this study, a total of 100 animals (cattle and buffaloes) were screened for bTB by comparative intradermal tuberculin test (CITT) and interferon-γ (IFN-γ) test and in serum samples obtained from 100 screened animals, ADA seric activity was evaluated using ADA-MTB kit procured from Tulip Diagnostics. RESULTS: A total of 18 animals were positive TB reactors by CITT, 8 were positive by IFN-γ, and 4 animals were positive by both CITT and IFN-γ. The average ADA value of bTB-positive animals either by CITT, IFN-γ, or both CITT and IFN-γ was 12.55 U/L, 14.8 U/L, and 18.36 U/L, respectively, in CID negative, it was 10.57 U/L and in IFN-γ negative, it was 10.59 U/L. CONCLUSION: The average ADA value of bTB-positive animals positive either by CITT, IFN-γ, or both CITT and IFN-γ was more than the average ADA value in animals negative for bTB by either of the tests.

ISMap02 element targeted nested polymerase chain in the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples of cattle and buffaloes.

BACKGROUND AND AIM: Johne's disease is chronic granulomatous enteritis which affects ruminants. There are many diagnostic approaches for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) of which molecular detection methods using various elements are less time consuming and more accurate. The present study was conducted using ISMap02 element for nested polymerase chain reaction (nPCR) based detection of MAP in fecal samples. The aim was to test the sensitivity and specificity of the ISMap02 element and also to use this element for the detection of MAP in fecal samples of cattle and buffaloes. MATERIALS AND METHODS: A total of 211 fecal samples of cattle and buffaloes from different herds around Ludhiana aged between 2 and 13 years were collected, and DNA extraction was done from these samples. The nPCR was carried out for the detection of MAP in fecal samples. RESULTS: The ISMap02 element was specific for the detection of MAP only and showed a sensitivity of detection of 7.6 fg/µL of the standard genomic DNA. Among the 211 fecal samples of cattle and buffaloes tested for the ISMap02 element, 18 samples (8.5%) were positive for MAP. CONCLUSION: The ISMap02 element is specific and sensitive for the detection of MAP in various samples, and when used in nPCR format, it can increase the sensitivity of detection.

Antimicrobial Resistance: Its Surveillance, Impact, and Alternative Management Strategies in Dairy Animals.

Antimicrobial resistance (AMR), one among the most common priority areas identified by both national and international agencies, is mushrooming as a silent pandemic. The advancement in public health care through introduction of antibiotics against infectious agents is now being threatened by global development of multidrug-resistant strains. These strains are product of both continuous evolution and un-checked antimicrobial usage (AMU). Though antibiotic application in livestock has largely contributed toward health and productivity, it has also played significant role in evolution of resistant strains. Although, a significant emphasis has been given to AMR in humans, trends in animals, on other hand, are not much emphasized. Dairy farming involves surplus use of antibiotics as prophylactic and growth promoting agents. This non-therapeutic application of antibiotics, their dosage, and withdrawal period needs to be re-evaluated and rationally defined. A dairy animal also poses a serious risk of transmission of resistant strains to humans and environment. Outlining the scope of the problem is necessary for formulating and monitoring an active response to AMR. Effective and commendably connected surveillance programs at multidisciplinary level can contribute to better understand and minimize the emergence of resistance. Besides, it requires a renewed emphasis on investments into research for finding alternate, safe, cost effective, and innovative strategies, parallel to discovery of new antibiotics. Nevertheless, numerous direct or indirect novel approaches based on host-microbial interaction and molecular mechanisms of pathogens are also being developed and corroborated by researchers to combat the threat of resistance. This review places a concerted effort to club the current outline of AMU and AMR in dairy animals; ongoing global surveillance and monitoring programs; its impact at animal human interface; and strategies for combating resistance with an extensive overview on possible alternates to current day antibiotics that could be implemented in livestock sector.

Multiplex real-time PCR for identification of canine parvovirus antigenic types.

Canine parvovirus (CPV) is an important disease causing gastroenteritis and/or haemorrhagic gastroenteritis in dogs. There are four antigenic types of CPV reported worldwide viz. CPV 2, CPV 2a, CPV 2b and CPV 2c. The diagnosis of CPV with the identification of the antigen type responsible remains problematic. In the present study, identification as well as antigenic typing of CPV was done using a de novo multiplex real time PCR to combat the problem of antigenic type identification. From the study it could be concluded that the here developed multiplex real time PCR assay could be used for rapid detection of CPV as well as typing of its three antigenic types.

