Evaluation of immunogenicity and protective efficacy of bacteriophage conjugated haemagglutinin based subunit vaccine against equine influenza virus in a murine model.

Equine influenza (EI) is a highly contagious acute respiratory disease of equines caused by the H3N8 subtype of Influenza A virus i.e. equine influenza virus (EIV). Vaccination is an important and effective tool for the control of EI in equines. Most of the commercial influenza vaccines are produced in embryonated hen's eggs which has several inherent disadvantages. Hence, subunit vaccine based on recombinant haemagglutinin (HA) antigen, being the most important envelope glycoprotein has been extensively exploited for generating protective immune responses, against influenza A and B viruses. We hypothesized that novel vaccine formulation using baculovirus expressed recombinant HA1 (rHA1) protein coupled with bacteriophage will generate strong protective immune response against EIV. In the present study, the recombinant HA1 protein was produced in insect cells using recombinant baculovirus having cloned HA gene of EIV (Florida clade 2 sublineage) and the purified rHA1 was chemically coupled with bacteriophage using a crosslinker to produce rHA1-phage vaccine candidate. The protective efficacy of vaccine preparations of rHA1-phage conjugate and only rHA1 proteins were evaluated in mouse model through assessing serology, cytokine profiling, clinical signs, gross and histopathological changes, immunohistochemistry, and virus quantification. Immunization of vaccine preparations have stimulated moderate antibody response (ELISA titres-5760 ± 640 and 11,520 ± 1280 for rHA1 and rHA1-phage, respectively at 42 dpi) and elicited strong interferon (IFN)-γ expression levels after three immunizations of vaccine candidates. The immunized BALB/c mice were protected against challenge with wild EIV and resulted in reduced clinical signs and body weight loss, reduced pathological changes, decreased EIV antigen distribution, and restricted EIV replication in lungs and nasopharynx. In conclusion, the immune responses with moderate antibody titer and significantly higher cytokine responses generated by the rHA1-phage vaccine preparation without any adjuvant could be a novel vaccine candidate for quick vaccine preparation through further trials of vaccine in the natural host.

Knowledge, awareness and perception about equine glanders among veterinarians and medical professionals in India.

Glanders is a highly infectious and notifiable disease of equines that occurs due to Burkholderia mallei. In India, glanders re-emerged in 2006 and thereafter regular outbreaks have been reported in various states (n = 14). Frequent and prolonged contact with equids with glanders may transmit B. mallei infection to humans. This study was designed to learn more about the Knowledge, Awareness and Perception (KAP) of veterinarians, para veterinarians, and physicians about equine glanders, which will help in enhancing the nation-wide glanders eradication programme. A total of 165 respondent's from 11 Indian states and one union territory were surveyed. Most of the respondents (n = 160) were from equine glanders affected or endemic states. Knowledge gap analysis revealed that 40.3 and 22% of the participants were not aware of government regulations and the transmission of glanders, respectively. These are major concerns given the wide spread occurrence of disease in the country. Awareness test on glanders revealed that 65(39.4%) participants would collect biological samples for laboratory confirmation, 67(40.6%) would inform the concerned authorities and 106 (64.2%) replied that they would eliminate the glanders infected equines. Analysis of perception towards equine glanders showed that majority of the participants (n = 113, 68.4%) observed that equine keepers were reluctant to disclose the clinical symptoms of B. mallei infection. Furthermore, non-co-operation and unwillingness by superiors (33.9%), financial (31%), administrative (28.4%), and technical limitations (27.8%) were major constraints under the perception analysis. This study reveals that veterinarians need to be educated on governmental policies and guidelines on equine glanders with regular training and awareness programs. Intersectoral co-ordination to investigate human glanders is also needed.

miRNA profiling of primary lamb testicle cells infected with lumpy skin disease virus.

