Contagious bovine pleuropneumonia (CBPP) is a highly contagious disease of cattle caused by Mycoplasma mycoides subsp. mycoides. It is characterized by anorexia, fever, dyspnea, polypnea, cough, and nasal discharges. Gross lesions in the lung such as marbling, sequestra, thickening of interlobular septa, and consolidation are evident. Serological tests including complement fixation test and competitive enzyme-linked immunosorbent assay and molecular tests such as polymerase chain reactions are used for diagnostic purposes. In this study, lung samples of suspected large ruminants (cattle n=560, buffalo n=293) were collected from abattoirs of three districts of Punjab namely Lahore, Kasur and Jhang. PCR was performed with specific primers, targeting the 16S ribosomal RNA gene to detect the positive cases. The results indicated that 49 samples (8.75%) of cattle were positive, with maximum prevalence was observed in Jhang with 16 positive samples (10.06%), but CBPP was not detected in any buffalo sample. High prevalence of disease was seen in cattle of more than seven years of age, in female cattle, and in cross-bred cattle. Age and gender were found significantly associated (P<0.05) with the prevalence of the disease. Gene sequencing of identified 5 isolates of Mycoplasma mycoides subsp. mycoides had more than 99% similarities with the strains isolated from China, Italy, Australia and Tanzania and were categorized into a monophyletic group but strain isolated from Portugal had more than 55% variable regions, hence clustered separately. This study confirms the presence of contagious bovine pleuropneumonia in the country which can be a threat to the livestock export market and warrants the implementation of control measures to mitigate the economic losses associated with the disease.
Battling malaria will be a persistent struggle without the proper means to diagnose the parasitic infection. However, the inherent limitations of microscopy, the conventional method of diagnosing malaria, affect the accuracy of diagnosis. The present study aimed to compare the accuracy of two different set of primers targeting the small subunit ribosomal RNA (ssRNA) and the dihydrofolate reductase-thymidylate synthase linker region (dhfr-ts) in detecting species specific malaria infections by nested PCR. The sensitivity and specificity of nested PCR assay using the two primers were calculated with reference to microscopy as the 'gold standard'. The results show that 18S rRNA primers had 91.9% sensitivity and 100% specificity in detecting human Plasmodium species as opposed to dhfr-ts primers which had 51.4% sensitivity and 100% specificity. The higher sensitivity of 18S rRNA primers suggests that it may be a better diagnostic tool for detecting human malaria.
Klebsiella pneumoniae is a healthcare-associated bacterial pathogen which causes severe diseases in immunocompromised individuals. Concanavalin A (conA), a lectin which recognizes proteins with mannose or glucose residues, has been reported to agglutinate K. pneumoniae and hence, is postulated to have therapeutical potential for K. pneumoniae-induced liver infection. This study investigated the conA binding properties of a large collection of clinical isolates of K. pneumoniae. ConA agglutination reaction was demonstrated by 94 (51.4%) of 183 K. pneumoniae isolates using a microtiter plate assay. The conA agglutination reactions were inhibited in the presence of 2.5 mg/ml D-mannose and 2.5 mg/ml glucose, and following pretreatment of the bacterial suspension with protease and heating at 80ºC. Majority of the positive isolates originated from respiratory specimens. Isolation of conA-binding proteins from K. pneumoniae ATCC 700603 strain was performed using conA affinity column and the conA binding property of the eluted proteins was confirmed by western blotting analysis using conA-HRP conjugates. Proteins with molecular weights ranging from 35 to 60 kDa were eluted from the conA affinity column, of which four were identified as outer membrane protein precursor A (37 kDa), outer membrane protein precursor C (40 kDa), enolase (45 kDa) and chaperonin (60 kDa) using mass spectrometry analysis. Several conA binding proteins (including 45 and 60 kDa) were found to be immunogenic when reacted with rabbit anti-Klebsiella antibody. The function and interplay of the conA binding proteins in bacterium-host cell relationship merits further investigation.
