Prevalence and Genotyping of Echinococcus Species from Livestock in Kajiado County, Kenya

Cystic Echinococcosis (CE) is a widespread neglected zoonotic disease and is caused by the larval stage of the dog tapeworm Echinococcus granulosus sensu lato. CE is more frequent in livestock-rearing areas and where people live a nomadic or seminomadic lifestyle such as in Kajiado County, Kenya. There is limited data on CE disease situation in the county of Maasailand; the present study, therefore, reports on the prevalence of CE in cattle, sheep, and goats and their relative importance in CE transmission in Kajiado County. In total, 1,486 livestock (388 cattle, 625 sheep, and 473 goats) slaughtered in two abattoirs were examined for the presence of hydatid cysts in various organs. Cyst isolates were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the NADH dehydrogenase subunit 1 gene (nad1). The overall prevalence of CE was 14.8% (220/1486), while prevalence per livestock species was 15.2% (72/473) in goats, 14.9% (93/625) in sheep, and 14.2% (55/388) in cattle. Out of the 421 cysts isolated, 389 cysts were successfully characterized to be either E. granulosus sensu stricto (s. s.), 356/389 (91.5%), E. canadensis (G6/7), 26/389 (6.7%), or E. ortleppi, 7/389 (1.8%). This record confirms predominance of E. granulosus s. s. in Maasailand and other parts of Kenya, while the importance of E. ortleppi and E. canadensis (G6/7) to the general CE burden in Maasailand might be higher than previously thought. More so, a higher infection pressure for humans by E. granulosus s. s. based on its abundance could be speculated. The study sheds significant light on CE situation in livestock in the nomadic/seminomadic society of the Maasai in Kajiado County and provides good bases to investigate human CE in the area.


Introduction
Cystic Echinococcosis is caused by the larval stage of the dog tapeworm Echinococcus granulosus sensu lato (s. l.) and is currently recognized by World Health Organization (WHO) as a neglected tropical disease [1]. CE is a common zoonotic disease of great public health significance globally due to its associated economic losses [2]. Approximately US$ 3 billion are lost annually on treatment of CE in humans and losses incurred due to the condemnation of infected organs in livestock [3]. Dogs and to a lesser extent other canids and felids are the primary definitive hosts of Echinococcus species, with herbivores acting as the intermediate hosts and the humans as aberrant intermediate hosts. The outcome of the infection in livestock and human is cyst development in the liver, lungs, or other organs [4]. E. granulosus s. l. consists of at least five species, namely, E. granulosus sensu stricto (s. s.), E. equinus, E. ortleppi, E. canadensis (G6-G10), and E. felidis [5,6].
East Africa and Kenya, in particular, have long been known to be of the world's largest foci of CE in humans [7][8][9]. Previous data from Kenya have centered their focus on CE situations in Turkana and Maasailand [8]. Data from other endemic areas are available but only sparingly. Previous CE studies in Kenya, thus far, indicate the presence of all five E. granulosus s. l. and the recently discovered Gomo genotype [10][11][12][13][14][15]. To appreciate the CE situation in the whole of Kenya, epidemiological data from all endemic localities including Kajiado County is required. The only available data from this area is nearly three decades old and did not report Echinococcus spp. in livestock [16]. Furthermore, the recent study examined livestock originating mainly from Bissil area  (Kajiado South) [10]. Therefore, this study focused on the two main slaughterhouses receiving livestock from the wider scope of the Kajiado County. We report here the prevalence of CE in cattle, sheep, and goats and the Echinococcus spp. causing CE in Kajiado County. Findings from the study will improve our knowledge of CE in this county and establish the relative contribution of each livestock species in the distribution and transmission of CE.  Figure 1). A total of 1486 carcasses of livestock were inspected for the presence of hydatid cysts in all organs (lungs, liver, heart, spleen, as well as the kidneys) of the pleural and abdominal cavities. Visual inspection, palpation, and incision were done for all of the organs for the presence and cyst distribution. The lesions were carefully excised from all infected organs. Individual cysts were identified as those that had a continuous cyst wall while multiple cysts had a visibly separate cyst wall even for the calcified cysts. The isolated cysts were packed in clean polythene bags placed in cooler boxes and transported to the parasitology laboratory of the Kenya Medical Research Institute, for examination and further analysis. Cysts were dissected using a sterile scalpel blade and each cyst material was fixed and preserved in 70% Ethanol in individual tubes. The contents of the cysts were examined microscopically for the presence of protoscoleces (PS). Cysts were classified as fertile (with protoscoleces) sterile (fluid-filled without protoscoleces), degenerated (collapsed cyst walls with caseated protoscoleces and soft cheesy debris without calcification), and calcified (hard solid appearance of the ectocyst). All the cysts from the same organ were examined individually to confirm mixed infections.

DNA Extraction.
DNA was obtained from cyst material and protoscoleces by lysing in 0.02 M NaOH at 99 ∘ C for 10 minutes. In a few instances where the above process failed to yield adequate DNA, genomic DNA was extracted using DNeasy Blood & Tissue Kit5 (Qiagen, Hilden, Germany). The germinal layers or cyst walls were cut into small pieces and lysed in ATL lysis buffer (180 l) and proteinase K (20 l), and DNA was subsequently extracted using the manufacturer's protocol. Extracted DNA was eluted in 50 L of elution buffer.

