Impact of pH on the viability and morphology of Blastocystis isolates.

Blastocystis sp. is ubiquitous in avian, mammalian and human hosts and propagates in either neutral or slightly alkaline conditions within the host's gastro-intestinal tract. Of the few previous studies on this enteric protozoan parasite in feline and canine hosts, prevalence values have been shown to range between 0 to 70.8%. In view of the close association between humans, and canine and feline hosts as companion animals, faecal samples of 180 Felis catus and 82 Canis lupus, collected from Penang and Kuala Lumpur, Malaysia, were initially screened by in vitro cultivation followed by molecular characterization. No positive isolates were identified in culture but in 12 feline samples DNA barcoding detected a zoonotic subtype Blastocystis ST1 for the first time. Consequently, avian and human isolates, which had previously been successfully cultured, were used to investigate the impact of pH on the viability and morphology of Blastocystis sp. The use of Trypan blue showed that the number of viable cells increased when exposed to pH 4 and a significant increase in viability occurred in pH values of 5 to 7. Development of Blastocystis cells in both isolates was suppressed in media less than pH 5 followed by the disappearance of viable cells from avian isolates in more acidic media below pH 4. Morphologically at pH 4 cells from avian isolates were less rounded, and with wrinkled / shrunken surfaces, than the more normal rounded cells from human isolates. On the other hand, at values below pH 3, no viable cells in human isolates were visible. The present findings therefore confirm that gastro-intestinal pH is an important determinant of Blastocystis viability and consequently influences the epidemiology of infection within avian, mammalian and human hosts.

Current status of Blastocystis in cockroaches.

There are few reports on Blastocystis spp. infections in invertebrate hosts namely, cockroaches. Due to their close proximity to humans especially to their dwellings prompted this study as these organisms could possibly play a role in human transmission. A total of 151 cockroaches consisted predominantly of nymph and adult stages were captured from several types of dwellings in the state of Perak and Selangor, Malaysia. Approximately half (40.4%) of the cockroach intestinal contents screened were positive and were found associated to two main factors, host-stage and types of dwellings. The granular and vacuolated forms were the most common cell form found in the in vitro cultures and were morphologically similar to B. hominis. However, the surface coat observed was thick with an electron lucent area observed in the central vacuole. The isolates grew in room temperature but optimal growth was observed at a 24ºC similar to the reptilian Blastocystis with a high number of cells were recovered. Using the DNA barcoding method, two isolates were identified as ST3 (allele 56), one isolate was consider as the new subtype with close relation to allele 114.

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.