PCR-ELISA for diagnosis of Trypanosoma evansi in animals and vector

A highly sensitive and specific polymerase chain reaction (PCR) based assay for the detection of Trypanosoma evansi present in the blood of different animals and vector was developed. A simple lysis method was used to remove of the red blood cells to facilitate direct input of samples into the PCR reactions. The primer set was designed and synthesized to amplify a single band of 257 bp PCR product that was subsequently examined by enzyme-linked immunosorbent assay (ELISA). The sensitivity limit of PCR-ELISA was 0.01 pg that was corresponded to 1 parasite/ml of blood. No cross-reactivity of the assay was observed against Babesia bovis, B. bigemina, Anaplasma marginale,Theileria sp. and host DNA. The PCR-ELISA was shown to detect 33 samples of T. evansi infected blood of animals and 10 mosquitoes from different geographical area in Thailand. The results were corresponded to those of the PCR and mouse inoculation. This implies that the technique of PCR-ELISA is not only beneficial for diagnosis of the parasite but also useful for epidemiological study and designing rational trypanosomiasis control program.     

Effect of surface charge on the stability of oil/water emulsions during steam sterilization

Intravenous lipid emulsions are used for total parenteral nutrition and as carriers for lipophilic drugs. Exposure to the high temperature (121 degrees C) required for steam sterilization may cause coalescence and an increase in droplet size. The purpose of this study was to investigate whether an increase in the electrostatic repulsive force between oil droplets produced by formulation modification improves the thermal stability of lipid emulsions during autoclaving. The addition of a small amount, 0.66 or 1.32 mmol/kg (mm), of purified anionic phospholipid fractions (phosphatidic acid, phosphatidylglycerol, or phosphatidylinositol) to the standard formula increased the zeta potential from its normal value of -11 mV to -39 mV. Emulsions with the larger negative zeta potential did not exhibit any change in oil droplet size or distribution during steam sterilization at 121 degrees C for 15 min. The autoclaved emulsions having the larger negative zeta potential did not exhibit any evidence of coalescence when samples were stored for 1 month at 4 degrees C, room temperature, or 40 degrees C. Reduction of the negative surface charge of the oil droplets by the addition of stearylamine confirmed that the surface charge was an important factor, as emulsions having a reduced negative surface charge separated into two phases during autoclaving.     

Susceptibility of mansonia indiana (Diptera: Culicidae) to nocturnally subperiodic Brugia malayi (Spirurida: Filariodea)

Mosquitoes, Mansonia indiana Edwards, 1930, were collected from non-endemic area of human lymphatic filariasis and tested for their susceptibility of infection using nocturnally subperiodic Brugia malayai Buckley & Edeson, 1956. Three cats naturally infected with B. malayi were used in the experiment for mosquitoes feeding. The data revealed that the susceptibility of mosquito infection ranged from 30 to 70%. The results also revealed that the susceptibility rates were not linearly correlated to the microfilarial densities in the cat at the time of feeding. The microfilarial density in cats ranged from 15 to 27 per 10 microl of blood whereas the mean number of third stage larvae in the infective mosqiitoes ranged from 21.6 to 26.8. In addition, statistical analysis showed no significant difference (P > 0.05) between the mean number of third-stage larvae in mosquitoes and the density of microfilaria in cats. The study indicated that Ma. indiana, collected from non-endemic areas, is capable for transmitting the nocturnally subperiodic B. malayi.     

Intraspecies variation of Brugia spp. in cat reservoirs using complete ITS sequences

The internal transcribed spacer (ITS) region was used to study the intraspecies variation of Brugia spp. in cat reservoirs. Blood specimens from seven naturally infected cats were collected from two different geographical brugian-endemic areas in Thailand. The DNAPAR tree of these Brugia spp. was constructed using a maximum likelihood approach based on ITS nucleotide sequences and was compared to those of Brugia malayi, Brugia pahangi, and Dirofilaria immitis that were previously reported in GenBank. The phylogenetic trees inferred from ITS1, ITS2, and complete ITS sequences indicated that B. malayi and B. pahangi were separated into two clades, and subgroups were generated within each clade. The data revealed that ITS2 sequences were less informative than ITS1 for studying intraspecies variation of Brugia spp. Our results are primary data for intraspecies variation of B. malayi and B. pahangi in cat reservoirs. The information could be applicable for studying the molecular epidemiology and the dynamic nature of the parasites. GenBank accession numbers of Brugia malayi and Brugia pahangi complete ITS regions using in this study were EU373601-EU373625 and EU373626- EU373655, respectively.     

