Occurrence and phylogenetic description of cystic echinococcosis isolate from Egyptian camel (Camelus dromedarius)

A cross sectional study was conducted in Aswan city, which is located at a latitude of 24° 5′ 20.18″ N and a longitude of 32° 53′ 59.39″ E. Aswan is famously for its beautiful Nile Valley scenery, important archaeological sites and peaceful atmosphere. The climate is warm throughout the year, making it an ideal winter destination. Furthermore, agriculture is the main source of employment opportunities in the province, with approximately 29 % of the province’s population working in agriculture.

The sample size was calculated according to the method previously described by Charan (2013). The parameters of the formula (n=Z²×P (1-p) /d²) were as follows: Z = Is standard normal variate at 5 % type 1 error (P<0.05), it is 1.96, P= the expected prevalence of HC based on previous studies was 9.7 % (Zienab 2021), and d = is ±5 % desired level of precision. The formula was n = (1.96)² × 0.097 × (1 – 0.097)/0.05. The required sample size n was determined to be 110 camels. To determine the prevalence of hydatid cyst infection in the camels of the southern region of Egypt, the visceral organs (liver, lungs, kidneys, and spleen) of 110 slaughtered camels were inspected over the course of one year from June, 2018 to May, 2019 in Daraw Abattoir, Aswan, Egypt. During the sampling gender, age, and the seasonal dynamics were recorded.

All organs or tissues containing HCs were collected and all HCs found were carefully removed and separately collected, kept in sterile saline solution, 70 % ethanol and transported within 2 hours to the Parasitology laboratory, Faculty of Veterinary Medicine, South Valley University in ice box for further cyst characterization to assess their status and for genetic strain typing of E. granulosus sensu lato (s.l). Data related to the cyst size, distribution, seasonal dynamics, viability and fertility was recorded. A total of 35 HCs were randomly selected from lungs and livers and was measured and categorized as small (< 4 cm in diameter), medium (4 – 8 cm in diameter) and large (>8 cm) (Oostburg et al. 2000). The cyst fertility was assessed based on the presence or absence of brood capsules containing protoscolices in hydatid fluid examined microscopically (Kebede et al., 2009). Additionally, fertile cysts were subjected to viability test. A drop of sediment containing the protoscoleces was placed on the microscopic glass slide and a drop of 0.1 % eosin solution was added and covered with cover slip and then examined under a high power microscopy (40x) after 5 min. with the principle that viable protoscoleces should completely or partially exclude the dye, while the dead ones take it up (Daryani et al., 2007). During these investigations, the biosecurity rules were strictly observed and all examined biological material and the remains of hydatid cysts were incinerated.

Five samples of hydatid fluids were collected from the lung cysts of the screened hosts (camels and cattle), approximately 25ml of the collected fluids (HCF) was centrifuged at 15000 rpm at 4 ºC for 30 min and the supernatant was analysed for various biochemical parameters. The flame photometry method was utilized to measure the amount of Sodium and Potassium, copper and magnesium by the spectrophotometry method. Creatinine, Calcium and Protein were analyzed by the colorimetric method. The measurements of Glucose, cholesterol, triglycerides, and urea was made by enzymatic methods (SYNCHRON CX 7 PRO, USA 2009). These parameters were estimated by a commercially available diagnostic Kits from Sigma, Germany. Each electrolyte and biochemical profile was prepared and quantified according to the manufacturer’s instructions.

Statistical significance differences were assessed with a Chisquare using a statistical package program (Sigma Plot version 11.0). Statistical significance was considered with P values < 0.05.

