Calcium phosphate nanoparticles conjugated with outer membrane vesicle of Riemerella anatipestifer for vaccine development in ducklings.

Biodegradable calcium phosphate nanoparticles offer a viable substitute for traditional adjuvants such as aluminum in vaccine production. Calcium phosphate nanoparticle adjuvanted with outer membrane vesicle (OMV) of gram negative bacteria may induce efficient immune response in the host. The present study was carried out to evaluate the potential of a mucosal vaccine formulation of calcium phosphate (CAP) nanoparticle using OMV of Riemerella anatipestifer (RA) as antigen against New Duck disease in ducks. The work was initiated with isolation, identification of RA, followed by OMV production and extraction. The CAP-OMV nanoparticle was prepared and characterized. The efficacy of the vaccine formulation and toxicity were studied in ducks. The average OMV yield in terms of protein concentration was found to be 122.33 ± 3.48 mg per liter of BHI broth. In SDS-PAGE, isolated OMVs exhibited presence of 16 distinct protein bands with molecular weight ranging from 142.1 to 12.1 kDa. Seven protein bands of 74.1, 69.3, 55.5, 50.6, 45.6, 25.1 and 13.1 kDa were detected relatively more distinct. The major bands detected in our findings were 42 kDa, 37 kDa and 16 kDa that corresponds to OmpA, OmpH, P6 respectively. The mean zeta size (±SD) and potential of the nanoparticle were 246.20 ± 0.53 nm and -25.60 ± 5.97 respectively. In transmission electron microscopy (TEM), the nanoparticles exhibited an average diameter of 129.80 ± 11.10 nm and displayed spherical morphology. The median protective dose (PD50) of CAP-OMV nanoparticle was 1881.10 µg of protein. Group I ducks received 3762 µg of protein (entrapped protein in CAP-OMV nanoparticle) via intra nasal route and it showed the highest serum IgG and secretory IgA level than other immunized groups. All experimental ducks were challenged with 10 × LD50 on 35 days of post primary immunization. Group I showed 100 % survivability in the challenge study. No gross and biochemical indication of acute or chronic toxicity were recorded. In conclusion, our results suggest that CAP-OMV nanoparticle can be a safe and efficient mucosal vaccine delivery system for RA, eliciting strong immune response in the host.

Prevalence of bovine tuberculosis and analysis of risk factors among the dairy farms in and around Guwahati metropolitan city, India.

Bovine tuberculosis (bTB), a neglected zoonotic disease caused by Mycobacterium bovis is being reported worldwide. The present work was carried out from December 2020 to November 2021 to assess the prevalence and risk factors of bTB in peri-urban and urban dairy farms of Guwahati, Assam, India. A questionnaire was used to collect data on knowledge about bTB on 36 farms, and ten animals per farm were screened by single intradermal comparative cervical tuberculin test (SICCT) to determine the prevalence of bTB, giving a total of 360 animals. The demographic data of the farmers revealed that 61.1% respondents were illiterate, 66.7% had no awareness about bovine tuberculosis and 41.7% consumed unpasteurised milk and milk products. SICCT showed that 38 cattle from 18 of the farms were positive reactors for bTB, yielding an overall animal level prevalence of 10.55% (95% confidence interval (CI = 7.58-14.2%) and a 50% herd prevalence (95% CI 32.9-67.1%). Animals 5 years and above were found to be more likely to be positive for bTB (17.18%). The study highlighted the widespread prevalence of bovine tuberculosis in peri-urban and urban dairy farms of Guwahati which gives a picture also about other major cities of India. Hence, it is of utmost importance to undertake a comprehensive epidemiological study in such cities for effective control and prevention of bTB in a one health approach.

RIG-I expression pattern and cytokine profile in indigenous ducks infected with duck plague virus.