Since a safe and effective mass vaccination program against dengue fever is not presently available, a good way to prevent and control dengue outbreaks depends mainly on controlling the mosquito vectors. Aedes aegypti mosquito populations can be monitored and reduced by using ovitraps baited with organic infusions. A series of laboratory experiments were conducted which demonstrated that the bacteria in bamboo leaf infusion produce volatile attractants and contact chemical stimulants attractive to the female mosquitoes. The results showed that the female mosquitoes laid most of their eggs (59.9 ± 8.1 vs 2.9 ± 2.8 eggs, P<0.001) in bamboo leaf infusions when compared to distilled water. When the fresh infusion was filtered with a 0.45 μm filter membrane, the female mosquitoes laid significantly more eggs (64.1 ± 6.6 vs 4.9 ± 2.6 eggs, P<0.001) in unfiltered infusion. However when a 0.8 μm filter membrane was used, the female laid significantly more eggs (62.0 ± 4.3 vs 10.1 ± 7.8 eggs, P<0.001) in filtrate compared to a solution containing the residue. We also found that a mixture of bacteria isolated from bamboo leaf infusion serve as potent oviposition stimulants for gravid Aedes mosquitoes. Aedes aegypti laid significantly more eggs (63.3 ± 6.5 vs 3.1 ± 2.4 eggs, P<0.001) in bacteria suspension compared to sterile R2A medium. Our results suggest microbial activity has a role in the production of odorants that mediate the oviposition response of gravid mosquitoes.
Leishmaniasis is a neglected and endemic disease that affects poorest population mainly in developing countries. Thymus provides an essential complex environment for T cell maturation and differentiation during leishmania infection. The aim of this study was to investigate the pathological alterations of the Thymus during early Leishmania amazonensis murine infection. BALB/c mice were infected with 105 amastigotes for 24 h, 3 days, 7 days, 15 days or 30 days. At different times of infection, the relative weight of the Thymus was obtained, and the Thymus cellularity was determined by counting total cells of one thymic lobe. The thymic lobe was, alternatively, processed for standard Haematoxylin and Eosin protocol. Our results suggest thymic alteration during the early days of BALB/c mice infection with L. amazonensis. The thymic hypertrophy was accompanied by histological alterations in Thymus architecture with thickening cortex at 3 days p.i. and loss of an evident delimitation between the cortex and medulla at 7 days p.i. when compared to the control mice. That is the first time that Thymus hypertrophy was observed during the early leishmaniasis. However, how it may contribute to infection susceptibility requires further investigation.
One hundred and fourteen strains of Pasteurella multocida were isolated from different domestic animals species (cattle, buffalo, sheep, goat, pig, rabbit, dog, cat), avian species (chicken, duck, turkey) and wild animals (deer, tiger, orang utan, marmoset). The serogroups of P. multocida were determined by both conventional capsular serotyping and a multiplex PCR assay targeting specific capsular genes. Based on the conventional serotyping method, the 114 strains of P. multocida were subtyped into 55 species-specific (untypeable strains) P. multocida, 15 serogroup A, 23 serogroup B and 21 serogroup D. Based on the multiplex PCR assay on the specific capsular genes associated with each serogroup, the 114 strains were further divided to 22 species-specific P. multocida (KMT1 - 460 bp), 53 serogroup A (A - 1,044 bp), 33 serogroup B (B - 760 bp) and 6 serogroup D (D - 657 bp). No serogroup E (511 bp) or F (851 bp) was detected among the Malaysian P. multocida. PCR-based typing was more discriminative and could further subtype the previously untypeable strains. Overall, there was a significant and positive correlation between both methods in serogrouping P. multocida (r = 0.7935; p<0.4893). Various serogroups of P. multocida were present among the livestock with 75% of the strains belonging to serogroups A or B. PCR serotyping was therefore a highly species-specific, sensitive and robust method for detection and differentiation of P. multocida serogroups compared to conventional serotyping. To the best of our knowledge, this is the first report from Malaysia of the application of a PCR to rapidly define the species-specific P. multocida and its serogroups as an important zoonotic pathogen in Malaysia.
