Identification of Immunodominant Outer Membrane Proteins of Fusobacterium necrophorum from Severe Ovine Footrot By MALDI-TOF Mass Spectrometry.

The aim of this study was to identify the immunodominant outer membrane proteins (OMPs) of Fusobacterium necrophorum from sheep affected with severe foot-rot. The OMP profile of ovine strains of F. necrophorum has not been well studied. We analyzed the OMP profile of the most frequent lktA variant JKS-F3 of F. necrophorum associated with severe ovine foot-rot with lesion score 4 in order to identify its major immunodominant OMPs. Electrophoretic separations of extracted OMPs showed a number of spots in two-dimensional electrophoretic gels. Two immunoreactive proteins of size around 43 kDa were identified through western blotting using hyperimmune sera raised in rabbits. These two immunogenic OMPs were analyzed by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF/MS) which revealed that these two OMPs of lktA variant JKS-F3 of F. necrophorum showed 46 and 42 percent protein sequence coverage and scores of 125 and 114, respectively, with the reported 43 kDa outer membrane protein of F. necrophorum strain H05, a putative porin having properties similar to pore-forming proteins of anaerobic Gram-negative bacteria. These identified immunogenic OMPs will contribute to our understanding of the pathogenic role played by this organism in ovine foot-rot and could be exploited to devise an effective control strategy through development of an OMP-based recombinant vaccine to mitigate foot-rot in sheep and goats.

Genotyping and antibiotic resistance patterns of Campylobacter fetus subsp.venerealis from cattle farms in India.

Bovine genital campylobacteriosis caused by Campylobacter fetus subsp. venerealis (Cfv) is of considerable economic importance to the cattle industry worldwide. Cfv causes syndrome of temporary infertility in female cattle, early embryonic mortality, aberrant oestrus cycles, delayed conception, abortions and poor calving rates. In the present study, a total of 200 samples obtained from vaginal swabs, cervicovaginal mucous (CVM), preputial washes and semen straws were investigated that were obtained from organized cattle farm of MLRI, Manasbal and unorganized sectors. Out of a total of 200 samples, 49 (47·57%) vaginal swabs, 1 (3·33%) preputial wash and 8 (25%) carried out CVM samples were positive for Cfv, whereas none of the semen straws were positive for Cfv. A total of eleven isolates of Cfv were recovered. PFGE (Pulse field gel electrophoresis) analysis revealed four different pulsotypes (I-IV) circulating in the screened farms. A common pulsotype circulating among farms could not be established. Insertion element (ISCfe1), a 233 bp amplicon of Cfv, was sequenced and the sequence was deposited in GenBank (accession no: MK475662).

Enterotoxin gene profile and molecular epidemiology of Aeromonas species from fish and diverse water sources.

AIMS: This investigation was undertaken to study the prevalence, enterotoxin gene profile and molecular epidemiology of Aeromonads from various sources of water (182) and fish (173). METHODS AND RESULTS: A total of 116 Aeromonas sp. were isolated, of which 48 (26·37%) were from water and 68 (34·62%) were from fish samples collected from retail markets and fish farms. The Aeromonads were recovered from all types of water sources viz. drinking water (13%), surface waters (26%) and fish ponds (69%). The most prevalent species recovered from drinking water was A. hydrophila, from fish ponds it was A. caviae, from surface water sources A. hydrophila and A. caviae were recovered more frequently, and A. hydrophila and A. veronii bv. sobria were isolated predominantly from gills of fish samples. On multiplex PCR analysis for the detection of enterotoxin genes (act, alt, ast), the above mentioned Aeromonas species frequently contained enterotoxin genes, irrespective of their sources. From isolates across all the sources, act (63%) and alt (57%) genes were encountered more frequently than ast (6%). The enterobacterial repetitive intergenic consensus sequences polymerase chain reaction was used for typing of isolates and most of the isolates from water and fish were related, owing to similar ecosystem. CONCLUSION: A wide distribution of enterotoxin genes in Aeromonads from water and fish is a potential public health threat and molecular genotyping can be helpful to study epidemiology of the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: A high proportion of isolates recovered from diverse water sources, particularly potable drinking water and fish samples carried one or more enterotoxin genes thereby indicating a potential pathogenic nature of isolates from these sources. The genetic relatedness was detected amongst many isolates recovered from water sources and fish samples indicating circulation of familiar virulent clones in the aquatic environments.