In this study, miRNA profiling of cells infected with lumpy skin disease virus (LSDV) was conducted for the first time. When compared to mock-infected cells, LSDV-infected primary lamb testicle (LT) cells showed dysregulation of 64, 85, and 85 miRNAs at 12 hours postinfection (hpi), 48 hpi, and 72 hpi, respectively. While some of these miRNAs were found to be dysregulated at a particular time point following LSDV infection, others were dysregulated at all three time points. Analysis of the differentially expressed miRNA-mRNA interaction networks, Gene Ontology analysis of the predicted targets, and KEGG analysis of highly enriched pathways revealed several cellular factors/pathways involved in protein/ion/enzyme binding, cell differentiation, movement of subcellular components, calcium reabsorption, aldosterone synthesis and secretion, and melanogenesis. Some selected upregulated (oar-mir-379-5p, oar-let-7d, Chr10-18769, Chr2_5162 and oar-miR-493-5p) and downregulated (ChrX-33741, Chr3_8257 and Chr26_32680) miRNAs were further confirmed by quantitative real-time PCR. These findings contribute to our understanding of virus replication, virus-host interactions, and disease pathogenesis, and the differentially expressed miRNAs and their cellular targets may serve as biomarkers as well as novel targets for therapeutic intervention against LSDV.

Attenuation of equine herpesvirus 1 through deletion of gE gene and its pathological evaluation in murine model.

Equine herpesvirus 1 (EHV1) infection is a global health problem in equines and the virus is responsible for abortions, respiratory disease and myeloencephalitis in horses. Disease management requires proper biosecurity and immunoprophylactic measures. Vaccines strengthening both arms of immunity are essential for proper control and there has been a continuous focus in this area for generation of better vaccines. Here we report construction of bacterial artificial chromosome (BAC) clone of EHV-1 strain Tohana for mutagenesis of the virus and generation of gE gene deletion mutant EHV1. The BAC clone was generated by inserting the mini-F plasmid replacing ORF71 of EHV1 and transforming into E. coli for generation of EHV1-BAC. The infectious virus was regenerated from EHV-1 BAC DNA in RK13 cells. To check utility of EHV1-BAC, we have generated mutant EHV1 by deleting the virulence-associated gE gene. The mutant virus (vToHΔgE) showed significantly reduced plaque size without affecting replication efficiency. Pathological evaluation of lesions in BALB/c mice infected with vToHΔgE revealed reduction in clinical signs and pathology in comparison to the wild-type virus. Generation of infectious BAC of EHV1 and its usage in construction of attenuated viruses shows potential of the technology for development of indigenous modified live vaccine for EHV1. Keywords: quine herpesvirus 1; bacterial artificial chromosome (BAC); mutation; glycoprotein E; vaccine.

Role of ROCK signaling in virus replication.

Rho-associated protein kinase (ROCK) is a serine-threonine kinase and is a major downstream effector of the small GTPaseRhoA. Upon activation, Rho/ROCK cell signaling pathway regulates cell morphology, polarity, and cytoskeletal remodeling. Recent years have highlighted the role of ROCK signaling pathway in the replication of diverse group of viruses. Cell contractions and membrane blebbing induced by certain group of viruses is mediated via ROCK signaling and facilitates virus replication by sequestration of cellular factors and anchoring them at replication sites (viral factories). Besides, ROCK signaling also stabilizes the nascent viral mRNA for its efficient transcription and translation and, regulates trafficking of the viral proteins. In addition, ROCK signaling is also involved in modulating the immune response to viral infections. This review describes the regulation of virus replication by ROCK signaling with the basic aim of defining it as a target for the development of novel antiviral therapeutics.

Comparative Genome Analysis of 19 Trueperella pyogenes Strains Originating from Different Animal Species Reveal a Genetically Diverse Open Pan-Genome.

Trueperella pyogenes is a Gram-positive opportunistic pathogen that causes severe cases of mastitis, metritis, and pneumonia in a wide range of animals, resulting in significant economic losses. Although little is known about the virulence factors involved in the disease pathogenesis, a comprehensive comparative genome analysis of T. pyogenes genomes has not been performed till date. Hence, present investigation was carried out to characterize and compare 19 T. pyogenes genomes originating in different geographical origins including the draftgenome of the first Indian origin strain T. pyogenes Bu5. Additionally, candidate virulence determinants that could be crucial for their pathogenesis were also detected and analyzed by using various bioinformatics tools. The pan-genome calculations revealed an open pan-genome of T. pyogenes. In addition, an inventory of virulence related genes, 190 genomic islands, 31 prophage sequences, and 40 antibiotic resistance genes that could play a significant role in organism's pathogenicity were detected. The core-genome based phylogeny of T. pyogenes demonstrates a polyphyletic, host-associated group with a high degree of genomic diversity. The identified core-genome can be further used for screening of drug and vaccine targets. The investigation has provided unique insights into pan-genome, virulome, mobiliome, and resistome of T. pyogenes genomes and laid the foundation for future investigations.

