Comprehensive genetic diversity and molecular evolutionary analysis of Theileria annulata isolates based on TAMS 1 gene.

Molecular epidemiological studies related to the phylogenetic characterization of Theileria annulata are important in delineating the evolutionary history of the parasite. In the current study, the Theileria annulata (T. annulata) merozoite surface antigen 1 (TAMS 1) gene from 14 bovine isolates of T. annulata originating from semi-arid zone of northern India were amplified and sequenced. TAMS 1 gene sequences (n= 337) reported from 16 countries were subsequently analyzed for haplotype network along with genetic diversity. A total of five haplotypes out of the 14 sequenced isolates and 92 haplotypes out of 337 worldwide sequences are documented in this study. Phylogenetic and molecular evolutionary analyses based on TAMS 1 gene sequences showed that T. annulata is dissipated across different countries and numerous strains are closely linked, even though they belong to different geographical locations. The nucleotide homology between 14 isolates from northern India varied between 91.3 and 100%, whereas it was between 31.5 and 100% when sequences across the globe were compared. Haplotype 14 was recognized as most widely distributed haplotype, with 46 isolates circulating in 10 countries. Globally, negligible genetic distance (FST˂0.15) and very high gene flow (Nm˃1) was found in the five populations of the world (South Asia, East Asia, West Asia, Europe and Africa), supporting the absence of clearly defined subgroups in the phylogenetic analysis. Significant negative values of neutrality tests; Tajima's D (D) and Fu and Li's F (F) provided evidence for recent population expansion through positive selection of advantageous variations.

Phylogenetic characterization and analysis of Sarcocystis buffalonis.

Despite its frequent presence in buffaloes, Sarcocystis buffalonis remains as one of the most under studied parasite. In the present study, isolates of S. buffalonis from,Mathura, Uttar Pradesh India were characterized for 18S rRNA (MF595842-MF595844), cox 1 (MG792800-MG792802), 28S rRNA (MH793418-MH793420) and ITS 1 (MH793421-MH793423) genes. Analysis revealed multiple haplotypes for each individual gene viz., 18S rRNA (three haplotypes), cox 1 (two haplotypes), 28S rRNA (two haplotypes) and ITS 1 (single haplotype). The studied Indian sequences showed variable homologies for individual gene loci viz., 18S rRNA (99.3-99.9%); cox 1 (99.8-100.0%); 28S rRNA (99.9-100.0%) and ITS 1 (100.0%) The phylogenetic association between S. buffalonis and closely related Sarcocystis spp. infecting buffaloes and cattle was delineated. All these gene loci placed S. buffalonis close to S. hirsuta. The study has generated A vital phylogenetic data about this erstwhile neglected parasite.

An endpoint visualization loop-mediated isothermal amplification (LAMP) for detecting bubaline theileriosis.

Background: Tropical theileriosis is a significant disease affecting the health and production levels of buffaloes in India. It is caused by an apicomplexan-Theileria annulata. The timely and accurate detection of infection is vital for implementing a mass vaccination or control programme in a given area under outbreak. Most of the literature concerned with diagnosis of theileriosis revolves around cattle, and practically, there are very limited assays available for detecting bubaline theileriosis. Loop-mediated isothermal amplification (LAMP) assay certainly amplifies the targeted deoxyribosenucleic acid (DNA) with a comparatively higher efficacy, rapidity and sensitivity. Alongside, minimal use of sophisticated instruments in performing LAMP assay is certainly an add on. The present study describes the application of LAMP assay in diagnosing tropical theileriosis in buffaloes alongside, its comparison with polymerase chain reaction (PCR) and blood microscopical examination. Results: No cross-reaction was seen with DNA of other haemoprotozoan. LAMP was compared with blood microscopy and PCR. LAMP detected infection in 27 out of 100 buffaloes, while blood microscopy and PCR detected disease in 16 and 24 buffaloes, respectively. Conclusion: The sensitivity, specificity and kappa value prediction of LAMP were found to be much higher than the PCR and blood microscopy. The present communication reports the first use of LAMP in detecting theileriosis in buffaloes in the world.

Phylogenetics of Sarcocystis fusiformis isolates based on 18S rRNA and cox 1 genes.

