Tor tambroides, a common and appreciated cyprinid fish of the Tasik Kenyir water reservoir in Malaysia, is one of the species selected for propagation. This fish was first successfully propagated in Malaysia by the Department of Agriculture, Sarawak, Malaysia, and the breeding program continued throughout the country. The gills were frequently infected by a Myxobolus species to be described as Myxobolus tambroides sp. n. The small, 50 to 70 μm, round plasmodia of this species is located intralamellarly. Plasmodia were filled with pyriform myxospores, 9.9 and 7.4 μm wide. In sutural view, the caudal end of the myxospores had a distinctive valvular groove, parallel with the suture. Plasmodia caused deformations on the affected and the neighbouring gill lamellae. The 18S rDNA sequence of M. tambroides sp.n. did not show a close relationship with any other Myxobolus spp., represented in the GenBank. This might be an emerging parasite likely to impact the propagation of this fish.
The authors studied the myxosporean infection of wild gobiid fishes (Perciformes: Gobioidei) in the Merang Estuary of Terengganu, Malaysia, and described Myxobolus ophiocarae sp. n. in Ophiocara porocephala. Several myxosporean plasmodia were found intralamellarly within the gill filaments. The spores differed from those of other Myxobolus species previously recorded on gobiid fishes. They were round in valvular view and lens-shaped in sutural view, and had two equal-sized, pyriform polar capsules with polar filaments having six to seven turns. The spores measured 10.34 × 8.79 × 4.53 μm. The 18S rDNA sequence of M. ophiocarae sp. n., based on a contiguous sequence of 1,789 base pairs, differed from any other Myxobolus spp. in GenBank. Phylogenetic analysis of the 18S rDNA gene revealed that this species showed the closest similarity to Myxobolus nagaraensis, Myxobolus lentisuturalis, and Myxobolus cultus.
Trypanosoma evansi, the causative agent of "surra" is enzootic in Iran. The current study aimed to detect T. evansi in horses from different regions of Iran using morphological, serological, and molecular methods. In 2021, 400 blood samples were collected from horses in eight regions. Eighty horses showed clinical signs such as cachexia (n = 64), fever (n = 36), foot edema (n = 40), and abdominal edema (n = 32), and 320 horses appeared healthy. All samples from the studied regions were evaluated for the presence of trypanosomes using direct analysis of blood smears, mercuric chloride, and PCR-based tests. In total, 12% (95% CI: ± 3.1%), 21% (95% CI: ± 3.9%), and 21% (84) of animals were positive for Trypanosoma in microscopic, serologic, and molecular analyses, respectively. All animals positive for SSU rDNA PCR were from Qom, Semnan, and Golestan regions. Further molecular analyses on 84 PCR-positive horses revealed that 29 horses scored positive in PCR using primers of trypanozoon species and 5 scored positive in PCR using primers of Trypanosoma evansi type A. All samples (n = 5) were from Qom region. The 205-bp fragments of T. evansi RoTat 1.2VSG (accession numbers: ON017789-93) analyzed and compared to other isolates sequence from GenBank BLAST search. It has close similarities with isolates from Pakistan, Egypt, Malaysia, Kenya, and India. Data herein demonstrated that horses from Iran were at high risk of T. evansi infection. Comprehensive control programs, such as those based on the application of repellants and traps, and also, compliance with quarantine standards are recommended for minimizing the risk of the infection.