Characterization of immunoglobulin and cytokine responses in Burkholderia mallei infected equids.

Burkholderia mallei causes a highly fatal infectious disease in equines known as glanders. It is one of the OIE listed notifiable diseases, which entails strict control policy measures once B. mallei infection is confirmed in the susceptible hosts. Humans, especially equine handlers, veterinary professionals and laboratory workers are at greater risk to acquire the B. mallei infection directly through prolonged contact with glanderous equines, and indirectly through unprotected handling of B. mallei contaminated materials. Further, natural resistance of B. mallei to multiple antibiotics, aerosol transmission, lack of effective vaccine and treatment make this organism a potential agent of biological warfare. Results of experimental B. mallei infection in mouse and non-human primates and immunization with live attenuated B. mallei strains demonstrated that activation of early innate and adaptive immune responses play a critical role in controlling B. mallei infection. However, the immune response elicited by the primary hosts (equids) B. mallei infection is poorly understood. Therefore, we aimed to investigate immune responses in glanders affected horses (n = 23) and mules (n = 1). In this study, chronically infected equids showed strong humoral responses (IgM, IgG and IgA) specific to B. mallei type 6 secretory proteins such as Hcp1, TssA and TssB. The infected equids also elicited robust cellular responses characterized by significantly elevated levels of IFN-γ, TNF-α, IL-12, IL-17 and IL-6 in PBMCs. In addition, stimulation of equine PBMCs by Hcp1 resulted in the further elevation of these cytokines. Thus, the present study indicated that antibody response and T helper cell (Th) type 1-associated cytokines were the salient features of chronic B. mallei infection in horses. The immune responses also suggest further evaluation of these proteins as potential vaccine candidates.

Comparative genome analysis of Salmonella enterica serovar Gallinarum biovars Pullorum and Gallinarum decodes strain specific genes.

Salmonella enterica serovar Gallinarum biovar Pullorum (bvP) and biovar Gallinarum (bvG) are the etiological agents of pullorum disease (PD) and fowl typhoid (FT) respectively, which cause huge economic losses to poultry industry especially in developing countries including India. Vaccination and biosecurity measures are currently being employed to control and reduce the S. Gallinarum infections. High endemicity, poor implementation of hygiene and lack of effective vaccines pose challenges in prevention and control of disease in intensively maintained poultry flocks. Comparative genome analysis unravels similarities and dissimilarities thus facilitating identification of genomic features that aids in pathogenesis, niche adaptation and in tracing of evolutionary history. The present investigation was carried out to assess the genotypic differences amongst S.enterica serovar Gallinarum strains including Indian strain S. Gallinarum Sal40 VTCCBAA614. The comparative genome analysis revealed an open pan-genome consisting of 5091 coding sequence (CDS) with 3270 CDS belonging to core-genome, 1254 CDS to dispensable genome and strain specific genes i.e. singletons ranging from 3 to 102 amongst the analyzed strains. Moreover, the investigated strains exhibited diversity in genomic features such as virulence factors, genomic islands, prophage regions, toxin-antitoxin cassettes, and acquired antimicrobial resistance genes. Core genome identified in the study can give important leads in the direction of design of rapid and reliable diagnostics, and vaccine design for effective infection control as well as eradication. Additionally, the identified genetic differences among the S. enterica serovar Gallinarum strains could be used for bacterial typing, structure based inhibitor development by future experimental investigations on the data generated.

Cholesterol Loaded Cyclodextrin Supplementation Enhances the Cholesterol-to-Phospholipid Ratio and Diminishes Oxidative Stress in Jack Spermatozoa During Cryopreservation.