Sarcocystosis is a significant meat borne coccidian disease with immense zoonotic potential. Sarcocystis fusiformis is the most prevalent Sarcocystis spp. affecting buffaloes across the globe. Most of the molecular characterization works on S. fusiformis are from Egypt and there is no record of such work from India. In the present study, 21 isolates of S. fusiformis from Northern India were characterized for 18S rRNA (MF595821-MF595841) and cox 1 (MF423105-MF423119 and MH899162-MH899167) genes. S. fusiformis was seen as a monophyletic sister group to S. cafferi on the phylogenetic tree comprising of different Sarcocystis spp. Both genes placed S. fusiformis close to those Sarcocystis spp. which have felids as definitive hosts in comparison to those with canids as definitive host. A total of 15 and 7 haplotypes were noticed for both the genes, respectively. The studied Indian isolates showed 99.1-100.0% and 99.2-100.0% nucleotide homologies within themselves for both the respective gene loci. Over all, cox 1 gene was found to be better in delineating the evolutionary phylogenetics in comparison to 18S rRNA gene. The findings are important from evolutionary point of view.

Associative genetic diversity among Sarcocystis cruzi isolates from Northern India based on 18S ribosomal gene.

Sarcocystis cruzi is perhaps the most common Sarcocystis spp. affecting cattle throughout the world. Despite its wide presence across Indian subcontinent, very limited studies are reported from India describing the phylogenetics of the parasite. The present communication describes the phylogenetic characterization of various isolates of S. cruzi. The isolates were characterized at 18S gene locus. An appreciable among of genetic variability was noticed between various S. cruzi isolates. Sequences generated from the present study (MN121572-MN121576) represented two haplotypes with 99.0-100.0% nucleotide homology within themselves. Alongside, nucleotide homology of 98.36-100.0% was observed between Indian isolates and isolates across the globe. The two haplotypes were markedly distinct from each other with 10 nucleotide substitutions within themselves. Moreover, there was a deletion of G nucleotide at position 545 in one of the sequences (MN121575). In general, the Indian isolates were seen closer to isolates from China than to the isolates from Iran, Argentina, Japan, Australia and Netherlands. Results clearly depicted the presence of multiple lineages of S. cruzi in Indian cattle. The findings are very much important in delineating the evolutionary phylogenetics of S. cruzi from India and abroad.

Associative Genetic Diversity of RoTat 1.2 VSG in Different Trypanosoma evansi Isolates.

BACKGROUND: Numerous phylogenetic markers have been tested over a period of time for delineating evolutionary history of haemoflagellate-Trypanosoma evansi. PURPOSE: To find out the associative genetic diversity, within the various isolates of T. evansi across the globe, based on RoTat 1.2 VSG gene. METHODS: A total of 5 equine isolates of T. evansi from Northern India were characterized. PCR products were sequenced and sequences were compared with available sequences across India and world. Phylogenetic tree was constructed based on maximum parsimony (MP) method with the tree-bisection-regrafting (TBR) algorithm. RESULTS: Indian isolates formed multiple clades with two haplotypes. The present isolates showed 99.49-100.00% nucleotide homology within themselves. On broader line, Indian isolates were found to be closer to Egyptian isolates than the African counterparts. Few of the Indian isolates showed marked resemblance with a particular Egyptian isolate than with their Indian counter parts. Another remarkable finding is the close association of equine isolates from India with other equine isolates and their clear divergence from isolates of T. evansi affecting other hosts from India and abroad. CONCLUSION: Vast genetic divergence was seen between the isolates suggesting of multiple distinct lineages of T. evansi amongst the Indian livestock. Interestingly, variations in sequences were seen based on the host range of isolates. The findings are very important from molecular evolutionary point of view.

Phylogenetic characterization of Setaria equina and its association with other filarids.

Molecular characterization studies on Setaria equina are limited. The present study aimed to characterize S. equina at the cytochrome c oxidase gene and to examine its phylogenetic relationships with other filarid species. Sequence analysis showed 100% nucleotide homology with an S. equina sequence from Italy (AJ544873). However, both sequences exhibited 7 nucleotide substitutions from a S. equina donkey isolate from Egypt (MK541847). Overall, S. equina formed a monophyletic sister group to Setaria tundra. All Setaria spp. examined formed a separate group on the phylogenetic tree that was related to corresponding Onchocerca spp. and Dirofilaria spp. clades. Human filarid worms-Brugia spp. and Wuchereria spp. grouped in a separate clade alongside Theilezia spp. Dipetalonema spp.-formed a separate group at the top of the tree.

Rapid diagnosis of Theileria annulata by colorimetric loop-mediated isothermal amplification (LAMP) assay.

