Bolstering Buck Fertility: The Impact of Asparagus racemosus Aqueous Extract on Semen Cryopreservation and Antioxidant Defense System.

Importance of Study: Semen cryopreservation results in sperm damage due to lipid peroxidation or oxidative stress, leading to a decrease in conception rate. The sperm damage during cryopreservation can be minimized with the use of suitable antioxidant supplements in semen diluent. Some herbs have potent antioxidant potential and can be used in semen diluent to protect the spermatozoa. Objective: Hence, the investigation was planned to evaluate the effect of Asparagus racemosus (A. racemosus) aqueous extract on buck semen quality during cryopreservation. Methodology: In the current study, semen was collected from eight Sirohi bucks, and from each buck, 8 ejaculates were collected. Good-quality semen samples were pooled during each collection. Pooled semen samples were then divided into four equal parts and diluted in TRIS buffer containing different concentrations of A. racemosus aqueous extract (different groups, i.e., G I -5 mg, G II -2.5 mg, G III -1.25 mg, and G IV -0 mg of A. racemosus aqueous extract in 1 mL TRIS buffer). All the diluted semen samples were kept at equilibration temperature (5°C) for 2 hours and then cryopreserved by the manual method. Semen samples were evaluated for various sperm characteristics and antioxidant status before and after cryopreservation. Results: Asparagus racemosus aqueous extract showed significant (p < 0.05) enhancement of sperm viability, sperm motility, acrosomal integrity, and plasma membrane integrity, whereas it reduced sperm abnormality. Furthermore, in the experimental groups, the antioxidant gene expression was found to be increased compared to that of the treatment group. G III (p < 0.05) showed significantly better results in terms of sperm viability, sperm motility, acrosomal integrity, and plasma membrane integrity. Conclusion: Asparagus racemosus aqueous extract has the antioxidant potential to protect buck spermatozoa during semen cryopreservation.

Pan msr gene deleted strain of Salmonella Typhimurium suffers oxidative stress, depicts macromolecular damage and attenuated virulence.

Salmonella encounters but survives host inflammatory response. To defend host-generated oxidants, Salmonella encodes primary antioxidants and protein repair enzymes. Methionine (Met) residues are highly prone to oxidation and convert into methionine sulfoxide (Met-SO) which compromises protein functions and subsequently cellular survival. However, by reducing Met-SO to Met, methionine sulfoxide reductases (Msrs) enhance cellular survival under stress conditions. Salmonella encodes five Msrs which are specific for particular Met-SO (free/protein bound), and 'R'/'S' types. Earlier studies assessed the effect of deletions of one or two msrs on the stress physiology of S. Typhimurium. We generated a pan msr gene deletion (Δ5msr) strain in S. Typhimurium. The Δ5msr mutant strain shows an initial lag in in vitro growth. However, the Δ5msr mutant strain depicts very high sensitivity (p < 0.0001) to hypochlorous acid (HOCl), chloramine T (ChT) and superoxide-generating oxidant paraquat. Further, the Δ5msr mutant strain shows high levels of malondialdehyde (MDA), protein carbonyls, and protein aggregation. On the other side, the Δ5msr mutant strain exhibits lower levels of free amines. Further, the Δ5msr mutant strain is highly susceptible to neutrophils and shows defective fitness in the spleen and liver of mice. The results of the current study suggest that the deletions of all msrs render S. Typhimurium highly prone to oxidative stress and attenuate its virulence.

Pathology, virulence-associated gene profiling, antimicrobial susceptibility, and pathogenicity of untypeable capsular serotypes of Pasteurella multocida isolated from slaughtered pigs of India.

Pasteurella multocida is widely distributed in all pig-rearing countries, affecting the economic viability and profitability of pig production. The present research highlights the molecular characterization and pathology of untypeable capsular serotypes of P. multocida in slaughtered pigs from prominent pig-rearing states of India. The prevalence of Pasteurellosis was 27.17% by Pasteurella multocida specific Pasteurella multocida specific PCR (PM-PCR). assay, while isolation rate was 7.62%. The microscopic lesions of bronchopneumonia, tonsillitis, and the presence of bacterial antigens in immunohistochemistry confirmed P. multocida with pathologies. In capsular typing, the majority of the isolates were untypeable with prevalence of 52.15% and 43.58% in molecular and microbiological methods, respectively. All the isolates showed the uniform distribution of virulence genes such as exbB, nanB, sodC, plpB, and oma87 (100%), while the variations were observed in ptfA, hasR, ptfA, pfhA, hsf-1, and plpE genes. The untypeable isolates showed higher prevalence of hsf-1 gene as compared to others. The untypeable serotypes showed a higher degree of resistance to ampicillin, amoxicillin, and penicillin antibiotics. The mouse pathogenicity testing of untypeable capsular isolates confirmed its pathogenic potential. The higher frequency of pathogenic untypeable isolates with antibiotic resistance profile might pose a serious threat to the pigs, and therefore, preventive measures should be adopted for effective control.

