Molecular Detection and Phylogeny of Tick-Borne Pathogens in Ticks Collected from Dogs in the Republic of Korea.

Ticks are important vectors of various pathogens that result in clinical illnesses in humans and domestic and wild animals. Information regarding tick infestations and pathogens transmitted by ticks is important for the identification and prevention of disease. This study was a large-scale investigation of ticks collected from dogs and their associated environments in the Republic of Korea (ROK). It included detecting six prevalent tick-borne pathogens (Anaplasma spp., A. platys, Borrelia spp., Babesia gibsoni, Ehrlichia canis, and E. chaffeensis). A total of 2293 ticks (1110 pools) were collected. Haemaphysalis longicornis (98.60%) was the most frequently collected tick species, followed by Ixodes nipponensis (0.96%) and H. flava (0.44%). Anaplasma spp. (24/1110 tick pools; 2.16%) and Borrelia spp. (4/1110 tick pools; 0.36%) were detected. The phylogenetic analyses using 16S rRNA genes revealed that the Anaplasma spp. detected in this study were closely associated with A. phagocytophilum reported in humans and rodents in the ROK. Borrelia spp. showed phylogenetic relationships with B. theileri and B. miyamotoi in ticks and humans in Mali and Russia. These results demonstrate the importance of tick-borne disease surveillance and control in dogs in the ROK.

Molecular Detection and Phylogenetic Analysis of Anaplasma and Borrelia Species in Ticks Collected from Migratory Birds at Heuksan, Hong, and Nan Islands, Republic of Korea.

The extended distribution and potential introduction of exotic ticks and associated tick-borne pathogens along the northern and southern routes of migratory birds pose zoonotic tick-borne disease risks to wild and domestic animals and incidentally to humans. A knowledge of bird migratory patterns, species of attached ticks, and associated pathogens during their migrations to and from their feeding and nesting grounds is central to understanding associated tick-borne disease risks. Tick-borne disease surveillance was conducted from 2010 to 2011 and 2016 at Hong-do (do = island), Heuksan-do, and Nan-do, major stopovers for migratory birds in Republic of Korea (ROK), as part of the Migratory Birds Research Center bird-banding program for studying bird migration patterns in the ROK. A total of 877 ticks belonging to three genera and nine species were collected, Ixodes turdus (576, 65.7%), Haemaphysalis flava (134, 15.3%), H. longicornis (91, 10.4%), I. nipponensis (56, 6.4%), H. formosensis (7, 0.8%), H. ornithophila (6, 0.7%), H. phasiana (5, 0.6%), H. concinna (1, 0.1%), and Amblyomma testudinarium (1, 0.1%) were collected from 274 birds belonging to 20 genera and 41 species. A total of 15/380 pools (3.95%) were positive for Borrelia species (14 pools of I. turdus and 1 pool of H. flava), while only 1/380 pools (0.26%) was positive for Anaplasma phagocytophilum (1 pool of I. nipponensis). Our findings support the role of migratory birds as possible vectors for the introduction of tick-borne pathogens, which requires continuous monitoring for the potential introduction of ticks and their associated tick-borne pathogens.

Seroprevalence and B1 gene Phylogeny of Toxoplasma gondii of Dogs and Cats in Republic of Korea.

The outbreak of human toxoplasmosis can be attributed to ingestion of food contaminated with Toxoplasma gondii. Toxoplasmosis recently increased in domestic and stray dogs and cats. It prompted studies on the zoonotic infectious diseases transmitted via these animals. Sero- and antigen prevalences of T. gondii in dogs and cats were surveyed using ELISA and PCR, and B1 gene phylogeny was analyzed in this study. Toxoplasmosis antibodies were measured on sera of 403 stray cats, 947 stray dogs, 909 domestic cats, and 2,412 domestic dogs collected at nationwide regions, Korea from 2017 to 2019. In addition, whole blood, feces, and tissue samples were also collected from stray cats (1,392), stray dogs (686), domestic cats (3,040), and domestic dogs (1,974), and T. gondii-specific B1 gene PCR was performed. Antibody prevalence of stray cats, stray dogs, domestic cats, and domestic dogs were 14.1%, 5.6%, 2.3%, and 0.04%, respectively. Antigen prevalence of these animals was 0.5%, 0.2%, 0.1%, and 0.4%, respectively. Stray cats revealed the highest infection rate of toxoplasmosis, followed by stray dogs, domestic cats, and domestic dogs. B1 gene positives were 5 of stray cats, and identified to high/moderate pathogenic Type I/III group. These findings enforce that preventive hygienic measure should be strengthened at One Health level in dogs and cats, domestic and stray, to minimize human toxoplasmosis infections.

Prevalence of antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato, Babesia gibsoni, and Ehrlichia spp. in dogs in the Republic of Korea.