Lymnaeid snails play a crucial role in the transmission of trematode cercariae as an intermediate host that can infect humans, ruminants like buffalo, and other animals, resulting in serious economic losses. The purpose of the study was to identify the morphological and molecular characteristics of snails and cercariae collected from water bodies near buffalo farms that were integrated with palm oil in Perak, Malaysia. The presence or absence of snails in 35 water bodies was examined via cross-sectional study. From three marsh wetlands, 836 lymnaeid snails were gathered in total. Each snail's shell was morphologically identified to determine its family and species. The cercarial stage inside each snail's body was observed using the crushing method and trematode cercariae types were determined. In addition, the target gene Cytochrome c oxidase subunit 1 (Cox1) and the ribosomal internal transcribed spacer 2 (ITS2) region were used to identify the snail species and cercarial types according to the species level. The findings indicated that the collected snails belong to the family lymnaeidae and Radix rubiginosa species. In snails, the cercarial emergence infection rate was 8.7%. Echinostome, xiphidiocercariae, gymnocephalous, brevifurcate-apharyngeate distome cercariae (BADC), and longifurcate-pharyngeal monostome cercariae (LPMC) are the five morphological cercarial types that were observed. The cercariae were identified using morphological and molecular techniques, and they are members of the four families which are Echinostomatidae, Plagiorchiidae, Fasciolidae, and Schistosomatidae. Interestingly, this is the first study on R. rubiginosa and several trematode cercariae in Perak water bodies near buffalo farms that are integrated with palm oil. In conclusion, our research shown that a variety of parasitic trematodes in Perak use R. rubiginosa as an intermediate host.
The ornamental fish trade provides a pathway for the global translocation of aquatic parasites. We examined a total of 1020 fish imported from Singapore, Malaysia, Thailand, or Sri Lanka to Australia (including freshwater and marine fish species) for monogenean ectoparasites. Fish were received following veterinary certification that they showed no clinical signs of pests and diseases from the exporting country and visual inspection at Australian border control. Australian import conditions require mandatory treatment for goldfish with parasiticides (e.g. trichlorfon, formaldehyde, sodium chloride) for the presence of gill flukes (Dactylogyrus vastator Nybelin, 1924 and Dactylogyrus extensus Mueller and Van Cleave, 1932) prior to export. Over 950 individual parasites were detected in five imported fish species, representing 14 monogenean species. Seven Dactylogyrus spp. including D. vastator and three Gyrodactylus spp. infected goldfish, Carassius auratus Linnaeus, 1758, from Malaysia, Singapore, and Thailand. Dactylogyrus ostraviensis Řehulka, 1988, infected rosy barb, Pethia conchonius Hamilton, 1822, from Singapore, Sri Lanka, and Thailand while two Trianchoratus spp. infected three spot gourami, Trichopodus trichopterus Pallas, 1970 and pearl gourami Trichopodus leerii Bleeker, 1852, from Sri Lanka. Urocleidoides reticulatus Mizelle & Price, 1964, infected guppy, Poecilia reticulata Peters, 1859, from Sri Lanka. The discovery of D. vastator in goldfish, as well as 13 other monogenean species, shows that pre-export health requirements, which include chemical treatment of goldfish, and inspection of all ornamental fish species did not prevent infection by monogeneans. Inspection prior to exportation and at border control must account for the highly cryptic nature of monogenean parasites and consider alternatives to current pre-export conditions and visual inspection at border control.
To date, more than 50 Eimeria spp. have been isolated from marsupials of the family Macropodidae. Although 18 species of Eimeria have been previously detected from multiple animal species belonging to the genus Macropus of the family, limited genetic analyses of the parasites are available, and their pathogenicity remains unclear. Here, we report the isolation of Eimeria spp. from a zoo specimen of red-necked wallaby (Macropodidae; Macropus rufogriseus). Specifically, two distinct types of Eimeria oocysts were recovered, one from the feces before treatment with an anthelmintic and the second from the intestinal contents after death of the animal. The oocysts obtained from the two sources were morphologically identified as E. hestermani and E. prionotemni, respectively. We successfully determined partial gene sequences from the two isolates, including segments of the 18S rRNA genes, and for the first time have used phylogenetic analyses of these sequences to assign the species to distinct clades. In combination with further genetic data, these results are expected to help elucidate the pathogenicity and host ranges of Eimeria spp. within the respective family and genus.
