Entamoeba histolytica is the parasite responsible for amoebiasis, which can result in amoebic colitis or amoebic liver abscess. Metronidazole has been the conventional treatment for intestinal amoebiasis, but concerns regarding resistance have emerged due to the identification of resistance pathways in E. histolytica. This study investigates a novel anti-amoebic approach targeting the CDP-choline pathway. Inhibition studies were conducted using potential choline kinase (CK) inhibitors to inhibit the EhCK enzyme, and RNA interference was employed to knock down the EhCK gene. Km and Vmax of purified EhCK and hCKa2 proteins were determined by pyruvate kinase-lactate dehydrogenase (PK-LDH) coupled assay. The IC50 values for EhCK and hCKa2 were determined with several commercial CK inhibitors. Selected inhibitors were incubated with E. histolytica trophozoites for 48 hours to determine the EC50 for each inhibitor. Silencing of gene encoding EhCK was carried out using duplex siRNA and the gene expression level was measured by real-time qPCR. Based on the IC50 values, three of the inhibitors, namely CK37, flavopiridol and H-89 were more potent against EhCK than hCKa2. Trophozoites growth inhibition showed that only HDTAB, H-89 and control drug metronidazole could penetrate and induce cell death after 48-hour incubation. siRNA concentration of 10 µg/mL was used for the transfection of positive control GAPDH, EhCK, and non-targeting GFP siRNAs. RNAi experiment concluded with positive control GAPDH downregulated by 99% while the level of EhCK mRNA was downregulated by 47%. In this study, potential inhibitors of EhCK and siRNA have been identified, paving the way for further refinement and testing to enhance their potency against EhCK while sparing hCK. The utilization of these specific inhibitors and siRNA targeting EhCK represents a novel approach to impede the growth of E. histolytica by disrupting its phospholipid synthesis pathway.
The disease melioidosis, caused by the soil bacteria Burkholderia pseudomallei, often manifests as acute septicemia with high fatality. Glycogen synthase kinase-3β (GSK3β) plays a key role during the inflammatory response induced by bacteria. We used a murine model of acute melioidosis to investigate the effects of LiCl, a GSK3 inhibitor on experimental animal survivability as well as TNF-α, IL-1β, IFN-γ, IL-10 and IL-1Ra cytokine levels in blood, lung, liver and spleen of B. pseudomallei-infected mice. Our results showed that administration of 100 μg/g LiCl improved survivability of mice infected with 5 X LD50 of B. pseudomallei. Bacterial counts in spleen, liver and lungs of infected mice administered with LiCl were lower than non-treated controls. Our data also revealed that GSK3β is phosphorylated in the spleen, liver and lung of animals infected with B. pseudomallei. However in infected animals administered with LiCl, higher levels of pGSK3 were detected in the organs. Levels of proinflammatory cytokines (TNF-α, IL-1β and IFN-γ) and anti-inflammatory cytokines (IL-10 and IL-1Ra) in sera and organs tested were elevated significantly following B. pseudomallei infection. With GSK3β inhibition, pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β) were significantly decreased in all the samples tested whilst the levels of anti-inflammatory cytokines, IL-10 (spleen and lung) and IL-1Ra (spleen, liver and sera) were further elevated. This study represents the first report implicating GSK3β in the modulation of cytokine production during B. pseudomallei infection thus reiterating the important role of GSK3β in the inflammatory response caused by bacterial pathogens.
