The first detection of Brucella canis in cattle in the Republic of Korea.

Twenty mammary lymph node samples were collected from cattle on a farm in the Republic of Korea. These cattle were serologically negative for Brucella by tube agglutination test (≤ 1:50) and serum agglutination test (≤ 1:50). Out of 20 lymph node samples, two samples were positive for Brucella growth on Brucella agar as well as blood agar. Tests for urease, hydrogen sulphide and reactions against monospecific sera A and M indicated that these two isolates (No. 15 and 16) belong to the genus Brucella. Genus specific, AMOS (abortus, melitensis, ovis, suis) and Bruce-ladder multiplex polymerase chain reaction (PCR) assays confirmed the Brucella isolates as either a B. abortus or a B. canis strain. This is the first report of the occurrence of a B. canis infection in cattle in Korea. More survey data are needed to determine whether B. canis is a significant aetiology in the cases of cattle brucellosis in Korea.

Brucella abortus infection in indigenous Korean dogs.

Three dogs reared on a dairy farm with a high incidence for Brucella abortus were serologically positive for B. abortus and no other Brucella spp. The identity of the organism was confirmed to be B. abortus by AMOS (abortus melitensis ovis suis)-polymerase chain reaction with specific primers for B. canis. One hundred percent homology of the canine isolate and the bovine pathogen isolated from the farm was demonstrated. The only possible source of infection was infected cattle on the same farm. It is suggested that dogs be routinely included in brucellosis surveillance and eradication programs.

Characterization of the protective response against a homologous challenge infection with Strongyloides venezuelensis in rats.

The protective response in rats against a homologous challenge infection with Strongyloides venezuelensis was characterized. In an initial infection with 1000 filariform larvae and migrating larvae (L(3)) of S. venezuelensis, the population of L(3) in the lungs on day 3 postinfection (PI), and that of adult worms in the small intestine on day 7 PI, were 180.8+/-14.5 and 336.8+/-70.7, respectively. The latter were gradually expelled towards day 42 PI. After the initial infection, the rats developed strong immunity against a homologous challenge infection as manifested by a marked reduction in worm populations, stunted body length and width, damage to reproductive organs, impaired egg production and rapid expulsion of the worms by day 14 after challenge. Expulsion of the worms was preceded by a significantly elevated (P<0.05) peripheral blood eosinophil (PBE) count, both in the initial (200.0+/-26.5 x 10(3)ml) and the challenge infection (400.9+/-165.4 x 10(3)ml). These findings suggest that rats acquire strong homologous immunity following initial exposure to S. venezuelensis. It is suggested that PBEs are involved in worm expulsion. A major target of these effector mechanisms is the reproductive system of S. venezuelensis.

Examination of gastrointestinal helminth in livestock grazing in grassland of Bangladesh.

To determine association of grassland with parasitic diseases of livestock in Bangladesh, the 'Tracer' animals (two cow calves and two goats) were released for a month in a grassland used for communal grazing of livestock near school premise in Kanthal, Trishal, Mymensingh, Bangladesh. After slaughtering of the tracer animals, their gastrointestinal tract examination revealed six species of nematode and one cestode. The nematode species were Haemonchus contortus, Trichostrongylus axei, Mecistocirrus digitatus, Oesophagostomum spp., Trichuris spp. and Bunostomum sp. The cestode was one of the genus Moniezia. With this preliminary study, grasslands are thought to be one of the main sources of gastrointestinal parasitic diseases of livestock in Bangladesh.

Partial cross-resistance between Strongyloides venezuelensis and Nippostrongylus brasiliensis in rats.