Sarcocystis nesbitti, using snakes as the definitive host, is a causative agent of acute human muscular sarcocystosis in Malaysia. Therefore, it is important to explore the distribution and prevalence of S. nesbitti in snakes. Nevertheless, epizootiological information of S. nesbitti in snakes remains insufficient because few surveys have assessed Sarcocystis infection in snakes in endemic countries. In Japan, snakes are popular exotic pet animals that are imported from overseas, but the degree of Sarcocystis infection in them remains unclear. The possibility exists that muscular sarcocystosis by S. nesbitti occurs in contact with captive snakes in non-endemic countries. For a total of 125 snake faecal samples from 67 snake species collected at animal hospitals, pet shops and a zoo, this study investigated the presence of Sarcocystis using polymerase chain reaction (PCR) for the 18S ribosomal RNA gene (18S rDNA). Four (3.2%) faecal samples were positive by PCR. Phylogenetic analysis of the 18S rDNA sequences obtained from four amplification products revealed one isolate from a beauty snake (Elaphe taeniura), Sarcocystis zuoi, which uses rat snakes as the definitive host. The isolate from a Macklot's python (Liasis mackloti) was closely related with unidentified Sarcocystis sp. from reticulated pythons in Malaysia. The remaining two isolates from tree boas (Corallus spp.) were closely related with Sarcocystis lacertae, Sarcocystis gallotiae and unidentified Sarcocystis sp. from smooth snakes, Tenerife lizards and European shrews, respectively. This report is the first of a study examining the distribution of Sarcocystis species in captive snakes in Japan.
Enterocytozoon bieneusi is an emerging opportunistic pathogen infecting humans, and both domestic and wild pigs are known to harbour zoonotic genotypes. There remains a paucity of information on the prevalence and epidemiology of this enteropathogen in Southeast Asia. The present study was undertaken to determine the molecular prevalence and risk factors associated with E. bieneusi infection among commercially farmed pigs in Malaysia. Faecal samples were collected from 450 pigs from 15 different farms and subjected to nested PCR amplification of the ribosomal internal transcribed spacer (ITS) gene of E. bieneusi. Phylogenetic analysis involved 28 nucleotide sequences of the ITS region of E. bieneusi. An interviewer-administered questionnaire provided information on the animal hosts, farm management systems and environmental factors and was statistically analysed to determine the risk factors for infection. The prevalence of E. bieneusi infection was relatively high (40.7%). The highest prevalence (51.3%) was recorded among the piglets, while the adults showed the lowest level of infection (31.3%). Multivariate analysis indicated that age of the pigs, distance of the farm from human settlement and farm management system were significant risk factors of infection. Three genotypes (EbpA, EbpC and Henan-III) detected among the pigs are potentially zoonotic. The high prevalence of E. bieneusi among locally reared pigs, the presence of zoonotic genotypes and the spatial distribution of pig farms and human settlements warrant further investigation on the possibility of zoonotic transmission.
Dientamoeba fragilis, a trichomonad parasite is usually found in the gastrointestinal tract of human, and it is known to be the cause for gastrointestinal disease. The parasite is globally distributed and mostly found in rural and urban areas. The parasite is found in humans and nonhuman primates such as the macaques, baboons, and gorillas. Often, the parasite is confused with another largely found organism in stools called Blastocystis sp. especially when seen directly under light microscopy on culture samples containing both parasites. Both sometimes are seen with two nuclei with sizes tending to be similar which complicates identification. Stools were collected fresh from nine previously diagnosed persons infected with D. fragilis who also were found to be positive for Blastocystis sp. Samples were then cultured in Loeffler's medium and were stained with Giemsa, iron hematoxylin, and modified Fields' (MF) stain, respectively. D. fragilis was differentiated from Blastocystis sp. when stained with MF stain by the presence of a thinner outer membrane with clearly demarcated nuclei in the center of the cell whilst Blastocystis sp. had a darker and thicker stained outer membrane with the presence of two nuclei. The staining contrast was more evident with modified Fields' stain when compared with the other two. The simplicity in preparing the stain as well as the speed of the staining procedure make MF stain an ideal alternate. The modified Fields' stain is faster and easier to prepare when compared to the other two stains. MF stain provides a better contrast differentiating the two organisms and therefore provides a more reliable diagnostic method to precisely identify one from the other especially when cultures show mixed infections.
