Ticks Collected from Wild and Domestic Animals and Natural Habitats in the Republic of Korea

Ticks were collected from 35 animals from 5 provinces and 3 metropolitan cities during 2012. Ticks also were collected by tick drag from 4 sites in Gyeonggi-do (2) and Jeollabuk-do (2) Provinces. A total of 612 ticks belonging to 6 species and 3 genera were collected from mammals and a bird (n=573) and by tick drag (n=39). Haemaphyalis longicornis (n=434) was the most commonly collected tick, followed by H. flava (158), Ixodes nipponensis (11), Amblyomma testudinarium (7), H. japonica (1), and H. formosensis (1). H. longicornis and H. flava were collected from all animal hosts examined. For animal hosts (n>1), the highest Tick Index (TI) was observed for domestic dogs (29.6), followed by Siberian roe deer (17.4), water deer (14.4), and raccoon dogs (1.3). A total of 402 H. longicornis (adults 86, 21.4%; nymphs 160, 39.8%; larvae 156, 38.9%) were collected from wild and domestic animals. A total of 158 H. flava (n=158) were collected from wild and domestic animals and 1 ring-necked pheasant, with a higher proportion of adults (103, 65.2%), while nymphs and larvae only accounted for 12.7% (20) and 22.2% (35), respectively. Only 7 A. testudinarium were collected from the wild boar (6 adults) and Eurasian badger (1 nymph), while only 5 I. nipponensis were collected from the water deer (4 adults) and a raccoon dog (1 adult). One adult female H. formosensis was first collected from vegetation by tick drag from Mara Island, Seogwipo-si, Jeju-do Province.     

Ticks collected from selected mammalian hosts surveyed in the Republic of Korea during 2008-2009

A tick survey was conducted to determine the relative abundance and distribution of ticks associated with selected mammals in the Republic of Korea (ROK) during 2008-2009. A total of 918 ticks were collected from 76 mammals (6 families, 9 species) captured at 6 provinces and 3 Metropolitan Cities in ROK. Haemaphysalis longicornis (54.4%) was the most frequently collected tick, followed by Haemaphysalis flava (28.5%), Ixodes nipponensis (7.6%), Ixodes pomerantzevi (4.8%), Ixodes persulcatus (4.6%), and Haemaphysalis japonica (0.1%). Adults (57.0%) and nymphs (28.7%) of Ixodes and Haemaphysalis spp. were collected most frequently from medium or large mammals in this survey, while few larvae (14.3%) were collected. Hydropotes inermis was the most frequently captured mammal (52.6%), with a 16.4 tick index and 5 of 6 species of ticks collected during this survey. H. longicornis (69.7%) was the predominant tick collected from H. inermis, followed by H. flava (22.2%), I. persulcatus (6.1%), I. nipponensis (1.8%), and H. japonica (0.2%).     

No Detection of Severe Fever with Thrombocytopenia Syndrome Virus from Ixodid Ticks Collected in Seoul

Larvae, nymphs, and adult stages of 3 species of ixodid ticks were collected by tick drag methods in Seoul during June-October 2013, and their infection status with severe fever with thrombocytopenia syndrome (SFTS) virus was examined using RT-PCR. During the period, 732 Haemaphysalis longicornis, 62 Haemaphysalis flava, and 2 Ixodes nipponensis specimens were collected. Among the specimens of H. longicornis, the number of female adults, male adults, nymphs, and larvae were 53, 11, 240, and 446, respectively. Ticks were grouped into 63 pools according to the collection site, species, and developmental stage, and assayed for SFTS virus. None of the pools of ticks were found to be positive for SFTS virus gene.     

