Emergence of variant avian infectious bronchitis virus in India.

BACKGROUND: Infectious bronchitis virus (IBV) is the etiological agent of an acute and highly contagious disease. Infectious bronchitis (IB) affects chicken of all ages and poses major economic loses to the poultry industry worldwide. The continuous evolution of the spike protein (S1) of IBV is responsible for the prevalence of many serotypes/genotypes around the world. Multiple lineages of IBV strains have been detected in chicken flocks in India since 2003. AIMS: To detect IBV genotypes prevalent in India. METHODS: Organ samples from 20 IBV-positive flocks with variable clinical signs were used for the amplification of the S1 gene of IBV by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Positive PCR amplicons were sequenced. Sequence analysis showed that 14 field isolates belonged to the GI-1 genetic lineage (Mass 41 serotype), two field isolates belonged to the GI-13 (UK 4/91 variant IBV strain), one field isolate grouped with GIII, GV, and GVI genetic lineage and three belonged to a variant genotype unique to India (GI-24). Phylogenetic analysis also showed a similar type of grouping within the field isolates. Among the fourteen GI-1 isolates, 12 were isolated between 2003 and 2006 and only two were isolated between 2009 and 2011. The two field isolates belonging to GI-13 were isolated in 2007, another one belonging to GIII, GV, and GVI was isolated in 2010 and three field isolates were not close to any reference IBV sequences isolated in 2006 (IND-TN-168-06), 2010 (IND-TN-280-10) and 2011 (IND-TN-290-11). CONCLUSION: A unique variant of IBV is emerging in India (GI-24). Our findings will have important implications for future vaccine intervention.

Duck plague outbreak in a Chara-Chemballi duck farm.

BACKGROUND: Duck rearing is one of the important livelihoods of rural people. Duck plague is one of the diseases causing heavy mortality resulting in economic losses. CASE DESCRIPTION: An outbreak of duck plague in a farm in Kadavakathi Village near Tenkasi, Tirunelveli Dt., is reported. FINDINGS/TREATMENT AND OUTCOME: Two thousands out of 4500 Chara-Chemballi breed of ducks which were recently purchased from Chenganacherry in Kerala died, with a mortality rate of 44.4%. Clinical signs of inappetence, partial closure of eyelid, conjunctivitis, corneal opacity, oculo-nasal discharge, soiled vent with green white watery diarrhoea, ataxia, incoordination and sudden death were observed. Necropsy examination revealed diphtheritic membrane in the oesophagus, congestion, petechial haemorrhages and multifocal gray white areas on the surface of the liver, epicardial haemorrhages, congested trachea, lung, kidneys, splenomegaly with mottled appearance and enteritis. Microscopical examination revealed presence of eosinophilic intranuclear and intracytoplasmic inclusions in the epithelial cells of the intestine and hepatocytes, degeneration and necrosis of enterocytes, dilated crypt epithelial cells with presence of eosinophilic intranuclear and intracytoplasmic inclusions, congestion and lymphoid cell depletion in the spleen, vasculitis, congestion, and haemorrhages in the trachea and lungs, proventriculitis, and congested kidneys. Polymerase chain reaction (PCR) also confirmed the duck plague viral infection by the amplification of polymerase gene fragment (446 bp). CONCLUSION: Based on the above findings, the Chara-Chemballi duck disease outbreak was diagnosed as duck viral enteritis infection.

Avian parvovirus: classification, phylogeny, pathogenesis and diagnosis.

