Cloning and sequence analysis of IL-2, IL-4 and IFN-γ from Indian Dromedary camels (Camelus dromedarius).

The cDNAs of three cytokines, viz., IL-2, IL-4 and IFN-γ from Dromedary camels were amplified by PCR using Bactrian camel sequences and subsequently cloned for sequence analysis. Relationship based on amino acid sequences revealed that Dromedary camel IL-2 shared 99.5% and 99.3% identity at the nucleotide and amino acid levels with Bactrian camel IL-2. In the case of IL-4, the identity of Dromedary camel was 99.7% and 99.2% at the nucleotide and amino acid levels, respectively with that of Bactrian camel. The Dromedary camel IFN-γ shared 100% identity both at nucleotide and amino acid levels with Bactrian camel IFN-γ. Phylogenetic analysis based on amino acid sequences indicated the close relationship in these cytokine genes between the Dromedary camel and other camelids.

Sonographic monitoring of early follicle growth induced by melatonin implants in camels and the subsequent fertility.

The present study examined the effect of melatonin implants on follicle growth in dromedary camels two months ahead of their natural breeding season (December to March). Female camels (n = 6) were treated with melatonin implants at the dose rate of 1 implant per 28 kg body weight sc. Control camels (n = 6) were administered an SC placebo implant of 8 ml vitamin A. Ovarian ultrasonography was performed at weekly interval upto 7 weeks. Camels were mated with virile stud when a follicle (≥10 mm) was visible on either of the ovaries. Blood was collected on day 7, 9, 15, 20, 25 and 30 for assay of plasma progesterone and sonography performed at the same time. Small follicles (2-3 mm) appeared around the periphery of ovaries in 83.3% of camels by day 7 and in 100% camels by day 14. By the end of 7th week an ovulatory size follicle (≥1.0 cm) could be observed in 83.3% of treated camels, and these camels were mated with virile studs. In control group, small follicles appeared at the periphery of ovaries only in 66.6% camels but did not progress in growth except in one camel (16.6%) however, ovulating size (≥10 mm) follicle was not observed in any camel by the end of 7th week. All treated camels ovulated and one treated camel became pregnant while early embryonic death occurred in one camel. Non-pregnant camels of both groups were mated during the breeding season. All camels of treatment group and 33.33% camels of control group became pregnant by the end of breeding season (April 2010). It was concluded that melatonin implants can augment the follicle growth in lactating camels ahead of the breeding season and pregnancy can occur on mating. Fertility of treated camels during the breeding season is improved.

Zoonotic cases of camelpox infection in India.

This study reports the first conclusive evidence of zoonotic camelpox virus (CMLV) infection in humans associated with outbreaks in dromedarian camels (Camelus dromedaries) in northwest region of India during 2009. CMLV infection is usually restricted to camels and causes localised skin lesions but occasionally leads to generalised form of disease. However, the present outbreak involved camel handlers and attendants with clinical manifestations such as papules, vesicles, ulceration and finally scabs over fingers and hands. In camels, the pock-like lesions were distributed over the hairless parts of the body. On the basis of clinical and epidemiological features coupled with serological tests and molecular characterization of the causative agent, CMLV zoonosis was confirmed in three human cases. Clinical samples such as skin scabs/swabs and blood collected from affected animals and humans were analysed initially, for the presence of CMLV-specific antigen and antibodies by counter immunoelectrophoresis (CIE); serum neutralization test (SNT); plaque-reduction neutralization test (PRNT) and indirect immunoperoxidase test which was later confirmed by amplification of CMLV-specific ankyrin repeat protein (C18L) gene. Virus isolation was successful only from samples collected from camels. Further, sequence analyses based on three full-length envelope protein genes (A27L, H3L and D8L) revealed 95.2-99.8% and 93.1-99.3% homology with other Orthopoxviruses at nucleotide and amino acid levels, respectively. Phylogram of the three genes revealed a close relationship of CMLV with Variola virus (VARV). Considering the emerging and re-emerging nature of the virus, its genetic relatedness to VARV, zoonotic potential and productivity losses in camels; the control measures are imperative in curtailing economic and public health impact of the disease. This is the first instance of laboratory confirmed camelpox zoonosis in India.

Cloning and phylogenetic analysis of Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) from Indian dromedaries (Camelus dromedarius).

