Occurrence, Morphometric, and Molecular Investigation of Cattle and Buffalo Liver Adult Fluke in Peninsular Malaysia Main Abattoirs.

Fascioliasis is a parasitic disease of human and animal caused by Fasciola gigantica (F. gigantica) and Fasciola hepatica (F. hepatica). More than 700 million of grazing animals and over 180 million human population are at the risk of fascioliasis. This study was conducted in Banting, Ipoh, Shah Alam, and Taiping abattoirs within Peninsular Malaysia to determine the occurrence and identify the species of liver flukes, causing liver condemnation in cattle and buffaloes. Within a study period from January to December 2018, a total of 25 condemned bovine livers were collected from Banting, Ipoh, Shah Alam, and Taiping abattoirs and analyzed. Taiping abattoir had the highest occurrence of fascioliasis [14/1014 (1.38%)], and Shah Alam abattoir had the least occurrence [1/3377 (0.03%)]. From all the sampled livers, the average number of adult flukes recorded ranged from 1 to 83. A total of 440 adult flukes were studied morphometrically, using parameters such as body length (BL), body width (BW), cone length (CL), cone width (CL), body area (BA), body perimeter (BP), the distance between the ventral sucker and posterior end of body (VS-P), BL/BW ratio, BL/V-SP, and body roundness (BR). Furthermore, molecular analysis was conducted using PCR-RFLP methods to distinguish between F. gigantica and F. hepatica involving ITS1 primer and RsaI restriction enzyme. RFLP pattern with RsaI produced a consistent pattern of 360,100 and 60 bp fragments in F. hepatica, whereas F. gigantica worms had a profile of 360,170, and 60 bp in size. The morphometric and molecular analysis results indicated that cattle and buffaloes slaughtered at Banting, Shah Alam, Taiping, and Ipoh abattoirs were infected with F. gigantica.

A Retrospective Study on Bovine Fascioliasis in Veterinary Regional Laboratories in Peninsular Malaysia.

This is a retrospective study on bovine fascioliasis infection on cattle in Peninsular Malaysia, spanning from January 2007 to December 2017. Cattle were diagnosed with Fasciola based on the fecal examination and the results were reported to the Veterinary Regional Laboratories in Peninsular Malaysia. These records were analysed for the occurrence of bovine fascioliasis within that 11-year period. Records of annual diagnostic cases from five major Veterinary Regional Laboratories were examined: Bukit Tengah, Pulau Pinang (north); Kuantan, Pahang (east); Johor Bahru, Johor (south); Sepang, Selangor (west); and Kota Bharu, Kelantan (east). A positive fascioliasis infestation was calculated based on a number of positively infected cattle with Fasciola from a number of cattle examined. A total of 1988 cattle were examined during this period and 35 (1.76%) cattle were reported to be positive for bovine fascioliasis. Parasite infection was the highest at Bukit Tengah region (5.55%) where 19 cases were positive from 342 reported cases while, at Kuantan (4.96%), 15 positive cases were reported, unlike Johor Bahru (0.09%), with only 1 positive case from 1136 cattle examined. Sepang and Kelantan had no positive cases. These results showed that bovine fascioliasis was generally prevalent in the northern and southeast parts of the Peninsular Malaysia; however, there was no significant relationship between the region and the occurrence of fascioliasis.

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.

Therapeutic use of Lucilia sericata maggot in controlling bacterial bio-burden in Rat wound model.

Delayed wound healing due to extraneous bacterial contamination, antibacterial resistance and other associated factors are of great concern in dealing patients having chronically infected wound. Medicinal properties of certain maggots of Calliphoridae family are known for its effective wound debridement therapy. The objective of the study was to evaluate the wound healing potential of maggots of Lucilia sericata in an experimentally infected cutaneous wound model in Wistar rat. The study was carried out by using male Wistar rats (n=48) by creating excisional wounds and later contaminated with mixed population of gram positive and gram-negative bacteria. Animals were divided randomly in to four groups with 12 individuals each, being denominated as control, antibiotic treated, maggot treated, and antibiotic plus maggot combination treated group. Ten pre-sterilized maggots were applied per centimetre square wound bed for 24 hours. Different wound kinetics in L. sericata maggot treated wounds revealed significant reduction in wound area with maximum contraction, early elimination of bacterial bioburden as compared to group of infected control and group of rats receiving only antibiotic treatment. The histopathological examination of wounded tissue of maggot treated groups showed early and better epithelialization, collagenation and neovascularization with complete healing of wound in two weeks. The maggot effects on healing when used singly or in combination with antibiotic were recorded to be similar. The results of the present study clearly demonstrate that the maggots of L. sericata possesses a definite antibacterial action along with removal of dead tissues and effectively reduced the bacterial bio-burden in infected wound and induced wound healing quickly.