There is a lack of comprehensive studies on the seroprevalence of tick-borne pathogens in the Republic of Korea. Therefore, the aim of this study was to investigate the seroprevalences of Anaplasma spp. (A. phagocytophilum/A. platys), Borrelia burgdorferi sensu lato, Babesia gibsoni, Ehrlichia spp. (E. canis/E. ewingii), and Ehrlichia chaffeensis in dogs across the Republic of Korea in 2017 and 2018. A total of 2215 serum samples collected from 938 companion dogs, 969 shelter dogs, and 308 military working dogs were examined using commercial enzyme-linked immunosorbent assay (ELISA) and indirect fluorescence immunoassay (IFA) kits. Data collected for each animal, including breed, sex, age, region, season, and dog type, were used for statistical analysis. The overall seroprevalence was highest for Anaplasma spp. (15.1 %), followed by Ehrlichia spp. (10.3 %), B. burgdorferi sensu lato (6.4 %), E. chaffeensis (2.3 %), and B. gibsoni (1.7 %). One hundred and sixty-one dogs had antibodies against two or three different pathogens. The most common combinations were Anaplasma spp. - Ehrlichia spp. (2.1 %), Anaplasma spp. - E. chaffeensis (1.4 %), and Anaplasma spp. - B. burgdorferi sensu lato (1.2 %). Season was significantly associated with the seroprevalences of B. burgdorferi sensu lato and Ehrlichia spp., with dogs presenting the highest percentage of positive results during summer. Anaplasma spp. and B. gibsoni were significantly more prevalent in the northern and southern regions, respectively. The seroprevalences of Anaplasma spp., B. burgdorferi sensu lato, and Ehrlichia spp. were significantly higher in military working dogs, while the seroprevalence of E. chaffeensis was higher in companion dogs. The current findings are important for future surveillance of canine tick-borne pathogens and designing appropriate approaches for the diagnosis and control of these pathogens in the Republic of Korea.

Molecular Prevalence of Acarapis Mite Infestations in Honey Bees in Korea.

Acarapis mites, including Acarapis woodi, Acarapis externus, and Acarapis dorsalis, are parasites of bees which can cause severe damage to the bee industry by destroying colonies and decreasing honey production. All 3 species are prevalent throughout many countries including UK, USA, Iran, Turkey, China, and Japan. Based on previous reports of Acarapis mites occurring in northeast Asia, including China and Japan, we investigated a survey of Acarapis mite infestations in honey bees in Korean apiaries. A total of 99 colonies of Apis mellifera were sampled from 5 provinces. The head and thorax of 20 bees from each colony were removed for DNA extraction. PCR assays were performed with 3 primer sets, including T, A, and K primers. Results indicated that 42.4% (42/99) of samples were Acarapis-positive by PCR assay which were sequenced to identify species. Each sequence showed 92.6-99.3% homology with reference sequences. Based on the homology, the number of colonies infected with A. dorsalis was 32 which showed the highest infection rate among the 3 species, while the number of colonies infected with A. externus and A. woodi was 9 and 1, respectively. However, none of the Acarapis mites were morphologically detected. This result could be explained that all apiaries in the survey used acaricides against bee mites such as Varroa destructor and Tropilaelaps clareae which also affect against Acarapis mites. Based on this study, it is highly probable that Acarapis mites as well as Varroa and Tropilaelaps could be prevalent in Korean apiaries.

Efficacy of silver ions against Sacbrood virus infection in the Eastern honey bee Apis cerana.

Although silver is known to be a broad-spectrum biocidal agent, the effects of this metal against Sacbrood virus have not yet been investigated. In this study, we evaluated the efficacy of silver ions against natural Korean sacbrood virus (KSBV) infection of Apis (A.) cerana. Ten KSBV-infected colonies containing A. cerana with similar strength and activity were selected from an apiary located in Bosung-gun (Korea). Among these, five colonies were randomly assigned to the treatment group that was fed sugar syrup containing 0.2 mg/L silver ions. The other colonies were assigned to the untreated control group in which bees were given syrup without the silver ions. To assess the efficacy of the silver ions, colony strength, colony activity, and the number of dead larvae per hive were measured. During the experimental period, the test group maintained its strength and activity until day 32 while those of bees in the control group decreased sharply after day 8 to 16. Survival duration of the test group was significantly longer (40 days) than that of the control group (21 days). These results strongly indicated that silver ions are effective against KSBV infection in A. cerana.

Derivation of cell-adapted Sacbrood virus (SBV) from the native Korean honeybee.

