Evidence of G1-G3 Complex Life Cycle of Echinococcus granulosus sensu stricto Through Molecular Characterization (Genotyping) in Dogs, Sheep, Goats and Humans of North-western Himalayas, India

Echinococcosis is a helminthic zoonotic disease caused by tapeworms belonging to genus Echinococcus (Family-Taeniidae). Currently, recognized species of Echinococcus include E. granulosus, E. multilocularis, E. vogeli, E. oligarthrus and E. hiquicus. Among these, E. Granulosus and E.multilocularis are of zoonotic importance (Bowles and McManus, 1993; Malki et al., 2021) and responsible for causingcystic echinococcosis (CE) and alveolar echinococcosis (AE), respectively, in humans. E.granulosusis primarily transmitted by dogs and various intermediate hosts, but it is rarely involved in a predator-prey wildlife cycle that is not anthropogenic (Woolsey and Miller, 2021; Mares et al., 2023). The intermediate hosts consume the eggs released into the environment by adult worms that reside in the small intestine of dogs as they graze, drink, etc. (Chhabra and Singla, 2009) Echinococcosis in animals is linked to decreased meat, milk and wool supply and quality, decreased growth and birth rate and organ condemnation, particularly of the liver and lung, which results in significant financial losses (Singh et al., 2014). Furthermore, a prohibition on the export of animals and their products could result in financial losses. Echinococcocosis in humans is linked to fatalities and lost man-days. As per the WHO’s Food borne Disease Burden Epidemiology Reference Group (2010), echinococcosis causes 19300 deaths and 871000 disability-adjusted life-years in humans annually worldwide.  Further, the annual cost for treating cases and losses to the livestock industry is estimated to be US$ 3 billion. The estimated minimum global human burden ofCE catches up 285,500 disability-adjusted life years (DALYs), whereas AEresults in the annual loss of 666,434 thousand DALYs (Kurt et al., 2020). E. granulosus strains have been divided into 10 genotypes (G1-G10) that differ in their host range and infectivity as a result of genetic analysis of hydatid cysts across several different geographic regions, depending upon partial sequencing of the mitochondrial gene (Cytochrome oxidase 1) (Bowles and McManus, 1993; Metwally et al., 2018). The genotype strains of G1 for sheep, G2 for Tasmanian sheep, G3 for buffalo, G4 for horses, G5 for cattle, G6 for camels, G7 for pigs, G9 for humans, G8 for cervids and G10 for sheep. Genotypes G1 through G3 are collectively referred to as E. granulosus sensu stricto. It was suggested that the current study look into the prevalence and genotyping of E. granulosus in the North-Western Himalayas, taking into account the disease¢s importance to the economy and public health.

The present study was conducted in the Division of Veterinary Public Health and Epidemiology, Faculty of Veterinary Sciences and Animal Husbandry (F.V.Sc and A.H.), Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (SKUAST-Jammu) and collaboration with the Division of Veterinary Parasitology, F.V.Scand A.H., SKUAST-Jammu for a period of 12 months (July, 2021- June, 2022).
 
Extraction of cyst from the organs and tissue
 
A total of 137 and 72 cysts were collected from sheep and goats, respectively from various slaughter houses of North-Westen Himalayas. All the organs and tissues were examined for the presence of the cysts.To determine the frequency of CE in various organs, the collected cysts were tallied according to the organ from which they were extracted. The viability of the cysts/protoscolices (living/dead), size, fertility (fertile/sterile/calcified) and type (single/multiple) of the organs were also assessed. According to Rausch et al., (1990), the cysts that were removed from different organs were measured individually using a centimeter scale and they were then divided into three groups: small (diameter <4 cm), medium (diameter between 4 and 8 cm) and large (diameter >8 cm). Cysts without protoscolices were thought to be infertile, whilst those that did were thought to be fruitful. It was also believed that the calcified or suppurative cysts were sterile.
 
