Molecular detection of porcine Enterocytozoon bieneusi infection in Peninsular Malaysia and epidemiological risk factors associated with potentially zoonotic genotypes.

Enterocytozoon bieneusi is an emerging opportunistic pathogen infecting humans, and both domestic and wild pigs are known to harbour zoonotic genotypes. There remains a paucity of information on the prevalence and epidemiology of this enteropathogen in Southeast Asia. The present study was undertaken to determine the molecular prevalence and risk factors associated with E. bieneusi infection among commercially farmed pigs in Malaysia. Faecal samples were collected from 450 pigs from 15 different farms and subjected to nested PCR amplification of the ribosomal internal transcribed spacer (ITS) gene of E. bieneusi. Phylogenetic analysis involved 28 nucleotide sequences of the ITS region of E. bieneusi. An interviewer-administered questionnaire provided information on the animal hosts, farm management systems and environmental factors and was statistically analysed to determine the risk factors for infection. The prevalence of E. bieneusi infection was relatively high (40.7%). The highest prevalence (51.3%) was recorded among the piglets, while the adults showed the lowest level of infection (31.3%). Multivariate analysis indicated that age of the pigs, distance of the farm from human settlement and farm management system were significant risk factors of infection. Three genotypes (EbpA, EbpC and Henan-III) detected among the pigs are potentially zoonotic. The high prevalence of E. bieneusi among locally reared pigs, the presence of zoonotic genotypes and the spatial distribution of pig farms and human settlements warrant further investigation on the possibility of zoonotic transmission.

Control of Aedes aegypti and Ae. albopictus, the vectors of dengue and chikungunya, by using pheromone C21 with an insect growth regulator: Results of multicentric trials from 2007-12 in India.

BACKGROUND & OBJECTIVES: Aedes mosquito control has gained much importance nowadays in view of rise in number of reported cases of dengue and chikungunya in India and other countries. In the present study, C21 attracticide (containing a pheromone and an insect growth regulator­IGR, developed by Defence Research and Development Establishment (DRDE), Gwalior, India was tested for its feasibility for surveillance and control of Aedes mosquito in a multicentric mode from October 2007 to June 2012 in urban (Delhi, and Bengaluru district, Karnataka) and suburban (Alappuzha district, Kerala) settings of the country in three phases. METHODS: Across the randomly selected households in each study area, two to four containers treated with attracticide (experimental) and untreated (control) were placed and monitored by trained surveillance workers on weekly/ fortnightly basis for determining the presence of eggs, larvae and pupae. Container positivity, percent larvae, egg and pupae collected were determined during different phases and analyzed statistically using SPSS 18.0. RESULTS: Container positivity was found statistically significant at Bengaluru and Alappuzha, Kerala while in Delhi, it was found non-significant. Eggs collected from experimental containers were significantly higher in comparison to control at all the locations except Delhi. Also larvae collected from control containers were significantly higher at all the locations except Bengaluru. Pupae collected from control containers remained significantly higher at all the locations as no pupal formation was recorded from experimental containers. INTERPRETATION & CONCLUSION: The use of C21 attracticide hampered pupal formation, thus inhibiting adult population in the study areas. The study established that C21 attracticide was efficacious in the field conditions and has potential for use in surveillance and management of dengue and chikungunya mosquitoes.

Comparative efficacy of quadratus lumborum type-II and erector spinae plane block in patients undergoing caesarean section under spinal anaesthesia: a randomised controlled trial.

BACKGROUND: Quadratus lumborum and erector spinae plane blocks have been used to provide analgesia in patients undergoing thoracic or abdominal surgeries. Our study compared the analgesic efficacy of the quadratus lumborum type-II block (QLB-II) and the erector spinae plane block (ESPB) in parturients who underwent caesarean section under spinal anaesthesia. METHODS: Fifty-two patients with comparable demographic profiles were randomised into two groups, QLB-II (n = 26) and ESPB (n = 26). After the surgery, patients received either ultrasound-guided QLB-II or ESPB using 0.25% bupivacaine 0.3 mL/kg. Comparison of analgesic efficacy was in terms of fentanyl consumption (primary outcome), pain scores, incidence of complications in the 24-h postoperative period, and quality of recovery (QoR-15) on postoperative days one and two, and day of discharge. RESULTS: There was no significant difference in cumulative number of fentanyl doses (W = 349.000, P = 0.840), numerical rating score at rest (P = 0.648) or with movement (P = 0.520), QoR-15 scores on postoperative day one (P = 0.549), day two (P = 0.927) or day of discharge (P = 0.676). CONCLUSION: We concluded that patients who underwent QLB-II or ESPB reported similar analgesic efficacy, complications, and quality of recovery in the postoperative period.

