Wolbachia is an obligatory intracellular endosymbiotic bacterium, present in over 20% of all insects altering insect reproductive capabilities and in a wide range of filarial worms which is essential for worm survival and reproduction. In Egypt, no available data were found about Wolbachia searching for it in either mosquitoes or filarial worms. Thus, we aimed to identify the possible concurrent presence of Wolbachia within different mosquitoes and filarial parasites, in Assiut Governorate, Egypt using multiplex PCR. Initially, 6 pools were detected positive for Wolbachia by single PCR. The simultaneous detection of Wolbachia and filarial parasites (Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens) by multiplex PCR was spotted in 5 out of 6 pools, with an overall estimated rate of infection (ERI) of 0.24%. Unexpectedly, the highest ERI (0.53%) was for Anopheles pharoensis with related Wolbachia and W. bancrofti, followed by Aedes (0.42%) and Culex (0.26%). We also observed that Wolbachia altered Culex spp. as a primary vector for W. bancrofti to be replaced by Anopheles sp. Wolbachia within filaria-infected mosquitoes in our locality gives a hope to use bacteria as a new control trend simultaneously targeting the vector and filarial parasites.
Aedes ; Anopheles ; Bacteria ; Culex ; Culicidae* ; Dirofilaria ; Dirofilaria immitis ; Dirofilaria repens ; Egypt* ; Hope ; Insects ; Larva* ; Multiplex Polymerase Chain Reaction ; Parasites ; Polymerase Chain Reaction ; Reproduction ; Wolbachia* ; Wuchereria bancrofti

Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs.
Aedes/*parasitology ; Animals ; Anopheles/*parasitology ; Culex/*parasitology ; Dirofilaria immitis/genetics/*isolation & purification ; Dirofilaria repens/genetics/*isolation & purification ; Egypt ; Entomology/methods ; Female ; Multiplex Polymerase Chain Reaction/*methods ; Parasitology/methods ; Sensitivity and Specificity ; Wuchereria bancrofti/genetics/*isolation & purification

Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs.
Aedes/*parasitology ; Animals ; Anopheles/*parasitology ; Culex/*parasitology ; Dirofilaria immitis/genetics/*isolation & purification ; Dirofilaria repens/genetics/*isolation & purification ; Egypt ; Entomology/methods ; Female ; Multiplex Polymerase Chain Reaction/*methods ; Parasitology/methods ; Sensitivity and Specificity ; Wuchereria bancrofti/genetics/*isolation & purification

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5+/-0.2degrees C, 79.0+/-0.3degrees C, 76.8+/-0.1degrees C, and 79.9+/-0.1degrees C, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.
Animals ; Blood/*parasitology ; Brugia/classification/genetics/*isolation & purification ; Cats ; Culicidae/*parasitology ; Dirofilaria immitis/classification/genetics/*isolation & purification ; Dogs ; Humans ; Male ; Parasitology/*methods ; RNA, Helminth/genetics ; RNA, Ribosomal, 5S/genetics ; Real-Time Polymerase Chain Reaction/*methods ; Sensitivity and Specificity ; Transition Temperature ; Wuchereria bancrofti/classification/genetics/*isolation & purification

The morphological characters of the microfilaria of Brugia malayi collected in Cheju-Do, Korea were described and compared with those or other local strains, already reported from the Malay Archipelago(Brug, 1927), Huchow, South China (Feng, 1933), Hachijo Kojima, Japan (Hayashi, 1951) etc. And it was found that there are no marked difference among these kinds of Microfilaria malayi. The percentage measurements of the fixed points in the Microfilaria malayi from Korea are summarized as follows; B.N.C.: 3.38%(2.90-3.72%), N.R.: 22.64% (17.36-25.81%), E.P.: 32.26% (27.49-36.60%), E.C.: 38.78% (34.90-41.72%), Inn. K. begin: 53.95% (49.50-58.84%) end: 67.87%(63.02-72.97%), G-1: 70.42% (60.22-75.23%), G-2:76.70% (75.10-78.32%), G-3:78.84% (77.81-79.59%), G-4:80.84%(79.82-81.35%), A.P.:82.94%(77.32-89.72%), Length: 193.39(+-12.3 micro-meter)(170.77-233.08 micro-meter), Width: 7.04+-0.37 micro-meter(5.99-7.99 micro-meter), Sheath: 268.48+-17.07 micro-meter(218.72-286.47 micro-meter), Ratio(Cephalic space): 1: 0.78
parasitology-helminth-nematoda ; Brugia malayi ; microfilaria

