Hong-Kean Ooi

Serological survey and first finding of Neospora caninum in Taiwan, and the detection of its antibodies in various body fluids of cattle.

A serological survey for antibodies against Neospora caninum in cattle, goats and farm dogs in Taiwan was carried out. Sera of 613 cattle from 25 dairy farms, 24 goats from six goat farms and 13 dogs from six dairy cattle farms were tested for antibodies against N. caninum using indirect fluorescence antibody test (IFAT). The same sera were also tested for antibodies against Toxoplasma gondii using latex agglutination test. Of the 613 cattle sera, 44.9% (275/613) were found to have antibodies against N. caninum. Among these 275 positive cattle, 77 also possessed antibodies against T. gondii. Nevertheless, 92 cattle which were negative for N. caninum showed antibodies against T. gondii. Of the 24 goat sera tested, none was found to be positive for N. caninum but 50% (12/24) were positive for T. gondii. Of the 13 farm dogs tested, three were found to possess antibodies against N. caninum, two of which tested negative for T. gondii antibodies. Besides sera, antibodies to N. caninum in cattle could be observed in the milk, vaginal secretion and saliva. However, the order of higher frequency of antibodies detection is in sera, milk, vaginal secretion and saliva. This is the first demonstration of the presence of antibodies to N. caninum in vaginal secretion and saliva of cattle. A 50microm cyst was observed in the brain of one of the 13 prednisolone-treated SPF ICR mice which had been peritoneally inoculated 4 months earlier with the brain homogenate of a serologically N. caninum positive but T. gondii negative cattle. Thus, we have confirmed for the first time the presence of N. caninum in Taiwan and also observed that it is widespread among dairy cattle and farm dogs.

Competence of Aedes albopictus and Culex quinquefasciatus as vector of Dirofilaria immitis after blood meal with different microfilarial density.

Aedes albopictus and Culex quinquefasciatus were fed canine blood with different microfilarial density of Dirofilaria immitis ranging from 2500 to 25,000 mff/ml. Larval development in these two mosquito species did not differ significantly. Although C. quinquefasciatus ingested more microfilariae, the number of larvae which developed in A. albopictus was invariably greater than in C. quinquefasciatus. Mortality of the engorged A. albopictus was significantly greater than that of C. quinquefasciatus, and higher microfilarial density raised the mortality in both species. The vector efficiency index of A. albopictus was greater than C. quinquefasciatus at all microfilarial densities, but its survival time was much reduced. Thus, dogs with low microfilarial density are implicated as the main source for the transmission of D. immitis from dogs to mosquitoes.

Molecular identification of Anisakis type I larvae isolated from hairtail fish off the coasts of Taiwan and Japan.

Anisakid nematodes are known to cause the zoonotic disease, anisakiasis, through the consumption of raw or undercooked fish. The parasites most frequently associated with the disease in humans are categorized as Anisakis type I, which comprise several species of the genus Anisakis. The larvae show primitive forms and lack the detailed morphological characteristics required for precise species identification. Thus, molecular characterization is necessary for determining the species of Anisakis type I larvae and acquiring important clinical and epidemiological information. In this study, we isolated Anisakis type I larvae from hairtail fish caught off the coasts of Taiwan and Japan. The ribosomal DNA (rDNA) internal transcribed spacer (ITS) region was sequenced, and restriction fragment length polymorphism (RFLP) analyses using HinfI and HhaI was carried out for species identification. Most larvae isolated from hairtail caught in Taiwan were Anisakis typica (84%), while those isolated from hairtail caught in Japan were almost exclusively identified either as Anisakis simplex sensu stricto (65%) or Anisakis pegreffii (33%). This is the first report of A. typica in fish obtained from Taiwan. Our results shed the light on the epidemiology of Anisakis type I larvae, which is a potential cause of human anisakiasis in Taiwan and Japan.

Susceptibility of mosquitoes in central Taiwan to natural infections of Dirofilaria immitis

From October 1997 to September 1998, 3085 Culex quinquefasciatus (Say) (Diptera: Culicidae), 584 Cx. tritaeniorhynchus (Giles) (Diptera: Culicidae), 392 Cx. annulus (Theobald) (Diptera: Culicidae), 374 Aedes albopictus (Skuse) (Diptera: Culicidae) and 102 Armigeres subalbatus (Coquillet) (Diptera: Culicidae) were collected and examined for Dirofilaria immitis (Leidy) (Spirurida: Filariidae) infection. However, only Cx. quinquefasciatus and Ae. albopictus were infected, with a prevalence of 4.28% and 3.74%, respectively. The intensity of D. immitis found in Ae. albopictus (3.43 larvae/mosquito) was higher than that found in Cx. quinquefasciatus (2.89 larvae/mosquito). After being fed with canine blood containing 7500 microfilariae (mf) per mL, Cx. quinquefasciatus ingested approximately two times as many mf as Ae. albopictus (mean of 31.73 in comparison to 16.47). However, almost three times as many third‐stage infective larvae developed in Ae. albopictus as in Cx. quinquefasciatus (mean of 3.25 as compared with 1.10), with a vector efficiency index (VEI) of 19.73 and 3.47, respectively. The results showed that Cx. quinquefasciatus and Ae. albopictus served as natural vectors of D. immitis in central Taiwan. Although Ae. albopictus was more efficient for heartworm transmission, Cx. quinquefasciatus may play a more prominent role on the transmission of dirofilariasis in central Taiwan.

