This study was conducted to determine the prevalence of intestinal parasites among children and adult Orang Aslis (Aborigines) from different locations in Perak. Faecal samples were collected and analyzed using the direct smear and formal ether sedimentation technique. Some of the faecal samples were stained using the Modified Acid fast stain for Cryptosporidium. Nail clippings of the respondents and the soil around their habitat were also analyzed. Of the 77 stool samples examined, 39 (50.6%) were positive for at least one intestinal parasite. The most common parasite detected was Trichuris trichiura (39.0%) followed by Ascaris lumbricoides (26.9%), Entamoeba coli (5.2%), Giardia lamblia (5.2%), Blastocystis hominis (3.9%), hookworm (3.9%), Entamoeba histolytica (1.3%), Iodamoeba butschlii (1.3%) and Cryptosporidium sp. (1.3%) respectively. Some respondents had single parasites (24.7%), some with two parasites (18.2%). Some with three parasites (6.5%) and one had four parasites species (1.3%). The parasites were slightly more common in females (54.7%) than males ((41.7%). The parasites were more common in the 13-20 year age group (90.9%) followed by 1-12 years (69.6%), 21-40 year age group (34.8%) and least in the 41-60 year age group (27.8%). Nail examinations of the respondents did not show any evidence of parasites. One had a mite, three had pollen grains and one had yeast cells isolated from the finger nails. Soil samples taken around their houses showed only one sample with a nematode ova and one with oocyst which was of a non human origin.
GRA4 of Toxoplasma gondii has been shown to prompt IgG, IgM and IgA responses in previous studies and is thus considered one of the major immunogenic proteins from T. gondii that can be used for both diagnostics purposes and vaccine development. This study seeks to clone and express the GRA4 in Pichia pastoris, which has numerous advantages over other systems for expression of eukaryotic proteins. In order to achieve this, the gene was cloned into the pPICZα A expression vector, which was then incorporated into the P. pastoris genome via insertional integration for expression of the recombinant protein, under the AOX1 promoter. The antigen was expressed along with the prepro sequence of the α-factor of yeast so that it could be excreted out of the P. pastoris cells and obtained from the medium. Upon SDS-PAGE analysis it was found that the recombinant protein was expressed optimally as a 40 kDa protein after 96 hours of induction with 0.75% of methanol. The expressed GRA4 protein showed discrepancy in size with the calculated molecular mass. This may be attributed to the various posttranslational modifications including glycosylation and phosphorylation. Despite the difference in molecular weight, the recombinant protein was able to detect toxoplasmosis in Western blot format. The recombinant GRA4 was expressed with an intact polyhistidine-tag, which could be used for future purification of the antigen.
Toxoplasmosis is a protozoal infection of zoonotic potential with worldwide geographical distribution which affects nearly all warm-blooded animals including mammals and birds. Keeping in view, this study was conducted to determine the seroprevalence of toxoplasmosis along with associated risk factors and its haematological impacts in small ruminants of district Multan, Pakistan. In this study, a total of 250 sera samples collected from sheep (n=125) and goats (n=125) from three tehsils of Multan were examined using commercially available Latex agglutination test kit for the presence of anti-T. gondii antibodies. The haematological profiles of Toxoplasma seropositive and seronegative animals were determined by using automated haematology analyser. Overall seroprevalence of toxoplasmosis in small ruminants was 42.80% with a higher prevalence rate (44.80%) in sheep as compared to goats (40.80%). Sex, existence of co-morbid conditions, feeding pattern and presence of pet cats and dogs were identified as significant (P<0.05) risk factors associated with the presence of antibodies against toxoplasmosis. The breed was found to be a significant (P=0.026) risk factor for the seroprevalence of toxoplasmosis in goats but not in sheep. Haematological analysis revealed significantly altered leukocytic counts (P<0.05) in seropositive sheep and goats as compared to seronegative ones. Our findings showed that small ruminants of the Multan District in Pakistan are toxoplasma seropositive and may pose a serious threat of public health concern in the region.
Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64- Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.