Detection of Plasmodium falciparum using a cloned DNA probe: a simple procedure suitable for field application

A simple procedure was developed for spotting blood samples directly onto nylon membrane filter, without the necessity to treat samples with pronase or proteinase K, followed by hybridizing with 32P-labelled DNA probe, pUNK1-45. This probe detected specifically P. falciparum DNA and did not cross react with DNA from man, P. knowlesi, P. chabaudi or P. cynomolgi. The probe was sensitive to detect a parasitemia of 0.001% in 20 microliters of blood.     

Detection of Plasmodium falciparum and Wuchereria bancrofti infected blood samples using multiplex PCR

A rapid and sensitive multiplex PCR has been developed for the diagnosis of multiple parasitic infection in human blood. Infection is detected by a single multiplex PCR reaction containing two pairs of oligonucleotide primers whereby each primer is specific for each parasite species. These primer sets amplified 400 and 450-bp fragments for Wuchereria bancrofti and 208-bp fragment for Plasmodium falciparum. The PCR products derived from each parasite species were visualized in ethidium bromide-stained agarose gels, therefore allowing the rapid identification of any, or all, of the two human parasites, if present, in a single amplification reaction. This multiplex PCR was very sensitive with the ability to detect the presence of as little as 10 pg of parasite DNA. The primers used in this multiplex PCR also showed highly specific amplification of each respective parasite DNA without the presence of non-specific and non-target PCR products. This multiplex PCR system was used to analyse 36 human blood samples of Myanmar workers in the endemic area at Tak Province, Thailand. Two samples showed the multiple infection, 27 samples were either infected with W. bancrofti or P. falciparum and seven samples were negative for both methods. The high sensitivity, specificity and rapidity of this multiplex PCR method make it suitable for large-scale epidemiological studies and following of drug treatment.     

Expression and characterization of Cu/Zn superoxide dismutase from <Emphasis Type="Italic">Wuchereria bancrofti</Emphasis>

The Cu/Zn superoxide dismutase gene from Wuchereria bancrofti (Cu/Zn WbSOD) was isolated by PCR using degeneracy primers. The complete Cu/Zn WbSOD consisted of 1,032 nucleotides containing 4 exons (477 nucleotides) and 3 introns. The molecular phylogenetic analysis of the Cu/Zn WbSOD gene in comparison with those from other organisms revealed that the gene was classified in the same clade to those of filarial Brugia malayi and Brugia pahangi (bootstrap value at 90). The nucleotide and deduced amino acid sequences of Cu/Zn WbSOD exhibited the similarity to those of intracellular Cu/Zn SOD of B. malayi and B. pahangi. The amino acid comparison of Cu/Zn WbSOD to others revealed that the binding sites and active sites were conserved. The expression of this gene yielded 16.366 kDa in size. After Ni-IDA column purification, the enzyme showed specific activity of 8.5 U/mg and 42.1% yield. The enzyme activity was inhibited when 6 mM KCN was added.     

Differential diagnosis of human lymphatic filariasis using PCR-RFLP

Filariasis is still a public health problem in tropical countries. The most common causative agents of human filariasis are Wuchereria bancrofti and Brugia malayi. Traditional methods used to detect filarial parasites in human, animal and vector populations are tedious, time consuming, and confer little guarantee of sensitivity and species specificity. We have developed a rapid and specific method to detect filarial parasite DNAs in blood and mosquito samples using the polymerase chain reaction (PCR) technique. The primers used are MF/F and MF/R which amplify a 1.5 kb glutathione peroxidase gene of filarial worms. Using the restriction fragment length polymorphism (RFLP) technique, these PCR products will be further digested with restriction enzymes either Hpa I, Pst I, Alu I or Hinf I to differentiate the genus of filaria. This PCR-RFLP technique can be apply to use in diagnosis and to differentiate between species of filaria in humans the reservoir host and the mosquito vector in endemic areas Copyright 2000 Academic Press.     

PCR based method for identification of zoonostic Brugia malayi microfilariae in domestic cats

The survey of 326 human blood samples in the endemic area of Surat Thani and Narathiwat, the provinces in the south of Thailand, revealed that 5 of them were infected with Brugia malayi. Similarly, 53 feline blood samples were also investigated and found that 15 of the domestic cats were also infected with B. malayi. Upon the examination of human and feline blood specimens, a pair of human and domestic cat stayed in the same house and region. The periodicities of human B. malayi and feline B. malayi were similar as well as the results of Giemsa and acid phosphatase stained blood films of microfilaria positive cases. Likewise, the PCR-RFLP profile of Hha I repeat genes and PCR amplification of Trans-Spliced Leader Exon I (SLX) demonstrated that 15 samples the feline B. malayi were the same as those of human B. malayi. The data indicated that domestic cat plays an important role as the animal reservoir for B. malayi in the endemic areas of Thailand.