The present study was undertaken to elucidate mRNA expression pattern of RIG-I and serum cytokines profile alterations in indigenous ducks of Assam, India viz. Pati, Nageswari and Cinahanh in response to natural infections of duck plague virus. Field outbreaks of duck plague virus were attended during the study period for collection of tissue and blood samples. The ducks under study were divided into three distinct groups as per health status i.e. healthy, duck plague infected and recovered. Results from the study revealed that RIG-I gene expression was significantly upregulated in liver, intestine, spleen, brain and PBMC of both infected and recovered ducks. However, fold changes in RIG- I gene expression was lower in recovered ducks as compared to infected ones which indicated continued stimulation of RIG-I gene by the latent viruses. Both serum pro and anti-inflammatory cytokines were elevated in infected ducks as compared to healthy and recovered ducks, indicating activation of inflammatory reactions in the ducks due to virus invasion. The results from the study indicated that innate immune components of the infected ducks were stimulated in order to make an attempt to resist the virus from the infected ducks.

Evaluation of an in-house IgM/IgG lateral flow assay for serodiagnosis of human brucellosis.

This study aimed to evaluate the diagnostic efficacy of an in-house lateral flow assay (LFA) for the detection of IgM/IgG anti-Brucella antibodies for rapid serodiagnosis of human brucellosis. Three groups of sera samples including 476 from high-risk individuals, 27 from culture-confirmed patients, and 43 from healthy blood donors were used for evaluation of LFA. In comparison with iELISA, the sensitivity, specificity, and accuracy of LFA were >95%, >99%, and 99% respectively. Considering the very good agreement, accuracy, simplicity, and rapidity, LFAs might be useful as a point of care test for the diagnosis of human brucellosis in resource-limited laboratories.

Draft Genome Sequence Analysis of the Genotype II African Swine Fever Virus from India.

African swine fever virus (ASFV) entered the northeastern (NE) part of India early in 2020, causing huge economic loss to the piggery sector. Here, we are presenting a brief report on the draft genome sequence of an ASFV strain ABTCVSCK_ASF007 from Assam state of NE India belonging to genotype II.

Prevalence of leptospirosis among animal herds of north eastern provinces of India.

Serum samples from 840 animals were examined for Leptospira spp. antibodies by microscopic agglutination test (MAT) to assess the risk factors and the prevalence of leptospirosis among animal herds of Assam, Meghalaya, Mizoram the north eastern (NER) provinces. They were compared with Tamilnadu (TN) the southern province of India for the serovar and risk factor inconsistency. Serovar Ballum was reported to be prevalent (28.1 %) in Assam and Grippotyphosa (16.1 %) in Tamilnadu. The overall seropositivity observed was 36.8 %(206/560) from NER and 30.7 %(86/280) from TN. In this study, the higher seroprevalence was observed in pigs (42.6 %), cattle (39.8 %) and goats (26 %) in NER. Cattle (36.4 %) and goat (17.6 %) showed higher prevalence in TN. The presence of rodents in pig herds was found to be significant (P = 0.0088) in NER and it was for cattle in TN (P = 0.0063). We suggest that a program of rodent control should be included in the flock management practices aiming to reduce transmission of the leptospires.

Assessment of Risk Factors of African Swine Fever in India: Perspectives on Future Outbreaks and Control Strategies.

African swine fever (ASF) is one of the most important transboundary diseases of pigs. ASF has been identified in India for the first time in domestic pigs from outbreaks reported in two of the northeastern states, Arunachal Pradesh and Assam in 2020. A total of 11 ASF outbreaks in different regions killed over 3700 pigs and devastated the economy of small-scale livestock owners of both the states. Considering the first outbreak of ASF in India, a generic risk assessment framework was determined to identify potential risk factors that might favor future emergence of the disease. Based on the Indian scenario, we considered population density of host, farming practice, availability of biological vectors and wildlife reservoirs, epidemiological cycles, and international trade to analyze the possibility of future outbreaks of ASF and chances of establishment of endemism. On critical analysis of the identified risk factors associated with ASFV transmission, we observed that the risk factors are well preserved in the Indian geography and might participate in future outbreaks, further disseminating the disease to nearby countries. Since no vaccine is currently available against ASF, the domestic and the wild pigs (wild boars and the endangered pygmy hogs native to India) of this region are under constant threat of infection. For the near future, this region will have to continue to rely on the implementation of preventive measures to avoid the devastating losses that outbreaks can cause. The various adaptive control strategies to minimize the risks associated with the transmission of ASF, keeping our views to Indian settings, have been described. The risk-analysis framework presented in the study will give a further understanding of the dynamics of disease transmission and will help to design control strategies and corresponding measures to minimize the catastrophic consequences of ASF disease.

