Serological tests may yield false-negative results for specific antibodies detection before or at the early seroconversion phase. Tests that detect circulating antigens of Angiostrongylus cantonensis would therefore be of value in diagnosis to distinguish current or past infection. Here, a quick, easy to perform, portable and inexpensive diagnostic device for detection of 31-kDa A. cantonensis specific antigens had been developed. This sandwich dot-immunogold filtration assay (AcDIGFAAg), for detecting active angiostrongyliasis was produced using anti-A. cantonensis polyclonal antibody dotted on the nitrocellulose membrane as a capture agent and colloidal gold-labelled anti-31 kDa A. cantonensis antibody as a detection agent. A well-defined pink dot, indicating positivity, was seen readily by naked eye within 10-15 min. The AcDIGFAAg detected A. cantonensis-specific antigens in cerebrospinal fluid samples from 4 out of 10 serologically confirmed angiostrongyliasis cases and 2 out of 5 suspected cases with negative anti-A. cantonensis antibodies. Among the 19 patient sera with A. cantonensis infection, 2 showed positive reaction by AcDIGFAAg. No positive AcDIGFAAg reaction was observed in all the serum samples with other parasitic diseases, and the healthy controls. The present 'AcDIGFAAg' enables rapid qualitative detection of the specific 31-kDa antigens of A. cantonensis in clinical samples with potential for application even under resource-limited settings.
Daily intramuscular injection with thyroxine (T4) at a dose of 2.5 micrograms/100 g body weight decreased the larvae and adult worm burden of Parastrongylus malaysiensis in the brain and pulmonary arteries of male Sprague-Dawley albino rats. In contrast, rats treated with propyl thiouracil (PTU), an antithyroid drug, at a dose of 3.75 mg/100 g body weight retained greater numbers of larvae and adult worms. The results may reflect the contrasting immunomodulatory effects of T4 and PTU that influence the susceptibility of the host.
Angiostrongylus cantonensis is the main causative agent of human angiostrongyliasis. A sibling species, A. malaysiensis has not been unequivocally incriminated to be involved in human infections. To date, there is only a single report on the application of the partial 66-kDa protein gene sequence for molecular differentiation and phylogeny of Angiostrongylus species. Nucleotide sequences of the 66-kDa protein gene of A. cantonensis and A. malaysiensis from Thailand, as well as those of the laboratory strains of A. cantonensis from Thailand and Hawaii, A. cantonensis from Japan and China, A. malaysiensis from Malaysia, and A. costaricensis from Costa Rica, were used for the reconstruction of phylogenetic tree by the maximum likelihood (ML) method and the haplotypes by the median joining (MJ) network. The ML phylogenetic tree contained two major clades with a full support bootstrap value - (1) A. cantonensis and A. malaysiensis, and (2) A. costaricensis. A. costaricensis was basal to A. cantonensis and A. malaysiensis. The genetic distance between A. cantonensis and A. malaysiensis ranged from p = .82% to p = 3.27%, that between A. cantonensis and A. costaricensis from p = 4.90% to p = 5.31%, and that between A. malaysiensis and A. costaricensis was p = 4.49% to p = 5.71%. Both A. cantonensis and A. malaysiensis possess high 66-kDa haplotype diversity. There was no clear separation of the conspecific taxa of A. cantonensis and A. malaysiensis from different geographical regions. A more intensive and extensive sampling with larger sample size may reveal greater haplotype diversity and a better resolved phylogeographical structure of A. cantonensis and A. malaysiensis.
A rapid dot immunogold filtration assay (DIGFA) was adopted for specific immunodiagnosis of human cerebral angiostrongyliasis, using purified 31-kDa glycoprotein specific to Angiostrongylus cantonensis as diagnostic antigen and protein A colloidal gold conjugate as antigen-antibody detector. A total of 59 serum samples were assayed - 11 samples from clinically diagnosed patients with detectable A. cantonensis-specific antibody in immunoblotting; 23 samples from patients with other related parasitic diseases, i.e. gnathostomiasis (n= 8), cysticercosis (n= 5), toxocariasis (n= 2), filariasis (n= 4), paragonimiasis (n= 2) and malaria (n= 2); and 25 samples from normal healthy subjects. The sensitivity and specificity of DIGFA to detect anti-A. cantonensis specific antibodies in serologically confirmed angiostrongyliasis cases, were both 100%. No positive DIGFA was observed in cases with other parasitic diseases, and the healthy control subjects. The 3-min DIGFA is as sensitive and specific as the 3-h immunoblot test in angiostrongyliasis confirmed cases that revealed a 31-kDa reactive band. The gold-based DIGFA is more rapid and easier to perform than the traditional enzyme-linked immunosorbent assay (ELISA). The test utilizing purified A. cantonensis antigen is reliable and reproducible for specific immunodiagnosis of human infection with A. cantonensis - thus can be applied as an additional routine test for clinical diagnostic support. Large-scale sero-epidemiological studies in endemic communities in north-east Thailand are under way to evaluate its usefulness under field conditions.
