Revisiting the genotypes of Theileria equi based on the V4 hypervariable region of the 18S rRNA gene.

Introduction: Equine theileriosis, an economically important disease that affects horses and other equids worldwide, is caused by a tick-borne intracellular apicomplexan protozoa Theileria equi. Genotyping of T. equi based on the 18S rRNA gene revealed the presence of two, three, four or five genotypes. In previous published reports, these genotypes have been labelled either alphabetically or numerically, and there is no uniformity in naming of these genotypes. The present study was aimed to revisit the phylogeny, genetic diversity and geographical distribution of T. equi based on the nucleotide sequences of the V4 hypervariable region of the 18S rRNA gene available in the nucleotide databases. Methods: Out of 14792 nucleotide sequences of T. equi available in the GenBank™, only 736 sequences of T. equi containing the complete V4 hypervariable region of the 18S rRNA gene (>207 bp) were used in multiple sequence alignment. Subsequently, a maximum likelihood phylogenetic tree was constructed based on the Kimura 2-parameter model (K2+I). Results: The phylogenetic tree placed all the sequences into four distinct clades with high bootstrap values which were designated as T. equi clades/ genotypes A, B, C and D. Our results indicated that the genotype B of Nagore et al. and genotype E of Qablan et al. together formed the clade B with a high bootstrap value (95%). Furthermore, all the genotypes probably originated from clade B, which was the most dominant genotype (52.85%) followed by clades A (27.58%), and C (9.78%) and D (9.78%). Genotype C manifested a comparatively higher genetic diversity (91.0-100% identity) followed by genotypes A (93.2-99.5%), and B and D (95.7-100%). The alignment report of the consensus nucleotide sequences of the V4 hypervariable region of the 18S rRNA gene of four T. equi genotypes (A-D) revealed significant variations in one region, between nucleotide positions 113-183, and 41 molecular signatures were recognized. As far as geographical distribution is concerned, genotypes A and C exhibited far-extending geographical distribution involving 31 and 13 countries of the Asian, African, European, North American and South American continents, respectively. On the contrary, the genotypes B and D exemplified limited distribution with confinement to 21 and 12 countries of Asian, African and European continents, respectively. Interestingly, genotypes A and C have been reported from only two continents, viz., North and South America. It was observed that genotypes A and C, and B and D exhibit similar geographical distribution. Discussion: The present study indicated the presence of only four previously described T. equi genotypes (A, B, C and D) after performing the molecular analyses of all available sequences of the complete V4 hypervariable region of the 18S rRNA gene of T. equi isolates in the GenBank™.

Population genetic characterization of Theileria annulata based on the cytochrome b gene, with genetic insights into buparvaquone susceptibility in Haryana (India).

The present investigation was aimed at population genetic characterization of Theileria annulata on the basis of the cytochrome b (cyt b) gene along with the evaluation of status of buparvaquone resistance in Haryana (India). The sequences originating from China, Egypt, India, Iran, Iraq, Tunisia, Turkey and Sudan were included in the analysis. The maximum likelihood tree based on the Tamura-Nei (TN93+G) model placed all the sequences of T. annulata into a single clade. The median-joining haplotype network exemplified geographical clustering between T. annulata haplotypes originating from each country. Only five haplotypes (7.81 %) were shared between any two countries, while the remaining 59 haplotypes (92.19 %) were singleton and unique to one country. The values of pairwise genetic distance (FST) between all the populations indicated huge genetic differentiation (> 0.25) between different T. annulata populations, barring the FST value between Iraq and Turkey (0.14454) which suggested a moderate differentiation. Contrary to the FST index, the values of gene flow (Nm) between T. annulata populations were very low. The neutrality indices and mismatch distributions indicated a population expansion in the Indian T. annulata population. Furthermore, the secondary structure and homology modeling of the partial cyt b protein is also reported. The molecular analysis of newly generated sequences for buparvaquone resistance revealed that all the isolates were susceptible to buparvaquone treatment. However, two novel mutations at positions V203I and V219I in between the Q01 and Q02 drug-binding regions of the cyt b gene were observed for the first time.

Cladistics of Echinococcus granulosus Sensu Stricto Genotypes Infecting the Slaughtered Pigs.

