An improved visual closed tube Loop mediated isothermal amplification (LAMP) assay for rapid identification of orf virus in sheep and goats.

The orf virus (ORFV) is an epitheliotropic virus causing a highly contagious skin disease mainly in sheep and goats. Several diagnostics including molecular tools like Loop mediated isothermal amplification (LAMP) assay are available to detect ORFV in affected species. However, the carry-over contamination associated with LAMP as open tube format prevents the assay applicability as point of care test in field diagnostic settings. In this study, the B2L gene based LAMP assay was optimized in a closed tube format using hydroxynaphthol blue (HNB) and calcein as pre-addition dyes and it has shown a clear positive and negative signal at 60 °C using 4 and 5 mM concentrations of MgSO4 respectively for these dyes. Optimitimzed assay that could reveal the result within one hour is highly specific and senstive with a limit of detection at 12.5 femtogram of viral genomic DNA or ~85 virus genome equivalent. This improved method prevented the cross-contamination of future LAMP reactions in the laboratory without compromising diagnostic sensitivity (100%) and specificity (100%) when compared to open tube system. This closed tube LAMP method has potential to act as a simple visual detection assay for the rapid and specific diagnosis of ORFV in sheep and goats.

Comparative efficacy of chemical stabilizers on the thermostabilization of a novel live attenuated buffalopox vaccine.

In the present investigation, the thermostability of a live attenuated buffalopox vaccine prepared with an indigenous baffalopox virus isolate (BPXV Vij/96) and freeze-dried under conventional lyophilizing conditions is described. Three different stabilizer combinations like LS (lactalbumin hydralysate + sucrose), LHT (lactalbumin hydralysate + Trehalose dihydrate) and TAA (Trehalose dihydrate + l- Alanine + l-Histidine) were used to prepare the vaccine. The study indicated that the LS stabilizer was found to be the stabilizer of choice followed by LHT and TAA for buffalopox vaccine at all temperatures studied. The presence of stabilizers has beneficial influence in preserving the keeping quality of the vaccine. Further, among the diluents used to reconstitute the freeze-dried buffalopox vaccine, double distilled water, 0.85% normal saline solution and phosphate buffer saline were the choice of diluents in that order. However, 1M MgSO4 did not perform well at higher temperatures. Investigation suggests for using LS as a stabilizer for freeze-drying and any of the three diluents except 1MgSO4 for reconstitution of buffalopox vaccine.

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.

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.

Classical Swine Fever in Wild Hog: Report of its Prevalence in Northeast India.

Classical swine fever virus (CSFV) is the causative agent of a highly contagious disease, hog cholera in pigs. The disease is endemic in many parts of the world and vaccination is the only way to protect the animals from CSFV infection. Wild hogs belong to the species Sus Scrofa Cristatus under the family Suidae are quite susceptible to CSFV infection. The epidemiological role concerning classical swine fever (CSF) in India is largely unknown. We report here the three isolated cases of CSF in wild hogs from three National parks, namely Kaziranga National Park, Manas National Park and Jaldapara National Park, from north-east part of India. The post-mortem and histopathological findings were clearly indicative for CSFV infection. The presence of CSFV genome was demonstrated in several organs and tissues collected from hogs died due to viral infection. In addition, CSF-specific antibodies were detected in two wild hogs as well as in eighteen feral pigs from the same locations. The phylogenetic analysis of the partial E2 protein gene and 5' untranslated region of CSFV isolates from the wild hog showed identities with genotype 2.2 of the Indian isolates. Occurrence of CSF in wild hogs may pose a potent threat in the epidemiology of the virus in Northeast part of India. To the best of our knowledge, the report presented in the manuscript is the first comprehensive report on CSF in wild hogs form Northeast India. The findings reported would help us to understand the epidemiology and biology of CSFV in wild animals.

Evaluation of stability of live attenuated camelpox vaccine stabilized with different stabilizers and reconstituted with various diluents.

In this study, thermostability of a Vero cell attenuated live camelpox vaccine under conventional lyophilization conditions has been evaluated. Three stabilizers were used separately for freeze-drying the vaccine and the stability of the vaccine, both in freeze-dried and reconstituted forms at different temperatures was assessed. The study revealed that the camelpox vaccine lyophilized with TAA stabilizer found superior with a shelf life of 44 months, 227 days, 22 days and 20 days at 4, 25, 37 and 45 °C, respectively followed by LS stabilizer. In terms of half-life, TAA stabilizer proved better followed by LS and BUGS stabilizers at all temperatures except at 25 °C in which LS found relatively superior. Among the four diluents viz. 1x PBS (phosphate buffered saline, pH 7.4), 0.85% NaCl, distilled water and 1 M MgSO4, PBS was a better diluent followed by 0.85% NaCl. Both the diluents maintained the infectivity titer more than the minimum effective dose (3 log10TCID50 with a maximum titre of 6.53 log10TCID50 in both the diluents) for 60 h at 37 and 45 °C. However, 1 M MgSO4 found less suitable for camelpox vaccine dilution. The study indicates that the TAA and 1× PBS are the choice of stabilizer and diluent, respectively for camelpox vaccine.

Real time PCR: a rapid tool for potency estimation of live attenuated camelpox and buffalopox vaccines.

