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Abstract Ticks are blood-sucking ectoparasites that act as vectors for transmission of various pathogens. The purpose of this study was to assess tick-borne bacteria, whether pathogenic or not, in ticks distributed in Korea using 16S rRNA metabarcoding and to confirm the results by PCR. Questing ticks were collected from four provinces in Korea in 2021 using the flagging method. After pooling the DNAs from the 61 tick pools (including 372 ticks), the bacterial 16S rRNA V3–V4 hypervariable region was amplified and sequenced using the MiSeq platform. Rickettsia, Ehrlichia, and the endosymbiont Wolbachia were confirmed by conventional PCR and molecular analysis. In total, 6907 ticks (534 pools) were collected and identified as belonging to five species (Haemaphysalis spp., H. longicornis, H. flava, I. nipponensis, and A. testudinarium). Through 16S rRNA metabarcoding, 240 amplicon sequence variants were identified. The dominant taxa were Rickettsiella and Coxiella. Additionally, pathogenic bacteria such as Rickettsia and Ehrlichia, endosymbiotic bacteria such as Wolbachia and Spiroplasma were identified. Polymerase chain reaction (PCR) was performed to confirm the presence of Rickettsia, Ehrlichia, Bartonella, and Wolbachia in individual ticks. Overall, 352 (65.92%) of 534 pools tested positive for at least one of the screened tick-borne bacteria. Rickettsia was the most prevalent (61.42%), followed by Wolbachia (5.05%). Ehrlichia was detected in 4.86% of tested samples, whereas Bartonella was not detected. In this study, 16S rRNA metabarcoding revealed the presence of Rickettsia, Wolbachia, and Ehrlichia, in that order of abundance, while showing absence of Bartonella. These results were confirmed to exhibit the same trend as that of the conventional PCR. Therefore, large-scale screening studies based on pooling, as applied in this study, will be useful for examining novel or rare pathogens present in various hosts and vectors.

Abstract Apis mellifera filamentous virus (AmFV) is a double-stranded DNA virus that infects Apis mellifera bees. To our knowledge, this is the first comprehensive study aiming to detect and analyse the genetic diversity and prevalence of AmFV in Korean honeybee colonies. Phylogenetic analysis based on baculovirus repeat open reading frame–N gene (Bro–N) sequences revealed that AmFV isolates from the Republic of Korea (ROK) fell into two distinct lineages, with genetic origins in Switzerland and China, with nucleotide similarities of 98.3% and 98.2%, respectively. Our prevalence analysis demonstrated a noteworthy infection rate of AmFV in 545 honeybee colonies, reaching 33.09% in 2022 and increasing to 44.90% by 2023. Intriguingly, we also detected AmFV in Varroa destructor mites, highlighting their potential role as vectors and carriers of AmFV. The presence of AmFV was correlated with an increased infection rate of sacbrood virus, deformed wing virus, Lake Sinai virus 2, black queen cell virus, and Nosema ceranae in honeybee colonies. These findings provide valuable insight into the prevalence and potential transmission mechanisms of AmFV in honeybee colonies in the ROK. The results of this study may be instrumental in the effective management of viral infections in honeybee apiaries.

Abstract Mites of the genus Tyrophagus (Acari: Acaridae) are among the most widely distributed mites. The species in this genus cause damage to stored products and crops, and pose a threat to human health. However, the influence of Tyrophagus spp. in apiculture remains unknown. In 2022, a study focusing on the identification of Tyrophagus species within five apiaries was conducted in Chungcheongnam Province, Republic of Korea. Its specific objective was to investigate the presence of Tyrophagus mites in response to the reported high mortality of honey bee colonies in this area. Morphological identification and phylogenetic analysis using the mitochondrial gene cytochrome-c oxidase subunit 1 (CO1) confirmed for the first time the presence of the mite species Tyrophagus curvipenis in a honey bee colony in the Republic of Korea. Two honey bee pathogens were detected in the mite, a viral pathogen (deformed wing virus, DWV) and a protozoal pathogen (Trypanosoma spp.). The presence of the two honey bee pathogens in the mite suggests that this mite could contribute to the spread of related honey bee diseases. However, the direct influence of the mite T. curvipenis on honey bee health remains unknown and should be further investigated.