PURPOSE: Echinococcus granulosus sensu stricto (s. s.) consists of the most widespread genotypes (G1, G3) implicated in human cystic echinococcosis worldwide. The present study aimed to evaluate the role of pigs in the transmission dynamics of E. granulosus s. s. genotypes, including the phylogenetics, evolutionary divergence and haplotype network analyses of north Indian pig isolates along with GenBank™ archived sequences. METHODS: In totality, 223 slaughtered pigs were thoroughly screened for the presence of hydatid cysts. The amplification of the partial mitochondrial cytochrome C oxidase subunit 1 gene was performed for the molecular confirmation and phylogenetics of the retrieved metacestodes. RESULTS: The molecularly confirmed and sequenced present study isolates (n = 2) clustered with the E. granulosus genotype G3. The very low evolutionary divergence values (< 0.0027) of the present study isolates with E. ganulosus genotype G3 indicated the absence of diverged lineages. The significantly negative values of Tajima's D (- 2.598) and Fu and Li's D (- 4.054) of the overall dataset and for the Asian sequences signified an expansion of population size. The overall dataset exhibited low nucleotide (0.067 ± 0.055) and high haplotype (0.574 ± 0.015) diversities, also alluding to demographic expansion. The haplotype network showed that the pig isolates from South America and Europe constituted the predominant haplotype, Hap_2 along with Hap_3 and Hap_6, primarily associated to E. granulosus genotype G1; whereas, the Indian isolates formed different haplotypes (Hap_1 and Hap_5) belonging to genotype G3. CONCLUSIONS: The present study highlighted the important role of pigs in the transmission of E. granulosus s. s., which is of paramount significance given the public health and economic impact of cystic echinococcosis.

Phylogeography and demographic dynamics of Rhipicephalus microplus from North India.

Rhipicephalus microplus, a hematophagous vector prevalent in the tropics and subtropics, is responsible for huge economic losses throughout the globe. However, the taxonomy of the tick species, especially prevalent in north India and south China has been challenged in the recent past. The present study attempted to assess the cryptic status of R. microplus ticks of north India based on two mitochondrial markers; the 16S rRNA and cox1 gene sequences. The phylogenetic tree corresponding to both markers demonstrated the presence of three distinct genetic assemblages/ clades of R. microplus. The present study isolates (n = five and seven for the cox1 and 16S rRNA gene sequences, respectively) from north India along with other isolates from India assorted in the R. microplus clade C sensu. Based on the median joining network analysis corresponding to the 16S rRNA gene sequence, 18 haplotypes were recorded, exhibiting a stellate shape, which was indicative of rapid population expansion. For the cox1 gene, the haplotypes corresponding to clades A, B and C were distantly placed with two exceptions. While performing the population structure analysis, low nucleotide (0.04745 ± 0.00416 and 0.01021 ± 0.00146) and high haplotype diversities (0.913 ± 0.032 and 0.794 ± 0.058) were recorded for the different clades of R. microplus based on the cox1 and 16S rRNA mitochondrial markers, respectively. Eventually, high genetic differentiation and low gene flow were recorded among the different clades. A negative value for the neutrality indices (Tajima's D = -1.44125, Fu's Fs = -4.879, Fu and Li's D = -2.78031 and Fu and Li's F = -2.75229) corresponding to the 16S rRNA gene for the overall dataset evinced an expansion of population size. Based on the detailed studies, it was inferred that the R. microplus tick species circulating in north India belonged to clade C sensu, similar to that of the species prevalent in the other parts of the country as well as in the Indian subcontinent.

First record of molecular and pathological investigations of physalopterid larvae infection in northern palm squirrel Funambulus pennantii.

Around 100 reported species of Physaloptera commonly infect mammals, reptiles, birds, and amphibians. The identification of Physaloptera species solely on morphological characteristics is difficult, especially in the case of larval and congeneric infections. The present study is an attempt to identify molecularly and to perform phylogeny and pathology of natural Physaloptera larval infection in northern palm squirrels. The molecular confirmation of the recovered parasitic stages was performed by targeting the nuclear 18S rRNA gene sequence. Phylogenetic analysis and evolutionary divergence of the present study isolate with GenBank™ archived Physaloptera sequences were performed. The cysts (containing the larval stages) were subjected to histopathological examination. Morphological identification of the larval stages revealed the presence of pseudolabia, two spines, and a collar-like projection at the anterior end. Histopathology of the cysts revealed transverse sections of parasites in the lumen along with the thickened cystic wall, infiltration of mononuclear cells, fibrous tissue proliferation in the wall, and cellular debris in the cystic lumen. The molecularly confirmed and sequenced present study isolate was submitted to GenBank™ under the accession number LC706442. Blast analysis revealed 96.82-98.64% nucleotide homology of the present study isolate to the GenBank™ archived Physaloptera sequences. The isolate of the present study was monophyletic with Physaloptera sp. and P. praeputialis recovered from the cats of Haryana, India. Also, evolutionary divergence studies revealed no difference among these sequences. The present study evinced the most probable role of the northern palm squirrel Funambulus pennantii as an aberrant or second intermediate host for P. praeputialis.

