The Importance of Habitat in the Ecology of Decomposition on Rabbit Carcasses in Malaysia: Implications in Forensic Entomology.

The stages of decomposition and the faunal succession on rabbit carcasses in three different habitats, namely jungle, rural, and highland areas, were studied. Three New Zealand White rabbit (Oryctolagus cuniculus) carcasses weighing ∼2 kg were sampled daily until the decomposition process was completed. Representative specimens of adult flies, larvae, pupa, and mites were collected from the carcasses and processed in the laboratory. There were differences in decomposition rate and faunal succession between the carcasses. The fastest rate of decomposition was recorded in rural area, and the slowest rate of decomposition was recorded in highland area. The carcasses exhibited the same pattern of colonization by adult flies, but the dominant species of larvae and adult flies on each carcass in specific habitats were different. The primary species of flies recorded in jungle were Chrysomya megacephala F., Chrysomya rufifacies (Macquart), Chrysomya chani Kurahashi, Chrysomya villenuevi Patton, Chrysomya nigripes Aubertin, Chrysomya pinguis (Walker), Hemipyrellia ligurriens (Wiedemann), Hemipyrellia tagaliana (Bigot), Hypopyiopsis fumipennis (Walker), Hypopygiopsis violacea (Macquart), and Hydrotaea spinigera Stein represented by both adults and larvae. Musca domestica L., Atherigona sp., Lioproctia pattoni (Senior-White), Lioproctia saprianovae Pape & Bänziger, and Seniorwhitea princeps (Wiedemann) were represented by adults only. The biodiversity of flies in the rural area were C. megacephala, C. rufifacies, H. ligurriens, Fannia canicularis L., Hydrotaea chalcogaster (Wiedemann), and Hyd. spinigera represented by both adults and larvae, meanwhile M. domestica, Atherigona sp., Boettcherisca peregrina (Robineau-Desvoidy), Parasarcophaga taenionota Wiedemann, Parasarcophaga scopariiformis Senior-White, and S. princeps were represented by adults only. The species of flies collected in the highland area were Lucilia porphyrina (Walker), C. megacephala, C. rufifacies, C. villenuevi, C. pinguis, H. ligurriens, Hyd. spinigera, Hyd. chalcogaster, F. canicularis, and Boettcherisca highlandica Kurahashi & Tan represented by both adults and larvae, whereas C. nigripes, Chrysomya thanomthini Kurahashi & Tumrasvin, M. domestica, Atherigona sp., Parasarcophaga albiceps Meigen, P. taenionota, Sepsidae, Phoridae, and Millichidae were represented by adults only. Faunal succession followed the sequence of dominant flies, i.e., Calliphoridae, Sarcophagidae, Muscidae, Sepsidae, and lastly Stratiomyidae for jungle, or Sepsidae for rural and highland studies. Mites, from suborders Mesostigmata, Prostigmata, Astigmatina, and Oribatida, were also recovered throughout decomposition, which could be used for future implementation in forensic investigations. The data obtained from this study could provide more accurate indicators for local forensic scientists in solving criminal cases especially on the determination of time and primary location of death.

Detection of sarcocystosis in goats in Malaysia by light microscopy, histology, and PCR.

A number of methods have been used for the detection of the presence of microsarcocysts in animals, but little information exists on the value between the various methods. This study therefore examined for Sarcocystis spp. using three different methods in 105 samples of skeletal muscle collected from goats slaughtered in an abattoir in Selangor, Malaysia from January to February 2014. Three methods were used, direct light microscopy of squashed fresh muscle tissues; histological examination of fixed, sectioned, and hematoxylin and eosin (H&E)-stained samples of muscle; and molecular identification by polymerase chain reaction (PCR). Of the 105 tissue samples, 55 (52.38 %) were positive by light microscopy (LM), 46 (43.8 %) by histology, and 95 (90.48 %) by PCR. Only 29 (27.6 %) and 5 (4.76 %) samples were positive and negative, respectively, by all three methods. The cysts were elongated to a spindle shape with a mean size of 393.30 × 81.6 µm and containing banana-shaped bradyzoites of size 12.32 × 2.08 µm. The wall of the cyst was radially striated with a thickness of 2.83 µm. Samples were tested for the presence of Sarcocystis-specific 18S rRNA and were identified as Sarcocystis capracanis. Of the three methods used, the PCR test appears to be the most useful method for the diagnosis of sarcocystosis especially for species identification.

Comparison of the Anaerocult A and the oil blocking methods for the in vitro cultivation of Entamoeba histolytica.

