Molecular and epidemiological updates on cystic echinococcosis infecting water buffaloes from Egypt

Aim: Cystic echinococcosis (CE) represents a serious parasitic disease at both animal and public health levels. The majority of reports negated the CE infection in buffaloes from Egypt; however, one study illustrated their infection with G6 genotype (camel strain). The present work contributed to update the epidemiological and molecular knowledge about CE infecting this economically important animal for better understanding of its role in maintaining the Echinococcus life cycle. Materials and Methods: A total of 120 slaughtered water buffaloes at Mansoura abattoir, Dakahlia province, Egypt, were inspected for the existence of hydatid cysts. Cysts location and fertility were examined. Five out of 27 revealed cysts were tested molecularly using both cytochrome C oxidase subunit 1 and nicotinamide adenine dinucleotide hydrogen subunit 1 (nadh1) genes. Results: Low prevalence (4.2%) as well as considerably low fertility rate (14.8%) of buffaloes CE was noted. G1 genotype (common sheep strain) was revealed from the five examined cysts. At the level of nadh1 partial sequences, a globally singleton G1 haplotype was reported. Conclusion: This the first report about the G1 infection in buffaloes from Egypt. This study proposed the minimized role of this animal in echinococcosis transmission. These findings could provide preliminary data for the local control of this disease.


Introduction
Cystic echinococcosis (CE) is an important parasitic infection which caused by the larval stages (hydatid cyst) of the genus Echinococcus (a dog tapeworm) [1]. This parasite has a two-host life cycle in which canines serve as definitive hosts, while the intermediate host role is played by the domestic and wild ungulates [2]. Transmission of infection is initiated by ingestion either of Echinococcus eggs by the intermediate host or the larval stage by the definitive host.
CE constitutes a serious animal health concern in many rural areas of the world altogether with its public health hazards. From the economic perspective, CE causes important economic losses originated from decreased productivity and viscera condemnation in livestock species. In Ismailia city abattoir (a small Egyptian abattoir), the estimated annual loss in livestock due to CE was 36,480 Egyptian pounds which represented by total or partial condemnation of 1216 kg of meat and offal [3].
Buffaloes are widely reared in Egypt. Economically, it considered the second important animal, providing meat, milk, skin, and hides. In Egypt, Dyab et al. [4] did not detect CE infection in buffaloes, while Haridy et al. [5] recorded 6.4% infection rate.
Reports about the genotypes of CE infecting buffaloes are considerably low due to the scarcity number of countries in which buffaloes can accommodate to living. The majority of studies illustrated the susceptibility of this animal species to the G1-3 cluster [7,8] although cattle strain (G5) was reported in India [9,10]. Molecular studies on hydatid cyst isolates from the Egyptian animals and human illustrated the presence of G1, G4, G5, and G6, which considered the most prevalent genotype [11][12][13]. Previously, 2 buffalo samples, obtained from Cairo, were genotyped as G6 (camel strain) [13]. Herein, we examine the slaughtered water buffaloes at Dakahlia province for hydatid cysts to update the CE epidemiology and to clarify if there are genotypes other than G6 infecting water buffaloes from Egypt.

Ethical approval
This study was conducted after getting approval from both Scientific and Animal Ethics Committees of Faculty of Veterinary Medicine, Mansoura University, Egypt.

Samples collection and DNA isolation
A 120 water buffaloes slaughtered at Mansoura Abattoir, Dakahlia Province, Egypt, were examined for the presence of hydatid cysts. Cysts were dissected out and washed with phosphate buffer saline (PBS). Cysts fertility was assessed by examining the hydatid fluid and the germinal layer for protoscolices. The germinal layer, as well as the protoscolices, was harvested. Both were washed 3 times in PBS (pH 7.2) and stored at −20°C until used. Total DNA of protoscolices and/or germinal layers was extracted using the NucleoSpin ® Tissue kit (Macherey-Nagel, Düren, Germany), according to the manufacturer's instructions.
PCR amplification was carried out in 35 µL final mixture containing 2 µL of template DNA, 1 µL (25 µM) of each primer, 0.7 µL (10 mM) deoxynucleotide triphosphate mix, 3.5 µL of Taq buffer (×10), 0.35 µL Taq polymerase (5 Prime Perfect Taq TM ), and 26.45 µL nuclease free water. PCR products were separated on agarose gels (1%) stained with ethidium bromide (Figure-1). Gel bands were cut out, and DNA was purified using QIAquick gel extraction kits (Qiagen, Germany) following the manufacturer's instruction. The purified DNA was commercially sequenced in both directions. Genotypes identification and the reference sequences (Tables-1 and 2) were achieved using GenBank with the BLAST system. Bioedit software was used to align the different sequences, while the phylogenetic analysis was initiated using the neighbor-joining method of the software Mega (version 6) with Kimura 2-parameter model.

Results
Examination of 120 slaughtered water buffaloes in Mansoura Abattoir, Dakahlia province, Egypt, demonstrated the presence of hydatid cysts in 5 carcasses (4.2%). One infected animal harbored 14 sterile cysts in its liver (Figure-2), while the lung is the affected tissue in four animals (two of them had single fertile cyst infection, one had 2 fertile cysts, and the last one had 9 caseated sterile cysts). Collectively, 4 (14.8%) out of 27 collected cysts were fertile.