Isolation and characterization of two lytic bacteriophages against Staphylococcus aureus from India: newer therapeutic agents against Bovine mastitis.

Bovine mastitis causes severe economic losses to dairy farmers. Staphylococcus aureus, is one of the most important pathogen implicated in etiology of clinical and subclinical mastitis in bovines. In view of increasing antimicrobial resistance alternatives to antibiotic therapy are much needed. The present decade has witnessed a renewed interest in phage based therapeutics and diagnostics. The present study, describes isolation and characterization of two lytic phages SAJK-IND and MSP against Staphylococcus aureus having a potential to be used in therapy against mastitis. SAJK-IND and MSP phages belonged to Myoviridae and Podoviridae families, respectively. TEM imaging of the two phages revealed an iscosahedral head. MSP phage has a short non contractile tail. SAJK-IND and MSP have a burst size of 44 ± 3 and 25 ± 5 PFU/ infected cell, respectively. SAJK-IND and MSP phages revealed Ì´ 12 and Ì´16 proteins, respectively on SDS-PAGE analysis. The lytic activity of the phages was specific for Staphylococcus aureus. SAJK-IND revealed 100% lytic activity against several strains of Staphylococcus aureus isolated from mastitis milk samples whereas, MSP had only 40% lytic activity. SAJK-IND phage genome was sequenced, assembled and deposited in Genbank under accession no MG010123.

The detection and prevalence of leukotoxin gene variant strains of Fusobacterium necrophorum in footrot lesions of sheep in Kashmir, India.

The objective of this study was to determine the prevalence and identification of leukotoxin gene, lktA, variant strains of Fusobacterium necrophorum in the footrot lesions of sheep. The detection of F. necrophorum was carried out by PCR targeting the lktA gene fragment and identification of lktA variant strains was done by PCR-single stranded conformational polymorphism (PCR-SSCP) and gene sequencing. Of the 450 swabs collected from footrot lesions of sheep, 117 were lktA-positive for F. necrophorum. Of the 50 swabs collected from apparently asymptomatic sheep, only one was lktA-positive for F. necrophorum. The overall prevalence of F. necrophorum in footrot affected sheep in Kashmir valley was 26%, and ranged from 20 to 34.8%, respectively. PCR-SSCP of lktA gene fragment analysis revealed three lktA variants, designated as JKS-F1/F2/F3, while two samples (1.7%) showed multiple lktA variant strains of F. necrophorum in a single footrot-affected sheep hoof. This appears to be the first report on the presence of more than one lktA variant of F. necrophorum in a footrot lesion of sheep. The JKS-F3 lktA variant was the most frequent (75.4%), followed by JKS-F2 (14.4%) and JKS-F1 (8.4%), respectively. Among the three lktA variants identified, JKS-F3 was detected in 74 (86.0%) samples from severe footrot affected sheep with a lesion score of 4. The data suggest that JKS-F3 is the predominant lktA variant of F. necrophorum and is associated with severe footrot in sheep. Hence, JKS-F3 may be a significant variant contributing to the severity and duration of the disease in sheep.

A multiplex PCR for detection of enterotoxin genes in Aeromonas species isolated from foods of animal origin and human diarrhoeal samples.