For elucidation of the taxonomic status of the Japanese Fasciola species, whole mitochondrial DNA of Fasciola hepatica from Australia, F. gigantica from Malaysia, and Fasciola sp. from Japan was digested with three four-base-cutting endonucleases: HinfI, MspI, and RsaI. The resulting digestion patterns showed that for each enzyme there were some bands specific for each geographical isolate and that the Japanese Fasciola sp. shared more bands with F. gigantica than with F. hepatica. Nucleotide sequences of two regions, the second internal transcribed spacer (ITS2) of the nuclear ribosomal RNA cluster and mitochondrial cytochrome c oxidase subunit I (COI), were also compared among them. The ITS2 sequence was highly conserved among the three isolates. F. gigantica and the Japanese Fasciola sp. were identical, but they differed from the Australian F. hepatica at six sites, one of which was a deletion. The COI sequence was less conserved but implied a similar relationship between the isolates. There seems no reason to regard the Japanese Fasciola sp. as anything other than a strain of F. gigantica.
A main challenge in parasitology is the development of reliable tools to prevent or treat mosquito-borne diseases. We investigated the toxicity of magnetic nanoparticles (MNP) produced by Magnetospirillum gryphiswaldense (strain MSR-1) on chloroquine-resistant (CQ-r) and sensitive (CQ-s) Plasmodium falciparum, dengue virus (DEN-2), and two of their main vectors, Anopheles stephensi and Aedes aegypti, respectively. MNP were studied by Fourier-transform infrared spectroscopy and transmission electron microscopy. They were toxic to larvae and pupae of An. stephensi, LC50 ranged from 2.563 ppm (1st instar larva) to 6.430 ppm (pupa), and Ae. aegypti, LC50 ranged from 3.231 ppm (1st instar larva) to 7.545 ppm (pupa). MNP IC50 on P. falciparum were 83.32 μg ml(-1) (CQ-s) and 87.47 μg ml(-1) (CQ-r). However, the in vivo efficacy of MNP on Plasmodium berghei was low if compared to CQ-based treatments. Moderate cytotoxicity was detected on Vero cells post-treatment with MNP doses lower than 4 μg ml(-1). MNP evaluated at 2-8 μg ml(-1) inhibited DEN-2 replication inhibiting the expression of the envelope (E) protein. In conclusion, our findings represent the first report about the use of MNP in medical and veterinary entomology, proposing them as suitable materials to develop reliable tools to combat mosquito-borne diseases.
Established criteria for morphological typing of sarcocysts was applied to a large series of cases of human skeletal muscle sarcocystosis in Malaysia to determine the type of sarcocyst present. We also wanted to test the general usefulness of this classification and to determine if there are any new cyst types. Three-dimensional (3-D) reconstruction was done to see if the sarcocyst has a distinct 3-D morphology. A total of 66 sarcocysts from 21 cases of human muscle sarcocystosis obtained from a previous prevalence study were examined. Tissue sections (5 microns thick) were stained with haematoxylin and eosin and studied under the light microscope. For 3-D reconstruction, an image analyser was used to align and reconstruct the sarcocyst after microscopic images had been captured with a charge-coupled device (CCD) camera. All the cysts best fit into the type 4 category. This classification is generally useful, although cyst wall characteristics and zoite size appear to be the most reliable criteria for classification. The cyst width averaged 77 microns (range, 30-137.5 microns). Cyst walls were smooth, had no cytophaneres and were less than 1 micron thick. No secondary cyst wall or surrounding inflammation was evident. Numerous cyst merozoites with diameters averaging 1 micron filled the cyst lumen. Although septa were not apparent, in many cysts, zoites were arranged in a unique, curvilinear fashion that suggested their presence. 3-D reconstruction showed the sarcocyst to be a long, tortuous "cylinder" with no branching or other distinguishing feature.
