Immunohistochemical and molecular detection of natural cases of bovine rotavirus and coronavirus infection causing enteritis in dairy calves.

Bovine rotavirus (BRoV) and bovine coronavirus (BCoV) are major enteric viral pathogens responsible for calve diarrhoea. They are widespread both in dairy and beef cattle throughout the world and causing huge economic losses. The diagnosis of these agents is very difficult due to non-specific nature of lesions and the involvement of some intrinsic and extrinsic risk factors. We performed postmortem of 45 calves, which was below three months of age. Out of 45 necropscid calves, three (6.66%) cases were positive for BRoV and four (8.88%) cases were found positive for BCoV, screened by reverse transcriptase polymerase chain reaction (RT-PCR). Further RT-PCR positive cases were confirmed by immunohistochemistry (IHC) in paraffin-embedded intestinal tissue sections. Three cases of enteritis caused by BRoV showed the hallmark lesions of the shortening and fusion of villi, denudation and infiltration of mononuclear cells in the lamina propria. The BRoV antigen distribution was prominent within the lining epithelium of the villi, peyer's patches in the ileum and strong immunoreactions in the lymphocytes and some macrophages of the mesenteric lymph nodes. Four cases in which BCoV was detected, grossly lesions characterized by colonic mucosa covered with thick, fibrinous and diphtheritic membrane. Histopathologically, jejunum showed skipping lesion of micro-abscesses in crypts. The BCoV antigen distribution was prominent within the necrotic crypts in the jejunum and cryptic micro-abscesses in the colon and ileum. It is the first report of BRoV and BCoV antigen demonstration in the jejunum, colon, ileum, Peyer's patches and mesenteric lymph nodes of naturally infected calves from India by using IHC.

Co-infection of Newcastle disease virus genotype XIII with low pathogenic avian influenza exacerbates clinical outcome of Newcastle disease in vaccinated layer poultry flocks.

Newcastle disease (ND) and avian influenza (AI) are economically important infectious diseases of poultry. Sometime, concomitant secondary viral/or bacterial infections significantly alters the pathobiology of ND and AI in poultry. As of now, the disease patterns and dynamics of co-infections caused by ND virus (NDV, genotype XIII) and Low Pathogenic AI viruses (LPAI, H9N2) are explicitly elusive. Thus, we examined the clinicopathological disease conditions due to these two economically important viruses to understand the complex disease outcomes by virus-virus interactions in vaccinated flocks. The findings of clinicopathological and molecular investigations carried on 37 commercial ND vaccinated poultry flocks revealed simultaneous circulation of NDV and AIV in same flock/bird. Further, molecular characterization of hemagglutinin (HA) and neuraminidase (NA) genes confirmed that all the identified AIVs were of low pathogenicity H9N2 subtype and fusion (F) gene analysis of detected NDVs belong to NDV class II, genotype XIII, a virulent type. The NDV and H9N2 alone or co-infected flocks (NDV + LPAI) exhibit clinical signs and lesions similar to that of virulent NDV except the degree of severity, which was higher in H9N2-NDV co-infected flocks. Additionally, avian pathogenic E. coli and mycoplasma infections were detected in majority of the ailing/dead birds from the co-infected flocks during progression of the clinical disease. Overall, the findings highlight the multi-factorial disease complexity in commercial poultry and suggest the importance of NDV genotype XIII in intensifying the clinical disease in vaccinated birds.

Changes of haemogram and serum biochemistry in neonatal piglet diarrhoea associated with porcine rotavirus type A.

