Genetic characterization of hydatid cysts of different intermediate hosts

Cystic echinococcosis (CE) is one of the most widespread parasitic zoonotic diseases in the world. Identification of the cyst genotype would be beneficial for prevention and control of the disease (Ahmed et al., 2017). Four species of Echinococcus were recognized until a few years ago, namely, E. granulosus, E. multilocularis, E. vogeli, and E. oligarthus (Thompson & McManus, 2002).

Recently, molecular characterization based on genome pattern, morphology and host specificity differentiated E. granulosus species into 10 genotypes (G1-G10) (Madawy et al., 2011): E. granulosus sensu stricto (E. granulosus s. s.) (G1, a common sheep strain; G2, a Tasmanian sheep strain; and G3, a buffalo strain), E. equinus (G4, a horse strain), E. ortleppi (G5, a cattle strain) and E. canadensis (G6, a camel strain; G6/G7, cattle strain; G7, a pig strain; G8, a cervid strain; and G10, a Fennoscandian cervid strain); the G9 strain remains unidentified (Nakao et al., 2010, Ohiolei et al., 2019). Phylogenetic analysis of nuclear genes such as internal transcribed spacer 1 (ITS1) (Espinoza et al., 2014) was used to identify E. granulosus genotypes. In North Africa, Eastern Africa and Tunisia, the sheep strain (G1) and camel strain (G6) were found to be the two main genotype groups (Madawy et al., 2011). In Sudan, Omer et al., 2010 reported that the camel strain was the predominant strain and was infective towards humans. In Egypt, most samples collected from camels slaughtered at the Toukh abattoir (Qalyubia Governorate) were identified as E. canadensis (G6), and a few of these strains were identified as E. ortleppi (G5), isolated from newly imported Sudanese camels (Abdel Aziz et al., 2016). Abbas et al., 2016 reported, for the first time, a cattle HC formed by the G1 sheep strain and another cyst formed by the G5 strain (a cattle strain). Additionally, the G1 sheep strain was detected in both Egyptian camel and sheep (Amer et al., 2015). Molecular identification of HCs collected from Egyptian donkeys revealed that these cysts were formed by E. equinus (G4) (Aboelhadid et al., 2013). The present study was undertaken to identify genotypes of HCs DNA isolated from camels, cattle, sheep and donkeys by sequencing of PCR-amplified nuclear ITS1 gene products. Additionally, identification of the genotype of secondary HCs developed in rabbits intra-peritoneally injected with protoscolices (PSCs) of HCs (of camel origin) to detect the presence of any genetic mutation or variation that occurred during parasite transmission between two intermediate hosts.

All animal research reported in the present study were followed animal care and handling were carried out accordance with the institutional guidelines of Cairo University, Egypt.

CE is a cyclo-zoonotic disease that is distributed worldwide (Ahmadi, 2005). The first step in the prevention and clearance of infections from a specific area is identification of the strain(s) responsible for dissemination of infection and determination of the life cycle of the strain (Abdel Aziz et al., 2016). Therefore, in this study, the collected HCs were subjected to molecular characterization. Amplification of ITS1 by PCR showed a DNA band of the same molecular size of 1115 bp for all the isolates. This result was consistent with the results of Madawy et al., 2011 and Harandi et al., 2002, who found that the amplified DNA bands of the ITS1 gene had molecular sizes of 1115 bp in different HCs isolates (cattle and sheep in Egypt and sheep, goat, cattle and camel in Iran, respectively).

The partial nucleotide sequence of the ITS1 gene of the three isolates obtained from HCs camels belonged to E. canadensis (G6, camel strain). This result was consistent with the results of Khalifa et al., 2014 that studied a camel lung HCs in Egypt and identified G6 by gene sequencing of the NADH 1 gene. Abdel Aziz et al., 2016, found that 90.5 % of the HCs isolates collected from the lungs of slaughtered camels at the Toukh abattoir, Qalyubia Governorate, Egypt, were identified as E. canadensis (G6), and 9.5 % was identified as E. ortleppi (G5), which were from newly imported Sudanese camels. Amer et al., 2015, demonstrated the presence of E. canadensis (G6) in 26 out of 28 Egyptian camel HCs, while the 2 remaining cysts belonged to E. granulosus s. s. (G1) and E. ortleppi (G5). Additionally, in Sudan, Omer et al., 2010, recorded the predominance of the camel strain (G6) and the infectivity of this strain towards humans. Moreover, Abushhewa et al., 2010, detected G6 in camels in Libya, and Harandi et al., 2002 and Hosseinzadeh et al., 2012, detected the camel strain in Iranian camels.

