Epidemiological investigations and molecular characterization of Toxocara canis from Jammu region

Introduction

Toxocara canis is one of the most prevalent and harmful parasite that infect dogs globally. (Chattha et al., 2009; Traversa et al., 2012) [6,39] . The nematode is a member of the family Ascaridae, found in the small intestine of the dog (Glickman and Schantz, 1981; Yarsan et al., 2003) [12,42] . It causes a common helminthic disease in dogs that has been seen in few different parts of the nation, with varying range of clinical signs in infected animals. Foetal pups acquire T. canis infection in utero via trans-placental route when hypobiotic larvae from the somatic tissue stores are activated in the late period of gestation. The larva can also enter mammary glands and then spread via colostrum and milk in newborn pups (Overgaauw et al., 1997) [23] . In pups (≤6 months of age), ingestion of infective eggs of T. canis most likely leads to hepato-tracheal migration of larva followed by a patent infection while the older dogs are less likely to get patent infection as they develop immunity against tracheal migration of larvae (Greve, 1971 and Dubey, 1978) [13,9] . Somatic migration occurs in older dogs (Sprent, 1958) [34] . However, few older dogs ≥6 months do shed T. canis eggs, posing a risk of zoonosis (Sager et al., 2006; Claerebout et al., 2009; Barutzki and Schaper, 2011) [27,7,3] . Confirmatory diagnosis of the infection can be done by qualitatively detecting ascarid eggs in faeces using the floatation method and quantitatively estimating the eggs per gram using the Mc. Master method (Habluetzel et al., 2003) [15] . However, precise identification of Toxocara species and their clear distinction from one another, as well as from other ascaridoid nematodes responsible for visceral larva migrans (VLM) and ocular larva migrans (OLM), is essential for investigations related to taxonomy, epidemiology, population genetics, diagnosis, and disease control. Molecular detection of Toxocara infection using ELISA and PCR techniques have also proven to be the diagnostic tools (Elsemore et al., 2017; Oge et al., 2019) [10,22] .

PCR technique has the ability to selectively amplify DNA from nematode eggs, larval or adult stages and therefore can be employed for rapid and specific diagnosis (Smith et al., 2009; Gaser, 2013) [31] . Despite the veterinary and zoonotic importance of T. canis information on its prevalence, associated risk factors, clinical manifestation, and molecular characteristics in young dogs from Jammu region remains limited. Therefore, the present study was undertaken to determine the prevalence of T. canis infection and associated risk factors in dogs from the selected area of Jammu, to assess the major clinical manifestations associated with infection, and to characterize the parasite molecularly using ITS-2 and COI gene markers. The study also aimed to evaluate the phylogenetic relationship of the Jammu isolate with previously reported T. canis isolates from different geographical regions.

Materials and Methods

The research was approved by IAEC (Institutional Animal Ethics Committee) registered with CPCSEA (Committee for the purpose of control and supervision of experiments in animal) under no. 08/IAEC-20/2021.

Experimental design:

The epidemiological investigations of Toxocara canis infection in canines was conducted on domesticated and stray pups from various rural and urban areas of Jammu district of Union Territory of Jammu & Kashmir. Sick and healthy pups presented at the small animal medicine OPD under division of teaching veterinary clinical complex (TVCC), SKUAST-J and private based veterinary clinics in Jammu region were included in this study. Prevalence of Toxocara canis infection was studied on the basis of qualitative and quantitative faecal examination. A total of 300 pups aged ≤ 3 months using simple random sampling method were considered. The calculation of sample size was done based on formula described by (Thrushfield, 2007) [36] .

Evaluation of epidemiological data:

The evaluation of epidemiological data viz; age, gender, faecal consistency, body weight, body coat and co-infection for analysis of association with T. canis infection was done. The research period was divided into four seasons, according to the Indian Meteorological Department, Pune: summer (March-June), monsoon (July-September), post monsoon (October-November), and winter (December-February). Along with that the consistency of the faecal sample was scored using the Waltham faeces scoring chart (https://www.waltham.com/dyn/_assets/_pdfs/other-resources/waltham-scoring.pdf). And, body weight of pups was measured with digital weighing scale.

Faecal processing and examination:

The examinations of faecal samples were done grossly for adult worm, microscopically by floatation method as per method described by (Soulsby, 1982) [33] . Quantitative faecal examination was done to obtain accurate information with regard to severity of an infection. Egg counting method was used to determine number of eggs per gram of faeces (EPG), for this purpose, Mc-Master technique was performed as per method described by (Soulsby, 1982) [33] .