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.

Genetic diversity of rodent Blastocystis sp. from Peninsular Malaysia.

Rodents are ubiquitous zoonotic vectors for many human pathogens including Blastocystis sp. In this study, we examined the prevalence and subtypes of Blastocystis sp. in rodents captured from Peninsular Malaysia. A total of 293 rodents predominantly brown rat (Rattus norvegicus) (290 of 293, 99.0%) and house shrew (Suncus murinus) (3 of 293, 1.0 %), were captured in the vicinity of popular eateries in two cities (Kuala Lumpur and Ipoh) in Peninsular Malaysia. In vitro cultivation method showed presence of Blastocystis sp. in approximately half (133 of 290, 45.9%) of the brown rats tested. Among the 47 Blastocystis isolates subtyped using partial small subunit ribosomal RNA gene analysis, ST4 was the most abundant (43 of 47, 91.5%) followed by ST1 (2 of 47, 4.3%), ST5 (1 of 47, 2.1%) and ST7 (1 of 47, 2.1%). Our findings highlighted the importance of rodents as a source of Blastocystis sp. infection in Malaysia and showed the high prevalence of ST4 within the rodent population infected with Blastocystis sp.

Molecular Characterization of Avian Infectious Bronchitis Virus Isolated in Malaysia during 2014-2016.

Avian Infectious Bronchitis (IB) is a highly contagious disease which can cause huge economic losses to the poultry industry. Forty five IB viruses (IBV) were isolated from poultry in Malaysia during 2014-2016. Phylogenetic analysis of the spike glycoprotein 1 (S1) gene revealed that all isolates were clustered into five distinct groups. The predominant type of IBV isolated was QX strains (47%), second was 4/91 type (27%), followed by Malaysian strain MH5365/95 (13%), Massachusetts type (11%) and finally Taiwanese strains (2%). Four types of S1 protein cleavage recognition motifs were found among the isolates which includes HRRRR, RRSRR, RRFRR and RRVRR. To our knowledge, this is the first report describing the motif RRVRR and are unique to Malaysian strains. Six IBVs were grouped in Malaysian MH5365/95 strains. Among these, one isolate was different from others where it only shared 82% identity with MH5365/95 and to others. It formed its own branch in the Malaysian cluster suggesting it may be a variant unique to Malaysia. Alignment analysis of the S1 amino acid sequences indicated that point mutations, insertions and deletions contribute to the divergence of IB variants. This study indicated at least five groups of IBV are circulating in Malaysia with most of the isolates belonged to QX strains. As new IBV variants continue to emerge, further study need to be carried out to determine whether the current available vaccine is able to give protection against the circulating virus.

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.

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.

Anthelmintic resistance in sheep and goat farms on Peninsular Malaysia.

The faecal egg count reduction test (FECRT) was conducted on 39 sheep farms and 9 goat farms located in Peninsular Malaysia. The anthelmintic groups used in these tests were the benzimidazoles, levamisole, the benzimidazole/levamisole combination, macrocyclic lactones and closantel. Results indicated that the prevalence of resistance to the benzimidazole group was high, with approximately 50% of the sheep farms and 75% of the goat farms having resistant nematode parasite populations present. Resistance to levamisole, closantel and ivermectin was also detected. Differentiation of the infective larvae derived from faecal cultures indicated that by far the most predominant parasite species was Haemonchus contortus.

The control of the free-living stages of Strongyloides papillosus by the nematophagous fungus, Arthrobotrys oligospora.

Two laboratory trials were conducted to determine the effect of the addition of spores (conidia) of the nematophagous fungus, Arthrobotrys oligospora, on the development of the ruminant parasite, Strongyloides papillosus, in cultures of bovine faeces. Both studies showed that at a concentration of 2000 conidia/g faeces virtually eliminated infective larvae (> 99% reduction), following 14 days incubation under ideal conditions (25 degrees C and saturated humidity) for free-living development of this parasite species. In one trial, a high level of control was also observed at a 10-fold decrease in conidia concentration (200 spores/g faeces). This work has demonstrated, in principle, that A. oligospora could provide a practical biological control agent against S. papillosus infecting intensively raised young ruminants in the humid tropics/subtropics.