Babesia: the protective effects of killed Propionibacterium acnes on the infections of two rodent Babesia parasites in mice

In the present study, we investigated the protective effects of killed Propionibacterium acnes on the infections of two rodent Babesia parasites in mice. Pre-treatment with "EqStim" (a commercially available immunostimulant containing killed P. acnes) showed significant resistance to both infections. To elucidate the immunological status in the mice, the concentrations of multiple cytokines were measured in serum collected from infected mice. After B. microti infection, the levels of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12p70, and tumor necrosis factor (TNF)-alpha in the treated group were significantly lower than in the control group. In contrast, after B. rodhaini infection, only IL-12p70 and TNF-alpha were detectable at significantly higher levels in the treated group than in the control group. The present findings indicated the protective effects of killed P. acnes on rodent babesiosis even with different immune responses between the B. microti and B. rodhaini infections. Killed P. acnes might be a powerful tool for the control of serious livestock babesiosis.     

Proteomic Screening of Antigenic Proteins from the Hard Tick,Haemaphysalis longicornis (Acari: Ixodidae)

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis.     

Screening and Molecular Cloning of a Protective Antigen from the Midgut of Haemaphysalis longicornis

Vaccination is considered a promising alternative for controlling tick infestations. Haemaphysalis longicornis midgut proteins separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane were screened for protective value against bites. The western blot demonstrated the immunogenicity of 92 kDa protein (P92). The analysis of the P92 amino acid sequence by LC-MS/MS indicated that it was a H. longicornis paramyosin (Hl-Pmy). The full lenghth cDNA of Hl-Pmy was obtained by rapid amplification of cDNA ends (RACE) which consisted of 2,783 bp with a 161 bp 3'' untranslated region. Sequence alignment of tick paramyosin (Pmy) showed that Hl-Pmy shared a high level of conservation among ticks. Comparison with the protective epitope sequence of other invertebrate Pmy, it was calculated that the protective epitope of Hl-Pmy was a peptide (LEEAEGSSETVVEMNKKRDTE) named LEE, which was close to the N-terminal of Hl-Pmy protein. The secondary structure analysis suggested that LEE had non-helical segments within an α-helical structure. These results provide the basis for developing a vaccine against biting H. longicornis ticks.     

PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle

PCR and nested-PCR methods were used to assess the frequency of Babesia bovis and Babesia bigemina infection in Boophilus microplus engorged females and eggs and in cattle reared in an area with endemic babesiosis. Blood and the engorged female ticks were from 27 naturally infested calves and 25 crossbred cows. The frequency of both Babesia species was similar in calves and cows (P>0.05). Babesia bovis was detected in 23 (85.2%) calves and in 25 (100%) cows and B. bigemina was detected in 25 (92.6%) calves and in 21 (84%) cows. Mixed infections with the both Babesia species were identified in 42 animals, 21 in each age category. Of female ticks engorged on calves, 34.9% were negative and single species infection with B. bigemina (56.2%) was significantly more frequent (P<0.01) than with B. bovis (4.7%). Most of the females (60.8%) engorged on cows did not show Babesia spp. infection and the frequency of single B. bovis infection (17.6%) was similar (P>0.05) to the frequency of single B. bigemina infection (15.9%). Mixed Babesia infection was lower (P<0.01) than single species infection in female ticks engorged either in cows (5.7%) or in calves (4.3%). An egg sample from each female was analysed for the presence of Babesia species. Of the egg samples from female ticks infected with B. bovis, 26 (47.3%) were infected while from those from female ticks infected with B. bigemina 141 (76.6%) were infected (P<0.01). The results showed that although the frequency of both species of Babesia was similar in calves and cows, the infectivity of B. bigemina was higher to ticks fed on calves while to those ticks fed on cows the infectivity of both Babesia species was similar.     

Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis

A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.     

Detection of IgG antibody against Neospora caninum in cattle in Korea

A total of 492 cattle sera was screened by IgG-ELISA against Neospora caninum (Nc-1 strain and a Korean isolate, KBA-2) and Toxoplasma gondii. Out of 492, 113 sera (23.0%) reacted positively to either Nc-1 or KBA-2 strains of N. caninum. Among the 113 positive sera, 92 sera (81.4%) reacted with antigens of both strains, but 6 sera (5.3%) with Nc-1 and 15 sera (13.3%) with KBA-2 strain only. And with T. gondii antigen, 6 sera (1.2%) were positive but all reacted with N. caninum antigen also. Western blot revealed typical binding pattern according to ELISA values, such that high OD group reacted specifically to the major surface proteins including 43 kDa protein. Seroprevalence of 23.0% indicates that neosporosis seemed to be one of major causes of abortion in cattle. It is suggested here to establish more epidemiological researches nationwide systematically.     