Extraction of taeniid egg from feces
 
Faecal samples from 305 dogs, including 122 pet dogs visiting small animal clinics, 183 stray dogs having access to condemned meat/offals residing near slaughter shops/post mortem areas in Jammu division were collected. The faecal samples were collected aseptically in stool collection bottles. Samples were examined fresh and were also preserved separately in 5 per cent formal saline (1 part faeces: 4 parts formal saline) for microscopic analysis. Four hours after the samples were collected, this was completed. As previously reported (Ito 1980, Theinpont et al., 1979), the taeniid eggs were found. In a nutshell, a test tube containing 10-15 milliliters of water and 2 grams of feces were centrifuged at 1000 gram for 10 minutes. After combining the sediment with a sucrose solution (Sp. gr. 1.27), the mixture was centrifuged once more for ten minutes at 1000 g. After completely filling the test tube, the cover glass was put on top of it.After a whole day, the cover glass was inspected under a microscope. Every fecal sample that tested positive for Taenia eggs was kept at -20°C. Echinococcus Ganulosus cysts were removed surgically from five human patients at the Govt. Medical College in Jammu¢s surgery department. After being extracted, the cyst fluid was aseptically transferred to a lab and kept there for processing at 4°C.
 
Molecular characterization
 
In order to extraction of the genomic DNA from hydatid cyst, Qiagen, Hilden, Germany,s DNAse Blood and Tissue kit was utilised. After being preserved in 70% ethanol, the contents of the cyst were centrifuged at 447×g for 15 minutes, 131 times and then cleaned with PBS (pH 7.4). DNA was extracted using the pellet that was found in the silt. After carefully gathering the cysts, they were cleaned with regular saline. The hydatid fluid that was aspirated from the cysts contained the genomic DNA. For five minutes, the fluid was centrifuged at 5,000 rpm and the pellet in the sediment was screened to check for the presence of protoscolices.The Qiagen stool Kit (Qiagen, Hilden, Germany) was used to extract DNA in accordance with the manufacturer's instructions. The eluted DNA was kept at -20°C till further use.Following the manufacturer’s recommendations, genomic DNA was extracted from each sample using the DN easy Blood and Tissue kit (Qiagen, Hilden, Germany) using protoscolices that were collected from cyst fluid and three PBS washes to remove ethanol.
 
Amplification of gene by PCR
 
Mitochondrial gene was amplified using specific primers for cytochrome C oxidase 1 (cox1) (Shahzad et al., 2014) as: Forward (JB3) 5-TTT TTT GGG CAT CCT GAG GTT TAT-3; Reverse (JB 4.5) 5-TAA AGA AAG AAC ATA ATG AAA ATG-3. All PCR amplification reactions, including negative control samples were carried out in a final volume of 25 ml with12.5 ml of commercially available PCR master mix (Thermo Scientific™ DreamTaq™ Green PCR Master Mix-2X). The PCR program was carried out in temperature gradient thermocycler as per the protocol described by Shahzad et al., (2014) with the following program: Initial denaturation at 94°C for 2 minutes followed by 38 cycles of each denaturation at 95°C for 30 seconds an annealing at 51°C for 45 seconds. Amplification was done at 72°C for 30 seconds followed by final extension at 72°C for 5 minutes. PCR product was analyzed using 1.5% agarose gel. For this, 1% agarosegel was prepared in 1X TAE stained with ethidium bromide @ 0.5 mg/ml. The 5 ml of the samples and negative control was loaded in the wells of the gel and run at 80V for 1hour. Finally, the gel was analyzed in a UV trans illuminator for visualization of PCR product. Gel extraction kit (Promega) was used for purification of the PCR product as per manufacturer protocol. Approximately, 100 ml of PCR product was run in 1.5% agarose gel in 1X TAE buffer. The gel containing the DNA fragment was cut un 162 der a UV trans illuminator and was placed into asterile 1.5 ml microfuge tube.The eluted purified PCR products of cox1 gene amplification from protoscolices of hydatid cysts collected from sheep, goat, human and from dog faecal eggs from Jammu division,were sent for sequencing to Biokart India Pvt. Ltd. Kasturi Nagar, East of NGEF, Bangalore-560043. The sequencing data received was analysed using DNA star, Laser gene software and Basic Local Alignment Search Tool (BLAST, NCBI).