AIMS: The present study describes incidence and enterotoxin gene profile of Aeromonas spp. from human diarrhoeal samples (83) and raw meats (171). METHODS AND RESULTS: The samples were screened for isolation of Aeromonads. Aeromonas spp. contaminated raw meats of all kinds under the study and per cent contamination in chicken, mutton and beef was 14·03, 22·89 and 19·35, respectively. Of the 83 diarrhoeal samples from children, 6 (7·22%) were positive for presence of Aeromonas spp. Seven different species of Aeromonas (Aer. hydrophila, Aer. caviae, Aer. veronii bv sobria, Aer. trota, Aer. schubertii, Aer. jandaei and Aer. allosaccharophila) could be identified from foods and from diarrhoeal samples two species (Aer. caviae and Aer. hydrophila) were encountered. Unique primers were designed, and a multiplex PCR was standardized for detection of three enterotoxin genes (act, alt, ast) in the Aeromonas spp. Of the 39 isolates, 35 (89·74%) carried one or more enterotoxin genes: act, alt and ast genes were detected in 30 (76·92%), 31 (79·48%) and 4 (10·25%) isolates, respectively. The enterotoxin genes from a strain recovered from mutton were sequenced and submitted to GenBank and the accession no.s KC687135, KC633828 and KC687134 were provided for alt, ast and act, respectively, by the GenBank. CONCLUSIONS: The occurrence of enterotoxigenic Aeromonads in raw meats and diarrhoeal samples is a public health concern. SIGNIFICANCE AND IMPACT OF THE STUDY: Given the increasing evidence of involvement of Aeromonads in foodborne outbreaks, the standardization of single-step multiplex PCR will be helpful tool for detection of enterotoxin genes in Aeromonas spp.

Non-specificity of primers used for PCR based serogrouping of Dichelobacter nodosus and identification of a novel D. nodosus strain.

The present study records the first case of non-specificity of typing primers developed by Dhungyel et al. A strain of Dichelobacter nodosus (JKS-20G) isolated from ovine footrot in Kashmir, India, showed specificity for serogroup C and G primers. The fimA sequence of the strain turned out to be closer to serogroup G than C. The nucleotide sequence showed maximum homology of 92% with that of serotype G1 strain 238 and 95% with partial sequence available for serotype G2 strain VCS 1004. However, the deduced amino acid sequence of the fimbrial subunit gene of JKS-20G differed from strain 238 by 16 amino acids and by four amino acids from that of partial sequence of strain VCS 1004. This variation indicates towards declaring this isolate as a new serotype (G3) but just insufficient to classify this into a new serogroup. Some of the amino acid substitutions were located within three hypervariable regions a characteristic of different serogroups. However, to ascertain whether this isolate deserves a new serotype status, there is a need to go for antigenic characterisation of this isolate using the tube and cross tube agglutination test.

Diarrhoeagenic Escherichia coli and salmonellae in calves and lambs in Kashmir absence, prevalence and antibiogram.

Polymerase chain reaction assays and culture were used to investigate 728 faecal samples from 404 calves (286 diarrhoeic, 118 healthy) and 324 lambs (230 diarrhoeic, 94 healthy) in Kashmir, India, for the presence of enterotoxigenic Escherichia coli (ETEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC) and salmonellae. Antimicrobial sensitivity patterns were also investigated. In total, 23 ETEC isolates were obtained from the diarrhoeic calves and 12 from diarrhoeic lambs. Most (74%) of the isolates from calves harboured the gene encoding heat-labile enterotoxin I, whereas 75% of the isolates from lambs possessed only the gene encoding for heat-stable enterotoxin a. The ETEC isolates belonged to 20 serogroups, among which serogroups O15 (five isolates) and O8 (four isolates) were the most frequent. Salmonella Typhimurium or S. Enteritidis was identified in three samples from diarrhoeic lambs. The ETEC isolates and the salmonellae showed multidrug resistance. No EAEC or DAEC was detected in any of the samples.

Prevalence of ovine footrot in the tropical climate of southern India and isolation and characterisation of Dichelobacter nodosus.

The present communication records the first determination of the prevalence of footrot in the unexpected situation of the tropical climate of Andhra Pradesh and Tamil Nadu, two states in southern India where the maximum temperature rises to 42 degrees C. In total, 73 outbreaks of footrot in Nellore brown sheep were investigated in 11 districts of Andhra Pradesh and one district of Tamil Nadu during the period March 2009 to March 2011.The overall prevalence of ovine footrot was 15%, with severity scores of 2 to 4 (lesion severity scale 0 to 4). The outbreaks occurred mostly during the rainy season, which is usually from June to December. From a total of 1,050 samples of lesions in naturally infected sheep, 478 (45.5%) were positive for Dichelobacter nodosus. Serogrouping of the isolates revealed six serogroups: A, B, C, E, F and I. Among the positive samples, 448 (93.7%) were a single serogroup and 30 (6.3%) carried a mixed infection with two serogroups. Taking single and mixed infections together, serogroup B was most frequent at 50.4% and was found in all districts, followed by serogroup I in 29.3% of samples, A in 14%, F in 6.7% and C in 5.6%. Serogroup E was detected in only one sample. Serogroups A and F were detected for the first time in India. All of 58 D. nodosus isolates in a sub-sample representing different serogroups were found to be virulent, based on the production of thermostable proteases and the presence of the integrase A gene intA. Thus, the present paper reporting isolation and characterisation of D. nodosus confirms the occurrence of virulent footrot in the tropical climate of southern India.

