Association between levels of insulin-like growth factor-1 in serum and seminal plasma with fresh and frozen-thawed semen characteristics in Beetal bucks.

The aim of this study was to investigate the relationship between the levels of insulin-like growth factor-1 (IGF-1) in serum and seminal plasma and the characteristics of semen in Beetal bucks (Capra hircus). A total of 12 adult Beetal bucks were involved in the study, with each buck providing six ejaculates collected using a standard artificial vagina (n = 72 total). Only qualified semen samples (volume of 0.7 mL, a mass motility rating of 3+ or higher on a 0-+ scale, and individual progressive motility of 80% or more) divided into three fractions were processed for estimation of IGF-1 and other seminal parameters like motility, viability, acrosome integrity, sperm abnormality and superoxide dismutase (SOD) activity. The first and second fraction were diluted and extended with Optixcell extender (1:15 ratio). The first ejaculate fraction was processed for studying fresh semen parameters and the second fraction was cryopreserved for evaluating frozen semen parameters. French mini straws (0.25 mL) were used for semen filling, and polyvinyl alcohol powder of different colours was used for sealing the extended semen. The third fraction of each ejaculate was centrifuged at room temperature (1100 × g for 7 min) to separate the seminal plasma. Additionally, blood samples were taken from each buck on the same day as semen collection, resulting in a total of 36 blood samples. The results revealed a significant positive correlation (r = .4243; p < .05) between the concentration of IGF-1 in both serum and seminal plasma of the Beetal bucks. Furthermore, the concentration of IGF-1 in serum showed significant positive correlations with sperm viability (r = .554; p < .05), acrosome integrity (r = .527; p < .05), post-thaw sperm motility (r = .407; p < .01), post-thaw sperm viability (r = .426; p < .01) and post-thaw acrosome integrity (r = .333; p < .05). However, it had a significant negative correlation with SOD activity in fresh semen (r = -0.458; p < .01). Moreover, the concentration of IGF-1 in seminal plasma demonstrated significant positive correlations with individual progressive motility (r = .341; p < .05), sperm viability (r = .527; p < .05), acrosome integrity (r = .539; p < .05), sperm plasma membrane integrity (r = .464; p < .05), post-thaw sperm motility (r = .644; p < .01), post-thaw sperm viability (r = .643; p < .01), post-thaw acrosome integrity (r = .487; p < .01) and post-thaw sperm plasma membrane integrity (r = .521; p < .01). Additionally, it showed a significant negative correlation with SOD activity in both fresh semen (r = -0.714; p < .01) and frozen semen (r = -0.558; p < .01) of Beetal bucks. Based on these findings, IGF-1 in seminal plasma can be considered as a potential biomarker for the selection of bucks for breeding purposes.

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.

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.

Allelic variability of estrogen receptor (ESR) gene and its effect on litter traits of Doom pigs.

The present study was conducted to identify the polymorphism of estrogen receptor (ESR) gene and its biological association with litter traits (litter size at birth, litter size at weaning, litter weight at birth and litter weight at weaning) of Doom pigs native to Assam. A total of 50 adult pigs (12 males and 38 females) chosen randomly from three different herds (Herd I, Herd II, and Herd III) were utilized in the present study. Detection of polymorphism of ESR gene was done by means of PCR-RFLP method. The amplified PCR product was digested with Pvu II restriction enzyme. PCR-RFLP analysis of ESR gene revealed polymorphic banding pattern. Two genotypes viz. AA and AB were identified. AA genotype yielded one fragment (120 bp) and AB genotype yielded three fragments (120, 65, and 55 bp). BB genotype was not found in the population under study. The frequencies of A and B alleles were found to be 0.650 and 0.350, respectively, and the genotypic frequencies of ESR gene were found to be 0.300 and 0.700 for AA and AB genotypes, respectively. There was no significant (P > 0.05) effect of ESR genotype on litter traits and the population under study was not in Hardy-Weinberg Equilibrium for ESR gene. Clustal W Multiple alignment of partial sequence of ESR gene revealed single nucleotide changes at 33, 65, 70, 83, and 92nd nucleotide positions. The presence of Pvu II polymorphism and identification of single nucleotide variation of ESR gene opens interesting prospects for improvement of litter traits in Doom pig through selective breeding program, especially based on marker-assisted selection.

