Understanding the role of pigs in the transmission of zoonotic Echinococcus ortleppi in Haryana, India.

The etiological agents of zoonotic cystic echinococcosis comprise the Echinococcus granulosus sensu lato (s.l.) species complex. The present study was aimed at investigating the zoonotic genotypes of Echinococcus granulosus s.l. circulating in the pig population of Haryana, India. Out of 253 slaughtered pigs screened, 5 showed the presence of hydatid cysts. The amplification of the partial mitochondrial NADH dehydrogenase subunit 1 (nad1) gene for the molecular confirmation and phylogenetics of the retrieved metacestodes (n = 2) revealed the presence of E. ortleppi. The sequences generated herein exhibited 99.80% homology to the GenBank archived E. ortleppi sequences. Cladistics targeting genetic diversity and haplotype network analysis involved 37 E. granulosus s.l. GenBank archived sequences from India corresponding to different hosts (large and small ruminants and humans) along with the sequences (n = 2) generated in the present study. Overall, 14 haplotypes with high haplotype (0.780 ± 0.059) and low nucleotide (0.033 ± 0.010) diversities were recorded for the overall data set, which evinced a population expansion. The median-joining haplotype network revealed a stellate shape of E. granulosus sensu stricto (s.s.) sequences, which was indicative of rapid population expansion. High genetic differentiation (FST = 0.840 - 0.983) and low gene flow (Nm = 0.003 - 0.047) were recorded between the pig intermediate hosts infected with E. ortleppi and other hosts infected with E. granulosus s.s. The findings are of paramount significance for the formulation of effective control strategies considering the public health and economic impact of cystic echinococcosis.

Population structure and haplotype network analyses of Hyalomma anatolicum based on the large subunit ribosomal RNA (16S rRNA) gene.

Hyalomma anatolicum, an Anatolian hard tick is a well-recognized vector involved in the transmission of various pathogens to animals and humans. The present study elucidated the population structure and haplotype network of H. anatolicum based on the mitochondrial large subunit ribosomal RNA (16S rRNA) gene sequence. The population structure and haplotype network analysis of 75 sequences archived in the GenBank, including the 15 sequences generated herein, yielded 24 haplotypes. Haplotype 1 (Hap_1) was the predominant haplotype consisting of 45 sequences from India, China, Pakistan, Turkey, Egypt, Iraq, and Tajikistan. The complete haplotype network exhibited a stellate conformation, highlighting a recent population expansion. The overall dataset, together with the sequences corresponding to India, China, and Pakistan, showed a high haplotype (0.638 ± 0.065, 0.671 ± 0.103, 0.753 ± 0.099, and 0.854 ± 0.061, respectively) and low nucleotide (0.00407 ± 0.00090, 0.00525 ± 0.00196, 0.00680 ± 0.00233, and 0.00453 ± 0.00056, respectively) diversity, further emphasized a recent population expansion. The neutrality indices including Tajima's D, Fu and Li's D, and Fu and Li's F for the complete dataset (- 2.661, - 6.008, and - 5.649, respectively) as well as for the sequences from India (- 2.223, - 3.414, and - 3.567, respectively) were negative, suggesting deviation from neutrality and a recent population expansion. The present study provided novel insights into the population structure and haplotype networks of H. anatolicum based on the mitochondrial 16S rRNA gene, and the different tests inferred a low genetic differentiation and suggested a recent population expansion of this economically important tick species.

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.

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.

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.

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.

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.

Sub-lineages of Taenia solium Asian Genotype Recorded in North India.

PURPOSE: Porcine cysticercosis is a neglected zoonotic disease of significant veterinary and medical importance owing to its economic impact and public health significance. The present study aimed at genetic characterization of Taenia solium metacestodes in slaughtered pigs of Haryana (North India). METHODS: A total of 213 (160 and 53 from Chandigarh and Hisar, respectively) slaughtered pigs intended for human consumption were screened for the presence of T. solium metacestodes. The retrieved metacestodes were confirmed molecularly based on the partial amplification of mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. Evolutionary divergence, haplotype and nucleotide diversities and neutrality indices of the retrieved isolates were also assessed. RESULTS: Out of the 213 pigs, 2 (0.94%) revealed the presence of metacestodes involving 1 pig each from Chandigarh (0.62%) and Hisar (1.9%). The sequences obtained after custom sequencing were submitted to GenBank under the accession numbers LC661682-83. The present study haplotype clustered with haplotypes of Asian origin and showed variation from other haplotypes by 1-23 mutational steps. However, the present study isolates also showed nucleotide polymorphisms (A198T, A199G, A201T, G204A, T206A, C210T, T212G, T213A, T216G/A, T217C, T221C, C524T, G994A) at different positions, which indicated the presence of sub-lineages. Low nucleotide diversity (π = 0.020) and negative value of Tajima's D (- 1.304) observed for the haplotypes under consideration was indicative of purifying selection and recent population expansion. CONCLUSIONS: Our study confirms the circulation of T. solium Asian genotype (with distinct sub-lineages) in study area and recommends strict control measures to contain the zoonotic disease.

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.

First record of Toxoplasma gondii antibodiesin Royal Bengal tigers (Panthera tigris tigris) and Asiatic lions (Panthera leo persica) in India.

The purpose of this study was to detect the antibodies against Toxoplasma gondii in Royal Bengal tigers (Panthera tigris tigris), Asiatic lions (Panthera leo persica), leopards (Panthera pardus), and elephants (Elephas maximus indicus) residing in the Mahendra Chaudhury Zoological Park, in Chhatbir, Punjab (India) during winter and monsoon seasons. Using  indirect ELISA, 20 serum samples were analysed during the winter season. Results indicated that 1 lion (5%) tested seropositive, and 3 tigers and 1 lion (20%) were considered suspect. During the monsoon, 4 individuals (2 tigers and 2 lions, 20%) were seropositive, whereas only 1 tiger (5%) gave suspected results. Significantly higher globulin, creatinine, blood urea nitrogen, phosphorus, and creatine kinase values were recorded in seropositive and suspected groups. Levels of albumin, glucose, calcium, sodium, and iron decreased significantly in the seronegative group. Results from sero-testing 40 rodents trapped in and around the park depicted the presence of antibodies against Toxoplasma gondii in 1 individual. This study reveals the haemato-biochemical alterations in both seropositive and suspected wild felids for toxoplasmosis. Moreover, it provides the first serological evidence of T. gondii exposure in wild felids, notably Royal Bengal tigers and Asiatic lions, in India.

Cyathostomum catinatum infection in a mule of Palam Valley, India: a case report.

The small strongyles also called as cyathostomes, cyathostomins or trichonemes, affect the health status of equines, leading to morbidity and mortality in heavily infested individuals. The present study was carried out with the aim to identify the cause of colic in a mule of Palam valley, Himachal Pradesh, India. The detailed faecal sample examination revealed heavy intensity of strongyle eggs with an egg per gram value of 2300. The collected faecal sample also exhibited presence of adult worms. The morphological observations after clearing the retrieved parasites revealed the presence of adults of Cyathostomum catinatum species. The haematological and serum biochemical studies revealed alterations related to parasitic invasion. Fenbendazole used for the treatment proved completely effective. To the best of our knowledge and thorough literature search this is the first report of C. catinatum infestation in equines of India.