In silico mutation of aromatic with aliphatic amino acid residues in Clostridium perfringens epsilon toxin (ETX) reduces its binding efficiency to Caprine Myelin and lymphocyte (MAL) protein receptors.

Enterotoxaemia (ET) is a severe disease that affects domestic ruminants, including sheep and goats, and is caused by Clostridium perfringens type B and D strains. The disease is characterized by the production of Epsilon toxin (ETX), which has a significant impact on the farming industry due to its high lethality. The binding of ETX to the host cell receptor is crucial, but still poorly understood. Therefore, the structural features of goat Myelin and lymphocytic (MAL) protein were investigated and defined in this study. We induced the mutations in aromatic amino acid residues of ETX and substituted them with aliphatic residues at domains I and II. Subsequently, protein-protein interactions (PPI) were performed between ETX (wild)-MAL and ETX (mutated)-MAL protein predicting the domain sites of ETX structure. Further, molecular dynamics (MD) simulation studies were performed for both complexes to investigate the dynamic behavior of the proteins. The binding efficiency between 'ETX (wild)-MAL protein' and 'ETX (mutated)-MAL protein complex' interactions were compared and showed that the former had stronger interactions and binding efficiency due to the higher stability of the complex. The MD analysis showed destabilization and higher fluctuations in the PPI of the mutated heterodimeric ETX-MAL complex which is otherwise essential for its functional conformation. Such kind of interactions with mutated functional domains of ligands provided much-needed clarity in understanding the pre-pore complex formation of epsilon toxin with the MAL protein receptor of goats. The findings from this study would provide an impetus for designing a novel vaccine for Enterotoxaemia in goats.Communicated by Ramaswamy H. Sarma.

Temperature response of enriched pre-pubertal caprine male germline stem cells in vitro.

The present study aims to evaluate culture temperature-dependent variation in survival, growth characteristics and expression of stress, pluripotency, apoptosis, and adhesion markers in enriched caprine male germline stem cells (cmGSCs). For this, testes from pre-pubertal bucks (4-5 months; n = 4) were used to isolated cells by a two-step enzymatic digestion method. After enrichment of cmGSCs by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), viability of CD90+ cells was assessed before co-cultured onto the Sertoli cell feeder layer at different temperatures (35.5, 37.0, 38.5, and 40.0 °C). The culture characteristics of cells were compared with MTT assay (viability); cluster-forming activity assay, SA-ß1-gal assay (senescence), BrdU assay (proliferation), and transcript expression analyses by qRT-PCR. Moreover, the co-localization of pluripotency markers (UCHL-1, PLZF, and DBA) was examined by a double-immunofluorescence method. The cells grown at 37.0 °C showed faster proliferation with a significantly (p < 0.05) higher number of viable cells and greater number of cell clusters, besides higher expression of pluripotency markers. The transcript expression of HSPs (more noticeably HSP72 than HSP73), anti-oxidative enzymes (GPx and CuZnSOD), and adhesion molecule (ß1-integrin) was significantly (p < 0.05) downregulated when grown at 35.0, 38.5, or 40.0 °C compared with 37.0 °C. The expression of pluripotency-specific transcripts was significantly (p < 0.05) lower in cmGSCs grown at the culture temperature lower (35.5 °C) or higher (38.5 °C and 40.0 °C) than 37.0 °C. Overall, the culture temperature significantly affects the proliferation, growth characteristics, and expression of heat stress, pluripotency, and adhesion-specific markers in pre-pubertal cmGSCs. These results provide an insight to develop strategies for the improved cultivation and downstream applications of cmGSCs.

Semen quality and total microbial load: An association study in important Indian Goat breeds during different seasons.

The invasion of the male urogenital tract by microorganisms, and its subsequent effects on sperm fertilising ability, has not been well discussed in bucks. The present study was conducted to assess the bacterial load in fresh semen of the 2-6 years old bucks. For conducting the experiment, semen ejaculates from 18 bucks (6 from each breed namely Jakhrana, Jamunapari and Barbari) were used. We collected 5 ejaculates from each buck in each season (Summer-April to June, Rainy-July to Sept and Winter-November to January). Semen was collected with the artificial vagina (AV) method, and separate AV was used for each buck every time. The semen collection frequency was once in a week. Immediately after initial evaluation, collected semen samples were transferred to the microbiology laboratory of the institute. Thereafter, the semen samples were subjected to bacteriological examination to assess the microbial load. The results of the current study indicate that the microbial load in the semen was significantly (p < 0.05) higher in the Jamunapari bucks and in aged bucks. Bacteriospermia in different seasons was not significantly varied; however, nonsignificant increase in microbial load during the rainy season was observed. Overall, the average bacterial load in the semen of Jamunapari, Barbari and Jakhrana bucks was found 540.50 ± 55.88 CFU/ml, 391.81 ± 46.33CFU/ml and 388.93 ± 44.71 CFU/ml respectively. No significant difference in bacterial counts in the subsequent ejaculates among bucks was observed. Moreover, correlation analysis revealed that the proportions of motility, viability, plasma membrane integrity and acrosomal integrity were negatively influenced by the increased bacterial contamination of buck semen.

