Double and quadruple deletion mutant of EHV-1 is highly attenuated and induces optimal immune response.

Equid alphaherpesvirus 1 (EHV-1) infection causes significant health problems in equines. The EHV-1 infection leads to abortion storm in mares, respiratory disease and myeloencephalopathy. Despite the wide use of vaccines, the outbreaks of EHV-1 infections keep occurring globally, suggesting the need for the development of improved vaccines. Gene deletion attenuated mutant viruses could be a good candidate for the development of modified live vaccines. Here, we report the generation of mutant EHV-1 by deleting virulence (glycoprotein E & internal repeat 6; IR6) and immune evasive (pUL43 & pUL56) associated genes either individually or in combinations; and comprehensive evaluation of mutants through in vitro characterization followed by in vivo study in murine model to adjudge the attenuation of the virus and immune responses generated by mutants vis-à-vis wild type (wt) virus. The EHV-1 mutants with deletion of IR6 and gE genes (vToH-DMV) and four genes (i.e., gE, IR6, pUL43 and pUL56) (vToH-QMV) revealed a significant reduction in plaque size with minimal loss in replication efficiency in comparison to the wt virus. Further, in vivo studies showed virus attenuation adjudged through significant reduction in clinical signs, weight loss, gross and histopathological lesions in comparison to wt virus also revealed improved immune responses estimated through serum neutralization and flow cytometric analysis of CD4 + and CD8 + cell populations. Thus it can be concluded that EHV-1 mutants viz. vToH-DMV and vToH-QMV (novel combination) are promising vaccine candidates and qualify to be studied for adjudging the protective efficacy with wt virus challenge.

Tracking the phage trends: A comprehensive review of applications in therapy and food production.

In the present scenario, the challenge of emerging antimicrobial resistance is affecting human health globally. The increasing incidences of multidrug-resistant infections have become harder to treat, causing high morbidity, and mortality, and are posing extensive financial loss. Limited discovery of new antibiotic molecules has further complicated the situation and has forced researchers to think and explore alternatives to antibiotics. This has led to the resurgence of the bacteriophages as an effective alternative as they have a proven history in the Eastern world where lytic bacteriophages have been used since their first implementation over a century ago. To help researchers and clinicians towards strengthening bacteriophages as a more effective, safe, and economical therapeutic alternative, the present review provides an elaborate narrative about the important aspects of bacteriophages. It abridges the prerequisite essential requirements of phage therapy, the role of phage biobank, and the details of immune responses reported while using bacteriophages in the clinical trials/compassionate grounds by examining the up-to-date case reports and their effects on the human gut microbiome. This review also discusses the potential of bacteriophages as a biocontrol agent against food-borne diseases in the food industry and aquaculture, in addition to clinical therapy. It finishes with a discussion of the major challenges, as well as phage therapy and phage-mediated biocontrols future prospects.

Pathological and immunological protection induced by inactivated reverse genetics-based H3N8 equine influenza vaccine candidate in murine model.

Equine influenza (EI) is an important viral respiratory disease of equines caused by influenza A virus (IAV). The antigenic drift in IAVs necessitates regular updating and harmonization of vaccine strain with the circulating virus. The reverse genetics-based recombinant viruses could be easy instrument in generating vaccine against circulating virus in a quick and effective manner. Present study has been envisaged to evaluate the immunogenicity and protective efficacy of inactivated recombinant equine influenza virus (rgEIV) vaccine candidate having six segments from H1N1 virus (A/WSN/33/H1N1) and HA (hemaglutinin) and NA (neuraminidase) segments from H3N8 equine influenza virus [(A/eq/Jammu-Katra/06/08) of clade 2 of Florida sublineage] generated through reverse genetic engineering. BALB/c mice were immunized with inactivated rgEIV adjuvanted with aluminium hydroxide gel and challenged with H3N8 virus (A/eq/Jammu-Katra/06/08). The protective efficacy was evaluated through serology, cytokine profiling, clinical signs, gross and histopathological changes, immunohistochemistry and residual virus quantification. Immunizations induced robust humoral immune response as estimated through hemagglutination inhibition assay (HAI). The antibodies were isotyped and the predominant subclass was IgG1. The vaccine candidate produced mixed Th1 and Th2 responses through stimulation of IFN-γ, IL-2, IL-4 and IL-6 expression. Immunization protected mice against challenge as reflected through reduction in clinical signs and body weight loss, early recovery, mild pathological changes (gross and histopathological lesions) as evident through scoring of lesions, low residual virus in nasopharynx and lungs quantified through egg titration and quantitative reverse transcriptase PCR (qRT-PCR). The study demonstrates that inactivated recombinant EIV generated through reverse genetic approach provides equivalent protection to that observed with inactivated whole H3N8 EIV vaccine. Keywords: equine influenza; reverse genetics; vaccine; pathology; murine model.

