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.

Development of a peptide based latex agglutination assay for serotype identification of foot and mouth disease virus.

Out of 200 serum samples collected from cattle (142) and buffaloes (58) of various ages and sexand subjected to latex agglutination test (LAT) using serotype specific peptides (O, A, Asia 1) and also with peptide for non-structural protein 2B (NSP-2B), 114 (70%) samples were positive against FMDV type ‘O’, 102 (51%) against serotype ‘A’ and 104 (52%) against serotype ‘Asia 1’. With NSP-2B peptide a total of 71 (35.5%) samples were positive. The results suggest that LAT could be used for the diagnosis of foot and mouth disease virus as it is easy, cheap and effective test.

Comparative analysis of protein profiles of wild virulent (E156) and aroA-htrA double deletion mutant vaccine strain (S30) of Salmonella enterica subsp. enterica serovar Abortusequi under in vivo and in vitro growth conditions.

In the present study, cell lysate and cell supernatant of the both strains i.e., virulent wild type (E156) and mutant (S30) vaccine strains of Salmonella enterica subspecies enterica serovar Abortusequi (S. Abortusequi), grown under varied in vivo and in vitro conditions were subjected to SDS PAGE and western blotting (using rabbit hyperimmune serum). Variation in growth conditions did not have any significant effect on expression of different proteins. SDS PAGE of E156 and S30 cell lysate (CL) revealed 26 and 28 bands, respectively with 3 prominent proteins of 71, 46 and 42 kDa in cell lysate of E 156 and 4 prominent proteins 71, 65, 46 and 40 kDa in S30 strain. The cell supernatant (CS) from both the strains, subjected to SDS PAGE, exhibited similarity in protein profile among these strains, however three bands of 65, 53 and 40 kDa were more prominent in CS preparation of S30, whereas a 56 kDa protein was prominent in CS of E156. Western blotting of E156 and S30 revealed 3 unique proteins of 65, 53 and 40 kDa present in CS preparation of S30 strains which could be used for differentiation of mutant and wild strains and also in development of test for differentiating vaccinated animals from naturally infected.

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.

Antigenic competition among different 'O' antigens of Salmonella enterica subspecies enterica serovars during hyperimmunization in pony mares.

The present study on antigenic competition among somatic 'O' antigens of different Salmonella groups (A, B, C1, C2, D and E1) in mares revealed that the immune response to most of the antigens was not (A, B, C2) or little (C1, D) affected by antigenic competition. However, E1 group antigen, which induced high antibody titres (Avg. 12967.3) when given alone, produced almost 3.5 log2 lower antibody titres on giving with other antigens, indicating the antigenic competition among some Salmonella group antigens. The antigenic competition varied for different antigens even of the similar chemical nature. Therefore, antigens belonging to different somatic groups should not be given together for the purpose of raising polyvalent serum or for immunization using multivalent Salmonella vaccines prepared from strains of different 'O' groups revealing antigenic competition.

Novel haemolysins of Salmonella enterica spp. enterica serovar Gallinarum.

Haemolysins of Salmonella are important due to their probable role in pathogenesis of systemic salmonellosis and use in sub-serovar level typing. The present study was undertaken to determine haemolytic potential of Salmonella Gallinarum strains through phenotypic and genotypic methods. Amplification of haemolysin gene (clyA) and cytolysin gene (slyA) was attempted in order to determine their role in haemolysin production. Study on 94 strains of S. Gallinarum revealed the production of two types of haemolysis viz., beneath the colony haemolysis (BCH) or contact haemolysis and clear zone haemolysis (CZH). Haemolysis was observed on blood agar prepared with blood of cattle, buffalo, sheep, goat, horse, rabbit, guinea pig, fowl, and human blood group A, B, AB and O. Although, haemolysis was also observed on blood agar prepared with whole blood, clarity of zone was more evident on blood agar made from washed erythrocytes. Clear zone haemolysis was best observed on blood agar prepared with washed erythrocytes of goat and a total of 12% (11 of 94) S. Gallinarum strains under study produced CZH on it. The clyA gene could not be detected in any of the 94 strains under study, while slyA gene could be amplified uniformly irrespective of haemolytic potential (CZH) and haemolytic pattern (BCH) of the strains. The study suggested that the two types of haemolysis (CZH and BCH) observed among S. Gallinarum strains may not be due to either slyA or clyA gene products and thus there may be some other gene responsible for haemolytic trait in Gallinarum serovar. Different haemolytic patterns of strains under study indicated multiplicity of haemolysins in S. Gallinarum.