In vitro and in vivo evaluation of efficacy of berberine chloride: Phyto-alternative approach against Trypanosoma evansi infection.

Current chemotherapy against the Surra organism, Trypanosoma evansi has several limitations in terms of efficacy, toxicity, availability and emerging resistance. These reasons make the search of new chemo-preventive and chemo-therapeutic agent with high potency and low toxicity. Alkaloid phyto-molecules, berberine has shown promising anti-kinetoplastid activity against T. cruzi, T. congolense, T. brucei, Leishmania donovani and L. tropica. However, till date, there is no investigation of therapeutic efficacy of berberine chloride (BC) against T. evansi. The IC50 value of BC for growth inhibition of T. evansi at 24 h of culture was calculated as 12.15 µM. The specific selectivity index (SSI) of BC was calculated as 19.01 and 10.43 against Vero cell line and Equine PBMC's, respectively. Thirteen drug target genes affecting various metabolic pathways were studied to investigate the mode of trypanocidal action of BC. In transcript analysis, the mRNA expression of arginine kinase 1 remained refractory to exposure with BC, which provides metabolic plasticity in adverse environmental conditions. In contrary, rest all the drug target gene were down-regulated, which indicates that drug severely affect DNA replication, cell proliferation, energy homeostasis, redox homeostasis and calcium homeostasis of T. evansi, leading to the death of parasite in low concentrations. It is the first attempt to investigate in vitro anti-trypanosomal activity of BC against T. evansi. These data imply that phytochemicals as alternative strategies can be explored in the future as an alternative treatment for Surra in animal.

Identification, molecular characterization and risk factors of Theileria infection among sheep: a first comprehensive report from North India.

The present study was aimed at the identification, molecular characterization, and risk factor assessment of Theileria infection among sheep of Haryana province, north India. A total of 402 blood samples were collected from three different climatic zones of Haryana from March 2020 to September 2021. Light microscopy of blood smears revealed Theileria spp. infection in 47.26% (n = 190), while 60.94% (n = 245) of blood samples were positive using nested PCR. Extensive molecular characterization of Theileria infection using four pairs of species-specific primers indicated the dominance of T. ovis (29.1%) followed by T. lestoquardi (12.69%), T. luwenshuni (5.97%) and T. annulata (1.49%). Mixed infection was detected in 11.69% of cases. Bidirectional sequencing and phylogeny further confirmed the presence of these four Theileria spp. in the investigated area under study. Hematology indicated a significant (p < 0.01) reduction in various haematological indices of animals infected with T. luwenshuni and T. lestoquardi compared to the healthy control group. Risk factors like age, sex, and zone were significantly associated with Theileria infection in sheep. The present investigation depicts the first comprehensive molecular report of ovine Theileria spp., which warrants further study to develop suitable control strategies against these haemoparasitic infections.

Molecular characterization of Hyalomma dromedarii and evaluation of acaricidal potential of herbal methanolic extracts against H. dromedarii larvae in comparison to synthetic acaricides.

The present study had two aims: molecular characterization of Hyalomma dromedarii infesting one-humped camels of Haryana (North India), and assessment of the acaricidal potential of herbal methanolic extracts against H. dromedarii larvae in comparison to synthetic acaricides. Phylogenetics and population neutrality indices were assessed by targeting partial amplification of mitochondrial 16S rDNA sequences. Larval packet test (LPT) was performed to evaluate the acaricidal efficacy of herbal extracts (Ferula asafoetida and Trachyspermum ammi) and synthetic acaricides (deltamethrin and fipronil). Phylogenetic studies established the collected ticks to be H. dromedarii, exhibiting a homology of 99.8-100%. However, the present study isolates formed a different sub-clade compared to H. dromedarii sequences from Egypt, Senegal, Tunisia and Saudi Arabia. Nucleotide and haplotype diversity values were indicative of demographic expansion and low gene flow. Negative values of Tajima's D (-0.612) and Fu and Li's Fst (-0.479) highlighted deviations from neutrality and emphasized recent population expansion. The median lethal concentration (LC50) values recorded for T. ammi, F. asafoetida and their combination were 3.68, 2.87 and 2.59 mg/mL, respectively, whereas the 90% lethal concentration (LC90) values were 4.09, 3.58 and 3.35 mg/mL, respectively. It was also observed that the H. dromedarii population under study was completely susceptible to both the formulated synthetic acaricides. In conclusion, combination of methanolic extracts of F. asafoetida and T. ammi could provide a potential substitute to toxic synthetic chemical acaricides and might prove a valuable component of integrated tick management strategies.

