First Report on Molecular Identification of Moniezia expansa in Sheep from Mannavanur, Palani Hills, Tamil Nadu, India.

PURPOSE: Tape worm infection is common among sheep at SRRC, Mannavanur, Palani hills, Tamil Nadu, India. The aim of the present study is to find out the cestode species infecting the sheep being maintained at SRRC, Mannavanur, by means of molecular method. METHODS: During the second week of June 2021, the hogget flock of sheep (comprising both Bharat Merino and Avikalin sheep breeds) was drenched on empty stomach with commercial preparation of anthelmintic drug containing Niclosamide plus Albendazole, as per the standard dose specified by the manufacturer (Niclozole™: each 5 ml contains 500 mg of Niclosamide and 150 mg of Albendazole: dose for sheep-10 ml/15 kg body weight). The tapeworms expelled in dung by the drug-treated sheep were collected, washed in PBS (pH 7.2), and fixed in between two glass slides using 10% formalin. Furthermore, cytochrome c oxidase subunit I (Cox-I) gene-based PCR was carried out. Only partial sequence (1593 bp) of Cox-I gene of Moniezia expansa from Sheep at SRRC, Mannavanur, Tamil Nadu, India was obtained by PCR. The PCR amplified fragment was cloned into pGEM-T vector and the recombinant plasmid was sequenced. The obtained nucleotide sequences of Cox-I gene of the M. expansa from Indian sheep were analysed with that of 27 more cestode species from different mammalian species (available in GenBank) using bioinformatics tools. RESULTS: The species of the tapeworm was identified as Moniezia species by the Department of Veterinary Parasitology, VC& RI, Orathanadu, TANUVAS by the standard Acidic alum carmine staining method. Due to the ambiguity in the conventional method, Cox-I gene-based PCR and subsequent gene sequencing protocols were used for the identification of the species of cestode infecting sheep at SRRC, Mannavanur, and it was confirmed as M. expansa upon BLAST analysis. Moniezia expansa from SRRC, Mannavanur is having 100% sequence identity at nucleotide level with that of M. expansa from Sengal/Ethiopia. M. benedeni shared 87-88% nucleotide identity with Indian M. expansa. With taenids, the share of percent nucleotide identity of Indian M. expansa ranged from 79 to 81%. M. expansa from Indian sheep was clustering with other anaplocephalids from various mammalian species in the analysis of phylogenetic tree based on Cox-I nucleotide sequences. CONCLUSION: From the present study, it is concluded that M. expansa is the anoplocephalid cestode infecting the sheep at Mannavanur, Tamil Nadu, India. To our knowledge, this is the first report on partial nucleotide sequences of Cox-I gene of M. expansa from Sheep of Indian peninsula. An investigation on the involvement of oribatid mites as the vector in the transmission of M. expansa among sheep at SRRC, Mannavanur needs to be carried out.

Biology and pathology of caecal nematode Subulura brumpti in Japanese quails (Coturnix coturnix japonica).

The occurrence of caecal nematode, Subulura brumpti has become more common in quails being maintained in commercial farms in Tamil Nadu, India. Two trials were carried out to study the biology and pathology of S. brumpti in quails. In the first trial, eight grower quails were divided into two groups (T1 and T2) comprising of four birds each. The birds belonged to the group T1 was infected with 20 cysts collected from beetle and birds of T2 group were kept as control. The beetle was identified as Tenebrionid species. Prevalence of S. brumpti in beetle was 89 per cent and intensity ranged from 1-27 cysts. The eggs of S. brumpti were observed in droppings on 30 - 32 DPI. In the second trial, 16 birds were divided to four groups viz., T1, T2, T3 and T4. The birds of T1, T2, and T3 were infected by gelatin capsule method. All the birds were sacrificed on 30 DPI. The caeca from infected group did not show any gross and histopathological changes.

Ground beetle, Opatroides frater (Coleoptera) as natural intermediate host for the poultry tapeworm, Raillietina cesticillus.

Poultry farms in and around Namakkal with a history of tapeworm infection were surveyed for the presence of beetles which could act as intermediate host for the tapeworms. Beetles collected from different poultry farms with suspected tapeworm infection were examined for the presence of metacestode stage of the parasite. A total of 1,880 beetles were collected from 12 poultry farms with suspected tapeworm infection to study the vector potentiality. Out of these, 205 beetles (10.9 %) from nine farms were found to harbour cysticercoids. The percentage of cysticercoid infection in beetles was 8.24, 10.34 and 16.66 % respectively in three different surveys. The beetles harbouring the cysticercoids were identified as Opatroides frater, which may be a natural intermediate host for Raillietina cesticillus. Infection free young chicks (4 weeks old) were experimentally infected with specific number of cysticercoids and prepatent period of tapeworms was found to be between 12 and 13 days. Gravid segments were expelled between 3 and 4 p.m. consistently. The results of this study would help to formulate suitable control measures against the above tapeworm infection.