Association of Myroides odoratimimus in immunocompromized piglets with post weaning multisystemic wasting syndrome.

AIM: To study the association of opportunistic infection due to Myroides odoratimimus in piglets immunocompromised by porcine circovirus type 2 (PCV2) infection. METHODS AND RESULTS: The clinical samples (n = 101) were analysed bacteriologically. The isolates were identified by their phenotypes and MALDI TOF-MS analysis as Myroides species. The phylogram constructed based on nucleotide sequences of the 16S rRNA gene showed identity (~99%) with the M. odoratimimus isolates. The minimum inhibitory concentration values for antibiotics revealed M. odoratimimus to be resistant against carbapenem, cephalosporins, aminoglycosides and fluoroquinolones. The presence of PCV2 in affected tissue samples was confirmed by amplification of the 565 bp region of ORF2 of the PCV2 genome. The topology of the phylogenetic tree grouped the PCV2 with cluster-2d. CONCLUSIONS: PCV2 being immunosuppressive in nature might have impaired the immunity thereby increasing the susceptibility of immunocompromised piglets to opportunistic pathogens such as M. odoratimimus leading to disease severity and high mortality. The M. odoratimimus isolates were found to be multidrug resistant and evidenced for uncertain clinical relevance and hence could act as hidden source of public health hazard. SIGNIFICANCE AND IMPACT OF THE STUDY: Myroides odoratimimus is a rarely reported human pathogen. We reported the incidence of infection due to seemingly rare isolates of M. odoratimimus causing an outbreak of pneumonia in piglets. This appears, to the best of authors' knowledge, to be the first outbreak due to Myroides recorded in animal clinical cases described in the literature.

Isolation and characterization of multidrug-resistant Leclercia species from animal clinical case.

Leclercia adecarboxylata, a Gram-negative bacillus of family Enterobacteriaceae, is an uncommonly identified pathogen isolated from environmental and clinical specimens. Most of the human infections are polymicrobial and commonly occur in immunocompromised hosts, although nosocomial infections in immunocompetent hosts have been documented. Here, we describe the case of isolation of Leclercia species as polymicrobial infection from bovine suffering from respiratory distress in Chhattisgarh state of India. The isolates were identified by their phenotypes, 16S rDNA sequencing and MALDI-TOF-MS. The isolate was found to be resistant to aminoglycosides and fluoroquinolone antibiotics and intermediate resistant to cephalosporins and evidenced for uncertain clinical relevance and could act as hidden source of public health hazard. SIGNIFICANCE AND IMPACT OF THE STUDY: Leclercia adecarboxylata is a rarely reported human pathogen. We report here the case from bovine suffering from respiratory distress; the sample yielded Leclercia species as polymicrobial culture. The isolate was found to be multidrug resistant and evidenced for uncertain clinical relevance and could act as hidden source of public health hazard. The limited literature available on this organism is reviewed, and the potential implications of findings are discussed. To the best of our knowledge, this is the first report of isolation and characterization of multidrug-resistant Leclercia species from animal clinical case from India.

Antigenic characterization of 52-55kDa protein isolated from Trypanosoma evansi and its application in detection of equine trypanosomosis.