Poultry parvoviruses identified during the early 1980s are found worldwide in intestines from young birds with enteric disease syndromes as well as healthy birds. The chicken parvovirus (ChPV) and turkey parvovirus (TuPV) belong to the Aveparvovirus genus within the subfamily Parvovirinae. Poultry parvoviruses are small, non-enveloped, single-stranded DNA viruses consisting of three open reading frames, the first two encoding the non-structural protein (NS) and nuclear phosphoprotein (NP) and the third encoding the viral capsid proteins 1 (VP1 and VP2). In contrast to other parvoviruses, the VP1-unique region does not contain the phospholipase A2 sequence motif. Recent experimental studies suggested the parvoviruses to be the candidate pathogens in cases of enteric disease syndrome. Current diagnostic methods for poultry parvovirus detection include PCR, real-time PCR, enzyme linked immunosorbent assay using recombinant VP2 or VP1 capsid proteins. Moreover, sequence-independent amplification techniques combined with next-generation sequencing platforms have allowed rapid and simultaneous detection of the parvovirus from affected and healthy birds. There is no commercial vaccine; hence, the development of an effective vaccine to control the spread of infection should be of primary importance. This review presents the current knowledge on poultry parvoviruses with emphasis on taxonomy, phylogenetic relationship, genomic analysis, epidemiology, pathogenesis and diagnostic methods.

Sequence analysis of VP2 hypervariable region of the field isolates of infectious bursal disease viruses from southern region of India.

Infectious bursal disease virus isolates obtained from southern parts of India were subjected to comparative sequencing and phylogenetic analysis of 743bp hypervariable region of VP2. The sequence analysis showed that among eight isolates, only HY12 showed the characteristic conserved amino acid residues at 256I, 294I, and 299S of vvIBDV. Six isolates BGE14, PY12, NKL14, VCN14, RPM14 and EDE14 had conserved amino acid residues at 256I and 299S, whereas at residue 294, isoleucine was substituted by valine. The remaining isolate MB11 had leucine at residue 294 and asparagine at residue 299 similar to classical strain 52/70. The serine-rich heptapeptide sequence SWSASGS adjacent to the second hydrophilic region was conserved in all seven Indian IBDV isolates except isolate MB11. Conservation of this sequence was earlier reported to be an indication of a virus isolate being pathogenic in nature. The reported heptapeptide sequence of the classical strain is 'SWSARGS'. In the present study, 'SWSARGS' heptapeptide sequence was observed in MB11 isolate. The pathogenicity trials conducted with these isolates further confirmed the genome analysis in classification. This study further reveals that the circulating IBDV strains in India could be diverse in nature.

Equipping providers with principles, knowledge and skills to successfully integrate behaviour change counselling into practice: a primary healthcare framework.

OBJECTIVES: There is an urgent need for healthcare providers and healthcare systems to support productive interactions with patients that promote sustained health behaviour change in order to improve patient and population health outcomes. Behaviour change theories and interventions have been developed and evaluated in experimental contexts; however, most healthcare providers have little training, and therefore low confidence in, behaviour change counselling. Particularly important is how to integrate theory and method to support healthcare providers to engage in behaviour change counselling competently. In this article, we describe a general training model developed from theory, evidence, experience and stakeholder engagement. This model will set the stage for future evaluation research on training needed to achieve competency, sustainability of competency, as well as effectiveness/cost-effectiveness of training in supporting behaviour change. DESIGN AND METHODS: A framework to support competency based training in behaviour change counselling is described in this article. This framework is designed to be integrative, sustainable, scalable and capable of being evaluated in follow-up studies. RESULTS AND DISCUSSION: Effective training in behaviour change counselling is critical to meet the current and future healthcare needs of patients living with, or at risk of, chronic diseases. Increasing competency in establishing change-based relationships, assessing and promoting readiness to change, implementing behaviour modification and addressing psychosocial issues will be value added to the healthcare system.

Evaluating the efficacy of LaSota vaccination induced protection in chickens upon challenge with a genotype IV strain of Newcastle disease virus.

Newcastle disease (ND) is a major risk to the poultry industry which results in severe economic loss throughout the world even with vaccination. The vaccine viruses that are used in many countries include the LaSota and other live viruses that were isolated in the early and late 1950s. Reports from several laboratories including ours indicate a greater variance of the circulating strains and recent classification indicates the existence of XVIII different genotypes of NDV strains. The efficiency of the LaSota vaccination in inducing protective immunity to different heterologous strains has been a question and its efficacy upon exposure to a virulent genotype IV strain has not been reported after 1989 world-wide except for India. Serum antibody negative (SAN) chicks of either sex obtained by hatching specific-pathogen-free (SPF) eggs were vaccinated with increasing doses of the vaccine virus from 101 to 107 EID50 per bird delivered through occulo-nasal route and challenged 20 days later with NDV-2K3 (genotype IV) strain of virus isolated in the year 2000 from pigeon in India. The birds were monitored for serum antibody titers and following challenge for morbidity, mortality, viral load in the cloacal swab and different tissues. We could clearly show that a minimum vaccine titre of 104 EID50 could establish protective antibody levels and also prevent viral replication post challenge upon exposure to the virulent genotype IV strain. We conclude based on our results and previous observation that there do exist differences in the levels of the antibody that could limit viral replication and shedding upon exposure to different heterologous genotype of NDV. Developing a strain matched vaccine might less potential to result in better protection by limiting the viral shedding.