Porcine rotavirus type A (RVA) is a major cause of neonatal piglet mortality in India. The effect of the disease on haemogram and serum biochemical profile is not well established in piglets. Accordingly, we assessed the haemogram and serum biochemical profile in the neonatal piglet diarrhoea with RVA infection (n = 17). The diagnosis of RVA was confirmed using RNA-polyacrylamide gel electrophoresis (RNA-PAGE), commercially available enzyme-linked immunosorbent assay (ELISA) kit and reverse transcription-polymerase chain reaction (RT-PCR). Non-infected healthy piglets (n = 6) served as control. The concentrations of total protein, albumin, alanine amino transaminase (ALT), aspartate amino transaminase (AST), blood urea nitrogen (BUN) and creatinine in serum were measured by spectrophotometric method. Haemogram was done in the blood using sodium ethylenediaminetetraacetic acid (Na2 EDTA) as anticoagulant. The mean values of total protein, albumin and globulin concentrations were significantly (P < 0.001) decreased and concentrations of ALT, AST, BUN and creatinine were significantly increased (P < 0.001) in the RVA-infected piglets. Haemogram showed marked haemoconcentration (P < 0.001), leukopenia (P < 0.01) and neutropenia (P < 0.01) in the presence of RVA infection than healthy piglets. The results indicated a possible extra-intestinal spread of RVA in piglets during neonatal diarrhoea. The finding might be helpful to clinicians and while treating such type of clinical cases, incorporation of organ protective drugs will be helpful for better response in the treatment schedule.

Spatial molecular epidemiology of carbapenem-resistant and New Delhi metallo beta-lactamase (blaNDM)-producing Escherichia coli in the piglets of organized farms in India.

AIM: A cross-sectional study was conducted in 10 government-organized pig farms between 2014 and 2016 representing seven states of India to understand the epidemiology of carbapenem resistance in the Escherichia coli. METHODS AND RESULTS: In this study, fecal sample (n = 673) from non-diarrheic (n = 501) and diarrheic (n = 172) piglets were processed for isolation of carbapenem resistant E. coli. Of 673, E. coli isolate (n = 112) was genotyped for confirming the carbapenem resistance and associated virulence factors. Of the 112 isolates, 23 were phenotypically resistant to carbapenem and 8 were carrying the New Delhi metallo beta-lactamase (blaNDM) gene. The carbapenem-resistant isolates also produced extended spectrum beta-lactamases and were multidrug resistant. The PCR-based pathotyping revealed the presence of stx1, stx2, eae and hlyA genes. The enterobacterial repetitive intergenic consensus PCR dendrogram analysis of the isolates yielded three distinct clusters. The statistical analysis revealed no association between carriages of carbapenem-resistant E. coli in different breed of piglets however, location, sex, health status of piglets and age showed significant difference. The spatial analysis with SaTScan helped in identification of carbapenem-resistant clusters. CONCLUSIONS: The presence of carbapenem resistant E. coli isolates with virulence genes in the piglet poses a potential public health risk through possible access and spread via the food chain and environment. Efflux pump may also play an important role in carbapenem resistance in piglet E. coli isolates. Furthermore, identification of risk factors in relation to spatial clusters will help in designing preventive strategies for reducing the risk of spread of carbapenem resistant bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: 1. Piglets harbor carbapenem resistant E. coli and have great public health significance. 2. Apart from carbapenemase, efflux pump is also important for carbapenem resistance. 3. This is the first report of blaNDM in the piglets from India.

Evidence for Occurrence of Human group B rotavirus in Central India Based on Characterization of NSP2 Gene.