Entamoeba histolytica, the causative agent for human amoebiasis, is among the most deadly parasites, accounting for the second highest mortality rate among parasitic diseases. Because this parasite dwells in low oxygen tension, for its cultivation, microaerophilic conditions are required to mimick the human gut environment. Several methods developed for optimal growth environment are commercially available and some are conventionally modified in-house which include the Anaerocult A and oil blocking preparation methods. This study was undertaken to compare the reliability of the Anaerocult A and the oil blocking methods in generating anaerobic environment for cultivation of E. histolytica. The trophozoites of E. histolytica HM1: IMSS strains were axenically cultivated in TYI-S-33 medium in culture incubated anaerobically by using Anaerocult A (Merck) and mineral oil blocking method. The outcomes of both methods were determined by the minimum inhibitory concentration (MIC) of metronidazole against E. histolytica by giving a score to the growth pattern of the trophozoites. The reliability of both methods was assessed based on susceptibility testing of E. histolytica to metronidazole. The MIC obtained by both anaerobic condition methods was 6.25 ug/ ml, thus showing that oil-blocking method is comparable to the Anaerocult A method and therefore, considered as a reliable method for generating an anaerobic environment for the cultivation of E. histolytica.

Pentaplex PCR assay for detection of hemorrhagic bacteria from stool samples.

Diarrheal diseases cause illness and death among children younger than 10 years in developing countries. Conventional testing for the detection of hemorrhagic bacteria takes 2 to 5 days to yield complete information on the organism and its antibiotic sensitivity pattern. Hence, in the present study, we developed a molecular-based diagnostic assay that identifies common hemorrhagic bacteria in stool samples. A set of specific primers were designed for the detection of Salmonella spp., Shigella spp., enterohemorrhagic Escherichia coli (EHEC), and Campylobacter spp., suitable for use in a one-tube PCR assay. The assay in the present study simultaneously detected five genes, namely, ompC for the Salmonella genus, virA for the Shigella genus, eaeA for EHEC, 16S rRNA for the Campylobacter genus, and hemA for an internal control. Specific primer pairs were successfully designed and simultaneously amplified the targeted genes. Validation with 20 Gram-negative and 17 Gram-positive strains yielded 100% specificity. The limit of detection of the multiplex PCR assay was 1 × 10(3) CFU at the bacterial cell level and 100 pg at the genomic DNA level. Further evaluation of the multiplex PCR with 223 bacterium-spiked stool specimens revealed 100% sensitivity and specificity. We conclude that the developed multiplex PCR assay was rapid, giving results within 4 h, which is essential for the identification of hemorrhagic bacteria, and it might be useful as an additional diagnostic tool whenever time is important in the diagnosis of hemorrhagic bacteria that cause diarrhea. In addition, the presence of an internal control in the multiplex PCR assay is important for excluding false-negative cases.

Sarcocystosis among wild captive and zoo animals in Malaysia.

Sarcocystis sp. infection was investigated in 20 necropsied captive wild mammals and 20 birds in 2 petting zoos in Malaysia. The gross post-mortem lesions in mammals showed marbling of the liver with uniform congestion of the intestine, and for birds, there was atrophy of the sternal muscles with hemorrhage and edema of the lungs in 2 birds. Naked eye examination was used for detection of macroscopic sarcocysts, and muscle squash for microscopic type. Only microscopically visible cysts were detected in 8 animals and species identification was not possible. Histological examination of the sections of infected skeletal muscles showed more than 5 sarcocysts in each specimen. No leukocytic infiltration was seen in affected organs. The shape of the cysts was elongated or circular, and the mean size reached 254 x 24.5 µm and the thickness of the wall up to 2.5 µm. Two stages were recognized in the cysts, the peripheral metrocytes and large numbers of crescent shaped merozoites. Out of 40 animals examined, 3 mammals and 5 birds were positive (20%). The infection rate was 15% and 25% in mammals and birds, respectively. Regarding the organs, the infection rate was 50% in the skeletal muscles followed by tongue and heart (37.5%), diaphragm (25%), and esophagus (12.5%). Further ultrastructural studies are required to identify the species of Sarcocystis that infect captive wild animals and their possible role in zoonosis.

Schistosomiasis in Malaysia: A review.

