Integration of Microscopic, Serologic and Molecular Techniques for Detection of Filarial Parasites in Dogs in Malaysia.

PURPOSE: Canine filariosis in domestic dogs caused by several species of filarids is an emerging vector-borne disease and the spread of this disease remains a global veterinary and public health concern. However, information regarding these filarids and their epidemiological patterns remains scarce in Malaysia. The present study aimed to determine the infection rate and associated risk factors of filarial parasites in dogs in Malaysia. METHODS: A total of 399 dog blood samples were collected from veterinary hospitals and animal shelters in Malaysia to determine the infection rate and associated risk factors via a combination of microscopic, serologic and molecular diagnostic techniques. RESULTS: Two species of canine filariae identified in this study were Dirofilaria immitis (6.5%) and Brugia pahangi (1.3%), and their infections were associated with cross breed, medium size and short hair (p < 0.05). CONCLUSIONS: A new pair of primers was developed to complement the recovery of the 12S rRNA gene fragment of filarial parasites. This study represents the first molecular evidence of B. pahangi in dogs in Malaysia.

Effect of Brugia pahangi co-infection with Plasmodium berghei ANKA in gerbils (Meriones unguiculatus).

Malaria and lymphatic filariasis (LF) are two leading and common mosquito-borne parasitic diseases worldwide. These two diseases are co-endemic in many tropical and sub-tropical regions and are known to share vectors. The interactions between malaria and filarial parasites are poorly understood. Thus, this study aimed at establishing the interactions that occur between Brugia pahangi and Plasmodium berghei ANKA (PbA) co-infection in gerbils. Briefly, the gerbils were matched according to age, sex, and weight and grouped into filarial-only infection, PbA-only infection, co-infection, and control group. The parasitemia, survival and clinical assessment of the gerbils were monitored for a period of 30 days post Plasmodium infection. The immune responses of gerbils to both mono and co-infection were monitored. Findings show that co-infected gerbils have higher survival rate than PbA-infected gerbils. Food and water consumption were significantly reduced in both PbA-infected and co-infected gerbils, although loss of body weight, hypothermia, and anemia were less severe in co-infected gerbils. Plasmodium-infected gerbils also suffered hypoglycemia, which was not observed in co-infected gerbils. Furthermore, gerbil cytokine responses to co-infection were significantly higher than PbA-only-infected gerbils, which is being suggested as a factor for their increased longevity. Co-infected gerbils had significantly elicited interleukin-4, interferon-gamma, and tumor necrotic factor at early stage of infection than PbA-infected gerbils. Findings from this study suggest that B. pahangi infection protect against severe anemia and hypoglycemia, which are manifestations of PbA infection.

Pathogenesis of Plasmodium berghei ANKA infection in the gerbil (Meriones unguiculatus) as an experimental model for severe malaria.

BACKGROUND: As the quest to eradicate malaria continues, there remains a need to gain further understanding of the disease, particularly with regard to pathogenesis. This is facilitated, apart from in vitro and clinical studies, mainly via in vivo mouse model studies. However, there are few studies that have used gerbils (Meriones unguiculatus) as animal models. Thus, this study is aimed at characterizing the effects of Plasmodium berghei ANKA (PbA) infection in gerbils, as well as the underlying pathogenesis. METHODS: Gerbils, 5-7 weeks old were infected by PbA via intraperitoneal injection of 1 × 106 (0.2 mL) infected red blood cells. Parasitemia, weight gain/loss, hemoglobin concentration, red blood cell count and body temperature changes in both control and infected groups were monitored over a duration of 13 days. RNA was extracted from the brain, spleen and whole blood to assess the immune response to PbA infection. Organs including the brain, spleen, heart, liver, kidneys and lungs were removed aseptically for histopathology. RESULTS: Gerbils were susceptible to PbA infection, showing significant decreases in the hemoglobin concentration, RBC counts, body weights and body temperature, over the course of the infection. There were no neurological signs observed. Both pro-inflammatory (IFNγ and TNF) and anti-inflammatory (IL-10) cytokines were significantly elevated. Splenomegaly and hepatomegaly were also observed. PbA parasitized RBCs were observed in the organs, using routine light microscopy and in situ hybridization. CONCLUSION: Gerbils may serve as a good model for severe malaria to further understand its pathogenesis.