Sacbrood virus (SBV), a causative agent of larval death in honeybees, is one of the most devastating diseases in bee industry throughout the world. Lately the Korean Sacbrood virus (KSBV) induced great losses in Korean honeybee (Apis cerana) colonies. However, there is no culture system available for honeybee viruses, including SBV, therefore, the research on honeybee viruses is practically limited until present. In this study, we investigated the growth and replication of SBV in cell cultures. The replication signs of KSBV after passages from mammalian cells was identified and confirmed by using combined approaches with nested, quantitative, negative-strand PCR and electron microscopy along with in vivo experiment. The results revealed that mammalian cell lines, including Vero cells could support the replication KSBV. Although there were no signs of cytopathic effect (CPE) in cells, it was for the first time demonstrated that SBV could be replicated in cells through the sequential passages linked with cell adaptation. KSBV from the present study would be a valuable source to understand the mechanism of pathogenicity of sacbrood virus in the future.

Reverse transcription loop-mediated isothermal amplification for sensitive and rapid detection of Korean sacbrood virus.

Sacbrood virus (SBV) is one of the most serious honeybee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Recently, the Korean sacbrood virus (KSBV) caused great losses in Korean honeybee (Apis cerana) colonies. Although KSBV shows high homology with SBV strains, it has unique motifs and causes different symptoms. Therefore, a simple, sensitive and specific method for detecting KSBV is needed urgently. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting KSBV using total RNA extracted from honeybees (A. cerana) infected with SBV. The LAMP and the polymerase chain reaction (PCR) methods were then compared for their ability to detect KSBV in clinical samples. The virus was detected in RT-LAMP reactions containing 10(3) copies of pBX-KSBV within 30min, which was comparable to RT-PCR. In addition, the LAMP was able to distinguish between KSBV and other closely-related SBV strains, indicating a high degree of specificity. This simple and sensitive RT-LAMP assay is a useful method for the rapid diagnosis of KSBV infection in honeybees.

Genetic and phylogenetic analysis of South Korean sacbrood virus isolates from infected honey bees (Apis cerana).

Sacbrood virus (SBV) is one of the most destructive honey bee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Genetic analysis of SBV infected honey bees (Apis cerana) from five different provinces was carried out based on three nucleotide sequences; one partial structural protein coding sequence and two non-structural protein coding sequences. Sequences amplified by three specific primer pairs were aligned and compared with reference sequences deposited in the GenBank database. Sequence alignments revealed a low level of sequence variation among Korean isolates (≥ 98.6% nucleotide identity), regardless of the genome regions studied or the geographic origins of the strains. Multiple sequence comparisons indicated that Korean SBV isolates are genetically closely related to Chinese and other Asian strains. Interestingly, the Korean SBV isolates showed a number of unique nucleotides and amino acids that had not been observed in other published strains. Korean and other Asian isolates from the host A. cerana and the UK, European and Japanese strains from the host Apis mellifera showed differences in nucleotide and deduced amino acid identities. This suggests that host-specificity exists among SBV strains isolated from different species. Phylogenetic relatedness between compared sequences was analyzed by MEGA 4.1 software using the neighbor-joining (NJ) method with a boot-strap value of 1000 replicates. Obtained topologies were in agreement with previous studies, in which a distinct group of SBV was formed by UK and European genotypes and another group was comprised of Asian genotypes including strains that originated from China, Japan (japonica), India and Nepal. However, phylogeny based on a partial protein structural coding sequence grouped all Korean SBV isolates identified in A. cerana as a separate cluster. Our findings suggest that further study, including Korean SBV isolated from A. mellifera, is needed.

Effects of ginsenosides on glycine receptor alpha1 channels expressed in Xenopus oocytes.

Ginsenosides, major active ingredients of Panax ginseng, are known to regulate the excitatory ligand-gated ion channel activity. Recent reports showed that ginsenosides attenuate nicotinic acetylcholine and NMDA receptor channel activity. However, it is not known whether ginsenosides also affect the inhibitory ligand-gated ion channel activity. We investigated the effect of ginsenosides on human glycine alpha1 receptor channel activity expressed in Xenopus oocytes using a two-electrode voltage clamp technique. Treatment of ginsenoside Rf enhances glycine-induced inward peak current (IGly) with dose dependent and reversible manner but ginsenoside Rf itself did not elicit membrane currents. The half-stimulatory concentrations (EC50) of ginsenoside Rf was 49.8 +/- 8.9 microM. Glycine receptor antagonist strychnine completely blocked the inward current elicited by glycine plus ginsenoside Rf. Cl- channel blocker 4,4'-disothiocyanostilbene-2,2'-disulfonic acid (DIDS) also blocked the inward current elicited by glycine plus ginsenoside Rf. We also tested the effect of eight individual ginsenosides (i.e., Rb1, Rb2, Rc, Rd, Re, Rg1, Rg2, and Ro) in addition to ginsenoside Rf. We found that five of them significantly enhanced the inward current induced by glycine with the following order of potency: Rb1 > Rb2 > Rg2 > or = Rc > Rf > Rg1 > Re. These results indicate that ginsenosides might regulate gylcine receptor expressed in Xenopus oocytes and this regulation might be one of the pharmacological actions of Panax ginseng.