Rats were immunized through an initial infection with 1,000 filariform larvae (L3) of Nippostrongylus brasiliensis and after complete expulsion of worms they were challenged with 1,000 L3 of Strongyloides venezuelensis to investigate whether cross-resistance developed against a heterologous parasite. Nippostrongylus brasiliensis-immunized rats developed a partial cross-resistance against S. venezuelensis migrating larvae (MSL3) in the lungs and adult worms in the small intestine. The population of MSL3 in the lungs were significantly lower (P < 0.05) in immunized rats (22.0 +/- 7.4) compared with controls (105.0 +/- 27.6). The populations of adult worms, egg output and fecundity were initially decreased but from day 14 post-challenge they did not show any significant difference between immunized and control rats. However, the length of worm in immunized rat was revealed as retardation. Peripheral blood eosinophilia was significantly decreased (P < 0.05) on day 7 post-challenge and then gradually increased, which peaked on day 42 post-challenge when most of the worms were expelled. These results suggest that peripheral blood eosinophilia is strongly involved in the worm establishment and expulsion mechanisms.

Effects of in vitro culture methods on morphological development and infectivity of Strongyloides venezuelensis filariform larvae.

The effects of in vitro culture methods on morphological development and infectivity of Strongyloides venezuelensis filariform larvae (L3) to rats were investigated. A significantly higher body length was observed in L3 from filter paper culture (597.3 +/- 32.2 microns) than those in fecal (509.9 +/- 35.0 microns) and nutrient broth culture (503.3 +/- 31.0 microns) (P < 0.05). Larval infectivity was assessed by exposing rats to 1,000 L3 from each culture and worms were recovered from the lungs and small intestines. Recovery rate of these worms did not show any significant difference. A significantly greater body length of adults was recorded in those corresponding to the L3 harvested from filter paper (2,777.5 +/- 204.4 microns) and nutrient broth culture (2,732.5 +/- 169.8 microns) than those corresponding to the L3 obtained from fecal culture (2,600.5 +/- 172.4 microns) (P < 0.05). Although worm fecundity and EPG counts differed among culture methods but worm burdens and course of infection did not. These findings suggest that the methods of cultures have a significant effect on the morphological development of the larvae to the L3 stage, but do not influence the infectivity to rats.

Detection of Staphylococcus aureus in milk by use of polymerase chain reaction analysis.

OBJECTIVE: To evaluate polymerase chain reaction (PCR) analysis for detection of Staphylococcus aureus (nuc gene) in fresh and formalin-preserved milk. SAMPLE POPULATION: Samples from 80 lactating sheep and 100 lactating dairy cows. PROCEDURE: 4 lactating sheep were inoculated with S aureus by intramammary infusion. A set of primers specific for the nuc gene of S aureus was used to develop a PCR technique, and modification of the rapid boil method was used to isolate bacterial DNA. Milk was obtained from experimentally infected sheep before and after infusion with S aureus, and from the 100 cows and remaining 76 sheep. Samples were screened by bacteriologic culture and PCR. To validate the PCR assay, S aureus or other pathogens were added to distilled water and "normal" sheep milk samples, with and without formalin. RESULTS: The PCR assay was 100% specific for S aureus when known negative and positive samples were tested. Sensitivity was 100% for samples with added S aureus or other pathogens. Sensitivity was lower for samples obtained from experimentally infected sheep, but increased from 53% to 90% with increased washing of target DNA. CONCLUSIONS: The PCR technique based on the nuc gene is able to detect S aureus in sheep milk yields results faster than does traditional culturing, is highly specific, and is able to detect S aureus in formalin-fixed milk samples. CLINICAL RELEVANCE: The assay is particularly suitable for analysis of samples shipped or stored without refrigeration. Although antibiotics in milk may inhibit growth in culture, they should not affect the results of the PCR assay.

Viability of eggs, filariform larvae and adults of Strongyloides venezuelensis (Nematoda: Strongyloidea) maintained in vitro.

