Durlav Prasad Bora

Identification and phylogenetic analysis of orf viruses isolated from outbreaks in goats of Assam, a northeastern state of India

Two outbreaks of orf virus (ORFV) (a parapoxvirus) infection in goats, which occurred in Golaghat and Kamrup districts of Assam, a northeastern part of India, were investigated. The disease was diagnosed by standard virological and molecular techniques. The entire protein-coding region of B2L gene of two isolates were cloned and sequenced. Phylogenetic analysis based on B2L amino acid sequences showed that the ORFVs identified in these outbreaks were closely related to each other and both were closer to ORFV-Shahjahanpur 82/04 isolate from north India. The present study revealed that the precise characterization of the genomic region (B2L gene) might provide evidence for the genetic variation and movement of circulating ORFV strains in India.

Isolation and characterization of Shiga toxigenic Escherichia coli of animal and bird origin by multiplex polymerase chain reaction

Aim: The purpose of this study was to determine the virulence genes and serotype of Shiga toxin producing Escherichia coli (STEC) strains isolated from animals and birds. Materials and Methods: A total of 226 different samples viz., fecal, intestinal content, rectal swab and heart blood were collected from different clinically affected/healthy animals and birds and were streaked on McConkeys’ lactose agar and eosin methylene blue agar for isolation of E. coli, confirmed by staining characteristics and biochemical tests. By polymerase chain reaction (PCR) all the E. coli isolates were screened for certain virulence genes, viz., Shiga toxin 1 (stx1), stx2 and eae and enterohemolytic (Ehly) phenotype was observed in washed sheep blood agar plate. All the isolated E. coli strains were forwarded to the National Salmonella and Escherichia Centre, Central Research Institute, Kasauli (Himachal Pradesh) for serotyping. Results: Out of 226 samples 138 yielded E. coli. All the isolates were screened for molecular detection of different virulent genes, viz. stx1, stx2 and eae, based on which 36 (26.08%) were identified as STEC. Among those STEC isolates, 15 (41.67%), 14 (38.89%), 1 (2.78%) exhibited eae, stx2, stx1 alone, respectively, whereas 4 (11.11%) and 2 (5.56%) carried both stx1 and stx2, stx2 and eae, respectively. Among the STEC isolates 22 were belonged to 15 different sero-groups, viz., O2, O20, O22, O25, O43, O60, O69, O90, O91, O95, O106, O118, O130, O162 and O170 and others were untypable. Ehly phenotype was observed in 10 (27.78%) the STEC isolates. Conclusion: The present study concluded that STEC could be isolated from both clinically affected as well as healthy animals and birds. Regular monitoring of more samples from animal and bird origin is important to identify natural reservoir of STEC to prevent zoonotic infection.

Seroprevalence of contagious ecthyma in goats of Assam: An analysis by indirect enzyme-linked immunosorbent assay.

Aim: The objective of this study was to screen the prevalence of contagious ecthyma (CE) among the goat population of Assam owing to its high prevalence rate. Materials and Methods: In this study, a total of 231 serum samples were collected from 12 districts of Assam during September 2013 to July 2014. The serum samples were tested for the presence of antibodies against Orf virus (ORFV) by indirect enzyme-linked immunosorbent assay (ELISA). Indirect ELISA was standardized using purified Orf reference virus produced in bulk in primary lamb testes cells. Results: Studies on seroprevalence showed 76.62% of goats were seropositive. The total number of animals were divided into different age groups starting from 0-2 months, 2-4 months, 4-6 months, and above 8 months and accordingly highest prevalence of antibodies against ORFV was recorded in the age-group above 8 months of age. Significantly, lower rates of infection were observed in goats of age group 2-4 months. This study recorded that seropositivity from naturally infected animals and in contact apparently healthy animals to be 53.67% and 46.32%, respectively. Conclusion: The results indicated that CE is a prevalent infection in goats of Assam, and the healthy population is at increased risk of infection.

Seroprevalence of Rotavirus infection in pig population of Arunachal Pradesh

Aim: This study was conducted to find out the seroprevalence of Rotavirus(RV) infection among the pig population of Arunachal Pradesh. Materials and Methods: Serums samples were collected from piglets of age ranging from 1 week to 6 months and the sows associated with the piglets that were reared under organized and unorganized system of management in six different districts of Arunachal Pradesh. The prevalence of RV specific antibodies was detected using a polyclonal antibody-based indirect enzyme-linked immunosorbent assay (i-ELISA). Results: The study revealed that out of 394 serum samples, 255 (64.72%) samples were found to be positive for RV-specific antibody in i-ELISA. Considering the samples from different districts, Papumpare district of Arunachal Pradesh showed highest numbers of seropositive animals (68.75%) followed by upper Subansiri (64.91%) while West Siang district showed lowest positivity rate (61.22%). Conclusion: As considerable seropositivity was recorded among pig population of Arunachal Pradesh in this study, there is urgent need to establish high-impact and cost-effective public health intervention tools, key among them being the introduction of strict hygiene practice and RV vaccination program, to greatly reduce the number of deaths due to diarrheal diseases. To the authors’ knowledge, this is the first report on the prevalence of RV infection from pigs of Arunachal Pradesh.

