Genetic assemblage of Sarcocystis spp. in Malaysian snakes

Background Sarcocystis species are protozoan parasites with a wide host range including snakes. Although there were several reports of Sarcocytis species in snakes, their distribution and prevalence are still not fully explored. Methods In this study, fecal specimens of several snake species in Malaysia were examined for the presence of Sarcocystis by PCR of 18S rDNA sequence. Microscopy examination of the fecal specimens for sporocysts was not carried as it was difficult to determine the species of the infecting Sarcocystis. Results Of the 28 snake fecal specimens, 7 were positive by PCR. BLASTn and phylogenetic analyses of the amplified 18S rDNA sequences revealed the snakes were infected with either S. nesbitti, S. singaporensis, S. zuoi or undefined Sarcocystis species. Conclusion This study is the first to report Sarcocystis infection in a cobra, and S. nesbitti in a reticulated python.


Background
Sarcocystis is an intracellular protozoan belonging to the family Sarcocystidae and is closely related to medically important protozoa, such as Toxoplasma gondii and Neospora caninum. Its life cycle involves intermediate and definitive hosts and is based on predator-prey relationships [1]. Asexual stages develop in intermediate hosts, ultimately resulting in the formation of intramuscular cysts (Sarcocysts). Following ingestion of tissue containing sarcocysts by a definitive host, the sexual stages of the life cycle are initiated in the intestine, producing oocysts that are excreted in feces [1]. Sarcocystosis is a zoonotic disease that is found mostly in tropical and sub-tropical countries, which usually affects wild and domestic animals, such as cows and pigs [2]. Notably, humans may serve as definitive hosts for Sarcocystis hominis and Sarcocystis suihominis after consuming raw, infected meat from cattle and pigs, respectively. However, humans can also be intermediate hosts for some Sarcocystis species of unknown origin. In such cases, humans can be infected through food or drinks that are contaminated with feces containing sporocyst from infected animals. In fact, a study identified sarcocystosis antibodies in 20% of Malaysians [3], and tissue autopsies showed 21 out of 100 individuals contained sarcocysts [4]. In December 2011, the surveillance program of the International Society of Travel Medicine and Centers for Disease Control and Prevention (CDC) reported an outbreak of acute muscular sarcocystosis, which involved 32 travellers to Tioman Island off the coast of peninsular Malaysia [5]; however, the Sarcocystis species involved was not identified.
Muscular sarcocystosis can be diagnosed through microscopic examination of histologic sections stained with hematoxylin and eosin. Nevertheless, technical variability in staining and tissue sectioning procedures might be anticipated [6]. Additionally, the ultrastructure of many species is inadequately described. Thus, definitive diagnosis of sarcocystosis requires identification of sporocysts in feces. However, the sporocysts of different species are similar in size and shape, making species identification almost impossible by microscopy. Therefore, sequencing of the small subunit ribosomal RNA (18S rRNA) gene was introduced as an ideal means for species-specific detection [7]. In fact, this gene contains hypervariable regions interspersed within highly conserved DNA sequences, making it ideal for differentiation between species. For this reason, the 18S rRNA gene has been frequently used in phylogenetic analyses of species within the Apicomplexa [8][9][10][11][12][13][14]. Moreover, the large number of 18S rRNA gene sequences available for different Sarcocystis species should enable construction of an informative phylogenetic tree of the Sarcocystidae. Indeed, the utility of 18S rRNA gene sequencing for differentiation of Sarcocystis species and for phylogenetic studies has been demonstrated by several researchers [15][16][17][18].
SP Kan and R Pathmanathan [19] have highlighted five well-defined (complete life cycle) Sarcocystis species in Malaysia. Notably, it was identified that three out of five of these Sarcocystis species (i.e., S. singaporensis, S. villivillosus, and S. zamani) circulate between pythons (definitive hosts) and rats (intermediate hosts) [20], indicating the high diversity of Sarcocystis species infecting snakes. During their research in Thailand, T Jakel, Y Khoprasert, I Sorger, D Kliemt, V Seehabutr, K Suasa-ard and S Hongnark [21] confirmed that Broghammerus/ Python reticulatus [22] served as a suitable definitive host for S. singaporensis and S. zamani. Furthermore, in other countries, P Daszak and A Cunningham [23] as well as Šlapeta and his collaborators (2003) discovered Sarcocystis species parasitizing a bullsnake (Pituophis melanoleucus sayi) and African tree vipers (Atheris spp. and Bitis spp.), respectively.
Here, we have performed phylogenetic analyses based on sequencing of the 18S rRNA gene in order to determine evolutionary relationships between Sarcocystis species isolated from Malaysian snakes.

Sample processing and examination
The preserved samples were examined under a microscope. Fecal samples were concentrated by formalinether concentration and filtered to remove the course debris followed by microscopic examination.

DNA isolation
Total DNA from fecal samples was isolated using the PowerSoil® DNA Isolation Kit (MO BIO Laboratories) according to the manufacturer's protocol. Briefly, 0.25 g of the fecal sample was lysed by adding the solution containing SDS (Solution C1). The non-DNA organic and inorganic materials in the lysed sample were removed by adding Solution C2 and Solution C3. Following centrifugation, the supernatant was mixed with high salt solution (Solution C4) and loaded into the Spin Filter to allow binding of DNA to the silica membrane. The bound DNA was cleaned with ethanol wash solution (Solution C5) and followed by elution of the DNA by using 30 μl elution buffer (Solution 6).

