Morphological and molecular detection of Blastocystis in wildlife from Tioman Island, Malaysia.

Blastocystis infection is widely reported in wildlife, livestocks and in non-human primates however, occurrence in Malaysian wildlife is scarce. A wildlife survey on Tioman Island captured six water monitor lizard (Varanus salvator), four mouse-deer (Tragulus sp.) and one Malayan porcupine (Hystrix brachyura) based on convenience sampling. Intestinal contents from each animal were subjected to in vitro cultivation method using Jones medium supplemented with 10% horse serum. Low prevalence of infections was detected with only 1/6 (16.7%) water monitor lizard and 1/4 (25%) mouse-deer infected. The vacuolated form was the most common cell form found in both cultures with similar morphology to B. hominis. However, the monitor lizard isolate propagated well in the laboratory for several months using Jones medium while mouse-deer isolate could not be maintained for more than a week. The reptilian isolates grew optimally at a lower temperature of 24ºC compared to 37ºC for the mouse-deer isolate. Using the DNA barcoding method, both isolates were confirmed to be Blastocystis sp. Sequence obtained from a monitor lizard isolate has 94% sequence identity to B. lapemi, an isolate recovered from a reptile sea-snake whereas a mouse-deer isolate has 99% sequence identitical to B. hominis HJ01-7. The phylogenetic tree revealed that the monitor lizard isolate were positioned within the herptiles clade (clade VIII) while the mouse deer isolate located at the homoithermal clade (clade IV). The present paper is the first report on the presence as well as genetic characteristics of Blastocystis in wildlife captured from Tioman Island, Pahang.

Sarcocystis infection in Kedah-Kelantan crossbred cattle and Murrah Buffalo slaughtered in abattoir in Perak, Malaysia.

Sarcocystis infection, sarcocystosis or sarcosporidiosis is the disease caused by zoonotic intracellular protozoan parasite with an obligatory two-host life cycle namely Sarcocystis spp. The affected animals are mostly asymptomatic and the parasite is discovered only at slaughter. The aim of this study is to determine the status of sarcocystis infection in large ruminants slaughtered in four abattoirs in the state of Perak. A total of eighty-six fresh heart muscle and oesophagus samples were collected between February 2013 to October 2013. The samples were examined macroscopically to look for - sarcocyst. Digestion technique was also used to detect the sarcocyst bradyzoites. Part of the samples were preserved in formalin for histological examination. Out of the 86 animals, 19 (22.0%) animals were infected with Sarcocystis spp. 22.5% (16 of 71) of cattle and 20.0% (3 of 15) of buffalo were diagnosed with sarcocystis infection. Four animals were detected positive from Ipoh abattoir, followed by 9, 4 and 2 animals from Taiping, Teluk Intan and Tapah abattoir respectively. It is strongly recommended to cook meat thoroughly to reduce the incidence of the disease in humans.

Species identification of Malayan Gaur, Kedah-Kelantan and Bali cattle using polymerase chain reaction-restricted fragment length polymorphism.

Mitochondrial DNA (mtDNA) is a useful genetic marker that can be used for species identification. The cytochrome b (Cyt b) gene is a suitable mtDNA candidate gene for use in phylogenetic analyses due to its sequence variability, which makes it appropriate for comparisons at the subspecies, species, and genus levels. This study was conducted to develop a rapid molecular method for species identification of Malayan gaur (Bos gaurus hubbacki), Kedah-Kelantan (KK) (Bos indicus), and Bali (Bos javanicus) cattle in Malaysia. DNA was extracted from blood samples of 8 Malayan gaurs, 30 KK, and 28 Bali cattle. A set of both specific and universal primers for the Cyt b gene were used in PCR amplification. DNA sequences obtained were then analyzed using BioEdit and Restriction Mapper softwares. The PCR products obtained from Cyt b gene amplification were then subjected to restriction enzyme digestion. The amplification, using both specific and universal primers, produced a 154- and a 603-bp fragment, respectively, in all three species. Two restriction enzymes, NlaIV and SspI, were used to obtain specific restriction profiles that allowed direct identification of Malayan gaur, KK, and Bali cattle. Our findings indicate that all three species can be identified separately using a combination of universal primers and the restriction enzyme SspI.

The molecular phylogenetic signature of Bali cattle revealed by maternal and paternal markers.

Bali cattle is a domestic cattle breed that can be found in Malaysia. It is a domestic cattle that was purely derived from a domestication event in Banteng (Bos javanicus) around 3,500 BC in Indonesia. This research was conducted to portray the phylogenetic relationships of the Bali cattle with other cattle species in Malaysia based on maternal and paternal lineage. We analyzed the cytochrome c oxidase I (COI) mitochondrial gene and SRY of Y chromosome obtained from five species of the Bos genus (B. javanicus, Bos gaurus, Bos indicus, Bos taurus, and Bos grunniens). The water buffalo (Bubalus bubalis) was used as an outgroup. The phylogenetic relationships were observed by employing several algorithms: Neighbor-Joining (PAUP version 4.0), Maximum parsimony (PAUP version 4.0) and Bayesian inference (MrBayes 3.1). Results from the maternal data showed that the Bali cattle formed a monophyletic clade, and together with the B. gaurus clade formed a wild cattle clade. Results were supported by high bootstrap and posterior probability values together with genetic distance data. For the paternal lineage, the sequence variation is low (with parsimony informative characters: 2/660) resulting an unresolved Neighbor-Joining tree. However, Bali cattle and other domestic cattle appear in two monophyletic clades distinct from yak, gaur and selembu. This study expresses the potential of the COI gene in portraying the phylogenetic relationships between several Bos species which is important for conservation efforts especially in decision making since cattle is highly bred and hybrid breeds are often formed. Genetic conservation for this high quality beef cattle breed is important by maintaining its genetic characters to prevent extinction or even decreased the genetic quality.