An insight into misidentification of the small-subunit ribosomal RNA (18S rRNA) gene sequences of Theileria spp. as Theileria annulata.

BACKGROUND: There had been isolated reports of the presence of novel Theileria annulata genotypes based on the 18S rRNA gene sequence data from India, Pakistan and Saudi Arabia; but, these studies were restricted to limited field samples. Additionally, no comparative study has been conducted on all the isolates of this parasite from different countries whose sequences are available in the nucleotide databases. Therefore, we aimed to study the genetic diversity of T. annulata based on all available nearly complete 18S rRNA gene sequences in the GenBank™. Out of a total of 312 gene sequences of T. annulata available in the NCBI database, only 70 nearly complete sequences (> 1527 bp) were used for multiple sequence alignment. RESULTS: The maximum likelihood tree obtained using TN93 + G + I model manifested two major clades. All the valid host-cell transforming Theileria species clustered in one clade. The T. annulata designated sequences occupying this clade clustered together, excluding two isolates (DQ287944 and EU083799), and represented the true T. annulata sequences (n = 54). DQ287944 and EU083799 exhibited close association with Theileria lestoquardi. In addition, 14 Indian sequences formed a large monophyletic group with published Theileria orientalis sequences. The broad range of sequence identity (95.8-100%) of T. annulata designated sequences indicated the presence of different Theileria spp. A closer analysis revealed the presence of three Theileria spp., namely, T. annulata, T. orientalis, and two isolates (DQ287944 and EU083799) closely related to T. lestoquardi. The true T. annulata sequences manifested 98.8-100% nucleotide identity within them. EU083799 and 14 misidentified Indian T. annulata sequences exhibited the highest similarity with T. lestoquardi (98.6-98.8%) and T. orientalis (98.0-99.9%) in comparison with the other Theileria spp. of domestic and wild ruminants. CONCLUSION: In the course of analyzing the genetic diversity of T. annulata, we identified the nearly complete 18S rRNA gene sequences of other Theileria spp. that have not only been misidentified as T. annulata in the GenBank™, but are also published as T. annulata. Moreover, a high level of sequence conservation was noticed in the 18S rRNA gene of true T. annulata and T. orientalis sequences.

In vitro anti-trypanosomal effect of ivermectin on Trypanosoma evansi by targeting multiple metabolic pathways.

High cytotoxicity and increasing resistance reports of existing chemotherapeutic agents against T. evansi have raised the demand for novel, potent, and high therapeutic index molecules for the treatment of surra in animals. In this regard, repurposing approach of drug discovery has provided an opportunity to explore the therapeutic potential of existing drugs against new organism. With this objective, the macrocyclic lactone representative, ivermectin, has been investigated for the efficacy against T. evansi in the axenic culture medium. To elucidate the potential target of ivermectin in T. evansi, mRNA expression profile of 13 important drug target genes has been studied at 12, 24, and 48 h interval. In the in vitro growth inhibition assay, ivermectin inhibited T. evansi growth and multiplication significantly (p < 0.001) with IC50 values of 13.82 µM, indicating potent trypanocidal activity. Cytotoxicity assays on equine peripheral blood mononuclear cells (PBMCs) and Vero cell line showed that ivermectin affected the viability of cells with a half-maximal cytotoxic concentration (CC50) at 17.48 and 22.05 µM, respectively. Data generated showed there was significant down-regulation of hexokinase (p < 0.001), ESAG8 (p < 0.001), aurora kinase (p < 0.001), casein kinase 1 (p < 0.001), topoisomerase II (p < 0.001), calcium ATPase 1 (p < 0.001), ribonucleotide reductase I (p < 0.05), and ornithine decarboxylase (p < 0.01). The mRNA expression of oligopeptidase B remains refractory to the exposure of the ivermectin. The arginine kinase 1 and ribonucleotide reductase II showed up-regulation on treatment with ivermectin. The ivermectin was found to affect glycolytic pathways, ATP-dependent calcium ATPase, cellular kinases, and other pathway involved in proliferation and maintenance of internal homeostasis of T. evansi. These data imply that intervention with alternate strategies like nano-formulation, nano-carriers, and nano-delivery or identification of ivermectin homologs with low cytotoxicity and high bioavailability can be explored in the future as an alternate treatment for surra in animals.

