Serodiagnosis of surra, caused by Trypanosoma evansi, is still based on native antigens purified from bloodstream form of T. evansi grown in rodents. In order to investigate prospective diagnostic possibilities as an alternative for native antigens, we cloned, expressed 26 kDa calflagin protein containing 218 amino acids from T. evansi (Indian Strain) in Escherichia coli. The potential of recombinant calflagin (rCLF) protein as diagnostic antigen was evaluated in immunoblot and indirect ELISA using experimentally infected equine serum samples from 0 to 84 days post infection. The antibodies against T. evansi were detected with rCLF antigen in serum samples of experimentally infected equines as early as 10 days and 14 days post infection, using immunoblot and ELISA respectively. No cross-reactivity was observed with rCLF antigen in ELISA with different serum samples of equines positive for Equine herpesvirus 1, Burkholderia mallei, and Theileria equi infections. Several immunoreactive regions ranging from 10 to 28 kDa were detected using distinct T. evansi isolates (pony, cattle, donkey and camel origin) indicating presence of multiple calflagin family members in a single trypanosome. Indirect immunofluorescence antibody test with anti-CLF rabbit hyperimmune serum showed localisation of native immunogenic protein near attachment of flagellum. The rCLF protein was found to be a potential diagnostic candidate for distinguishing T. evansi positive and negative equine serum sample, suggesting that it could be used for serological surveys in animals for surra. In addition, it could be used with other potential diagnostic candidates to improve the diagnostic efficiency.

Trypanosoma evansi, an extracellular haemoprotozoan parasite, causes surra in a wide range of domestic and wild animals. In the present study, a diagnostic PCR assay was developed using primers targeting invariable surface glycoprotein (ISG) gene of T. evansi, which amplified a 196 bp product. The DNA was extracted from purified trypanosomes and T. evansi infected mice blood, and serially diluted ranging from 20 ng/µl to 0.002 pg/µl and from 90 ng/µl to 0.009 pg/µl, respectively. The diagnostic sensitivity of the PCR assay was 0.02 ng/µl (2×101 parasites) and 0.09 ng/µl (1×102 parasites) with purified parasite DNA and infected mice blood DNA, respectively. The PCR assay was also performed on extracted genomic DNA from 86 blood samples from the field out of which 3 animals were found positive by ISG-PCR assay. The developed ISG gene based PCR assay could be employed for sensitive detection of early infection, sub-clinical status of trypanosomosis and drug efficacy studies in animals.

Trypanosoma evansi causes an economically important disease called surra, which is responsible for significant losses to livestock productivity in Asia, Africa, Central and South America. In the present study, a SYBR Green based quantitative PCR (qPCR) assay was optimized using primers targeting the internal transcribed spacer 1 (ITS–1) region of rRNA gene for quantitative estimation of T. evansi infection in experimentally infected mice. The sensitivity of qPCR assay was found approximate to the gold standard TBR1/2 primers based PCR assay and two times more than the conventional parasitological techniques, viz. wet blood film (WBF) stained thin blood smear (TBS) and microhaematocrit centrifugation test (MHCT). The detection limit of the assay was 1.5 parasite equivalence or 0.15 pg T. evansi DNA. On fourth day post infection, 100% (6/6) mice were detected positive by all parasitological techniques with parasitaemia ranging from 1.6×105 to 2.1×108 parasites. The study indicated that this assay can be applied for quantitative estimation of parasitaemia in animals suffering from surra and may be helpful for understanding disease status, risk analysis and efficacy of drug used in treatment of disease.

Burkholderia mallei is the causative agent of glanders which is a highly contagious and fatal disease of equines. Considering the nature and severity of the disease in equines, and potential of transmission to human beings, glanders is recognised as a 'notifiable' disease in many countries. An increasing number of glanders outbreaks throughout the Asian continents, including India, have been noticed recently. In view of the recent re-emergence of the disease, the present study was undertaken to estimate the prevalence of glanders among indigenous equines from different parts of India. Serum samples were analysed by complement fixation test (CFT) and ELISA for the detection of B mallei specific antibodies. A total of 7794 equines, which included 4720 horses, 1881 donkeys and 1193 mules were sampled from April 2011 to December 2014 from 10 states of India. Serologically, 36 equines (pony=7, mules=10, horses=19) were found to be positive for glanders by CFT and indirect-ELISA. The highest number of cases were detected in Uttar Pradesh (n=31) followed by Himachal Pradesh (n=4) and Chhattisgarh (n=1). Isolation of B mallei was attempted from nasal and abscess swabs collected from seropositive equines. Four isolates of B mallei were cultured from nasal swabs of two mules and two ponies. Identity of the isolates was confirmed by PCR and sequencing of fliP gene fragment. The study revealed circulation of B mallei in northern India and the need for continued surveillance to support the eradication.