The objectives of this study were to investigate the prevalence and associated risk factors for gastrointestinal (GI) parasites in buffaloes from various areas of Sarawak, and to assess current management practices of GI parasites among farmers. Faecal samples were collected from 15 farms and 129 animals, as well as data on farm and animal-based characteristics. A total of 129 faecal samples were examined for GI parasites using a modified McMaster and sedimentation. Association between potential risk factors and the prevalence of GI parasites was investigated using Chi-square statistic. The prevalence of Paramphistomum sp., strongyles, and coccidia were 75.2% (95% CI±7.5), 52.7% (95% CI±8.6) and 48.1% (95% CI±8.6), respectively. Farms which had a grazing area less than 50 acres in size had significantly higher prevalence of strongyles (70.5%, χ2 = 8.34, P = 0.004) and paramphistomes (88.6%, χ2 = 6.46, P = 0.01) relative to farms with a larger grazing area (43.5% and 68.2%, respectively). Prevalence of strongyles was lower in farms that did not implement a cut- and-carry system (45.6%, χ2 = 4.17, P = 0.04) in comparison to those that did (64%). The prevalence of paramphistomes was higher on farms with more than 40 animals (80.6%, χ2 = 3.18, P = 0.05) relative to farms with fewer animals. The majority of farmers surveyed (67.9%) showed awareness of GI parasite infection and reported that they recognized the associated symptoms. Most farmers practised deworming, and ivermectin was the most commonly used anthelminthic (60.4%); only 1.9% of farmers used albendazole. Overall this study revealed a high prevalence of GI parasites in buffalo in Sarawak. Although farmers report they are aware of parasitic diseases, further education is still required. This could include how they can successfully implement on-farm changes to reduce the prevalence of GI parasites in their herds.
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.
This study was aimed to identify and characterize Leishmania amastigote, and axenic form antigens. Two in vitro techniques were used to change leishmania parasite isolates from promastigote form to amastigotes and amastigote like (axenic) forms. The main strategy relied upon in vitro infection of murine macrophages cell line J774 with leishmania promastigote, at 37°C with 5% CO2, while the second technique relied upon the culture of promastigote at 37°C with low pH (5.5), and 5-10% CO2. Proteins were extracted and fractionated utilizing 12% Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE). Antigens were recognized using both immune dot blot and western blot procedures. PCR was performed for recognition of leishmania parasites in infected J774 macrophages. L. major was quicker in infectivity of macrophages cell line than L. donovani. Shared proteins ranging from 26-116 kDa were identified by SDS PAGE in all stages. Immune Dot-blot method showed positive outcomes, while western blot identified an exceptional antigen band of 16 kDa in amastigote, this unique band could be of value in diagnosis and vaccination of leishmaniasis. PCR results confirmed presence of both isolates demonstrating that coinfection is conceivable, and no indications of hereditary recombination at kinetoplast DNA (kDNA) were identified in macrophages simultaneously infected by L. major and L. donovani.
Toxocara canis is a major parasite that infects many animals with high risk of human infections. This study aims at assessing the immunization with gamma radiationattenuated infective stage on rats challenged with non-irradiated dose. Level of vaccine protection was evaluated in liver and lung regarding parasitological, histopathological, biochemical and molecular parameters. Fifty rats were enrolled in three groups: group A (10 rats) as normal control; group B (20 rats) subdivided into subgroup B1 (infected control) and subgroup B2 infected then challenged after 14 days with the same dose of infection (challenged infected control); and group C (20 rats) subdivided into subgroup C1 vaccinated with a dose of 800 gray (Gy) gamma-radiated infective eggs (vaccine control) and subgroup C2 vaccinated then challenged on 14th day with same number of infective eggs (vaccinated-challenged). Tissues were stained with Haematoxylin and Eosin (H and E) for histopathological studies. Biochemical studies through detection of nitric oxide (NO) and Caspase-3 were conducted. Extent of DNA damage by Comet assay was assessed. Vaccinated-challenged subgroup revealed a marked reduction in larvae in tissues with mild associated histological changes. In addition there was accompanied reduction of NO, Casepase-3 level and DNA damage compared to the control infected group. It could be concluded that vaccination of rats with a dose of 800Gy gamma radiation-attenuated infective stage improves immune response to challenge infection and drastically reduces the morbidity currently seen.
Hemoprotozoans are important pathogens of animals and humans, among which some species have zoonotic significance. The prevalence of different hemoprotozoa and Anaplasma spp. in larger mammals have been reported from different regions of the world. But, very few studies have been conducted to estimate the prevalence of hemoprotozoa in rodents and shrews of South-East Asia. The study assessed the prevalence of hemoprotozoa and Anaplasma spp. in rodents and shrews of Bangladesh. Blood samples (n=451) were collected from rodents and shrews between June 2011 and June 2013 and July-December 2015 from 4 land gradients of Bangladesh. Giemsa-stained blood smears revealed that 13% of animals were harboring hemoprotozoa (4.7% Babesia spp., 0.67% Plasmodium spp.), and Anaplasma spp. (7.5%). The study may serve as a guide for future hemoparasitic research of rodents and shrews.