Schistosomiasis, a neglected tropical parasitic disease caused by the trematode flatworms of the genus Schistosoma, affects approximately 207 million people worldwide. Among the five main species infecting humans, Schistosoma mansoni and S. japonicum are responsible for the majority of hepatointestinal schistosomiasis. Human settlements near fresh water sites that lack proper sanitary systems often contribute to the transmission of disease. This risk particularly impacts on travellers or immigrants who come into contact with larvae-contaminated water. This review discusses the central features of schistosomiasis; including clinical manifestations, diagnosis, treatments, and the preventive measures available for the control of this disease. The description of the Malaysian schistosome species Schistosoma malayensis and the current status of schistosomiasis in Malaysia including the compilation of cases diagnosed from 1904 to 2015 are also discussed in this paper.

Evaluation of PCR-ELISA as a tool for monitoring transmission of Wuchereria bancrofti in District of Gampaha, Sri Lanka.

OBJECTIVE: To compare Wuchereria bancrofti (W. bancrofti) infection rates of Culex quinquefasciatus, using dissection and PCR-ELISA in two consecutive time periods (from 2007 to 2008 and from 2008 to 2009). METHODS: Mosquitoes were collected in 30 sentinel and 15 non-sentinel sites in 15 Medical Officer of Health areas of Gampaha District known for the presence of W. bancrofti transmission in two consecutive time period of 2007 to 2008 and 2008 to 2009. Captured mosquitoes were dissected to determine the W. bancrofti larvae (L1, L2, L3). PCR was carried out using DNA extracted from mosquito pools (15 body parts/pool) utilizing the primers specific for Wb-SspI repeat. PCR products were analyzed by hybridization ELISA using fluorescein-labeled wild type specific probes. The prevalence of infected/infective mosquitoes in PCR pools (3 pools/site) was estimated using the PoolScreen™ algorithm and a novel probability-based method. RESULTS: Of 45 batches of mosquitoes dissected, W. bancrofti infected mosquitoes were found in 19 and 13 batches, with an infection rate of 13.29% and 3.10% with mean larval density of 8.7 and 1.0 larvae per mosquito for two study periods in the Gampaha District. Total of 405 pools of head, thorax and abdomen were processed by PCR-ELISA for each year. Of these, 51 and 31 pools were positive for W. bancrofti in the two study periods respectively. The association of dissection based prevalence rates with PCR based rates as determined by the Pearson correlation coefficient were 0.176 and 0.890 respectively for the two periods. CONCLUSIONS: Data indicate that PCR-ELISA is more sensitive than the traditional dissection techniques for monitoring transmission intensity.

Human pentastomiasis caused by Armillifer moniliformis in Malaysian Borneo.

We report a case of visceral pentastomiasis caused by Armillifer moniliformis in a 70-year-old aboriginal farmer from rural Malaysian Borneo. The patient complained of upper abdominal pain, jaundice, and loss of weight. Radiological investigations and subsequent histopathological examination revealed an adenocarcinoma of the pancreas with an adjacent liver nodule containing a nymph of A. moniliformis. This report constitutes the first documented human pentastomid infection in the whole of Malaysia after nearly 40 years, and it is the third description from Malaysian Borneo. Cases of human and animal pentastomiasis in Malaysia are discussed.

Determination of the specificities of monoclonal and polyclonal antibodies to Neospora, Toxoplasma and Cryptosporidium by fluorescent antibody test (FAT).

Flourescent antibody test (FAT) was applied to determine the cross-reactivities of monoclonal (mAb), polyclonal (pAb) antibodies to Neospora, Toxoplasma and Cryptosporidium and antisera from cattle naturally infected with Neospora canium against antigens from a number of sources. Both mAb and pAb to Neospora reacted strongly (FAT titre up to 2560) with the homologous antigens and demonstrated weak titre (80) or no reaction with both Toxoplasma and Cryptosporidium antigens. Also mAb and pAb to Toxoplasma gondii reacted at titres of 80 - 640 with homologous antigens and at titres of 10-40 with N. caninum. No cross-reactions with either mAb or pAb antibodies to N. caninum and T. gondii were observed with Cryptosporidium parvum. The same results were observed with C. parvum mAb when tested with both N. caninum and T. gondii antigens. Sera from cattle naturally infected with N. caninum had titres ranging from 80- 640 with N. caninum antigens, and 10- 40 with T. gondii and C. parvum antigens. At low dilutions, the complete surfaces of Neospora and Toxoplasma parasites were fluorescent, while in higher dilutions only dotted fluorescence appeared on the apical complex. These results indicated the presence of cross-reactivity between Neospora and Toxoplasma but not with Cryptosporidium. Accordingly the recommended cut-off antibody titre for diagnosis of neosporosis is 80.