Theileria annulata the causative agent of bovine tropical theileriosis (BTT) is globally distributed. Rapid and accurate detection of the parasite is essential for the implementation and evaluation of mass drug administration and planned vaccination programs for controlling BTT. Loop mediated isothermal amplification (LAMP) amplifies targeted nucleic acid with a high efficacy, sensitivity and rapidity under isothermal conditions. In the present study, the internal transcriber space (ITS) gene of T. annulata was targeted for the development of a LAMP assay using pH-sensitive dye (phenol red) for enhanced visual detection of amplification. This method employed a set of specially designed four primers that recognized six distinct regions on the targeted gene. No amplification was detected with the DNA of other tested haemoprotozoans. Positive LAMP products were identified by a colour change from pink to yellow, and then rechecked by specific ladder pattern upon agarose gel electrophoresis. LAMP was able to detect infection in 63 out of 85 animals compared with blood microscopy, simple PCR and nested PCR which detected infection in 40, 49 and 64 animals, respectively. No difference in detection was seen in the colorimetric assay and the classical UV based LAMP assay.

Coexistence of Multiple Theileria annulata Genotypes Circulating in Neonatal Calves in Semi-arid India.

BACKGROUND: Knowledge of local isolates and strains is a prerequisite for the development of either effective mass vaccination strategy or a suitable molecular marker-based diagnostic tool. PURPOSE: The pathogenesis of Bovine tropical theileriosis (BTT), caused by Theileria annulata in susceptible ruminants, is known to vary depending upon the nature of isolate and strain involved. Therefore, RFLP and sequencing-based characterization of Indian isolates of T. annulata were attempted using TAMS gene. METHOD: In the present study, TAMS 1 gene of T. annulata was amplified from 25 naturally infected calves from the BTT endemic semi-arid zone of Northern India. The amplified products were then digested with three restrictions enzymes viz., Taq I, Rsa I, and Alu I to find out the variations in pattern of restriction digests, so as to have an idea about the various isolates of T. annulata present in the studied area. Around 14 samples covering all the variants (from the PCR-RFLP patterns) were sequenced and submitted in NCBI (MH277607-MH277620). RESULT: Coexistence of 4 variant genotypes was detected upon in-silico analysis of RFLP and sequence variations. CONCLUSION: The nucleotide variations alongside the chromatogram analysis revealed point mutations leading to presence of noticeable genetic diversity among the isolates.

Babesia (Theileria) equi genotype A among Indian equine population.

Equine piroplasmosis, caused by Babesia (Theileria) equi, is well reported from many parts of India. However, literature regarding its prevalence from semi arid India is limited. Alongside, there is complete absence of information about genetic characterization of B.(T.) equi and the associated genotypes from India. In the present study, the prevalence of B.(T.) equi was studied from semi arid India using 18S ribosomal gene based PCR assay. An overall prevalence rate of 10.46% was recorded. PCR was more sensitive and specific in comparison with blood smears. The found isolates were sequenced. These sequences were aligned and submitted into NCBI. All the isolates showed 100% homology with each other and represented a single haplotype. When compared with other isolates of B.(T.) equi across India and globe, using Genetool and Mega 6 softwares, they showed 99.5-99.2% and 95.7-97.0% homologies, respectively. A unique substitution of G by A at 206 nucleotide position was reported in all found isolates in comparison to earlier reported isolates from India. The studied isolates were found to be phylogenetically closer to isolates from USA and Trinidad than to other parts of the world. All the Indian isolates belonged to genotype A of B.(T.) equi.

First report of molecular characterization and phylogenetic analysis of Sarcocystis tenella from India.

Sarcocystis tenella is a common tissue coccidian parasite of sheep. It is reported worldwide with high prevalence rate ranging from 9 to 100%. However, there are very limited reports of this parasite from the Indian context and those reports are totally based on the morphology alone. When it comes to molecular characterization, such studies are absent from India. The present communication reports the first characterization study of S. tenella from India. 18S rRNA ribosomal gene and mitochondrial cytochrome c oxidase subunit I (cox1) genes were used for molecular characterization and phylogenetic analysis alongside standard histopathology of sarcocysts. Five Indian isolates were characterized for each gene, and respective sequences were submitted in the NCBI. Two haplotypes were noticed, both for the 18S rRNA and cox1 gene showing 99.8-100.0% and 99.7-100.0% nucleotide homologies within themselves, respectively. When compared with other sequences of S. tenella across the globe, the present isolates showed 93.3-99.9% nucleotide homology based on 18S rRNA gene and 95.2-99.8% nucleotide homology based on cox1 gene, respectively. In both the 18S and cox1 phylogenetic trees, respective sequences of S. tenella were placed with monophyletic cluster which was sister to a cluster comprising of sequences of S. gracilis and S. alces.

Comparison of different PCR protocols and respective primer sets from pool of TAMS 1 gene for diagnosis of calf theileriosis from semi arid India.

