O.K. Raina

Prevalence and molecular characterization of bovine Cryptosporidium isolates in India.

A survey based on PCR assay of 18S SSU rRNA gene revealed a 30.2% infection with Cryptosporidium spp., out of 457 faecal samples collected from neonatal bovine calves across three different regions of India. The PCR-RFLP pattern of the gene in all the positive cases established the species as Cryptosporidium parvum. Highest prevalence was recorded in the monsoon months (37.3%) and in the calves showing acute diarrhoea (32.3%). The calves below 15 days of age were mostly affected (45.1%). The infection was more prevalent in the northern parts (35.4%) of the country than in the eastern or southern parts. Results indicated that C. parvum was the only species of Cryptosporidium prevalent in bovine calves in three different geographical regions of India.

Prevalence of Cryptosporidium andersoni: A molecular epidemiological survey among cattle in India

Cryptosporidiosis is an important and established cause of calfhood morbidity in bovines. The present communication reports the prevalence of Cryptosporidium infection among juvenile and adult cattle (6-24 months old) in India based on examination of faecal samples collected from 350 animals across three different agro-climatic regions of the country and further confirmation by a two-step nested PCR assay targeting 18S ssu rRNA gene. A total of 45 samples were positive for Cryptosoridium species by nested PCR assay. The PCR products were subjected to restriction fragment length polymorphism (RFLP) analysis using SspI and VspI restriction enzymes for species differentiation. The results showed that the species involved in all the samples found positive was Cryptosporidium andersoni. The overall prevalence rate was 12.85%, with highest occurrence in the northern states (14.37%) of the country. The animals between age group of 6-12 months were mostly affected (21.67%) and the season wise prevalence of infection was more during the hot and humid monsoon season (20.16%). The results clearly demonstrated that C. andersoni is the major Cryptosporidium species affecting juvenile and adult cattle in three agro-climatically different geographical regions of India. This is the first report on prevalence of C. andersoni in bovines from India the confirmation of which is based on application of nested PCR and PCR-RFLP based molecular tools.

Detection of antibodies to Toxoplasma gondii in domesticated ruminants by recombinant truncated SAG2 enzyme-linked immunosorbent assay

An antibody detection recombinant enzyme-linked immunosorbent assay (ELISA) specific for Toxoplasma gondii was laboratory standardized using recombinant truncated surface antigen 2 (SAG2) protein of T. gondii. A 483-bp sequence coding for truncated tachyzoite stage-specific SAG2 protein was amplified and ligated in pPROExHT-b expression vector to transform Escherichia coli DH5α cells. A high-level expression of the histidine-tagged fusion protein was obtained after 8xa0h of incubation. The recombinant protein was affinity purified using Ni-NTA agarose column and characterized by SDS-PAGE and Western blot analysis. Subsequently, the diagnostic potential of the recombinant protein was assessed with 168 field sera samples from sheep, goats and cattle. Among the small ruminants, 50xa0% (nu2009=u200960) sheep sera samples and 41.26xa0% (nu2009=u200963) goat samples were detected positive for T. gondii-specific antibodies. As far as seroprevalence of toxoplasmosis in cattle is concerned, 64.44xa0% (nu2009=u200945) of sera samples assayed were found to be positive. When compared to indirect fluorescent antibody test (IFAT), the sensitivity of the recombinant truncated SAG2 antigen-based ELISA (rec-SAG2-ELISA) ranged from 81.25 to 87.10xa0% while the specificity was 85.71 to 91.43xa0% with substantial agreement between the tests.

Establishment of Ascaridia galli in betamethasone-treated chickens

Twenty-five day-old White Leghorn chickens were each infected orally with 500 (Group I), 1000 (Group II) and 2000 (Group III) infective eggs of Ascaridia galli and were killed 30 days after the infection. A high percentage of the infecting dose (6.5%) established as adult worms in the intestine of chickens receiving the lowest level of primary infection, but as the amount of primary infection given to birds increased, there was a significant fall in the percentage establishment of adult worms in the intestine. A similar pattern of worm establishment was shown by chickens of the same age and receiving similar levels of primary infections, but which were treated with betamethasone at a dose of 2 mg/kg body weight commencing 5 days before and continuing up to 15 days after the infection. Betamethasone-treated birds, however, showed more establishment of worms in the intestine, but lower weight gains in comparison to the birds which were not treated. Different levels of primary infections given, with or without treatment with betamethasone, had no effect on the sex ratio of the resultant male/female worm populations, which became established in almost equal numbers in the intestine of chickens.

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.

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.