Present address: 1National Fellow (rkg@scientist.com), 2,4Research Associate (parvati.hsr@gmail.com, sachingl76 @gmail.com), 3Senior Research Fellow (deepakgr21 @gmail.com), 5Principal Scientist (kumarsanjay66@yahoo.com. Trypanosomosis (surra) caused by Trypanosoma evansi is one of the important diseases of livestock including equines. The disease has the widest geographical range of all the pathogenic trypanosome species and is a major constraint to livestock productivity on the three continents, Asia, Africa and South America. T. evansi infects a wide range of domestic and wild animals in India, viz. camels, horses, donkeys, mules, cattle, buffaloes, dogs, pigs, goats, tigers and elephants. It is hypothesized that T. evansi has evolved from T. brucei when infected camels moved to tse tse free areas and adapted to survive without natural biological vector and transformed to mechanical transmission through haematophagus biting flies and thus existence of sexual recombination in this species is highly unlikely and follow clonal propagation population structure. The variations in host susceptibility, virulence of different isolates are commonly observed conditions in animals suffering from surra. Therefore, study of genetic variability among different T. evansi isolates is very important to know molecular epidemiology, and formulation of prevention and control strategies of the disease. For specific detection and molecular typing of blood protozoan, random amplification of polymorphic DNA (RAPD) technique has gained importance in recent years. The present study was carried out to know genetic variability among Indian T. evansi isolates of different hosts and agro-ecological regions using RAPDPCR approach with new and already reported informative primers. Six isolates of T. evansi were used in the study, collected from different livestock species (horse, donkey, camel and cattle) inhabiting different agro-ecological regions of India (Table 1), were propagated in vivo in Swiss albino mice. Initial inoculation to mice was done with 1 × 105 trypanosomes by intra-peritoneal route. Thereafter, the parasitaemia was monitored regularly. At peak parasitaemia, mice were bled by cardiac puncture for isolation and purification of parasites. The trypanosomes from infected Indian Journal of Animal Sciences 85 (11): 1205–1208, November 2015/Short communication https://doi.org/10.56093/ijans.v85i11.53261

Cytokines play very important roles in the development of protective immune responses against a variety of pathogens. During the last decade rapid progress has been made in the cloning and characterization of cytokines from livestock and companion animals. However, no such work was carried out on cytokines of Indian horse breed. The present work was undertaken with the objective of generating sequence information of cytokines of Marwari horse. The cDNAs encoding interleukin (IL)-2, IL-4, IL-10 and IL-18 of Marwari horse were cloned, sequenced and subsequently compared with respective interleukin sequences of mammalian species including zebra, donkey, cattle, buffalo, sheep, goat, camel and pig available in GenBank database. Horse IL-10 and IL-18 shows nearly 90% sequence homology both at nucleotide and amino acid level with major livestock species. But horse IL-2 and IL-4 had significant sequence divergence with corresponding interleukins of domestic animals. Structural and functional conservation of horse interleukins in respect of cysteine residues, glycosylation, myristoylation, and phosphorylation motifs revealed that IL-10 and IL-18 molecules are evolutionary conserved across species. Nucleotide and amino acid sequences identity and phylogenetic analyses of the 4 cytokine genes in the present study indicated that horse cytokines are closely related to that of pig and camel cytokines.

Objective . To express truncated TssB protein of Burkholderia mallei and to evaluate its diagnostic efficacy for serological detection of glanders among equines. Materials and Methods . In an attempt to develop recombinant protein based enzyme-linked immunosorbent assay (ELISA), N-terminal 200 amino acid sequences of B. mallei TssB protein—a type 6 secretory effector protein—were expressed in prokaryotic expression system. Diagnostic potential of recombinant TssB protein was evaluated in indirect ELISA using a panel of glanders positive (n=49), negative (n=30), and field serum samples (n=1811). Cross-reactivity of the assay was assessed with equine disease control serum and human melioidosis positive serum. Results . In comparison to CFT, diagnostic sensitivity and specificity of ELISA were 99.7% and 100%, respectively. Conclusions . The indirect ELISA method using the truncated TssB offered safer and more rapid and efficient means of serodiagnosis of glanders in equines. These data highlight the use of TssB as potential diagnostic antigen for serological diagnosis of glanders.