Genotype identification and sequence analysis
Clear and readable nucleotide sequences were revealed from the 5 examined cysts (four fertile lung cysts and one sterile liver cyst). Alignment of the 5 samples with each other indicated that all of them were belonged to the same haplotype, and they were typed under the E. granulosus G1 genotype (common sheep strain).
At the level of cox1 gene, single nucleotide substitution (C257T) was noted by alignment of this study haplotype and the reference sequence M84661 (the first described G1 by Bowles et al. [14]) (Figure-3). Nevertheless, complete identity was found with a number of G1 isolates from different countries and host species like sheep from Tibet (KJ628364), goats from Iran (KR337824), and human from Mongolia (AB893250).

Phylogenetic analysis
Using the G5 and G6 genotypes from the Egyptian camels and buffaloes, respectively, as an out group, the neighbor-joining phylogenetic trees indicated the clustering of our G1 haplotype within the other G1 haplotypes from variable host species in different geographical regions ( Figures-5 and 6). Despite the nadh1 illustrated the separation of our haplotype in a unique branch within the same G1 clade (Figure-6).

Discussion
CE is an important economic and life-threatening zoonotic disease. In view of its economic importance, we aimed to update the knowledge about CE from water buffaloes slaughtered in Egypt.
Furthermore, lungs appeared to be the predilection site for CE in buffaloes (80% out of the examined animals), coinciding with results from studies conducted in Turkey [8] and Italy [22].
On the other hand, the fertility rate of the harvested cysts was considerably low (14.8%). Our molecular results indicated the homogeneity of those cysts with G1 genotype (common sheep strain). Furthermore, the previous reports discussed the low fertility rate of  hydatid cysts of buffaloes (7.3%) and their relation to the G1 [18,20]. While in Pakistan, where the G3 (buffalo strain) was the prevalent genotype, the fertility rate was high (84.3%) [21]. Therefore, this could be explained by the low adaptation of buffaloes to the common sheep strain which increases the tendency of sterile cysts formation [23]. Consequently, our results assumed the reduced role played by buffaloes in maintaining the Echinococcus life cycle in this geographical area. The analyzed sequences of the 5 hydatid cyst isolates in the present work indicated the infection of the Egyptian buffaloes with the G1 genotype. Previously, 2 samples from the Egyptian buffaloes were characterized as G6 which not described before from buffaloes worldwide [13]. Considering reports from buffalo rearing countries (Table-3), G1 was the widely dispersed genotype in Iran [24]; India [25], Pakistan [26], Italy [27], Turkey [8], and Greece [18]. Although, G3 (buffalo strain) and G5 (cattle strain) were recorded but in few cases [7,21,25,28]. This is the first report about the existence of the G1 genotype in buffaloes from Egypt.
The alignment results of the partial cox1 sections ( Figure-3) showed the complete matching of the G1 isolates from buffaloes in this work with that of sheep (AB921090) and camels (AB921054) from Egypt, which represented phylogenetically by Available at www.veterinaryworld.org/Vol.9/December-2016/4.pdf  their sharing of the same branch. On the contrary, our isolates evoked a singleton G1 haplotype, at the level of nadh1 partial sections ( Figure-4), which not previously reported from Egypt and worldwide. This haplotype was clustered in a separate branch and nearer to the G1 haplotype from the Indian cattle (EF125693) and buffaloes (EF179167), which assuming the introduction of this haplotype to Egypt through the imported cattle and buffaloes meat from India. Moreover, the previous results illustrated the more usefulness of nadh1 than cox1 in studying Echinococcus haplotypes diversity [29], although Bowles and McManus [15] found identical sequences of G2 and G3 for nadh1 gene in the same tested samples with cox1which showed 2 mutational sites between both genotypes (Figure-4).
Little is known about the genotypes of Echinococcus from the Egyptian animals and human. Studies illustrated the wide domination of the G6 (camel strain), which favors the role the camel-dog cycle in the transmission of echinococcosis [11,13,30,31]. Here, we have notice that all the collected samples in these studies were originated from the same area (Cairo and Qalubiya provinces), which considered the main localities in Egypt for importing camels and consuming their meats. Furthermore, G1 genotype (common sheep strain) was described in one camel as well as 4 sheep samples [13], and in one out of 31 human samples [11]. In addition, Abd El Baki et al. [32] stated that 80% of human and camel isolates were belonged to G1. From the zoonotic point of view, it is well known that the majority of human cysts were typed under the G1 and its variants [1,18,33], and thus emphasis the great role of the sheep-dog cycle in echinococcosis transmission for different reasons, like the wide use of dogs with sheep flocks for protection, the commonness of the out-abattoir sheep slaughter in the rural communities specially in the ceremonies like Al-Adhua feast in Muslims countries and the unsatisfactory offal disposal which enable dogs to access easily the hydatid cysts. This assumption is clearly evident in our results which illustrated the commonness of G1 genotype in buffaloes from Dakahkia province, Egypt.

Conclusion
In spite of the low infection and fertility rates of buffalo, hydatid cysts recorded in this study, the mission, even a weak, of this animal for keeping G1 genotype life cycle is still important.