Trypanosoma evansi is a haemo-protozoan parasite responsible for the disease surra, an economically important disease of wide range of domestic and wild animals. The present diagnostic methods using soluble antigens have inherent problems like lack of standardized and reproducible antigens, as well as ethical issues. This entails further efforts for search of defined antigenic molecules with satisfying sensitivity and specificity for sero-epidemiology of trypanosomosis. In present investigation, we have identified and purified 52-55kDa immuno-dominant protein cluster in molecular mass ranges by preparatory SDS-PAGE methods from T. evansi proteome. The purified protein was further characterized by hyper immune serum raised in rabbits and also further evaluated for its immunodiagnostic potential using experimentally infected horse serum samples by different immunological tests. The immunoblot, ELISA and dot blot assay using purified cluster in infected pooled serum samples showed detection of infection early as 10th days post infection till termination of experiment. The observations revealed that purified cluster is expressed not only at early stage but also persisted and detected throughout course of infection. Further, whole cell lysate antigen separated out and detected 141 spots by 2-D gel electrophoresis. The isoelectric focussing (PI) of 52-55kDa was determined in pH range between 6.9 and 7.5 along with two other cluster of proteins recognised by immune sera of ponies infected with T. evansi. MS/MS analysis of the purified protein cluster identified five proteins i.e. pyruvate kinase 1, beta tubulin, paraflagellar rod protein, alanine aminotransferase and variable surface glycoprotein showing homology to protein present in Trypanosome database. These identified proteins may be useful for development of vaccines and diagnostic targets against animal trypanosomosis.

First Draft Genome Sequence of Salmonella enterica Serovar Gallinarum Strain VTCCBAA614, Isolated from Chicken in India.

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum causes fowl typhoid (FT), which results in huge economic losses to poultry farmers in India. We report the draft genome sequence of Salmonella biovar Gallinarum strain VTCCBAA614, isolated from a chicken in an FT affected broiler flock.

Genetic characterization and phylogenetic analysis of host-range genes of Camelpox virus isolates from India.

Camelpox virus (CMLV), a close variant of variola virus (VARV) infects camels worldwide. The zoonotic infections reported from India signify the need to study the host-range genes-responsible for host tropism. We report sequence and phylogenetic analysis of five host-range genes: cytokine response modifier B (crmB), chemokine binding protein (ckbp), viral schlafen-like (v-slfn), myxomavirus T4-like (M-T4-like) and b5r of CMLVs isolated from outbreaks in India. Comparative analysis revealed that these genes are conserved among CMLVs and shared 94.5-100 % identity at both nucleotide (nt) and amino acid (aa) levels. All genes showed identity (59.3-98.4 %) with cowpox virus (CPXV) while three genes-crmB, ckbp and b5r showed similarity (92-96.5 %) with VARVs at both nt and aa levels. Interestingly, three consecutive serine residue insertions were observed in CKBP protein of CMLV-Delhi09 isolate which was similar to CPXV-BR and VACVs, besides five point mutations (K53Q, N67I, F84S, A127T and E182G) were also similar to zoonotic OPXVs. Further, few inconsistent point mutation(s) were also observed in other gene(s) among Indian CMLVs. These indicate that different strains of CMLVs are circulating in India and these mutations could play an important role in adaptation of CMLVs in humans. The phylogeny revealed clustering of all CMLVs together except CMLV-Delhi09 which grouped separately due to the presence of specific point mutations. However, the topology of the concatenated phylogeny showed close evolutionary relationship of CMLV with VARV and TATV followed by CPXV-RatGer09/1 from Germany. The availability of this genetic information will be useful in unveiling new strategies to control emerging zoonotic poxvirus infections.

Draft Genome Sequence of Pasteurella multocida subsp. multocida B:2 Strain VTCCBAA264 Isolated from Bubalus bubalis in North India.

The Pasteurella multocida subsp. multocida B:2 serotype causes hemorrhagic septicemia in bubalines with high morbidity and mortality in the Indian subcontinent. We report the draft genome sequence of Pasteurella multocida strain VTCCBAA264 isolated from the small-intestine of a buffalo calf that died of high fever.

Identification of immuno-dominant antigens of Trypanosoma evansi for detection of chronic trypanosomosis using experimentally infected equines.