Molecular characterisation and genetic affinities of Cyclophyllidean cestodes infecting wild rats in Peninsular Malaysia.

Cestode infections is widely reported in rodents, however species identification remains problematic due to the genetic or interspecies variation. Therefore, this study was aimed to verify the Cyclophyllidean parasites recovered from wild rats captured from different forest types using molecular based methods. Maximum-likelihood (ML) and neighbour-joining (NJ) trees were constructed inferred from 18 small subunit nuclear ribosomal RNA gene (18SrDNA) and mitochondrial cytochrome c oxidase subunit one gene (COX1) sequences of cestode worms recovered from 124 individuals from four rat species. Sequences obtained from both Hymenolepis diminuta and Hydatigera parva represents the first records in Malaysia. All the sequences were successfully amplified with product with total length of 205 and 1202 base pairs (bp), respectively. Three cestode species from the Family Hymenolepididae (Hymenolepis diminuta) and Family Taeniidae (Hydatigera parva; Hydatigera taeniaeformis) were successfully characterized using phylogenetic analyses and haplotype networking. Phylogenetic analysis showed that H. diminuta, Hydatigera parva (Hy. parva) and Hydatigera taeniaeformis (Hy. taeniaeformis) formed its own monophyletic clade in 18SrDNA analyses. Results also showed that Hy. taeniaeformis shared the same haplotype group with Hy. taeniaeformis from China (COX1) and linked with Hy. taeniaeformis from Japan (18SrDNA) while the Malaysian H. diminuta clearly formed a separate haplotype and networked with other regions. The Malaysian Hy. parva isolation, on the other hand, appeared to be genetically distinct from the European Hy. parva (Spain) strain, but closely linked to the local isolates. Molecular methods employed successfully improved in the detection of complex species in this group. The findings showed that molecular data can be useful to deeply study intra-specific variation in other cestode worms.

Prevalence of Toxoplasma gondii in pet and stray cats 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.

First molecular detection of Tritrichomonas foetus in domestic cats in Klang Valley, Malaysia.

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.

Antitrypanosomal and cytotoxic activities of botanical extracts from Murraya koenigii (L.) and Alpinia mutica Roxb.

Four carbazoles (girinimbine, mahanimbine, murrayafoline and murrayanine), isolated from Murraya koenigii, and one kavalactone (5,6-dehydrokawain) and one flavonoid (pinostrobin) isolated, from Alpinia mutica, were tested for their antitrypanosomal activity using in vitro cultured Trypanosoma evansi cell lines. The cytotoxic activities of these compounds were also investigated against mammalian Vero cells using the MTT (3-(4,5- Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide)-cell proliferation assay. Three carbazole compounds, namely mahanimbine, murrayafoline, and girinimbine, showed a potent antitrypanosomal activity, scoring a median inhibitory concentration (IC50) of 3.13, 6.35 and 10.16 µg/ml, respectively. Girinimbine was the least toxic to Vero cells, and the mean cytotoxic concentration (CC50) and the selectivity index (SI) of this compound were 745.58 ± 42.38 µg/ ml and 73.38, respectively. Girinimbine and the other carbazole compounds possess potential antitrypanosomal activity with comparably low toxicity against mammalian cells. Girinimbine, in particular, is a good candidate to be further investigated as a potential antitrypanosomal agent using in vivo models.

Toxoplasma gondii infection in native village chickens (Gallus domesticus) in Selangor and Melaka, Malaysia.

Toxoplasmosis is a worldwide zoonosis caused by the protozoa Toxoplasma gondii which affects human and animals. Village chickens (Gallus domesticus) most commonly known as Ayam Kampung or free-range chickens, have been suggested to play a role in the epidemiology of toxoplasmosis. This study determines the presence of T. gondii in the village chicken populations in two states of Malaysia. A total of 50 serum samples from the chickens from Selangor (n=20) and Melaka (n=30) were collected and analysed using commercial serological kits. T. gondii antigen was detected in 20% (Selangor 30%; Melaka 13%) samples using ELISA test and anti-T. gondii antibody was detected in all positive ELISA samples using the indirect haemagglutination test (IHAT). Histopathological examination revealed tissue changes such as inflammation and degeneration in brain and liver of seropositive chickens. This is the first report of T. gondii infection in the village chickens in Malaysia.

