Reuben Sunil Kumar Sharma

The heart of darkness: growth and form of Trypanosoma brucei in the tsetse fly.

The first description of African trypanosomes was made over a century ago. The importance of the tsetse in transmission and cyclic development of trypanosomes was discovered soon afterwards, and has been the focus of numerous studies since. However, investigation of trypanosomes in tsetse flies requires high resource investment and unusual patience; hence, many facets of trypanosome biology in the tsetse remain to be characterised despite the long history of research. Here, current knowledge and questions about some of the developmental changes in trypanosomes that occur in tsetse flies are summarised, along with recent technical advances that can now be used to provide some answers.

Functional characterization of cohesin subunit SCC1 in Trypanosoma brucei and dissection of mutant phenotypes in two life cycle stages

In yeast and metazoa, structural maintenance of chromosome (SMC) complexes play key roles in chromosome segregation, architecture and DNA repair. The main function of the cohesin complex is to hold replicated sister chromatids together until segregation at anaphase, which is dependent on proteolytic cleavage of the cohesin subunit SCC1. Analysis of trypanosomatid genomes showed that the core cohesin and condensin complexes are conserved, but SMC5/6 is absent. To investigate the functional conservation of cohesin in eukaryotes distantly related to yeast and metazoa, we characterized the Trypanosoma brucei SCC1 orthologue. TbSCC1 is expressed prior to DNA synthesis at late G1, remains in the nucleus throughout S‐ and G2‐phases of the cell cycle and disappears at anaphase. Depletion of SCC1 by RNAi or expression of a non‐cleavable SCC1 resulted in karyokinesis failure. Using the dominant negative phenotype of non‐cleavable SCC1 we investigated checkpoint regulation of cytokinesis in response to mitosis failure at anaphase. In the absence of chromosome segregation, procyclic trypanosomes progressed through cytokinesis to produce one nucleated and one anucleate cell (zoid). In contrast, cytokinesis was incomplete in bloodstream forms, where cleavage was initiated but cells failed to progress to abscission. Kinetoplast duplication was uninterrupted resulting in cells with multiple kinetoplasts and flagella.

Structure of a Glycosylphosphatidylinositol-anchored Domain from a Trypanosome Variant Surface Glycoprotein

The cell surface of African trypanosomes is covered by a densely packed monolayer of a single protein, the variant surface glycoprotein (VSG). The VSG protects the trypanosome cell surface from effector molecules of the host immune system and is the mediator of antigenic variation. The sequence divergence between VSGs that is necessary for antigenic variation can only occur within the constraints imposed by the structural features necessary to form the monolayer barrier. Here, the structures of the two domains that together comprise the C-terminal di-domain of VSG ILTat1.24 have been determined. The first domain has a structure similar to the single C-terminal domain of VSG MITat1.2 and provides proof of structural conservation in VSG C-terminal domains complementing the conservation of structure present in the N-terminal domain. The second domain, although based on the same fold, is a minimized version missing several structural features. The structure of the second domain contains the C-terminal residue that in the native VSG is attached to a glycosylphosphatidylinositol (GPI) anchor that retains the VSG on the external face of the plasma membrane. The solution structures of this domain and a VSG GPI glycan have been combined to produce the first structure-based model of a GPI-anchored protein. The model suggests that the core glycan of the GPI anchor lies in a groove on the surface of the domain and that there is a close association between the GPI glycan and protein. More widely, the GPI glycan may be an integral part of the structure of other GPI-anchored proteins.

Molecular Detection of Ehrlichia canis in Dogs in Malaysia

An epidemiological study of Ehrlichia canis infection in dogs in Peninsular Malaysia was carried out using molecular detection techniques. A total of 500 canine blood samples were collected from veterinary clinics and dog shelters. Molecular screening by polymerase chain reaction (PCR) was performed using genus-specific primers followed by PCR using E. canis species-specific primers. Ten out of 500 dogs were positive for E. canis. A phylogenetic analysis of the E. canis Malaysia strain showed that it was grouped tightly with other E. canis strains from different geographic regions. The present study revealed for the first time, the presence of genetically confirmed E. canis with a prevalence rate of 2.0% in naturally infected dogs in Malaysia.