This study was conducted to determine the occurrence of Anaplasma spp. in the blood samples of cattle, goats, deer and ticks in a Malaysian farm. Using polymerase chain reaction (PCR) and sequencing approach, Anaplasma spp. was detected from 81(84.4%) of 96 cattle blood samples. All blood samples from 23 goats and 22 deer tested were negative. Based on the analysis of the Anaplasma partial 16S ribosomal RNA gene, four sequence types (genotypes 1 to 4) were identified in this study. Genotypes 1-3 showed high sequence similarity to those of Anaplasma platys/ Anaplasma phagocytophilum, whilst genotype 4 was identical to those of Anaplasma marginale/ Anaplasma centrale/ Anaplasma ovis. Anaplasma DNA was detected from six (5.5%) of 109 ticks which were identified as Rhipicephalus (formely known as Boophilus) microplus ticks collected from the cattle. This study reported for the first time the detection of four Anaplasma sequence types circulating in the cattle population in a farm in Malaysia. The detection of Anaplasma DNA in R. microplus ticks in this study provides evidence that the ticks are one of the potential vectors for transmission of anaplasmosis in the cattle.
To evaluate the effects of the juvenile hormone analogue pyriproxyfen on colonies of the Pharaoh ant Monomorium pharaonis (L.), peanut oil containing different concentrations (0.3, 0.6, or 0.9%) of pyriproxyfen was fed to monogynous (1 queen, 500 workers, and 0.1 g of brood) and polygynous (8 queens, 50 workers, and 0.1 g of brood) laboratory colonies of M. pharaonis. Due to its delayed activity, pyriproxyfen at all concentrations resulted in colony elimination. Significant reductions in brood volume were recorded at weeks 3 - 6, and complete brood mortality was observed at week 8 in all treated colonies. Brood mortality was attributed to the disruption of brood development and cessation of egg production by queens. All polygynous colonies exhibited significant reduction in the number of queens present at week 10 compared to week 1. Number of workers was significantly lower in all treated colonies compared to control colonies at week 8 due to old-age attrition of the workers without replacement. At least 98.67 ± 1.33% of workers were dead at week 10 in all treated colonies. Thus, treatment with slow acting pyriproxyfen at concentrations of 0.3 - 0.9% is an effective strategy for eliminating Pharaoh ant colonies.
The hepatitis C virus (HCV) is a blood-borne pathogen that causes acute or chronic infection of the liver, sometimes leading to serious liver damage and fatality. The objective of this study was to evaluate HCV prevalence in patients attending the Regional Training and Research Hospital for Medical Examination and Surgery in Samsun Province of Turkey between 2014 and 2017. Blood specimens taken from 152 596 patients were screened for HCV infection by using the anti-HCV assay. Seropositive samples were subjected to polymerase chain reaction (PCR) testing in order to determine whether the HCV infection was active. Genotyping was then performed. Overall, HCV seropositivity and active HCV infection were 2.76% and 2.05%, respectively. Foreign nationals accounted for 5.61% of the seropositive samples and 1.37% of active HCV infective samples. We further report that 2017 was the year with the highest seroprevalence which was 3.64%. HCV genotype 1 was the most common genotype detected in residents of Samsun Province at 89.86%, followed by Genotype 3 at 4.54%. This study provides important information on the levels of HCV infection in the Samsun region of Turkey. The data indicate that there was a rising trend of HCV infection between 2014 and 2017.
Sarcocystosis, an unusual parasitic zoonotic disease, is caused by coccidian/ apicomplexan protozoa in humans and animals. The parasites usually develop in a heteroxenous predator-prey life-cycle involving final (carnivore) and intermediate (omnivore/herbivore) hosts. Besides the intestinal, non-invasive form of the disease in which humans and animals are the definitive hosts for certain Sarcocystis spp., the invasive form has come to recent attention. In the latter, humans and animals serve as intermediate host harbouring sarcocysts in their muscle tissue. Already in 1991 sarcocystosis was seen as a potential emerging food borne zoonosis in Malaysia, and in 2011 and 2012 the largest cluster of symptomatic human muscular sarcocystosis world-wide was reported from Tioman Island, Pahang state. In this review, we focus on invasive sarcocystosis in humans and animals in Malaysia, review the recorded cases and epidemiology, and present hypotheses.