The present study was conducted with the hypothesis that addition of cholesterol to the extender would stabilize the sperm membranes by increasing the cholesterol-to-phospholipid (C:P) ratio and would result in an improved post-thaw semen quality and reduce oxidative stress in the jack (Martina franca) semen. Forty-eight ejaculates from six jacks were collected and analyzed for the present study. The freshly collected semen sample of each jack stallion was divided into five equal fractions after addition of the primary extender without cholesterol-loaded cyclodextrin (CLC) (C) and with 1, 1.5, 2, and 3 mg/mL CLC to obtain 120 × 106 sperm/mL spermatozoa concentration. The semen was cryopreserved using customized freezing protocols. Evaluation of seminal parameters, the C:P ratio, and the oxidative status of jack spermatozoa was analyzed at all stages of cryopreservation. The oxidative status in the jack semen was evaluated by measuring malondialdehyde, glutathione and total antioxidant capacity levels. The results indicated that the mean percent values for various seminal quality parameters and the oxidative parameters were found to be significantly higher (P < .05) in CLC-treated groups with the highest values for 2 mg of CLC/120 × 106 spermatozoa. In conclusion, the present study revealed that the supplementation of CLC before cryopreservation has significantly reduced the oxidative stress and also increased the C:P ratio during semen cryopreservation process. Furthermore, a reduction in lipid peroxidation levels, reduced damage to the sperm plasma and acrosome membranes and improvement in the post-thaw sperm integrity as well as stability were recorded.

Leaf Extract of Aerva javanica Suppresses Excessive Growth of Granulation Tissue in Horses.

Wound healing in horses is complicated by the excessive growth of granulation tissue, commonly known as proud flesh and is similar to keloids in human beings. At present, there is no satisfactory treatment for proud flesh in horses. In this study, we, for the first time, demonstrated that leaf extract of Aerva javanica suppresses excessive growth of granulation tissue in horses. Many plant flavonoids are claimed to have antiproliferative properties. Kaempferol is a natural flavonoid containing 3-hydroxy flavone backbone found in many plants in its aglycone form and attached with various sugars. Ecdysteroids are steroidal analogs of invertebrate steroidal hormones found in plants. Both flavonoids and ecdysteroids accumulate more in plants during abiotic stress. We hypothesized that Aerva javanica may have high levels of ecdysteroids and kaempferols for surviving in stressful conditions of desert. Those kaempferols may suppress the growth of granulation tissue by their antiangiogenesis property. Ecdysteroids may control the larvae of habronema if associated with proud flesh. Extract was prepared using solvent-based fractionation and silica gel column flash chromatography. Application of the leaf extract in horses suppressed growth of granulation tissue along with restoration of normal skin function. Various purification steps and mass spectrometry were used to identify the active components in the study.

Serological Survey of Humans Exposed to Burkholderia mallei-Infected Equids: A Public Health Approach.

Glanders is a fatal bacterial infection of equids caused by Burkholderia mallei. The infection can be transmitted to humans through prolonged direct contact with glanderous equids. Recently, reemergence of equine glanders has been reported in many countries. To investigate zoonotic transmission of B mallei infection, sera were collected from 538 humans including equine handlers and veterinary professionals exposed to glanderous equids. Samples were tested by ELISA (enzyme-linked immunosorbent assay) and complement fixation test and found negative for B mallei-specific antibodies. Even though there was no incidence of human glanders during this survey period, occupational exposure will continue to remain a serious concern and a key risk factor. Therefore, we emphasize the need for intersectoral collaboration and coordination among veterinary, human, and public health authorities for continuous surveillance and monitoring of human glanders under one health concept.

Serological surveillance and clinical investigation of glanders among indigenous equines in India from 2015 to 2018.