Immune responses to polyethylenimine-mannose-delivered plasmid DNA encoding a Fasciola gigantica fatty acid binding protein in mice.

Fasciola gigantica fatty acid binding protein (FABP) was evaluated for evoking an immune response in mice, by delivering the gene coding for this protein with mannosylated-polyethylenimine (PEI) to peritoneal cells. Mice were immunized with 50 microg recombinant plasmid DNA (Group I) or DNA-PEI-mannose (a 22 kDa linear cationic polymer with mannose ligand) (Group II) via the intraperitoneal route. Antibody studies showed no significant humoral immune response evoked to this DNA immunization with either PEI-mannose-delivered or naked DNA. However, on protein boosting of these DNA-primed mice there was a significant enhancement of antibody titre. Flow cytometric bead array was used to measure quantities of interleukin (IL)-2, IL-4, IL-5, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) cytokines. Overexpression of T-helper 1 (Th1) cytokines such as IFN-gamma and TNF, with a lower but significant expression of the T-helper 2 (Th2) cytokine IL-5 was detected. Gene delivery using polyethylenimine-mannose ligand showed significant expression of IFN-gamma and TNF (P < 0.05), but no significant difference in IL-2, IL-4 and IL-5 (P>0.05) cytokine expression was observed between naked-DNA- and mannosylated PEI-DNA-delivered mice. Naked- or PEI-delivered-DNA immunization produced insignificant levels of IL-2 and IL-4 (P>0.05) cytokines in both groups of mice.

Prepatent detection of Fasciola gigantica infection in bovine calves using metacercarial antigen.

Metacercarial antigen of Fasciola gigantica was evaluated for early immunodiagnosis of experimental bovine fasciolosis using ELISA and Western blot. In ELISA, the experimental F. gigantica infection was detected as early as 2 weeks post-infection (WPI). The gradual increasing trend of antibody level was observed from 2 to 7 WPI, followed by a plateau, which was maintained up to 14 WPI. In Western blot, sera from experimentally infected calves recognized one distinct polypeptide of 21 kDa in fractionated metacercarial antigen as early as 10th day post infection. From 2 WPI, more polypeptide bands were reacting. Recognition of these protein bands persisted till the end of the experiment (14 WPI). Cattle sera collected from the field showed 34.5% seroprevalence of fasciolosis by ELISA using MAg. Comparative immunoblot studies of metacercarial antigen with anti-Gigantocotyle explanatum and anti-Paramphistomum epiclitum sera revealed that 21 and 25 kDa polypeptides of metacercarial antigen did not cross-react with any of these sera and appear to be unique to F. gigantica and having the desirable qualities of early and specific immunodiagnosis.

Cathepsin L cysteine proteinase in the diagnosis of bovine Fasciola gigantica infection.

Cathepsin L cysteine proteinase from Fasciola gigantica was evaluated for its potential in the early prepatent detection of this helminth infection in bovine calves. Five cross-bred bovine calves were experimentally infected with 400 metacercariae/calf and evaluated for anti-cathepsin L antibody response. F. gigantica infection in these calves could be detected 4 weeks post-infection using an ELISA, dipstick ELISA and Western blotting with 100% sensitivity. The antigen was also used to detect F. gigantica field infection in cattle, by screening 256 sera of these animals by an ELISA, which demonstrated an overall infection rate of 26.95%. Preliminary studies showed that F. gigantica cathepsin L cysteine proteinase does not cross-react with Paramphistomum epiclitum, Gigantocotyle explanatum and hydatid cyst antigens. However, extensive studies on the cross-reactivity of this antigen with related helminth parasites of cattle and buffaloes are required, before this antigen can be considered suitable for immuno-diagnosis of fasciolosis in these ruminants.

Detection of circulating 54 kDa antigen in sera of bovine calves experimentally infected with F. gigantica.

The antibody response and circulating antigen levels in bovine calves, infected experimentally with Fasciola gigantica, were monitored using enzyme-linked immunoelectrotransfer blot (EITB) and sandwich ELISA, respectively. By EITB, the infected calves' sera recognized the polypeptides in the range of 54-58 kDa as early as 2 weeks post-infection. By 12th week post-infection, the lower two polypeptides of 12 and 8 kDa had disappeared. In sandwich ELISA, the circulating 54 kDa and whole worm antigen of F. gigantica were detected in the sera samples of infected calves as early as 2 weeks post-infection and persisted until the end of experiment (26th week PI). The 54 kDa antigen of F. gigantica appears to be specific and possesses promising immunodiagnostic potential for early prepatent diagnosis of bovine fasciolosis.