Molecular detection and epidemiological risk factors associated with Cryptosporidium infection among cattle in Peninsular Malaysia.

Enteric protozoa infection among cattle may pose a threat to productivity and survival leading to negative impacts on the livestock industry. A number of these pathogens are also known to be zoonotic and are of public health concern. Despite the importance of these enteric protozoa to both animal and human health, there remains a paucity of published information on the epidemiological risk factors that may be associated with bovine cryptosporidiosis in Southeast Asia. The present study was undertaken to determine the molecular prevalence and associated risk factors for Cryptosporidium infection among beef and dairy cattle in Peninsular Malaysia. Faecal samples were collected from 824 cattle in 39 farms (526 beef and 298 dairy) situated in 33 locations throughout the country, and subjected to PCR detection for Cryptosporidium using primers targeting the 18S SSUrRNA gene. Epidemiological variables including host, environment and management factors were subjected to univariate and multivariate logistic regression analyses to determine the potential risk factors for infection. The prevalence of Cryptosporidium among the cattle was 12.5%, with significant difference in the infection rate among the various breeds. There was no significant effect of gender, and both the beef and dairy cattle were at similar odds for infection. The younger cattle had a significantly higher infection rate compared to the older animals. Multivariate analysis revealed that deworming practice, distance to human settlement, geographical location (zone) and farm management system were significant risk factors associated with Cryptosporidium infection. The cattle that were reared on farms located in the northeast of the country, closest (≤200 m) to human settlements, reared extensively, and dewormed every four months were at highest risk of infection. The present study constitutes the first attempt to analyze the multivariable epidemiological risk factors involved in bovine cryptosporidiosis in Malaysia and in Southeast Asia. It is envisaged that the data obtained will facilitate better control and prevention measures for Cryptosporidium infection among cattle in the region. Due to the potential zoonotic nature of the infection, serious steps should be instituted for animal treatment and biohazard waste management on local cattle farms.

Molecular and serological prevalence of Anaplasma and Ehrlichia sp. among stray dogs in East Malaysia.

Anaplasma and Ehrlichia species are important bacterial pathogens of various animal species including dogs and humans. They constitute a major health problem worldwide, especially in the tropics and sub-tropics. In order to identify the prevalence of Anaplasma and Ehrlichia sp. from dogs in the selected area, a total of 100 randomly selected clinically healthy rescued dogs from animal shelters in Sarawak and Sabah, East Malaysia (50 dogs per State) were used for this study. Whole blood was collected from these dogs and screened for presence of Anaplasma and Ehrlichia spp. using polymerase chain reaction (PCR) and commercial ELISA test (SNAP 4Dx® Plus) for detection of antigen and antibody against the two pathogens respectively. 27% (A. platys) and 33% (E. canis) of the sampled population were positive using PCR; while the serological prevalence was 29% for Anaplasma sp. and 55% for Ehrlichia sp. The results of this investigation revealed that stray dogs are potential reservoir hosts of Anaplasma and Ehrlichia sp. infection with a high prevalence in the area. The high prevalence rates detected highlights the urgent need to address the growing population of possible vectors and reservoirs for transmission in the region and also to look into the public health aspect of the disease.

Occurrence of gastro-intestinal parasites among small ruminants in Malaysia: highlighting Dicrocoelium infection in goats.

The aim of the present study was to determine the gastro-intestinal (GI) parasitic infections among small ruminants (i.e., goats, sheep, deer) in Malaysia through formalin-ether concentration technique. Overall, 70.9% or 302 out of 426 small ruminants (79.4% or 251/316 goats; 87.5% or 35/40 sheep; 22.9% or 16/70 deer) were infected with at least one species of GI parasites. Overall, ten types of GI parasites [Helminth: strongyle (57.7%), Moniezia spp. (5.4%), Paramphistomum spp. (4.5%), Strongyloides spp. (4.2%), Dicrocoelium spp. (2.3%), Trichuris spp. (2.3%); Protozoa: Eimeria spp. (23.7%), Entamoeba spp. (18.8%), Giardia spp. (1.9%), Cryptosporidium spp. (0.2%)] were detected in this study. Among the studied animals, goats harboured the highest diversity of GI parasites (ten types), followed by sheep (six types) and deer (two types). Polyparasitism was observed in goats (43.7% or 138 of 316) and sheep (15.0% or 6 of 40). Cumulatively, a total of 32 combinations of coinfections (Helminth+Helminth: 8 combinations; Helminth+Protozoa: 20 combinations; Protozoa+Protozoa: 4 combinations) between detected parasites with up to quintuple infections were reported. Among these parasites, "strongyle + Eimeria spp." and "Moniezia spp. + strongyle" were the commonest infections in goats (13.5% or 34 of 251) and sheep (5.7% or 2 of 6), respectively. This study is a comprehensive documentation on multiple GI parasitisms among small ruminant in Malaysia, and the findings are crucial for effective farm management, especially for the formulation of parasitic control and elimination strategies.