Complete Genome Sequence Analysis of a Naturally Reassorted Infectious Bursal Disease Virus from India.

The novel infectious bursal disease virus (IBDV) isolate BGE14/ABT1/MVC/India is a very virulent IBDV that was isolated from broiler flocks in southern parts of India during 2014. Here, we report, for the first time in India, the complete genome sequence of BGE14/ABT1/MVC/India, a reassortment strain with segments A and B derived from a very virulent IBDV strain and an attenuated IBDV, respectively. The findings from this study provide additional insight into the genetic exchange between attenuated and very virulent strains of IBDV circulating in the field.

Complete Genome Sequence of a Chicken Embryo Fibroblast-Adapted Attenuated Infectious Bursal Disease Virus Isolate from India.

Infectious bursal disease virus is an avian pathogen that causes huge morbidity and mortality in the poultry sector all over the world. Here, we report the full-length genome sequence of an Indian strain, MB11/ABT/MVC/2016, isolated from a commercial broiler flock. This is a first report of a complete genome sequence of infectious bursal disease virus from India.

Genotypic characterization of Indian isolates of infectious bursal disease virus strains by reverse transcription-polymerase chain reaction combined with restriction fragment length polymorphism analysis.

The reverse transcription PCR (RT-PCR) combined with restriction fragment length polymorphism (RFLP) is used for the differentiation of classical virulent (cv), virulent (v) and very virulent (vv) strains of infectious bursal disease virus (IBDV) isolates from chicken bursal tissues in southern states of India. In the present study, six different isolates (MB11, HY12, PY12, BGE14, VCN14 and NKL14) of IBDV strains were subjected for genotyping along with vaccine virus (Georgia, intermediate strain) using RT-PCR for amplification of a 743 bp sequence in the hypervariable region of VP2 gene followed by restriction enzyme digestion with 5 different restriction enzymes (BspMI, SacI, HhaI, StuI and SspI). The RT-PCR products obtained from vvIBDV strains were digested by SspI enzyme except PY12, BGE14 and MB11 isolates. The SacI digested the isolate MB11, PY12 and the vaccine strain, but it did not cleave the very virulent isolates of IBDV. HhaI cleaved all the isolates with different restriction profile patterns. StuI digested all the vvIBDV isolates and BspMI was not able to differentiate field isolates from vaccine strain. Though RT-PCR combined with RFLP is a genotypic method, further confirmation of serotypes to distinguish the vvIBDV from cvIBDV has to be carried out using pathogenicity studies.

Emergence of Porcine Circovirus 2 Associated Reproductive Failure in Southern India.

Incidence of unusually high numbers of stillbirths was observed at a piggery unit at the Veterinary University research farm in Tamil Nadu State of India. Systematic examination of the tissue from stillborn piglets led to the identification of presence of Porcine circovirus 2 (PCV2). Detailed analysis utilizing electron microscopy, polymerase chain reaction and sequencing confirmed the presence of PCV2 in the tissue of affected piglets. Histopathology analysis of the affected piglet tissue showed lymphoid cell depletion of lymphnodes, spleen and infiltration of liver, kidney, myocardium, etc. Retrospective examination of the morbidity and mortality history in the farm revealed high mortality in young and weanling piglets suggestive of PCV2 infection-induced diseases. This is the first report of emergence of major disease incidence in farmed swine due to PCV2 infection in India.

Natural influence of season on follicular, luteal, and endocrinological turnover in Indian crossbred cows.