Carrier status of Streptococcus suis in the palatine tonsils of apparently healthy slaughtered pigs of India.

Streptococcus suis is an emerging bacterial pathogen of huge economic impact to the swine industry worldwide. The information regarding the carrier status of S. suis in the slaughtered pigs along with its genetic characterization is not available in Indian pig population, which needs to be addressed for the therapeutic and preventive measures. In the present study, 563 palatine tonsils of apparently healthy slaughtered pigs were probed for the prevalence, and genetic characterization of S. suis and prevalence were found to be 15.45% and 32.68% by bacteriological and molecular methods, respectively. In 87 isolates recovered, 6 cps-types were detected showing the predominance of serotype 7 (24.13%) and 5 (18.39%), whereas 11 cps-types were detected in tonsillar DNA involving cps-types 9 (28.26%) and 7 (14.13%) as the major serotypes with arcA+/sly+/epf+/mrp- being the prevalent genotype. The histopathological changes with the immunodetection of S. suis antigen confirmed its persistence in asymptomatic carriers. Of 87 bacterial isolates, 7 isolates (serotypes 7 & 2) were pathogenic to Swiss albino mice showing the classical lesions of meningitis and septicemia. The presence of virulent serotypes of S. suis in healthy slaughtered pigs suggests a great health risk to the people engaged in piggery operations and in-contact pigs.

Evaluation of real-time PCR as an alternative for potency testing of Brucella abortus vaccines.

The aim of the research was to evaluate real-time PCR (qPCR) as an alternate method for quantitative detection of Brucella abortus strain 544 (S544) in the spleen of mice for potency testing of live B. abortus strain 19 (S19) vaccine. IS711 and eryC gene-based qPCR were optimized for calculating copy number. The copy number was further correlated with live Brucella count in the spleen by standard plate count (SPC) method. The mice were immunized with S19 and challenged with S544 on 30th Day post-immunization. The spleen of mice was collected at 15th, 21st, and 30th days post challenge (DPC) for estimation of S19 and S544 load via SPC as well as qPCR. The noteworthy difference was observed between immunized and unimmunized group by both methods at all time points. The maximum correlation between SPC and qPCR method was observed at 15th DPC in both immunized and unimmunized group. Repeated experiments at 15th DPC gave the parallel significant difference between immunized and unimmunized group by both methods. Thus novel, risk-free qPCR method can be used for the indirect culture-free potency evaluation of S19 vaccine in order to preclude the cultivation of zoonotic Brucella organisms from spleen samples.

Non-infectious outer membrane vesicles derived from Brucella abortus S19Δper as an alternative acellular vaccine protects mice against virulent challenge.

The prime human and animal safety issues accentuate the search of promising newer alternative vaccine candidates to resolve complications associated with the live attenuated Brucella abortus strain19 (S19) vaccine. Outer membrane vesicles (OMVs S19 Δper) extracted from Brucella abortus S19Δper (S19Δper) as an alternative subunit vaccine candidate has been explored in the present study as OMVs are endowed with immunogenic molecules, including LPS and outer membrane proteins (OMPs) and do not cause infection by virtue of being an acellular entity. The LPS defective S19Δper released a higher amount of OMVs than its parent strain S19. Under transmission electron microscopy (TEM), OMVs were seen as nano-sized outward bulge from the surface of Brucella. Dynamic light scattering analysis of OMVs revealed that OMVs S19Δper showed the less polydispersity index (PDI) than OMVs S19 pointing towards relatively more homogenous OMVs populations. Both OMVs S19Δper and OMVs S19 with or without booster dose and S19 vaccine were used for immunization of mice and subsequently challenged with 2 × 105 CFU virulent Brucella abortus strain 544 (S544) to assess protective efficacy of vaccines. The less splenic weight index and less S544 count in OMVs immunized mice in comparison to unimmunized mice after S544 challenge clearly indicated good protective efficacy of OMVs. OMVs S19 Δper induced relatively high titer of IgG than OMVs S19 but conferred nearly equal protection against brucellosis. An ELISA based determination of IgG and its isotype response, Cytometric Bead Array (CBA) based quantitation of serum cytokines and FACS based enumeration of CD4+ and CD8+ T cells revealed high titer of IgG, production of both Th1 (IgG2a) and Th2 (IgG1) related antibodies, stimulation of IL-2, TNF (Th1) and IL-4, IL-6, IL-10 (Th2) cytokines, and induced T cell response suggested that OMVs S19Δper elicited Th1 and Th2 type immune response and ensured protection against S544 challenge in murine model.