Trypanosoma evansi is the most extensively distributed trypanosome responsible for disease called surra in livestock in many countries including frequent outbreaks in India. The prevalence of this disease is most commonly reported by standard parasitological detection methods (SPDM); however, antibody ELISA is being in practice by locally produced whole cell lysate (WCL) antigens in many countries. In the present investigation, we attempted to identify and purify immuno dominant, infection specific trypanosome antigens from T. evansi proteome using experimentally infected equine serum by immuno blot. Three immuno dominant clusters of proteins i.e. 62-66 kDa, 52-55 kDa and 41-43 kDa were identified based on their consistent reactivity with donkey sequential serum experimentally infected T. evansi up to 280 days post infection (dpi). The protein cluster of 62-66 kDa was purified in bulk in native form and comparatively evaluated with whole cell lysate antigen (WCL). ELISA and immuno blot showed that polypeptide of this cluster is 100% sensitive in detection of early and chronic infection. Further, this protein cluster was also found immuno reactive against hyper immune serum raised against predominantly 66 kDa exo antigen, revealed that this is a common immunodominant moieties in proteome and secretome of T. evansi.

Genetic Analysis of the Neuraminidase (NA) Gene of Equine Influenza Virus (H3N8) from Epizootic of 2008-2009 in India.

The neuraminidase (NA) gene sequences of four Indian equine influenza viruses (EIVs) isolated from epizootic in 2008 and 2009 were analyzed. The phylogenetic relationship and selection pressure of NA genes were established in comparison to other EIVs circulating worldwide along with the domains and motifs of the encoded protein to find out the significance of mutational changes. Among Indian isolates, two amino acid (aa) changes each in Mysore/12/08 (Asn67Tyr & Asp396Gly), Gopeshwar/1/09 (Ile49Val & Asp396Gly), and Uttarkashi/1/09 (Ile49Val & Asp396Gly) isolates were observed in respect to Jammu-Katra/06/08 isolate. Amino acid (aa) sequence analysis also revealed five consistent aa residue changes viz, Gly/Arg40Glu, Tyr66His, Val191Ile, Val209Ile and Asp235Asn in Asian including Indian isolates, Spain/07 and Spain/09 isolates in comparison to other EIVs circulating worldwide. The topology of the phylogenetic tree revealed that the Indian, Chinese, Mongolian and Kazakhstan isolates together formed a subgroup with Yokohama/10 isolate. Spain/07 & Spain/09 isolates showed closest clustering with Asian isolates. This indicates that non-synonymous mutations in Asian isolates with temporal pattern originating from Spain/07, led to the subgroup of the Asian isolates within Florida clade 2 sublineage. The analysis of the predicted secondary structure has not shown any significant difference in the NA proteins of all Indian isolates. Fixed-effects likelihood (FEL) analysis of the selection pressure revealed three codons (43, 355 & 434) under positive selection pressure. The overall evolutionary changes (ω value) of 3.4 indicates NA gene to be under strong selection pressure. Further, seven putative N-glycosylation sites were observed in the NA protein. The mapping of specific aa changes, their mutational and functional analysis need to be carried out to ascertain their role in pathogenecity of the virus.

Genetic analysis of the matrix and non-structural genes of equine influenza virus (H3N8) from epizootic of 2008-2009 in India.

India faced an epizootic of equine influenza in 2008-2009. The isolated viruses were typed as H3N8 and grouped with the clade 2 viruses of Florida sublineage on the basis of haemagglutinin (HA) gene sequence analysis. This report describes the genetic analysis and selection pressure of matrix (M) and non-structural 1 (NS1) genes of the Indian isolates. All isolates shared 98.41% and 99.54% homology with other clade 2 viruses of Asian origin for M1 and M2 amino acid (aa) sequences, respectively. There were 3 and 4 unique aa residue changes respectively in M1 and M2 proteins in all Asian isolates. Phylogenetic analysis revealed clustering of Indian and Chinese isolates in a separate group designated here as Asian clade for M gene. Indian and Chinese isolates shared homology ranging from 98.17% to 99.08% at aa level. The M and NS1 genes were under negative selection pressure with estimated magnitude of pressure (ω) 0.054, 0.581 and 0.30 for M1, M2 and NS1, respectively.