Biological control of nematode parasites of small ruminants in Malaysia using the nematophagous fungus Duddingtonia flagrans.

Control of nematode parasites of small ruminants in a wet, tropical environment using the nematophagous fungus, Duddingtonia flagrans, was assessed in this study. Two methods of fungal delivery were tested, namely as a daily feed supplement, or incorporated into feed blocks. Initially, pen trials were conducted with individually penned groups of sheep and goats at dose rates of 125,000 spores and 250,000 spores/kg live weight per day. At the lower dose rate this reduction was between 80 and 90% compared with the pre-treatment levels. At the higher dose rate, there was virtually complete suppression (>99% reduction) of larval recovery. Trials using the fungal feed blocks, showed that when animals were individually penned, they consumed only small amounts of the block (particularly goats), hence little effect on larval recovery in faecal cultures was observed. Grouping animals according to species and dose rate induced satisfactory block consumption and subsequent high levels of larval reduction in faecal cultures. These larval reductions were mirrored by the presence of fungus in faecal cultures. This work was followed by a small paddock trial, whereby three groups of sheep were fed either a feed supplement without fungal spores, supplement with spores, or offered fungal blocks. The dose rate of spores in the latter two groups was 500,000 spores/kg live weight per day. Egg counts were significantly reduced in the two fungal groups, compared with the control group and the latter required two salvage anthelmintic treatments to prevent mortality due to haemonchosis. Pasture larval numbers on the two fungal group plots were also much lower than on the control plot.

Field studies on the biological control of nematode parasites of sheep in the tropics, using the microfungus Duddingtonia flagrans.

Long-term field studies were conducted on two government managed small ruminant research farms, located in different geo-climatic regions and approximately 300 km separate from each other, on Peninsula Malaysia. The Infoternak trial (48 weeks) and the Chalok trial (43 weeks) each compared nematode parasite control in separately managed groups of young sheep, either short-term rotationally grazed around a suite of 10 paddocks in addition to receiving a daily supplement of Duddingtonia flagrans spores (Fungus Group); or similar groups of sheep being rotationally grazed alone (Control Group). The prevailing weather conditions at Infoternak farm were of below average rainfall conditions for the most of the trial. As a consequence, only very low worm infections (almost exclusively Haemonchus contortus) were acquired by the 17 sets of tracer lambs that grazed sequentially with the experimental lambs. However on all except 2 occasions in the early part of the trial, the mean tracer worm burdens were significantly lower (P < 0.05) and the experimental lambs grew significantly better (P = 0.054) in the Fungus Group. Rainfall at Chalok farm during the course of the trial was also below average. As a consequence infectivity of pastures was assumed to be relatively low based on faecal egg counts (epg) of the experimental sheep, which following an anthelmintic treatment prior to allocation, remained very low in both treatment groups. Faecal egg counts of undosed replacement lambs in the latter half of the Chalok study, showed a progressive increase in the Control Group to levels exceeding 3000 epg, whereas the Fungus Group remained static at approximately 500 epg. These results show that the deployment of the nematophagous fungus, D. flagrans, can improve the level of parasite control of sheep in the tropics above that which can be achieved by the short-term rotational grazing strategy alone.

Isolation of Neospora caninum from a calf in Malaysia.

In order to attempt isolate the protozoan parasite Neospora caninum, an N. caninum seropositive pregnant Sahiwal Friesian cross heifer from a large-scale dairy farm in Malaysia was kept for observation until parturition at the Veterinary Research Institute, Ipoh. The heifer gave birth to a female calf that was weak, underweight and unable to rise. Precolostral serum from the calf had an N. caninum indirect fluorescent antibody test titre of 1:3200. It died 12 h after birth and necropsy was performed. Brain homogenate from the calf was inoculated into 10 BALB/c mice that were kept for 3 months after which brain tissue from the mice was inoculated onto 24 h fresh monolayer Vero cell lines. The cell cultures were examined daily until growth of intracellular protozoa was observed. DNA of the organisms from the cell cultures was analyzed by PCR and DNA sequencing. DNA fragments of the expected size were amplified from the isolate using N. caninum-specific primers, and sequence analysis of ITS1 clearly identified the isolate as N. caninum. This is the first successful isolation of N. caninum from a bovine in Malaysia, and the isolate is designated Nc-MalB1.

Seroepidemiological studies of bovine babesiosis caused by Babesia ovata, B. bigemina and B. bovis in Peninsular Malaysia.