Comparative mutant prevention concentration and antibacterial activity of fluoroquinolones against Escherichia coli in diarrheic buffalo calves.

Owing to emerging threat of antimicrobial resistance, mutant prevention concentration (MPC) is considered as an important parameter to evaluate the antimicrobials for their capacity to restrict/allow the emergence of resistant mutants. Therefore, MPCs of ciprofloxacin, enrofloxacin, levofloxacin, moxifloxacin, and norfloxacin were determined against Escherichia coli isolates of diarrheic buffalo calves. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were also established. The MICs of ciprofloxacin, enrofloxacin, levofloxacin, moxifloxacin and norfloxacin were 0·009, 0·022, 0·024, 0·028, and 0·036 µg/ml, respectively. The MBCs obtained were very close to the MICs of respective drugs that suggested a bactericidal mode of action of antimicrobials. The MPCs (µg/ml) of ciprofloxacin (4·2×MIC), moxifloxacin (4·8×MIC), and norfloxacin (5·1×MIC) were approximately equal but slightly lower than enrofloxacin (7·6×MIC) and levofloxacin (8·5×MIC) against clinical isolates of E. coli. The MPC data suggested that enrofloxacin has the potential for restricting the selection of E. coli mutants during treatment at appropriate dosing.

Isolation of Canine parvovirus with a view to identify the prevalent serotype on the basis of partial sequence analysis.

AIM: The aim of this study was to isolate Canine parvovirus (CPV) from suspected dogs on madin darby canine kidney (MDCK) cell line and its confirmation by polymerase chain reaction (PCR) and nested PCR (NPCR). Further, VP2 gene of the CPV isolates was amplified and sequenced to determine prevailing antigenic type. MATERIALS AND METHODS: A total of 60 rectal swabs were collected from dogs showing signs of gastroenteritis, processed and subjected to isolation in MDCK cell line. The samples showing cytopathic effects (CPE) were confirmed by PCR and NPCR. These samples were subjected to PCR for amplification of VP2 gene of CPV, sequenced and analyzed to study the prevailing antigenic types of CPV. RESULTS: Out of the 60 samples subjected to isolation in MDCK cell line five samples showed CPE in the form of rounding of cells, clumping of cells and finally detachment of the cells. When these samples and the two commercially available vaccines were subjected to PCR for amplification of VP2 gene, a 1710 bp product was amplified. The sequence analysis revealed that the vaccines belonged to the CPV-2 type and the samples were of CPV-2b type. CONCLUSION: It can be concluded from the present study that out of a total of 60 samples 5 samples exhibited CPE as observed in MDCK cell line. Sequence analysis of the VP2 gene among the samples and vaccine strains revealed that samples belonged to CPV-2b type and vaccines belonging to CPV-2.

Antigenic typing of canine parvovirus using differential PCR.

Canine parvovirus (CPV) is an enteric pathogen causing hemorrhagic enteritis in pups of 3-6 months of age and is mainly transmitted via feco-oral route. In the present study, a total of 85 animals rectal swabs suspected of CPV were tested using a PCR, nested PCR and a newly designed differential PCR. Using PCR 7 (8.23 %) animals were positive whereas 39 (45.88 %) were positive by using nested PCR and 40 (47.05 %) were positive for either one or more than one antigenic types of CPV using differential PCR. Using differential PCR it was found that CPV-2a and CPV-2b were the most prevailing antigenic types. Also it was found that dogs that were vaccinated too yielded positive CPV indicating a possible presence of additional CPV antigenic types. Thus, the primers used in differential PCR can be used in a single PCR reaction to detect various antigenic types of CPV.

Evaluation of vaccine candidate potential of deltaaroA, deltahtrA and deltaaroAdeltahtrA mutants of Salmonella enterica subspecies enterica serovar Abortusequi in guinea pigs.