2.4: Molecular characterization of Toxocara canis of dogs in Jammu and subsequent phylogenetic analysis with other Toxocara species

The infected pups were given oral piperazine citrate @ 200mg/kg body weight (Actipar) and adult worms were collected from their faecal samples. The worms were thoroughly washed in PBS and fixed in 70% ethanol and later stored at -20°C until genomic DNA was extracted. Isolation of DNA was done from small portion of adult T.canis specimen using the Wizard® genomic DNA purification kit (Promega, USA, Cat. no. A1120) with few modifications. The ITS-2 (internal transcribed spacer) located within nuclear ribosomal gene was selected and analyzed (Table1) (Mahdy et al., 2020) [20] . Additionally, Cytochrome C Oxidase subunit I (COI) primers were retrieved from the national centre for Biotechnology information (NCBI) database and utilized for the analysis (Table 1). The reaction mixture was prepared with PCR master mix (Thermo scientific, K0171) and supporting components required for the amplification of the desired DNA segments (Table 2). The DNA amplification was done with conventional PCR assay using T100 TM thermal cycler (Bio-rad, USA, Serial no. 621BR41692) using specefic cyclic conditions for each primer set (Table 3). The amplified PCR products were confirmed with gel electrophoresis using 1.5% of molecular grade agarose. The molecular weight were computed with 100-1500 bp DNA Ladder and visualized under UV exposure in the gel documentation system (E-BOX, Vilber). The positive PCR products were purified using SureExtract ® PCR clean-up/ gel extraction kit (Genetix Biotech Asia Pvt Ltd., and subsequently sent to Biokart India Pvt. Ltd. for Sanger sequencing. PCR master mix (Thermo scientific, K0171) 30 µl 3.

Nuclease-free water 23 µl Final elongation 72 o C for 5 minutes

Statistical analysis

The data obtained on the prevalence of Eimeria spp. infection in relation to age, season and sex of dogs were compared by Chi square test. The mean oocyst per gram of faeces was compared by ANOVA test using SPSS 16.0.

Here, p value of <0.05 was considered significant.

Results and Discussion Prevalence of Toxocara canis in dogs of Jammu region based on faecal floatation examination