Cloning and sequencing
Amplicons were cloned using pGEM®-T Vector System (Promega) and positive clones from each amplicon were sequenced in both directions by using M13 universal primers. Sequence analysis was performed using the BioEdit software.

Nucleotide search and phylogenetic analysis
Each 18S rRNA gene sequence was searched for match in the GenBank nucleotide database using the Basic Local Alignment Search Tool (BLASTn). Two to three sequences were chosen to represent each amplicon in phylogenetic analysis. The sequences were then compared and aligned with 18S rRNA gene sequences of Sarcocystis species available in GenBank. Multiple sequence alignment was carried out using ClustalW. A phylogenetic tree was constructed based on the 18S rRNA gene sequences by Neighbor-Joining method (bootstrap = 1000) available in MEGA4 [24]. The 18S rRNA gene sequence of Eimeria tennella was used as outgroup.

Result
In this study, 28 snake fecal samples were obtained but no oocyst/sarcocyst was found in the stools using microscopy. Seven snakes (25%) were found to be positive for Sarcocystis 18S rRNA by PCR. The 7 infected snakes included 4 reticulated pythons from Langkawi Wildlife Park; 1 Malayan brown pit viper from Pangkor; 1 Monocled cobra from Pangkor and 1 Malayan keeled rat snake from Kuala Kerai (Table 1). Amplicons from the PCR reaction were cloned into pGEM®-T and sequenced. Twenty 18S rRNA sequences were obtained and each sequence was searched for match in the GenBank database using BLASTn. Once the species was determined, a phylogenetic tree was constructed based on these 20 sequences plus 34 other Sarcocystis 18S rRNA sequences ( Figure 1). The phylogenetic tree shows two major clades (A, B) of Sarcocystis. Clade A is divided into two subclades (I, II). Subclade I consisted of species that infect ruminants. Subclade II is further divided into two groups, one consisting of species whose hosts are animals such as birds, opossum, treeshrew and lizard. The other group consisted of Sarcocystis species in which snakes (python, ratsnake, viper) are the definitive hosts, such as S. singaporensis and S. zuoi. Interestingly, clade B contains only one group. The members of this group, S. nesbittii and S. atheridis, use snakes (python, cobra, viper) as definitive hosts. The Sarcocystis detected in the snake fecal samples of our study are distributed in both clades A and B.
(See figure on previous page.) Figure 1 Phylogenetic tree based on 18S rRNA sequences of Sarcocystis species. The tree is constructed using the Neighbour Joining method. The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (1000 replicates) are shown next to the branches [27]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The phylogenetic analysis was conducted using MEGA4 [24].

Discussion
This is the first report identifying infection of a monocled cobra (N. kaouthia) with Sarcocystis species. In addition, phylogenetic analysis based on sequencing of the 18S rRNA gene indicated that the Sarcocystis species isolated from the monocled cobra was most closely related to S. nesbitti, which was first discovered by [25] in rhesus monkey muscle. Mandour's work had been supported years later with the proof of light and transmission electron micrograph [26]. In 2011, Tian and his collaborators hypothesized that a snake might serve as the definitive host for S. nesbitti based on phylogenetic analyses [17]. This was due to the fact that S. nesbitti closely resembled certain Sarcocystis species that cycle between rodent intermediates and snakes (e.g., African tree vipers) [8]. Moreover, in our study, S. nesbitti was also found to infect reticulated pythons from Langkawi. Our findings have thus confirmed that snakes are likely to be the definitive host for S. nesbitti. Four infected reticulated pythons were examined in this study: two with S. singaporensis, one with S. nesbitti, and the last with an unknown Sarcocystis species. Similarly in other studies, boid snake (Broghammerus/Python reticulatus) has shown to be the natural definitive host of S. singaporensis. S. singaporensis develops sexually in the intestine of the snake to produce sporocysts, which are released in the faces [20]. Suitable intermediate hosts for S. singaporensis include all of Rattus and Bandicota species as well as Nesokia indica [20,[28][29][30]. Furthermore, S. singaporensis is endemic in Southeast Asia [31][32][33][34], and considered to be highly pathogenic. In fact, it can be lethal due to extensive development of schizonts in rat endothelial cells [28]. As a result, it could potentially be used as a biological agent for controlling wild rodent populations in non-native environments [35].
S. zuoi was detected in Malayan keeled rat snake. Indeed, S. zuoi is commonly found in Norway rats (Rattus norvegicus), and transmission experiments carried out in China have indicated that the king rat snake is the definitive host of S. zuoi [16].
So far, phylogenetic relationships among the majority of analyzed Sarcocystis species have suggested their coevolution with final rather than intermediate hosts [8].
Our results are consistent with this notion because all of the Sarcocystis species that we have isolated from snakes are related to S. nesbitti, S. singaporensis, and S. zuoi, which are Sarcocystis species using snakes as definitive hosts.

Conclusion
We have demonstrated a high (25%) prevalence of Sarcocystis infection in snake populations in peninsular Malaysia. Taken together, we have identified snakes infected by S. nesbitti, S. singaporensis, S. zuoi, and some undefined species. These findings contribute to our phylogenetic understanding of the Sarcocystidae and provide valuable information to build upon for future research.