In the present study, SYBR Green and TaqMan real time PCRs (rt-PCR) based on the C18L gene (encodes ankyrin repeat protein) of camelpox (CMLV) and buffalopox viruses (BPXV) were, respectively employed for potency evaluation of live attenuated camelpox and buffalopox vaccines. Cells infected with the respective vaccine viruses were harvested at critical time points and subjected to respective PCRs. The critical time points of harvests for CMLV and BPXV respectively, were 36 and 30 h post infection and were respectively determined based on maximum slopes of (-3.324) and (-3.321) standard curves. On evaluation of eight batches of camelpox and seven batches of buffalopox vaccines, the results indicated that the titres estimated by respective rt-PCRs were well comparable to the conventional TCID(50) method. The rt-PCR assays were found relatively more sensitive, specific and rapid than end point dilution assay. Thus, they could be used as additional tools for estimation of live CMLV and BPXV particles in camelpox and buffalopox vaccines.

Classical swine fever in the pygmy hog.

The pygmy hog is a rare, small and highly endangered mammal belonging to the Suidae family, and it is presently found only in the Assam state of India. While investigating the cause of death of pygmy hogs housed at a conservation centre for captive breeding and research at Basistha, Assam, it was confirmed that they were susceptible to and died as a result of contracting classical swine fever (CSF), caused by CSF virus (CSFV), which is a highly infectious endemic disease of domestic pigs in India. The post-mortem findings and serum CSFV-specific antibody titres, along with the isolation of CSFV from two pygmy hogs, and further confirmation by CSFV genomic E2 and 5' untranslated region (UTR) gene amplification in PCR (polymerase chain reaction), clearly established the cause of death of the pygmy hogs. Further, on phylogenetic analysis, the pygmy hog CSFV 5' UTR sequences were grouped in the genotype 1.1 cluster of Indian CSFVs, and hence the strains causing infection were closely related to CSFV isolates circulating in domestic pigs. Therefore, the occurrence of CSF in this endangered species may pose a potent threat to their existence unless properly controlled, and thus it needs urgent attention. To the authors' knowledge this is the first report on CSF in pygmy hogs.

Comparative efficacy of live replicating sheeppox vaccine strains in Ovines.

In the present study, two sheeppox vaccines made from strains [sheeppox virus-Srinagar (SPPV-Srin) and Ranipet (SPPV-R)] indigenous to India and adapted to Vero cells were compared in terms of their safety, potency, efficacy and antigenic value with the commercial in-use Roumanian Fanar (SPPV-RF) vaccine, a foreign strain adapted in primary lamb testes cells. The safety test indicated that the SPPV (Sri and RF) vaccines were safe while SPPV-R was not completely attenuated and caused excessive adverse reactions at the passage level tested. The immunized animals showed DTH reaction and resisted virulent SPPV challenge, while control animals developed disease. Specific virus could be detected in the controls and animals immunized with lower dilutions of vaccines after challenge but not in any of the sheep immunized with 1 and 100 doses of each vaccine. All vaccines were found potent and the PD(50) was highest for SPPV (Srin and R) followed by RF. The immunized animals were seroconverted following vaccination with sustained antibody responses after challenge. In conclusion, indigenous SPPV-Srin vaccine was found to be as efficacious as SPPV-R and SPPV-RF vaccines. Thus, there is potential benefit in replacing the currently used commercial vaccine SPPV-RF with indigenous SPPV-Srin vaccine for use in India.

TaqMan real-time PCR assay based on DNA polymerase gene for rapid detection of Orf infection.

Both conventional and real time PCR (rt-PCR) assays based on the amplification of a 103bp fragment from the DNA polymerase (DNA pol) gene (conserved, non-structural) of Orf virus (ORFV) were developed for detection and semi-quantitation of ORFV DNA from infected cell culture and clinical samples. The latter technique was based on TaqMan chemistry. The rt-PCR assay was specific and sensitive as it could detect as low as 3.5fg or 15 copies of ORFV genomic DNA. Both intra- (0.38-1.0%) and inter-assay (0.53-2.87%) variabilities of rt-PCR were within the acceptable range meaning the high efficiency and reproducibility of the assay. The rt-PCR was applied successfully to detect ORFV DNA from suspected clinical samples. Further, the assay has shown a relative diagnostic sensitivity and specificity of 100% and 93.5%, respectively, when compared to B2L gene based semi-nested PCR implying a wide potential of this rt-PCR for rapid field diagnosis of Orf in sheep and goats.

Molecular Characterization of Classical swine fever virus Involved in the Outbreak in Mizoram.

Classical swine fever is the most insidious and devastating disease of pigs and wild boars. The virus is closely related to the other members of the genus Pestivirus. The outbreak recorded in Mizoram, India was strategically important as the state shares porous international boundary with East Asian countries. Both immunodiagnostic and molecular techniques were used to confirm the involvement of Classical swine fever virus (CSFV) in this outbreak. Sandwich ELISA and direct FAT could detect CSFV in the tissue samples. RT-nPCR specifically amplified E2 and 5'NTR product of 271 bp. Phylogenetic analysis showed, that the Mizoram isolate (MZ4/69) was very close to the Chinese strain Shimen-HVRI (93.0%) rather than other Indian isolate (CSF-30-03). Present study provides a valuable sequence based molecular data on Indian isolate of CSFV and will be useful in investigation on transmission of such disease from neighbour countries.