Immunization effect of recombinant P27/30 protein expressed in Escherichia coli against the hard tick Haemaphysalis longicornis (Acari: Ixodidae) in rabbits

We investigated the induction of resistance to Haemaphysalis longicornis infestation in rabbits that had been immunized with recombinant H. longicornis P27/30 protein. The success of immunological control methods is dependent upon the use of potential key antigens as tick vaccine candidates. Previously, we cloned a gene encoding 27 kDa and 30 kDa proteins (P27/30) of H. longicornis, and identified P27/30 as a troponin I-like protein. In this study, rabbits that were immunized with recombinant P27/30 expressed in Escherichia coli showed the statistically significant longer feeding duration for larval and adult ticks (P<0.05), low engorgement rates in larval ticks (64.4%), and an apparent reduction in egg weights, which suggest that H. longicornis P27/30 protein is a potential candidate antigen for a tick vaccine. These results demonstrated that the recombinant P27/30 protein might be a useful vaccine candidate antigen for biological control of H. longicornis.     

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.     

Seroprevalence of antibodies against Anisakis simplex larvae among health-examined residents in three hospitals of southern parts of Korea

The present study was performed to estimate the seroprevalence of larval Anisakis simplex infection among the residents health-examined in 3 hospitals in southern parts of Korea. A total of 498 serum samples (1 serum per person) were collected in 3 hospitals in Busan Metropolitan city, Masan city, and Geoje city in Gyeongsangnam-do (Province) and were examined by IgE-ELISA and IgE-western blotting with larval A. simplex crude extract and excretory-secretory products (ESP). The prevalence of antibody positivity was 5.0% and 6.6% with ELISA against crude extracts and ESP, respectively. It was also revealed that infection occurred throughout all age groups and higher in females than in males. A specific protein band of 130 kDa was detected from 10 patients with western blot analysis against crude extract and ESP among those who showed positive results by ELISA. Our study showed for the first time the seroprevalence of anisakiasis in Korea. The allergen of 130 kDa can be a candidate for serologic diagnosis of anisakiasis.     

Morphologic and Genetic Identification of Diphyllobothrium nihonkaiense in Korea

Diphyllobothrium nihonkaiense was first described by Yamane in 1986 but the taxonomical features have been obscure due to lack of critical morphologic criteria in its larval and adult stages. In Korea, this tapeworm had long been known as Diphyllobothrium latum. In this study, we observed 62 specimens collected from Korean residents and analyzed them by morphological features and nucleotide sequences of mitochondrial cox1 gene as well as the ITS1 region. Adult tapeworms were examined after carmine or trichrome stain. Longitudinal sections of the gravid proglottids showed an obtuse angle of about 150 degree between the cirrus sac and seminal vesicle. This angle is known as a major differential point compared with that of D. latum. Nucleotide sequence differences between D. latum and the specimens from Koreans represented 17.3% in mitochondrial DNA cox1 gene. Sequence divergence of ITS1 among 4 Korean isolates was 0.3% and similarity was 99.7% with D. nihonkaiense and D. klebanovskii. All of the Korean specimens analyzed in this study were identified as being D. nihonkaiense (n = 62). We propose its Korean name as "Dong-hae-gin-chon-chung" which means ''long tapeworm of the East Sea'' for this newly analyzed diphyllobothriid tapeworm in Korea.     

