Evaluation of humoral immune responses against C. perfringens epsilon toxin in Iranian sheep and goats after vaccination.

Clostridium perfringens is a common cause of death in domestic animals worldwide. However, vaccination on a regular basis is an economically beneficial means for controlling clostridial contamination.The objective of the current investigation was to evaluate the humoral immune responses using iELISA in Iranian sheep and goats following the vaccination programs administered by the bacterin-toxoid polyvalent entrotoxemia vaccine. A total of one-hundred-and-twenty animals, consisting of sixty sheep and sixty goats, were randomly divided into three groups. These animals were vaccinated with clostridial vaccine on days 0 and 14 using two different dosages. Blood samples were collected on day zero, 15, 30, 45, 60, 75, and 90 following vaccination. The sera samples were then separated and antibody titers were measured using an in-house enzyme-linked immunosorbent assay (ELISA) against C. perfringens epsilon toxin. The titers of antibodies in sheep were notably higher than those in goats, particularly after receiving the booster dose. No statistically significant variations were identified in the immune responses of Iranian sheep and goat breeds. (p>0.05). Overall, the duration of the humoral immune response in goats upon administration of the clostridial vaccine was relatively brief, requiring multiple booster injections.

Shedding rate of Brucella spp. in the milk of seropositive and seronegative dairy cattle.

Brucellosis in cattle herds has caused severe economic losses in many regions worldwide. A cross-sectional study was performed to investigate the presence of Brucella spp. in industrial dairy cattle farms in Iran. For this purpose, 935 blood and 935 milk samples were randomly collected from industrial dairy cattle farms in Iran's Alborz and Tehran provinces. Blood and milk samples were collected on the same day from each cow. Serological, bacteriological, and molecular characterization of Brucella isolates were performed using standard methods. Our results revealed the seroprevalence of brucellosis in dairy cattle farms in the Alborz and Tehran provinces, reaching 19.8%, 6.7%, 5.1%, 14.1%, and 13.1% using the Rose Bengal plate test (RBPT), serum agglutination test (SAT), 2-mercaptoethanol test (2-ME), indirect enzyme-linked immunosorbent assay (i-ELISA) and milk ring test (MRT), respectively. Furthermore, the results of bacterial culture and PCR analyses showed the presence of Brucella abortus among dairy cattle in the Alborz province and Brucella melitensis and B. abortus among dairy cattle in the Tehran province. Moreover, statistical analysis with Cohen's Kappa has highlighted the near-perfect agreement between RBPT and i-ELISA (k = 0.86). In contrast, substantial agreement was shown between RBPT and SAT performance (k = 0.70) and moderate agreement between RBPT and 2-ME (k = 0.67). The findings of this investigation showed shedding of Brucella in the milk of seropositive cows, which is a serious problem involving the maintenance and further spread of Brucella infection on the farm. Therefore, for brucellosis detection or eradication in dairy cattle farms, bacteriological and serological tests of milk samples should be performed along with blood analysis to inhibit the uncontrolled spread of the disease in animals and humans.

Development of an in-house Indirect ELISA for detection of bovine viral diarrhoea virus antibodies in bovine sera.

Bovine viral diarrhoea virus (BVDV) infection is a worldwide distributed animal disease. BVDV is the causal agent of congenital defects, diarrhea, reproductive failure, and contaminating biological products. Virus Neutralization test (VNT) as a gold standard method is used for detection of BVDV. Although this assay is very sensitive and specific, it has disadvantages including requires to an experienced person and cell culture facilities. VNT is time-consuming. It is important to design a method that does not have the mentioned disadvantages. So, in-house indirect enzyme linked immunosorbent assay (i-ELISA) was developed for laboratories where it is not possible to perform VNT. The system was made using NADL strain of BVDV and MDBK cell line. This ELISA system was compared with a commercial ELISA kit using 99 bovine sera. Coefficient of variation (CV) was calculated 3.9% and 4.8% for the positive and negative control, respectively for the designed i-ELISA system. The sensitivity, specificity, and accuracy of i-ELISA system was 88%, 53.6%, and 70.7% respectively. Based on our result correlation between in- house and commercial ELISA kit for detection of antibody against BVDV in bovine sera was significant (Kappa coefficient =0.41, p < 0.05). Results of the present study suggested that an in-house ELISA as an affordable and confident system for primary screening of the sera used for biological product.

A simple method for purification of epsilon toxin of Clostridium perfringens type D for serum neutralization assay.

Enterotoxaemia, a disease that affects domestic ruminants, is caused by the epsilon toxin of Clostridium perfringens type D and B. Control and prophylaxis are based on systemic vaccination of small ruminant herds with epsilon toxoid. Purified epsilon toxin is an essential material for vaccine evaluation. It is also necessary for diagnosis of enterotoxaemia disease in the field by in vitro tests including ELISA. The aim of this study was to set up a method for preparation of functional purified epsilon toxin of C. perfringens type D to be used in serum neutralization test. In this study, epsilon toxin was prepared from C. perfringens type D culture precipitated with ammonium sulfate, dialyzed against phosphate buffered saline (PBS) buffer and then, purified using chromatography system. Then, the purified epsilon toxin was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Toxin function was confirmed by cell culture and minimum lethal dose (MLD) assays. Also rabbits were immunized by vaccine in two turns with a 28-day interval. Then, blood samples were collected, and serum neutralization (SN) test was carried out. Results showed that the purified toxin was suitable for SN assay. Our purification method was simple, fast and cost-effective for preparation of epsilon toxin.