Humoral and cytokine response elicited during immunisation with recombinant Immune Mapped protein-1 (EtIMP-1) and oocysts of Eimeria tenella.

Eimeria tenella, the causative agent of caecal coccidiosis, is a pathogenic gut dwelling protozoan which can cause severe morbidity and mortality in farmed chickens. Immune mapped protein-1 (IMP-1) has been identified as an anticoccidial vaccine candidate; in the present study allelic polymorphism was assessed across the IMP-1 coding sequence in E. tenella isolates from four countries and compared with the UK reference Houghton strain. Nucleotide diversity was low, limited to expansion/contraction of a CAG triplet repeat and five substitutions, three of which were non-synonymous. The EtIMP-1 coding sequence from a cloned Indian E. tenella isolate was expressed in E. coli and purified as a His-tagged thioredoxin fusion protein. An in-vivo vaccination and challenge trial was conducted to test the vaccine potential of recombinant EtIMP-1 (rEtIMP-1) and to compare post-vaccination immune responses of chickens to those stimulated by live oocyst infection. Following challenge, parasite replication measured using quantitative PCR was significantly reduced in chickens that had been vaccinated with rEtIMP-1 (rIC group; 67% reduction compared to UC or unimmunised controls; 79% reduction compared to rTC group or recombinant thioredoxin mock-immunised controls, p<0.05), or the birds vaccinated by infection with oocysts (OC group, 90% compared to unimmunised controls). Chickens vaccinated with oocysts (OC) had significantly higher levels of interferon gamma in their serum post-challenge, compared to rEtIMP-1 vaccinated birds (rIC). Conversely rEtIMP-1 (rIC) vaccinated birds had significantly higher antigen specific serum IgY responses, correlating with higher serum IL-4 (both p<0.05).

Oral route of transmission: Trypanosoma evansi in a mice model experiment.

Twelve Swiss albino mice of either sex and equal body weight were randomly divided in 2 groups (I and II), consisting of 9 and 3 mice respectively and were used to conduct the study. A dose of 2.5 × 104 number of Trypanosoma evansi was instantly fed to each mouse of group I. Each mouse of group II was inoculated intraperitoneally with same dose of parasites through infected mice blood and kept separate. The tail blood of each mouse was examined daily up to 30 days post infection by examination of wet blood film and Giemsa-stained blood smears for presence of any trypanosomes. Out of 9 mice of group I those were infected orally, 3 (33.33%) mice became positive for presence of T. evansi both by examination of wet blood film and Giemsa-stained blood smears after 4, 6 and 7 days post infection. After 2 days post infection all intraperitoneally infected mice were found positive for T. evansi. Thus incubation period in orally infected mice was longer than the intraperitoneally infected mice. All the positive mice of both the groups died with high parasitaemia after 3-4 days of first appearance of parasitaemia. From the present study, it can be concluded that besides mechanical or parenteral means of transmission, T. evansi could also be transmitted through oral route. Thus zoo carnivores might be infected with T. evansi and develop disease by eating infected blood or flesh of the infected animals, as a prey and predator relationship.

Development of recombinant BgP12 based enzyme linked immunosorbent assays for serodiagnosis of Babesia gibsoni infection in dogs.

Indirect ELISA and dot-ELISA using recombinant BgP12 (rBgP12) were developed for the diagnosis of Babesia gibsoni infected dogs. The complete open reading frame of BgP12 gene (378bp) was cloned in pET-32a(+) expression vector and expressed in Escherichia coli as a soluble thioredoxin (Trx) fusion protein. The purified rBgP12 was used for production of anti-rBgP12 rabbit serum, which recognized a native 12-kDa protein in B. gibsoni infected erythrocyte by Western blot analysis. To evaluate the potential of rBgP12 for the serodiagnosis of B. gibsoni, a panel of serum/plasma samples from dogs infected with B. gibsoni (n=13), uninfected sera (n=13) and sera from dogs infected with other haemoparasites viz., Babesia canis vogeli (n=3), Ehrlichia canis (n=3), Hepatozoon canis (n=1) and Dirofilaria immitis (n=1) were used in ELISA formats. In addition, the performance of rBgP12 based indirect ELISA and dot-ELISA were evaluated using 75 serum/plasma samples collected from suspected dogs, in respect to the nested PCR as reference test. The diagnostic sensitivities of indirect ELISA and dot-ELISA were 94.59% and 89.18%, respectively, while their specificities were 84.21% and 81.57%, respectively. Moreover, both the assays using rBgP12 showed no cross reaction with sera from dogs infected with other common haemoparasites indicating their high specificity. High kappa values of indirect ELISA and dot-ELISA indicated the potentials of these assays with substantial agreement at 95% confidence level. It is concluded that indirect ELISA and dot ELISA using rBgP12 might be used in large scale epidemiological surveys and clinical diagnosis of B. gibsoni infection in dogs.

Cloning and sequencing of beta-tubulin and internal transcribed spacer-2 (ITS-2) of Eimeria tenella isolate from India.

