Anaplasma spp. infects a wide variety of wildlife and domestic animals. This study describes the identification of a novel species of Anaplasma (Candidatus Anaplasma pangolinii) from pangolins (Manis javanica) and Anaplasma bovis from wild boars (Sus scrofa) in Malaysia. Based on 16S rRNA gene sequences, Candidatus Anaplasma pangolinii is identified in a distinct branch within the family Anaplasmataceae, exhibiting the closest sequence similarity with the type strains of Anaplasma bovis (97.7%) and Anaplasma phagocytophilum (97.6%). The sequence also aligned closely (99.9%) with that of an Anaplasma spp. (strain AnAj360) detected from Amblyomma javanense ticks. The nearly full length sequence of the 16S rRNA gene derived from two wild boars in this study demonstrated the highest sequence similarity (99.7%) to the A. bovis type strain. Partial 16S rRNA gene fragments of A. bovis were also detected from a small population of Haemaphysalis bispinosa cattle ticks in this study. Our finding suggests a possible spread of two Anaplasma species in the Malaysian wildlife and ticks. The zoonotic potential of the Anaplasma species identified in this study is yet to be determined.
A specialized DNA extraction method and a SYBR Green quantitative polymerase chain reaction (SyG-qPCR) assay were combined to generate a ready-to-use kit for rapid detection of porcine admixtures in processed meat products. Our qPCR assay utilized repetitive LINE-1 elements specific to the genome of Sus scrofa domesticus (pig) as a target and incorporated internal controls. We improved the genomic DNA extraction method, and reduced extraction times to the minimum. The method was validated for specificity, sensitivity (0.001% w/w) and robustness, and values were compared with those of a commercially available kit. We also tested our method using 121 processed food products and consistently detected amplification only in samples containing pork. Due to its efficiency and cost-effectiveness, our method represents a valuable new method for detecting food adulteration with pork that is superior to existing quality control approaches.
Serum specimens were collected from 6 species of animals living in 9 states of Malaysia including Sabah, North Borneo in 1993. Antibodies against Japanese encephalitis (JE) virus in these sera were detected by means of hemagglutination-inhibition (HI) and neutralization (NT) tests. By HI test, 702 of 2,152 (32.6%) sera showed positive results. Higher positive rates were obtained by the NT test, in which 1,787 of 1,927 (92.7%) sera had antibodies against JE virus. All serum specimens with positive HI were confirmed as positive by the NT. Swine sera showed especially higher rates of antibody positive and higher antibody titers compared with other animals. These results suggest that JE infections are widely distributed among many animals of Malaysia, and pig is the most susceptible amplifier host for JE virus.
The C-terminal domain of Nipah virus (NiV) nucleocapsid protein (NP₄₀₁₋₅₃₂) was inserted at the N-terminus and the immunodominant loop of hepatitis B core antigen (HBc). The stability of NP₄₀₁₋₅₃₂ increased tremendously when displayed on the HBc particles. These particles reacted specifically with the swine anti-NiV and the human anti-HBc antisera.
The broad species tropism of Nipah virus (NiV) coupled with its high pathogenicity demand a rapid search for a new biomarker candidate for diagnosis. The matrix (M) protein was expressed in Escherichia coli and purified using a Ni-NTA affinity column chromatography and sucrose density gradient centrifugation. The recombinant M protein with the molecular mass (Mr) of about 43 kDa was detected by anti-NiV serum and anti-myc antibody. About 50% of the M protein was found to be soluble and localized in cytoplasm when the cells were grown at 30 degrees C. Electron microscopic analysis showed that the purified M protein assembled into spherical particles of different sizes with diameters ranging from 20 to 50 nm. The purified M protein showed significant reactivity with the swine sera collected during the NiV outbreak, demonstrating its potential as a diagnostic reagent.
A sequential solid-phase extraction (SPE) method was developed and validated using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for the detection and quantification of salbutamol enantiomers in porcine urine. Porcine urine samples were hydrolysed with β-glucuronidase/arylsulfatase from Helix pomatia and then subjected to a double solid-phase extraction (SPE) first using the Abs-Elut Nexus SPE and then followed by the Bond Elut Phenylboronic Acid (PBA) SPE. The salbutamol enantiomers were separated using the Astec CHIROBIOTIC™ T HPLC column (3.0mm×100mm; 5μm) maintained at 15°C with a 15min isocratic run at a flow rate of 0.4mL/min. The mobile phase constituted of 5mM ammonium formate in methanol. Salbutamol and salbutamol-tert-butyl-d9 (internal standard, IS) was monitored and quantified with the multiple reaction monitoring (MRM) mode. The method showed good linearity for the range of 0.1-10ng/mL with limit of quantification at 0.3ng/mL. Analysis of the QC samples showed intra- and inter-assay precisions to be less than 5.04%, and recovery ranging from 83.82 to 102.33%.