Localization of Pasteurella multocida antigens in the brains of pigs naturally infected with Pasteurellosis revealing a newer aspect of pathogenesis.

Pasteurella multocida is an economically important respiratory pathogen of pigs confronting swine industry worldwide. Despite extensive research over the decades, its pathogenesis is still poorly understood. Recent reports have demonstrated the nervous system affection as a newer aspect of pathogenesis by Pasteurella multocida type B:2 in Haemorrhagic Septicemia, but there are no reports of the involvement of nervous system by P. multocida in pigs. Therefore, the study was aimed to explore the neurovirulence of Pasteurella multocida in naturally infected pigs. A total of 15 brains were collected from the natural cases of pig mortality suggestive of Pasteurellosis. Grossly, the leptomeninges were markedly congested and brains were oedematously swollen. Histologically, there was moderate to severe fibrinohaemorrhagic and mononuclear cells exudates present in the leptomeningeal tissue and cerebrospinal spaces. Similar vascular inflammatory lesions (perivascular and perineuronal) along with gliosis, neuronal degeneration and necrosis were noted in various subanatomical sites of the brain (cerebrum, cerebellum, brainstem and spinal cord). The culture and biochemical tests showed the presence of P. multocida within the brain tissue. P. multocida type specific antibody staining in the brain tissues revealed intense distribution of antigens in the inflammatory exudates of meningeal vessels, neurons, glial cells and endothelial cells of the blood vessels contributing its association with neuropathological lesions. Pasteurella multocida specific PCR amplification of capsular polysaccharide gene yielded 460 bp and multiplex PCR showed the involvement of capsular serogroups A &D. All the isolates showed the presence of 10 genes for virulence factors. The disease confirmation of both serotypes was proven by Koch's postulates using Swiss albino mice. Further, histopathological brain lesions along with the immunohistochemical detection of bacterial antigens were corroborated with natural cases of P. multocida as described above. To the best of our knowledge, we first time report the neuroinvasion of P. multocida in naturally infected pigs.

Association of ACK1, TFRC polymorphism with diarrhoeagenic E. coli adhesion patterns and their jejunal expression profile in Indian Ghurrah pigs.

A total of 9 SNPs located in TFRC and ACK1 genes of SSC13q41 genomic region were examined for their association with the adhesion pattern of native Indian pigs using local isolate of diarrhoeagenic E. coli. Phenotypic evaluation of adhesion pattern of 150 pigs revealed 116 animals positive for adhesion, whereas 34 animals had non-adhesive phenotype. Among the adhesive animals, 6, 87 and 23 pigs were strongly adhesive, weakly adhesive and adhesive, respectively. PCR-RFLP study revealed 8 polymorphic SNPs with low to moderate PIC ranging from 7.39 to 37.25% and low to high heterozygosities (8-70%). The loci g.291 C > T, rs81218930 C > T, rs318751568 C > T of TFRC and g.93222 C > A g.94600 C > T of ACK1 showed significant departure from HWE. The genotypic frequencies of the SNPs as well as the haplotypes did not differ significantly (P > 0.05) across the adhesion patterns except one SNP (ACK1-g.107371 A > C). Among the g.107371 A > C genotypes observed, CA was associated with non-adhesive phenotype. Furthermore, TFRC mRNA expression levels were found to be significantly (P < 0.05) different among various adhesive phenotypes, whereas that of ACK1 was significantly (P < 0.05) different between non-adhesive and adhesive groups. The significant association of SNP (ACK1-g.107371 A > C), which was also previously reported to influence ETECF4 mediated diarrhoea susceptibility, implicates its wider application in genetic control of piglet diarrhoea. Furthermore, the up-regulation of TFRC gene expression in adhesive group supports its proposed role in activation of immune cells against E. coli and intracellular iron transport.

Resistance to ETEC F4/F18-mediated piglet diarrhoea: opening the gene black box.