The rat lungworm Angiostrongylus malaysiensis is a metastrongyloid nematode parasite. It has been reported in Malaysia, Thailand, Laos, Myanmar, Indonesia and Japan. In this study, A. malaysiensis adult worms recovered from the lungs of wild rats in different geographical regions/provinces in Thailand were used to determine their haplotype by means of the mitochondrial partial cytochrome c oxidase subunit I (COI) gene sequence. The results revealed high COI haplotype diversity of A. malaysiensis from Thailand. The geographical isolates of A. malaysiensis from Thailand and other countries formed a monophyletic clade distinct from the closely related A. cantonensis. In the present study, five new haplotypes were identified in addition to the four haplotypes reported in the literature. Phylogenetic analysis revealed that four of these five new haplotypes - one from Mae Hong Song (northern region), two from Tak (western region) and one from Phang Nga (southern region) - formed a distinct clade with those from Phatthalung (southern region) and Malaysia. The haplotype from Malaysia was identical to that of Phatthalung (haplotype AM1). In general, the COI sequences did not differentiate unambiguously the various geographical isolates of A. malaysiensis. This study has confirmed the presence of high COI genetic diversity in various geographical isolates of A. malaysiensis. The COI gene sequence will be suitable for studying genetic diversity, population structure and phylogeography.
With many non-human primates (NHPs) showing continued population decline, there is an ongoing need to better understand their ecology and conservation threats. One such threat is the risk of disease, with various bacterial, viral and parasitic infections previously reported to have damaging consequences for NHP hosts. Strongylid nematodes are one of the most commonly reported parasitic infections in NHPs. Current knowledge of NHP strongylid infections is restricted by their typical occurrence as mixed infections of multiple genera, which are indistinguishable through traditional microscopic approaches. Here, modern metagenomics approaches were applied for insight into the genetic diversity of strongylid infections in South-East and East Asian NHPs. We hypothesized that strongylid nematodes occur in mixed communities of multiple taxa, dominated by Oesophagostomum, matching previous findings using single-specimen genetics. Utilizing the Illumina MiSeq platform, ITS-2 strongylid metabarcoding was applied to 90 samples from various wild NHPs occurring in Malaysian Borneo and Japan. A clear dominance of Oesophagostomum aculeatum was found, with almost all sequences assigned to this species. This study suggests that strongylid communities of Asian NHPs may be less species-rich than those in African NHPs, where multi-genera communities are reported. Such knowledge contributes baseline data, assisting with ongoing monitoring of health threats to NHPs.
The prevalence of gastrointestinal (GI) nematodes and total worm burden of Damara and Barbados Blackbelly cross sheep was investigated among smallholder farms in Salak Tinggi district of Selangor, Malaysia. A total of 50 sheep raised in smallholder farms comprising of 27 Damara cross and 23 Barbados Blackbelly cross were categorized based on their age into young and adults. Fecal samples were collected and examined for strongyle egg count by using modified McMaster technique. Severity of infection was categorized into mild, moderate, and heavy, based on egg per gram (EPG). Five sheep were randomly selected and slaughtered to examine the presence of adult gastrointestinal (GI) nematodes through total worm count (TWC). Faffa Malan Chart (FAMACHA) score was used for investigation of worm load based on the degree of anemia. The study revealed an overall EPG prevalence of 88 %, of which 84.1 % had mild infection. There was a significant difference (p = 0.002) in EPG among the two breeds. Based on age, significant difference (p = 0. 004) in EPG was observed among Barbados Blackbelly cross, but not for Damara cross (p = 0.941). The correlation between severity of infection and the FAMACHA score was significant (r = 0.289; p = 0.042). Haemonchus spp. were the most predominant nematode found in the gastrointestinal tract, followed by Trichostrongylus and Oesophagostomum spps. EPG and TWC for Haemonchus were positively correlated, but not significant (r = 0.85, p = 0.066). From regression analysis, 73 % of the variability in TWC for Haemonchus could be explained by EPG. Thus, it can be concluded that FAMACHA score correlates well with severity of infection of a nematode and can be used to assess the strongyle nematode burden in the different sheep crosses.
Gonadectomized male laboratory rats were given 0.06 mg/kg estradiol benzoate daily for 14 days before being inoculated with 50 third-stage larvae of Parastrongylus malaysiensis. Hormone treatment was continued until the rats were killed. The numbers of larvae in the brain and of adult worms in the pulmonary area of the rats were determined every 7 days after the inoculation. It was found that the rats treated daily with estradiol benzoate had significantly and consistently higher numbers of larvae and adult worms as compared with the controls. The number of total leukocytes increased significantly after the rats were infected. The results show that estradiol-treated rats become susceptible to P. malaysiensis infection, which may indicate that the immunosuppressive effects of testosterone observed in earlier studies may partly be caused by estradiol that was peripherally aromatized from testosterone.