Morphological and molecular characteristics of Malayfilaria sofiani Uni, Mat Udin & Takaoka n. g., n. sp. (Nematoda: Filarioidea) from the common treeshrew Tupaia glis Diard & Duvaucel (Mammalia: Scandentia) in Peninsular Malaysia.

BACKGROUND: The filarial nematodes Wuchereria bancrofti (Cobbold, 1877), Brugia malayi (Brug, 1927) and B. timori Partono, Purnomo, Dennis, Atmosoedjono, Oemijati & Cross, 1977 cause lymphatic diseases in humans in the tropics, while B. pahangi (Buckley & Edeson, 1956) infects carnivores and causes zoonotic diseases in humans in Malaysia. Wuchereria bancrofti, W. kalimantani Palmieri, Pulnomo, Dennis & Marwoto, 1980 and six out of ten Brugia spp. have been described from Australia, Southeast Asia, Sri Lanka and India. However, the origin and evolution of the species in the Wuchereria-Brugia clade remain unclear. While investigating the diversity of filarial parasites in Malaysia, we discovered an undescribed species in the common treeshrew Tupaia glis Diard & Duvaucel (Mammalia: Scandentia). METHODS: We examined 81 common treeshrews from 14 areas in nine states and the Federal Territory of Peninsular Malaysia for filarial parasites. Once any filariae that were found had been isolated, we examined their morphological characteristics and determined the partial sequences of their mitochondrial cytochrome c oxidase subunit 1 (cox1) and 12S rRNA genes. Polymerase chain reaction (PCR) products of the internal transcribed spacer 1 (ITS1) region were then cloned into the pGEM-T vector, and the recombinant plasmids were used as templates for sequencing. RESULTS: Malayfilaria sofiani Uni, Mat Udin & Takaoka, n. g., n. sp. is described based on the morphological characteristics of adults and microfilariae found in common treeshrews from Jeram Pasu, Kelantan, Malaysia. The Kimura 2-parameter distance between the cox1 gene sequences of the new species and W. bancrofti was 11.8%. Based on the three gene sequences, the new species forms a monophyletic clade with W. bancrofti and Brugia spp. The adult parasites were found in tissues surrounding the lymph nodes of the neck of common treeshrews. CONCLUSIONS: The newly described species appears most closely related to Wuchereria spp. and Brugia spp., but differs from these in several morphological characteristics. Molecular analyses based on the cox1 and 12S rRNA genes and the ITS1 region indicated that this species differs from both W. bancrofti and Brugia spp. at the genus level. We thus propose a new genus, Malayfilaria, along with the new species M. sofiani.

Prevalence of filarial parasites in domestic and stray cats in Selangor State, Malaysia.

OBJECTIVE: To determine the prevalence of the filarial parasites,ie.,Brugia malayi, Brugia, Brugia pahangi(B. pahangi), Dirofilaria immitisandDirofilaria repens (D. repens) in domestic and stray cats. METHODS: A total of 170 blood sample were collected from domestic and stray cats and examined for filarial worm parasites in two localities, Pulau Carey and Bukit Gasing, Selangor State, Malaysia. RESULTS: The overall prevalence of infection was 23.5% (40/170; 95% CI = 17.4-30.6). Of this, 35% (14/40; 95% CI = 22.1-50.5) and 50% (20/40; 95% CI = 35.2-64.8) were positive for single B. pahangi nd D. repens, respectively. The remaining of 15% (6/40; 95% CI = 7.1-29.1) were positive for mixed B. pahangi and D. repens. In addition, 75% of the infected cats were domestic, and 25% were strays. No Brugia malayi and Dirofilaria immitis was detected. Eighty-four cats were captured at Pulau Carey, of which 35.7% (30/84) were infected. Among the cats determined to be infected, 93% (28/30; 95% CI = 78.7-98.2) were domestic, and only 6.7% (2/30; 95% CI = 19.0-21.3) were strays. Conversely, the number of infected cats was three times lower in Bukit Gasing than in Pulau Carey, and most of the cats were stray. CONCLUSIONS: B. pahangi and D. repens could be the major parasites underlying filariasis in the study area. Adequate prophylactic plans should be administrated in the cat population in study area.