DNA was successfully extracted from two fertile HCs out of fourteen cysts using QIAamp DNA Mini Kit (Qiagen, Germany, Catalogue no.51304,) according to the manufacturer’s recommendation. DNA was stored at -20 °C until being used for DNA amplification. For all DNA extracts, PCR were conducted on the gene encoding cytochrome c oxidase subunit 1 (cox1) and for the gene encoding NADH dehydrogenase subunit 1 (nad1). Fragments of mitochondrial genes amplified with specific primers that previously described as JB3 primer (5´-TTTTTTGGGCATCCTGAGGT-TTAT-3´) and JB4.5 (5´-TAAAGAAAGAACATAATG AAAATG-3´) for cox1 and JB11 (5´-AGATTCGTAAGGGGCCTAATA-3´) and JB12 (5´-ACCACTAACTAATTCACTTTC-3´) for nad1, as forward and reverse primers respectively (Aboelhadid et al., 2013) . PCR products were visualized using electrophoresis with 1.5 % Agarose gel in TAE buffer and stained with 0.5 μg/ml ethidium bromide (cat. no. SM0243). A 100-bp molecular ladder was used as DNA size marker in each gel for estimating the size of the bands. Gels were observed and photographed using a gel documentation system and the data was analyzed through computer software. The obtained DNA sequences were subsequently subjected to BLASTn (www. ncbi. nlm.nih.gov/BLAST/) analysis to check for similarity of the sequence against the GenBank database and compared with verified sequences of E. granulosus strains available in GenBank. Sequences with higher identity were downloaded from GenBank for phylogenesis and Taenia saginata (GenBank accession number MK644934) was used as an outgroup species (Table 1). Sequences were aligned using the Muscle alignment tool in MEGA X software (https://www. megasoftware.net/) as per Kumar et al. (2018). A phylogenetic tree was constructed using the maximum likelihood method and the Tamura Nei model (Tamura & Nei, 1993). The sequences analyzed in the present study were finally deposited in GenBank. Furthermore, reference sequences were compiled from previous studies (Table 1), with Taenia saginata as an outgroup.

All applicable institutional, national and international guidelines for the care and use of animals were followed regarding the National Research Ethics Committee of the South Valley University and Veterinary authorities in South Valley University, Egypt.

Echinococcus granulosus (s.l.), one of the smallest cestodes, displays a global public health and veterinary interest because its metacestode causes the life threatening cyclo-zoonotic disease (Eckert & Deplazes, 2004).

The current survey declared that the overall prevalence of HC was 10 % among slaughtered camels. The current findings were nearly close to the previous reports carried out in Assiut, Egypt (9 %) (Khalifa et al., 2014). Other reports in several Egyptian governorates reported relatively higher prevalence rates among slaughtered camels e.g. 16.25 % in Qalyubia (Mahmoud, 2012) and 39.5 % Beni-Suef (El-Dakhly et al., 2019). However, the infection rates of 2.5 %, 7.7 %, 5.6 % and 5 % and 8.32 % has been reported for camel hydatidosis from Mansura city (Haridy et al., 2006), Assiut and Aswan (Dyab et al., 2005), Giza (El-Dakhly et al., 2019) and Aswan (Omar et al., 2013 & Dyab et al., 2018), respectively. The infection rate of 59 %, 29.7 % 32.8 %, 30.1 % and 61.4 % has been reported from Sudan (Omer et al., 2010; Ibrahim et al., 2011), Saudi Arabia (Ibrahim, 2010), Mauritania, and Kenya (Njoroge et al., 2002), respectively. Additionally the present finding was also greater than worldwide reports done in Libya (3.6 %) and Tunisia (6.5 %), respectively (Kassem & Gdoura, 2006; Lahmar et al., 2004). This variation in prevalence could be due to several factors including differences in environmental conditions that are conducive for the perpetuation of the parasite, social activities and culture, approach towards dogs and difference in husbandry and hygienic system. Other reasons like the personal behavior of workers about the elimination of infected offal in abattoirs and the nature of the pasture may contribute to this variation (Salih et al., 2011; Abdelbaset et al., 2021).

