Enzyme-linked immunosorbent assay for detection of Trichinella spiralis antibodies and the surveillance of selected pig breeding farms in the Republic of Korea.

Trichinellosis is a parasitic zoonosis of public health importance. It is caused by Trichinella spiralis which has a wide host range including humans. In the present communication, the ELISA technique was employed on a total of 803 blood samples from 7 selected pig breeding farms in 1996 for diagnosis and surveillance of trichinellosis. Out of the entire 803 samples, nine were found to be suspected while one was positive by ELISA. But western blot analyses employed for further confirmation have shown that all of 10 samples did not react to larval excretory-secretory product antigens. These results indicate that pig breeding farms included in the present study are free from trichinellosis. However, it does not mean Korea is free from trichinellosis since human trichinellosis has recently been reported. The necessity of continued surveillance for trichinellosis in both pigs and wild animals was discussed.

Toxoplasma antibody titers by ELISA and indirect latex agglutination test in pregnant women.

The seroepidemiologic studies on anti-Toxoplasma antibody titers were carried out using ELISA and indirect latex agglutination test. Among 899 sera prepared from pregnant women, 39 cases (4.3%) revealed positive reaction and 218 sera from middle school students showed 4 positive reaction (1.8%) by ELISA. By LAT (newly established by National Veterinary Research Institute. Korea), the sera of 7 pregnant women (0.8%) showed positive reaction. When 80 sera showing > or = 1:8 by LAT were used for comparing the results obtained from LAT and Toxotest-MT (Eiken Chemical Co., Japan), 7 cases and 8 sera were positive, respectively. All of 11 sera of proven toxoplasmosis patients showed positive reaction in both tests. Overall proportion of agreement between LAT kit and Toxotest-MT was 0.94 (kappa-index = 0.632, p < 0.011, and LAT was considered to be useful for the screening of toxoplasmosis.

Setaria marshalli infection in neonatal calves.

A total of 50 filariid worms of Setaria spp. was recovered from the peritoneal cavity of three neonatal calves infected with the Akabane virus. The parasites were identified as S. marshalli by their morphological characteristics. Males were 41-52 mm long and females 68-98 mm. Most of them were fully matured, indicating that the calves were infected prenatally. This is the first report of prenatal infection in calves by S. marshalli in Korea.

Evaluation for detection of Cryptosporidium oocysts in diarrheal feces of calves.

For the detection of Cryptosporidium oocysts, fecal samples were collected from 201 calves which showed diarrhea. Among the 201 samples, 29 samples (14.4%) were positive for Cryptosporidium spp. by the DMSO-modified acid-fast stain (MAFS), 23 samples (11.4%) were positive by commercial kit (Meridian Diagnostics, Cincinnati, Ohio) and 23 by the indirect immunofluorescence antibody (IFA) assay employing the monoclonal antibody (mAb C6). When tested by both IFA and MAFS, 20 fecal samples were positive for Cryptosporidium oocysts whereas 169 fecal samples were negative. If the MAFS is considered a standard method for oocyst detection, the IFA showed 69% of sensitivity and 98% of specificity. When tested by both IFA and commercial kit, 22 fecal samples were positive for Cryptosporidium oocysts while 177 samples were negative. One sample tested by IFA was found to be false negative, when compared with the results by commercial kit. The sensitivity of IFA was calculated as high as 96%; the specificity as 99% and the predictive value was also 99%. In the present study, IFA employing the mAb C6 revealed that 23 samples (11.4%) were positive among the 201 calves showing diarrhea. Of 23 IFA positive samples, 4 samples (5%) showed cryptosporidial oocysts more than 10(5) OPG. Therefore, it is concluded that the calves showing cryptosporidial oocysts more than 10(5) OPG in the feces were highly associated with clinical cryptosporidiosis.

Diagnosis of bovine cryptosporidiosis by indirect immunofluorescence assay using monoclonal antibody.

Two hybridoma cell lines against Cryptosporidium parvum oocysts (VRI-CN91) were produced. The isotype of these 2 monoclonal antibodies (mAbs) was IgG2b (1E7.2) and and IgM (C6). Enzyme immuno-transfer blotting analysis showed that 1E7.2 reacted specifically to 36 kDa protein and C6 reacted to 67 and 70 kDa proteins. C. parvum was bound specifically to the surface region of oocysts by these mAbs. No cross-reactivity was observed with tachyzoites of Toxoplasma gondii and oocysts of Eimeria zuernii, E. bovis and E. canadensis of bovine origin. The indirect immunofluorescence assay (IIF) using mAb C6 was successful with counterstain. With the IIF using mAb C6, oocysts appeared as 3 to 5 microns spherical objects fluorescing bright apple green against a reddish dark background. The IIF using mAb C6 was agreed in specificity and sensitivity with those of a commercial diagnostic kit. These results demonstrated that the produced mAbs were specific to C. parvum and that the mAb C6 could be used for diagnosis of cryptosporidiosis.

[Isolation of Cryptosporidium parvum oocysts from fecal samples--the combination of ether extraction and discontinuous sucrose gradients].

A calf and 50 mice were infected with Cryptosporidium parvum, and their fecal materials were collected and treated with ether extraction (EE), followed by discontinuous sucrose gradients (DSG) method. EE method was to remove some of fat or lipid from feces. Sediments were washed by centrifugation (1,500 x g for 10 min., 3 times) in phosphate-buffered saline and then these washed sediments were sieved sequentially through stainless steel screens with a final mesh of 250 (61 microns porosity) to remove other debris. After sieving, the materials were suspended in 2.5% potassium dichromate solution. Oocysts were counted by using a hemocytometer and the recovery rate of pure oocysts was calculated on the basis of the count. Following centrifugation (1,500 x g for 30 min.) by DSG method, most oocysts were recovered at the interface between a gravity of 1.103 and 1.064. The recovery rates of pure oocysts from the fecal suspension of the calf (3.8 x 10(7)/ml) and the mouse (3.2 x 10(6)/ml) treated with EE method were 81.6% and 51.6%, respectively. It is suggested that the recovery rate was dependent on the number of oocysts in each suspension treated with EE method. To get the 50% recovery rate, there must be more than 2 x 10(6) oocysts per ml of the fecal suspension treated with EE method. By the combination of the two methods it was possible to isolate C. parvum oocysts from normal feces of the calf and mouse as well as from diarrheic feces.

[Experimental Cryptosporidium parvum infection in a Korean native calf isolated from a Korean mouse].

This study was performed to investigate experimental transmission of Cryptosporidium parvum in a calf. A 25-day-old Korean native calf was inoculated per os with 1 x 10(6) C. parvum oocysts isolated from a Korean mouse. The calf commenced oocyst discharge in feces on post-inoculation day 4, and continued until the day 11. The number of discharged oocysts peaked (4.9 x 10(5)) on post-inoculation day 6. However, the calf did not show signs of diarrhea. The present results indicate that C. parvum is cross-transmissible between the calf and the mouse.