Human group B rotavirus (HuGBR) was first described as the causative agent of severe gastroenteritis, affecting millions of people in China during 1982-1983. In spite of serological evidences for the presence of HuGBR in many countries of the world, the virus has only been detected from China, Bangladesh and some parts of India. The present study describes a HuGBR (designated as MP-1 isolate) which was confirmed in an adult patient suffering from gastroenteritis in 2008 in Madhya Pradesh, central India. The RNA electrophoresis in polyacrylamide gel (RNA-PAGE) and NSP2 gene based RT-PCR assays and later sequencing was used to confirm the isolate. The nucleotide and deduced amino acid sequences of this HuGBR (MP-1) isolate were analyzed and their relationship with corresponding gene of other Indian, Bangladeshi and Chinese HuGBR and animal group B rotaviruses (AnGBR) was determined. The isolate showed a typical RNA banding pattern of 4:2:2:3 in RNA-PAGE which was indicative of group B rotaviruses (GBR). The sequence comparison of MP-1 isolate with NSP2 gene revealed that MP-1 isolate had 98.6 and 97.7% nucleotide sequence homology and 93.8% amino acid similarity with Bang373 and CAL-1 strains, respectively. The nucleotide and amino acid sequence similarity of MP-1 isolate with one of the Chinese ADRV (WH-1) strain was 92.8 and 92.5%, respectively. While sequence homology with another Chinese strain ADRV (J19) was considerably lower (45.6 and 48.3%, respectively). The percent identity with AnGBRs (porcine and murine) was also lower at nucleotide and amino acid level (66 to 80%). The phylogenetic analysis suggested that MP-1 isolate is closer to Bangladeshi (Bang373) as compared to Indian strain (CAL-1). Our findings indicated that MP-1 isolate might have originated from a common ancestral HuGBR virus but distinct from AnGBR lineage. Occurrence of GBR in other parts of India warrants further epidemiological and molecular studies to develop effective control strategies for GBR infection in adults as well as children.

Production and characterization of monoclonal antibodies against NS1 protein of H5N1 avian influenza virus.

A panel of 11 murine monoclonal antibodies (MAbs) was generated against NS1 (non-structural) protein of highly pathogenic avian influenza H5N1 virus using whole viral antigen as immunogen and as antigen in the preliminary screening of clones followed by a more specific screening through recombinant NS1 protein-based ELISA. Isotyping showed two clones 10E2 (IgG2b) and 5H8 (IgG3) to be IgG type and 6B9 and 4C4 to be IgA type, while the rest could not be isotyped due to weak reactivity with NS1 antigen. The subclone 10E2E7G3 was selected for subsequent work, which showed comparatively higher OD in NS1-based ELISA. The reactivity of 10E2E7G3 with H5N1 virus as well as recombinant NS1 protein was confirmed in Western blot analysis. The anti-NS1 MAbs produced in the present work may be valuable in developing an immunodiagnostic assay as a DIVA test for differentiating infected from vaccinated birds in AIV.

Detection of Rotavirus Infection in Bovine Calves by RNA-PAGE and RT-PCR.

A total of 128 diarrhoeic faecal samples were collected from cattle and buffalo calves from Pantnagar and Dehradun during winter months. Of the 110 cattle calves screened by RNA-PAGE, rotavirus was detected in 13 samples (11.81%) while no sample from buffalo calves was found positive. All samples were found to have long electropherotype and two distinct electropherotypes having segment variation were observed. The overall prevalence of rotavirus was 10.15% (13/128). RT-PCR targeting group specific VP6 gene confirmed Group A rotavirus in 10 out of 13 samples, while three samples remained un-groupable.

Immunogenicity and protective efficacy of the E. coli-expressed domain III of Japanese encephalitis virus envelope protein in mice.

Domain III of Japanese encephalitis virus (JEV) envelope protein (E-DIII) was synthesized in E. coli as a fusion protein containing maltose-binding protein (MBP-E-DIII) or six contiguous histidine residues (His-E-DIII) at its N-terminus. MBP-E-DIII was found both in the soluble as well as the insoluble fraction of the bacterial lysate, while His-E-DIII was found exclusively in the inclusion bodies. These purified proteins were examined in mice for their immunogenicity in presence of an aluminium hydroxide based-adjuvant Alhydrogel and Freund's adjuvant. While both proteins generated anti-JEV antibodies that neutralized JEV activity in vitro, His-E-DIII generated higher antibody titers than MBP-E-DIII. Mice immunized with His-E-DIII in presence of Alhydrogel generated antibody titers similar to those induced by the commercial vaccine and protected mice against lethal JEV challenge.