The present study was performed to check the viability of eggs, filariform larvae and adults of Strongyloides venezuelensis exposed to various conditions for an in vitro maintenance. The eggs in the feces remained viable for about 25 days at 4 degrees C and 15 days at room temperature. However, the isolated eggs in sterile saline lost their viability within 24 hr at 4 degrees C. The eggs in morula stage were very sensitive to air drying and rapidly lost their viability (< or = 12 hr). Filariform larvae survived for a maximum period of 45 days in fecal suspension and 28 days in 0.12% nutrient broth in polyvinyl culture bags maintained at 20 degrees C. On the other hand, those isolated from nutrient broth cultures survived for a maximum period of 32 days in tap water and 22 days in sterile saline at 20 degrees C. The mature adult worms obtained from experimentally infected rats survived maximally for 9 days in serum supplemented (10% rat-serum) 0.12% nutrient broth and 4 days in serum free nutrient broth at 37 degrees C while the culture media were changed at an alternate day. The adult female worms deposited fertile eggs in serum supplemented and serum free nutrient broth cultures, however, the hatched larvae (L1) were not able to develop to the filariform stage in the culture media and found to die within 24 hr of maintenance. The present findings on an in vitro maintenance of different stages of S. venezuelensis may provide useful information for biological and biochemical studies with Strongyloides species.

Development of an in vitro culture method for harvesting the free-living infective larvae of Strongyloides venezuelensis.

An in vitro culture technique was established for harvesting Strongyloides venezuelensis free-living infective larvae using a nutrient broth medium as a substitute for rat-feces in polyvinyl culture bags (10 x 12 cm). The egg hatch rate (Y) in sterile saline at different incubation temperatures (X) was expressed as the quadratic function, Y = -0.192X2 + 8.673X - 19.550 (r = 0.901). The highest (100%) egghatch rate was observed at 25 degrees C. A significant difference (p < 0.05) in development rate (Y) of free-living infective larvae was observed between different concentrations of nutrient broth (X) which was highest (20.6%) in 0.12% nutrient broth concentrations, incubated at 20 degrees C for 5 days [Y = -864.032X2 + 245.995X - 0.560 (r = 0.875)]. Yields (Y) of infective larvae were observed relatively high when the culture medium was incubated at higher temperatures (X) which peaked at 25 degrees C (20.0%) than at lower temperatures, 15 degrees C (10.9%) and 20 degrees C (18.1%) [Y = -0.189X2 + 8.387X - 72.795 (r = 0.981)]. The period (Y) required for the development of infective larvae decreased with higher incubation temperatures (X) [Y = 0.035X2 - 2.025X + 32.375 (r = 0.995)]. The highest yield (19.2%) of infective larvae was obtained from culture bag inoculated with 15,000 eggs than with below and over 15,000 eggs in 0.12% nutrient broth and incubated at 25 degrees C for 4 days. The newly adapted culture method (from egg to third-stage larva) may be useful as a bio-bar/bioassay system for screening new chemical products, anthelmintics and pesticides, as well as for parasito-immunological studies with Strongyloides species.

Analysis of immunodominant piroplasm surface protein genes of benign Theileria parasites distributed in China and Korea by allele-specific polymerase chain reaction.

Benign Theileria species distributed in China and Korea were characterized by allele-specific polymerase chain reaction (PCR), based on the sequences of major immunodominant piroplasm surface protein genes. In China, all the isolates contained Chitose (C) type parasites. One out of 5 isolates tested was a mixed population of Ikeda (I), C and B-2 types, whereas, all the isolates from Korea consisted of I type parasites. Except for 4 isolates, 29 isolates from Korea consisted of more than two types of parasites. The present data showed that benign Theileria species distributed in these countries were mixed parasite populations.

Immunogenicity and protective efficacy of solubilized merozoite-enriched Theileria sergenti immunogens. III. Characterization of immunodominant peptides.