The life history of the male and female of the indoor forensic fly, Synthesiomyia nudiseta was studied under fluctuating temperature of indoor environments and analysed based on the age-stage and two sex life table. The life cycle of S. nudiseta was 14.0±1.0 days from the egg stage to adult emergence. The population parameters calculated were; net reproduction rate (R(o)= 108.6), mean generation time (T(o)= 12.2), intrinsic rate of increase (r(m)= 0.38), and finite rate of increase (λ= 1.46). The pre-oviposition period (APOP) was 6.0± 0.1 days. All population parameters suggested that S. nudiseta exhibits the r-strategist characteristics.
Legionella pneumophila are intracellular pathogens, associated with human disease, attributed to the presence and absence of certain virulent genes. In this study, virulent gene loci (lvh and rtxA regions) associated with human disease were determined. Thirty-three cooling tower water isolates, isolated between 2004 to 2006, were analyzed for the presence of these genes by PCR method. Results showed that 19 of 33 (57.5%) of the L. pneumophila serogroup 1 isolates have both the genes. Six (18.2%) of the isolates have only the lvh gene and 2 (6.1%) of the isolates have only the rtxA gene. However, both genes were absent in 6 (18.2%) of the L. pneumophila isolates. The result of our study provides some insight into the presence of the disease causing L. pneumophila serogroup 1 in the environment. Molecular epidemiological studies will provide better understanding of the prevalence of the disease in Malaysia.
The need for an intensive care protocol, sometimes weekly or biweekly, has led to a significant increase in laboratory costs for kidney recipients. In the present study, an inhouse tetraplex nested PCR assay was developed and validated for the specific detection of BKV, JCV, HCMV and EBV in clinical samples. We determined the Limit of Detection (LOD) and analytical specificity. To demonstrate the diagnostic performance of the assay, a total of 102 archival plasma samples were tested and compared with a commercial uniplex real-time PCR kits. The analytical sensitivity of the in-house tetraplex nested PCR assay was 173 copies/ml, when all four viruses were present in the specimens. These values were 79.2, 58.7, 87.6 and 96.1 copies/ml when only, BKV, JCV, HCMV and EBV respectively, were present. The cross-reactivity assays were shown no detectable signal in the tetraplex PCR results. The estimated diagnostic sensitivities were 92.6% for BKV, 92.3% for JCV and 100% for both HCMV and EBV as compared with commercial kits. Regarding the sensitivity and specificity, it seems that the developed Multiplex Nested PCR assay could be used as a reliable virusassociated renal rejection (VRR) panel in post renal transplant surveillance.
Cryptosporidiosis is a particular concern in immunocompromised individuals where symptoms may be severe. The aim of this study was to examine the epidemiological and molecular characteristics of Cryptosporidium infections in HIV/AIDS patients in Malaysia in order to identify risk factors and facilitate control measures. A modified Ziehl-Neelsen acid fast staining method was used to test for the presence of Cryptosporidium oocysts in the stools of 346 HIV/AIDS patients in Malaysia. Standard coproscopical methods were used to identify infections with other protozoan or helminths parasites. To identify the species of Cryptosporidium, DNA was extracted and nested-PCR was used to amplify a portion of the SSU rRNA gene. A total of 43 (12.4%) HIV-infected patients were found to be infected with Cryptosporidium spp. Of the 43 Cryptosporidium-positive HIV patients, 10 (23.3%) also harboured other protozoa, and 15 (34.9%) had both protozoa and helminths. The highest rates of cryptosporidiosis were found in adult males of Malay background, intravenous drug users, and those with low CD4 T cell counts (i.e., < 200 cells/mm3). Most were asymptomatic and had concurrent opportunistic infections mainly with Mycobacterium tuberculosis. DNA sequence analysis of 32 Cryptosporidium isolates identified C. parvum (84.3%), C. hominis (6.3%), C. meleagridis (6.3%), and C. felis (3.1%). The results of the present study revealed a high prevalence of Cryptosporidium infection in hospitalized HIV/AIDS patients. The results also confirmed the potential significance of zoonotic transmission of C. parvum in HIV infected patients, as it was the predominant species found in this study. However, these patients were found to be susceptible to a wide range of Cryptosporidium species. Epidemiological and molecular characterization of Cryptosporidium isolates provides clinicians and researchers with further information regarding the origin of the infection, and may enhance treatment and control strategies.