Equine glanders is an infectious and notifiable bacterial disease caused by Burkholderia mallei. The disease has been reported in South American, African and Asian countries including India. Here, we present the outcome of glanders serosurveillance carried out between January 2015 and December 2018 to know the status of equine glanders among different states in India. A total of 102,071 equid sera from 299 districts of twenty-one states and one union territory were tested for glanders. Samples were screened with Hcp1 indirect ELISA followed by confirmatory diagnosis by CFT. During this four-year surveillance, a total of 932 glanders-positive cases were detected from 120 districts of 12 states. The study also revealed increasing trend of glanders from 2016 onwards with maximum occurrence in northern India. Overall seroprevalence ranged between 0.62% (95% CI, 0.52-0.72) and 1.145% (95% CI, 1.03-1.25). Seasonal shifting from winter to summer (March to June) coincided with highest number glanders incidence with corresponding seroprevalences of 1.2% (95% CI, 1.09-1.30). The present surveillance unveils territorial ingression of glanders to six states like Jammu & Kashmir, Gujarat, Rajasthan, Madhya Pradesh, Delhi and Tamil Nadu. In addition, re-emerging cases have been reported in Maharashtra, Haryana and Punjab after a gap of 10 years. Lack of awareness, little veterinary care and unrestricted movement of equids across state borders might have led to the introduction and establishment of the infection to these states. We believe that information from this study will provide a baseline data on glanders for devising surveillance and control strategies in India. Being a zoonotic disease, the persistence of glanders poses a potential threat to occupationally exposed humans especially equine handlers and veterinarians. Therefore, targeted surveillance of human population from each glanders outbreak is also recommended.

Detection and genetic characterization of porcine circovirus 3 (PCV3) in pigs in India.

Porcine circovirus type 3 (PCV3), a novel circovirus, has been reported recently from major swine growing countries globally, and the virus is associated with diseases like porcine dermatitis, nephropathy syndrome and reproductive failure. This report describes the identification of PCV3 associated with reproductive failure in sows and piglet mortality and circulation of the virus in healthy pigs in India. The pathological changes in various tissues from stillborn piglet and characterization of the virus genomes were reported. The genome sequences of Indian PCV3 strains showed 91.4%-99.8% nucleotide identity with other sequences of PCV3 strains circulating worldwide. The phylogenetic analysis showed clustering of Indian strains into a separate group with the isolate from USA (MN/2016) under PCV3a genotype. The results confirmed the circulation of PCV3 in Indian pigs and its association with clinical cases. This study speculates emergence of PCV3 as an important pig pathogen in the country, which warrants the thorough investigation on PCV3 epidemiology, pathogenesis and to implement the control measures.

Protective efficacy of inactivated reverse genetics based equine influenza vaccine candidate adjuvanted with MontanideTM Pet Gel in murine model.

Equine influenza is a leading cause for respiratory illness in equines. Major control measures involve vaccination which requires continuous harmonization owing to antigenic drift. The present study focused on assessing the protective efficacy of an inactivated recombinant equine influenza virus (rgEIV) vaccine candidate adjuvanted with MontanideTM Pet Gel in murine model. The rgEIV was generated using reverse genetics by incorporating HA and NA segments from EIV/H3N8, clade 2-Florida sublineage in an A/WSN/33 /H1N1 backbone and inactivated by formalin. The vaccine was prepared by mixing inactivated rgEIV with MontanideTM Pet Gel adjuvant followed by intranasal inoculation into BALB/c mice intranasally. The immune responses and protective efficacy of the vaccine was evaluated by measurement of antibody titer, immunoglobulin subtyping, cytokines, clinical signs and pathological lesions after immunization and challenge with wild EIV. Serology and cytokine expression pattern indicated that the vaccine activated mixed Th1- and Th2-like responses of vaccine. Booster immunization stimulated strong antibody responses (HAI titre: 192 ± 28.6) at 42 days post immunization and the predominant antibody subtype was IgG1. Upregulation of interferon (IFN)-gamma, interleukin (IL)-12 and IL-2 levels indicates effective induction of Th1 type response. We found that vaccination has protected mice against equine influenza virus challenge as adjudged through a lack of nonappearance of visible clinical signs of disease, no loss of body weight loss, reduced pathology in the lungs and markedly reduced virus shedding from the respiratory tract. Therefore, we conclude that recombinant EIV vaccine candidate adjuvanted with MontanideTM Pet Gel could aid in quick harmonization of the vaccines through replacement of HA and NA genes for control of EIV outbreaks.

Evaluation of the comparative accuracy of the complement fixation test, Western blot and five enzyme-linked immunosorbent assays for serodiagnosis of glanders.