Immunoblot analysis utilizing bovine sera from naturally or experimentally infected with Theileria sergenti were used to determine the immunodominant polypeptides of T. sergenti (Korean isolate). The previously recognized major bands, 18 kDa, 29 kDa, 34 kDa and 45 kDa, were excised after electrophoresis and transfer to PVDF membrane. The individual bands were sequenced. The 34 kDa polypeptide which was the most antigenic and immunogenic peptide was observed in the Western blot. However, Chou-Fasman prediction sites (antigenic site) for antigen determinants of the 45 kDa, 24 kDa, 29 kDa and 18 kDa polypeptide were 6, 4, 2 and 0, respectively. However, the 45 kDa polypeptide showed no reaction with anti-T, sergenti hyperimmune serum.

Cultural, molecular, and immunological characterization of the etiologic agent for atypical canine ehrlichiosis.

More than 100 cases of canine ehrlichiosis, with three fatalities, were serologically negative by the indirect immunofluorescent antibody (IFA) test with Ehrlichia canis or E. sennetsu antigen but were reactive at titers of 10 to 640 with E. risticii. Ehrlichia-like agents were isolated from three such cases. The agents isolated from those cases were morphologically indistinguishable from each other and from a prototype, E. risticii, the etiologic agent of equine monocytic ehrlichiosis, in terms of growth characteristics and by light or electron microscopy. The patterns of and products from PCR were identical to those of E. risticii. The 16S rRNA sequences were distinct from those of E. canis and E. ewingii but were identical to those of E. risticii. A PCR product corresponding to the 5' half of the 16S rRNA gene was obtained from amplification of DNA from E. risticii and both sources of the atypical canine ehrlichiosis agent but was not obtained from uninfected host cells. The entire sequence of 719 nucleotides was identical for all three sources. The percentages of relatedness of the partial 16S rRNA gene of the atypical canine ehrlichiosis agent to E. risticii, E. sennetsu, E. platys, E. equi, E. phagocytophila, E. canis, E. chaffeensis, and E. ewingii were 100.0, 98.9, 83.7, 83.0, 83.0, 82.2, 81.8, and 81.5, respectively. These data are consistent with the identity of these isolates as E. risticii. The caninotropic characteristics of naturally acquired infections due to E. risticii are herein described for the first time, and the epizootiological implications are discussed in relation to the host range of E. risticii, which may include dogs as reservoirs.

Detection of Theileria sergenti infection in cattle by polymerase chain reaction amplification of parasite-specific DNA.

A pair of synthetic oligonucleotide primers, designed from the gene encoding a 32-kDa intraerythrocytic piroplasm surface protein of Theileria sergenti, were used to amplify parasite DNA from the blood of T. sergenti-infected cattle by means of the polymerase chain reaction (PCR). PCR-amplified DNA was examined by electrophoresis and by dot blot or microplate hybridization using a parasite-specific cDNA probe. PCR was specific for T. sergenti, since no amplification was detected with DNA from Anaplasma centrale, Babesia ovata, uninfected erythrocytes, and leukocytes. This method was sensitive enough to detect about 4.5 parasites per microliters of blood with a 10-microliters sample volume. Moreover, of 66 specimens from grazing cattle, 40 were microscopically positive, whereas PCR revealed that 54 samples were positive. Therefore, PCR provides a useful diagnostic tool for detecting T. sergenti-infected cattle, and it is significantly more sensitive than the current methods.

Studies on isosporosis in dogs. I: Isolation and sporulation of Isospora ohioensis.

Based on the morphologic characteristics of oocysts of the genus Isospora, we have demonstrated that I. ohioensis is a relatively common Isospora species in dogs which are resident in Chonbuk province, Korea. The prepatent period of I. ohioensis was four days. The size of the unsporulated oocysts in fresh stool specimens was 22.9 x 19.8 microns (R = 1.16). The size of the contracted sporonts was 17.4 x 16.3 microns (R = 1.06). By 96 hours, sporulation is complete and the ratio of length/width was constant relatively. And the sizes of oocysts and sporocysts were 22.8 x 20.5 microns (R = 1.11) and 15.0 x 10.8 microns (R = 1.39), respectively. At this time it is most reliable for the measurements of oocyst sizes of I. ohioensis to provide with the identifiable clues against the others. It is therefore recommended that the clinical fecal specimens suspected of isosporosis should first be incubated and aerated for 96 hours before a definitive parasitological diagnosis can be reached. These and other observations contribute to the understanding of the biological characteristics and laboratory and clinical diagnosis of isosporosis.