Efficacy of Pulsed-Field Gel Electrophoresis and Repetitive Element Sequence-Based PCR in Typing of Salmonella Isolates from Assam, India.

A total of 12 Salmonella isolates belonging to different serovars, viz, Salmonella enterica serovar Enteritidis (n = 4), Salmonella enterica serovar Weltevreden (n = 4), Salmonella enterica serovar Newport (n = 1), Salmonella enterica serovar Litchifield (n = 1), and untypeable strains (n = 2) were isolated from 332 diarrheic fecal samples collected from animals, birds, and humans. Of the two molecular typing methods applied, viz, repetitive element sequence-based PCR (REP-PCR) and pulsed-field gel electrophoresis (PFGE), PFGE could clearly differentiate the strains belonging to different serovars as well as differentiate between strains of the same serovar with respect to their source of isolation, whereas REP-PCR could not differentiate between strains of the same serovar. Thus, it can be suggested that PFGE is more useful and appropriate for molecular typing of Salmonella isolates during epidemiological investigations than REP-PCR.

Livestock, pets and humans as carriers of methicillin-resistant Staphylococcusaureus and comparative evaluation of two PCR protocols for detection.

Staphylococcus aureus are Gram positive bacteria known to acquire antibiotic resistance rapidly and pose a major challenge to clinicians worldwide. Infections by methicillin resistant Staphylococcus aureus (MRSA) are usually associated with increased mortality and prolonging of treatment. Samples (n = 706) from diverse sources (livestock, pets, animal handlers, human hospital) were collected and screened for the presence of MRSA by phenotypic and genotypic methods. The incidence of Staphylococcus aureus was greater in goats (42.00%; 28.20 - 56.80%, confidence interval [CI] 95.00%) followed by cattle (13.50%; 9.20 - 18.80%, CI 95.00%), humans (12.90%; 9.30 - 17.40%, CI 95.00%) and dogs (12.90%; 8.10 - 19.20%, CI 95.00%). Significantly higher incidence of MRSA was observed in dogs (65.00%; 40.80 - 84.60%, CI 95.00%), compared to other hosts namely cattle (48.00%; 26.50 - 64.30%, CI 95.00%), humans (35.00%; 20.20 - 52.50%, CI 95.00%) and goats (10.00%; 1.20 - 30.40%, CI 95.00%). All the S. aureus isolates were further screened for thermostable nuclease (nuc gene) by polymerase chain reaction (PCR). The incidence of nuc gene in cattle, dog, goat and human were found to be 3.30% (1.30 - 6.60%, CI 95.00%), 5.20% (2.30 - 9.90%, CI 95.00%), 28.00% (16.20 - 42.50%, CI 95.00%) and 9.10% (6.00 - 13.00%, CI 95.00%), respectively. Comparative evaluation of two PCR primers (mecA-162 and mecA-310) indicated the former one as more rational choice for detection of MRSA. Overall, the results of our study indicated possible risk of zoonotic transmission of MRSA from canines.

In Vitro and In Vivo Studies on the Efficacy of Zinc-Oxide and Selenium Nanoparticle in Cryopreserved Goat (Capra hircus) Spermatozoa.