A total of 1254 animals (sheep = 723 and goats= 531) were examined, out of which 209 animals (sheep= 137 and goats = 72) were found infected with hydatidosis with the overall infection rate of 16.67% (sheep= 18.95% and goats= 13.56%). Among 180 different organs examined, the highest infection rate was recorded in liver as 56.94% (sheep= 59.85% and goats= 51.39%), followed by lungs as 31.58% (sheep= 29.93% and goats= 34.72%) and least as mixed infection in liver and lungs as 11.48% (sheep= 10.22% and goats= 13.89%). Out of the total 209 infected organs, 150 had single cysts (71.77%) and 59 had multiple cysts (28.23%). Out of 82 infected liver of sheep, 61 had single cyst (74.39%) and 21 multiple cysts (25.61%). Of 41 infected lungs, 32 were found to be harbouring single form of cysts (78.05%) and 9 had multiple cysts (21.95%). From 14 infected lungs and liver, 11 had single cysts (78.57%) and 3 had multiple cysts (21.43%). Out of 37 infected liver of goats, 26 had single cysts (70.27%) and11 had multiple cysts (29.73%). From 25 infected lungs, 16 had single cysts (64.0%) and 9 had multiple cysts (36.0%). From 10 infected liver and lungs, 4 had single cysts (40.0%) and 6 had multiple cysts (60.0%). The overall fertility rate of hydatid cysts from slaughtered sheep and goats was found to be 66.03%. A total of 23.44% of the cysts were found to be sterile and10.52% cysts were calcified. It was observed that there was a consequential difference in fertility rates of cysts recuperated from lungs and livers 193 of slaughtered animals. When it came to the hydatid cyst fertility rate in sheep, it was found that it was greater in liver cysts-65.85%-than in lungs (63.41%) and mixed organs-liver and lung (64.29%). Conversely, the majority of the calcified cysts were discovered in the liver (13.42%), lungs (9.76%) and mixed organs, which includes the liver and lungs (7.14%). Liver and lungs, or co-mixed organs, had the highest percentage of sterile cysts (28.57%), followed by lungs (26.83%) and liver (20.73%). In case ofthe fertility rate for hydatid cysts from goats, it was observed that in liver cysts, it was 67.57%, which was lower than in lungs (68.0%) and co-mixed organs i.e., liver and lung (70.0%). However, most of the calcified cysts were found in co-mixed organs i.e. liver and lungs (10.0%), followed by liver (8.11%) and lungs (8.0%). Highest sterile cysts were found in liver (24.32%), followed by lungs (24.0%) and co-mixed organs i.e., liver and lung (20.0%). Out oftotal 138 fertile cysts, 88 were found viable with the overall rate of 63.31%. The organs with the highest viability among the several diseased sheep organs with viable cysts were the combined liver and lung (88.89%), followed by the liver (68.52%) and the lungs (50.0%). Goats co-mixed organs, such as their liver and lung, had the highest prevalence rate (85.71%), followed by their liver (64.0%) and lungs (47.06%). The reproductive rate of hydatid cysts in slaughtered sheep with varying cyst sizes was 60.87%, 75.42% and 50.0% for <4 cm, 4-8 cm and >8 cm, respectively. >8 cm cysts accounted for the majority of the calcified cysts (36.67%), followed by <4 cm (6.52%) and 4-8 cm (3.27%). Sterile cysts were found in the following order: less than 4 cm (32.61%), 4-8 cm (21.31%) and more than 8 cm (13.33%). The fertility rates of hydatid cysts in killed goats with varying cyst sizes were 52.38%, 84.21% and 46.15% for <4 cm, 4-8 cm and > 8 cm, respectively. >8 cm cysts accounted for the majority of the calcified cysts (30.77%), followed by <4 cm (4.76%) and 4-8 cm (2.64%). The percentage of sterile cysts observed was <4 cm (42.86%), then >8 cm (23.08%) and >4-8 cm (13.15%). It was found that 63.77% of the viable protoscolices recovered from fertile protoscolices of all animals that were killed were viable. Interms of the different size of fertile cysts from slaughtered sheep, the viability of protoscolices was 57.14%, 82.61% and 26.67% for cyst size <4 cm, 4-8 cm and >8 cm, respectively. In terms of the different size of fertile cysts from slaughtered goats, the viability of protoscolices was 36.36%, 75.0% and 33.33% for cyst size <4 cm, 4-8 cm and >8 cm, respectively. A total of 305 dog faecal samples were examined for the presence of taenid eggs. 66 samples were found positive with the prevalence of 21.63%. High prevalence was observed in stray dogs (34.43%) ascompared to pet/companion dogs (2.45%). Molecular prevalence based on copropcr was conducted, in which the overall prevalence of Echinococcus was found to be 14.09% [stray (22.95%); pet (0.82%)]. Molecular characterization of taenid eggs was carried out on positive faecal samples of dogs. Confirmation of Echinococcus species was ascertained by amplifying the entire Cox1 gene sequence using PCR. Clear amplification was observed for Echinococcus granulosus (440 bp) (Fig 1). For the identification of Echinococcus species from cyst of sheep, goats and humans, a PCR protocol employing the Cox1 primers was used. The amplified products of PCR were electrophoretically separated on agar gels (1%) and analysed in a gel documentation system. Clear amplification was noticed at 440 bp for sheep, goat and human (Fig 2). In the present study, a total of five sequences were submitted to NCBI Gene Bank. The generated Cox1 partial sequences were assigned the accession numbers as: dog (ON427821), goat (ON427822), sheep (ON427823), human1 (OP215294) and human 2 (OP215295). In the present study, The Echinococcus Cox1 sequences of dog, goat, sheep and human showed high homology with sequences from respective Echinococcus species present in the Gene Bank as follows: Echinococcus in dogs as Jammu isolate (ON427821) showed identity with other isolates of Echinococcus [100% with Haryana, India (LC721083); 100% with Iran (MN807919); 99.73% with Turkey (KX874707) and 99.73% with Greece (DQ856467)]. Echinococcusin goats as Jammu isolate (ON427822) showed identity with other isolates of Echinococcus [100% with Meghalaya, India (KR297266); 100% with Portugal (HF947568); 100% with Turkey (MT318688) and 100% with China (MK310277)]. Echinococcus in sheep as Jammu isolate (ON427823) showed identity with other isolates of Echinococcus [100% with Chandigarh, India (JX854028); 100% with West Bengal, India (DQ269946); 100% with Iran (MJ792561) and 100% with France (MJ548753)]. Echinococcus in humans as Jammu isolate (OP215294/OP215295) showed identity with other isolates of Echinococcus [100% with Azerbaijan (KJ540230); 100% with Turkey (MW421883) and 100% with Morocco (EF367291)]. The phylogenetic tree was constructed by the Maximum Likelihood method using the Tamura 3-parameter model based on five sequences generated in the present study with the available database of Cox1 sequences of Echinococcus isolates from India, Turkey, Iran, China, Azerbaijan, Morocco, Greece, Portugal and France (Fig 3). All the Cox1 sequences from dog, goat, sheep and humans were clustering in the same clade, irrespective of their geographical location, representing the cycle of transmission in the Jammu region as E. granulosus G1-G3 complex.
 