Salmonella enterica subspecies enterica serovar Abortusequi (S. Abortusequi), a host adapted Salmonella causes abortions, still births and foal mortality in equids. Though known since more than 100 years, it is still a problem in many of the developing countries including India. There is dearth of really good vaccine affording immunity lasting at least for one full gestation. In search of a potential vaccine candidate, three defined deletion mutants (deltaaroA, deltahtrA and deltaaroAdeltahtrA) of S. Abortusequi were tested in guinea pig model for attenuation, safety, immunogenicity, humoral immune response, protective efficacy and persistence in host. The deltahtrA and deltaaroAdeltahtrA mutants were found to be safe on oral inoculation in doses as high as 4.2 x 10(9) cfu/animal. Also through subcutaneous inoculation deltaaroAdeltahtrA mutant did not induce any abortion in pregnant guinea pigs. All the three mutants did not induce any illness or death in 1-2 week-old baby guinea pigs except deltahtrA mutant which caused mortality on intraperitoneal inoculation. Inoculation with mutants protected against challenge and increased breeding efficiency of guinea pigs. After >4.5 months of mutant inoculation, guinea pigs were protected against abortifacient dose of wild type S. Abortusequi and mother guinea pigs also conferred resistance to their babies to the similar challenge. Early humoral immune response of S. Abortusequi mutants was characteristic. Faecal excretion of deltaaroA and htrA mutants was detected up to 45 days of inoculation in guinea pigs while deltaaroAdeltahtrA mutant could not be detected after 21 days of inoculation. The results indicated that the double deletion mutant (deltaaroAdeltahtrA) was the most effective and safe candidate for vaccination against S. Abortusequi through mucosal route of inoculation.

Development of a peptide based latex agglutination assay for serotype identification of foot and mouth disease virus.

Out of 200 serum samples collected from cattle (142) and buffaloes (58) of various ages and sexand subjected to latex agglutination test (LAT) using serotype specific peptides (O, A, Asia 1) and also with peptide for non-structural protein 2B (NSP-2B), 114 (70%) samples were positive against FMDV type 'O', 102 (51%) against serotype 'A' and 104 (52%) against serotype 'Asia 1'. With NSP-2B peptide a total of 71 (35.5%) samples were positive. The results suggest that LAT could be used for the diagnosis of foot and mouth disease virus as it is easy, cheap and effective test.

A single step multiplex PCR for identification of six diarrheagenic E. coli pathotypes and Salmonella.

E. coli is generally a commensal but includes some highly pathogenic strains carrying additional genes in plasmids and/or the chromosome. Based on these genes the pathogenic strains are divided into pathotypes including enteropathogenic (EPEC), enterohemorrhagic (EHEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), enteroinvasive (EIEC) and diffusely adherent (DAEC) E. coli. Here, previously developed multiplex PCR strategies for these strains were integrated into one single step multiplex that differentiates all these E. coli pathotypes, usually based on multiple characteristic PCR products. This multiplex PCR works reliably for colony PCR. Two additional markers were added: one to detect most Enterobacteriacea, which acts as a positive control for successful PCR, and one to distinguish Salmonella. The multiplex correctly classified a set of 45 reference strains by colony PCR and 71 (45+26) strains by in silico PCR. It was then used to interrogate 44 clinical strains from bovine hosts resulting in detection of EAEC and DAEC determinants.

Plasmid profile and drug resistance pattern of zoonotic Salmonella isolates from Indian buffaloes.

BACKGROUND: Buffalo is the major source of animal protein in south-east Asia, including India; therefore, the presence of multiple drug resistance in Salmonella strains of buffalo meat and milk products is of immense public health concern. METHODOLOGY: Forty-six strains of Salmonella enterica subspecies enterica belonging to eight serovars (S. Anatum, 13; S. Weltevreden, 13; S. Rostock, 6; S. Typhimurium, 5; S. Gallinarum, 5; S. Stockholm, 1; S. Dublin, 1; and S. Orion, 2), isolated from buffalo meat and diseased buffaloes were studied for their antibiotic sensitivity and plasmid profile. RESULTS: All except six strains of Salmonella had one or more plasmids. Virulence plasmid of ~35MdA was present in 39 isolates while 19 strains had one to six additional plasmids with molecular weight ranging from 1 Mda > 35 Mda. A plasmid-free S. Anatum strain was resistant to seven drugs including fluoroquinolones, while strains having six to seven plasmids were resistant to fewer antimicrobial drugs. One S. Anatum isolate, resistant to 11 antibiotics, had only one plasmid. Eight serovars of Salmonella could be divided into 28 resistotypes on the basis of antimicrobial sensitivity assay. Most strains were resistant to streptomycin (84.8%) followed by kanamycin (58.7%), gentamicin (52.2%), ampicillin (50%) and oxytetracycline (50%). Few strains were resistant to cefotaxime (2.2%), amoxycillin (2.2%) and newer fluoroquinolones (6.5%). CONCLUSION: Multiple drug resistance was common among Salmonella isolates of buffalo origin, particularly against aminoglycosides, oxytetracycin, ampicillin and cephalexin. Presence of plasmids is not mandatory for occurrence of multiple drug resistance in S. enterica strains.

