Specific polymerase chain reaction for differential diagnosis of Dirofilaria immitis and Dipetalonema reconditum using primers derived from internal transcribed spacer region 2 (ITS2).

Both Dirofilaria immiti and Dipetalonema reconditum may be found in blood of infected dogs but it is not easy to distinguish D. immitis from D. reconditum in morphology. We cloned and sequenced the contiguous internal transcribed spacer (ITS) region, ITS1-5.8S-ITS2, of these two different parasites and published on GenBank as AF217800 for D. immiti and AF217801 for D. reconditum in this study. We designed two pairs of specific primers derived from ITS2 being used for polymerase chain reaction (PCR). The amplicons of ITS2 from D. immiti and D. reconditum are 302 and 348bp, respectively. Moreover, the limitation for amplifying ITS2 gene using this PCR demonstrated that 1 x 10(-2) microfilaria of each species of parasite smashed or even with mixed samples could be detected and the PCR products were predicted as the same as that described above. Thus, D. immiti and D. reconditum could be differentially diagnosed by this specific PCR. Seventeen clinical cases were evaluated and all of them were correctly identified. In this study, ITS1-5.8S-ITS2 of D. immiti or D. reconditum were the first time sequenced and analyzed. No significant similarity of ITS1 and ITS2 between D. immiti and D. reconditum could be observed.

Antifilarial activity of some 2H-1-benzopyran-2-ones (coumarins).

Six synthetic 2H-1-benzopyran-2-one (cournarin) derivatives (CDRI compounds # 1, 2, 3, 4, 5 and 6) were evaluated for filaricidal activity against Litomosoides carinii and Acanthocheilonema viteae infections in cotton rats (Sigmodon hispidus) and Mastomys coucha respectively. Significant effects on macrofilariae (>80% death/sterilisation) were detected with compounds #2, 3 and 6 against L. carinii and/or A. viteae. Thus detection of filaricidal activity in benzopyrones, which are so far known for anti-inflammatory activity, provides a new lead for development of better filaricidal agents for combating filariasis.

Millardia meltada, a new host for Acanthocheilonema viteae and a simple technique for separation of microfilariae from peripheral blood.

Millardia meltada were infected with Acanthocheilonema viteae and examined for their susceptibility. The morbidity of infected M. meltada was low compared with that of jirds. On day 47 post-infection (p.i.), 13 of 14 M. meltada developed microfilaremia. Male M. meltada then showed gradually increasing microfilaremia with a peak level of 7000 per 30 microliters blood at week 20 p.i., which was much higher than that (3000) of male jirds. In contrast, microfilarial densities of female M. meltada were markedly low with a peak level of 200 during weeks 10-12 p.i. A simple centrifugation technique with Lympholyte-M was devised for microfilarial separation from the peripheral blood of infected M. meltada and yielded approximately 17 x 10(5) viable microfilariae from 1 ml of blood. This method also makes it possible to collect microfilariae from the same individuals repeatedly. M. meltada, coupled with this microfilarial separation technique, serves as a useful animal model for microfilarial studies of A. viteae.

Transplantation into jirds as a method of assessing the viability and reproductive integrity of adult Acanthocheilonema viteae from culture.

The reproductive integrity and viability of adult female Acanthocheilonema viteae (syn. Dipetalonema viteae) maintained in culture for relatively long periods were assessed by transplantation into jirds. Worms cultured in chemically defined NI medium for approximately 3-4 wk remained active, but microfilarial release declined to barely detectable levels. Microfilarial production, however, was restored when the worms were transplanted subcutaneously into jirds. When cultured in NI medium beyond 4 wk no restoration of microfilarial production occurred on transplantation, presumably due to irreversible injury to the reproductive system. However, when NI medium was supplemented with fetal bovine serum resumption of microfilarial production occurred in transplanted females that had been in culture for as long as 2 1/2 mo. The addition of serum to NI medium played an important role in maintaining and protecting the functional integrity of the reproductive system.

Immunity to Dipetalonema viteae (Filarioidea) infections in resistant and susceptible mice.