Different nanoparticles (NPs) are currently being investigated for their potential role as cryoprotectant during semen cryopreservation in several mammalian species. It may be possible to improve semen quality following cryopreservation by supplementation of NPs in the freezing extenders. The present study was carried out in semen collected from four (4) Assam Hill Goat bucks (10 ejaculates per buck) to investigate the effect of supplementing zinc oxide (ZnO) and selenium (Se) NPs in Tris-citric acid-fructose yolk (TCFY) extender on in vitro sperm quality and in vivo fertility rate after freeze-thawing. The size morphology and zeta potential of ZnO and Se NPs were evaluated prior to its incorporation in the freezing extender. Qualified semen samples (> 70% progressive motility) were divided into five (5) aliquots and then diluted in TCFY extender containing ZnO and Se NP supplementation at different concentrations (T0, control; T1, 0.1 mg/mL ZnO NPs; T2, 0.5 mg/mL ZnO NPs; T3, 0.5 µg/mL Se NPs; and T4, 1 µg/mL Se NPs). Diluted semen was packed in 0.25 mL straws and then stored in liquid nitrogen. After thawing, post-thaw in vitro sperm attributes were evaluated. Finally, the effect of NPs on in vivo fertility rate was checked in heat-synched does (n = 70) by artificial insemination (AI) using straws that showed superior results during the in vitro study. Results showed that ZnO and Se NPs were poly-crystalline in nature with particle size below 100 nm (nm). The evaluated post-thaw sperm in vitro attributes were significantly (p < 0.001) higher in T1 in comparison to T0. The antioxidant enzyme activities were significantly (p < 0.001) higher in T1. Lipid peroxidation (LPO) profile was significantly (p < 0.001) lower in T1. Sperm motility and mitochondrial membrane potential (MMP) had a highly significant (r = 0.580, p < 0.05) association in T1. No significant (p > 0.05) differences in pregnancy rates were recorded after AI in the different treatments. In conclusion, extender supplemented with 0.1 mg/mL ZnO NPs improved post-thaw semen quality of goat spermatozoa consequently by increasing activities of endogenous antioxidant enzymes thereby lowering LPO levels. However, improved in vitro outcomes might not correspond to improved field fertility outcomes.

Immunopotential of Pasteurella multocida bivalent outer membrane protein-based vaccine entrapped in aluminum hydroxide nanoparticles.

Swine pasteurellosis is one of the most economically important diseases of pig caused by Pasteurella multocida (P. multocida) capsular types A and D. These organisms are commensals and opportunistic pathogens in the upper respiratory tract in pig. In the present study, we extracted whole outer membrane proteins (OMP) from P. multocida capsular types A and D and were mixed together in the ratio of 1:1 forming bivalent outer-membrane proteins. The bivalent OMP was adsorbed onto aluminum hydroxide nanoparticles. The size of aluminum hydroxide nanoparticles adsorbed outer membrane protein was found to be in the range of 125 to 130 nm. We observed that aluminum hydroxide nanoparticles adjuvanted bivalent OMP-based vaccine elicited quicker immune kinetics in terms of IgG response as compared to aluminum hydroxide microparticles adjuvanted bivalent bacterin vaccine against P. multocida capsular type A and D.

A GMMA-CPS-Based Vaccine for Non-Typhoidal Salmonella.

Non-typhoidal Salmonella are a major cause of gastroenteritis worldwide, as well as causing bloodstream infections in sub-Saharan Africa with a high fatality rate. No vaccine is currently available for human use. Current vaccine development strategies are focused on capsular polysaccharides (CPS) present on the surface of non-typhoidal Salmonella. This study aimed to boost the amount of CPS purified from S. Typhimurium for immunization trials. Random mutagenesis with Tn10 transposon increased the production of CPS colanic acid, by 10-fold compared to wildtype. Immunization with colanic acid or colanic acid conjugated to truncated glycoprotein D or inactivated diphtheria toxin did not induce a protective immune response in mice. However, immunization with Generalized Modules for Membrane Antigens (GMMAs) isolated from colanic acid overproducing isolates reduced Salmonella colonization in mice. Our results support the development of a GMMA-CPS-based vaccine against non-typhoidal Salmonella.

Identification of swinepox virus from natural outbreaks in pig population of Assam.