The study was aimed at investigating the effect of seasonal changes on follicular and luteal dynamics in vivo in normally cycling crossbred cows during summer and winter months of the year. Six healthy regularly cycling Jersey crossbred nonlactating pluriparous cows were used for the study. Follicular and luteal developmental pattern was studied every other day throughout the estrous cycle by scanning the ovaries during two periods of a year viz., hot season (April to June; n = 16) and cold season (December to February; n = 12). Plasma progesterone (P4) concentrations were measured on Days 0 (estrus), 6, and 12 of the estrous cycle. Among the 12 cycles studied during the cold season, 11 (91.7%) had three waves and one had two waves. Of 16 cycles studied during the hot season, eight (50%) had two waves, four (25%) had three waves, and the remaining four cycles had single (n = 2) and four waves (n = 2). High P4 concentrations during the midcycle would have suppressed the dominant follicle of the second follicular wave and induced the emergence of the third wave during the cold season. The first follicular wave (wave I) of the cycle emerged much earlier (Day 0.5 ± 0.3) during the cold season than that in the hot season (Day 1.7 ± 0.4). The ovulatory wave emerged significantly earlier during the hot season (Day 11.5 ± 1.3) than in the cold season (Day 14.8 ± 0.4), and hence, the growth phase of ovulatory follicle significantly increased during the former season (11.0 ± 1.4 days) than the latter (5.8 ± 0.2 days). The ovulatory follicle attained a significantly larger diameter (12.8 ± 0.8 mm) to express the estrus during the hot season when compared to the cold season (11.3 ± 0.4 mm), which might be indicative of alterations in steroidogenic activity within the follicular microenvironment. During the midphase of the cycle, a period critical for embryonic sustenance, the P4 level was significantly reduced in the hot months indicating suppression of luteal activity during hot period of the year. Thus, it could be concluded that increased incidence of two follicular waves associated with a prolonged growth phase of the ovulatory follicle, and altered luteal endocrine activity during the hot season might be associated with decreased fertility in crossbred cattle.

Next generation sequencing technologies: tool to study avian virus diversity.

Increased globalisation, climatic changes and wildlife-livestock interface led to emergence of novel viral pathogens or zoonoses that have become serious concern to avian, animal and human health. High biodiversity and bird migration facilitate spread of the pathogen and provide reservoirs for emerging infectious diseases. Current classical diagnostic methods designed to be virus-specific or aim to be limited to group of viral agents, hinder identifying of novel viruses or viral variants. Recently developed approaches of next-generation sequencing (NGS) provide culture-independent methods that are useful for understanding viral diversity and discovery of novel virus, thereby enabling a better diagnosis and disease control. This review discusses the different possible steps of a NGS study utilizing sequence-independent amplification, high-throughput sequencing and bioinformatics approaches to identify novel avian viruses and their diversity. NGS lead to the identification of a wide range of new viruses such as picobirnavirus, picornavirus, orthoreovirus and avian gamma coronavirus associated with fulminating disease in guinea fowl and is also used in describing viral diversity among avian species. The review also briefly discusses areas of viral-host interaction and disease associated causalities with newly identified avian viruses.

Seroepidemiology of infectious bovine rhinotracheitis infection in unvaccinated cattle.

AIM: The present study aimed to investigate the seroepidemiology of infectious bovine rhinotracheitis (IBR) infection in the non-vaccinated cattle population in northern part of Tamil Nadu, India. MATERIALS AND METHODS: A total of 255 sera samples were collected from cattle having the history of respiratory and reproductive disorder from cattle of different age, breeds, and sex. All the sera samples were subjected to indirect ELISA for the diagnosis of IBR antibodies. RESULTS: Results revealed that the seroprevalence of IBR infection among non-vaccinated cattle population was of 65.88%. No significant difference was noticed in the prevalence of IBR infection between cattle showing respiratory (63.64%) and reproductive form (70.89%) (p≥0.05). A higher prevalence was noticed in animals above 3 years of age (59.60%) and in crossbred animals (71.26%) than young and non-descript animals. This study showed the higher prevalence of IBR infection in female (67.92%) than in male (33.33%). CONCLUSION: Cattle population in this part can better be protected with vaccination than leaving them unvaccinated and sero-monitoring shall have to be stressed with regular attempts to isolate and characterize the causative agent for IBR.