The circulation of the camel strain (G6) in camels suggests that specific mechanisms are responsible for the persistence of this strain in this area. This effect is probably due to close relationships between dogs and camels in the study area (Dinkel et al., 2004). In poor communities, the slaughtering process is performed in open spaces, so dogs can be easily fed the viscera of slaughtered animals carrying the HCs strains, which then complete their life cycles and become adults inside the intestine of the dogs (Abdel Aziz et al., 2016).

The HCs isolates of cattle and sheep were identical to E. granulosus s. s. (G1, sheep strain). This result was consistent with the results of Madawy et al., 2011 and Kandil et al., 2016 who detected the sheep strain (G1) in both cattle and sheep in Egypt. Amer et al., 2015, identified that G1 and G6 from sheep in Egypt. Abbas et al., 2016, detected E. granulosus s. s. (G1) and E. ortleppi (G5) from cattle slaughtered at the Mansoura abattoir, Egypt. So, G1 is the most common genotype in grazing animals (cattle and sheep) due to its direct contact with dogs. In Iran, Jamali et al., 2004 and Parsa et al., 2011, identified sheep and cow isolates as sheep strains, and Hosseinzadeh et al., 2012, identified G1 in sheep and G6/7 in cattle. Bardonnet et al., 2003 and Busi et al., 2006, identified the sheep strain (G1) in cattle and sheep in North Algeria and Italy, respectively. In Pakistan, Ali et al., 2015, found that the E. granulosus s. s. (G1) and G6 genotypes were responsible for cattle infections. Also, Barghash et al., 2017, recorded the existence of two main genotypes; G6 (camel strain) followed by G1 (Sheep strain) in the majority of the studied human and animal isolates ( camels, cattle, buffaloes, sheep, goats, pigs and donkeys) from different regions of Egypt. While, Sofi et al., 2019, recorded that G6 genotype of C.E. in buffaloes was the first report in the Northern India. Also, in Nigeria, Ohiolei et al., 2019, recorded that E. canadensis (G6/G7) were the isolate of cattle which showed 99 – 100% identical to previously reported G6/G7 haplotypes across Europe, Asia, North and East Africa.

The three examined isolates from donkey HCs belonged to E. equinus (G4, horse strain). This result was consistent with the results of (Aboelhadid et al., 2013), who found that HC isolates from donkeys in Beni-Suef, Egypt, belonged to E. equinus. In Southern Germany, Blutke et al., 2010, identified the genotypes of HCs isolates from mare lungs to belong to E. equinus.

Donkeys with E. equinus-containing HCs may act as sources of infections for dogs and foxes in Egypt via unhygienic disposal of donkey carcasses near water canals or cultivable land, making the carcasses easily accessible to stray dogs or foxes. Additionally, feeding of donkeys to carnivores in zoos may cause parasite transmission. Both pathways play an important role in the maintenance of the Echinococcus species life cycle, making the carcasses of these infected equines, especially donkeys, major sources of infection for dogs and foxes (Aboelhadid et al., 2013). Therefore, post-mortem examination of donkeys before use as food for wild animals, hygienic disposal of the carcasses of these animals and proper destruction of the infected offal are recommended.

The rabbit secondary HCs isolates belonged to E. canadensis (G6, camel strain). Partial nucleotide sequences of the ITS1 gene of rabbit secondary HCs isolates and camel HCs isolates were aligned and compared with those of the G6 genotype (camel strain) in GenBank. Both the camel HCs and rabbit secondary HCs showed the same sequence identity matrix, indicating the absence of mutation in the rabbit secondary HCs. Only a single nucleotide substitution of adenine to guanine at position 47 (A47G) occurred in the rabbit secondary HCs sequence. M’rad et al., 2010, recorded a single nucleotide substitution (C44T) in one bovine isolate compared to the G3 genotype in GenBank and explained that this substitution represents either a genotypic variant or a distinct strain that resembles the G3 genotype. Therefore, this aspect requires further investigation.

According to the results of our study, the E. granulosus genotype is not mainly host dependent. The same genotype was isolated from different intermediate hosts. In this study, G1 was collected from both cattle and sheep. In addition, G6 exhibited no changes when transmitted from camel to rabbit.