Platelet kinetics and other hematological profiles in experimental Plasmodium falciparum infection: a comparative study between Saimiri and Aotus monkeys.

Levels of platelets and other hematological values were monitored in 21 Saimiri and 12 Aotus monkeys over a period of three weeks post-infection with monkey-adapted Indochina CDC-1 strain of Plasmodium falciparum. In both Saimiri sciureus boliviensis and Aotus nancymai karyotype-1 monkeys the severest thrombocytopenia was observed at 14 days post-infection coinciding with peak parasitemia, neutropenia, lymphocytosis, and anemia associated with severe hemoglobinemia and elevated fibrinogen degeneration products(FDP's). MCH and MCV profiles in Aotus monkeys decreased with ascending parasitemia. In contrast, these parameters in Saimiri were characterized by a significant compensatory increase correlating with parasitemia. In general, thrombocytopenia was one of the earliest clinical manifestations of the infection with the platelets returning to normal levels shortly after peak parasitemia at 14 days. Platelet kinetics had a strong correlation with hematologic and parasitologic values in the Aotus model. No consistent associations were observed between platelet kinetics and other parameters in the Saimiri model. These data indicate that the Aotus model for malaria is more predictable than the Saimiri. Further, platelet turnover rates and recovery provide a useful prognostic parameter during malaria infection. The results are discussed in relation to the value of the two species of monkeys as models for the pathogenesis of human malaria.

Immunogenicity and protective efficacy of solubilized merozoite-enriched Theileria sergenti immunogens. II: Protection against natural exposure under field conditions.

A Theileria sergenti soluble merozoite preparation containing the 29, 34, 35 and 105 KD as the immunodominant polypeptides, was evaluated for efficacy, safety and protectivity in Holstein calves against virulent field tick challenge. The soluble antigens (100 mg/dose) were fortified with either complete or incomplete Freund's adjuvant. Twenty naive calves, aged one month, were subcutaneously inoculated with the preparation and a booster dose was administered 4 weeks later. Twenty additional calves served as controls. Five weeks after the booster dose, vaccinates and uninoculated controls were moved to a pasture, a heavily tick infested area in Cheju-do, Korea. The vaccinates showed negligible change in hematocrit and total RBC count whereas control animals showed significant (p < 0.05) hematological changes and associated anemia. Only 30% of vaccinates required chemotherapy after the experiment was terminated. All control animals required chemotherapy and 25% received blood transfusion. The highest percent parasitized erythrocytes in vaccinated cattle was 0.4% as compared with 3.6% among controls during the month of July. A significant difference (p < 0.05) was observed in the rate of body weight increase. Significant differences were also noted in serum albumin, aspartate aminotransferase, total protein and bilirubin. Significantly more vaccinated cattle maintained normal ranges of hematological and biochemical values as compared with the control group. It is suggested that soluble merozoite T. sergenti antigens may be potential vaccine candidates for developing a genetic vaccine in Korea.

Immunogenicity and protective efficacy of solubilized merozoite-enriched Theileria sergenti immunogens. I: Protection against homologous stabilate challenge.