The present study revealed that the prevalence of HCs varies greatly among screened age groups; those with age of 3 years and over (>3) were highly infected (12.3 %) with cysts as compared to the age group of 1 – 2 years old (6.7 %). These findings were in line with the previous studies (Azlaf & Dakkak, 2006; Regassa et al., 2010). This could be mainly due to the fact that aged animals have longer exposure time to eggs of E. granulosus in addition to weaker immunity to combat against the infection (Regassa et al., 2014). Furthermore, the prevalence of hydatid cysts was recorded slightly higher in male animals (10.1 %) as compared to females (9.1 %), in contrast to the results observed by previous reports in Zambia and eastern Ethiopia (Banda et al., 2013; Lemma et al., 2014). This might be due to few number of tested female. Differences in livestock management for males and females in Egyptian community could be another issue.

Fig. 5

The current figures indicated that the rate of infection in lungs (72.7 %) was higher than that of the liver (27.3 %). Our observation was in accordance with those noticed in camels in several African countries, such as Mauritania (Bardonnet et al., 2003), Sudan (Ibrahim et al., 2011), and Ethiopia (Salih et al., 2011). This might be due to the fact that camels are slaughtered at an older age, during which period the liver capillaries are dilated and most oncospheres pass directly to the lungs; additionally, it is possible for the Echinococcus oncosphere to enter the lymphatic circulation and be carried via the thoracic duct to the heart and lungs in such a way that the lung may be infected before or instead of the liver (Arene, 1985).

The fertility of cysts is an important factor affecting the spread of E. granulosus. The current finding showed that the fertility rate among the screened organs was found higher in lungs (54.3 %) compared to liver which was Zero. It has been stated that the fairly softer consistency of lung tissue allows the easier development of the cyst (Himonas et al., 1987). Our result was also in consistent with earlier research which demonstrated that the fertility of the cyst from lungs was 69.7 % as compared to liver in slaughtered camel in five different abattoirs from Iran (Ahmadi, 2005). The peak prevalence and high fertility rate of lung cyst over hepatic one of camel specify the importance of viscera as a potential source of infection for dogs. Additionally, the higher yield of calcified cysts in the liver could be attributed to the relatively higher reticuloendothelial cells and abundant connective tissue reaction of the organ. Similarly, the high percentage of small cysts may be due to the immunological response of the host which might prevent their development (Lahmar et al., 1999). In the present study, viable protoscoleces in lung cysts were higher than the previous report in Saudi Arabia (Mohamed, 2010). Moreover, previous report on the viability of cysts, recovered from the camel lungs, have shown a higher viability in comparison with the results of the present study (Lahmar et al., 2013). The difference in viability of protoscoleces could be due to the numerous factors, such as temperature and humidity, and genetic variations of E. granulosus (Diker et al., 2007).

The current data revealed a vital seasonal fluctuations for hydatid cyst in camel among the various seasons. The peak infection rate was observed in winter and summer seasons. Likewise, several previous reports found a seasonal variation in infection prevalence, with the highest trend of occurrence in winter (Daryani et al., 2007; Almalki et al., 2017). The seasonal variation in infection prevalence maybe related to the differences in the environmental conditions that are responsible for the dissemination of the parasite, the availability of infected final hosts, and the nature of the pasture among seasons (Ernest et al., 2009).

In this study, the existence of E. granulosus can be explained by different hypotheses. One possibility is that some uncontrollable slaughtering (i.e. done outside the slaughterhouse without veterinary supervision), without removing all the infected organs. Second, infected dogs can contaminate pastures. Finally, the dogs that die near the lakes, rivers or on roads and their bodies were not collected and processed properly were considered as a main source of infection. Research on camel-dog cycle will assess the persistence of E. granulosus in Egypt.