Hemoglobin-A2-Coburg or alpha2delta2116Arg leads to His (G18).

Hemoglobin-A2-Coburg or alpha2delta2-116 Arg leads to His (G18) has been found in members of a family of Sicilian origin. The propositus is heterozygous for hemoglobin-A2-Coburg as well as for beta-thalassemia, and family data indicate that the gene for the delta-Coburg chain is in trans of the beta-thalassemia determinant.

Evaluation of bifenthrin treated mosquito nets against anopheline and culicine mosquitoes.

BACKGROUND AND OBJECTIVES: The main rural malaria vector Anopheles culicifacies has developed resistance to dichloro diphenyl trichloroethane (DDT), hexachloro cyclo hexane (HCH) and malathion in the state of Haryana in northern India. An alternative synthetic pyrethroid insecticide bifenthrin was therefore evaluated on mosquito nets against anopheline and culicine mosquitoes, in two villages Jagdishpur and Garh Mirakpur of Community Health Center (CHC) Badhkhalsa in district Sonipat, Haryana state. METHODS: Two formulations of bifenthrin, suspension concentrate (SC) and micro-emulsion (ME) were compared with micro-capsule suspension (CS) of lambdacyhalothrin. The impact of three doses of bifenthrin (10, 25 and 50 mg/m(2)) impregnated on mosquito nets was compared with lambdacyhalothrin (25 mg/m(2)) and untreated control. Quality assessment of treatment on treated nets was carried out by residue analysis and the persistence of the insecticide on nets was determined by contact bioassays. Efficacy of treated nets on mosquito density was assessed by calculating mosquito entry rate, immediate mortality, delayed mortality and excito-repellency to the insecticides. RESULTS: In susceptibility tests An. culicifacies was susceptible to bifenthrin (0.1% test papers) and to lambdacyhalothrin (0.05% test papers). Bioassays on treated nets against A. culicifacies recorded 100 per cent mortality up to tenth fortnight for all the doses of impregnation with bifenthrin (SC and ME) and lambdacyhalothrin (CS). Ring-net bioassays against An. culicifacies showed median knock-down time between 3.1 to 11.4 min. Behavioural indices were also studied for anopheline and culicine mosquitoes. The reduction in entry rates of anopheline and culicine mosquitoes into the rooms with treated nets compared to control indicated good efficacy with all the formulations and doses of the insecticides. INTERPRETAION AND CONCLUSION: Indoor (immediate) mortality of mosquitoes with bifenthrin ME formulation was relatively lower compared to SC fomulation of bifenthrin and based on delayed mortility and continued susceptibility in bioassays, bifenthrin ME at the rate of 10 mg/m(2) dose was found suitable for the impregnation of mosquito nets for phase III trial.

Seasonal fluctuations of dengue fever vector, Aedes aegypti (Diptera: Culicidae) in Delhi, India.

Studies on the seasonal fluctuation of Aedes aegypti were undertaken in different localities of Delhi, during 2000. The Aedes aegypti population was found to be prevalent in all the localities in Delhi. Water coolers and tires were found to be the preferred breeding habitats of Aedes mosquitos in the city. Aedes aegypti, being hygroscopic, showed a phenomenon of annual pulsation. It tends to move to mother foci in the central areas of the city, which are humid in the dry season, and spread out during the wet season. Out of 103,778 houses surveyed, 20,513 houses and 3,547 containers were reported positive for Aedes aegypti. The house container, and Breteau indices were very high during the post-monsoon season. The container indicies was very high (17.7%) in the defence area in September 2000. The container index in the areas of the Municipal Corporation of Delhi (MCD) and the New Delhi Municipal Committee (NDMC) were found to be high during the same period. The house index forAedes aegypti ranged from 0.1 to 7.4, 0.1 to 11.3, and 0.1 to 11.1 in the MCD, NDMC, and Defence areas, respectively.

Semen characteristics: Advancement in andrological assessment.