Identification of Single Meloidogyne Juveniles by Polymerase Chain Reaction Amplification of Mitochondrial DNA

Polymerase chain reaction (PCR) was used to amplify a specific 1.8-kb sequence of mitochondrial DNA from single juveniles and eggs from 17 populations of Meloidogyne incognita, M. hapla, M. javanica, and M. arenaria. Approximately 2 μg amplified product were produced per reaction. Restriction digestion of the amplified product with HinfI permitted discrimination of clonal lineages of the four species. Meloidogyne javanica, however, could not be separated from M. hapla by the enzymes used in these experiments. Various amplification conditions and nematode lysis procedures were examined in order to optimize the speed and quality of identifications.     

Axenic cultivation and characterization of Giardia lamblia isolated from humans in Korea.

Inoculating of human fecal cysts to suckling Mongolian gerbils, two Giardia lamblia isolates, K1 and K2, were established as axenic cultures. Using this in vitro culture, both isolates were characterized as a "medium-rate grower" upon its growth pattern. These two Giardia isolates were grouped by using two genetic analysis. With genetic analysis of SSU-rDNA sequences, both K1 and K2 were found as members of Hopkins' group 1, despite some nucleotide differences noticed in K2 (5 differences/292 bases). The other genetic study used PCR-RFLP of the tim (triose phosphate isomerase) gene. Both of K1 and K2 were found to belong to Nash's group 2. Our results suggest that Nash's group 2 can not be a separate group, but a part of Hopkins' group 1.     

Rodent model for long-term maintenance and development of the viable cysticerci of Taenia saginata asiatica

Although oncospheres of Taenia saginata asiatica can develop into cysticerci in immunodeficiency, immunosuppressed, and normal mice, no detailed information on the development features of these cysticerci from SCID mice is available. In the present study, the tumor-like cyst was found in the subcutaneous tissues of each of 10 SCID mice after 38-244 days inoculation with 39,000 oncospheres of T. s. asiatica. These cysts weighed 2.0-9.6 gm and were 1.5-4.3 cm in diameter. The number of cysticerci were collected from these cysts ranged from 125 to 1,794 and the cysticercus recovery rate from 0.3% to 4.6%. All cysticerci were viable with a diameter of 1-6 mm and 9 abnormal ones each with 2 evaginated protoscoleces were also found. The mean length and width of scolex, protoscolex, and bladder were 477 x 558, 756 x 727, and 1,586 x 1,615 microns, respectively. The diameters of suckers and rostellum were 220 microns and 70 microns, respectively. All cysticerci had two rows of rostellar hooks. These findings suggest that the SCID mouse model can be employed as a tool for long-term maintenance of the biological materials for advanced studies of immunodiagnosis, vaccine development, and evaluation of cestocidal drugs which would be most benefit for the good health of the livestocks.     

A Hospital-Based Serological Survey of Cryptosporidiosis in the Republic of Korea

The seroprevalence of cryptosporidiosis was examined using patients'' sera collected from hospitals located in 4 different areas of the Republic of Korea. ELISA was used to measure antibody titers against Cryptosporidium parvum antigens from a total of 2,394 serum samples, which were collected randomly from patients in local hospitals; 1) Chungbuk National University Hospital, 2) Konkuk University Hospital, 3) local hospitals in Chuncheon, Gangwon-do (province), 4) Jeonnam National University Hospital, from 2002 through 2003. Of the 2,394 samples assayed, 34%, 26%, and 56% were positive for C. parvum-specific IgG, IgM, and IgA antibodies, respectively. Positive IgG titers were most common in sera from Jeonnam National University Hospital, Gwangju, Jeollanam-do, and positive IgM titers were most common in sera from Chungbuk National University Hospital, Cheongju, Chuncheongbuk-do. The seropositivity was positively correlated with age for both the IgG and IgA antibodies but was negatively correlated with age for the IgM antibodies. Western blotting revealed that 92%, 83%, and 77% of sera positive for IgG, IgM, and IgA ELISA reacted with 27-kDa antigens, respectively. These results suggested that infection with Cryptosporidium in hospital patients occurs more commonly than previously reported in the Republic of Korea.