Identification of swinepox virus from natural outbreaks in pig population of Assam

Outbreaks of swinepox [caused by a swinepox virus (SWPV)] in pigs were investigated in 3 districts of Assam, a north eastern state of India. Diagnosis of the disease was carried out employing both standard virological as well as molecular methods. Three representative isolates from different places were selected for inoculation into confluent monolayers of Porcine Kidney-15 (PK-15) cell line. The cytopathic effects were characterized by cell rounding, nuclear vacuolation, cell fusion, granulation of cells and finally detachment from third blind passage onwards. The three genes viz., SPV18–20 and P42 of SWPV was targeted for confirmation of the virus. Swinepox virus was successfully adapted to the PK-15 cell line from seventh passage onwards. The isolated viruses were characterized by sequencing and phylogenetic analysis of P42 gene (extracellular envelope protein), a homologue of vaccinia virus F13L gene. In India, studies on swine pox are very limited. This is the first report on successful isolation of swinepox virus from north eastern region of India. Assam and the other north-eastern states of India being a hub for pig husbandry, isolation of swinepox virus will help in developing and formulating control strategies against the disease.

Goatpox outbreak at a high altitude goat farm of Mizoram: possibility of wild life spill over to domestic goat population

In this study, pox-like outbreaks in goat population was investigated that occurred in a high altitude goat farm located in Mizoram, a hilly state of North eastern India. The outbreak initially involved the serows, an wild animal belonging to the family Bovidae, subfamily Caprinae and genus Capricornis, the state animal of Mizoram. Later, the disease affected the domestic goat population. The disease was diagnosed on the basis of gross lesions and PCR amplification of partial P32 gene of capripox virus. The virus was isolated in vero cells. The full length P32 gene was sequenced and phylogenetic tree was constructed. It was revealed that the capripox virus isolated from the outbreak was closely related to the Chinese strain of goatpox virus at both amino acid and nucleotide level. To the authors’ knowledge, this is the first report on isolation and characterization of capripoxvirus from north eastern region of India.

Virulence gene profiling of porcine Pasteurella multocida isolates of Assam

Aim: The present study was conducted to detect and identify the virulence genes in Pasteurella multocida isolates of porcine origin from Assam. Materials and Methods: A total of 21 porcine P. multocida isolates were subjected to capsular typing and detection of virulence-associated genes (pfhA, tbpA, hgbB, toxA, oma87, ompH, and nanB) using various polymerase chain reaction (PCR) methods reported elsewhere. Further, pathogenicity of the porcine isolates of P. multocida was studied in mice. For each strain of P. multocida selected for pathogenicity trial, the group of mice was injected intraperitoneally (i/p) with 0.1 ml of the inoculum prepared from respective field isolates, containing 109 organisms per ml. Results: Capsular typing of the isolates by multiplex PCR showed two capsular types, type A (66.66%) and type D (33.33%). All the isolates were positive for outer membrane protein genes, oma87 and ompH genes. Iron acquisition genes, tbpA and hgbB, were detected in 14.28% and 19.04% of the isolates. The dermonecrotoxin encoding gene, toxA, was present in 23.80% of the isolates. Filamentous hemagglutinin encoding gene, pfhA, was detected in 28.57%. The virulence gene distribution pattern of the isolates indicates the important role of the genes in disease pathogenesis. Conclusion: From the present study, it can be concluded that toxA gene is an important marker gene for defining the pathogenic potential of P. multocida strains in swine.

Heterologous DNA prime-protein boost immunization with RecA and FliD offers cross-clade protection against leptospiral infection

The emergence of >300 serovars of Leptospira confounded the use of generalized bacterin, the whole cell lysate, as vaccines to control leptospirosis. Because of substantial genetic and geographic heterogeneity among circulating serovars, one vaccine strain per serovar cannot be efficacious against all the serovars. We have performed heterologous DNA prime-protein boost vaccination challenge studies in hamsters using in vivo expressed, leptospiral recombinase A (RecA) and flagellar hook associated protein (FliD). We prepared the monovalent recombinant protein, plasmid DNA, and DNA prime protein boost adjuvant vaccines. The whole cell bacterin served as a control. Our data show that (i) RecA and FliD have multiple immunogenic B and T-cell epitopes with highly conserved domains among most prevalent pathogenic Leptospira spp., (ii) humoral and cell mediated immune responses were induced remarkably, (iii) provides significant protection against homologous (Autumnalis strain N2) and cross-clade heterologous (Canicola strain PAI-1) challenge infection for the heterologous prime-protein boost (∼91–100%) and, the DNA vaccine (∼75–83%). Recombinant protein vaccine shows only partial protection (∼58–66%), (iv) RecA prime-protein boost vaccine shows sterilizing immunity, with heterologous protection. This RecA/FliD prime-protein boost strategy holds potential for vaccination against animal leptospirosis and for a better control of zoonotic transmission.