The present study revealed an overall prevalence of GI parasitic infection from 305 faecal samples of dogs aged ≤ 3 months in Jammu area was 52.13%. Among the individual parasites detected, Toxocara canis (21.97%) infection showed the highest prevalence followed closely by Ancylostoma caninum (20.65%) while 9.51% of the dogs harboured mixed infections (Table 4 and Figure 1). The relatively high prevalence of T.canis in young dogs may be attributed to their increased susceptibility to infection and the efficient transmission of this parasite through multiple routes, particularly transplacental and transmammary transmission (Soulsby, 1982) [33] . The predominance of T. canis in the present study therefore highlights its epidemiological importance in young dog and its potential zoonotic significance. A distinct seasonal variation was observed in the occurrence o GIT parasite infections. Among the four seasons, Monsoon (69.35%) had the highest occurrence of overall parasitic infection followed by post-monsoon (45.57%), winter (41.18%), and summer (33.82%). The quantitative analysis of infection showed highest EPG (egg per gram) in monsoon period followed by the post-monsoon season, summer and winter (Table 5 and figure 2). The likelihood of contracting T. canis infection during the monsoon was 4.74 and 2.66 times greater significantly (p<0.05) than it was during the winter and summer, respectively. The higher prevalence of endoparasitic infections in dogs recorded during the months from July to September might be associated with the role of rainfall in the translocation and acquisition of L3 infective larvae in the environment as opined by (Soulsby, 1994) [32] . The seasonal pattern observed in the present study, is however not consistent with all the previous reports. Different investigators have documented peak prevalence in winters (Agnihotri et al., 2008; Kumar et al., 2014) [2,17] , autumn (Totkova et al., 2006) [37] , monsoon (Utaaker et al., 2018) [41] and summer (Moudgil et al., 2016) [21] while some workers have reported influence of season on infection as insignificant (Gugsa et al., 2015) [14] . The discrepancies among studies in the seasonal prevalence pattern of Toxocara canis infection in different areas may be attributed due to reasons like variation in sampling, geo-climatic conditions, degree of contamination of environment with infective stages of parasite, etc. Therefore, the observed seasonal variations in T. canis prevalence appears to be region-specefic and influenced by the interaction of environmental, epidemiological and host related factors. Age wise analysis revealed the highest EPG recorded in age group between 31 and 60 days followed by 61-90 days and up to 30 days (Table 6, Figure 3). The probability of T. canis infection was shown to be significantly (p<0.05) greater in the age groups up to 30 days and 31-60 days by 3.67 and 3.12 times, respectively, when compared with 61-90 days age group. Similar findings of low prevalence of infection with advancing age in dogs have been reported by several workers (Daryani et al., 2009; Abrere et al., 2013; Rostami et al., 2020) [8,25] . The higher prevalence of toxocarosis in very young puppies might be attributed to the age susceptibility and lack of premunity. Moreover, puppies acquire the infections at the early age through prenatal and transmammary routes while the adult dogs develop immunity which decreases the establishment as well as fecundity of the parasites (Soulsby, 1982; Urquhart et al., 1996) [33,40] . The comparatively high prevalence observed in puppies upto 30 days age may be attributed to their dependence on dam for feeding which facilitates the acquisition of infective larvae through milk. In contrast the pet puppies are commonly weaned by approximately one month of age and are often dewormed at this stage. This practice could be the reason for subsequent decline in the prevalence with the advancement of age. The sex-wise analysis revealed that female dogs exhibited a higher prevalence of infection (23%) than male dogs (21.23%) (Table 7, Figure 4). However, no significant difference (p>0.05) was observed in the mean EPG values of Toxocara canis between the two sex groups. Previous studies have reported variable findings regarding sex-related prevalence of Toxocara spp. infection in dogs. A higher prevalence in female dogs was documented by Satyal et al., (2013) [29] and Sahu et al., (2010) [28] , whereas Swai et al., (2010) [35] reported a greater prevalence in male dogs. In contrast, several studies have reported no significant association between sex and the occurrence of parasitic infections in dogs (Swai et al., 2010; Bhangale and Khillare, 2018) [35,4] . The marginally higher prevalence recorded in female dogs in the present study may be attributed to the greater number of faecal samples collected from female animals. An association between clinical manifestations and T. canis infection was identified in this study. Among the infected dogs, the most frequently clinical manifestations were pasty faeces (47.76%), rough hair coat (77.61%), and pale mucous membranes (85.07%) (Table 8). These observations are in agreement with the findings of Lefkaditis et al. (2006) [18] and Adolph et al. (2017) [1] . The occurrence of pale mucous membranes may be attributed to blood loss resulting from extensive intestinal tissue damage and the blood-feeding activity of infective Toxocara larvae in puppies. Similarly, pasty faeces could be due to increased intestinal secretion and reduced absorption caused by the presence of adult parasites, as suggested by Rao et al. (1995) [24] , Bowman (2021) [5] , and Sharma et al. (2010) [30] . The rough hair coat observed may reflect poor nutritional status resulting from impaired nutrient absorption, along with the physiological stress associated with parasitic infection.

Gene sequence of Toxocara canis infecting dogs of Jammu:

In the present study, only one sequence was submitted to NCBI Gene Bank as shown in [19] who observed specific amplification of ITS-1 and ITS-2 using species-specific primers for Toxocara canis.

Additionally, PCR amplification of the ITS-2 gene was carried out by Khademvatan et al. (2014) [16] after comparing the obtained sequence with several isolates of T. canis observed significant homology. Phylogenetic classifications are a helpful tool for understanding the links between different species during evolution (Mahdy et al. 2020) [20] . In addition to ITS-2, the COI region might also help in the accurate identification of Toxocara species using PCR.

The phylogenetic tree was built using Maximum Likelihood method using Tamura canis isolates from Jammu region was performed using the ITS-2 and COI genes. The COI sequence generated in the present study was submitted to Gen Bank and assigned accession number ON911829, representing the Jammu isolate. The sequence exhibited high nucleotide identity with other Toxocara isolates, showing 98.25% identity with T. canis infection from Russia (KX365898) and 98.25% with T. canis from Heilongjiang, China (KC998807). These findings further support the molecular identification of the Jammu isolate as T. canis.

Acknowledgement

AcknowledgementThe authors are thankful to the Department of Veterinary Medicine, TVCC and Department of Veterinary Parasitology, Sher-e-Kashmir University of Agricultural Sciences and Technology for providing all the infrastructure and support during the research.

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