Co-infection of Haemonchus contortus and Trichostrongylus spp. among livestock in Malaysia as revealed by amplification and sequencing of the internal transcribed spacer II DNA region

BackgroundHaemonchus contortus and Trichostrongylus spp. are reported to be the most prevalent and highly pathogenic parasites in livestock, particularly in small ruminants. However, the routine conventional tool used in Malaysia could not differentiate the species accurately and therefore limiting the understanding of the co-infections between these two genera among livestock in Malaysia. This study is the first attempt to identify the strongylids of veterinary importance in Malaysia (i.e., H. contortus and Trichostrongylus spp.) by amplification and sequencing of the Internal Transcribed Spacer II DNA region.ResultsOverall, 118 (cattle: 11 of 98 or 11.2%; deer: 4 of 70 or 5.7%; goats: 99 of 157 or 63.1%; swine: 4 of 91 or 4.4%) out of the 416 collected fecal samples were microscopy positive with strongylid infection. The PCR and sequencing results demonstrated that 93 samples (1 or 25.0% of deer; 92 or 92.9% of goats) contained H. contortus. In addition, Trichostrongylus colubriformis was observed in 75 (75.8% of 99) of strongylid infected goats and Trichostrongylus axei in 4 (4.0%) of 99 goats and 2 (50.0%) of 4 deer. Based on the molecular results, co-infection of H. contortus and Trichostrongylus spp. (H. contortus + T. colubriformis denoted as HTC; H. contortus + T. axei denoted as HTA) were only found in goats. Specifically, HTC co-infections have higher rate (71 or 45.2% of 157) compared to HTA co-infections (3 or 1.9% of 157).ConclusionsThe present study is the first molecular identification of strongylid species among livestock in Malaysia which is essential towards a better knowledge of the epidemiology of gastro-intestinal parasitic infection among livestock in the country. Furthermore, a more comprehensive or nationwide molecular-based study on gastro-intestinal parasites in livestock should be carried out in the future, given that molecular tools could assist in improving diagnosis of veterinary parasitology in Malaysia due to its high sensitivity and accuracy.

Two New Species of Hapalorhynchus (Digenea: Spirorchiidae) from Freshwater Turtles (Testudines: Geoemydidae) in Malaysia

Abstract Hapalorhynchus snyderi n. sp. and Hapalorhynchus tkachi n. sp. are described from the circulatory systems of the Malayan snail-eating turtle, Malayemys subtrijuga (Testudines: Geoemydidae) and the black marsh turtle, Siebenrockiella crassicollis (Testudines: Geoemydidae), respectively. Both of the new species are described from hosts in the family Geoemydidae, while previous species reported from Malaysia (Hapalorhynchus macrotesticularis and Hapalorhynchus rugatus) were described from soft-shelled turtles (Family Trionychidae). Both new species can be differentiated from H. rugatus Brooks and Sullivan (1981) in that they lack the rugae on the hindbody that is characteristic of H. rugatus. Hapalorhynchus snyderi n. sp. is most similar to H. macrotesticularis (Rohde, Lee, and Lim, 1968) but is much smaller and differs from that species in a suite of morphometric criteria. In addition, H. snyderi possesses intestinal cecae that are simple and straight while the cecae in H. macrotesticularis are highly sinuous, particularly in the posttesticular region. Hapalorhynchus tkachi n. sp. can be differentiated from H. snyderi by its much smaller size (<1 mm in total length), the presence of a distinct constriction at the level of the acetabulum, and the more-posterior location of the ventral sucker.

Two New Species of Camallanus (Nematoda: Camallanidae) From Freshwater Turtles in Queensland, Australia

Abstract We describe 2 new species of Camallanus (Nematoda: Camallanidae) from freshwater turtles collected in Queensland, Australia: Camallanus nithoggi n. sp. from Elseya latisternum (Gray) and Camallanus waelhreow n. sp. from Emydura krefftii (Gray), Emydura macquarrii (Gray), and Em. macquarrii dharra Cann. The only Camallanus sp. previously reported from turtles is C. chelonius Baker, 1983 (all other species in the family have been transferred to Serpinema). The 2 new species described here differ from C. chelonius in the number of male preanal papillae (7 vs. 6 in C. chelonius), the number of male postanal papillae (5 vs. 4 in C. chelonius), and the number of buccal capsule ridges. Additionally, we removed the tissues overlying the buccal capsule and used scanning electron micrographs (SEM) to show that the peribuccal shields extend laterally from the buccal capsule, the basal ring is separated from the buccal capsule by a narrow isthmus, and there is a buttress along the lateral margin of the buccal capsule that has not previously been observed in species of Camallanus.

Seroepidemiology of Toxoplasmosis among People Having Close Contact with Animals.