Parasitic nematodes are among the most common and economically important infectious diseases of grazing livestock, especially in small ruminants in the tropics and subtropics in Kenya the control of gastrointestinal nematode infections in sheep and goats is usually made with synthetic anthelmintics but substantial levels of anthelmintic resistance have been recorded. A number of medicinal plants, that may provide possible alternatives, and are used by pastoralists and smallholder farmers in Kenya as deworming agents for their livestock and equines, namely Aframomum sanguineum, Dodonea angustifolia, Hildebrandtia sepalosa, Myrsine africana, Rapanea melanophloeos from Kenya, and Azadirachta indica from Kenya and Malaysia, together with the chemicals embelin and santonin that occur in some of these plants, were evaluated against Heligmosomoides polygyrus in mice. Commercial anthelmintics, namely ivermectin, pyrantel and piperazine, were also investigated, both to validate the mouse model system and to assess efficacy of these drugs against H. polygyrus. Pyrantel and ivermectin were highly effective in reducing the numbers of H. polygyrus worms as well as eggs in faeces of the mice, but piperazine had a lower activity. Application of santonin and M. africana significantly reduced the number of total worm counts (TWC) but not faecal egg counts (FEC). The use of embelin, R. melanophloeos and A. indica reduced FEC but not TWC. In all cases, however, reductions were well below the a priori level of 70% required for biological significance. A. sanguineum, D. angustifolia and H. sepalosa had no effect on either TWC or FEC. In conclusion, none of the plant preparations had any biologically significant anthelmintic effect in this monogastric host-parasite model system.
The rat lungworm Angiostrongylus cantonensis is a food-borne zoonotic parasite of public health importance worldwide. It is the primary etiologic agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans in many countries. It is highly endemic in Thailand especially in the northeast region. In this study, A. cantonensis adult worms recovered from the lungs of wild rats in different geographical regions/provinces in Thailand were used to determine their haplotype by means of the mitochondrial partial cytochrome c oxidase subunit I (COI) gene sequence. The results revealed three additional COI haplotypes of A. cantonensis. The geographical isolates of A. cantonensis from Thailand and other countries formed a monophyletic clade distinct from the closely related A. malaysiensis. In the present study, distinct haplotypes were identified in seven regions of Thailand - AC10 in Phitsanulok (northern region), AC11 in Nakhon Phanom (northeastern region), AC15 in Trat (eastern region), AC16 in Chantaburi (eastern region), AC4 in Samut Prakan (central region), AC14 in Kanchanaburi (western region), and AC13 in Ranong (southern region). Phylogenetic analysis revealed that these haplotypes formed distinct lineages. In general, the COI sequences did not differentiate the worldwide geographical isolates of A. cantonensis. This study has further confirmed the presence of COI haplotype diversity in various geographical isolates of A. cantonensis. The COI gene sequence will be a suitable marker for studying population structure, phylogeography and genetic diversity of the rat lungworm.
A dot-blot ELISA was compared with a previously performed sandwich ELISA for the detection of Parastrongylus cantonensis antigens in sera from patients. Using the same monoclonal antibody and the same sera, 6 of 10 sera (60%) from parastronglyiasis patients were positive in dot-blot ELISA, whereas with sandwich ELISA, 5 of the same patient sera (50%) were positive. The specificity in both assays was 100% using 50 sera from patients with other parasitic diseases; of these, 10 each were from patients with cysticercosis, filariasis, gnathostomiasis, malaria and toxocariasis. The control group consisted of 53 sera from normal health Thais and Malaysians. The sensitivity of the assays was, however, slightly better with dot-blot ELISA and because it is simple, quick and cost-effective, it may be a test of choice for specific diagnosis of human parastrongyliasis.
A study was conducted to evaluate the anthelmintic properties of enhanced virgin coconut oil (EVCO) and senduduk (Melastoma malabathricum) plant against strongyle nematodes in goats. Two preparations of 10% EVCO dissolved in 90% virgin coconut oil and 10% EVCO dissolved in 90% palm oil, were given orally to two groups of mixed breeds goats. The efficacy test indicated that EVCO was insufficiently active as an anthelmintic. Four concentrations of senduduk solution (1.25, 2.5, 5.0 and 10 mg ml(-1)) were compared with a control and albendazole in an in vitro test for larvicidal effect. There was no significant larval mortality using senduduk solution. An in vivo test of senduduk was conducted by comparing three groups of goats, namely control, levamisole and treatment groups that were given a daily oral dose of senduduk crude extract with 1g kg(-1) from Day 0 to Day 12 and 2 g kg(-1) from Day 13 to Day 30. This efficacy test with senduduk also gave negative results. The findings obtained indicated that EVCO and senduduk were ineffective as anthelmintics against caprinestrongyle nematodes at the concentrations used.