A clinical trial comparing parenteral oxytetracyline and enrofloxacin on time to recovery in sheep lame with acute or chronic footrot in Kashmir, India.

BACKGROUND: No clinical trials have been conducted in India on the efficacy of parenteral antibacterials to treat footrot in sheep. In addition, there are no studies worldwide on the efficacy of parenteral antibacterials to treat chronic footrot. Sixty two sheep with acute footrot and 30 sheep with chronic footrot from 7 villages in Kashmir, India were recruited into two separate trials. Sheep with acute footrot were allocated to one of three treatments using stratified random sampling: long acting parenteral oxytetracycline, long acting parenteral enrofloxacin and topical application of potassium permanganate solution (a traditional treatment used by sheep farmers in India). In a quasi pre-post intervention design, sheep with chronic footrot that had not responded to treatment with potassium permanaganate were randomly allocated to treatment with one of the two parenteral antibacterials mentioned above. Sheep with acute footrot were treated on day 0 and those with chronic footrot on days 0, 3, 6 and 9. Sheep were monitored for up to 28 days after treatment. Time to recovery from lameness and initial healing of lesions was assessed using Kaplan-Meier survival curves, nonparametric log-rank and Wilcoxon sign-rank tests. RESULTS: There was significant correlation in recovery from lameness and presence of healing lesions in sheep with acute (r = 0.94) or chronic (r = 0.98) footrot. Sheep with acute footrot which were treated with parenteral antibacterials had a significantly more rapid recovery from lameness and had healing lesions (median = 7 days) compared with those treated with topical potassium permanganate solution (less than 50% recovered in 28 days). The median time to recovery in sheep with chronic footrot treated with either antibacterial was 17 days; this was significantly lower than the median of 75 days lame before treatment with antibacterials. The median time to recovery for both acute and chronic footrot increased as the severity of lesions increased. There was no difference in time to recovery by age, body condition score, duration lame, or presence of pus in the foot within acute and chronically affected sheep. CONCLUSIONS: We conclude that use of parenteral antibacterials to treat sheep lame with either acute or chronic footrot in India is highly effective. This is likely to improve welfare and give economic benefits to the farmers.

Putative Virulence Genes and Biofilm Production Among Typical Enteroaggregative Escherichia coli Isolates from Diarrhoeic Children in Kashmir and Andhra Pradesh.

Fifty-eight typical EAEC isolates from children with diarrhoea were examined for HEp-2 cell adherence assay, presence of dispersin (aap), yersiniabactin (irp2), plasmid encoded toxins (pet), Shigella enterotoxin1 (set1A) and cryptic open reading frame (shf) putative virulence genes by polymerase chain reaction as well as for biofilm production. All the isolates showed aggregative adherence pattern on HEp-2 cells. All but five isolates (91.3 %) carried aap gene. While irp2, pet, set1A and shf genes were detected in 68.9, 5.1, 39.6, and 60.3 % isolates, respectively. Thirty-three (64.7 %) isolates out of 51 tested were found to produce biofilm which was found to be significantly associated only with set1A virulence gene (P = 0.025). Highest amount of biofilm was produced by a strain that possessed all the genes studied. Out of 14 isolates in which the most frequent gene combination (aap, irp2 and shf) was observed, only six produced biofilm. It is concluded that there is significant heterogeneity in putative virulence genes of EAEC isolates from diarrhoeic children and biofilm formation is associated with multiple genes.