The polymorphic nature of Theileria annulata merozoite surface antigen (TAMS 1) attributes to limitation in PCR based detection of various T. annulata genotypes present in different geographical domains across the globe. Multiple reports of failure of detection of tropical theileriosis using classical N516/517 primer set in the studied area were noticed. Hence, three single PCR protocols using N516/517, TAMS F/R and NTA F/R primer sets encoding different portions of TAMS 1 gene and two nested protocols, using combinations of these three primers, were compared to find out the most suitable primer set for diagnosis of calf theileriosis in studied area. The studied area constitutes the semi-arid theileriosis endemic area of Northern India. The various PCR protocols were tested on 75 clinically confirmed cases of calf theileriosis. Alongside, 25 confirmed theileriosis negative blood samples and DNA of other haemoprotozoa were also tested for specificity of these primer sets. Results revealed that the primer set NTA F/R to be more suitable in detecting the circulating T. annulata genotypes in the studied area in comparison to the classical N516/517 primer set. None of the primers gave false positive amplification with negative samples and/or DNA of other haemoprotozoa.

First report of molecular characterization and sequence phylogenetic analysis of Linguatula serrata from India.

Linguatula serrata is a pentastomid which is worldwide in distribution. However, a very few references are reported from India. In the present study, the cox I gene of L. serrata nymphs, originally isolated from mesenteric lymph nodes of buffaloes, was amplified and custom sequenced. Based on sequence analysis, two haplotypes were noticed and were subsequently submitted in NCBI database. The sequences were also compared with the other sequences available in the pubmed and phylogenetic analysis coupled with nucleotide homologies were commutated. The studied Indian isolates were found closer to Bangladesh and Iran isolates. This is the first report of molecular characterization of L. serrata from India.

Diagnostic potential of low molecular weight excretory secretory proteins of Paramphistomum epiclitum for caprine amphistomosis.

In the present study, the 75% alcoholic fractionation of excretory-secretory (ES) antigen isolated from 200 to 300 live P. epiclitum was assessed for its diagnostic potential for the detection of caprine amphistomosis by using antibody detection enzyme immunoassay. Prior to enzyme immunoassay, 75% alcoholic fractionation of excretory-secretory (ES) antigen was subjected to SDS- PAGE and western blot analysis for the presence of immunoreactive polypeptides. SDS-PAGE analysis of ES antigen resolved a total 7 polypeptides bands of size 56, 27, 25, 22.5, 12, 11 and 10 kDa. Western blot analysis revealed only two immunoreactive polypeptides (11 kDa and 12 kDa) when polypeptides resolved in SDS-PAGE were probed with known positive pooled serum. None of the polypeptides showed reactions with pooled known negative serum. The working dilutions of antigen, sera and conjugates were determined by checkerboard titration for employing ELISA and cut-off O.D. was calculated 0.616 by using the mean absorbance value of 11 negative kid sera. The sensitivity and specificity of ELISA was found to be 100% and 86.76%, respectively. As per kappa value estimation, the strength of agreement was found to be good. Antibodies to 75% alcoholic fractionation of ES antigen was detected in 20% goats (n = 160) of either sex, although faecal examination detected 10.6% goats to be infected with amphistomosis. The study confirmed that 75% alcoholic fractionation of ES antigen of P. epiclitum based ELISA had good value for serodiagnosis of caprine amphistomosis.

Variation in cardiac markers and electrocardiographic alterations in young calves naturally infected with bovine tropical theileriosis.

The present study was designed to assess the deleterious effects of bovine tropical theileriosis on the cardiovascular system and the consequent myocardial involvement in young calves. Myocardial effects in parasitic diseases are often neglected. Hemolytic anemia, associated secondary hypoxia, and vasculitis are cardinal features of bovine theileriosis. In the present study, electrocardiogram (ECG) alongside serum cardiac troponin I (cTnI) and creatinine phosphokinase-myocardial band (CPK-MB) concentrations were analyzed in infected, treated, and control groups of young calves. Non-significant alterations were noticed in ECG. However, certain signs like sinus tachycardia, first-degree AV block, atrial premature complex, left atrial hypertrophy, and right atrial hypertrophy were found on consistent basis in infected calves. A significant increase in the serum concentration levels of cTnI and CPK-MB was noticed in infected calves followed by significant fall in both these biomarkers post treatment. cTnI and CPK-MB can definitely be used as myocardial markers in theileriosis-affected animals.

First report of molecular characterization and phylogenetic analysis of RoTat 1.2 VSG of Trypanosoma evansi from equine isolate.