The course of infection with Dipetalonema viteae in mice shows marked genetically-determined strain variation. Subcutaneous implantation of 5 female D. viteae into C57BL/10 (B10) mice results in a short term, low level microfilaraemia compared with that seen in similar infections in BALB/c mice. Adult worm survival is similar, thus the different patterns of infections reflect responses directed against the microfilariae larvae (mf). A number of immunological parameters have been monitored during infection in an attempt to identify strain differences which may be correlated with levels of resistance. Blast cell activity in the spleen and lymph nodes showed little strain difference, peaking on day 10 and declining as mf disappeared from the circulation. Total serum IgG levels doubled in both strains during infection, the response being more rapid in B10 mice. Serum IgM levels increased threefold in BALB/c mice but fourteen-fold in B10. Radiosorbent assays identified comparable anti-adult antibody and anti-mf homogenate IgM antibody responses in both strains. Immunofluorescent assay showed that the appearance of IgM antibodies directed against the mf surface correlated with the clearance of mf from the blood of B10 mice, whereas similar antibodies were not detected in BALB/c mice. It is proposed that the efficient clearance of mf in B10 is mediated through an IgM-dependent mechanism and that the chronic microfilariaemia seen in BALB/c mice is facilitated by the absence of a specific IgM response to mf surface antigens.

Longevity of microfilariae following removal of the adult worms.

The present study was undertaken to determine the longevity of a population of microfilariae in a natural host following the removal of the adult worms without drug intervention. Four squirrel monkeys previously infected with Dipetalonema gracile were allowed to develop stable microfilaremias. All adult worms were then removed surgically from the peritoneal cavity. Weekly microfilaria counts were made on each animal and the decline in microfilariae recorded. At the time of adult worm removal, microfilaremias ranged from 750 to 12,500 mf/ml. The observed decline in microfilaria densities was gradual, but steady, in all animals. Microfilariae persisted in 1-ml blood samples for 60, 62, 91, and 101 weeks following removal of adult worms. The results indicate clearly that in a naturally produced population, in a natural definitive host, microfilariae survive for 60 to 100 weeks. The gradual decrease in microfilarial densities would appear to be the result of the death of specific batches or broods of microfilariae. The microfilariae which persisted in the blood the longest undoubtedly represent those which were produced just prior to the removal of the adult worms. It is postulated that because of the long life-span of microfilariae, female worms are not called upon to produce a continual supply of microfilariae, nor is the need for mating as frequent as might be expected. Equally important, the number of microfilariae which the host is called upon to phagocytize is considerably smaller than previously suggested.

Transplanted Dipetalonema viteae in the jird: effect of worm burden on parturition rates and microfilaremia.

Dipetalonema viteae was studied in the jird, Meriones unguiculatus, to determine the mechanism controlling the level of peripheral microfilaremia. Jirds killed 40 days after infection served as donors of female worms of known age and reproductive status. These worms were transplanted into uninfected jirds and the resultant microfilaremias were monitored. After approximately 100 days, the recipient jirds were killed and 58% of the transplanted worms were recovered alive but depleted of sperm and microfilariae, regardless of the total number implanted in a given host. A direct linear relationship between microfilaremia and the number of recovered adult worms was found. Based on the uniform absence of sperm and microfilariae in the recovered worms it was concluded that female worms, under the conditions of the present study, do not control the peripheral microfilaremia in multi-worm infections through a reduced parturition rate.

Soleus muscle adenosine-5'-triphosphate (ATP) levels in Mastomys natalensis with Dipetalonema viteae infections: effect of diethylcarbamazine.

Adenosine-5'-triphosphate (ATP) levels in the soleus muscles of Mastomys natalensis during the patent phase of Dipetalonema viteae infection were studied. Decreased ATP levels were found in the infected animals as compared with the uninfected controls. Diethylcarbamazine citrate produced an 'anaphylactic reaction' and enhanced microfilaraemia in the infected animals, but did not cause any lowering of the soleus muscle ATP in the infected as well as in the uninfected animals.

Separation of viable microfilariae free of blood cells on Percoll gradients.

A consistent and reproducible method is described for isolating pure populations of microfilariae of Litomosoides carinii, Brugia pahangi, B. malayi and Dipetalonema viteae, free of cells, from blood, by density gradient centrifugation on Percoll in 0.25 M sucrose. The recovery of the microfilariae was 85 to 97%.

Clinical responses of dogs to experimentally induced Dipetalonema reconditum infection.