Zoonotic cases of camelpox infection in India.

This study reports the first conclusive evidence of zoonotic camelpox virus (CMLV) infection in humans associated with outbreaks in dromedarian camels (Camelus dromedaries) in northwest region of India during 2009. CMLV infection is usually restricted to camels and causes localised skin lesions but occasionally leads to generalised form of disease. However, the present outbreak involved camel handlers and attendants with clinical manifestations such as papules, vesicles, ulceration and finally scabs over fingers and hands. In camels, the pock-like lesions were distributed over the hairless parts of the body. On the basis of clinical and epidemiological features coupled with serological tests and molecular characterization of the causative agent, CMLV zoonosis was confirmed in three human cases. Clinical samples such as skin scabs/swabs and blood collected from affected animals and humans were analysed initially, for the presence of CMLV-specific antigen and antibodies by counter immunoelectrophoresis (CIE); serum neutralization test (SNT); plaque-reduction neutralization test (PRNT) and indirect immunoperoxidase test which was later confirmed by amplification of CMLV-specific ankyrin repeat protein (C18L) gene. Virus isolation was successful only from samples collected from camels. Further, sequence analyses based on three full-length envelope protein genes (A27L, H3L and D8L) revealed 95.2-99.8% and 93.1-99.3% homology with other Orthopoxviruses at nucleotide and amino acid levels, respectively. Phylogram of the three genes revealed a close relationship of CMLV with Variola virus (VARV). Considering the emerging and re-emerging nature of the virus, its genetic relatedness to VARV, zoonotic potential and productivity losses in camels; the control measures are imperative in curtailing economic and public health impact of the disease. This is the first instance of laboratory confirmed camelpox zoonosis in India.

Equine influenza outbreak in India (2008-09): virus isolation, sero-epidemiology and phylogenetic analysis of HA gene.

An outbreak of equine influenza (EI) was reported in India in June, 2008 after a gap of two decades. The outbreak started from Jammu and Kashmir (Katra), northern state of India and spread to the other parts of the country affecting equines in 11 states. The virus (H3N8) was isolated from nasal swabs obtained from clinical cases in various locations in the country including Katra (Jammu and Kashmir), Mysore (Karnataka) and Ahmedabad (Gujarat) using embryonated chicken eggs. The virus isolates were identified as H3N8 by haemagglutination inhibition (HI) test titration with standard serum and by sequencing of full-length haemagglutinin (HA) gene and partial sequence of neuraminidase (NA) gene. Paired serum samples (n=271) showing more than fourfold rise in antibody titres tested from 11 states confirmed equine influenza. Serum samples (n=2517) of equines from 13 states of the country screened by HI test revealed 687 (26.85%) samples positive for antibodies to EI (H3N8). Phylogenetic analysis of the haemagglutinin (HA) gene confirmed the virus to be closely related to Clade 2 of the Florida sublineage in American lineage. Comparison of deduced amino acid sequence of HA gene with EIV isolates from various lineages showed substitutions in the antigenic regions C and D. HA1 gene sequence had highest amino acid identity to A/eq/Gansu/7/08 and A/eq/Hubei/6/08 isolates from China and Inner-Mongolia isolate, while the complete HA gene sequence was closest to A/eq/A/eq/Newmarket/5/03, A/eq/Bari/05 and A/eq/Kentucky/05/02 isolates. Recent outbreaks of Mongolia, China and India by clade 2 EI viruses imply their predominance in Asia in addition to Europe.

Molecular appraisal of Indian animal isolates of Echinococcus granulosus.