Theileria sergenti were isolated from infected erythrocytes by hypotonic lysis, and soluble merozoite antigens were purified by sonication and differential centrifugation. The preparation contained 29, 34, 35 and 105 kD immuno-dominant poly-peptides. The soluble antigens (0.5 mg/ml) were prepared and fortified with Freund's adjuvant. Five month old naive Korean calves were subcutaneously inoculated with the preparation and a booster dose was administered 4 weeks later. Nine weeks after the booster dose, vaccinates and controls were challenged with a homologous stabilate (5.6 x 10(6) RBC/dose, 40% Parasitemia). All animals were monitored for hematocrit, total erythrocyte count, parasitemia and for the specific antibody by Western immunoblot (WB) and indirect immuno-fluorescent antibody (IFA) test. By 18 weeks after vaccination (6 weeks after the challenge), vaccinated cattle had an average IFA titer of 1:10,240 compared with 1:1,280 of the controls. The vaccinates showed negligible change in hematocrit and total RBC count whereas control animals showed significant (p less than 0.05) hematological changes and associated anemia. After vaccination and challenge, the antibody responses demonstrated that vaccination had induced significant production of antibody to the 29 and 35 kD polypeptides. The latter polypeptide was much more strongly recognized by the vaccinated animals, and thus it may be a potential candidate for the vaccine.

Efficacy of purified Anaplasma marginale initial bodies as a vaccine against anaplasmosis.

Anaplasma marginale initial bodies of the Florida strain were purified from infected erythrocytes using a combination of ultrasonic disruption, nonionic detergent and differential centrifugation. Immunochemical analysis revealed at least 12 A. marginale proteins in the molecular mass (m) range 81-15 kDa with a prominent band at 38 kDa. Several of these proteins remained insoluble in the presence of nonionic detergent. Preparations of purified Anaplasma initial bodies contained negligible erythrocytic contamination, as confirmed by the minimal induction of isoantibodies against bovine blood group antigens and the absence of delayed-type hypersensitivity to erythrocytic antigens in immunized animals. A total of 33 crossbred and purebred Holstein cattle were vaccinated with either 1.5, 1.0, or 0.1 mg protein of intact initial bodies, or with 1.0 mg of solubilized Anaplasma protein. The immunogens were supplemented with 3.0 mg Quil-A saponin adjuvant and administered in 2 subcutaneous injections given at a 4-week interval. A similar number of nonvaccinated cattle served as controls. Three months after vaccination, all cattle were challenged by inoculation of 10(9) virulent A. marginale of either the homologous (Florida) or heterologous (Venezuelan) strains. Vaccinated cattle showed solid protection after homologous and heterologous challenge, characterized by parasite clearance and minimal hematocrit reductions. Initial data from four field vaccine trials revealed a reduced incidence of clinical anaplasmosis among immunized animals. Use of immunogens consisting of purified A. marginale initial bodies offers a potential immunoprophylactic approach to control of bovine anaplasmosis.

Structural investigation on the effects of the herbs on Clonorchis sinensis in rabbits.

The effects of boiled water extracts of clonorchicidal raw drugs screened by the EPG counts in vivo on the structure of Clonorchis sinensis were investigated. The extracts of Cassia obutusifolia and Dictamnus dasycarpus did not seem to induce the morphological changes of the worms, and in those of Machilus thunbergii and Prunús mume, widening of bladder to lower level of seminal receptacle was visible without any other changes. Those of Inula helenium and Saussurea lappa, however, disclosed regressive and progressive changes as degeneration, atrophy, necrosis, dilatation, etc. of viscera of the worms. The recover rates of the worms from experimentally infected rabbits administered with the extracts of I. helenium and S. lappa for 30 days, beginning at the 3rd day of inoculation, were as low as 2% and 2.8%, respectively.

Alternations of Clonorchis sinensis EPG by administration of herbs in rabbits.

In order to investigate clonorchicidal activity in vivo, boiled water extracts of 32 species of clonorchicidal raw drugs in vitro were orally administered into rabbits infected with Clonorchis sinensis. The results of the observation of EPG variation were as follows: Suppression effects of egg-laying capacity from the rabbits administered Prunus mume and Inula helenium were greatest. Those from Dictamnus dasycarpus and Saussurea lappa were somewhat effective. Machilus thunbergii and Cassia obutusifolia, however, were less effective.