Co-culture: A quick approach for isolation of street rabies virus in murine neuroblastoma cells.

BACKGROUND: Laboratory detection of rabies in most cases is based on detection of the antigen by fluorescent antibody test, however, in weak positive cases confirmative laboratory diagnosis depends on widely accepted mouse inoculation test. Cell lines like neuroblastoma have been used to isolate the virus with greater success not only to target for diagnosis, but also for molecular studies that determine the epidemiology of the circulating street rabies strains and in studies that look at the efficiency of the developed monoclonal antibodies to neutralize the different rabies strains. Due to the recent issues in obtaining ethical permission for mouse experimentation, and also the passages required in the cell lines to isolate the virus, we report herewith a co-culture protocol using the murine neuroblastoma (MNA) cells, which enable quicker isolation of street rabies virus with minimum passages. OBJECTIVE: This study is not to have an alternative diagnostic assay, but an approach to produce sufficient amount of rabies virus in minimum passages by a co-culture approach in MNA cells. MATERIALS AND METHODS: The MNA cells are co-cultured by topping the normal cells with infected cells every 48 h and the infectivity was followed up by performing direct fluorescent-antibody test. RESULTS: The co-culture approach results in 100% infectivity and hence the use of live mouse for experimentation could be avoided. CONCLUSION: Co-culture method provides an alternative for the situations with limited sample volume and for the quicker isolation of virus which warrants the wild type strains without much modification.

Rapid Detection of Avian Infectious Bronchitis Virus by Reverse Transcriptase-Loop Mediated Isothermal Amplification.

A reverse-transcription loop mediated isothermal amplification (RT-LAMP) was developed for rapid diagnosis of infectious bronchitis (IB) in poultry by targeting the spike protein 2 gene (S2). RT-LAMP primers were designed for IBV-S2 targets and optimized to run at 60 °C for 45 min. As compared with RT-PCR, RT-LAMP was 100 times more sensitive for IBV-S2 gene. RT-LAMP showed specific amplification with IB viral genome but not with other avian respiratory pathogens due to their mismatching with IBV-S2-RT-LAMP primers. RT-LAMP reaction products were visually detected by the addition of propidium iodide stain. Out of 102 field samples tested for detection of IBV, RT-LAMP detected IBV in 12 samples for S2 gene whereas RT-PCR detected IBV in six samples for S2 gene. The sensitivity of the RT-LAMP was 100 % and the specificity was 94 % for S2 gene. Since the developed RT-LAMP to detect IBV is simple, rapid, sensitive and specific, it can be a useful diagnostic tool for detection of IB in poultry in less equipped laboratories and in field conditions.

Comparison of Immunoprotection of Leptospira Recombinant Proteins with conventional vaccine in experimental animals.

Leptospirosis is a bacterial disease caused by bacteria of the genus Leptospira affecting humans and animals. Untreated leptospirosis may result in severe kidney damage, meningitis, liver failure, respiratory distress, and even death. Virulent leptospirosis can rapidly enter kidney fibroblasts and induce a programmed cell death. Thus, it is a challenge for immunologists to develop an effective and safe leptospirosis vaccine. Here, we compared the commercial canine leptospira vaccine and recombinant proteins (OmpL1 and LipL41) with and without adjuvant in terms of immune response and challenge studies in hamsters and immune response studies alone in experimental dogs. The outer membrane proteins viz., lipL41 and OmpL1 of leptospira interrogans serovars icterohaemorrhagiae were amplified. The primers were designed in such a way that amplified products of OmpL1 and lipL41 were ligated and cloned simultaneously into a single vector. The cloned products were expressed in E. coli BL21 cells. The immunoprotection studies were conducted for both recombinant proteins and commercial vaccine. The challenge experiment studies revealed that combination of both rLip41 and rOmpL1 and commercial vaccine gave 83% and 87% protection, respectively. Histopathological investigation revealed mild sub lethal changes were noticed in liver and kidney in commercially vaccinated group alone. The immune responses against recombinant leptospiral proteins were also demonstrated in dogs.