Diarrhoea, a significant problem in pig rearing industry affecting pre- and post-weaning piglets is caused by enterotoxigenic Escherichia coli (ETEC). The ETEC are classified as per the fimbriae types which are responsible for bacterial attachment with enterocytes and release of toxins causing diarrhoea. However, genetic difference exists for susceptibility to ETEC infection in piglets. The different phenotypes found in pigs determine their (pigs') susceptibility or resistance towards fimbrial subtypes/variants (F4ab, F4ac, F4ad and F18). Specific receptors are present on intestinal epithelium for attachment of these fimbriae, which do not express to same level in all animals. This differential expression is genetically determined and thus their genetic causes (may be putative candidate gene or mutations) render some animals resistant or susceptible to one or more fimbrial subtypes. Genetic linkage studies have revealed the mapping location of the receptor loci for the two most frequent variants F4ab and F4ac to SSC13q41 (i.e. q arm of 13th chromosome of Sus scrofa). Some SNPs have been identified in mucin gene family, transferring receptor gene, fucosyltransferase 1 gene and swine leucocyte antigen locus that are proposed to be linked mutations for resistance/susceptibility towards ETEC diarrhoea. However, owing to the variety of fimbrial types and subtypes, it would be difficult to identify a single causative mutation and the candidate loci may involve more number of genes/regions. In this review, we focus on the genetic mutations in genes involved in imparting resistance/susceptibility to F4 or F18 ETEC diarrhoea and possibilities to use them as marker for selection against susceptible animals.

Protective role of Brucella abortus specific murine antibodies in inhibiting systemic proliferation of virulent strain 544 in mice and guinea pig.

AIM: The major objective of the investigation was to evaluate the hitherto uncharacterized potential of Brucella-specific antibodies to win the battle against virulent Brucellaabortus infection. MATERIALS AND METHODS: Brucella-specific immune serum was raised in mice. The antibody titer of serum was determined by standard tube agglutination test and indirect enzyme-linked immunosorbent assays (iELISA). Groups of mice and guinea pigs were passively immunized with serum containing specific agglutinin titers. 24 h after immunization, all animals along with unimmunized controls were challenged with B. abortus S544. Total B. abortus S544 counts in the spleen of each animal collected on the 7th day of challenge was determined to evaluate the protective index (PI) of anti-Brucella serum by statistical analysis. RESULT: A dose-dependent protective response to immune mice serum was observed in both experimental models though the values of PI of mice were higher than those obtained for guinea pigs. The PI values in mice passively immunized with 50 IU or 25 IU antibodies were 1.38 and 0.69, respectively. In guinea pigs, however, animals passively immunized with 50 IU or 25 IU antibodies showed PI values equivalent to 0.79 and 0.41, respectively. CONCLUSION: The observations support our hypothesis that the presence of antibodies inhibits the initial multiplication and eventual colonization of systemic organs by B. abortus. Therefore, a predominant antibody-mediated response induced by a vaccine is expected to protect the animal against the most severe clinical outcome of infection.

Novel Polymerase Spiral Reaction (PSR) for rapid visual detection of Bovine Herpesvirus 1 genomic DNA from aborted bovine fetus and semen.

Bovine herpesvirus-1 (BHV-1) is a major viral pathogen affecting bovines leading to various clinical manifestations and causes significant economic impediment in modern livestock production system. Rapid, accurate and sensitive detection of BHV-1 infection at frozen semen stations or at dairy herds remains a priority for control of BHV-1 spread to susceptible population. Polymerase Spiral Reaction (PSR), a novel addition in the gamut of isothermal techniques, has been successfully implemented in initial optimization for detection of BHV-1 genomic DNA and further validated in clinical samples. The developed PSR assay has been validated for detection of BHV-1 from bovine semen (n=99), a major source of transmission of BHV-1 from breeding bulls to susceptible dams in artificial insemination programs. The technique has also been used for screening of BHV-1 DNA from suspected aborted fetal tissues (n=25). The developed PSR technique is 100 fold more sensitive than conventional PCR and comparable to real-time PCR. The PSR technique has been successful in detecting 13 samples positive for BHV-1 DNA in bovine semen, 4 samples more than conventional PCR. The aborted fetal tissues were negative for presence of BHV-1 DNA. The presence of BHV-1 in bovine semen samples raises a pertinent concern for extensively screening of semen from breeding bulls before been used for artificial insemination process. PSR has all the attributes for becoming a method of choice for rapid, accurate and sensitive detection of BHV-1 DNA at frozen semen stations or at dairy herds in resource constrained settings.