Previously, we have identified a gene encoding thrombospondin-related anonymous protein of Babesia gibsoni (BgTRAP), and have shown that the antisera raised against recombinant BgTRAP expressed in Escherichia coli inhibited the growth of parasites. In the present study, a recombinant vaccinia virus expressing the BgTRAP (VV/BgTRAP) was constructed. A specific band with a molecular mass of 80 kDa, which is similar to that of native BgTRAP on the merozoites of B. gibsoni, was detected in the supernatant of VV/ BgTRAP-infected RK13 cells. Mice inoculated with VV/BgTRAP produced a specific antiBgTRAP response. The antiserum against VV/BgTRAP showed reactivity against the native BgTRAP on parasites. These results indicated that the recombinant vaccinia virus expressing BgTRAP might be a vaccine candidate against canine B. gibsoni infection.
Sudanese mucosal leishmaniasis (ML) is a rare clinical form of leishmaniasis and characterized by persistent ulcer of the oral and/or the nasal mucous membranes caused by Leishmania donovani. No data is available about the systemic and local immune responses in mucosal leishmaniasis. This study aimed to measure the systemic and the local cytokines responses of Sudanese ML patients compared to cured cutaneous leishmaniasis patients (Leishmanin skin test positive, LST+ve) and unexposed healthy controls (Leishmanin skin test negative, LST-ve). Six parasitological confirmed ML patients, 7 LST+ve, and 6 LST-ve were enrolled. Systemic Th-1 (IFN-γ and TNF-α), Th-2 (IL-10 and IL-13), Treg (TGF-β1), and inflammatory cytokines IL-6 and IL-8 concentration were measured in the supernatant of whole blood samples following stimulation with live L. donovani promastigotes using ELISA. Local intralesion IL-10, IFN-γ, and IL-13 expression was measured using Real Time PCR. A significant high concentrations of IFN-γ, TNFα, IL-10, TGFβ, IL-6, and IL-8 were detected in the supernatant of stimulated whole blood samples of ML patients compared with the LST+ve and LST-ve controls. Using Real Time-PCR and primers for various cytokines, a significant high expression of TH2 cytokines IL-10 and IL-13 mRNA was detected in contrast to a low TH1 cytokine IFN-γ mRNA in the mucosal lesion. There is a clear dichotomy in the cytokine response during Mucosal leishmaniasis. A significantly high TH1, inflammatory and Treg cytokines response is produced systemically, in contrast to a significant high TH2 cytokines response in the mucosal lesion.
Ever since the first reported case series on SARS-CoV-2-induced neurological manifestation in Wuhan, China in April 2020, various studies reporting similar as well as diverse symptoms of COVID-19 infection relating to the nervous system were published. Since then, scientists started to uncover the mechanism as well as pathophysiological impacts it has on the current understanding of the disease. SARS-CoV-2 binds to the ACE2 receptor which is present in certain parts of the body which are responsible for regulating blood pressure and inflammation in a healthy system. Presence of the receptor in the nasal and oral cavity, brain, and blood allows entry of the virus into the body and cause neurological complications. The peripheral and central nervous system could also be invaded directly in the neurogenic or hematogenous pathways, or indirectly through overstimulation of the immune system by cytokines which may lead to autoimmune diseases. Other neurological implications such as hypoxia, anosmia, dysgeusia, meningitis, encephalitis, and seizures are important symptoms presented clinically in COVID-19 patients with or without the common symptoms of the disease. Further, patients with higher severity of the SARS-CoV-2 infection are also at risk of retaining some neurological complications in the long-run. Treatment of such severe hyperinflammatory conditions will also be discussed, as well as the risks they may pose to the progression of the disease. For this review, articles pertaining information on the neurological manifestation of SARS-CoV-2 infection were gathered from PubMed and Google Scholar using the search keywords "SARS-CoV-2", "COVID-19", and "neurological dysfunction". The findings of the search were filtered, and relevant information were included.