The purification of parasite-infected erythrocytes from whole blood containing leucocytes is crucial for many downstream genetic and molecular assays in parasitology. Current methodologies to achieve this are often costly and time consuming. Here, we demonstrate the successful application of a cheap and simple Non-Woven Fabric (NWF) filter for the purification of parasitized red blood cells from whole blood. NWF filtration was applied to the malaria-parasitized blood of three strains of mice, and one strain of rat, and to Babesia gibsoni parasitized dog blood. Before and after filtration, the white blood cell (WBC) removal rates and red blood cell (RBC) recovery rates were measured. After NWF filter treatment of rodent malaria-infected blood, the WBC removal rates and RBC recovery rates were, for Kunming mice: 99.51%±0.30% and 86.12%±8.37%; for BALB/C mice: 99.61%±0.15% and 80.74%±7.11%; for C57 mice: 99.71%±0.12% and 84.87%±3.83%; for Sprague-Dawley rats: 99.93%±0.03% and 83.30%±2.96%. Microscopy showed WBCs were efficiently removed from infected dog blood samples, and there was no obvious morphological change of B. gibsoni parasites. NWF filters efficiently remove leukocytes from malaria parasite-infected mouse and rat blood, and are also suitable for filtration of B. gibsoni-infected dog blood.
Black soldier fly (Hermetia illucens (Linnaeus, 1758)) larvae inhibit oviposition of house fly (Musca domestica Linnaeus, 1758) by releasing a semiochemical, although in some situations, inhibition is only partial. We hypothesized that there is a certain period in the life cycle of black soldier fly when it can cause antibiosis of the house fly. Choice and non-choice tests were used to separately examine the effectiveness of each developmental stage of black soldier fly (egg, larval (phase I, II, and III), prepupal and pupal stages) and crude extract of larvae on house fly oviposition. Tests using each developmental stage were compared with controls lacking black soldier fly. The effects of black soldier fly on the number of newly hatched house fly larvae were evaluated and there was a significant difference between the test and control in the number of eggs laid by house fly for all phases of the black soldier fly larval stage. Strong inhibition was found in some black soldier fly larval phases. Significant differences in the numbers of house fly eggs oviposited in food containers treated with crude extract were found when compared with a control, confirming that chemicals from black soldier fly larvae resulted in inhibition of oviposition of house fly. The results from experiment also indicated that chemicals from black soldier larvae influenced the number of house fly larvae newly hatched from eggs.
House dust mites and storage mites are well-known causes for allergenic diseases. The aim of this study was to investigate the immunogenic sites of Blomia tropicalis, Aleurogyphus ovatus and Glycycometus malaysiensis. The mites were maintained in a culture medium at 25ºC and 75% relative humidity. Mites were harvested either with heat escape or floatation method, purified, homogenized, quantified and used for the production of polyclonal antibody and immunostaining. For each species of mites, five male mice and five male rats were randomly selected and immunized intraperitoneally with respective crude mite extract at two-weekly intervals. Blomia tropicalis, A. ovatus or G. malaysiensis whole mites and paraffin-embedded mite sections were immunostained with the respective polyclonal antibody. The faecal pellets of mites were intensely stained for all the three species in the present study. The legs of sectioned A. ovatus were not immunogenic as compared with those of G. malaysiensis and B. tropicalis. The outer layer (cuticle) of whole mites and the eggs for these species were very immunogenic. Hence, the polyclonal antibodies obtained in this study may serve as potential tools in detecting the eggs and immature mites in environmental samples. Future studies should focus on the antigenic components of eggs since they were relatively abundant in dust and highly antigenic as seen in the present study.
