Molecular characterisation and genetic affinities of Cyclophyllidean cestodes infecting wild rats in Peninsular Malaysia.

Cestode infections is widely reported in rodents, however species identification remains problematic due to the genetic or interspecies variation. Therefore, this study was aimed to verify the Cyclophyllidean parasites recovered from wild rats captured from different forest types using molecular based methods. Maximum-likelihood (ML) and neighbour-joining (NJ) trees were constructed inferred from 18 small subunit nuclear ribosomal RNA gene (18SrDNA) and mitochondrial cytochrome c oxidase subunit one gene (COX1) sequences of cestode worms recovered from 124 individuals from four rat species. Sequences obtained from both Hymenolepis diminuta and Hydatigera parva represents the first records in Malaysia. All the sequences were successfully amplified with product with total length of 205 and 1202 base pairs (bp), respectively. Three cestode species from the Family Hymenolepididae (Hymenolepis diminuta) and Family Taeniidae (Hydatigera parva; Hydatigera taeniaeformis) were successfully characterized using phylogenetic analyses and haplotype networking. Phylogenetic analysis showed that H. diminuta, Hydatigera parva (Hy. parva) and Hydatigera taeniaeformis (Hy. taeniaeformis) formed its own monophyletic clade in 18SrDNA analyses. Results also showed that Hy. taeniaeformis shared the same haplotype group with Hy. taeniaeformis from China (COX1) and linked with Hy. taeniaeformis from Japan (18SrDNA) while the Malaysian H. diminuta clearly formed a separate haplotype and networked with other regions. The Malaysian Hy. parva isolation, on the other hand, appeared to be genetically distinct from the European Hy. parva (Spain) strain, but closely linked to the local isolates. Molecular methods employed successfully improved in the detection of complex species in this group. The findings showed that molecular data can be useful to deeply study intra-specific variation in other cestode worms.

Molecular characterisation and genetic affinities of Cyclophyllidean cestodes infecting wild rats in Peninsular Malaysia

@#Cestode infections is widely reported in rodents, however species identification remains problematic due to the genetic or interspecies variation. Therefore, this study was aimed to verify the Cyclophyllidean parasites recovered from wild rats captured from different forest types using molecular based methods. Maximum-likelihood (ML) and neighbour-joining (NJ) trees were constructed inferred from 18 small subunit nuclear ribosomal RNA gene (18SrDNA) and mitochondrial cytochrome c oxidase subunit one gene (COX1) sequences of cestode worms recovered from 124 individuals from four rat species. Sequences obtained from both Hymenolepis diminuta and Hydatigera parva represents the first records in Malaysia. All the sequences were successfully amplified with product with total length of 205 and 1202 base pairs (bp), respectively. Three cestode species from the Family Hymenolepididae (Hymenolepis diminuta) and Family Taeniidae (Hydatigera parva; Hydatigera taeniaeformis) were successfully characterized using phylogenetic analyses and haplotype networking. Phylogenetic analysis showed that H. diminuta, Hydatigera parva (Hy. parva) and Hydatigera taeniaeformis (Hy. taeniaeformis) formed its own monophyletic clade in 18SrDNA analyses. Results also showed that Hy. taeniaeformis shared the same haplotype group with Hy. taeniaeformis from China (COX1) and linked with Hy. taeniaeformis from Japan (18SrDNA) while the Malaysian H. diminuta clearly formed a separate haplotype and networked with other regions. The Malaysian Hy. parva isolation, on the other hand, appeared to be genetically distinct from the European Hy. parva (Spain) strain, but closely linked to the local isolates. Molecular methods employed successfully improved in the detection of complex species in this group. The findings showed that molecular data can be useful to deeply study intra-specific variation in other cestode worms.

Molecular detection of porcine Enterocytozoon bieneusi infection in Peninsular Malaysia and epidemiological risk factors associated with potentially zoonotic genotypes.

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.

Prevalence of Toxoplasma gondii in pet and stray cats in Klang Valley, Malaysia.