Between February and April, 1999, an outbreak of viral encephalitis occurred among pig-farmers in Malaysia. We report findings for the first three patients who died.
A convenient acidimetric assay for phospholipase A using egg yolk suspension as substrate has been developed. The substrate mixture consists of 1 part egg yolk, 1 part 8.1 mM sodium deoxycholate, and 1 part 18 mM calcium chloride. Phospholipase A activity is measured by following the initial rate of pH change, which is linear between pH 8.0 and 7.75 and is proportional to enzyme concentration over a wide range. The assay is highly reproducible, with a coefficient of variation of 3%, and as sensitive as most established assays for phospholipase A. The assay uses inexpensive and easily available substrate and is simple to perform. It is particularly useful for monitoring phospholipase A activity in chromatography fractions.
The Nipah virus is highly virulent to swine and humans. The envelope attachment glycoprotein (G) of Nipah virus
plays a key role in viral entry and induction of neutralizing antibody in mammalian hosts, thus is considered a good
candidate for vaccine development. Plant transient expression systems are gaining recognition as a viable alternative
for the production of vaccine antigens. In this study, we expressed the Nipah virus G protein heterologously in Nicotiana
benthamiana using an agroinfiltration approach. The highest expression of recombinant G protein in N. benthamiana at
RNA and protein levels was detected on day 9 post-infiltration. Western blot analysis demonstrated that the purified G
protein reacted specifically with rabbit anti-Nipah Virus serum, indicating its potential for vaccine use.
Rotavirus is the leading causative viral agent of pediatric acute gastroenteritis globally, infecting mostly children 5 years old and below. Data on rotavirus prevalence in Malaysia is scarce, despite the WHO's recommendation for continuous rotavirus surveillance, and has underestimated the need for national rotavirus vaccination. Characteristics of the current rotavirus strains in Malaysia have to be determined to understand the rotavirus epidemiology and vaccine compatibility. This study sought to determine the genetic relatedness of Sarawak rotavirus strains with global strains and to determine the antigenic coverage and epitope compatibility of Rotarix and RotaTeq vaccines with the Sarawak rotavirus strains via in silico analysis. A total of 89 stool samples were collected from pediatric patients (<5 years old) with acute gastroenteritis at private hospitals in Kuching, Sarawak. Rotavirus was detected using reverse transcription-polymerase chain reaction. Positive amplicons were analyzed using nucleotide sequencing before phylogenetic analyses and assessment of epitope compatibility. Genotyping revealed G1P[8] (1/13; 7.7%), G3P[8] (3/13; 23%), G9P[4] (1/13; 7.7%), and G9P[8] (3/13; 23%), G9P[X] (1/13; 7.7%), GXP[4] (1/13; 7.7%), and GXP[8] (3/13; 23%) in samples. All wild-type Sarawak rotavirus strains, with the exception of G1, showed variations in their phylogenetic and antigenic epitope characteristics.
The Nipah virus is a newly identified paramyxovirus responsible for an outbreak of fatal encephalitis in Malaysia and Singapore. This paper reports the follow up clinical and magnetic resonance imaging findings in 22 affected subjects. Of 13 patients with encephalitis, one died, one was lost to follow up, and seven recovered. Among the four remaining patients, one had residual sixth nerve palsy, another suffered from severe clinical depression, and a third patient had evidence of retinal artery occlusion. One patient with delayed onset Horner syndrome had a single lesion in the cervical spinal cord. The brain magnetic resonance findings were stable or improved in nine patients over 18 months of follow up. Among a second group of nine asymptomatic seropositive abattoir workers, magnetic resonance examination in seven subjects revealed discrete small lesions in the brain; similar to those detected in encephalitis patients. These findings suggest that in addition to encephalitis, the newly discovered Nipah virus affects the spinal cord and the retina. Late clinical and radiological findings can occur in Nipah virus infections as with other paramyxoviruses.
Zoonotic infections with Onchocerca species are uncommon, and to date only 25 clinical cases have been reported worldwide. In Japan, five previous zoonotic infections were concentrated in Oita, Kyushu (the southern island), with one previous case in Hiroshima in the western part of Honshu (the main island). The causative agent in Japan was identified as Onchocerca dewittei japonica Uni, Bain & Takaoka, 2001 from Japanese wild boars (Sus scrofa leucomystax Temminck, 1842). Here we report two infections caused by a female and male O. dewittei japonica, respectively, among residents of Hiroshima and Shimane Prefectures in the western part of Honshu.