Outbreaks of swinepox [caused by a swinepox virus (SWPV)] in pigs were investigated in 3 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. Three representative isolates from different places were selected for inoculation into confluent monolayers of Porcine Kidney-15 (PK-15) cell line. The cytopathic effects were characterized by cell rounding, nuclear vacuolation, cell fusion, granulation of cells and finally detachment from third blind passage onwards. The three genes viz., SPV18-20 and P42 of SWPV was targeted for confirmation of the virus. Swinepox virus was successfully adapted to the PK-15 cell line from seventh passage onwards. The isolated viruses were characterized by sequencing and phylogenetic analysis of P42 gene (extracellular envelope protein), a homologue of vaccinia virus F13L gene. In India, studies on swine pox are very limited. This is the first report on successful isolation of swinepox virus from north eastern region of India. Assam and the other north-eastern states of India being a hub for pig husbandry, isolation of swinepox virus will help in developing and formulating control strategies against the disease.

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.

Cloning, sequencing, and in silico characterization of Omp 28 of Salmonella Typhi (strain MTCC 733) to develop r-DNA vaccine for typhoid fever.

BACKGROUND: Typhoid is one of the most important diseases of human beings caused by Salmonella Typhi. There are many vaccine reported against Salmonella Typhi, but search for new candidate vaccine antigens is still going on because presently available vaccines have several limitations such as short-term immunity, high cost, and allergic reaction. Several approaches such as subunit vaccines, Vi polysaccharide, mutant vaccines, and r-DNA vaccines have been tested. r- DNA vaccines have shown some promising potential (targeted Omp). Omp 28 had shown very promising results and suggests that it should be used in further studies of animal protection against the disease. OBJECTIVE: Cloning, Sequencing and In silico analysis of Omp 28 gene to develop r-DNA vaccine of S. Typhi. MATERIALS AND METHODS: Omp 28 is made up of three identical subunits of 9.6 kDa showing PCR amplicon of 330 bp which has been cloned in the pJET vector. Recombinant clones has been sequenced, and data submitted to NCBI. Secondary structure was deduced by the Chou Fasman and Garnier method. The sequence of Omp 28 was studied for antigenic indexing, epitope mapping, and MHC mapping using various bioinformatics tool. RESULTS AND CONCLUSION: The sequence of Omp 28 has been assigned accession no GQ 907044.1 by NCBI. Secondary structure has shown it has more alpha region. Hydrophobic plot and surface probability plot shows most amino acids are surface exposed which is a requirement to develop a r-DNA vaccine. Antigenic sites are located within surface exposed regions and eight antigenic determinants are present in Omp 28. On Prosite analysis of Protein shown two motifs i.e. anaphylatoxin domain signature motif at position 219-252 and other one was iron sulphur binding region signature motif at position 36-44. On epitope analysis total six major B cell epitopes were observed which can provoke humoral immunity. On T cell epitope mapping several major epitopes has been found in case of MHC class I and MHC class II. It indicates that Omp 28 can provoke cell mediated as well as humoral immunity and can be proven a promising candidates of Salmonella Typhi.

Cloning, sequencing and in silico analysis of omp C of salmonella typhimurium.

Salmonella Typhimurium is an important pathogen having a broad host range. In human population it causes mostly gastroenteritis but there are reports in which it was found to be responsible to cause several lethal diseases like endocarditis and meningitis. Poultry products are the major sources of this organism in India as these are consumed at various stages of cooking. The available vaccines have their own limitations such as short-term immunity. Outer membrane proteins have shown some promising potential, so in the present study Omp C of Salmonella Typhimurium was cloned and sequenced to explore the possibility of development of r-DNA vaccine against Salmonella Typhimurium for poultry. The sequence of Omp C was studied for antigenic indexing, epitope mapping, and MHC mapping using various bioinformatic tools. The ORF analysis revealed a complete coding region of approximately 1000 bp. Five major and 13 minor B-cell epitopes were identified having an antigenic index of 1.7. The sequences also showed major histocompatibility complex (MHC) class I and class II binding region indicating a potential of eliciting cell-mediated immune response. The findings indicate that Omp C may be proven as promising candidate for development of r-DNA vaccine against Salmonella Typhimurium.