Toxoplasma gondii, a zoonotic protozoan that has a worldwide distribution, is known to infect many warm-blooded vertebrates. The feline species including domestic cats are the definitive hosts for Toxoplama gondii and shed the infective oocyst. There is lack of information on the prevalence of Toxoplasma gondii in cats in Malaysia. The objective of this study was to determine both the seroprevalence of T. gondii and the prevalence of T. gondii DNA in cats' feces in Klang Valley, Malaysia. 198 blood and 201 fecal samples were collected from pet and stray cats from the local council, Dewan Bandaraya Kuala Lumpur (DBKL) and University Veterinary Hospital, Universiti Putra Malaysia respectively. The overall seroprevalence of Toxoplasma gondii in cats in the Klang Valley was found to be 5.5%. There was a high prevalence (10.5%) of T. gondii DNA detected in the cat fecal samples in both pet and stray cats suggestive of T. gondii oocyst shedding. Stray cats showed a higher seroprevalence and molecular prevalence of T. gondii than the pet cats. However, comparative analysis using Chi-square test showed no significant difference between both groups (P>0.05). Higher prevalence (10.5%) of cats shedding T. gondii DNA as compared to the seroprevalence (5.5%) was found in the cat population in the Klang Valley. The high prevalence of cats shedding T. gondii DNA is alarming as this may directly reflect the number of oocysts excreted into the environment posing a significant public health hazard.

First molecular detection of Tritrichomonas foetus in domestic cats in Klang Valley, Malaysia.

Tritrichomonas foetus is known to cause chronic diarrhea in the feline species in many different regions of the world. However, there is a paucity of information on T. foetus among cats in Malaysia. This study was conducted to determine the prevalence of Tritrichomonas foetus in the pet and stray cat population in Klang Valley, Malaysia. A total of 201 pet and stray cats' fecal samples were collected in Klang Valley. 24 samples were cultured in the InPouch® TF Feline to observe for motile trophozoites. A nested PCR protocol was used to screen for T. foetus in the collected samples. The prevalence of T. foetus in the cat population in Klang Valley was 33%. There was no association between Tritrichomonas infection and age, sex, breed or management of the cats. However, statistical analysis revealed that stray cats were more likely to be infected with T. foetus compared to pet cats. This study confirmed for the first time the presence of T. foetus among the cat population in Klang Valley, Malaysia.

First molecular detection of Tritrichomonas foetus in domestic cats in Klang Valley, Malaysia

@#Tritrichomonas foetus is known to cause chronic diarrhea in the feline species in many different regions of the world. However, there is a paucity of information on T. foetus among cats in Malaysia. This study was conducted to determine the prevalence of Tritrichomonas foetus in the pet and stray cat population in Klang Valley, Malaysia. A total of 201 pet and stray cats’ fecal samples were collected in Klang Valley. 24 samples were cultured in the InPouch® TF Feline to observe for motile trophozoites. A nested PCR protocol was used to screen for T. foetus in the collected samples. The prevalence of T. foetus in the cat population in Klang Valley was 33%. There was no association between Tritrichomonas infection and age, sex, breed or management of the cats. However, statistical analysis revealed that stray cats were more likely to be infected with T. foetus compared to pet cats. This study confirmed for the first time the presence of T. foetus among the cat population in Klang Valley, Malaysia.

Prevalence of Toxoplasma gondii in pet and stray cats in Klang Valley, Malaysia

@#Toxoplasma gondii, a zoonotic protozoan that has a worldwide distribution, is known to infect many warm-blooded vertebrates. The feline species including domestic cats are the definitive hosts for Toxoplama gondii and shed the infective oocyst. There is lack of information on the prevalence of Toxoplasma gondii in cats in Malaysia. The objective of this study was to determine both the seroprevalence of T. gondii and the prevalence of T. gondii DNA in cats’ feces in Klang Valley, Malaysia. 198 blood and 201 fecal samples were collected from pet and stray cats from the local council, Dewan Bandaraya Kuala Lumpur (DBKL) and University Veterinary Hospital, Universiti Putra Malaysia respectively. The overall seroprevalence of Toxoplasma gondii in cats in the Klang Valley was found to be 5.5%. There was a high prevalence (10.5%) of T. gondii DNA detected in the cat fecal samples in both pet and stray cats suggestive of T. gondii oocyst shedding. Stray cats showed a higher seroprevalence and molecular prevalence of T. gondii than the pet cats. However, comparative analysis using Chi-square test showed no significant difference between both groups (P>0.05). Higher prevalence (10.5%) of cats shedding T. gondii DNA as compared to the seroprevalence (5.5%) was found in the cat population in the Klang Valley. The high prevalence of cats shedding T. gondii DNA is alarming as this may directly reflect the number of oocysts excreted into the environment posing a significant public health hazard.