Gene expression study to elucidate the anti-trypanosomal activity of quinapyramine methyl sulphate (QPS).

The kinetoplastid protozoan parasite, Trypanosoma evansi causes a fatal disease condition known as Surra in equines throughout the globe. Disease condition being acute in nature, entrust a huge economic and health impact on the equine industry. Till date, quinapyramine methyl sulphate (QPS) is the first line of treatment and a panacea for the T. evansi infection in equines. Still after the >70 years of its discovery, there is no clue about the mode of action of QPS in T. evansi. The establishment of in vitro cultivation of T. evansi in HMI-9 media has provided opportunity to study the alteration in mRNA expression of parasite on exposure to the drug. With this research gap, the present study aimed to investigate the relative mRNA expression of 13 important drug target genes to elucidate the anti-trypanosomal activity of QPS against T. evansi. The IC50 of QPS against a pony isolate of T. evansi was determined as 276.4 nM(147.21 ng/ mL) in the growth inhibitory assay. The in vitro cultured T. evansi population were further exposed to IC50 of QPS and their relative mRNA expression was studied at 12 h, 24 h and 48 h interval.The mRNA expression of several genes such as hexokinase, trypanothione reductase, aurora kinase, oligopeptidase B and ribonucleotide reductase II were found refractory (non-significant, p > 0.1234) to the exposure of QPS. Significant up-regulation of trans-sialidase (p < 0.0001), ESAG8 (p < 0.0021), ribonucleotide reductase I (p < 0.0001), ornithine decarboxylase (p < 0.0001), topoisomerase II (p < 0.0021) and casein kinase I (p < 0.0021) were recorded after exposure with QPS. The arginine kinase 1 and calcium ATPase I showed highly significant (p < 0.0001) down-regulation in the drug kinetics. Therefore, the arginine kinase 1 and calcium ATPase I can be explored further to elucidate the trypanocidal activity of QPS. The preliminary data generated provide the potential of arginine kinase 1 and calcium ATPase I mRNA mediated pathway of trypanocidal action of QPS. Further, transcriptomics approach is required to investigate the possible mechanism of action of drugs at molecular level against the targeted organism.

Prevalence and phylogeography of Taenia hydatigena metacestodes from goats of India.

The study determined the prevalence and genetic population structure relationships of Cysticercus tenuicollis (Taenia hydatigena metacestode) retrieved from the goats slaughtered in north India. An overall prevalence of 9.62% (59/613) was recorded. Genetic population structure relationships were assessed by targeting partial cytochrome c oxidase subunit 1 mitochondrial gene sequence. Phylogenetic tree analysis revealed that all the present study representative isolates (n = 7) formed a major clade and grouped with T. hydatigena isolates retrieved from sheep, goats, pigs and dogs, originating from China, Iran, Nigeria, Ghana and Poland. However, a single isolate from Himachal Pradesh (isolate 3) formed a subgroup within the clade. The neutrality and diversity indices revealed high values of haplotype diversity [Hd = 0.99695 (0.95238­1.0000)] and low nucleotide diversity (π = 0.49276), which was indicative of demographic expansion and low gene flow, suggesting that Indian T. hydatigena isolates were not genetically differentiated. Tajima's D (−1.26988) and Fu and Li's D statistics values (−0.74556) were negative, demonstrating deviations from neutrality and both propounded recent population expansion or purifying selection. Results highlighted a low genetic diversity of T. hydatigena metacestodes across the geographical range of north India.

Molecular Detection and Differentiation of Different Theileria Species in Naturally Infected Goats Using Nested PCR-RFLP: A First Report from Northern India.