Thermostabilizing effect of heavy water (D2O) or deuterium oxide has been demonstrated previously on several enzymes and vaccines like oral poliovirus vaccine and influenza virus vaccine. In view of the above observations, effect of heavy water on in situ thermostabilization of recombinant p26 protein on enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of equine infectious anemia virus (EIAV) infection was investigated in the present study. The carbonate-bicarbonate coating buffer was prepared in 60% and 80% D2O for coating the p26 protein in 96-well ELISA plate and thermal stability was examined at 4 °C, 37 °C, 42 °C, and 45 °C over a storage time from 2 weeks to 10 months. A set of positive serum (n = 12) consisting of strong, medium, and weak titer strength (4 samples in each category) and negative serum (n = 30) were assessed in ELISA during the study period. At each time point, ELISA results were compared with fresh plate to assess thermal protective effect of D2O. Gradual increase in the stabilizing effect of 80% D2O at elevated temperature (37 °C < 42 °C < 45 °C) was observed. The 80% D2O provides the thermal protection to rp26 protein in ELISA plate up to 2 months of incubation at 45 °C. The findings of the present study have the future implication of adopting cost effective strategies for generating more heat tolerable ELISA reagents with extended shelf life.

Trypanosoma evansi (T. evansi) is a causative agent of disease called surra, affecting wide range of domestic and wild animals. In this study, a PCR assay was developed using primers targeting ITS-1 region flaking between 5.8 S and 18 S subunits of rRNA. The test was employed using serially diluted DNA extracted from purified trypanosomes ranging from 200 ng/μl to 0.00002 pg/μl and T. evansi infected mice blood ranging from 350 ng/μl to 0.00035 ng/μl. The diagnostic sensitivity of PCR assay was found to be 0.2 pg/μl and 0.035 ng/μl with purified parasite DNA and infected mice blood DNA, respectively.

Equine infectious anemia (EIA)—a retroviral disease caused by equine infectious anemia virus (EIAV)—is a chronic, debilitating disease of horses, mules, and donkeys. EIAV infection has been reported worldwide and is recognized as pathogen of significant economic importance to the horse industry. This disease falls under regulatory control program in many countries including India. Control of EIA is based on identification of inapparent carriers by detection of antibodies to EIAV in serologic tests and “Stamping Out” policy. The current internationally accepted test for diagnosis of EIA is the agar gel immune-diffusion test (AGID), which detects antibodies to the major gag gene (p26) product. The objective of this study was to develop recombinant p26 based in-house immunoassays [enzyme linked immunosorbent assays (ELISA), and AGID] for EIA diagnosis. The synthetic p26 gene of EIAV was expressed in Escherichia coli and diagnostic potential of recombinant p26 protein were evaluated in ELISA and AGID on 7,150 and 1,200 equine serum samples, respectively, and compared with commercial standard AGID kit. The relative sensitivity and specificity of the newly developed ELISA were 100 and 98.6 %, respectively. Whereas, relative sensitivity and specificity of the newly developed AGID were in complete agreement in respect to commercial AGID kit. Here, we have reported the validation of an ELISA and AGID on large number of equine serum samples using recombinant p26 protein produced from synthetic gene which does not require handling of pathogenic EIAV. Since the indigenously developed reagents would be economical than commercial diagnostic kit, the rp26 based-immunoassays could be adopted for the sero-diagnosis and control of EIA in India.

Equine piroplasmosis is a tick-transmitted protozoan disease caused by Theileria equi and/or Babesia caballi. In the present study, we expressed a 53kDa protein from the truncated EMA-2 gene of T. equi (Indian strain) and developed EMA-2ELISA using this expressed protein. This ELISA is able to detect T. equi-specific antibodies in experimentally infected animals as early as 9 days post-infection. The assay developed was validated with the OIE recommended competitive ELISA (cELISA) on 120 serum samples and significant agreement (kappa=0.93) was observed between results of both the ELISAs which indicates suitability of EMA-2ELISA for use in sero-diagnosis. Diagnostic sensitivity and specificity of EMA-2ELISA - as compared with cELISA - were 0.97 and 0.96, respectively. Analysis of 5651 equine serum samples - collected during 2007-2012 from 12 states of India representing eight agro-climatic zones - by EMA-2ELISA revealed 32.65% seroprevalence of T. equi in India. In conclusion, the EMA-2ELISA developed using the T. equi EMA-2 recombinant protein as antigen for detecting T. equi-specific antibodies has good diagnostic potential for sero-epidemiological surveys.

Equine infectious anemia (EIA) is a retroviral infection of horses. Horses infected by EIA virus (EIAV) become inapparent carriers that remain asymptomatic for the remainder of their life span and serve as infection source to other horses. In this study, agar gel immunodiffusion test and ELISA were used to investigate the presence of antibodies to EIAV in equines. A total of 67,042 equine serum samples from 19 states and two union territories were tested during April 1999 to September 2012. The results revealed that none of the animals were positive for antibodies to EIAV from 1999 to December 2009. However, two EIAV sero-positive cases one each from indigenous and thoroughbred equines were detected in 2010 and 2012, respectively. Occurrence of EIA after a long gap of 11 years is indicative of reemergence of EIA in India which warrants concerted efforts in nationwide surveillance and monitoring for detection and elimination of EIAV carrier animals to prevent EIA outbreak.