Beta-tubulin is an important multifunctional protein of eukaryotes abundant in the cytoskeleton and responsible for the formation of tubulin, structures responsible for cell morphology and which aid in motility and intracellular transportation. It has been used as a genotypic marker for studying the evolutionary history and phylogenetic relationships between eukaryotic organisms. Internal transcribed spacers of the ribosomal RNA genes have been widely used for typing inter-species and intra-species variation. An Indian isolate of Eimeria tenella was genotyped following the cloning and sequencing of beta-tubulin and internal transcribed spacer-2 (ITS-2) and compared with other reference isolates of E. tenella. The ß-tubulin has 99 % intra-species similarity at the gene level and the functional homology of the protein is very high with more than 95 % amino-acid similarity across the Apicomplexa. The ITS-2 sequence had a similar pattern of nucleotide base arrangement with 99 % homology to Houghton and Nippon strains of E. tenella.

Development of loop-mediated isothermal amplification (LAMP) for detection of Babesia gibsoni infection in dogs.

Diagnosis of canine babesiosis, caused by Babesia gibsoni is difficult, especially in chronically infected dogs. A loop mediated isothermal amplification (LAMP) assay was developed and standardized by using four oligonucleotide primers targeting the hypervariable region of 18S rRNA gene (GenBank Acc. no. KC461261). The primers specifically amplified B. gibsoni DNA, while no amplification was detected with DNA from non-infected dogs as well as from dogs infected with Babesia canis vogeli, Hepatozoon canis, Ehrlichia canis and Trypanosoma evansi. The assay could detect 1.35 × 10(-7) parasitaemia and 10(-4) dilution of recombinant plasmid, equivalent to 12 pg of target DNA. All the samples were tested by nested PCR as well as LAMP assay. LAMP was found to be 10 times more sensitive than nested PCR targeting the same gene. Out of 75 suspected field samples, collected from different parts of the country, LAMP could detect B. gibsoni in 43 samples, while nested PCR and microscopy could detect 37 and 23 samples, respectively. High sensitivity, specificity and rapidity of LAMP assay may be exploited for screening large number of samples in a field setting.

Experimental studies on survivality and degenerative changes of Trypanosoma evansi after death of host.

Twenty adult Swiss albino mice, 20 rats and 10 rabbits were artificially infected with Trypanosoma evansi and killed at the peak of parasitaemia to know the period of survivality of T. evansi and degenerative changes of the parasite after death of these hosts. Examination of Giemsa stained blood smears and wet blood smears revealed the presence of parasites and live trypanosomes along with motility in the heart blood of mice and rats up to 14 h and in rabbits up to 13 h post death. Mouse inoculation test (MIT) conducted with heart blood up to 13 h post death of mice and rabbits became positive. MIT with both heart blood and portal blood of rats became positive up to 14 h post death. The liver and lung impression smears could detect the parasites up to 14 h of death of mice and rats and up to 13 h post death of rabbits whereas spleen impression smears revealed the presence of parasites up to 12 h post death of these animals. It is confirmed that T. evansi infection in animals may be diagnosed after post mortem examination of hosts by demonstration of parasites.

Development and evaluation of serodiagnostic assays with recombinant BgSA1 of Babesia gibsoni.

Indirect ELISA, dot-ELISA and double antibody sandwich ELISA (DAS-ELISA) using truncated recombinant BgSA1 (rBgSA1) were developed for detecting Babesia gibsoni infection in naturally infected dogs. Truncated BgSA1 gene of 858 bp, encoding 32 kDa protein was cloned in pET-32a(+) expression vector, expressed in Escherichia coli and the recombinant protein was purified under native conditions. To evaluate the ability of the truncated rBgSA1as serodiagnostic reagent for B. gibsoni infection, a panel of sera/plasma samples from dogs infected with B. gibsoni (n = 13), uninfected sera (n = 13) and sera from dogs infected with other haemoparasites namely, Babesia canis vogeli (n = 3), Ehrlichia canis (n = 3), Hepatozoon canis (n = 1) and Dirofilaria immitis (n = 1) were used. Besides these, 75 samples collected from dogs suspected for babesiosis were used to evaluate the performance of rBgSA1 based serological assays in comparison to nested PCR. Based on the results, the diagnostic sensitivity of indirect ELISA, dot-ELISA and DAS-ELISA were 97.3%, 91.9% and 100%, respectively, when nested PCR was taken as a reference test, while their specificities were 81.6%, 84.2% and 97.4%, respectively. Further, DAS-ELISA had a quantitation limit of 0.03 µg/ml of the rBgSA1. High kappa values of indirect ELISA, dot-ELISA and DAS-ELISA were recorded, indicating that these assays had substantial to almost perfect agreement at 95% confidence level. There was no cross-reactivity with sera from dogs infected with B. canis vogeli, E. canis, H. canis and D. immitis. The results suggest that the indirect ELISA, dot-ELISA and DAS-ELISA with rBgSA1 may be used in large scale epidemiological surveys and clinical diagnosis of B. gibsoni infection in dogs. DAS-ELISA has advantages over indirect or dot-ELISA in the detection of current infection as well as monitoring the parasite burden.