Characterization of rDNA sequences from Syphacia obvelata, Syphacia muris, and Aspiculuris tetraptera and development of a PCR-based method for identification

To differentiate the morphologically similar pinworms of the common laboratory rodents, such as Syphacia obvelata and Syphacia muris, we amplified and sequenced the region spanning the internal transcribed spacer 1 (ITS-1), 5.8S gene, and ITS-2 of the ribosomal DNA followed by designing of species-specific primers for future use in the identification of the worms. It was observed that S. obvelata, S. muris and Aspiculuris tetraptera can be differentiated from each other based on their rDNA sequences. This is the first report of the ITS-1, 5.8S, and ITS-2 of the rDNA of the three aforementioned rodent pinworm species. The use of restriction endonucleases, AluI or RsaI, further allowed the delineation of the three species. Moreover, we also constructed species-specific primers that were designed for unique regions of the ITS-2 of the three species. This approach allowed their specific identification with no amplicons being amplified from heterogenous DNA samples, and sequencing confirmed the identity of the sequences amplified. Thus, the use of these specific primers along with PCR-RFLP can serve as useful tools for the identification of pinworms in rats, mice, and wild rodents.

Fecal occult blood manifestation of intestinal Eimeria spp. infection in rabbit

The relationship between oocysts production and fecal occult blood manifestation was studied in rabbits experimentally infected with a single species of Eimeria. Pure single Eimeria species infection in non-infected rabbits for 6 species of Eimeria, namely: Eimeria media, Eimeria magna, Eimeria perforans, Eimeria coecicola, Eimeria piriformis, and Eimeria exigua were produced using a single oocyst. Fecal samples from these single Eimeria species infected rabbits were collected daily from day 1 through day 23 post-infection (DPI) to observe for oocysts shedding and also to test for presence of fecal occult blood (FOB). Rabbits infected with E. perforans were positive for FOB test (FOBT) reaction when their OPG exceeded 2.2x105 at 11 and 12 DPI. FOBT positive reactions were also seen in naturally infected rabbits with concurrent infections, of E. perforans and E. media as well as of E. perforans and E. magna. Thus, E. perforans was observed to cause intestinal hemorrhage during the period of massive oocyst shedding. This phenomenon was not seen in the other Eimeria species that we had examined. This is also the first report on the association of E. perforans infection with intestinal hemorrhage in rabbit.

Infectivity and development of Metagonimus yokogawai in experimentally infected domestic ducks (Cairina moschata).

Information concerning whether fowl such as duck is a suitable reservoir host of Metagonimus yokogawai is largely unclear to date. In the present study, the growth and development of M. yokogawai metacercaria (Mc) in domestic duck (Cairina moschata) was determined by worm recovery rate (WRR) and morphological changes e.g., the size of flukes body as well as their internal organs was assessed by using Semichons acetocarmine staining. Each duck was orally inoculated with 50 Mcs of M. yokogawai and infected ducks were deeply anesthetized with ether and killed at 1, 2, 3, 4, 5, 6, 7, and 14 days post-infection (DPI). On each date, two infected ducks were killed and the small intestines of each duck were separated into four parts then they were opened longitudinally to harvest the flukes. Results revealed that WRR of M. yokogawai from inoculated ducks increased during early infection with a peak as seen at 4 DPI (28.5+/-6.9%); thereafter it gradually decreased and a drastic decline was observed in 14 DPI (2.0+/-1.1%) in the trial. The preference sites for M. yokogawai were low portions of the small intestine; nevertheless the size of flukes body and organs developed increasingly with time and they maturated to produce ova from 4 DPI onward in the trial. However, present results indicated that ducks, based on the findings of this study, are not suitable hosts for establishment of M. yokogawai infection because most flukes were expelled from ducks intestine within 14 days. Nevertheless, it was proposed that ducks might play a certain role in transmitting M. yokogawai when they deposited the ova via feces into marsh where snails and fish were abundant since they could presumably establish transient and possibly patent infections with this parasite.