Stress exacerbates infectivity and pathogenicity of Blastocystis hominis: in vitro and in vivo evidences.

BACKGROUND: Stress alters the oxidant-antioxidant state and immune cell responses which disrupts its function to combat infection. Blastocystis hominis, a common intestinal protozoan has been reported to be opportunistic in immunocompromised patients namely cancer. B. hominis infectivity in other altered immune system conditions especially stress is unknown. We aimed to demonstrate the stress effects towards the susceptibility and pathogenicity of B. hominis infection. METHODS/FINDINGS: Three-week-old Wistar rats were divided into four groups: (a)control; (b)stress-induced; (c)B. hominis infected; (d)stress-induced with B. hominis infection; (n = 20 respectively). Stress was induced for an hour daily (30 days) using a Belly Dancer Shaker. Weight gain was monitored, stool samples were collected for B. hominis screening and blood for the determination of differential count, levels of immunoglobulin, oxidative damage, and peripheral blood mononuclear cell (PBMC) proliferation upon induction with solubilized antigen of B. hominis (Blasto-Ag). Group (b) exhibited the highest level of weight gain. Group (d) had higher levels of parasite cyst count in stools, serum IgE, oxidized protein and lipid compared to the group (c). Levels of monocyte and antioxidant in group (d) were decreased and their PBMCs showed highest inhibition of proliferation level when exposed to Blasto-Ag. Monocyte level in Group (b) showed insignificant difference compared to group (a) but was significantly lower compared to group (c). Antioxidant levels in group (c) were generally lower compared to group (a) and (b). Inhibition level exhibited by Blasto-Ag treated PBMCs of group (c) was higher compared to group (a) and (b). CONCLUSION: The pathogenicity and augmentation of B. hominis infection is enhanced when stress is present. Lifestyles today are becoming increasingly stressed and the present findings suggest that the parasite which has been reported to be one of the most common organisms seen in stool surveys, namely in developing countries, may tend to be more pathogenic in stressful situations.

Armigeres subalbatus incriminated as a vector of zoonotic Brugia pahangi filariasis in suburban Kuala Lumpur, Peninsular Malaysia.

BACKGROUND: In 2011, we reported occurrence of natural human infections with Brugia pahangi, a filarial worm of dogs and cats, in a surburb of Kuala Lumpur, the capital city of Malaysia. Our preliminary entomological survey at that time suggested the mosquito species Armigeres subalbatus as the vector of the zoonotic infections. In this present report, we provide biological evidence to confirm our preliminary finding. FINDINGS: A total of 1798 adult female Ar. subalbatus mosquitoes was caught in the vicinity of the suburb, and 1599 were dissected for the presence of filarial larvae. Sixty-two mosquitoes were positive, and 27 of these were infected with L3 larvae. The L3 were inoculated into male gerbils. Microfilariae could be detected in the gerbils 92 days post-infection. Post-mortem on the gerbils recovered adult worms in the peritoneal cavity, heart, lungs, tail and testis. Male adult worms were confirmed to be B. pahangi by the ratio length of their spicules (left spicule: right spicule). Female adult worms were confirmed by the absence of minute cuticular bosses in the tail region. The worms were further confirmed to be B. pahangi by PCR. CONCLUSIONS: Our results showed that Ar. subalbatus was the vector for the zoonotic Brugia pahangi infections. This mosquito species should now be categorised as a medically important mosquito species in Malaysia. Its role in the transmission of zoonotic B. pahangi must therefore be considered in future studies on filarial infections.