Pathogenic effects of Salmonella enterica subspecies enterica serovar Typhimurium on sprouting and growth of maize.

The study was undertaken to understand effects and survival of S. enterica subspecies enterica serovar Typhimurium (S. Typhimurium), a zoonotic serovar, on maize seed germination and plant growth. All the four strains of S. enterica subspecies enterica serovar Typhimurium significantly reduced germination of maize seeds in sprouting plates as well as in soil. About > or =2.7x10(3) Salmonella cfu ml(-1) of soaking water, while > or =2.7x10(7) Salmonella cfu g(-1) soil were required to significantly inhibit germination of maize. Similar inhibition of germination could be observed using > or = 16 mg of bacteria free Salmonella cell lysate (CL) protein per g of soil or > or =0.5 mg of CL protein per ml of soaking water in sprouting plates. At the constant dose of 3.6x10(7) to 3.8x10(7) Salmonella cfu or 5 mg cell lysate protein ml(-1) of soaking water, four strains of Salmonella significantly reduced germination, however difference between strains was insignificant. After germination too, maize growth was affected both by Salmonella organism and CL with little strain-to-strain variation. All Salmonella persisted in growing plants from 15 to 35 days of plant age and up to 190 days in soil. Maize plants once grown for a week in sterile soil were resistant to invasion of S. enterica subspecies enterica serovar Typhimurium in their leaves even in doses as high as 7.6x10(9) cfu g(-1) of soil. Salmonella persisted better and longer in plants grown from contaminated seed sown in loam soil, but rarely in plants grew in sandy soil. All maize plants had Salmonella in their stumps even after 35 days of sowing irrespective of kind of soil, primary source of infection (soil or seed) and type of S. enterica subspecies enterica serovar Typhimurium strain. The study revealed that Salmonella is not only zoonotic but a phytopathogen also.

Haemolysins of Salmonella, their role in pathogenesis and subtyping of Salmonella serovars.

Haemolysin patterns of 175 strains of different Salmonella enterica subspecies enterica serovars isolated from different animal sources and places were determined using 11 different blood agar media made with either non-washed horse/sheep erythrocytes or with washed erythrocytes of cattle, sheep, horse, goat, rabbit, guinea pig, and human A, O and B blood groups. Study on 47 strains belonging to 10 serovars of Salmonella from buffalo meat (buffen), 42 strains of 11 serovars from goat meat (chevon): 16 strains of Salmonella enterica serovar Paratyphi B and 25 of S. enterica serovar Paratyphi B var Java from fish, meat, meat products and clinical cases; 45 isolates of S. Abortusequi from aborted mares (18), fetal contents (21), aborted donkey mares (2) and 4 reference strains, revealed that all host restricted Salmonella namely, S. enterica serovar Gallinarum, S. enterica serovar Anatum, S. enterica serovar Abortusequi and S. enterica serovar Paratyphi B could be divided into different haemolysin types based on their inability to produce haemolysis on one or more types of blood agar, while strains of all zoonotic Salmonella serovars induced haemolysis on all the 9 types of blood agar made of washed erythrocytes. None of 175 Salmonella could produce hemolytic colonies on blood agar made of non-washed horse/ sheep erythrocytes. Haemolysin type I (lysing all types of washed erythrocytes) was the commonest one among all serovars except S. Abortusequi, none of which lysed horse erythrocytes. Salmonella enterica serovar Abortusequi having hemolytic activity against sheep erythrocytes were more invasive but had lesser ability to survive in sheep mononuclear cells than non-hemolytic strains. Multiplicity of haemolysins appeared significant epidemiological tool.