Cattle in Peninsular Malaysia were examined for evidence of infection with Babesia ovata, B. bigemina and B. bovis by an enzyme-linked immunosorbent assay for detection of antibody to the three Babesia species. All of the test samples when assayed with B. ovata antigen, resulted in low value indicating low probability of cattle infected with B. bigemina, 74.4% were positive for B. bovis and 72.6% were positive for both Babesia species. In addition, a serological survey with regard to age difference was carried out on a milk production farm. High reactivity antibody to B. bigemina and B. bovis was detected in calves less than 1 month of the age. The reactivity decreased in calves 1-3 months of the age. Then, the reactivity increased for both Babesia species in 6 months old calves. These results suggested that cattle infected with B. bigemina and B. bovis were widespread throughout Peninsular Malaysia and that both parasites might exist as an enzootical parasite.

Total anthelmintic failure to control nematode parasites of small ruminants on government breeding farms in Sabah, East Malaysia.

Government-owned small-ruminant breeding farms in Malaysia provide the source of sheep and goats to smallholder farmers in the country. In the eastern Malaysian state of Sabah, high-level stock losses have been recorded on these farms for several years, frequently accompanied by clinical signs indicating pathogenic levels of infections with the nematode parasite Haemonchus contortus. This suggests that their dependence on chemotherapy to control parasite infections had failed. Accordingly, tests for anthelmintic efficacy using the faecal egg count reduction test (FECRT) on the range of drugs used to control nematode parasites were carried out on the five government small-ruminant breeding farms in Sabah. These tests showed a total failure of the benzimidazole, imidothiazole, macrocyclic lactone and salicylanilide groups of anthelmintics to control H. contortus infections of sheep and goats on all farms. Drastic changes in animal management need to be made in an attempt to deal with this situation, for which suggestions are made.

A case of human enteric myiasis due to larvae of Hermetia illucens (Family: Stratiomyiadae): first report in Malaysia.

A case of true enteric myiasis in a 7-year-old girl is reported. Two larvae were obtained from the vomitus of the patient. After processing and identification, the larvae were found to be those of Hermetia illucens (Soldier Fly). This is the first case of true enteric myiasis due to these larvae in Malaysia.

Evaluation of enhanced virgin coconut oil and senduduk (Melastoma malabathricum) as anthelmintics against caprine strongyle nematodes.

A study was conducted to evaluate the anthelmintic properties of enhanced virgin coconut oil (EVCO) and senduduk (Melastoma malabathricum) plant against strongyle nematodes in goats. Two preparations of 10% EVCO dissolved in 90% virgin coconut oil and 10% EVCO dissolved in 90% palm oil, were given orally to two groups of mixed breeds goats. The efficacy test indicated that EVCO was insufficiently active as an anthelmintic. Four concentrations of senduduk solution (1.25, 2.5, 5.0 and 10 mg ml(-1)) were compared with a control and albendazole in an in vitro test for larvicidal effect. There was no significant larval mortality using senduduk solution. An in vivo test of senduduk was conducted by comparing three groups of goats, namely control, levamisole and treatment groups that were given a daily oral dose of senduduk crude extract with 1g kg(-1) from Day 0 to Day 12 and 2 g kg(-1) from Day 13 to Day 30. This efficacy test with senduduk also gave negative results. The findings obtained indicated that EVCO and senduduk were ineffective as anthelmintics against caprinestrongyle nematodes at the concentrations used.

Zoonotic parasites from exotic meat in Malaysia.

Four zoonotic parasites, Sarcocystis spp., Toxoplasma gondii, Trichinella spp. and Taenia spp were screened in exotic meats. A total of forty-six (n=46) meat samples from various species of exotic animals were received from all the 14 states in Malaysia from January 2012 to April 2012. All exotic meat samples were examined macroscopically and histologically for the four zoonotic parasites. Results by histological examination of exotic meats showed the presence of Sarcocystis and Toxoplasma cysts at 8.7% (n=4) and 4.3% (n=2) respectively. No Trichinella spp. and Taenia spp. were found.

Comparative seroprevalences of bovine trypanosomiasis and anaplasmosis in five states of Malaysia.

A comparative seroprevalence study on bovine trypanosomiasis and anaplasmosis was conducted. Sera of adult cattle and buffaloes of different breeds from farms from five different states in Malaysia were collected and tested for the presence of Trypanosoma evansi antibodies by CATT and Anaplasma marginale antibodies by c-ELISA. Of the 116 samples, 14.7% tested positive for bovine trypanosomiasis and 77.6% for bovine anaplasmosis.