PURPOSE: Theileriosis is an economically important tick-borne pathogen with a serious impact on livestock health and productivity. Despite the fact that bovine theileriosis has been widely investigated, there exists a paucity of information on these infections in small ruminants, especially in India. The present study was carried out to detect and differentiate different Theileria spp. in goats using nested PCR RFLP. METHODS: Blood samples and ticks were collected from 405 goats in various agro-climatic zones of Haryana state, India. The blood samples were screened by microscopy, nested PCR-RFLP, and sequence analysis. The nested PCR-RFLP was performed with four restriction enzymes viz., Hpa II, Bsh 1285I, Hae II and Rsa I. Six nested PCR amplicons with different RFLP patterns were sequenced and submitted to NCBI (OM666861, MZ220430, OM666628, MZ220437, OM666637, OM721806). RESULTS: Microscopy revealed 18.27% (n = 74) infection with Theileria spp., while 33.58% (n = 136) of blood samples were confirmed positive by nested PCR. Out of 136 positive samples, 43.38% (n = 59), 11.02% (n = 15) and 20.58% (n = 28), were positive for T. ovis, T. lestoquardi and T. luwenshuni (Theileria sp. China 1), respectively. Mixed infection was detected in 25% (n = 34) cases. Based upon Hpa II digestion pattern, 13 samples with T. lestoquardi and T. ovis, and 21 samples with T. ovis and T. luwenshuni were detected. Sequence study further confirmed their identity. The majority of ticks collected from goats were identified as Rhipicephalus spp., Hyalomma anatolicum and Hemaphysalis spp. CONCLUSION: This study represents the first confirmed molecular report of goats infected with T. ovis, T. lestoquardi, and T. luwenshuni from northern India.

Molecular identification of Theileria species in naturally infected sheep using nested PCR-RFLP.

Ovine theileriosis is an important tick-borne haemoprotozoan disease of sheep in tropical and subtropical regions, causing severe productivity and economic loss. There is a paucity of information related to molecular studies of ovine theileriosis from India. The present study identified different Theileria spp. in naturally infected sheep using nested PCR-restriction fragment length polymorphism (nPCR-RFLP). Blood samples and ticks were collected from 204 sheep in different agro-climatic zones of Haryana state, India, during the tick active season. Microscopic examination of thin blood smears revealed 33.3% (68/204) infections with Theileria spp., while 44.6% (91/204) of blood samples were positive by nPCR assay. Different Theileria spp. were identified based upon RFLP patterns using four restriction enzymes: Hpa II, Bsh 1285I, Hae II and Rsa I. Out of 91 positive samples, 50.5% (46/91), 23.08% (21/91), 11% (10/91) and 2.2% (2/91) were positive for T. ovis, T. lestoquardi, T. luwenshuni (Theileria sp. China 1/Theileria sp. China) and T. annulata, respectively. Mixed infection was detected in 13.2% (12/91) of cases. Based upon HpaII enzymatic digestion pattern, two samples with T. lestoquardi and T. annulata, nine samples with T. lestoquardi and T. ovis and one sample with T. ovis and T. annulata were detected. The presence of these Theileria spp. was further confirmed by sequence analysis. The majority of ticks collected from sheep were identified as Rhipicephalus spp. followed by Hyalomma anatolicum and Hemaphysalis spp. The present investigation depicts the first comprehensive molecular report of naturally infected sheep with T. ovis, T. lestoquardi, T. annulata and T. luwenshuni from northern India.

Molecular insights into the population structure and haplotype network of Theileria annulata based on the small-subunit ribosomal RNA (18S rRNA) gene.

The present study was conducted to elucidate the population genetic diversity and haplotype network of Theileria annulata based on all available nearly complete 18S rRNA gene sequences in the GenBank™. In total, 52 sequences of the nuclear 18S rRNA gene used to assess the relationship of T. annulata with their country of origin identified 34 haplotypes. Haplotype 4 was widespread, occurring in India, China, Turkey and Iran, while the remaining haplotypes were singleton and unique to one country. Haplotype 4 displayed numerous single haplotypes around it and the stellate shape of the network suggested a rapid population expansion. India exhibited the largest number of haplotypes (h = 25) followed by Turkey (h = 6), China (h = 4), and Iran and Italy (h = 1). No geographical clustering of haplotypes was recorded. Nucleotide diversity was the highest in the Turkish followed by the Indian and Chinese populations. Similarly, haplotype diversity was the highest in China followed by Turkey, and the lowest in India. The overall dataset exhibited a low nucleotide diversity (0.00253 ± 0.00035), but high haplotype diversity (0.917 ± 0.034). It suggested the presence of only minor differences (01-11 nucleotide) between haplotypes which was also evident from the haplotype network. A high level of genetic diversity was documented within the Indian, Chinese and Turkish populations of T. annulata, whereas little genetic differentiation was noticed among these populations with a very high level of gene flow. Analysis of molecular variance (AMOVA) of T. annulata sequences revealed higher genetic variation within countries (83.58%) as compared to the variation among countries (16.42%). Neutrality indices, viz., Tajima's D, Fu and Li's F, Fu's Fs, and R2, along with the unimodal mismatch distributions demonstrated a recent population expansion of T. annulata in India and the overall dataset. However, the non-significant values of Tajima's D, Fu and Li's F, and Fu's Fs for the Chinese population along with a bimodal mismatch distribution signified a constant population size. For the Turkish population, the neutrality and mismatch distribution tests either indicated a constant or a slight increase in population size. The present study provides novel insights into the population genetics and haplotype network of T. annulata based on the 18S rRNA gene for the first time.