Temporal changes in plasma profile of pregnancy-associated glycoprotein, progesterone and estrone sulfate associated with fetal number during early- and mid-pregnancy in goats.

This study was designed to investigate plasma profile of pregnancy-associated glycoprotein (PAG), progesterone (P4) and estrone sulfate (E1S) during early- and mid-pregnancy. The goal was to explore the relationships with values for reproductive variables, to detect the most reliable predictor variable, and to identify the most desirable time point for blood collection for determining fetal number in goats. After ultrasonographic examination at d35-40 post-mating, blood sampling of 15 pregnant goats (total 18) was continued until d114. The PAG profile was characterized by gradual increase during early pregnancy from d26 to d51 and thereafter concentrations were relatively constant until d114 of gestation. The effect of fetal number on plasma PAG, P4 and E1S was first evident on d28, d51 and d26, respectively. During mid-pregnancy, does with twins had a greater (P < 0.05) PAG (S-N = 2.54 ± 0.12 compared with 1.59 ± 0.11), P4 (18.91 ± 0.67 compared with 14.51 ± 0.47 ng/mL) and E1S (16.34 ± 0.76 compared with 11.32 ± 0.44 ng/dL) as compared with does with a singleton fetus. Plasma PAG but not P4 and E1S was positively correlated with fetal number and birth weight of kids during early pregnancy. Multivariate linear regression and discriminant function analyzes allowed for identification of plasma PAG as the most reliable predictor for fetal number and birth weight of kids. Furthermore, d58 was the most suitable single time point for prediction of fetal number using PAG as a biomarker. In conclusion, plasma profile of PAG, P4 and E1S was affected by fetal count. Plasma PAG was identified as the most reliable predictor variable of fetal number and birth weight of kids as compared to plasma P4 and E1S.

Comparative molecular characterization and phylogenetic analysis of cerebral and non-cerebral coenurosis in Indian goats.

Coenurus cerebralis is the larval stage of Taenia multiceps, which infects the muscles and brain of goats and, to a lesser extent, sheep. The resulting cerebral and non-cerebral infections caused by the larval form (metacestode) of this cestode are commonly known as coenurosis. A weak emaciated carcass of five months old female goat, on necropsy, revealed numerous parasitic cysts (n = 56, grossly visible) in the visceral cavity including heart, diaphragm, thoracic cavity, abdominal cavity and pelvic inlet. A large number of variable sized parasitic cysts were also observed embedded in the pericardium and myocardium causing functional damage to the heart. The parasite caused extensive tissue damage at gross and microscopic levels in the heart including traumatic destruction of the myocardium with degenerative and necrotic changes and infiltration of mononuclear cells. On parasitological examination, the cysts were identified as Coenurus cerebralis, as the scolices had characteristic four suckers and a rostellum with a double crown of hooks. Further confirmation was done using polymerase chain reaction targeting specific ND1 and CO1 genes. Phylogenetic analysis of CO1 and ND1 genes showed a major branch comprising two clades of T. multiceps grouped as separate entities with the first clade showing T. multiceps/Coenurus cerebralis native CIRG strain (cerebral) being placed in proximity to T. multiceps/Coenurus cerebralis CIRG strain (non-cerebral/visceral) compared to the Chinese strains of T. multiceps. The phylogenetic analysis of ND1 and CO1 genes of C. cerebralis of cerebral and non-cerebral isolates revealed close proximity but expressed in two different disease forms (i.e., visceral coenurosis and neural coenurosis) which indicated that they were very close divergent from a common ancestor. On the basis of the observations it was concluded that goat died due to cardiac dysfunction resulting from severe systemic infection of metacestode of T. multiceps was closely related to isolate that caused neural coenurosis in another goat. Based on the sequencing analysis and phylogenetic information, the possible differences in the clinical manifestation (neural or visceral) could be attributed to the pathogenesis.