Identification of two new serotypes within serogroup B of Dichelobacter nodosus.

The present study records the strain-specific molecular typing system for Dichelobacter nodosus (D. nodosus) based on genetic analysis of fimA locus. Based on the study two new serotypes B5 and B6 are reported within the serogroup B. Out of 200 swab samples collected randomly from foot lesions of footrot affected sheep from all the districts of Kashmir, India, 122 (61.0%) detected positive for D. nodosus. Serogroup B was predominantly prevalent in 83.60% of positive samples. Restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) amplified fimA gene of D. nodosus serogroup B revealed only two fingerprint patterns (FP) designated as FP1 and FP2. The FP1 was most prevalent and depicted by 82.35% of the samples with serogroup B while, FP2 was depicted by rest (17.65%) of the samples. Though the FP1 fimA sequence had the homology of 95% to D. nodosus fimA of serotype B4 isolate VRS 54, but there were 14 nucleotide differences and four nucleotide insertions/deletions in the coding sequence between these two strains resulting in eight amino acid substitutions in the fimbrial subunit. Similarly the FP2 fimA showed the sequence homology of 97% with D. nodosus fimA of serotype B2 isolate 183, with 10 nucleotide differences and three nucleotide insertions/deletions between these two sequences. This resulted in six amino acid substitutions, plus an amino acid length variation in the subunit protein. Thus it was presumed that these FP1 and FP2 strains represented new serotypes (B5 and B6, correspondingly) within the B serogroup as the degree of amino acid sequence difference with their nearest homologous strains was much greater than that within a serotype (0-5 amino acid differences), but comparable to that between serotypes (8-15 amino acid differences). This presumption was confirmed by cross tube agglutination test.

Detection & characterization of Shiga toxin producing Escherichia coli (STEC) & enteropathogenic Escherichia coli (EPEC) in poultry birds with diarrhoea.

BACKGROUND & OBJECTIVES: Limited information is available on shiga toxin producing Escherichia coli (STEC) in animals and birds from India. An outbreak of acute diarrhoea in poultry birds at Aizawl, Mizoram was investigated for detection and characterization of STEC and enteropathogenic E. coli (EPEC). METHODS: E. coli was isolated and identified from rectal swabs, intestinal contents, heart blood and spleen of 19 poultry birds that died due to acute diarrhoea during the outbreak. Phenotypic characterization was done by standard bacteriological and biochemical techniques. All the isolates were serotyped based on their somatic antigens. Virulence genes (stx 1, stx 2, eaeA and hlyA) were detected by multiplex PCR assay. RESULTS: A total of 42 E. coli isolates were obtained, of which 24 belonged to 3 serogroups (O64, O89 and O91) and the remaining 18 were untypable (UT). Altogether, 14 (33.33%) isolates carried at least 1 virulence gene, of which 10 (23.81%) and 4 (9.52%) were recorded as STEC and EPEC, respectively. Of the 10 STEC isolates, one carried only stx2 , one carried stx 2 and hlyA, four carried stx1 , stx2 and hlyA, two carried stx 1, eaeA and hlyA genes and two carried stx 1 and eaeA. Of the four EPEC isolates, two carried eaeA and hlyA, one carried only eaeA gene and 1 carried only hlyA gene. INTERPRETATION & CONCLUSIONS: This is the first report on the involvement of STEC in poultry in India.

Determination of prevalence and economic impact of ovine footrot in central Kashmir India with isolation and molecular characterization of Dichelobacter nodosus.