Human zoonotic onchocercosis is caused by Onchocerca dewittei japonica, parasitic in wild boars (Sus scrofa leucomystax) in Japan. Previously, microfilariae longer than those of Onchocerca dewittei japonica were observed in skin snips from wild boars during the study of O. dewittei japonica. Moreover, the third-stage larvae (L3) of these longer microfilariae were obtained from the blackfly Simulium bidentatum after experimental injections. Based on morphometric and molecular studies, similar L3 were found in blackflies during fieldwork in Oita, Japan. However, except for O. dewittei japonica, adult worms of Onchocerca have not been found in wild boars. In this study, we discovered adult females of a novel Onchocerca species in the skin of a wild boar in Oita, and named it Onchocerca takaokai n. sp. Females of this new species had longer microfilariae and differed from O. dewittei japonica in terms of their morphological characteristics and parasitic location. The molecular characteristics of the cytochrome c oxidase subunit 1 and 12S rRNA genes of the new species were identical to those of the longer microfilariae and L3 previously detected, but they differed from those of O. dewittei japonica at the species level. However, both species indicated a close affinity among their congeners and Onchocerca ramachandrini, parasitic in the warthog in Africa, was basal in the Suidae cluster of the 12S rRNA tree.
The transmission of zoonotic filarial parasites by black flies has so far been reported in the Chiang Mai and Tak provinces, Thailand, and the bites of these infected black flies can cause a rare disease-human zoonotic onchocerciasis. However, species identification of the filarial parasites and their black fly vectors in the Chiang Mai province were previously only based on a morphotaxonomic analysis. In this study, a combined approach of morphotaxonomic and molecular analyses (mitochondrial cox1, 12S rRNA, and nuclear 18S rRNA (SSU HVR-I) genes) was used to clarify the natural filarial infections in female black flies collected by using human and swine baits from two study areas (Ban Lek and Ban Pang Dang) in the Chiang Mai province from March 2018 to January 2019. A total of 805 and 4597 adult females, belonging to seven and nine black fly taxa, were collected from Ban Lek and Ban Pang Dang, respectively. At Ban Lek, four of the 309 adult females of Simulium nigrogilvum were positive for Onchocerca species type I in the hot and rainy seasons. At Ban Pang Dang, five unknown filarial larvae (belonging to the same new species) were detected in Simulium sp. in the S. varicorne species-group and in three species in the S. asakoae species-group in all seasons, and three non-filarial larvae of three different taxa were also found in three females of the S. asakoae species-group. This study is the first to molecularly identify new filarial species and their vector black fly species in Thailand.
Reports of zoonotic infections with Onchocerca japonica (Nematoda: Filarioidea), which parasitizes the Japanese wild boar, Sus scrofa leucomystax, have recently increased in Japan. To predict the occurrence of infection in humans, it is necessary to determine the prevalence of O. japonica infection in the natural host animals. We investigated the presence of adult worms in the footpads, and of microfilariae in skin snips, taken from the host animals, between 2000 and 2018. Onchocerca japonica was found in 165 of 223 (74%) Japanese wild boars in Honshu and Kyushu. Among the nine regions studied, the highest prevalence of O. japonica infection was found in Oita, Kyushu, where 47 of 52 (90.4%) animals were infected. The ears were the predilection sites for O. japonica microfilariae. Adult worms of O. japonica were found more frequently in the hindlimbs than in the forelimbs of the host animals. Onchocerca takaokai was found in 14 of 52 (26.9%) Japanese wild boars in Oita. In Kakeroma Island among the Nansei Islands, both O. japonica and O. takaokai were isolated from the Ryukyu wild boar, S. s. riukiuanus. These observations could help predict future occurrences of human zoonotic onchocercosis in Japan.
An 11-year-old boy living in Otsu City, Shiga Prefecture, Kansai Region, Western Honshu, Japan had zoonotic onchocercosis. The patient developed a painful swelling on the little finger of his left hand. The worm detected in the excised mass had external transverse ridges but did not have inner striae in the cuticle. On the basis of the parasite's histopathological characteristics, the causative agent was identified as a female Onchocerca dewittei japonica (Spirurida: Onchocercidae). The species of the filarial parasite was confirmed by sequencing the cox1 gene of the parasite. The Japanese wild boar Sus scrofa leucomystax is a definitive host for O. dewittei japonica, which is then transmitted by blackflies as the vector to humans. The current case described occurred in the Kansai Region, Western Honshu, where such infections were previously not reported.