Drug resistance patterns of Salmonella isolates of equine origin from India.

INTRODUCTION: Salmonellosis is a zoonosis, and one of the most serious public health and animal health problems. METHODOLOGY: We studied 111 isolates of Salmonella belonging to 14 S. enterica subspecies enterica serovars namely S. Abortusequi (45), S. Weltevreden (1), S. Dumfries (2), S. Tshiongwe (1), S.I. 4,5,12:r,i:1,5 (12), S. Bovismorbificans (3), S. Drogana (8), S. Lagos (4), S. Kottbus (3), S. Richmond (1), S. Typhimurium (6), S. Newport (7), S. Paratyphi B var Java (17) and S. Saintpaul (5) isolated from equids in India. RESULTS: All strains studied were resistant to one or more antimicrobials. Strains were resistant to ampicillin (18, 16%), ampicillin+cloxacillin (6, 5%), cefotaxime (6, 5%), chloramphenicol (2, 2%), ciprofloxacin (9, 8%), gentamicin (27, 24%), kanamycin (37, 33%), nalidixic acid (10, 9%), furazolidone (97, 87%), streptomycin (33, 30%), sulphamethoxazole (91, 82%), tetracycline (48, 43%) and trimethoprim (5, 4.5%). Multiple-drug-resistance was detected in 84 (75.7%) isolates and was seen in isolates of all serovars except of S. Kottbus, a rare serovar in India. Salmonella isolates could be classified into 51 resistotypes but 47 (42.3%) isolates belonged to six major resistotypes. Resistotype 13 (resistant to furazolidone, sulphamethoxazole and tetracycline) was most common, followed by resistotype 19 (resistant to nalidixic acid, sulphamethoxazole and tetracycline), resistotype 28 (resistant to furazolidone, streptomycine, sulphamethoxazole and tetracycline) and resistotype 40 (resistant to furazolidone, gentamycin, kanamycin, streptomycine, sulphamethoxazole and tetracycline) including 11, 8, 8 and 7 strains of different serovars, respectively. CONCLUSIONS: This study revealed that antimicrobial drug resistance was common in Salmonella isolates from equids even towards those drugs not used in equids.

Comparative analysis of protein profiles of wild virulent (E156) and aroA-htrA double deletion mutant vaccine strain (S30) of Salmonella enterica subsp. enterica serovar Abortusequi under in vivo and in vitro growth conditions.

In the present study, cell lysate and cell supernatant of the both strains i.e., virulent wild type (E156) and mutant (S30) vaccine strains of Salmonella enterica subspecies enterica serovar Abortusequi (S. Abortusequi), grown under varied in vivo and in vitro conditions were subjected to SDS PAGE and western blotting (using rabbit hyperimmune serum). Variation in growth conditions did not have any significant effect on expression of different proteins. SDS PAGE of E156 and S30 cell lysate (CL) revealed 26 and 28 bands, respectively with 3 prominent proteins of 71, 46 and 42 kDa in cell lysate of E 156 and 4 prominent proteins 71, 65, 46 and 40 kDa in S30 strain. The cell supernatant (CS) from both the strains, subjected to SDS PAGE, exhibited similarity in protein profile among these strains, however three bands of 65, 53 and 40 kDa were more prominent in CS preparation of S30, whereas a 56 kDa protein was prominent in CS of E156. Western blotting of E156 and S30 revealed 3 unique proteins of 65, 53 and 40 kDa present in CS preparation of S30 strains which could be used for differentiation of mutant and wild strains and also in development of test for differentiating vaccinated animals from naturally infected.