Blow flies, flesh flies, and house flies can provide excellent evidence for forensic entomologists and are also essential to the fields of public health, medicine, and animal health. In all questions, the correct identification of fly species is an important initial step. The usual methods based on morphology or even molecular approaches can reach their limits here, especially when dealing with larger numbers of specimens. Since machine learning already plays a major role in many areas of daily life, such as education, business, industry, science, and medicine, applications for the classification of insects have been reported. Here, we applied the decision tree method with wing morphometric data to construct a model for discriminating flies of three families [Calliphoridae, Sarcophagidae, Muscidae] and seven species [Chrysomya megacephala (Fabricius), Chrysomya rufifacies (Macquart), Chrysomya (Ceylonomyia) nigripes Aubertin, Lucilia cuprina (Wiedemann), Hemipyrellia ligurriens (Wiedemann), Musca domestica Linneaus, and Parasarcophaga (Liosarcophaga) dux Thomson]. One hundred percent overall accuracy was obtained at a family level, followed by 83.33% at a species level. The results of this study suggest that non-experts might utilize this identification tool. However, more species and also samples per specimens should be studied to create a model that can be applied to the different fly species in Thailand.
Entamoeba histolytica causes amoebic diarrhoea, colitis and liver abscess (ALA). Diagnosis of ALA is difficult, as most patients do not have simultaneous intestinal amoebic infection. At Hospital Universiti Sains Malaysia (HUSM), diagnosis of ALA relies on a combination of clinical findings, ultrasound examination of the liver and serodiagnosis using a commercial kit. In this study, two in-house indirect ELISAs were developed and evaluated. One of the in-house assays utilises E. histolytica crude soluble antigen (CSA) to detect serum IgG specific to the parasite whereas the other uses E. histolytica ether extract antigen (EEA). Preparation of CSA requires a sonicator to lyse the amoeba whereas EEA was prepared by chemically solubilizing the trophozoites. Based on the cut-off value of mean optical density + 3SD, CSA-ELISA showed 100% (24/24) sensitivity and 93.33% (210/225) specificity; while EEA-ELISA showed 91.67% (22/24) sensitivity and 95.11% (214/225) specificity. In conclusion, both the in-house indirect ELISAs were found to be efficacious for diagnosis of ALA; and the EEA is easier to prepare than the commonly used CSA.
Entamoeba histolytica causes about 50 million infections worldwide with a death rate of over 100,000 annually. In endemic developing countries where resources are limited, microscopic examinations based on Wheatley trichrome staining is commonly used for diagnosis of intestinal amoebiasis. Other than being a time-consuming method, it must be performed promptly after stool collection as trophozoites disintegrate rapidly in faeces. The aim of this study was to compare the efficacies of Eosin-Y, Wheatley trichrome and Iodine stains in delineating the diagnostic features of the parasite, and subsequently to determine the suitable microscopy observation period for detection of erythrophagocytic and non-erythrophagocytic trophozoites spiked in semi-solid stool sample. Wheatley trichrome staining technique was performed using the standard method while the other two techniques were performed on the slides by mixing the respective staining solution with the spiked stool sample. One million of axenically cultured non-erythrophagocytic E. histolytica and erythrophagocytic E. histolytica were separately spiked into 2 g of fresh semisolid faeces. Percentage viability of the trophozoites in the spiked stool sample was determined at 30 minute intervals for eight hours using the 0.4% Trypan blue exclusion method. The results showed that Eosin-Y and Wheatley trichrome stained the karyosome and chromatin granules better as compared to Iodine stain. The percentage viability of non-erythrophagocytic trophozoites decreased faster than the erythrophagocytic form in the first 5 hours and both dropped to ~10% in the 6th hour spiked sample. In conclusion, Eosin-Y staining technique was found to be the easiest to perform, most rapid and as accurate as the commonly used Wheatley trichrome technique; Eosin-Y stained slide sealed with DPX could also be kept as a permanent record. A period not exceeding 6 hours after stool collection was found to be the most suitable in order to obtain good microscopy results of viable trophozoites.