The present study determines the prevalence, economic impact of virulent footrot in central Kashmir, India, along with isolation and molecular characterization of Dichelobacter nodosus (D. nodosus) where so far no such work has been carried out. Over all 12.54% prevalence of footrot was recorded in central Kashmir with highest (15.84%) in district Srinagar, and least (10.89%) in district Budgam, while it was 13.28% in district Ganderbal. Overall economic impact of footrot was estimated to the tune of Rs 15.82 million annually to the sheep farming in central Kashmir. Out of 370 samples collected from footrot lesions of naturally infected sheep, 200 (54.05%) detected D. nodosus positive by polymerase chain reaction (PCR). Out of these, 132 (66.00%) samples carried serogroup B of D. nodosus, five (2.50%) serogroup E, one (0.50%) serogroup I, while, 53 (26.50%) had mixed infection of serogroups B and E, four (2.00%) of serogroups B and I, two (1.00%) of serogroups B and G and the remaining three (1.50%) samples harboured the mixed infection of serogroups B, E and I. Serogroup G was detected for the first time in India. Over all serogroup B was most frequent (97.0%) followed by E (30.5%), while serogoups I (4.0%) and G (1.0%) were least prevalent. A total of 265 D.nodosus strains were isolated out of which 194 (73.20%) were typed as serogroup B, 61 (23.01%) as serogroup E, eight (3.01%) as serogroup I and remaining two (0.75%) belonged to serogroup G. Out of 265 D. nodosus isolates, 164 (61.88%) possessed intA (integrase) gene, thus were considered as virulent strains. Serogroup wise intA gene was found in 121(62.37%) isolates of serogroup B, 36 (59.01%) of E, two (100%) of G and five (62.50%) of I. Out of 20 randomly selected isolates subjected to gelatin gel test, 16 isolates with intA gene produced thermostable protease while four isolates without intA gene revealed the production of thermolabile protease. This indicated a good co-relation between presence of intA gene and gelatin gel test in determination of the D. nodosus virulence. Thus the present investigation suggests the incorporation of serogroups B and E, based on their predominant prevalence, in the formulation of an effective bivalent vaccine to combat footrot in central Kashmir.

Determination of G and P type diversity of group A rotaviruses in faecal samples of diarrhoeic calves in Kashmir, India.

AIMS: To determine the genetic diversity of group A rotaviruses in bovine calves in Kashmir, India. METHODS AND RESULTS: Of 200 diarrhoeic faecal samples collected from calves, aged between 0 and 6 months and screened by polyacrylamide gel electrophoresis (PAGE), 31 were detected positive for group A rotaviruses. On G and P genotyping by reverse transcriptase-polymerase chain reaction (RT-PCR), G10P[11] turned out to be predominant (80·64%) combination followed by G8P[11] (7·7%). One (3·84%) sample carried mixed infection of G8+G10P[11]. Two (7·7%) samples belonged to P[11] genotype, but their G genotype specificity could not be established. This study revealed the ambiguity in RT-PCR typing method. All the samples that turned out to be G10 by Isegawa et al. (1993; Mol Cell Probes7, 277) primers could be amplified by G3 specific primers of Gouvea et al. (1990; J Clin Microbiol32, 1338). However, on homology study of their VP7 gene sequence, the strains turned out to be G10. CONCLUSIONS: Rotavirus is prevalent in diarrhoeic calves in Kashmir, India, and G10P[11] is the predominant genotype in circulation. There is evidence of mixed infection. Even though RT-PCR method is the quick way to type the strains, there is need to generate more sequence data to improve the specificity of typing primers. SIGNIFICANCE AND IMPACT OF THE STUDY: Rotavirus is a significant cause of diarrhoea in calves. RT-PCR typing method needs to be supported by the sequence data, and there is need to re-evaluate the primers used for typing.

Molecular epidemiology and in vitro antimicrobial susceptibility of Salmonella isolated from poultry in Kashmir.

A total of 480 samples, comprising 429 faecal samples from healthy adult birds and 51 tissue samples from dead birds, were collected from four government poultry farms in the Kashmir valley from September 2007 to April 2008. In all, 33 Salmonella isolates were obtained. Of these, 28 (84.85%) isolates were Salmonella Gallinarum, 3 (9.09%) were Salmonella Enteritidis and the remaining 2 (6.06%) were Salmonella Typhimurium. All the isolates harboured the invA, sefA, stn and spvC virulence-specific genes. However, the sopB gene was found in only 90.9% of the isolates. Pulsed-field gel electrophoresis analysis of representative isolates revealed that the majority were related but a few belonged to different clones. The majority of the isolates were resistant to cefpodoxime, nalidixic acid and sulphadiazine and sensitive to chloramphenicol, cefotaxime and tetracycline. Isolation of multidrug-resistant Salmonella, including the zoonotically important serovars, revealed a potential threat not only to poultry but also to human health in Kashmir.