Phylogenetic analysis, genetic diversity and geographical distribution of Babesia caballi based on 18S rRNA gene.

The present investigation was aimed to study the presence of Babesia caballi clades upon phylogenetic analysis of all available V4 hypervariable 18S rRNA gene sequences in GenBank in addition to the intra- and interclade genetic diversity in B. caballi and the distribution of parasite clades in different countries. Out of altogether 155 small-subunit ribosomal RNA gene sequences of B. caballi available in the database, only 92 sequences with a complete V4 hypervariable region (>293 bp) were used in multiple sequence alignment. The phylogenetic tree placed all the sequences into two distinct clades with high bootstrap values which are designated as B. caballi clades A and B. Clade A was further divided into two subclades A1 and A2 with 98% bootstrap support. On the contrary, clade B contained multiple small subclades which either lacked bootstrap support or did not have enough bootstrap support to further group them into subclades. All the sequences of B. caballi were 91.5-100% identical with each other. Clade B manifested a comparatively higher genetic diversity (95.2-100% identity) amongst sequences as opposed to clade A (97.3-100% identity). Moreover, it indicated 91.5-93.5%, 92.9-94.6% and 91.5-94.6% nucleotide identity with B. caballi subclades A1, A2, and clade A, respectively. Significant nucleotide variations were observed in one region, between nucleotide positions 126-178, in some of the sequences. A total of 21 molecular signature residues were identified in the V4 hypervariable region. The alignment report of the V4 hypervariable region of 18S rRNA gene of clades A and B exhibited nucleotide variation at nine and 24 places, respectively. The distribution map of all the clades of B. caballi is also reported. The number of 18S rRNA gene sequences employed in the study is relatively high compared to previous studies. Therefore, a fair comparison of definite genetic variations between isolates/sequences from different countries was carried out.

Phylogenetics and Pathology of Hydatid Disease in Slaughtered Buffaloes of North India.

PURPOSE: Hydatid disease is one of the neglected and challenging (for diagnosis as well as for treatment) parasitic diseases. Along with adverse effect on animal's health leading to production losses, hydatidosis is also associated with huge economic losses. The present study was envisaged with an aim to assess the phylogeny and pathological changes due to natural hydatid cysts in lungs and liver of slaughtered buffaloes in north India. METHODS: A total of 137 slaughtered buffaloes intended for human consumption were screened for the presence of cysts. The retrieved cysts were confirmed molecularly based on the amplification of mitochondrial cytochrome oxidase 1 gene (mtCO1), exhibiting a product size of approximately 446 bp. The samples collected from infected lungs and liver were subjected to histopathological examination. RESULTS: The hydatid cysts were recorded in 25 (18.2%) animals. Phylogenetic analysis revealed the isolated strain to be closely related to Echinococcus granulosus sensu stricto (G1) genotype. The nucleotide diversity (π) obtained was 0.014685, whereas, Tajima's D was negative (- 2.796053), which indicated purifying selection or recent population expansion. Histopathologically, in the infected lungs, fibrosis and inflammatory reaction comprising of mononuclear cells and fibroblasts around the thick coat of granulation tissue were observed. Marked calcified masses and necrosis were also observed in the calcified cysts. However, in case of infected livers, Kupffer cell hyperplasia, degeneration of hepatocytes, fibrosis and inflammatory cells were most commonly observed around the hydatid cysts. CONCLUSION: The findings of the present study are of significant veterinary and medical importance owing to economic and public health impact of G1 genotype of E. granulosus.