Molecular Characterization of Thermostable Newcastle disease virus Isolated from Pigeon.

The HN and L gene sequences of an Indian isolate of Newcastle disease virus was analyzed prior to and after exposure to 56 °C at tenth passage and fifteenth passage to study the variations at molecular level. In the HN gene sequence of progenitor and thermostable strain, substitution of K373I, F374L, M516R, D517V were considered to contribute to the increase in the stability of the protein. In the L gene of the thermostable strain, variations were observed at many positions and among these the substitutions at position P675H K677R, K893D, R1132K, had charged amino acids, and at L656A, F657V, F869L, T886I, M899I, G1131V, V1675L, had hydrophobic amino acids that could be related to increased stability of L protein at high temperatures. The changes in amino acid sequence in HN and L gene of the thermostable strain might render structural variations that might have contributed to the stability of the strain at higher temperature.

In vitro propagation of chicken anemia virus in chicken peripheral blood mononuclear cells.

KEYWORDS: chicken anemia virus; propagation; mononuclear cells; PCR.

Transcript profiling of pattern recognition receptors in a semi domesticated breed of buffalo, Toda, of India.

The primary objective of this study was to assess the expression profile and levels of toll-like receptor (TLR) mRNAs in the spleen, lung, mediastinal lymph node (MLN), jejunum, rectum, skin and peripheral blood mononuclear cells (PBMC) of Toda and Murrah buffalos. Spleen and PBMC had increased expression of TLR mRNAs 2, 4, 5, 6, 8, 9 and 10; lung had increased expression of TLR mRNAs 2, 4, 5, 6 and 8, MLN TLR mRNA 6, 9, 10 and decrease in TLR 3 and 7 mRNAs in skin. No significant differences were observed in the expression levels of any of the TLR mRNA in jejunum and rectum. Toda buffaloes showed significantly higher expression levels of TLR 9 mRNA in MLN, TLR mRNAs 1, 5, 6, 9 and 10 in skin and TLR mRNAs 2, 4, 7 and 9 in PBMC than Murrah buffaloes living in the vicinity. Toda and Murrah buffaloes were inoculated with TLR5 (flagellin) and TLR9 (CpG ODN) ligands in vivo and expression levels of the respective TLRs analyzed 12h later. Following CpG inoculation, Toda buffaloes had significantly higher levels of TLR 9 mRNA expression but not in Murrah. However, flagellin induction did not increase TLR 5 mRNA expression in both these breeds. Histological sections of the skin were made and infiltrating cell clusters were graded and quantified. Following CpG inoculation, Toda buffaloes showed higher numbers of infiltrating grade 1 and grade 3 cell clusters while Murrah showed lower numbers of infiltrating grade 1 cells as compared to mock-inoculated skin sections. Flagellin treatment revealed no significant differences in infiltrating cell clusters in both the breeds. The results have shown differential expression of TLR mRNAs in various tissues between two divergent buffalo breeds with the highest difference in TLR expression profile seen in the skin, the largest portal of entry of pathogens, of Toda.

A rapid and highly reliable field-based LAMP assay of canine parvovirus.

UNLABELLED: Loop-mediated isothermal amplification (LAMP) is known as a rapid and reliable alternative to conventional single-step or nested PCR for detection of genomic DNA of various pathogens in clinical samples. In this study, LAMP assay was developed for canine parvovirus (CPV) and compared with single-step and nested PCR assays. Out of 50 fecal samples from dogs clinically suspected for CPV infections, 19 were found positive by single-step PCR, 22 by nested PCR and 26 by LAMP. LAMP products were subjected to restriction analysis and sequencing to check their specificity. LAMP assay turned out to be a rapid and fairly reproducible method, did not amplify other common canine pathogens and was more sensitive than nested PCR assay. Therefore, it can be regarded as a highly reliable method for routine field diagnosis of CPV infection. KEYWORDS: canine parvovirus; nested polymerase chain reaction; loop-mediated isothermal amplification; sensitivity; specificity.