A 73-year-old man living in Kawamata-machi, Fukushima Prefecture, Northeastern Honshu, Japan, visited a hospital with complaints of a subcutaneous swelling that had developed on the back of his left hand. The nodule was surgically removed from the vagina fibrosa tendinis of his left forefinger. Based on the histopathological characteristics, the causative agent of this nodule was identified as a female Onchocerca dewittei japonica (Spirurida: Onchocercidae). The species identification was confirmed by cox1 gene sequencing of the worm tissues from paraffin-embedded sections of the nodule. Although 11 cases of zoonotic onchocercosis have previously been recorded in Kyushu and Western Honshu, Japan, the present findings represent the first human case of infection with O. dewittei japonica in Northeastern Honshu, Japan.
A hospital-based case-control study of viral encephalitis was carried out at Port Dickson Hospital, in the state of Negeri Sembilan, Malaysia. Between March and May 1999, 69 clinically diagnosed viral encephalitis cases and 31 controls were interviewed. Job histories on pig farming activities were assessed by a group of epidemiologists and veterinary surgeons. Results show that among clinical cases of viral encephalitis, 52 (75.4%) cases were diagnosed to have Nipah virus infection based on positive serology for antibodies to the cross-reacting Hendra virus antigen. The Nipah virus encephalitis was significantly associated with a history of working in pig farms (p < 0.001, OR = 196.0, 95% CI = 20.4-4741.6), history of contact with animals (p < 0.001, OR = 38.3, 95% CI = 8.2-209.0) and with history of direct contact with pigs (p = 0.002, OR = 34.4, 95% CI = 2.6-1,024.4). The Nipah virus infection was also significantly associated with history of feeding/cleaning pigs (p < 0.001, OR = 102, 95% CI = 11.9-2,271.5). These results provide evidence that involvement in pig farming activities is significantly associated with the risk of getting Nipah virus infection. They are potential risk factors for Nipah virus transmission in the major pig-producing area of Bukit Pelandok, Port Dickson Negeri Sembilan.
We constructed a novel chicken (Gallus gallus) lung cDNA library fused inside yeast acting domain vector (pGADT7). Using yeast two-hybrid screening with highly pathogenic avian influenza (HPAI) nucleoprotein (NP) from the strain (A/chicken/Malaysia/5858/2004(H5N1)) as bait, and the Gallus gallus lung cDNA library as prey, a novel interaction between the Gallus gallus cellular RNA export adaptor protein Aly/REF and the viral NP was identified. This interaction was confirmed and validated with mammalian two hybrid studies and co-immunoprecipitation assay. Cellular localization studies using confocal microscopy showed that NP and Aly/REF co-localize primarily in the nucleus. Further investigations by mammalian two hybrid studies into the binding of NP of other subtypes of influenza virus such as the swine A/New Jersey/1976/H1N1 and pandemic A/Malaysia/854/2009(H1N1) to human Aly/REF, also showed that the NP of these viruses interacts with human Aly/REF. Our findings are also supported by docking studies which showed tight and favorable binding between H5N1 NP and human Aly/REF, using crystal structures from Protein Data Bank. siRNA knockdown of Aly/REF had little effect on the export of HPAI NP and other viral RNA as it showed no significant reduction in virus titer. However, UAP56, another component of the TREX complex, which recruits Aly/REF to mRNA was found to interact even better with H5N1 NP through molecular docking studies. Both these proteins also co-localizes in the nucleus at early infection similar to Aly/REF. Intriguingly, knockdown of UAP56 in A549 infected cells shows significant reduction in viral titer (close to 10 fold reduction). Conclusively, our study have opened new avenues for research of other cellular RNA export adaptors crucial in aiding viral RNA export such as the SRSF3, 9G8 and ASF/SF2 that may play role in influenza virus RNA nucleocytoplasmic transport.
We describe Onchocerca dewittei japonica n. subsp. from the Japanese wild boar, Sus scrofa leucomystax, in Oita, Kyushu Island, where all seven animals examined were found to be infected. This study began with efforts to identify the causative species in a recent case of zoonotic onchocerciasis. Compared with Onchocerca dewittei dewittei from Sus scrofa jubatus in Malaysia, which was reexamined here, our new subspecies has much greater space between the ridges on the females. In addition, its microfilariae (from uteri) are shorter (192-210 microns compared with 228-247 microns), and only the posterior third of the microfilarial body is coiled, instead of the entire body. The Onchocerca species parasitic in suids (these two subspecies and O. ramachandrini from the warthog in the Ethiopian region) form a group sharing several characters. Among the most unusual characters are the body swellings (a specialized apparatus for mating, known in only a few other genera). In addition, longitudinal cuticular crests were found on males of both subspecies from wild boar and on females of O. ramachandrini.