Polymerase Chain Reaction and Restriction Fragment
Length Polymorphism (PCR-RFLP). Two nested PCR assays targeting part or the entire NADH dehydrogenase subunit 1 gene (nad1) were used for genotyping of cyst materials. The first nested PCR (entire nad1 gene) was performed as described by Hüttner and Nakao [17]. The cyst materials negative using the first PCR assay were genotyped using a second nested PCR as described by Mulinge and Magambo [18], which amplifies part of the nad1 gene (545-552 bp). In both PCR assays, the reaction mixture contained 2 l of the DNA, 1 × DreamTaq Green Buffer (20 mM Tris-HCl (pH 8.0), 1 mM DTT, 0.1 mM EDTA, 100 mM KCl, 0  (Table 1). Though CE infection in goat was the highest, infections with more than one cyst were higher in cattle and sheep than in goat ( Table 2). Majority of cysts of cattle origin were sterile (41.9%), while most of the calcified cysts were found in sheep (48.5%) and goats (52.2%) ( Tables 3 and 4). On average, sheep had the greatest number   (7) were not fertile (Table 4).
In addition to single infections, this study reports several cases of mixed infections. There were three cases of mixed infections in cattle and there was one case of all three Echinococcus spp. (E. granulosus s. s., E. ortleppi, and E. canadensis (G6/7) as well as two instances of E. granulosus s. s. and E. ortleppi and E. ortleppi and E. canadensis (G6/7). In goats, there were three cases of mixed infections, all with E. granulosus s. s. and E. canadensis (G6/7), and, in sheep, only one case of E. granulosus s. s. and E. canadensis (G6/7) mixed infection was observed (Table 5).

Discussion
This study reports the prevalence of cystic echinococcosis (CE) and Echinococcus spp. in cattle, goats, and sheep in Kajiado County, Kenya. The prevalence reported is within the range known of Maasailand from older accounts, such as in the works of Macpherson [16]  The liver was the most affected organ just as was known before from Maasailand [19]. The predilection site of E. granulosus s. l. is not fully understood and some studies [16,20,22] indicated the lungs to be the most affected. Cysts in the liver or the lungs could be either fertile containing protoscoleces/daughter cysts or nonfertile. The nonfertile cysts can further be divided into calcified, degenerated, or sterile. These nonfertile cysts are noninfectious and, therefore, have no epidemiological significance in CE transmission to definitive hosts. In this study majority of the cysts from livestock were nonfertile, and a recent survey reported 80% of cysts from sheep in Turkana being calcified (Zeyhle unpublished data). This observation is not clearly understood, because regular deworming of ruminants is less likely to have a significant effect on the calcification of cysts. Previous studies have shown that long-term treatment with high doses of anthelmintic drugs is required to arrest cyst development [23,24]. Sheep in which most fertile cysts were isolated in the present study would be more important in the transmission and maintenance of CE in Maasailand. The cysts fertility rates reported indicate the need for control measures such as health education, regular deworming of dogs, dog population control, good slaughter hygiene, and proper disposal of slaughter offal to avert transmission.
Majority of the cysts in this study were E. granulosus s. s. which confirms its predominance observed almost a decade ago in Maasailand [10] and in Kenya at large [11,13,15,25,26]. The high fertility rates of E. granulosus s. s. cysts in sheep indicate that they are important intermediate host of this taxon in this area. Sheep are also the most common home-slaughtered livestock species in Maasailand and that may enhance transmission of E. granulosus s. s. However, both cattle and goats may also play a role in transmission of E. granulosus s. s. based on the fertility rate in this study. Although goats are considered important intermediate host of E. canadensis (G6/7) in absence of camels, none of the cysts belonging to this taxon were fertile in this study [10,27]. Isolation of E. ortleppi from all three livestock species reveals a wider host range of the parasite, aspect that is less understood. Generally, E. ortleppi is a rare species even in cattle who are the principal intermediate hosts, possibly due to the fact that cattle are rarely slaughtered at home, and therefore dogs have less access to slaughter offal from cattle [10]. However, in a recent development, due to poor disposal of condemned viscera in poorly managed slaughter facilities in urban centres, dogs have readily access to slaughter offal and this might be a reason for the increased cases of E. ortleppi in our study [18]. Elsewhere in Brazil home slaughter of cattle is believed to be a factor that facilitates the recent rise of E. ortleppi prevalence in Brazil [28].

Conclusion
Cystic echinococcosis continues to persist in Maasailand with E. granulosus s. s. being the dominant species. The high fertility rate of cysts in sheep and its regular homeslaughter make it the most important intermediate host in the transmission of CE in Kajiado County of Kenya. Echinococcus ortleppi (G5) and E. canadensis (G6/7) may be important CE agents in Maasailand more than previously thought.

Data Availability
The prevalence and genotyping data used to support the findings of this study are included in the article.

Disclosure
The proceedings from this study were presented in the Kenya Veterinary Association 53rd Annual Scientific Conference and World Veterinary Day Celebrations and the abstract was published in the Conference's Book of Abstract.

Conflicts of Interest
The authors declare no conflicts of interest.