 

 

       
The transmission cycle in India is definitely aided by the unsanitary killing of food animals, dogs’ unrestricted access to slaughterhouses, the habit of letting dogs consume food animal offal and other activities (Singh et al., 2012; Mares et al., 2024). However, the completion of domestic cycles of transmission is largely attributed to inadequate meat inspection, incorrect offal disposal at illegally operated abattoirs and a lack of public knowledge on the life cycle and transmission of E. granulosus (Singh et al., 2012). Among different organs examined, the highest infection rate was recorded in liver as 56.94% (sheep = 59.85% and goats = 51.39%), followed by lungs as 31.58% (sheep = 29.93% and goats = 34.72%) and least as mixed infection in liver and lungs as 11.48% (sheep = 10.22% and goats= 13.89%). Several studies (Singh et al., 2014; Azami et al., 2013) reported similar findings. Though, CE development may also occur in other organs and tissues of the body when oncospheres reach the circulatory system (Kebede et al., 2009). For instance, some studies reported the involvement of other organs like the heart, kidney and spleen, etc. It might be due to the reason that the liver collects blood with the oncosphere through the bile duct after blood circulates from the duodenum and if the oncosphere is not filtered in the liver, it might be passed to other organs like the lungs, heart, kidneys and spleen, etc. The reason for harbouring of hydatid cyst in these organs can be related to factors such as physiological and anatomical characteristics of organ, host and strain of parasite (Polat and Atamanalp, 2009). In most cases, standard treatment-basically, deworming the dogs-is provided in exchange for the collection of samples. Furthermore, in addition to reports of infection in domestic ungulates, there have also been reports in wild ungulates, particularly bovids, as well as in primates, leporids and macropod marsupials (Meymerian and Schwabe, 1962). This means that stray dogs have greater access to infected carcasses and the wild intermediate host in the bush (Ajogi et al., 1995). These dogs are also more likely to be fed raw viscera which might be infected due to the lack of knowledge of rural dwellers. The molecular results of the present study were in agreement with Sharma et al., (2013) who examined 32 patients infected with CE in North India. Mitochondrial cytochrome-c oxidase subunit1 (cox1) gene was amplified and sequenced for molecular identification of the isolates. From all the isolates, G3 genotype was isolated from Punjab, Haryana and Himachal Pradesh while G1-G3 genotypes were isolated from the patients of our study area.
       
The G1-G3 complex genotype, which was also isolated during the current study from dogs, sheep, goats and humans, is fully supported by this work since it has zoonotic significance. Comprehensive data on the incidence and prevalence of E.granulosus infections as well as the genetic characteristics of cystic echinococcosis in dogs, humans, sheep and goats in the Jammu region of Jammu and Kashmir were produced by the current study. Based on the current study’s findings, it is clear that activities like allowing dogs to consume food animal offal, allowing them unrestricted access to slaughterhouses and the unsanitary killing of food animals all contribute to the cycle of transmission. On the other hand, the completion of domestic cycles of transmission is largely attributed to inadequate meat inspection, incorrect offal disposal at illegally operated abattoirs and a lack of public knowledge of the life cycle and transmission of E. granulosus.

1. Management of slaughter house hygiene and restriction of dogs to consume food animal offals to control hydatidosis in Jammu region is of utmost importance.
2. Awareness among farmers/government agencies and professionals for control of hydatidosis using rotational anthelmintics and management practices is of prime importance.

The study revealed hydatidosis as predominant infection in dog, human, sheep and goats of Jammu region. Unhygienic slaughter of food animals, unfettered access for dogs to slaughter houses, allowing dogs to ingest food animal offal and other practices contribute to the transmission cycle. The ethanolic extracts of Juglans regia showed significant scolicidal activity against E. granulosus, under in-vitro conditions with reference to the known standard drug “Praziquantel”. All the Cox1 sequences from Dog, goat, sheep and humans were clustering in same clade, irrespective of their geographical location, representing the cycle of transmission in Jammu region as Echinococcus granulosus G1-G3 complex.

The authors wish to express their heartfelt gratitude to the Voice Chancellor, Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (SKUAST-Jammu) and Dean, Division of Veterinary Science to successfully carrying out the present research work.

Authors declare no conflict of interest.