Progress in diagnosis of infertility, has been dramatically increased during the past decades with changes occurring in virtually all aspects of infertility research, thus providing innovative diagnostic testing and sophisticated instrumentation for improved management and treatment of infertility. There are about 50% of infertile couples who are suffering because of male infertility. Semen examination is a basic investigation for these infertile couples. It not only reveals the quantity and quality of sperm but also the quality of the seminal plasma, which is essential for normal sperm function. In this review, the recent advancement in investigation procedures has been analyzed which are very important in clinical practice to (a) evaluate the sperm fertilizing ability (Acrosin, aniline blue, HOS), (b) characterization of male accessory sex glands secretions (Fructose, alpha-glucosidase, PSA) and (c) the management of azoospermic patients. It is believed that use of such diagnostic procedures will facilitate wide selection of patients for whom an effective therapy might be then possible.

In vivo antitrypanosomal activity of Garcinia hombroniana aqueous extract.

The anti-Trypanosoma evansi activity of Garcinia hombroniana (seashore mangosteen) leaves aqueous extract was tested on experimentally infected Sprague-Dawley rats. Treatment of infected rats with G. hombroniana extract resulted in a significantly extended post-infection longevity (p < 0.05), compared to the untreated control group. The possible mode of antitrypanosomal effect of the plant extract was also investigated on cultured T. evansi in HMI-9 medium with the addition of 25 µg/ml G. hombroniana aqueous extract. It was observed that the addition of G. hombroniana extract resulted in the inhibition of trypanosomal kinetoplast division, with no significant inhibitory effect on nuclear division. It is concluded from the current study that the aqueous extract of G. hombroniana has a potential antitrypanosomal activity through the inhibition of kinetoplast division, as one of the possible mechanisms of its antitrypanosomal effect. This plant could serve as a possible source of new antitrypanosomal compounds.

Culture of prostate epithelial cells of the rhesus monkey on extracellular matrix substrate: influence of steroids and insulin-like growth factors.

Rhesus monkey prostate epithelial cells from the cranial lobe were isolated and cultured in flasks coated either with collagen IV or laminin. The effects of stromal cell medium, androgens and growth factors on cell number, thymidine incorporation and secretory activity were assessed. The results indicate that dihydrotestosterone (DHT) and androstenedione have stimulatory influences on cell proliferation and secretion in coated flasks. DHT was more effective in increasing cell number but the induction of secretory activity was similar with both steroids. The combination of IGF-I and -II resulted in inducing better cell proliferation and secretory activity than the individual IGFs but, of the two IGFs, IGF-I was more effective than IGF-II. DHT with IGFs was more potent in inducing proliferation, differentiation and secretion than androstenedione. Even in the absence of steroids or growth factors, colony formation and confluence occurred in coated flasks but cell differentiation and secretion only to a limited extent. In conclusion, we were able to establish an in vitro primary culture of prostate epithelial cells from rhesus monkey using extracellular matrix proteins, steroids and growth factors as additional supplements. This culture system may be useful to study prostate cell physiology and to identify drugs that can inhibit cell proliferation.

Molecular diversity of the plasmid genotypes among Rhizobium gene pools of sesbanias from different habitats of a semi-arid region (Delhi).

Plasmid genotypes of root nodulating rhizobial isolates of Sesbania, sampled from six ecologically distinct habitats, were characterized. Plasmid profile analysis revealed nine different plasmid types having molecular masses ranging from 30 to 300 MDa, distributed among six profile types that grouped the isolates into six plasmid classes. The six plasmid profiles were diverged from each other and lack many common plasmid types among them. Variation in number and types of symbiotic (Sym) plasmid was assessed by hybridization of plasmid profiles with sym gene probes. Relatedness among different plasmid types was assessed by hybridization of total DNAs as well as plasmid profiles of different isolates with labelled intact plasmid. Plasticity of plasmid genotype and possible recombination between different plasmid types is suggested from the results obtained. Structural diversity among sym plasmids was assessed by PCR amplified product profiles using primer corresponding to the reiterated nif promoter consensus element (NPC-PCR). A total of 26 NPC-PCR profile types were recognized. Genetic diversity among sym plasmids of isolates belonging to the same plasmid class and having similar sym plasmid suggested recombinations and rearrangements of sequences within the sym plasmids. Cluster analysis based upon similarity among profile types sorted the isolates across the ecological gradient. We suggest that habitat heterogeneity and plasticity of plasmid genotype together contribute for the generation of genetic diversity leading to strainal differentiation in rhizobia.