Human immunodeficiency virus (HIV)-infected individuals have greater susceptibility to infections by a myriad of microorganisms which can cause significant morbidity and mortality compared to immunocompetent individuals. Of these microbial infections, intestinal parasitic infections (IPIs) however are receiving less attention than bacterial and viral infections, hence, the lack of information of parasitic infections in HIV individuals. Prevalence of IPIs among 346 HIV-infected individuals in Malaysia was determined in this study. The overall prevalence of intestinal parasitic infections (IPIs) was 37.9% (131 of 346) with protozoa infections (18.8%) being more common compared to helminth infections (7.5%). Observed protozoa include Entamoeba histolytica/dispar (16.8%), Cryptosporidium parvum (12.4%), Isospora belli (10.1%), Cyclospora cayetanensis (4.9%) and Giardia duodenalis (intestinalis) (3.2%) whilst helminthes which were detected comprised of Ascaris lumbricoides (13.9%), Trichuris trichiura (6.4%) and hookworms (0.6%). Among those 131 infected, 50.4% had multiple infections and 48.9% had single parasitic infection. The CD4 counts were significantly lower (i.e., 200 cells/mm³) in patients harbouring IPIs. Of those individuals infected with intestinal parasites, 49% were intravenous drug users and 58% were not on any antiretroviral therapy. Most were asymptomatic and had concurrent opportunistic infections (OIs) mainly with Mycobacterium tuberculosis infection. These results confirmed that IPIs are ubiquitous among HIV-infected individuals, especially those presenting with low CD4 T cells counts, and provide useful insights into the epidemiology of these infections among HIV-infected patients in Malaysia. It is therefore recommended, that diagnosis of these intestinal parasitic pathogens should be conducted on a routine basis for better management of gastrointestinal illnesses among HIV individuals.
Bacteria of the genus Bartonella have been known as emerging zoonotic pathogens for several human diseases including cat scratch disease, Carrion's disease and trench fever. Numerous species of small mammals have been reported to play a role as a suitable reservoir to many pathogenic Bartonella. These infections are thought to be transmitted through blood-feeding arthropod vectors such as ticks, fleas and lice. The purpose of this study is to detect the presence of Bartonella species from tick samples collected from small mammals in mangrove forests of Peninsular Malaysia. Herein, 38 individual ticks and their small mammals host were evaluated for the presence of Bartonella DNA by conventional PCR targeting the 16S rRNA intergenic spacer region (ITS) and partial sequencing of 460 bp from this locususing Bartonella genus-specific primers. Two tick individuals from Dermacentor auratus and Haemaphysalis hystricis collected from Rattus tiomanicus (host), were PCR-positive for Bartonella DNA amplification. No Bartonella amplification was possible in other tick species (Amblyomma sp.). Phylogenetic analysis of ITS fragments demonstrated that the sequences from ticks were closely related to Bartonella phoceensis, a species that has been reported from black rats (Rattus rattus) in Australia. This is the first report of a Bartonella bacteria detected in ticks from small mammals in Malaysia. Further research should be warranted to investigate the transmission of Bartonella and the potential impact of this zoonotic pathogen in animals and humans as this mangrove ecosystem is significant for local economy and tourism.