Glanders is a zoonotic contagious disease of equids caused by Burkholderia (B.) mallei. Serodiagnosis of the disease is challenging because of false-positive and false-negative test results. The accuracy of the complement fixation test (CFT) which is prescribed for international trade by the World Organisation for Animal Health (OIE), five ELISAs and a Western blot (WB) were compared for serodiagnosis of glanders using sera from 3,000 glanders-free and 254 glanderous equids. Four ELISA tests are based on recombinant antigens (TssA, TssB, BimA and Hcp1), the IDVet ELISA is based on a semi-purified fraction of B. mallei and WB makes use of a purified LPS-containing B. mallei-antigen. Sensitivity and specificity of tests were estimated using cut-off values recommended by the test developers. The WB and all ELISAs, except BimA, were significantly more specific than the CFT. ELISAs based on TssA, TssB, and BimA antigens had significantly lower sensitivity compared to CFT while the sensitivities of the Hcp1-ELISA, the IDVet-ELISA and the WB did not differ significantly from that of the CFT. Given their comparable sensitivities and specificities, the CFT (98.0%, 96.4%), the WB (96.8%, 99.4%), the Hcp1-ELISA (95.3%, 99.6%) and the IDVet-ELISA (92.5%, 99.5%) should be further developed to meet OIE requirements.

Role of MAPK/MNK1 signaling in virus replication.

Viruses are obligate intracellular parasites; they heavily depend on the host cell machinery to effectively replicate and produce new progeny virus particles. Following viral infection, diverse cell signaling pathways are initiated by the cells, with the major goal of establishing an antiviral state. However, viruses have been shown to exploit cellular signaling pathways for their own effective replication. Genome-wide siRNA screens have also identified numerous host factors that either support (proviral) or inhibit (antiviral) virus replication. Some of the host factors might be dispensable for the host but may be critical for virus replication; therefore such cellular factors may serve as targets for development of antiviral therapeutics. Mitogen activated protein kinase (MAPK) is a major cell signaling pathway that is known to be activated by diverse group of viruses. MAPK interacting kinase 1 (MNK1) has been shown to regulate both cap-dependent and internal ribosomal entry sites (IRES)-mediated mRNA translation. In this review we have discuss the role of MAPK in virus replication, particularly the role of MNK1 in replication and translation of viral genome.

Characterization of isolates of Bordetella bronchiseptica from horses.

Bordetella bronchiseptica is a well-known Gram-negative bacterial pathogen causing a plethora of diseases in different animals. Although its infection has been reported from pigs and dogs in India, no report of B. bronchiseptica from horses is described. We report for the first time, isolation, identification and characterization of strains of B. bronchiseptica from respiratory infection in horses from different states in India. The antimicrobial susceptibility testing showed resistance to penicillins, ceftazidime, and chloramphanicol. The virulence capability of the strains was confirmed by sequencing genes such as adenylate cyclase toxin (cyaA), bordetella virulence gene (bvgA) and by PCR detection of flagellin gene (fla). We demonstrate the involvement of B. bronchiseptica strains in respiratory tract infection in horses in India.

Isolation and characterization of a novel, T7-like phage against Aeromonas veronii.

A virulent Aeromonas veronii biovar sobria and the corresponding novel, lytic bacteriophage (VTCCBPA5) were isolated from village pond water. The phage was found to belong to family Podoviridae. PCR analysis of major capsid protein gene confirmed its classification to T7-like genus. The protein profiling by SDS-PAGE indicated the major structural protein to be ~ 45 kDa. The phage (VTCCBPA5) is host specific and is stable over a range of pH (6-10) and temperatures (4-45 °C). On the basis of restriction endonuclease analysis combined with prediction mapping, it was observed to vary significantly from previously reported podophages of Aeromonas sp., viz. phiAS7 and Ahp1. The phylogenetic analysis on the basis of PCR-amplified segment of DNA polymerase gene of phage revealed it being an outgroup from podophages of Klebsiella sp. and Pseudomonas sp. though a small internal fragment (359 bp) showed the highest identity (77%) with Vibrio sp. phages. Thus, this is the first report of a novel Podoviridae phage against A. veronii. It expands the assemblage of podophages against Aeromonas sp. and BPA5 could be potentially useful in biocontrol of environmentally acquired Aeromonas veronii infections.