In this study, we identified a new strain of the genus Neocypholaelaps from the beehives of Apis mellifera colonies in the Republic of Korea (ROK). The Neocypholaelap sp. KOR23 mites were collected from the hives of honeybee apiaries in Wonju, Gangwon-do, in May 2023. Morphological and molecular analyses based on 18S and 28S rRNA gene regions conclusively identified that these mites belong to the genus Neocypholaelaps, closely resembling Neocypholaelaps sp. APGD-2010 that was first isolated from the United States. The presence of 9 of 25 honeybee pathogens in these mite samples suggests that Neocypholaelaps sp. KOR23 mite may act as an intermediate vector and carrier of honeybee diseases. The identification of various honeybee pathogens within this mite highlights their significance in disease transmission among honeybee colonies. This comprehensive study provides valuable insights into the taxonomy and implications of these mites for bee health management and pathogen dissemination.

Lake Sinai Virus (LSV) is an emerging pathogen known to affect the honeybee (Apis mellifera). However, its prevalence and genomic characteristics in the Republic of Korea (ROK) remain unexplored. This study aimed to assess the prevalence of and analyze the LSVs by examining 266 honeybee samples from the ROK. Our findings revealed that LSV exhibited the highest infection rate among the pathogens observed in Korean apiaries, particularly during the reported period of severe winter loss (SWL) in A. mellifera apiaries in 2022. Three LSV genotypes- 2, 3, and 4 -were identified using RNA-dependent RNA polymerase gene analysis. Importantly, the infection rates of LSV2 (65.2%) and LSV3 (73.3%) were significantly higher in colonies experiencing SWL than in those experiencing normal winter loss (NWL) (p < 0.03). Furthermore, this study provides the first near-complete genome sequences of the Korean LSV2, LSV3, and LSV4 strains, comprising 5,759, 6,040, and 5,985 nt, respectively. Phylogenetic analysis based on these near-complete genome sequences demonstrated a close relationship between LSVs in the ROK and China. The high LSV infection rate in colonies experiencing a heightened mortality rate during winter suggests that this pathogen might contribute to SWL in ROK. Moreover, the genomic characteristic information on LSVs in this study holds immense potential for epidemiological information and the selection of specific genes suitable for preventing and treating LSV, including the promising utilization of RNA interference medicine in the future.

Abstract Background American foulbrood (AFB) disease caused by Paenibacillus larvae is dangerous, and threatens beekeeping. The eco-friendly treatment method using probiotics is expected to be the prospective method for controlling this pathogen in honey bees. Therefore, this study investigated the bacterial species that have antimicrobial activity against P. larvae. Results Overall, 67 strains of the gut microbiome were isolated and identified in three phyla; the isolates had the following prevalence rates: Firmicutes 41/67 (61.19%), Actinobacteria 24/67 (35.82%), and Proteobacteria 2/67 (2.99%). Antimicrobial properties against P. larvae on agar plates were seen in 20 isolates of the genus Lactobacillus, Firmicutes phylum. Six representative strains from each species (L. apis HSY8_B25, L. panisapium PKH2_L3, L. melliventris HSY3_B5, L. kimbladii AHS3_B36, L. kullabergensis OMG2_B25, and L. mellis OMG2_B33) with the largest inhibition zones on agar plates were selected for in vitro larvae rearing challenges. The results showed that three isolates (L. apis HSY8_B25, L. panisapium PKH2_L3, and L. melliventris HSY3_B5) had the potential to be probiotic candidates with the properties of safety to larvae, inhibition against P. larvae in infected larvae, and high adhesion ability. Conclusions Overall, 20 strains of the genus Lactobacillus with antimicrobial properties against P. larvae were identified in this study. Three representative strains from different species (L. apis HSY8_B25, L. panisapium PKH2_L3, and L. melliventris HSY3_B5) were evaluated to be potential probiotic candidates and were selected for probiotic development for the prevention of AFB. Importantly, the species L. panisapium isolated from larvae was identified with antimicrobial activity for the first time in this study.