Comparing the efficiency of different Escherichia coli strains in producing recombinant capsid protein of porcine circovirus type 2.

The present study was aimed at comparing different E. coli strains in expressing the capsid protein of Porcine Circovirus 2 (PCV2). Full length capsid protein could be expressed only in Rosetta-gami 2 (DE3) pLysS strain using pET32b (+) vector. This confirmed that only those strains which possess tRNAs for rare codons can express the full length capsid protein. Purification of full length capsid protein could not be achieved even after several attempts using native and denaturing conditions. Subsequently, an attempt was made for expression of N-terminal truncated capsid protein using the same expression system. Truncated capsid protein was successfully expressed, purified and characterized by western blotting. The truncated capsid protein was also shown to be efficacious in testing serum samples using an optimized indirect ELISA, wherein a diagnostic sensitivity of 88.89% and specificity of 90.82% was obtained as compared to commercially available GreenSpring® porcine circovirus (PCV2) ELISA test kit. Thus, the expressed truncated capsid protein appears to be a promising diagnostic agent for PCV2. The comparative analysis suggests that cluster of arginine residues at N-terminal of capsid protein not only affects its expression in some E. coli strains but also its purification by Ni-NTA chromatography, when expressed as a histidine tagged fusion protein.

Meta-analysis of the prevalence of livestock diseases in North Eastern Region of India.

AIM: The study aimed to determine the overall prevalence of livestock diseases in North Eastern Region (NER) of India, through a systematic review and meta-analysis of published data. MATERIALS AND METHODS: The articles used for the study were retrieved from PubMed, J-Gate Plus, Indian Journals, and Google scholar, R open-source scripting software 3.4.3. Metafor, Meta. The Chi-square test was conducted to assess for the heterogeneity, forest plot (confidence interval [CI] plot) is a method utilized to present the results of meta-analysis, displaying effect estimate and their CIs for each study were used for searching and retrieval of livestock diseases prevalence data in India using a search strategy combining keywords and related database-specific subject terms from 2008 to 2017 in English only. RESULTS: The prevalence of various livestock diseases are foot-and-mouth disease (21%), bluetongue (28%), brucellosis in bovine (17%), brucellosis in caprine (2%), brucellosis in porcine (18%), brucellosis in sheep and goat (3%), babesiosis (6%), theileriosis (26%), porcine reproductive and respiratory syndrome (1%), porcine cysticercosis (6%), classical swine fever (31%), Porcine circovirus (43%), and Peste des petits ruminants (15%). This information helps policymakers to take appropriate measures to reduce the disease burden. CONCLUSION: This study indicates that the overall prevalence of various livestock diseases in NER of India.

Advances in Diagnostic Approaches for Viral Etiologies of Diarrhea: From the Lab to the Field.

The applications of correct diagnostic approaches play a decisive role in timely containment of infectious diseases spread and mitigation of public health risks. Nevertheless, there is a need to update the diagnostics regularly to capture the new, emergent, and highly divergent viruses. Acute gastroenteritis of viral origin has been identified as a significant cause of mortality across the globe, with the more serious consequences seen at the extremes of age groups (young and elderly) and immune-compromised individuals. Therefore, significant advancements and efforts have been put in the development of enteric virus diagnostics to meet the WHO ASSURED criteria as a benchmark over the years. The Enzyme-Linked Immunosorbent (ELISA) and Polymerase Chain Reaction (PCR) are the basic assays that provided the platform for development of several efficient diagnostics such as real-time RT-PCR, loop-mediated isothermal amplification (LAMP), polymerase spiral reaction (PSR), biosensors, microarrays and next generation sequencing. Herein, we describe and discuss the applications of these advanced technologies in context to enteric virus detection by delineating their features, advantages and limitations.

First report and genetic characterization of porcine astroviruses of lineage 4 and 2 in diarrhoeic pigs in India.