This study focuses on the larvicidal, oviposition, and ovicidal effects of a crude extract of Artemisia annua against Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus. Dried cells of Artemisia annua from cell suspension cultures were extracted using hexane. The extract showed moderate larvicidal effects against mosquitoes. At 24-h post treatment, the LC50 values for Anopheles sinensis, Aedes aegypti, and Culex quinquefasciatus were recorded as 244.55, 276.14, and 374.99 ppm, respectively. The percentage mortality of larvae was directly proportional to the tested concentration. Anopheles sinensis was found to be the most susceptible species, whereas Culex quinquefasciatus was the most tolerant to the Artemisia annua extract. The results indicated that the Artemisia annua extract showed concentration-dependent oviposition deterrent activity and had a strong deterrent effect. At 500 ppm, the percentage effective repellency was more than 85% compared with the control group for all the species, with oviposition activity index values of -0.94, -0.95, and -0.78 for Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus, respectively. In the ovicidal assay, the percentage hatchability of eggs after treatment with 500 ppm of Artemisia annua extract was significantly lower than the control, with values of 48.84 ± 4.08, 38.42 ± 3.67, and 79.35 ± 2.09% for Aedes aegypti, Anopheles sinensis, and Culex quinquefasciatus, respectively. Artemisia annua was found to be more effective against Aedes aegypti and Anopheles sinensis compared with Culex quinquefasciatus. This study indicated that crude extract of A. annua could be a potential alternative for use in vector management programs.
The free living Acanthamoeba spp. are ubiquitous amoebae associated with potentially blinding disease known as Acanthamoeba keratitis (AK) and a fatal central nervous system infection granulomatous amoebic encephalitis (GAE). With the inherent ability of cellular differentiation, it can phenotypically transform to a dormant cyst form from an active trophozoite form. Acanthamoeba cysts are highly resistant to therapeutic agents as well as contact lens cleaning solutions. One way to tackle drug resistance against Acanthamoeba is by inhibiting the formation of cysts from trophozoites. The biochemical analysis showed that the major component of Acanthamoeba cyst wall is composed of carbohydrate moieties such as galactose and glucose. The disaccharide of galactose and glucose is lactose. In this study, we analyzed the potential of lactase enzyme to target carbohydrate moieties of cyst walls. Amoebicidal assessment showed that lactase was ineffective against trophozoite of A. castellanii but enhanced amoebicidal effects of chlorhexidine. The lactase enzyme did not show any toxicity against normal human keratinocyte cells (HaCaT) at the tested range. Hence, lactase can be used for further assessment for development of potential therapeutic agents in the management of Acanthamoeba infection as well as formulation of effective contact lens disinfectants.
The shedding pattern of the protozoan parasite, Blastocystis hominis, is investigated in man and in experimental animal infections. The shedding pattern of the vacuolar and cystic forms of Blastocystis hominis in infected individuals have been shown in the present study to be irregular. The study shows that there is marked fluctuation in the shedding of the parasite from day to day, varying from as high as 17 to 0 per x40 microscopic field. The cystic stages when estimated in 8 Blastocystis-infected individuals ranged from as high as 7.4x10(5) cysts per gram of stool to 0. The shedding of cystic and vacuolar forms observed over a period of 20 days in experimentally-infected Wistar rats were not only shown to be irregular but the amount varied from host to host. The study has important diagnostic implications in that the stool samples must be collected more than once from patients showing clinical signs and symptoms to eliminate the cause of it to Blastocystis. The study also shows that there are asymptomatic individuals who pass a large amount of cysts as such individuals should be treated to prevent transmission to others.
The intraspecific variation of four laboratory-reared isolates of Taenia taeniaformis the SRN and KRN isolates from Norway rats, Rattus norvegicus, captured in Japan and Malaysia, respectively; the BMM isolated from a house mouse, Mus musculus, captured in Belgium; and the ACR isolate from a gray red-backed vole, Clethrionomys rufocanus bedfordiae, captured in Japan was examined by various criteria. Eggs of each of the four isolates were orally inoculated into several species of intermediate host. They were most infective to the rodent species from which the original metacestode of each isolate had been isolated in the field, and only the ACR isolate was infective to the gray red-backed vole. Although little difference was found between the SRN, KRN, and BMM isolates by the other criteria, including the morphology of rostellar hooks, the protein composition of the metacestode, and restriction endonuclease analysis of DNA, the ACR isolate was clearly different from the others. It was considered that the ACR isolate was independent as a strain distinct from the other three isolates.