The cDNAs of two proinflammatory cytokines viz., IL-6 and TNF-α from dromedarian camels were amplified by PCR using bactrian camel sequences and subsequently cloned for sequence analysis. Relationship based on amino acid revealed that dromedarian camel IL-6 shared 99.5% identity both at nucleotide and amino acid level with bactrian camel IL-6 and in case of TNF-α, the identity of dromedarian camel was 99.4% and 99.1% at nucleotide and amino acid level, respectively with that of bactrian camel. Phylogenetic analysis based on their amino acid sequences indicated the close relationship in these cytokine genes between dromedarian camel and other members of camelids.

Isolation and characterization of Indian isolates of camel pox virus.

In this study, we isolated and identified three camel pox viruses (CMLV) from two outbreaks of camel pox infection in camels associated with eruptions on cheeks, nostrils, limbs, scrotum, and sheath that occurred at different places of Bikaner district, Rajasthan (India). The scab specimens collected were subjected for virus isolation in Vero cell culture, and the isolated viruses were characterized by employing polymerase chain reaction (PCR) and sequencing. The causative agent was identified as CMLV, based on A-type inclusion, B5R and C18L genes-specific PCRs and partial sequencing of these genes, which clearly confirmed that the outbreaks were caused by CMLV and identity of CMLV isolates. Further, phylogenetic analysis of partial C18L gene sequences have showed that Indian CMLV are clustered together with other reported isolates/strains.

Detection of benzimidazole resistance in Haemonchus contortus using RFLP-PCR technique.

Benzimidazole (BZ) resistance in Haemonchus contortus is linked primarily with the mutation in the beta-tubulin isotype 1 gene that substitute phenylalanine (Phe) to tyrosine (Tyr) at 200 codon of the gene. In the present study, a new restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) technique has been developed for detection of BZ resistance in the beta-tubulin isotype 1 gene of H. contortus. The technique utilizes two primers viz. AvikaF and AvikaR to amplify the region containing mutation in the beta-tubulin gene followed by restriction digestion. After digestion, the 'rr' individuals (homozygous resistant) revealed 257 and 48 bp bands, the 'rS' individuals (heterozygous) showed 305, 257 and 48 bp bands, while 'SS' individuals (homozygous susceptible) revealed uncut 305 bp band. A total of 162 adult male H. contortus collected from Avikanagar, Jaipur and Bikaner regions (54 from each region) were genotyped for analyzing BZ resistance in the beta-tubulin gene. Out of which, 130 adults were 'rr' types, 20 'rS' types and 12 'SS' types. The results showed that genotypic frequencies of different genotypes (rr, rS and SS) were highly significant difference among the three regions (P<0.001). The 'rr' individuals were higher (98%) in Jaipur followed by Avikanagar (93%) and Bikaner (50%) regions. Overall, the prevalence of BZ resistant allele (r) was higher (86%) as compared to BZ susceptible allele (S) (14%). The technique was also found suitable for genotyping of larvae of H. contortus and yielded reproducible results. The study indicated that RFLP-PCR is an easy, reproducible and less expensive than allele specific PCR. This technique will be helpful in establishing the prevalence rate of BZ resistance in H. contortus and can also be utilized for existing worm control programme.

A comparative evaluation of parasitological, serological and DNA amplification methods for diagnosis of natural Trypanosoma evansi infection in camels.

A representative number of 217 camels (Camelus dromedarius) from different areas of western Rajasthan State, India, were examined from July 2002 to May 2003 for Trypanosoma evansi infection. The tests used were parasitological (wet blood film, WBF; stained thin blood smear, TBS), immunodiagnostic (double antibody sandwich enzyme linked immunosorbent assay for antigen detection, Ag-ELISA), and DNA amplification by polymerase chain reaction (PCR). These techniques were compared and the best efficiency was found for the last named (PCR). A prevalence of T. evansi infection was detected in 17.05, 9.67, 4.60 and 4.14% by PCR, Ag-ELISA, TBS and WBF with a sensitivity of 100, 56.75, 27.02 and 24.32%, respectively. PCR revealed a specific 227bp band in positive samples. The intensity of PCR bands was variable in different test samples depending upon the level of infection in the test samples. The history of intermittent fever, emaciation, oedema, poor body condition significantly correlated with positive serological status in ELISA as well as trypanosome DNA detection by PCR.