Evaluation of vaccine candidate potential of ΔaroA, ΔhtrA and ΔaroAΔhtrA mutants of Salmonella enterica subspecies enterica serovar Abortusequi in guinea pigs.

Salmonella enterica subspecies enterica serovar Abortusequi (S. Abortusequi), a host adapted Salmonella causes abortions, still births and foal mortality in equids. Though known since more than 100 years, it is still a problem in many of the developing countries including India. There is dearth of really good vaccine affording immunity lasting at least for one full gestation. In search of a potential vaccine candidate, three defined deletion mutants (ΔaroA, ΔhtrA and ΔaroAΔhtrA) of S. Abortusequi were tested in guinea pig model for attenuation, safety, immunogenicity, humoral immune response, protective efficacy and persistence in host. The ΔhtrA and ΔaroAΔhtrA mutants were found to be safe on oral inoculation in doses as high as 4.2×109 cfu/animal. Also through subcutaneous inoculation ΔaroAΔhtrA mutant did not induce any abortion in pregnant guinea pigs. All the three mutants did not induce any illness or death in 1-2 week-old baby guinea pigs except ΔhtrA mutant which caused mortality on intraperitoneal inoculation. Inoculation with mutants protected against challenge and increased breeding efficiency of guinea pigs. After >4.5 months of mutant inoculation, guinea pigs were protected against abortifacient dose of wild type S. Abortusequi and mother guinea pigs also conferred resistance to their babies to the similar challenge. Early humoral immune response of S. Abortusequi mutants was characteristic. Faecal excretion of ΔaroA and htrA mutants was detected up to 45 days of inoculation in guinea pigs while ΔaroAΔhtrA mutant could not be detected after 21 days of inoculation. The results indicated that the double deletion mutant (ΔaroAΔhtrA) was the most effective and safe candidate for vaccination against S. Abortusequi through mucosal route of inoculation.

Effect of pH and salinity on pathogenicity of Flavobacterium columnare and Myxobacterium sp. in Indian cat fish, Clarias batrachus (Linn) and Heteropneustes fossilis (Bloch).

Flavobacterium columnare (FC) and Myxobacterium sp. recorded persistently associated in fish hatchery and culture system of Himalayan and Sub - Himalayan regions were found to be pathogenic. The pH and salinity played a significant role on the pathogenicity of these potent pathogens in case of Clarias batrachus and Heteropneustes fossilis. LD50 value of FC was 104.5 CFU in both the fishes and those of Myxobacterium sp it was 106 CFU ml-1 fish-1. Fish challenged with F. columnare and Myxobacterium sp. (@ 0.2 ml fish-1) individually consisting 105-6cfu ml-1 exhibited explicit symptoms of columnaris disease and marked with ulceration and saddle back lesion on the dorsal side of body. Maximum reisolation of inoculated bacteria was recorded at pH 7.0 and 7.5 and at 0.0-0.5 (F. columnare) and 0.0-1.0% (Myxobacterium sp.) salinity. Foregoing results elucidated that F. columnare was more sensitive to salinity in comparison to Myxobacterium sp. and their pathogenicity significantly (p<0.05) depends on the salinity and pH that might be one of the physical factors to control their proliferation.

Infertility associated with sub clinical salmonellosis.

Subclinical infection of guinea pigs with isogenic wild type and aroA, htrA and aroA-htrA mutants of Salmonella enterica subspecies enterica serovar Abortusequi (S. Abortusequi) induced infertility, while mutants had little or no effect on conception rate in guinea pigs. Conception rate was significantly lower in guinea pigs inoculated with wild type (S-787) and aroA mutant of S. Abortusequi than those inoculated with intracellular survival deficient htrA or aroA-htrA mutants of S. Abortusequi. Chi-test analysis revealed that none of the three mutants could be attributed to low conception rate, but wild type Salmonella inoculation and chronic carriage of the pathogen were significant cause of low conception rate in guinea pigs. Role of S. Abortusequi in causation of infertility was proven from the experiment for the first time.