BACKGROUND & OBJECTIVE: Cystic echinococcosis (CE) has a wide host range and distinct entities, not only reflected phenotypically but also by genotypic variation. Considering this fact, this study was undertaken to characterize the Indian isolates of Echinococcus granulosus to find out difference between Indian cattle, buffalo and sheep isolates on the basis of random amplification of polymorphic DNA (RAPD) PCR and PCR mediated restriction fragment length polymorphism (PCRRFLP) of internal transcribed spacer gene 1 (ITS1). METHODS: A total of 22 isolates of E. granulosus obtained from Indian cattle, buffalo and sheep (December 2004 - November 2005) were analysed by 26 random primers of 8-10 mers. After isolation of protoscoleces from fertile cyst, DNA was extracted, quantified and amplified by random primers. Internal transcribed spacer gene 1 (ITS1) was amplified using specific primer and digested by two restriction enzymes (Msp1 and Rsa1). RESULTS: Of the 26 primers, only two primers (5'ACC TGG ACA C3' and 5' TCA TCC GAG G3') could discriminate cattle, buffalo and sheep isolates collected from eastern part of India. Samples were further analysed by PCR mediated RFLP of internal transcribed spacer gene1 (ITS1) using two restriction enzymes (Msp1 and Rsa1). No ITS1 variants could be detected. INTERPRETATION & CONCLUSION: Our findings showed genotypic variation among Indian animal isolates of E. granulosus on the basis of RAPD fingerprinting.

Cloning, sequencing and expression of cDNA of bovine neutrophil beta-defensin from water buffalo (Bubalus bubalis).

Neutrophil beta-defensins have been identified as naturally occurring potent antibacterial cationic peptides serving as effector molecules of innate immunity that provide a first line of defence against pathogens. Considering the broad-spectrum antimicrobial activity against microorganisms and role in innate immunity of the neutrophil beta-defensins, it has been characterized in many livestock species including cattle, sheep, caprine and porcines. Here we report the isolation, cloning, sequencing and expression of precursor bovine neutrophil beta-defensin isolated from Indian water buffalo. Full-length cDNA was amplified using reverse transcription polymerase chain reaction (RT-PCR). The cDNA contained an open reading frame of 192 bp encoding a putative polypeptide of 63 amino acids. Deduced amino acid sequence of buffalo BNBD4 showed varying amino acid identity with the published sequences of related beta-defensins of other domestic ruminant species ranging from 67.18 to 79.68%. Recombinant buffalo defensin was produced in Escherichia coli as fusion protein.

Genotypic characterisation of Indian cattle, buffalo and sheep isolates of Echinococcus granulosus.

Twelve isolates of Echinococcus granulosus, collected from domestic animals, including cattle, buffalo and sheep were analysed for DNA nucleotide sequence variation within mitochondrial cytochrome c oxidase I (coxI), NADH dehydrogenase subunit I (nadI) and internal transcribed spacer gene I (ITS1). After analysis of sequence information this was found that the fragment size of ITS1 of buffalo isolate was more in comparison to cattle and sheep isolates. Based on the nadI genotype this was found that Indian cattle, buffalo and sheep isolates could be grouped into E. granulosus sensu stricto. Based on coxI genotype two sheep isolates and one buffalo isolate were homologous to G2 genotype. Rests of the isolates were microvariants of G2 genotype. Presence of G2 genotype in buffalo is the first report of this genotype from this host.

Cloning and sequencing of Indian water buffalo interleukin-18 cDNA.

Summary Full-length cDNA (582 bp) of the interleukin-18 (IL-18) gene of the Indian water buffalo (Bubalus bubalis) was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequenced. The deduced amino acid sequence has 99% and 95% similarity with the IL-18 sequences of cattle and sheep, respectively. There are two amino acid substitutions at positions 132 and 182 in buffalo IL-18 compared with that of cattle. Phylogenetic analysis showed that the IL-18 sequence of fish forms a different lineage and is most divergent from that of cattle, buffalo, sheep, pig, dog, horse, human, monkey, mouse, rat and chicken.

Thermal analysis of some analogous thiols and their palladium complexes.

Differential thermal analysis (DTA) and thermogravimetric analysis (TGA) have been conducted on several analogous heterocyclic thiols and their complexes with palladium in air, over the temperature range of 25-800 degrees . A rough order of thermal stability of the ligands and the complexes has been deduced.