The chemical composition of hydatid cyst fluid (HCF) plays a significant role in the immunology, metabolism, physiology and existence of the cyst (Muhsin et al., 2015). Mover, the biochemical analysis of HCF collected from different intermediate hosts can provide evidence as an additional diagnostic tool to distinguish different circulating isolates in the area, and can help to identify common isolates that circulate in different hosts (Muhsin et al., 2015). The current study declared a quantitative variation in the biochemical profiles of hydatid fluids of camels from cattle, proposing the existence of camels strains of E. granulosus in the study area. Similar trend was reported in Iran, that found the higher values of magnesium, total protein, glucose, copper and creatinine in the camel hydatid cyst fluids as compared to cattle (Radfar & Iranyar, 2004). The level of urea was higher in the HCF collected from camels as compared with cattle. In contrast, a study in Iran has shown the same value of urea in HCF of both animals (Radfar & Iranyar, 2004). The values of calcium, triglyceride and sodium in camel HCF was lower in comparison with cattle HCF. On the contrary, compared to Iranian camels, these values were found to be higher for cattle cysts (Radfar &Iranyar, 2004). This variability could be attributed to the differences in metabolic activities, cyst status (degree of growth, localization/ fertility) and isolates of E. granulosus sensu lato prevailing.

The current sequence analysis of two camel hydatid cysts revealed the presence of one genotype of E. granulosus, pig strain (G7genotype) in camels for the first time in Egypt. This genotype has been previously described in European beaver, pig, wild boar, cattle, goats and sheep worldwide, notably in Poland, Slovakia, Ukraine, Spain and Greece (Kedra et al., 1999, Kedra et al., 2000; Turcekova et al., 2003; Mwambete et al., 2004; Roinioti et al., 2016).

Globally, several studies have been conducted on camel isolates from different countries, mainly in Africa. The previous report carried out in Mauritania found that G6 genotype in all twenty camel isolates (Bardonnet et al., 2002). In another DNA sequence-based revision in North Africa, all camel isolates belonged to G6 genotype (Bart et al., 2004). Earlier investigation, revealed that all camel isolates belonged to G1 and G6 genotypes in Libya and Algeria, respectively (Tashani et al., 2002; Bardonnet et al., 2003). Furthermore, E. granulosus s.s. (G1- G3 complex) is also the principal genotype in humans, cattle, sheep, camel and goats in various European and Latin American countries (Beato et al., 2010; Piccoli et al., 2013).

In Egypt, previous studies upon nuclear (Khalifa et al., 2014) and mitochondrial genes (Abdel-Aaty et al., 2012) indicated the presence of G6 genotype as the only predominant genotype in slaughtered camels in Qalyubia and Sohag Governorate, respectively. Another investigation demonstrated the presence of E. canadensis (G6) in 26 out of 28 Egyptian camel HCs (Amer et al., 2015), while the 2 remaining cysts belonged to E. granulosus s. s. (G1) and E. ortleppi (G5). Furthermore, the sequencing analysis of 40 Egyptian camels samples revealed the predominance of G6 genotype (camel strain) in camel isolates (Alam-Eldin et al., 2015) and more recent study revealed that G6 genotype was the predominant strain in Egyptian camels (Mousa et al., 2020).

The association with distinct host species, largely separate geographical distribution and limited rate of cross-fertilization are the main factors that have limited the gene flow between genotypic groups. G6 is commonly involved in a cycle between goats/camels–dogs and G7 mainly pigs–dogs, these two also share some overlap in their life cycles as both can infect the same intermediate hosts. Furthermore, G6/G7 share the same final host (dog) cross-fertilization has apparently been frequent enough to guarantee that G6/G7 have not diverged (Laurimäe et al., 2018).

In conclusion, the current findings report for the first time the dominance of E. granulosus (G7, pig strain) in Egyptian camels. The existence of this strain and especially the fact that the zoonotic concern, makes E. granulosus a major public health disquiet in Egypt. Additionally, the present study indicates that camel can be considered a suitable new host for the G7 genotype. For this reason, the role of camels in maintaining the transmission cycle of different E. granulosus genotypes warrants more investigations, with further epidemiological studies on human CE and the strains of the parasite infecting humans.