Bovine Visceral Schistosomosis Caused By Schistosoma Indicum in Migrant Cattle Slaughtered at Chennai City, Southern India


 One hundred and eighty mesentery samples of cattle were collected during a period of October 2019- March 2020 for screening visceral schistosomosis from Perambur slaughter house, Chennai, Tamil Nadu, India. Schistosoma indicum was identified in eleven mesenteries of Nellore breed of cattle based on morphology and worm number varied from 1-114 per mesentry. Molecular confirmation based on 16s RNA revealed it to be S. indicum. It was found that the infection of S. indicum in cattle was first report in Tamil Nadu in last two decades. The slaughtered cattle originated from neighboring states especially Andhrapradesh. The tract of infection needs to be traced accordingly for future control strategies. Awareness must be created among the livestock farmers to prevent production loss due to S. indicum infection while purchasing cattle from neighboring states within India.


Introduction Bovine Visceral schistosomosis (BVS) is water borne trematode infection which is considered as
Neglected Tropical disease. BVS is well recognized as the fth major helminthosis of domestic animals in the Indian Subcontinent (Sumanth, 2004). This infection is caused mainly by Schistosoma indicum and Schistosoma. spindale in India (Agrawal, 2000). Hence, the species need to be differentiated based on morphology of adult worm tegument and number of testes in male as well as the shape of ova in female (Roy and Tandon,1992;Agrawal, 2012).S. indicum is an obligate parasite of blood vascular system residing in the portal and mesenteric veins of ruminants. The blood uke infection causes chronic wasting illness and is characterized with haemorrhagic diarrhoea, emaciation, anemia which overlaps with other existing debilitating diseases (De Bont and Vercruyesse, 1998). It also causes reduced milk yield, severe mortality with outbreaks leading to high death rates in cattle (Agrawal, 2012). Diagnostic methods includes direct parasitological examination of ova and miracidium from faeces/rectal pinch which is time consuming and limited in sensitivity since, S. indicum ova in cattle are found mostly in the mucous membrane of intestine causing cellular lesions (Krishnamurthi, 1956). In erstwhile Madras province S.indicum infection was uncommon though infrequent and the cases were reported from Kurnool, Chittor and Nellore districts, currently in the state of Andhra Pradesh of India (Alwar, 1950).
Mitochondrial markers particularly the species barcoding gene cytochrome c oxidase subunit I (cox I), 16s and 12s ribosomal subunit RNA have been currently used as target gene for species identi cation and phylogenetics (Jones et al.,2020). The current study was carried out to know the occurrence of BVS due to S.indicum in Chennai, Tamil Nadu, India, which was not recorded since decades and to target species speci c 16sRNA for molecular con rmation and phylogenetic studies.

Worm collection
A total of 180 mesentery samples of cattle were randomly collected during a period of October-March 2020 in order to check active visceral schistosomosis from Perambur slaughter house, Chennai, Tamil Nadu, India (Latitude 13.1038̊ North, 80.2612̊ East) (Fig. 1). The mesentery was soaked in normal saline to collect the blood ukes present if any for two hours (Fig. 2). Also the veins of the mesentery were punctured under sunlight for recovery of the adult worms by the method of Sumanth et al (2004). The collected worms were subjected to microscopical examination and individually counted to ascertain the intensity of infection.

Morphological identi cation
The adult worms were examined for the structural characteristics such as size, tegument, sucker position, gynaecophoric canal, number of testes, and morphology of ova under an inverted microscope at 40X magni cation to con rm their identity (Singh, 1958 Negative controls (no DNA template) were included in the PCR reactions which were run in the same thermal cycler. Amplicons were resolved in ethidium bromide-stained agarose gel (1.2%) and sized by comparison with GeneDirex ® 100 bp DNA ladder as molecular marker. Gels were photographed using Gel Doc 2000 (BioRad, Hercules, CA, USA). Gels were puri ed and sequenced using the Sanger's method.
Their nucleotide sequence analysis was undertaken by BLAST algorithms and databases from the National Centre for Biotechnology (http://www.ncbi.nlm.nih.gov). Phylogenetic tree was constructed using Mega 7.0 software.

Result
The adult worms examined under the inverted microscope revealed presence of tuberculated body surface in male, presence of oral and ventral sucker in anterior end, male and female found in copulation (Fig. 3), oval shaped ova with terminal spine in uterus (Fig. 4). Based on these characters the worms were morphologically identi ed as Schistosoma indicum. Among 180 mesenteries, 11 showed presence of S. indicum with 6.11 percent infection and worm number varied from 1-114 in number per mesentery. Mixed infection with S. spindale was also noticed in 4 mesenteries.
Molecular identi cation revealed the band size of 606 bp speci c for S. indicum (Fig. 5). The sequence obtained showed 99.30 per cent homology to S. indicum sequence available in Genbank. The sequence was submitted to Genbank (accession No.M233263). The phylogenetic tree was constructed using maximum likelihood method with 1000 replicates (Fig. 6 )

Discussion
Bovine Visceral schistosomosis caused by S. indicum is a neglected tropical disease in southeast Asia. This was rst discovered in horse, donkey and sheep in north India ( Montgomery, 1906). It has also been recorded in cattle, buffaloes, goat and camel. The record of this infection in erstwhile Madras province is not uncommon though infrequent (Rao, 1939;Alwar, 1950  S. indicum Bangladesh isolate showed 99. 30% identity to the Tamil Nadu isolate. The phylogenetic tree was constructed based on character based method. The pylogenetic tree based analysis indicated that there could have existed a common ancestor for S. indicum of India and S. cf. indicum W528 Nepal isolate. The common ancestor of Tamil Nadu, India and Nepal isolate could have originated from S. spindale of Thailand and Sri Lankan isolate. Gene sequence of Indian isolate of S.indicum was so far not compared with other country isolates ( Jones et al., 2020). However, the earlier report revealed S. indicum from India forms a separate clade along with S. spindale of UK origin.
Livestock sector farmers of Tamil Nadu are unaware of this infection which is prevalent in most of the South Indian states except Tamil Nadu. It was also found that the infection of S. indicum was so far not reported from domestic cattle population of Tamil Nadu. This report also showed this infection in slaughtered migrant Nellore cattle breed from neighbouring states of India. The infection was seen more among the adult males and occurred mainly during the monsoons. Further studies are required so that in depth knowledge about epidemiology and bionomics of this infection in other parts of Tamil Nadu can help us to know the clarity of prevalence and take strategic preventive measures in better way.

Declarations
Funding Figure 1 Nellore breed of cattle in lairage of abattoir Page 9/12  Phylogenetic analysis of S. indicum based on 16SrRNA gene from Chennai, Tamil Nadu, India