Antitrypanosomal and cytotoxic activities of botanical extracts from Murraya koenigii (L.) and Alpinia mutica Roxb.

Four carbazoles (girinimbine, mahanimbine, murrayafoline and murrayanine), isolated from Murraya koenigii, and one kavalactone (5,6-dehydrokawain) and one flavonoid (pinostrobin) isolated, from Alpinia mutica, were tested for their antitrypanosomal activity using in vitro cultured Trypanosoma evansi cell lines. The cytotoxic activities of these compounds were also investigated against mammalian Vero cells using the MTT (3-(4,5- Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide)-cell proliferation assay. Three carbazole compounds, namely mahanimbine, murrayafoline, and girinimbine, showed a potent antitrypanosomal activity, scoring a median inhibitory concentration (IC50) of 3.13, 6.35 and 10.16 µg/ml, respectively. Girinimbine was the least toxic to Vero cells, and the mean cytotoxic concentration (CC50) and the selectivity index (SI) of this compound were 745.58 ± 42.38 µg/ ml and 73.38, respectively. Girinimbine and the other carbazole compounds possess potential antitrypanosomal activity with comparably low toxicity against mammalian cells. Girinimbine, in particular, is a good candidate to be further investigated as a potential antitrypanosomal agent using in vivo models.

Toxoplasma gondii infection in native village chickens (Gallus domesticus) in Selangor and Melaka, Malaysia.

Toxoplasmosis is a worldwide zoonosis caused by the protozoa Toxoplasma gondii which affects human and animals. Village chickens (Gallus domesticus) most commonly known as Ayam Kampung or free-range chickens, have been suggested to play a role in the epidemiology of toxoplasmosis. This study determines the presence of T. gondii in the village chicken populations in two states of Malaysia. A total of 50 serum samples from the chickens from Selangor (n=20) and Melaka (n=30) were collected and analysed using commercial serological kits. T. gondii antigen was detected in 20% (Selangor 30%; Melaka 13%) samples using ELISA test and anti-T. gondii antibody was detected in all positive ELISA samples using the indirect haemagglutination test (IHAT). Histopathological examination revealed tissue changes such as inflammation and degeneration in brain and liver of seropositive chickens. This is the first report of T. gondii infection in the village chickens in Malaysia.

Molecular detection and epidemiological risk factors associated with Cryptosporidium infection among cattle in Peninsular Malaysia.

Enteric protozoa infection among cattle may pose a threat to productivity and survival leading to negative impacts on the livestock industry. A number of these pathogens are also known to be zoonotic and are of public health concern. Despite the importance of these enteric protozoa to both animal and human health, there remains a paucity of published information on the epidemiological risk factors that may be associated with bovine cryptosporidiosis in Southeast Asia. The present study was undertaken to determine the molecular prevalence and associated risk factors for Cryptosporidium infection among beef and dairy cattle in Peninsular Malaysia. Faecal samples were collected from 824 cattle in 39 farms (526 beef and 298 dairy) situated in 33 locations throughout the country, and subjected to PCR detection for Cryptosporidium using primers targeting the 18S SSUrRNA gene. Epidemiological variables including host, environment and management factors were subjected to univariate and multivariate logistic regression analyses to determine the potential risk factors for infection. The prevalence of Cryptosporidium among the cattle was 12.5%, with significant difference in the infection rate among the various breeds. There was no significant effect of gender, and both the beef and dairy cattle were at similar odds for infection. The younger cattle had a significantly higher infection rate compared to the older animals. Multivariate analysis revealed that deworming practice, distance to human settlement, geographical location (zone) and farm management system were significant risk factors associated with Cryptosporidium infection. The cattle that were reared on farms located in the northeast of the country, closest (≤200 m) to human settlements, reared extensively, and dewormed every four months were at highest risk of infection. The present study constitutes the first attempt to analyze the multivariable epidemiological risk factors involved in bovine cryptosporidiosis in Malaysia and in Southeast Asia. It is envisaged that the data obtained will facilitate better control and prevention measures for Cryptosporidium infection among cattle in the region. Due to the potential zoonotic nature of the infection, serious steps should be instituted for animal treatment and biohazard waste management on local cattle farms.