Porcine astroviruses (PAstVs) have extended their distribution globally and have a high prevalence; however, their clinical significance is still under investigation. Thus far, information about their prevalence and diversity in the Indian pig population is unknown. This study is the first report on the prevalence and genetic characterization of PAstVs in diarrhoeic piglets in India. From January 2013 to December 2017, 757 samples were screened using an RT-PCR assay and PAstV infection was detected in 17.6% (133/757) pigs. Of the 133 positive samples, 79 (59.4%) were positive for PAstV alone, whereas 54 (40.6%) were found to be co-infected with porcine rotavirus A (PoRVA). Phylogenetic analysis of RdRp/capsid gene region revealed high genetic heterogeneity among PAstV sequences, with a predominance of PAstV lineage 4 and detection of lineage 2. The lineage 4 PAstVs exhibited 61.2%-94.5% sequence similarity at the nucleotide level to other reported sequences, whereas lineage 2 strain shared 66.0%-71.6% sequence identity with cognate sequences of the same lineage. This is the first report on PAstV and circulation of lineages 4 and 2 in India. Further, phylogenetic analysis indicates a multiphyletic origin of PAstV strains and suggests cross-border circulation of PAstVs with a similar genetic configuration in Asian countries.

Goatpox outbreak at a high altitude goat farm of Mizoram: possibility of wild life spill over to domestic goat population.

In this study, pox-like outbreaks in goat population was investigated that occurred in a high altitude goat farm located in Mizoram, a hilly state of North eastern India. The outbreak initially involved the serows, an wild animal belonging to the family Bovidae, subfamily Caprinae and genus Capricornis, the state animal of Mizoram. Later, the disease affected the domestic goat population. The disease was diagnosed on the basis of gross lesions and PCR amplification of partial P32 gene of capripox virus. The virus was isolated in vero cells. The full length P32 gene was sequenced and phylogenetic tree was constructed. It was revealed that the capripox virus isolated from the outbreak was closely related to the Chinese strain of goatpox virus at both amino acid and nucleotide level. To the authors' knowledge, this is the first report on isolation and characterization of capripoxvirus from north eastern region of India.

Heterologous DNA prime-protein boost immunization with RecA and FliD offers cross-clade protection against leptospiral infection.

The emergence of >300 serovars of Leptospira confounded the use of generalized bacterin, the whole cell lysate, as vaccines to control leptospirosis. Because of substantial genetic and geographic heterogeneity among circulating serovars, one vaccine strain per serovar cannot be efficacious against all the serovars. We have performed heterologous DNA prime-protein boost vaccination challenge studies in hamsters using in vivo expressed, leptospiral recombinase A (RecA) and flagellar hook associated protein (FliD). We prepared the monovalent recombinant protein, plasmid DNA, and DNA prime protein boost adjuvant vaccines. The whole cell bacterin served as a control. Our data show that (i) RecA and FliD have multiple immunogenic B and T-cell epitopes with highly conserved domains among most prevalent pathogenic Leptospira spp., (ii) humoral and cell mediated immune responses were induced remarkably, (iii) provides significant protection against homologous (Autumnalis strain N2) and cross-clade heterologous (Canicola strain PAI-1) challenge infection for the heterologous prime-protein boost (∼91-100%) and, the DNA vaccine (∼75-83%). Recombinant protein vaccine shows only partial protection (∼58-66%), (iv) RecA prime-protein boost vaccine shows sterilizing immunity, with heterologous protection. This RecA/FliD prime-protein boost strategy holds potential for vaccination against animal leptospirosis and for a better control of zoonotic transmission.

Virulence gene profiling of porcine Pasteurella multocida isolates of Assam.