The periodicity of the microfilariae of Brugia malayi was studied in 9 human carriers from Shin-san Ri, Seong-san Myon, Cheju Island. The periodicity pattern was markedly nocturnal and the peaks were observed between 21:30 p.m. and 5:30 a.m. The average peak count was 1:30 a.m. and the percentage of peak count at this time was 95.3. The ratio of minimum of the average peak count percentage to the maximum was 8.3. The periodicity pattern of B.malayi in Cheju Island was compared with that in Inland and no differences were found between two forms. From the above observations, it was concluded that the periodicity of B. malayi in Korea is markedly nocturnal and closely resembles that in the strain of Penang, Malaya.
parasitology-helminth-nematoda-Brugia malayi ; periodicity ; microfilaria

A survey of canine heartworm (Dirofilaria immitis) infections among German shepherds in five areas of South Korea was performed from October 1994 to August 1995 using a microfilarial test (modified Knott's test) and an antigen test (DiroCHEK , Synbiotics, USA). The infection rate of 127 German shepherds (71 males and 56 females) was 10.2% (13/127) by the microfilarial test, but was 28.3% (36/127) by the antigen test, revealing that 24 of the 36 antigen-positive dogs were microfilaria-negative in the peripheral blood. All dogs that were microfilaria-positive were also antigen-positive. Three of the microfilaria-negative and antigen-positive dogs contained 4 - 15 adult heartworms in the heart and pulmonary arteries upon necropsy. The infection rate among German shepherds was the highest in Hoengsong-gun (Kangwon-do, 84.4%), while those of Yechon-gun (Kyongsangbuk-do) and Chungwon-gun (Chungchongbuk-do) areas were 20.0% and 14.3%, respectively. None of the dogs in the Kimhae-shi (Kyongsangnam-do) and Kwangju areas was heartworm positive. The infection rates of heartworm in dogs at the age of 1-3, 4-6, and 7-11 years were 6.3%, 21.4%, and 56.4%, respectively. Based on the fact that the antigen test is more accurate than the microfilarial test, this study strongly indicates that the prevalence rate of canine heartworm in Korea may be higher than previously reported (3.1+=23.0%) which utilized microfilarial tests.
parasitology-helminth-nematoda ; Dirofilaria immitis ; dogs ; antigen ; microfilaria ; epidemiology

In filariasis the infectivity of the appropriate mosquito vector is not consistent with the microfilarial density of the host. The reason may be attributed such factors as the time of microfilarial appearance in the peripheral blood of the host, the time of maximum biting activity of the arthropod vector, or the morphological adaptation of the feeding mechanism of the vector. However, it is quite puzzling to see why the number of microfilariae taken up by mosquitoes is subjected such a great variation, even though the same batch of mosquitoes are fed on the same filarial host under same laboratory conditions. The experiment was designed to observe more detail aspect of this relation. Adult Aedes togoi (Theobald, 1907) mosquitoes were reared from egg rafes colonized in an insectary. Animals used were Taiwan monkeys, Macaca cyclopsis which had been artificially infected with Wucheria malayi. The animals showed the microfilarial counts as low as nil to ten per slide of 20 cmm3 of blood, which seem to be rather fortunate for this kind of work. The microfilarial density of each animal was counted by taking each ten smears of 20 cmm(3) of peripheral blood the ear lobes before and after mosquito bite. Feeding were done in two occations, during 1600-1630 and 1900-1930 hours of the same day. The monkeys were immobilized and a rayon cage, housed 100 female mosquitoes for two days starvation, was exposed to the shaved abdomen of each animal. Fully engorged mosquitoes were transferred to a square rearing cage, which was later placed in the insectary, where kept temperature of 23-27degree C and relative humidity of 80-85 per cent. It was found that filarial larvae of the mosquito body usually develop to the third or infective stage in about 10 days after blood meal under these conditions. Daily dissections were made of these mosquitoes, either living or dead, after one week of rearing. Analysing of the result, the following conclusion was made. The rate and intensity of infection in mosquitoes are not directly related to the blood counts of microfilariae of the host animals. This is perhaps due to fluctuations of microbial outflow in the peripheral blood of individual animals. The reason of this would be no doubt due to a patch type of microfilarial distribution in the host blood.
parasitology-arthropodology-mosquito ; Aedes togoi ; Macaca cyclopsis ; monkey ; protozoology ; Brugia malayi ; microfilaria ; animal