Antitrypanosomal and cytotoxic activities of botanical extracts from Murraya koenigii (L.) and Alpinia mutica Roxb

@#Four carbazoles (girinimbine, mahanimbine, murrayafoline and murrayanine), isolated from Murraya koenigii, and one kavalactone (5,6-dehydrokawain) and one flavonoid (pinostrobin) isolated, from Alpinia mutica, were tested for their antitrypanosomal activity using in vitro cultured Trypanosoma evansi cell lines. The cytotoxic activities of these compounds were also investigated against mammalian Vero cells using the MTT (3-(4,5- Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide)-cell proliferation assay. Three carbazole compounds, namely mahanimbine, murrayafoline, and girinimbine, showed a potent antitrypanosomal activity, scoring a median inhibitory concentration (IC50) of 3.13, 6.35 and 10.16 μg/ml, respectively. Girinimbine was the least toxic to Vero cells, and the mean cytotoxic concentration (CC50) and the selectivity index (SI) of this compound were 745.58 ± 42.38 μg/ ml and 73.38, respectively. Girinimbine and the other carbazole compounds possess potential antitrypanosomal activity with comparably low toxicity against mammalian cells. Girinimbine, in particular, is a good candidate to be further investigated as a potential antitrypanosomal agent using in vivo models.

Toxoplasma gondii infection in native village chickens (Gallus domesticus) in Selangor and Melaka, Malaysia

@#Toxoplasmosis is a worldwide zoonosis caused by the protozoa Toxoplasma gondii which affects human and animals. Village chickens (Gallus domesticus) most commonly known as Ayam Kampung or free-range chickens, have been suggested to play a role in the epidemiology of toxoplasmosis. This study determines the presence of T. gondii in the village chicken populations in two states of Malaysia. A total of 50 serum samples from the chickens from Selangor (n=20) and Melaka (n=30) were collected and analysed using commercial serological kits. T. gondii antigen was detected in 20% (Selangor 30%; Melaka 13%) samples using ELISA test and anti-T. gondii antibody was detected in all positive ELISA samples using the indirect haemagglutination test (IHAT). Histopathological examination revealed tissue changes such as inflammation and degeneration in brain and liver of seropositive chickens. This is the first report of T. gondii infection in the village chickens in Malaysia.

Molecular and serological prevalence of Anaplasma and Ehrlichia sp. among stray dogs in East Malaysia.

Anaplasma and Ehrlichia species are important bacterial pathogens of various animal species including dogs and humans. They constitute a major health problem worldwide, especially in the tropics and sub-tropics. In order to identify the prevalence of Anaplasma and Ehrlichia sp. from dogs in the selected area, a total of 100 randomly selected clinically healthy rescued dogs from animal shelters in Sarawak and Sabah, East Malaysia (50 dogs per State) were used for this study. Whole blood was collected from these dogs and screened for presence of Anaplasma and Ehrlichia spp. using polymerase chain reaction (PCR) and commercial ELISA test (SNAP 4Dx® Plus) for detection of antigen and antibody against the two pathogens respectively. 27% (A. platys) and 33% (E. canis) of the sampled population were positive using PCR; while the serological prevalence was 29% for Anaplasma sp. and 55% for Ehrlichia sp. The results of this investigation revealed that stray dogs are potential reservoir hosts of Anaplasma and Ehrlichia sp. infection with a high prevalence in the area. The high prevalence rates detected highlights the urgent need to address the growing population of possible vectors and reservoirs for transmission in the region and also to look into the public health aspect of the disease.

Occurrence of gastro-intestinal parasites among small ruminants in Malaysia: highlighting Dicrocoelium infection in goats.