An outbreak of dengue in Kudat, northern Sabah in 2016-2017 provided an opportunity to investigate the circulating serotypes of dengue viruses of cases at Hospital Kudat. Between September 2016 and December 2017, a total of 156 dengue positive sera (tested positive by either NS1 antigen, or IgM and IgG antibody rapid test) were collected from dengue patients who had acute fever and showed signs and symptoms suggestive of dengue. RNA was extracted from the sera using QIAamp RNA Blood Mini Kit, and molecular amplification was performed using one-step RT-PCR kit, followed by nested PCR using HotStart Taq master mix kit with the primers of the dengue C-prM gene. There were 81 (52%) male and 75 (48%) female cases. The age group with the highest number of cases was the 10-19 years old, while the youngest infected was 8 months old and the oldest was 83 years old. RT-PCR results showed 88 sera dengue positive, 48 infected with a single serotype while another 40 with multiple serotypes. All four DENV serotypes were co-circulating during the outbreak period and DENV-1 was predominant. Molecular analysis also indicated 69.2%, 50.0%, 51.9% and 48.9% respectively of the NS1, IgM, IgG and IgM and IgG positive sera were RT-PCR positive for dengue. High number of cases were seen in December 2016, February and May 2017. The dengue outbreak might be related to switching of predominant serotype from DENV 4 to DENV 1.
Some of Vibrio species is well known as pathogenic bacteria in aquaculture and the marine industry. Its infection is able to generate a massive outbreak and affect the fish population, especially for net caged fish such as seabass. This study was conducted to investigate the prevalence of Vibrio spp. isolated from seabass (Lates calcarifer) in Sri Tujuh Lagoon, Tumpat, Kelantan. Then, to determine the antibiotic resistance in Vibrio isolates. Polymerase chain reaction (PCR) was used to detect Vibrio species using specific primer VR169 and VR744 with estimation base pair size band, 597 bp and further identified by sequencing. On the other hand, antibiotic susceptibility tests were continued by using 13 types of antibiotics; kanamycin (K30), chloramphenicol (C30), neomycin (N10), ampicillin (AMP10), nitrofurantoin (F300), tetracycline (TE30), streptomycin (S10), norfloxacin (NOR10), ciprofloxacin (CIP5), nalidixic acid (NA30), gentamicin (CN10), doxycycline (DO30) and sulfamethoxazole (SXT100). As a result, 14 Vibrio isolates were identified, including Vibrio fluvialis (n=6), Vibrio parahaemolyticus (n=3), Vibrio harveyi (n=2) and each isolate for Vibrio vulnificus, Vibrio alginolyticus and Vibrio spp. The results showed that all isolates were sensitive to most antibiotics except ampicillin, neomycin and streptomycin. The MAR index value was ranging from 0 to 0.31. This study demonstrates the prevalence of Vibrio spp. in seabass and the report on multidrug resistance strains that could be of concern to the fish farmers. In addition, data from this study can be further used in fish disease management plans.
Residents in irrigated urban agricultural sites face numerous mosquito problems such as increased mosquito populations and reduced insecticides susceptibility due to the creation of mosquito breeding sites and agricultural use of insecticides and hence require effective protective products against them. In this study, the protection effectiveness of three pyrethroid formulated mosquito coils of Malaysian origin against Anopheles gambiae sensu lato from an irrigated urban agricultural site in Ghana were evaluated for their potential use. Sucrose fed An. gambiae s.l. were exposed to insecticide-containing coils in a 70 cm x 70 cm x 70 cm glass chamber to assess the insecticidal effect of the coils. The 0.005% metofluthrin coil caused the most rapid knockdown of 50% of the test mosquitoes. The mean lethal effect of the coils on An. gambiae s.l. were as follows; 0.005% metofluthrin (86%), 0.3% d-allethrin (74.33%), 0.15% d-trans allethrin (72%) and the 0.25% d-allethrin reference coil (69%). The 0.005% metofluthrin coil achieved the highest insecticidal effect on An. gambiae s.l. compared to the other coils and hence performed better than the others as an anti-mosquito product. All the three test coils were effective against An. gambaie s.l. from the irrigated agricultural site compared to the reference coil.