African swine fever (ASF) is a transboundary haemorrhagic viral disease that affected domestic and wild pigs of all ages. The disease is caused by African swine fever virus (ASFV) and was introduced to China in 2018 before spreading rapidly to neighbouring Asian countries. As such, putting countries free from ASF like Malaysia at risk. ASF is highly lethal with no vaccine or treatment available. In February 2021, we confirmed backyard pigs from various locations in Sabah were infected with ASF using real time polymerase chain reaction (real- time PCR). Further characterization of the Sabah ASFVs indicated that they were of p72 genotype II with intergenic region (IGR) variant II that displayed an addition tandem repeat sequence (TRS) insertion, similar to ASFV from Indonesia, V ietnam and China. These results indicate and support the transboundary expansion of a homogenotypic ASFV (p72 genotype II and IGR variant II) in the Europe and Asia-Pacific, emphasizing the need for a holistic international collaboration in control and preventing further spreading of the current ASF pandemic. Importantly, our results informed the first detection and characterization of ASF, a disease previously not detected in Malaysia. This information is crucial for further mitigation and preventive measures.
Toxoplasma gondii is an obligate intracellular protozoon which causes toxoplasmosis, an important zoonotic disease that is endemic worldwide. Common sources of T. gondii infection in humans are food or water contaminated with oocysts and raw or undercooked meat with cysts. In animals, common sources of infection include feed, water, or litter contaminated with oocysts. The diagnosis and molecular characterization of T. gondii infection in humans and animals is crucial due to public and veterinary health importance. Various traditional and serological methods have been used in clinical practice for toxoplasmosis diagnosis, but interpreting the results remains a challenge. Several molecular techniques have also been used for the detection and genetic characterization of T. gondii , but primarily in research settings. In this paper, we review the techniques that are currently used for the diagnosis and genetic characterization of T. gondii in humans and animals, along with their advantages and disadvantages. The techniques reviewed have laid the groundwork for the future development of more effective and precise detection and characterization of T. gondii . These advances will contribute to a better understanding of epidemiology, prevention and control of toxoplasmosis. Thus, this review would be of particular interest to clinical physicians, veterinarians and researchers.
Aedes albopictus poses a public health risk in tropical countries and temperate countries in recent decades due to its capability to transmit various human arboviruses including dengue, yellow fever, and chikungunya. Vector control is the key for preventing transmission of these pathogenic viruses. Improving the effectiveness of currently utilized collection methods, such as ovitraps, is important for best species abundance monitoring, assessment of the threat of arbovirus transmission, and optimizing control activities. Therefore, this study aimed to assess the potential use of lactic acid bacteria (LAB) waste as an infusion-baited ovitrap for Aedes collection. The performance of overnight tap water, grass hay infusion and LAB waste infusion were compared for their ability in attracting gravid female Ae. albopictus. In this study, the LAB waste infusion was substantially more alluring to Ae. albopictus mosquitoes than the two controls grass hay infusion and tap water.
Emerging cases of Fasciola and Paramphistomes co-infection have been reported, especially in tropical regions. Thisis due to Fasciola and Paramphistomes sharing biological factors which influence the pattern of transmission, especially in faecal egg shedding due to interaction and competition in the definitive host. Most reports surveyed the occurrence of fasciolosis in ruminants with a lack of observation of faecal egg distribution. Therefore, present study is aimed to assess the distribution of Fasciola and Paramphistomes faecal egg count (fec) in co-infected large ruminants in Larut, Matang, and Selama areas (Taiping). A total of 371 faecal samples were collected at random from 23 ruminant herds. Flukefinder® sedimentation was used to quantify the Fasciola and Paramphistomes eggs. Descriptive analyses were performed to determine the prevalence of co-infections, and Spearman correlation analysis was used to correlate the fec. Overall, the prevalence of Fasciola and Paramphistomes co-infection was 23.7% (n=89/371) in Taiping. Prevalence of paramphistomosis was always higher than fasciolosis in overall and single infection, with 46.9% (n=174/371) and 22.9% (n=85/371) compared to 36.9% (n=137/371) and 12.9% (n=48/371) respectively. Egg per gram (epg) of both parasites were positively skewed with a median of 1.5 epg in fasciolosis and 10.5 epg in paramphistomosis. Spearman correlation analysis of the epg in co-infected bovine was found to have a moderately positive correlation with rs=0.39 (p-value<0.01). The recent study observed a moderate prevalence of Fasciola and Paramphistomes coinfection in a large ruminant population from Taiping, with the prevalence of paramphistomosis being higher than fasciolosis. Hence, this suggests that infection with one of these parasites increases the chance of infection with another. There is a need to integrate fec in parasite surveillance to monitor the trend of parasite transmission. Findings in the present study could tailor control strategies, especially for fasciolosis to limit the economic loss and prevent zoonotic transmission.
