J. R. Rao

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.

High-level expression of SAG1 and GRA7 gene of Toxoplasma gondii (Izatnagar isolate) and their application in serodiagnosis of goat toxoplasmosis

Toxoplasmosis, caused by Toxoplasma gondii, is a disease of economic importance in livestock, especially in sheep and goats, where it causes abortion. Although several serological tests are in use for diagnosis of infection, production of reliable reagents is a constraint. An 814 bp sequence coding for a truncated surface antigen surface antigen 1 (SAG1), a tachyzoite stage-specific protein, as well as a 657 bp sequence coding for granule protein 7 (GRA7), a dense granule protein were PCR amplified from the genomic DNA of T. gondii. The amplified products were ligated in pET-32b(+) and pET-32c(+) expression vectors, respectively and subsequently transformed into BL21(DE3)pLysS cells. A high-level expression of the histidine-tagged SAG1 and GRA7 fusion proteins were obtained after 7h of incubation. The recombinant proteins were purified using Ni-NTA column and were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis using reference positive sera from goat, rabbit and humans at 1:100 dilution. Subsequently, the diagnostic efficiency of the recombinant proteins, either individually or as a cocktail of the recombinant proteins, was assessed with 56 reference goat sera by enzyme-linked immunosorbent assay (ELISA). The immunoreactivity of the refolded SAG1 and GRA7 was evidenced by high OD values. The reactivity of the recombinant proteins as a cocktail preparation was more than that of individual proteins in ELISA and could detect accurately the infection in goats. This is the first report of serological detection of caprine toxoplasmosis by ELISA using a cocktail of recombinant Toxoplasma proteins.

A rapid MTT colorimetric assay to assess the proliferative index of two Indian strains of Theileria annulata

A study was undertaken to compare the proliferative index of macroschizont-infected lymphoblastoid cells of two Indian strains [Izatnagar (IZT) and Parbhani (PBN)] of Theileria annulata by an in vitro MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide], colorimetric assay. Culture conditions were standardized to define the optimal cell concentration in 96-well microculture plates to yield nearly 100% living cells for measurement of the metabolized formazan activity. A cell concentration of 1.5x10(5) cells/ml was found to be optimal for effective discrimination of the parasite strains. On the basis of conversion of MTT by the actively proliferating lymphoblastoid cells, the PBN strain of T. annulata stimulated a 2.5-fold increase in formazan activity in comparison to the IZT strain. The in vitro MTT assay was found to be a simple and convenient method for assessing the cell activation rate and growth, obviating the need for radioactive material for the assay. The results of the proliferation assay are discussed in relation to previously documented information on the biological characteristics of this important pathogen of cattle.

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.

Comparative evaluation and economic assessment of coprological diagnostic methods and PCR for detection of Cryptosporidium spp. in bovines.

The role of Cryptosporidium spp. as a major cause of diarrhoea and gastrointestinal illness of protozoan origin in neonatal calves has been established. Many coprological and serological techniques have been described for detection of the parasites with the limitations of sensitivity and specificity. Polymerase chain reaction (PCR) technique offers a useful alternative to conventional diagnosis of Cryptosporidium spp. in bovines from both clinical and environmental samples. We compared four conventional coprological techniques, viz., direct faecal smear staining (DFSS), normal saline sedimentation staining (NSSS), Sheathers flotation (SF) and Sheathers flotation sedimentation staining (SFSS) with PCR directed against the 18S SSU rRNA gene as standard reference test for the diagnosis of cryptosporidiosis in bovines. Out of 457 faecal samples collected from neonatal bovine calves, specific PCR amplification was achieved in 138 samples, whereas, 65 samples turned positive by DFSS. Normal saline sedimentation staining, SF and SFSS could detect 92, 82 and 109 samples as positive, respectively. Sheathers flotation sedimentation staining was found to be the most sensitive (82.6%) and specific (98.76%) among the coprological techniques. On per sample processing based cost analysis, DFSS was found to be the most economical method (15 cents) followed by NSSS (19.6 cents), SF (23.6 cents) and SFSS (33.9 cents). The time taken for complete processing and diagnosis varied between 70 and 100 min. PCR based diagnosis of a sample took about 7.5-8h for completion and cost of diagnosis was estimated as approximately 7.604 US

Interaction between Eimeria uzura infection and aflatoxicosis in Japanese quail (Coturnix coturnix japonica).

per sample. Among the conventional coprological methods, SFSS provided the required sensitivity and specificity along with nominal cost for diagnosis on per sample basis, and may be considered as a viable diagnostic alternative when PCR is not an option for a particular laboratory setting, especially in developing countries. This is the first comparative study describing the sensitivity and specificities of four conventional coprological techniques altogether with respect to PCR along with the economic assessment and per sample diagnosis time of all the techniques for the diagnosis of cryptosporidiosis in bovines.