Six male and 6 female Beagles, 6 to 7 months old, were allotted to 2 groups: group I--inoculated subcutaneously with 30 Dipetalonema reconditum infective larvae/dog, and group II--noninoculated controls. Group comparisons were made in regard to hematologic values, Knott test results, body weights, blood urea nitrogen, total serum protein, serum albumin and alanine aminotransferase and creatine kinase activities. Routine urinalysis data were compared at 1 week before and at 28 weeks after the inoculations. Mean total leukocyte counts were significantly (P less than 0.05) greater in group I dogs than in group II dogs at postinoculation weeks (PIW) 4, 5, and 7 to 12, and mean eosinophil counts were significantly greater in group I dogs at PIW 3 to 11, 13 to 15, 20, and 23 to 24. Microfilariae were detected as early as the 10th week and sporadically thereafter. Only 1 D reconditum adult worm was recovered from all of the inoculated dogs. Five other dogs (group III) with chronic, patient experimentally induced dipetalonemiasis, were evaluated with the same tests at PIW 70 to 89. Eosinophilia (greater than 750 cells/microliter) was present in 4 of 5 dogs; lymphocytosis (greater than 4,800 cells/microliter) was evident in 1 dog. Proteinuria (greater than or equal to 30 mg/dl) was detected in 3 of 4 dogs with chronic dipetalonemiasis.

Filariasis en Colombia: presencia de Dipetalonema perstans en la Comisaria del Guainia / Filariasis in Colombia: presence of Dipetalonema perstans in the Comisaria del Guainia

Al examinar 75 muestras de sangre (metodo de Knott) colectadas en Puerto Inirida, Coco y Pajuil en la Comisaria del Guainia, Colombia, se descubrieron 26 portadores de micofilarias. En 18 personas habia unicamente Mansonella ozzardi; 3 tenian infeccion con M. ozzardi; 3 tenian infeccion con M. ozzardi y Dipetalonema perstans; y cinco solo con D. perstans. La infeccion por M. ozzardi se observo en blancos y en indigenas de las tribus curripacos, puinaves, tukanos, guananos y salivas, pero D. perstans se encontro solamente entre los curripacos. El numero de microfilarias (mf) circulantes fue bajo, 73% de los portadores tenian menos de 200 mf/ml de sangre; las personas que presentaron unicamente D. perstans tenian menos de 310 mf/ml. Estos resultados confirman la presencia de D. perstans en Colombia y sugieren que su prevalencia y distribucion en la Comisaria del Guainia y areas cercanas pueden ser mas altas de lo que se ha sospechado hasta el momento

Filariasis in Colombia: presence of Dipetalonema perstans in the Comisaría del Guainía.

Examination of 75 blood samples (Knott preparation) collected in Puerto Inrida, Coco, and Pajuil, in the Comisaría del Guainía, Colombia, disclosed 26 microfilaria carriers. Eighteen persons harbored only Mansonella ozzardi microfilariae, three were infected with M. ozzardi and Dipetalonema perstans and five harbored only D. perstans. M. ozzardi infections were found in whites, and in Indians belonging to the Curripaco, Puinave, Tukano, Guanano and Saliva tribes, but D. perstans was found only in the Curripaco Indians. Numbers of circulating microfilariae (mf) were low, 73% of the carriers had less than 200 mf/ml of blood: persons who harbored only D. perstans had less than 310 mf/ml. These results confirm the presence of D. perstans in Colombia, and suggest that its prevalence and distribution in the Comisarïa del Guainía and neighboring areas may be far greater than has been hitherto suspected.

[Demonstration of antigenicity in adult dipetalonema vitae by the indirect immunofluorescence test using serum from filariasis patients (author's transl)]. / Lokalisation von Antigenität adulter Dipetalonema viteae-Würmer in der indirekten Immunfluoreszenz mit Filariose-Patientenserum

The localization of the antigen-antibody reactions on methacrylate embedded sections of an adult male and female of the nematode Dipetalonema vitae by the indirect immunofluorescence test was found to be as follows: female D. vitae: in the egg-shell and circumference of sections of the microfilariae as well as amorphous structures in the vesica seminalis and vas deferens. In both sexes: partially in the somatic muscle cells according to their glycogen content, the interstitial spaces between the muscle cells and the connecting pseudocoelomic cavity. Reference to the possible application of this technique in the immunofluorescence diagnosis of filariasis is made.