Isolation of atypical enteropathogenic Escherichia coli and Shiga toxin 1 and 2f-producing Escherichia coli from avian species in India.

AIMS: To study the prevalence and characterize atypical enteropathogenic Escherichia coli (EPEC) and Shiga toxin producing E. coli (STEC) in avian species in India. METHODS AND RESULTS: Two hundred and twelve faecal samples collected from 62 chickens, 50 ducks and 100 pigeons were investigated for the presence of stx(1), stx(2), eae and ehxA virulence genes by multiplex PCR. In all, 42 E. coli isolates (25 chicken, 2 duck and 15 pigeon) possessed at least one virulence gene. Out of these, nine (4.24%) isolates were STEC and 33 (15.56%) were EPEC. All isolates from duck and chicken were EPEC while among 15 pigeon isolates nine (60%) were STEC and six (40%) were EPEC. Among the STEC isolates four each carried stx(1) or stx(2) and one possessed both stx(1) and stx(2). Subtype analysis of stx revealed the presence of stx(2f) in four STEC isolates. None of the STEC isolates carried stx(1c), stx(2c), stx(2d) or stx(2e). Isolates carrying stx(2f) demonstrated vero cell toxicity. One each belonged to serogroup O17 and O78, while one was rough and the other untypeable. All EPEC isolates were atypical as they lacked bfpA. This appears to be the first report of detection of stx(2f) from India. CONCLUSIONS: The study established the presence of stx(1) and stx(2f) containing E. coli in pigeons and atypical EPEC in poultry in India. Pigeons might serve as vectors for transmission of STEC to environment and humans. SIGNIFICANCE AND IMPACT OF THE STUDY: Taking into account the close contact between fanciers and pigeons, these findings warrant a more critical appraisal of these zoonotic pathogens in pigeons and humans.

Serological diversity and virulence determination of Dichelobacter nodosus from footrot in India.

One hundred and twenty-eight swab samples from footrot lesions of naturally infected sheep were examined for presence of Dichelobacter nodosus (D. nodosus). The detection of D. nodosus was carried out by polymerase chain reaction (PCR), directly from swabs or after isolation, using 16S rDNA specific primers. The isolation of the bacterium was carried out anaerobically on trypticase-arginine-serine (TAS) agar containing 4% hoof powder. Serogrouping of the D. nodosus was accomplished with multiplex PCR using nine (A-I) serogroup specific primers. The virulent and benign status of the isolates was ascertained by detection of virulence specific integrase A (intA) gene. Out of total 83 D. nodosus isolates, 62 (74.69%) belonged to serogroup B, 18 (21.68%) to serogroup E and three (3.62%) to serogroup I. Serogroup I was detected and isolated for the first time in India. All the positive samples revealed infection by single serogroup of D. nodosus except one which showed mixed infection of serogroups B and E. Sixty (72.28%) isolates possessed intA gene and thus were considered as virulent strains. Serogroupwise intA gene was found in 43 (69.35%) isolates of serogroup B, 14 (77.78%) of E and in all the three (100%) of I.

Subtype analysis of stx1, stx2 and eae genes in Shiga toxin-producing Escherichia coli (STEC) and typical and atypical enteropathogenic E. coli (EPEC) from lambs in India.

Seventy-five Escherichia coli isolates with at least one targeted virulence gene were recovered from 338 lambs with (n=230) and without (n=108) diarrhoea. The isolates belonged to 36 different serogroups. Shiga toxin-producing E. coli (STEC) was isolated from 9.6% of lambs with and 24.1% of lambs without diarrhoea. Enteropathogenic E. coli (EPEC) was isolated from 6.1% of lambs with and 11.1% of lambs without diarrhoea. Of 26 EPEC isolates, seven were typical (positive for bfpA), and, of 34 stx(1) positive isolates, 25 were subtyped as stx(1c). Five of 29 stx(2) positive isolates were subtyped as stx(2d) and two as stx(2c). Seven of 45 eae positive isolates were subtyped as eae subtype zeta (eaezeta). This appears to be the first report of the isolation of typical EPEC from sheep in India.