Plants contain bioactive compounds and are constantly explored as safer alternatives to conventional insecticides. Despite numerous studies on many plants, information on the insecticidal potential of underutilised plants like tiger nut, Cyperus esculentus L., are scant, although their pharmacological potentials are well known. Hence, this study investigated the larvicidal potential of crude aqueous extracts of two C. esculentus varieties (black and yellow) on the mosquitoes Aedes aegypti (L.) and Culex quinquefasciatus (Say). Mosquito larvae were exposed to C. esculentus crude extracts using the larval bioassay technique of the World Health Organization. Differential larvicidal responses were observed in the test mosquitoes and extracts of Black Dried Tiger nuts (BDT) were more larvicidal than Yellow Dried Tiger nuts (YDT). Acute larval toxicity of the extracts was more pronounced on Cx. quinquefasciatus than Ae. aegypti. The results indicate the potential of C. esculentus (particularly BDT) as a source of mosquito bioinsecticide and merits further studies as a safer alternative to conventional insecticide-based vector control.
Malaria caused by Plasmodium knowlesi species has become a public health concern, especially in Malaysia. Plasmodium knowlesi parasite which originates from the macaque species, infects human through the bite of the Anopheles mosquitoes. Research on malaria vaccine has been a continuous effort to eradicate the malaria infection, yet there is no vaccine against P. knowlesi malaria to date. Apical membrane antigen 1 (AMA1) is a unique surface protein of all apicomplexan parasites that plays a crucial role in parasite-host cell invasion and thus has been a long-standing malaria vaccine candidate. The selection of protective epitopes in silico has led to significant advances in the design of the vaccine. The present study aimed to employ bioinformatics tools to predict the potential immunogenic B- and T-cell epitopes in designing malaria vaccine targeting P. knowlesi AMA1 (PkAMA1). B-cell epitopes were predicted using four bioinformatics tools, i.e., BepiPred, ABCpred, BcePred, and IEDB servers whereas T-cell epitopes were predicted using two bioinformatics servers, i.e., NetMHCpan4.1 and NetMHCIIpan-4.0 targeting human major histocompatibility complex (MHC) class I and class II molecules, respectively. The antigenicity of the selected epitopes computed by both B- and T-cell predictors were further analyzed using the VaxiJen server. The results demonstrated that PkAMA1 protein encompasses multi antigenic regions that have the potential for the development of multi-epitope vaccine. Two B- and T-cell epitopes consensus regions, i.e., NSGIRIDLGEDAEVGNSKYRIPAGKCP (codons 28-54) and KTHAASFVIAEDQNTSY RHPAVYDEKNKT (codons 122-150) at domain I (DI) of PkAMA1 were reported. Advancement of bioinformatics in characterization of the target protein may facilitate vaccine development especially in vaccine design which is costly and cumbersome process. Thus, comprehensive B-cell and T-cell epitope prediction of PkAMA1 offers a promising pipeline for the development and design of multi-epitope vaccine against P. knowlesi.
Plasmodium knowlesi has been recently identified as the "fifth human malaria species" following the discovery in Malaysian Borneo of a large focus of this simian malaria parasite in humans. Even though it shares microscopic similarities with Plasmodium malariae, it may cause severe illness with risk of fatality. We describe a case of P. knowlesi infection causing multi-organ failure in a patient who was successfully managed due to early recognition of the infection. Clinicians in this region should be more aware of the infection as it is not as rare as previously thought. This case write up highlight the case of severe malaria infection which presented with multi organ involvement which is caused by P. knowlesi.
Trichuris dysentery syndrome is caused by Trichuris trichiura which contributes to one of the most common helminthic infections in the world. It is associated with heavy colonic infection that manifests as mucoid diarrhoea, rectal bleeding, rectal prolapse, iron deficiency anaemia, and finger clubbing. Here, we report a case of trichuris dysentery syndrome complicated with severe chronic iron deficiency anaemia in a 4-year-old girl who required blood transfusion. The nematode was visualized on stool microscopic and colonoscopic examination. A longer duration of anti-helminthic treatment is required to achieve effective and better outcome.
Plasmodium knowlesi is a simian malaria parasite and is recently recognized as the fifth malaria parasite infecting humans. Manifestation of the infection may resemble other infection particularly dengue fever leading to inappropriate management and delay in treatment. We reported three cases of naturally acquired P. knowlesi in Hospital Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia. Clinical manifestations were quite similar in those cases. Microscopically, the diagnosis might be challenging. These cases were confirmed by polymerase chain reaction method which serves as a gold standard.