AIM: The present study was conducted to detect and identify the virulence genes in Pasteurella multocida isolates of porcine origin from Assam. MATERIALS AND METHODS: A total of 21 porcine P. multocida isolates were subjected to capsular typing and detection of virulence-associated genes (pfhA, tbpA, hgbB, toxA, oma87, ompH, and nanB) using various polymerase chain reaction (PCR) methods reported elsewhere. Further, pathogenicity of the porcine isolates of P. multocida was studied in mice. For each strain of P. multocida selected for pathogenicity trial, the group of mice was injected intraperitoneally (i/p) with 0.1 ml of the inoculum prepared from respective field isolates, containing 109 organisms per ml. RESULTS: Capsular typing of the isolates by multiplex PCR showed two capsular types, type A (66.66%) and type D (33.33%). All the isolates were positive for outer membrane protein genes, oma87 and ompH genes. Iron acquisition genes, tbpA and hgbB, were detected in 14.28% and 19.04% of the isolates. The dermonecrotoxin encoding gene, toxA, was present in 23.80% of the isolates. Filamentous hemagglutinin encoding gene, pfhA, was detected in 28.57%. The virulence gene distribution pattern of the isolates indicates the important role of the genes in disease pathogenesis. CONCLUSION: From the present study, it can be concluded that toxA gene is an important marker gene for defining the pathogenic potential of P. multocida strains in swine.

Effect of different smoking methods on the quality of pork sausages.

AIM: An experiment was conducted to evaluate the nutritional, physicochemical, microbiological, and sensory attributes of pork sausages treated with conventional smoking (CS) and liquid smoke (LS). MATERIALS AND METHODS: Pork sausages were prepared by employing CS (T1) and by addition of LS at 3% (T2A), 5% (T2B), and 7% (T2C) while smoking was not done in control (C) sausages. The ready-to-eat pork sausages were evaluated in terms of proximate composition, emulsion stability (ES), cooking loss (CL), pH, water activity (aw), texture profile analysis (TPA), and shear force on the day of preparation and the shelf life of the sausages was evaluated on the basis of thiobarbituric acid reactive substance (TBARS) value, organoleptic qualities, total viable plate count, total psychrophilic count, and yeast and mold counts at 5-day interval up to 15 days under refrigerated storage (6±1°C). RESULTS: The mean percentage moisture and percentage ether extract contents of the conventionally smoked sausages (T1) exhibited significant difference (p≤0.01) with the rest of the formulations. However, in terms of mean percentage crude protein and percentage total solids, no significant difference (p≥0.05) was recorded between the treatment groups. The mean ES (ml of oil/100 g emulsion) of the different sausage emulsions ranged from 1.88 to 3.20, while the mean aw values among the sausage formulations were found to be non-significant. In terms of mean percentage, CL and pH values, significantly lowest (p≤0.01) values were recorded by the T1 sausages. The mean TBARS values recorded at different periods of time in respect of all the treatment groups ranged from 0.10 to 0.33 mg malanoldehyde [MDA]/kg of sausages which are well within the permissible limit. The highest shear force values (KgF) were recorded by the sausages of T1 formulation (p≤0.01), while TPA of the sausages did not record any significant difference (p≥0.05) among the treatments. Organoleptic studies revealed acceptability of the sausages up to 10 days of refrigerated storage irrespective of treatments employed; however, the sausages of T1 formulation scored significantly (p≤0.01) higher panel ratings. Microbiologically, sausages with different formulations were found to be within the acceptable limit up to the 15th day of refrigerated storage. CONCLUSION: The study revealed that traditional hot smoking has slightly higher edges over the LS-treated sausages in terms of lipid oxidation, microbiological safety, and sensory panel ratings. However, if not superior, the same was found to be well within the acceptable limit in case of LS-treated sausages proving the potentiality of the use of LS as a suitable replacement for the traditional hazardous hot smoking process.

Seroprevalence of contagious ecthyma in goats of Assam: An analysis by indirect enzyme-linked immunosorbent assay.

AIM: The objective of this study was to screen the prevalence of contagious ecthyma (CE) among the goat population of Assam owing to its high prevalence rate. MATERIALS AND METHODS: In this study, a total of 231 serum samples were collected from 12 districts of Assam during September 2013 to July 2014. The serum samples were tested for the presence of antibodies against Orf virus (ORFV) by indirect enzyme-linked immunosorbent assay (ELISA). Indirect ELISA was standardized using purified Orf reference virus produced in bulk in primary lamb testes cells. RESULTS: Studies on seroprevalence showed 76.62% of goats were seropositive. The total number of animals were divided into different age groups starting from 0-2 months, 2-4 months, 4-6 months, and above 8 months and accordingly highest prevalence of antibodies against ORFV was recorded in the age-group above 8 months of age. Significantly, lower rates of infection were observed in goats of age group 2-4 months. This study recorded that seropositivity from naturally infected animals and in contact apparently healthy animals to be 53.67% and 46.32%, respectively. CONCLUSION: The results indicated that CE is a prevalent infection in goats of Assam, and the healthy population is at increased risk of infection.