Comparison of indirect fluorescent antibody test (IFAT) and slide enzyme linked immunosorbent assay (SELISA) for diagnosis of Babesia bigemina infection in bovines

The clinical signs and gross lesions caused by Eimeria uzura (10(5) oocysts) in Japanese quail (Coturnix coturnix japonica) exhibited little or no influence in the face of intercurrent dietary aflatoxicosis (1 p.p.m. of aflatoxin B1 from Day 0 to 55). Similarly, no significant differences in the mucosal morphology of the intestine were evident histologically between the two groups of Japanese quail. The nervous signs of ataxia, leg weakness, incoordination of movement, torticollis and terminal opisthotonos were toxin-induced manifestations. In the aflatoxic quail, hypoplastic changes and selective depletion of lymphocytes were more prominent in the bursa of fabricius. Increased relative mean weights of liver, kidney, spleen, crop, proventriculus and gizzard were observed in birds due to aflatoxin sensitivity. The combination of E. uzura infection and aflatoxicosis in Japanese quail may cause significant weight loss, and increased oocyst production and reproductive potential.

Slide Enzyme-Linked Immunosorbent Assay for the Diagnosis of Babesia bigemina Infection in Bovines

An indirect fluorescent antibody test (IFAT) and slide enzyme linked immunosorbent assay (SELISA) were standardized for the detection of antibodies specific to Babesia bigemina in experimentally infected bovine calves and subsequently used for the screening of naturally infected bovine and bubaline sera. In experimentally infected calves positive reactivity was detected in sera at the earliest on day 7 by both the tests. Serological studies for detection of B. bigemina specific antibodies in 180 cow and 120 buffalo serum samples procured from endemic zones of Uttar Pradesh and Punjab revealed 56.11% and 23.33% seropositivity, respectively, both by SELISA and IFAT. Variation in the reactivity pattern between these tests was found to be non significant. The sensitivity of SELISA was determined to be 94.85% whereas the specificity was 90.85% in comparison to IFAT. The agreement between the two tests by kappa statistics at 95% confidence interval revealed κ- value of 0.853 that depicts almost a perfect degree of agreement. The findings employing experimental as well as test sera from cattle and buffalo from some of the tick infested zones of India suggested that SELISA could be a useful tool for seroprevalence studies on babesiosis, as the test is less cost intensive with high levels of sensitivity and specificity.

On the High Seroprevalence of Bovine Babesiosis in Wynad District of Kerala

A slide enzyme-linked immunosorbent assay (SELISA) for the diagnosis of Babesia bigemina infection in cattle was standardized. Acetone-fixed whole Babesia bigemina-infected erythrocytes on micro-slides were immunoreacted with bovine serum samples followed by antibovine horseradish peroxidase conjugate and developed using diaminobenzidine tetrahydrochloride as a substrate. The positive immunoreactivity (staining pattern) was visualized in the form of dark brown piroplasms. Using the laboratory-standardized SELISA with a sensitivity of 94.4%, the seroprevalence of babesiosis was studied in cattle from two endemic areas of the disease. In comparison to IFAT, SELISA detected higher number of serum samples positive for bovine babesiosis.

Influence of dietary aflatoxin on Eimeria uzura infection in Japanese quail (Coturnix coturnix japonica).

Abstract Ravindran, R., Mishra, A.K. and Rao, J.R. 2002. On the high seroprevalence of bovine babesiosis in Wynad district of Kerala. J. Appl. Anim. Res., 22: 43–48. An indirect fluorescent antibody test was used to study the seroprevalence of Babesia bigemina infection in apparently healthy crossbred bovids of Wynad district, Kerala. Screening of 93 sera samples by IFAT revealed parasite specific antibodies in 67.6per cent of the animals suggesting high carrier status of the disease.