An epidemiological study on brugian filariasis was carried out in endemic areas including Cheju Island in Korea, with a brief review of literatures.The results showed that the incidence among residents has remarkably decreased in Cheju Island, which was the main endemic area. Reviewing available informations on the prevalence of filariasis reported in recent years and also judging from the present socio-economic conditions which enable people to practice personal protection against mosquitos, it can be said with confidence that filariasis has almost disappeared from Cheju Island and inland areas. The disease is considered to remain at a low level of endemicity in Hugsan Islands. Certainly mass diethylcarbamazine (DEC) treatment carried out in Cheju Island in the 1960s and 1970s and remarkable economic growth followed by improved living standard and altered life-style of inhabitants could all have combined effects on the disappearance of this mosquito-borne diseae in this island. If the present trends go on, the possibility of resurgence of filariasis in Cheju Island is hardly postulated.
parasitology-helminth-nematoda ; Brugia malayi ; filariasis ; epidemiology

With a purpose to find out natural transition of endemicity of Malayan filariasis in inland Korea, a survey was conducted in June 1980 in Isan-Myeon of Yongpung-Gun (former Yongju-Gun) where an epidemiological investigation had been carried out in 1973 without any control activities such as chemotherapy. Five sample villages were surveyed for microfilaremia by 20 microliter night blood examination among inhabitants and the results of the surveys conducted in 1973 and 1980 were compared to determine natural transition of the endemicity of malayan filariasis during the period of the last 7 years. The current microfilaria rate among inhabitants in the 5 villages was 2.2 percent on the average (male: 1.6 percent, female: 2.8 percent) from 370 persons examined. By village, the rates were 5.9 percent (number of persons examined: 34) in Baranggol, 0 percent (30) in Guitonggl, 4.2 percent (72) in Alseonggol, 0 percent(65) in Jangjagol and 1.8 percent (169) in Saehae. Extremely low microfilaria rate was noted in young age groups. By age group, no positive case was found in those age groups below 30-39 years except 10-14 age group in which 2 positives (4.4 percent) were found. Two positives each were found in the respective age groups of 40-49(3.2 percent), 50-59 (4.4 percent) and over 60 (3.4 percent). In evaluation of the natural transition of the endemicity during the period of the last 7 years, the microfilaria rate turned out from 13.1 percent in 1973 to 2.2 percent in 1980. The difference in the microfilaria rate was 10.9 percent and the natural reduction rate per year was 1.6 percent on the average. From the examination of 35 cases which had revealed microfilaremia 7 years ago, 85.7 percent(30) of them were found to have converted to microfilaria negatives. On the other hand, from the 151 cases which had revealed no microfilaria in 1973, only 0.7 percent (1) of them was found to have converted to microfilaria positive. In the intensity of microfilaremia, the number of microfilaria/20 microliter blood per positive case was 11.0 in 1973 and 9.1 in 1980. The number of microfilaria/20 microliter per examinee was 1.4 in 1973 and 0.2 in 1980, thus reduced to 1/7 during the period of the 7 years. The retarding endemicity of malayan filariasis in inland Korea was considered to be resulted in by the gradual increase of environmental factors in relation to ecology of vector mosquitoes, which adversely affect to the transmission of malayan filariasis. Followings are suggested to be the factors which control the transmission of the malayan filariasis in this area: Inhabitants are the only natural final host of Brugia malayi infection in this area, gradual elevation of living standards of the inhabitants, gradual awakening of consciousness and behavior among inhabitants to protect themselves from mosquito biting using such as mosquito nets and insecticide sprays, preference of animal bait of vector mosquitoes, Anopheles sinensis, increase in number of domestic animals and fowls being raised in the village areas which play a major part of blood donors to vector mosquitoes, and relatively short (3-4 months) period of mosquito season in a year.
parasitology-helminth-nematoda ; Brugia malayi ; epidemiology