Isolation and molecular characterization of Orf virus from natural outbreaks in goats of Assam.

Outbreaks of contagious ecthyma (caused by a Parapox virus) in goats were investigated in 6 districts of Assam, a north eastern state of India. Diagnosis of the disease was carried out employing both standard virological as well as molecular methods. Four representative isolates from different places were selected for phylogenetic analysis. The major envelop protein (B2L) of Orf virus was targeted for molecular analysis. The sequencing and phylogenetic analysis of the selected sequences at nucleotide level revealed that the Orf virus isolates were closely related to each other (97.6-100 %) and showed highest similarity to the Orf virus isolate 82/04 (98.4 %), reported from Shahjahanpur, India. The data will provide an insight in transmission of the virus from northern to North eastern part of the country.

Circulation of group A rotaviruses among neonates of human, cow and pig: study from Assam, a north eastern state of India.

Rotavirus (RV) infections are worldwide in distribution causing high morbidity and mortality in human and animal neonates. Human settlements in close proximity of animals aids for genetic re-assortment of the virus by interspecies transmission and consequent emergence of new viral antigenic strain. Therefore, the present study was designed to explore RV incidence in a single approach from human and animal neonates sharing similar environment. Altogether, 200 diarrheal samples from children (50), piglets (80) and calves (70) were collected during the year of 2010-2012 from various locality, farms and hospitals, initially screened through monoclonal antibody based enzyme immunoassay followed by RNA-PAGE and VP7 gene amplification by Reverse transcription PCR. The overall prevalence of rotavirus was found to be 41.5 % (83/200) where maximum numbers of positive cases were found in piglets (46.3 %) followed by human (40 %) and cow (37.1 %). Majority of samples demonstrated characteristic group A rotavirus (RVA) electropherotype of 4:2:3:2 pattern. Moreover, RNA profiles of seven samples from piglets and calves revealed variation in the migration pattern of class II, III and class IV segments. The study, for the first time from the valley, detected 43.7 % of neonatal RVA positive cases from human and animal sharing similar setting. The variation in RNA migration pattern in seven cases signifies tentative cases of gene re-assortment that warrant further evaluation.

Identification and phylogenetic analysis of orf viruses isolated from outbreaks in goats of Assam, a northeastern state of India.

Two outbreaks of orf virus (ORFV) (a parapoxvirus) infection in goats, which occurred in Golaghat and Kamrup districts of Assam, a northeastern part of India, were investigated. The disease was diagnosed by standard virological and molecular techniques. The entire protein-coding region of B2L gene of two isolates were cloned and sequenced. Phylogenetic analysis based on B2L amino acid sequences showed that the ORFVs identified in these outbreaks were closely related to each other and both were closer to ORFV-Shahjahanpur 82/04 isolate from north India. The present study revealed that the precise characterization of the genomic region (B2L gene) might provide evidence for the genetic variation and movement of circulating ORFV strains in India.

Quantitative polymerase chain reaction: another tool to evaluate viable virus content in live attenuated orf vaccine.

A probe-based real-time polymerase chain reaction (PCR) assay based on the highly conserved DNA polymerase gene of orf virus (ORFV) for the quality control of attenuated orf vaccine is reported. Primary lamb testis (PLT) cells were infected with orf vaccine virus and harvested at a critical time point to obtain maximum viable virus content as determined by real-time PCR. DNA extracted from these harvests was subjected to real-time PCR. A critical time point for the harvesting of PLT cells infected with various log(10) dilutions of vaccine virus was found to be 42 h (highest slope of 3.335), which was obtained by comparing the slopes of standard curves of different time intervals. The assay was employed to evaluate viable virus content in different batches of orf vaccine. The titres estimated by real-time PCR and conventional TCID(50) were comparable with a correlation of 0.8169. Thus, the real-time PCR assay could provide an alternative method or supplementary tool to estimate live ORFV particles in attenuated orf vaccine.