In forensic entomology, breeding of fly larvae in a controlled laboratory environment using animal tissue is a common technique to obtain insect developmental time for the estimation of postmortem interval. Previous studies on growth media are mostly on the effect of different diets on fly development. However, the interaction effects between temperature and food type used have not been explored. The objective of this study was to compare the use of cow's liver agar and raw liver on the development of a forensically important fly, Megaselia scalaris (Loew). This study also determined the interaction between different temperatures and different food types on the growth of this species. A total of 100 M. scalaris eggs were transferred into each of the two media mentioned above. Liver agar was prepared by adding dried ground liver into nutrient agar, whilst raw liver was naturally prepared from the same animal source. This experiment was conducted at 27, 30 and 33 °C in an incubator in a continuously dark condition. Length and weight of larvae, puparia and adult samples were determined. Total developmental times for larvae feeding on liver agar at each temperature were approximately 7-15 h slower than those feeding on raw liver. Survival rates were almost equal in both diets but were lower at 33 °C. Mean larva length in both diets did not differ significantly at all temperatures, but larvae feeding on liver agar had lower mean weight values than those in raw liver at 30 and 33 °C. The effect of temperature was significant in female puparia weight and male adult weight whereas the effect of diet types was significant in both male and female puparia size and weight. Interaction effects of temperature and food type on M. scalaris puparium size and adult weight were significant, indicating that puparium size and adult weight depended on both food type and temperature. This experiment highlighted the use of cow's liver agar as an alternative diet to breed M. scalaris in the laboratory and the importance of considering the interaction effect between temperatures and food types when deciding the most suitable medium in fly larva rearing.
Dirofilaria immitis is a parasite of domestic and wild canids and felids in tropical, subtropical and temperate regions throughout the world. The canine heartworm (D. immitis) is the causative agent of canine and feline cardiopulmonary dirofilariasis. This parasite is known to cause a zoonotic disease, namely human pulmonary dirofilariasis. D. immitis is known to be endemic in several South and Southeast Asian countries (e.g. India and Malaysia), but there has previously been no information about the presence of this pathogen in Bangladesh. We present a case of canine dirofilariasis caused by D. immitis in rural southeastern Bangladesh. A male filaroid nematode (95 mm in length and 1.94 mm in width) was identified in the heart of a dog. Species classification was performed by microscopy and molecular tools. Sequence analysis revealed a 100 % identity within the mitochondrial cytochrome c oxidase I (CO1) gene to two Chinese and one Australian D. immitis samples. Usually, dogs stay outside overnight with a high risk to get infected with D. immitis via nocturnal mosquito vectors, which may lead to high prevalences of this pathogen in the canine population and thus increase the risk of human infections with this neglected parasitic disease.
Blastocystis sp., an intestinal organism is known to cause diarrhea with metronidazole regarded as the first line of treatment despite reports of its resistance. The conflicting reports of variation in drug treatment have been ascribed to subtype differences. The present study evaluated in vitro responses due to metronidazole on ST3 isolated from three symptomatic and asymptomatic patients, respectively. Symptomatic isolates were obtained from clinical patients who showed symptoms such as diarrhea and abdominal bloating. Asymptomatic isolates from a stool survey carried out in a rural area. These patients had no other pathogens other than Blastocystis. Ultrastructural studies using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed drug-treated ST3 from symptomatic patients were irregular and amoebic with surface showing high-convoluted folding when treated with metronidazole. These organisms had higher number of mitochondrion-like organelle (MLO) with prominent cristae. However, the drug-treated ST3 from asymptomatic persons remained spherical in shape. Asymptomatic ST3 showed increase in the size of its central body with the MLO located at the periphery.
Blastocystis from infected stools of a person who showed chronic symptoms of abdominal discomfort and diarrhea were examined over a 6-month period, using transmission electron microscopy, for the ultrastructural changes from vacuolar to cystic stage. The study confirms the irregular shedding phenomenon of the organism previously reported, and for the first time, records sequential changes in encystation in stools collected over a time period. The study also confirms the existence of a precystic stage which has an immature cell wall consisting of a layer of a homogenous electron-dense mass surrounding the cell which acts as a intermediatory stage between the vacuolar and cystic stage.