Serological and immuno-histopathological detection of Paramphistomum epiclitum infection in large ruminant population in Punjab, Pakistan

Recent molecular identification of Paramphistomum epiclitum in Pakistan raises concerns about its epidemiology and pathologies in infected tissues of ruminants. The present study aimed to find the seroepidemiological and histopathological record of Paramphistomum epiclitum from cattle and buffaloes. Indirect ELISA on animal sera and histology of infected rumen with hematological and biochemical analyses were performed. The overall prevalence of P. epiclitum was noted as 15.3% in the abattoir survey and 37.6% in the serological examination. The sensitivity and specificity of the diagnostic test were 100% and 83.3% respectively. The paramphistomosis was significantly (p= 0.001) higher during August (6.4%) followed by September (5.4%), whereas the lowest prevalence was recorded during April (0.4%). The hematological and biochemical variations showed significant increase in total leukocyte count (p= 0.002), alanine aminotransferase (p= 0.05), glucose (p= 0.01) and cholesterol (p = 0.024) levels. However, significant decrease in the level of erythrocyte (p= 0.019), hemoglobin (p= 0.001), mean corpuscular haemoglobin concentration (p= 0.05), mean corpuscular volume (p= 0.038) and platelets count (p= 0.048) was observed. The histopathology of rumen tissue showed haemorrhages, atrophy of ruminal papillae, sloughed mucosa, cellular vacuolation, and infiltration of lymphocytes inflammatory cells. The present study provides the prevalence and histopathological record of P. epiclitum in Pakistan for the first time in order to take control measures in the country.

Molecular Characterization of Paramphistomum cervi in Buffaloes

Background: Paramphistomiasis (Rumen fluke disease) in ruminants is a major health problem, while documented reports on Paramphistomum species are limited in Asian countries. The present study aimed to identify paramphistomoid flukes that infects buffaloes with the goal of characterization of prevalence in Pakistan and its comparison with neighbor countries.Materials, Methods & Results: In 2018, a total of 178 slaughtered buffaloes aged four to six years were examined and flukes were collected from their infected rumen and reticulum using sterilized forceps. After amplification and sequencing of 18S rRNA partial fragment, the generated sequences were edited (810bp) and aligned with the other sequences of Paramphistomum species retrieved from NCBI. A phylogenetic tree was constructed using maximum likelihood method in MEGA X. The 18S rRNA sequence was found 100% similar with Paramphistomum cervi of China and 98% with Paramphistomum epiclitum and other Paramphistomum species of India. The parasitic Pharamphistomum species was identified molecularly as P. cervi.Discussion: Molecular studies provide insight into the biology and phylogenetic relationship among various parasites. These studies are reliable in the genetic-based identification and description of several disease causing agents. The 18S rRNA sequence of P. cervi generated in this study was found closely identical to the P. cervi of the neighbor countries (China and India) which may be due to the similar geographical, environmental conditions and transboundary movement of infected hosts. This is the first nature of study which provides the molecular-based evidence of P. cervi existence in Pakistan and revealed the 18S rRNA as novel molecular marker for the identification and further characterization of Paramphistomum species across Pakistan. The submitted sequence of this study will provide a baseline for further molecular characterization and to compare with other Paramphistoma species from different regions of Pakistan.

Morphological and molecular characterization of Paramphistomum epiclitum of small ruminants

Morphological and molecular identification can pave the way to design the most effective control measures against the Paramphistomum epiclitum in small ruminants. Morphology of the flukes had described the features of Paramphistomum genus. Body was conical with concave ventral and convex dorsal surface, tegumental spines all around the body in the immature stage, terminal funnel shape oral sucker, sub-terminal acetabulum, blind caeca with a serpentine course touching the anterior level of the acetabulum. Vitelline glands were at the lateral margins of the body extended from the pharynx to the posterior sucker. Testes were lobed and tandem, wavy post-testicular uterus and genital pore behind intestinal bifurcation. Sequence analyses of internal transcribed spacer (ITS)-2+ (PCR products of approximately 500 bp) of 10 flukes yielded 2 genotypes, Navsari isolate 1 and 2. In BLAST analysis, ITS-2+ genotypes were 97.3–99% similar with published sequences (KF564870, JF834888, KF642983 and JX678254) of P. epiclitum of Paramphistomatidae. Two genotypes depicted 4 single nucleotide polymorphisms (NPs) in the form of transitions (C-T at 10 and 18; G-A at 255; A-G at 367 locus), 1 triple NPs (CGT-GAA between 21–23 loci) and missing A base at codon 40 in the genotype 1. Average AT and GC content was 49.61% and 50.38%, respectively. Trees topology inferred by Neighbor Joining and Maximum Likelihood methods of ITS2+ of trematodes were similar, with small difference of bootstrap values. Navsari genotypes formed a tight cluster with the P. epiclitum, originated from different location with high bootstrap value and 0.004–0.011 estimated evolutionary divergence.

Immunodiagnostic potency of homologous antigens for natural Paramphistomum epiclitum infection in small ruminants in plate and paper enzyme linked immunosorbent assay

The objective of the present work was to standardize and evaluate indirect plate and dot- enzyme linked immunosorbent assay (ELISA) using purified Paramphistomum epiclitum homologous antigens in the small ruminants. Electrophoretic separation of somatic antigen (PeSAg) in reducing condition on 15% polyacrylamide gel resolved into 16 proteins of the molecular weight ranging from 14 -100 kDa. Two step ethanolic precipitation of supernatant of in-vitro culture of the fluke yielded P. epiclitum excretory-secretory antigen (PeESAg) of molecular weight 28 kDa. The animals (Goats=123; Sheep=91) were broadly kept into post-mortem and faecal examined groups. At many occasion the PeSAg found to cross reacts with other helminths parasites thus minimizing the specificity of the tests and antigens. There was no any direct correlation between the parasites load and ELISA reactivity pattern. The noted prevalence rate after combining the results of post-mortem examination and PeESAg based ELISA (plate and paper/ dot) was 30.08% (37/123) in goats and 28.57% (26/91) in sheep. While using PeESAg, the calculated overall sensitivity% was 92.86 (goats)/ 100 (sheep) in both plate and dot-ELISA, specificity% was 91.58 (goats)/ 91.55 (sheep) in plate ELISA while 88.42 (goats)/ 92.96 (sheep) in dot-ELISA, positive predictive value% was 76.47 (goats)/ 76.92 (sheep) in plate ELISA while 70.27 (goats)/ 80 (sheep) in dot-ELISA and negative predictive value% was 97.75 (goats)/ 100 (sheep) in plate ELISA while 97.67 (goats)/ 100 (sheep) in dot-ELISA, these values were optimum for the field sera sample so the tests and PeESAg can be recommended for the detection P. epiclitum infection in the small ruminants.

Molecular characterization of veterinary important trematode and cestode species in the mithun Bos frontalis from north-east India

Helminth infections in the mithun Bos frontalis, including the liver fluke Fasciola gigantica, hepato-gastric amphistomes Explanatum explanatum, Paramphistomum epiclitum and Calicophoron calicophorum, and the cestodes Echinococcus granulosus and E. ortleppi were studied in north-east India over a 2-year period from 2012 to 2014. Cystic echinococcosis caused by E. granulosus and E. ortleppi was found to be highly prevalent in the mithun, with E. ortleppi being reported for the first time. Molecular markers, including the internal transcribed spacer 2 (ITS-2), 28S rDNA and mitochondrial NADH dehydrogenase sub-unit1 (nad1) were used to confirm the identification of the trematode and cestode species.