A cross-sectional study was conducted to determine the seroepidemiology of Toxoplasma infection and its risk association among people having close contact with animals. A total of 312 blood samples were collected from veterinary personnel (veterinarian, technicians, and students) and pet owners from veterinary clinics and hospitals in the area of Klang Valley, Malaysia. About 4 cc of blood samples drawn from agreed participants were processed for measurement of anti-Toxoplasma IgG and IgM antibodies as well as avidity test of Toxoplasma IgG by ELISA I, II, and III kits. Meanwhile, the demographic profiles and possible risk factors of these participants were also recorded in the standardized data collection sheets. Overall seroprevalence of toxoplasmosis was observed in 62 (19.9%) participants being 7 (18.4%) in veterinarians, 15 (33.3%) in veterinary technicians, 29 (14.9%) in veterinary students, and 11 (31.4%) in pet owners. Of 19.9% Toxoplasma seropositive samples, 18.3% was positive for IgG antibody, 1.0% for IgM antibody, and 0.6% for both IgG and IgM antibodies. Of three different IgG avidity ELISA kits, ELISA III showed high avidity in all five seropositive samples (IgM and IgG/IgM antibodies) indicating chronic Toxoplasma infection which is consistent with no evidence of clinical toxoplasmosis diagnosed during the time of this study. Univariate analysis showed that age group, gender, study population, gardening, task performance, and working duration were significantly associated with Toxoplasma seropositivity. Further analysis by multivariate analysis using logistic regression showed that age group of ≥30 years old (OR = 0.34, 95% CI = 0.18–0.63, p = 0.001) and working or study duration of >10 years having close contact with animals (OR = 5.07, 95% CI = 1.80–14.24, p = 0.002) were identified as significant risks for Toxoplasma infection. Based on the results obtained, a comprehensive Toxoplasma screening and health surveillance program on toxoplasmosis should be implemented among people having close contact with animals in general and confirmed Toxoplasma seronegative individuals in particular to prevent seroconversion.

Redescription of Neopolystoma liewi Du Preez and Lim, 2000 (Monogenea: Polystomatidae), from Cuora amboinensis (Testudines: Geomydidae) with Notes on Specimen Preparation

Abstract Neopolystoma liewi Du Preez and Lim, 2000, is redescribed on the basis of specimens collected from the type host, Cuora amboinensis, in Malaysia during 2008. Morphometric comparison of the new specimens with the original description revealed significant differences because of differences in specimen preparation. The current material was killed in hot formalin without coverglass pressure, whereas the specimens from the original description were killed and fixed with coverglass pressure. Coverglass pressure resulted in increases of 30%–70% in most measurements of body organs. Sclerotized structures (genital and haptoral hooks and eggs) or structures with sclerotized supports (haptoral suckers) were not affected by coverglass pressure. The coefficient of variation of measurements from worms killed with hot formalin was low (<10%) for most structures. The authors recommend discontinuing the use of flattening in the preparation of monogenetic trematodes, with the exception of a small number of specimens necessary to accurately view structures not easily seen in unflattened material. Measurements from flattened specimens should not be used in species descriptions.

Prevalence and risk factors of Japanese encephalitis virus (JEV) in livestock and companion animal in high-risk areas in Malaysia

Japanese encephalitis (JE) is vector-borne zoonotic disease which causes encephalitis in humans and horses. Clinical signs for Japanese encephalitis virus (JEV) infection are not clearly evident in the majority of affected animals. In Malaysia, information on the prevalence of JEV infection has not been established. Thus, a cross-sectional study was conducted during two periods, December 2015 to January 2016 and March to August in 2016, to determine the prevalence and risk factors in JEV infections among animals and birds in Peninsular Malaysia. Serum samples were harvested from the 416 samples which were collected from the dogs, cats, water birds, village chicken, jungle fowls, long-tailed macaques, domestic pigs, and cattle in the states of Selangor, Perak, Perlis, Kelantan, and Pahang. The serum samples were screened for JEV antibodies by commercial IgG ELISA kits. A questionnaire was also distributed to obtain information on the animals, birds, and the environmental factors of sampling areas. The results showed that dogs had the highest seropositive rate of 80% (95% CI: ± 11.69) followed by pigs at 44.4% (95% CI: ± 1.715), cattle at 32.2% (95% CI: ± 1.058), birds at 28.9% (95% CI: ± 5.757), cats at 15.6% (95% CI: ± 7.38), and monkeys at 14.3% (95% CI: ± 1.882). The study also showed that JEV seropositivity was high in young animals and in areas where mosquito vectors and migrating birds were prevalent.