The study was aimed to investigate the expression of cytosolic and thiolated proteins of Musca domestica larvae under oxidative stress. Proteins from acute treatment of hydrogen peroxide (LC50 = 21.52% (v/v)) on 3rd stage larvae of housefly were extracted and purified using an activated Thiol Sepharose® for thiolated protein purification. Two dimensional gel electrophoresis was used for visualizing and analyzing expression of cytosolic and thiolated proteins. Protein spots with more than 5 fold of expression change were identified using liquid chromatography- tandem mass spectrometry (LC-MS/MS). The cytosolic proteins were actin, tropomyosin, ubiquitin, arginine kinase, pheromone binding protein/general odorant binding protein, and ATP: guanidino phosphotransferase. The thiolated proteins with more than 5 fold change in expression as an effect to the acute treatment were fructose bisphosphate aldolase, short chain dehydrogenase and lactate/malate dehydrogenase. The proteins identified in the study should provide vital information for future reference in oxidative stress defence and response occurring in houseflies.
Iranihindia martellata (Senior-White, 1924) is recorded from peninsular Malaysia for the first time. Male and female specimens in the recent collections of forensically important sarcophagid flies were examined and identified based on morphology and DNA sequencing analysis. Male genitalia offer unambiguous species identification characteristics in the traditional taxonomy of flesh flies but the female flies are very similar to one another in general morphology. Female of I. martellata was determined by DNA sequencing (COI and COII) and PCR-RFLP (COI) analysis. Identified females were carefully examined and compared with the morphologically similar species, Liopygia ruficornis (Fabricius, 1794). Female genitalia are re-described and illustrated in this paper.
The mitochondiral DNA region encompassing the cytochrome oxidase subunit I (COI) and cytochrome oxidase subunit II (COII) genes of two Malaysian blow fly species, Chrysomya megacephala (Fabricius) and Chrysomya rufifacies (Macquart) were studied. This region, which spans 2303bp and includes the COI, tRNA leucine and partial COII was sequenced from adult fly and larval specimens, and compared. Intraspecific variations were observed at 0.26% for Ch. megacephala and 0.17% for Ch. rufifacies, while sequence divergence between the two species was recorded at a minimum of 141 out of 2303 sites (6.12%). Results obtained in this study are comparable to published data, and thus support the use of DNA sequence to facilitate and complement morphology-based species identification.
Canine babesiosis caused by Babesia spp. is a noteworthy tick-borne zoonotic disease of domestic dogs and wild canids. In present study, a total of 556 blood samples were randomly collected from pet dogs in eight cities of Hunan province, subtropical China. Genomic DNA was extracted and Babesia DNA was detected by amplification of partial 18S rRNA gene sequences. A total of 56 (10.1%) blood samples were tested positive for Babesia species. Sequence analysis showed that 29 dogs (5.2%) were positive for B. gibsoni, and other 27 dogs for B. vogeli (4.9%). The age and health status were considered as important risk factors for B. gibsoni and B. vogeli infections in pet dogs in this study (P<0.05). Phylogenetic analysis showed that the examined positive samples were highly clustered in the same branch with B. gibsoni and B. vogeli, respectively. This is the first molecular report of B. gibsoni infection in pet dogs in Hunan province, subtropical China. Our finding has provided a guide for the control of dog babesiosis in China and elsewhere.
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.
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.
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.
Circumsporozoite protein (CSP) central repeat region is one of the main target regions of the RTS,S/AS01 vaccine for falciparum infection as it consists of immunodominant B cell epitopes. However, there is a lack of study for P. knowlesi CSP central repeat region. This study aims to characterise the CSP repeat motifs of P. knowlesi isolates in Peninsular Malaysia. CSP repeat motifs of 64 P. knowlesi isolates were identified using Rapid Automatic Detection and Alignment of Repeats (RADAR). Antigenicity of the repeat motifs and linear B cell epitopes were predicted using VaxiJen 2.0, BepiPred-2.0 and BCPred, respectively. A total of 35 dominant repeat motifs were identified. The repeat motif "AGQPQAQGDGANAGQPQAQGDGAN" has the highest repeat frequency (n=15) and antigenicity index of 1.7986. All the repeat regions were predicted as B cell epitopes. In silico approaches revealed that all repeat motifs were antigenic and consisted of B cell epitopes which could be designed as knowlesi malaria vaccine.