Rotat 1.2 variant surface glycoprotein (VSG) is considered to be an important VSG expressed in most of the isolates of Trypanosoma evansi. This makes the molecule an important candidate for both molecular- and serological-based detection of surra. There are ample reports of existence of this gene in isolates from cattle, buffalo, and camel across the world. Of late, there are reports of its absence from a fewer isolates of T. evansi of murine and wildlife origin. Search of literature revealed no reports from horses. The present communication presents the first report of molecular cloning and characterization of Rotat 1.2 VSG from horse isolate of T. evansi from semi-arid region of India. Alongside, the gene was compared with various other isolates across the world. Interestingly, the isolate was found to be closer to camel isolates from Egypt than the other known isolates from India and Kenya.

Classico-molecular targeting of oligopeptidase B, cysteine protease and variable surface glycoprotein (VSG) genes of Trypanosoma evansi.

Trypanosomosis or Surra can rightly be attributed as the most economically important vector-borne haemoprotozoan disease encountering India. Surra infected chronic cases show almost similar types of signs and symptoms often confusing it with other haemoprotozoan infections, thereby, making it prerequisite for the development of aspecific and sensitive technique for its detection in susceptible animals. Blood microscopy and serology suffers from the hands of lack of sensitivity and specificity thereby leaving molecular detection techniques as one of the promising alternative. Alongside, there is utmost need for exploring of new molecular gene targets for the development of a putative alternative for diagnosis and immunoprophylaxsis. The present communication describes the identification and amplification of oligopeptidase B, cysteine protease and variable surface glycoprotein genes of T. evansi so as to exploit them in future as potential candidates for immune protection and/or molecular detection.

Standardization and validation of simple PCR, duplex PCR and RAPD in comparison to blood smear examination for diagnosing bovine tropical theileriosis.

Bovine Tropical Theileriosis (BTT) is an important vector-borne protozoan disease that imposing serious constraints on the health and productivity of domestic cattle. It is matter of common fact that following recovery from primary infection, cattle become persistent carriers and act as reservoirs of infection thereby, playing a critical role in disease epidemiology. The present study describes the comparative diagnostic efficiency of simplex PCR, duplex PCR and RAPD assays for detection of Theileria annulata in cattle. An optimized simple PCR and duplex PCR assay were established using TAMS F/R as primer sets encoding for 721 bp amplicon alongside a RAPD with arbitrary primer coding for 963 bp product of T. annulata. The simple PCR and duplex PCR detected pathogen with almost same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA of another pathogen without nonspecific amplifications. RAPD failed to give comparable results and suffered from limitations of sensitivity as well as specificity. The developed assays may be seen as a good tool for epidemiological studies aiming at assessing the burden of chronic infections and improving control of the associated diseases in endemic regions.

Evaluation of clinical, biochemical and haematological markers in natural infection of canine monocytic ehrlichiosis.

Caanine monocytic ehrlichiosis caused by Ehrlichia canis has gained wider significance owing to its potential to inflict significant deleterious effect on the health of companion animals. In the present study, 46 confirmed ehrlichiosis positive dogs were evaluated for the alterations in clinical, haematological and biochemical attributes. Depression, anorexia, pyrexia, anaemia, weakness, jaundice, melana, vomition and diarrhoea were the main clinical symptoms onserved. Haematological alterations included pancytopenia especially thrombocytopenia. Significant changes were noticed in WBC, RBC, Hgb, McHc, Platelets, ALT values while rest all the studied haematological and biochemical parameters showed non-significant alterations within normal range in comparison to normal healthy controls. The findings substantiate that ehrlichiosis cause significant clinical, haematological and biochemical alterations of the varied intensity in dogs, even with lower grades of parasitaemia.

Variation in clinical markers in cattle naturally infected with bovine tropical theileriosis.

124 cattle naturally infected with Theileria annulata were inspected for clinical markers. Clinical manifestations of general weakness, reduced appetite, pyrexia reaching 40-42 °C and lachrymal discharge were seen in more than 75 % of the infected animals. Presence of ticks on body, general dullness and depression, recumbency/prostration, oedema of dependent body parts, diarrhea, hypersalivation, pale mucous membranes and yellow colored urine were revealed by more than 50 % of the infected animals. Clinical manifestations of dehydration, abdominal distension/ascites, jaundice and haemoglobinuria were revealed by more than 25 % of the infected animals less than 25 % of the infected animals showed clinical manifestations of exophthalmia, constipation, melena, buccal cavity erosions, congested mucous membranes, nasal discharge and tachypnoea.