We have previously isolated a novel avian Orthobunyavirus, Kedah Fatal Kidney Syndrome (KFKS) virus from a broiler farm in Kedah, Malaysia in 2020 with a severe kidney lesion in chickens. The virus was designated as KFKS2_CS virus. Sequence analysis of partial nucleocapsid (N) and nonstructural (NSs) sequence of this virus showed the highest sequence identity with previous KFKS1 from Malaysia (100%) and 97% with a zoonotic Umbre (UMB) virus, which was reported to cause encephalitis in immunocompromised humans in India. Phylogenetic analysis revealed that this virus was clustered together with previous KFKS1 virus from Malaysia, UMB and Cristoli viruses. This study aimed to assess the zoonotic potential of this KFKS2_CS virus in vitro by determining its ability to inhibit the production of interferon (IFN) in human glioblastoma multiforme (GBM) brain cells using reverse-transcriptase polymerase reaction (RT-PCR). This virus blocked the production of interferon-a in this human brain cells. In conclusion, this KFKS2_CS virus may have a zoonotic potential and become a public health concern in the future.
The parasites which occurred most frequently in 175 owned or stray dogs in Sabah were Ancylostoma spp. present in 68% of the animals. Dirofilaria immitis occurred in 70% of the adult dogs but neither D. immitis nor Spirocerca lupi were present in puppies under four months of age. The latter attained a prevalence of 30% in the adults. In contrast Toxocara canis occurred in 81% of the puppies but infrequently in older dogs. Dipylidium caninum was moderately prevalent (15 to 25%) in dogs of all ages. Ticks were the most common arthropod parasite being present on 26% of the dogs and were mainly Rhipicephalus sanguineus. Demodectic and sarcoptic mange were confirmed and fleas and lice were also recovered.
An investigation into the seasonal changes in the population structure of Haemonchus and Trichostrongylus in tracer goats was conducted over 12 months at Serdang, an area in southern West Malaysia with year-round tropical rainfall. Successive groups of parasite-free tracer goats were grazed for a month alongside naturally infected adult goats and necropsied for worm counts 14 days after their removal from pasture. No hypobiotic larvae of Trichostrongylus were recovered. Hypobiotic larvae of Haemonchus were evident during each month of the year but accounted for only a very small proportion of the total Haemonchus burden. Very low levels were encountered from December through to June. Comparatively higher levels of hypobiosis were observed thereafter with a peak of 7.4% in September. The factors responsible for hypobiosis were not clearly defined but the phenomenon was associated with increasing levels of soil moisture storage. Host resistance, adult worm population of tracers and population of ingested L3 were ruled out as possible inducing factors.
Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis, a debilitating chronic disease of sheep and goats. Little is known about the buck's reproductive pathophysiology with respect to inoculation with Corynebacterium pseudotuberculois and its immunogen mycolic acid extract. Therefore, this present study was designed to determine the concentration of testosterone hormone, pro-inflammatory cytokines, and semen quality of the experimental animals. A total of 12 bucks, divided into groups 1, 2, and 3 (Negative control group, Positive control group and Mycolic acid group respectively), were enrolled in this study. Following inoculation, all goats were observed for clinical responses and monitored for 60 days post-challenge and were then sacrificed. Blood samples were collected via the jugular once before inoculation and on a weekly basis post-challenge. Semen samples were collected 2 weeks post-challenge and prior to the sacrifice of the experimental animals. During the post inoculation period of 60 days, the concentration of testosterone hormone for group 2 was increased significantly (p 0.05) but increased significantly (p 0.05) as compared to group 1. The concentration of interferon-γ (IFNγ) significantly increased (p 0.05) compared to group 1. Both group 2 and group 3 showed a reduction in semen qualities as compared to group 1, but the severity was more intense in group 2 if compared to group 3. In conclusion, therefore, the present study concluded that the mycolic acid group revealed significant responses of testosterone hormone concentration, semen quality, and its related pro-inflammatory cytokines in bucks following infection but the severity lesser compared to Corynebacterium pseudotuberculosis group.