The aim of the present study was to determine the gastro-intestinal (GI) parasitic infections among small ruminants (i.e., goats, sheep, deer) in Malaysia through formalin-ether concentration technique. Overall, 70.9% or 302 out of 426 small ruminants (79.4% or 251/316 goats; 87.5% or 35/40 sheep; 22.9% or 16/70 deer) were infected with at least one species of GI parasites. Overall, ten types of GI parasites [Helminth: strongyle (57.7%), Moniezia spp. (5.4%), Paramphistomum spp. (4.5%), Strongyloides spp. (4.2%), Dicrocoelium spp. (2.3%), Trichuris spp. (2.3%); Protozoa: Eimeria spp. (23.7%), Entamoeba spp. (18.8%), Giardia spp. (1.9%), Cryptosporidium spp. (0.2%)] were detected in this study. Among the studied animals, goats harboured the highest diversity of GI parasites (ten types), followed by sheep (six types) and deer (two types). Polyparasitism was observed in goats (43.7% or 138 of 316) and sheep (15.0% or 6 of 40). Cumulatively, a total of 32 combinations of coinfections (Helminth+Helminth: 8 combinations; Helminth+Protozoa: 20 combinations; Protozoa+Protozoa: 4 combinations) between detected parasites with up to quintuple infections were reported. Among these parasites, "strongyle + Eimeria spp." and "Moniezia spp. + strongyle" were the commonest infections in goats (13.5% or 34 of 251) and sheep (5.7% or 2 of 6), respectively. This study is a comprehensive documentation on multiple GI parasitisms among small ruminant in Malaysia, and the findings are crucial for effective farm management, especially for the formulation of parasitic control and elimination strategies.

In vivo antitrypanosomal activity of Garcinia hombroniana aqueous extract.

The anti-Trypanosoma evansi activity of Garcinia hombroniana (seashore mangosteen) leaves aqueous extract was tested on experimentally infected Sprague-Dawley rats. Treatment of infected rats with G. hombroniana extract resulted in a significantly extended post-infection longevity (p < 0.05), compared to the untreated control group. The possible mode of antitrypanosomal effect of the plant extract was also investigated on cultured T. evansi in HMI-9 medium with the addition of 25 µg/ml G. hombroniana aqueous extract. It was observed that the addition of G. hombroniana extract resulted in the inhibition of trypanosomal kinetoplast division, with no significant inhibitory effect on nuclear division. It is concluded from the current study that the aqueous extract of G. hombroniana has a potential antitrypanosomal activity through the inhibition of kinetoplast division, as one of the possible mechanisms of its antitrypanosomal effect. This plant could serve as a possible source of new antitrypanosomal compounds.

The influence of enclosure design on diurnal activity and stereotypic behaviour in captive Malayan Sun bears (Helarctos malayanus).

The effect of enclosure design on diurnal activity and stereotypic behaviour was assessed in 17 adult Malayan Sun bears (Helarctos malayanus), kept either in barren indoor enclosures or relatively enriched outdoor enclosures. Locomotion was the most frequent activity observed in the indoor bears, followed by resting. In contrast, conspecifics housed outdoors spent most of the time resting. Eleven forms of stereotypic behaviours were recorded in the bears, with pacing being the most common. The frequency and repertoire of stereotypies were significantly higher in the indoor bears irrespective of enclosure size. Novel forms of locomotor (forward-reverse pacing) and oral (allo-sucking) stereotypies were recorded. Oral stereotypies were predominant in the bears housed indoors, while patrolling was confined to the outdoor bears. Enclosure complexity significantly influences activity budget and occurrence of stereotypic behaviours, highlighting the importance of appropriate enclosure design and enrichment for the welfare of captive bears.

Redescription of Serpinema octorugatum (Baylis, 1933) nematoda: Camallanidae from the Malayan box turtle Cuora amboinensis Daudin Chelonia: Bataguridae.

We redescribe the camallanid nematode Serpinema octorugatum (Baylis, 1933) from the box turtle Cuora amboinensis (Daudin) collected in Malaysia. In this redescription, we amend the original description by noting that there are only four cephalic papillae and that there are five pairs of post-anal papillae, and propose that the name of this species be corrected from S. octorugatus to S. octorugatum. Additionally, we removed the tissues overlying the buccal capsule and have used SEM studies to show that the peribuccal shields extend laterally from the buccal capsule, forming a surface possibly used in muscle attachment. Furthermore, we show that the supposedly non-cuticularised cylinder connecting the buccal capsule to the oesophagus in the Camallanidae is part of the buccal capsule and is, therefore, likely to be cuticularised. We also examine morphological measurements of taxonomic interest for correlations with total body length and find that many characters traditionally used for inter- and intra-specific comparisons are correlated with total body length in adult female worms. This suggests that comparisons between samples of adult female worms that do not account for the potential effect of total body length may be misleading. However, we show that some features of taxonomic interest are not correlated with total body length.