Abstract Migratory birds disperse ticks and associated tick-borne pathogens along their migratory routes. Four selected pathogens of medical importance (Coxiella burnetii, Rickettsia spp., Francisella tularensis, and Toxoplasma gondii) were targeted for detection in 804 ticks (365 pools) collected from migratory birds at Hong and Heuksan Islands in the Republic of Korea (ROK) from 2010 to 2011 and 2016. Toxoplasma gondii and Rickettsia spp., were detected in 1/365 (0.27%) and 34/365 (9.32%) pools of ticks, respectively. T. gondii and five rickettsial species were recorded in ticks collected from migratory birds for the first time in ROK. The five rickettsial species (R. monacensis, Candidatus Rickettsia longicornii, R. japonica, R. raoultii, and R. tamurae) were identified using sequence and phylogenetic analysis using ompA and gltA gene fragments. Rickettsia spp. are important pathogens that cause rickettsiosis in humans, with cases recorded in the ROK. These results provide important evidence for the potential role of migratory birds in the introduction and dispersal of T. gondii and Rickettsia spp. along their migratory routes and raise awareness of potential transmission of zoonotic tick-borne pathogens associated with migratory birds in the ROK.

Abstract Honey bees are important pollinators for the conservation of the ecosystem and agricultural products and provide a variety of products important for human use, such as honey, pollen, and royal jelly. Sacbrood disease (SD) is a devastating viral disease in Apis cerana; an effective preventive measure for SD is urgently needed. In this study, the relationship between the gut microbiome of honey bees and SD was investigated by pyrosequencing. Results revealed that sacbrood virus (SBV)-resistant A. cerana strains harbour a unique acetic acid bacterium, Bombella intestini, and the lactic acid bacteria (LAB) Lactobacillus (unclassified)_uc, Bifidobacterium longum, B. catenulatum, Lactococcus lactis, and Leuconostoc mesenteroides in larvae and Hafnia alvei, B. indicum, and the LAB L. mellifer and Lactobacillus HM215046_s in adult bees. Changes in the gut microbiome due to SBV infection resulted in loss of bacteria that could affect host nutrients and inhibit honey bee pathogens, such as Gilliamella JFON_s, Gilliamella_uc, Pseudomonas putida, and L. kunkeei in A. cerana larvae and Frischella_uc, Pantoea agglomerans, Snodgrassella_uc, and B. asteroides in adult bees. These findings provide important information for the selection of probiotics for A. cerana larvae and adults to prevent pathogenic infections and keep honey bees healthy.

Abstract Background Rickettsia spp. are important tick-borne pathogens that cause various human and animal diseases worldwide. A tool for rapid and accurate detection of the pathogens from its vectors is necessary for prevention of Rickettsioses propagation in humans and animals, which are infested by ticks. Therefore, this study was conducted to evaluate a molecular tool, ultra-rapid real-time PCR (UR-qPCR), for rapid and accurate detection of Rickettsia spp. from 5644 ticks in 408 pools collected from livestock and their surrounding environments in Gangwon and Jeju province in South Korea. Results The UR-qPCR of Rickettsia DNA showed a limit of detection of 2.72 × 101 copies of Rickettsia DNA and no cross reaction with other tick-borne pathogens, namely Anaplasma phagocytophilum, Ehrlichia chaffeensis, E. canis, Toxoplasma gondii, and Borrelia burgdorferi. In addition, the PCR assay also showed possibility of various Rickettsia species detection including R. monacensis, “Candidatus R. longicornii”, R. japonica, R. roultii, and R. tamurae. The collected ticks were identified with major species belonged to Haemaphysalis longicornis (81.62%), followed by H. flava (15.19%), and Ixodes nipponensis (3.19%). Rickettsia detection from tick samples using the UR-qPCR showed that the minimum infection rate (MIR) of Rickettsia in collected ticks was 1.24‰ and that all positive pools contained H. longicornis, equal to the MIR of 1.39‰ of this species. Additionally, MIR of Rickettsia spp. detected in ticks collected in Gangwon and Jeju was 1.53‰ and 0.84‰, respectively. Furthermore, the sequencing results of the 17 kDa protein antigen gene and ompA gene showed that Rickettsia spp. sequences from all pools were related to “Candidatus R. longicornii” and “Candidatus R. jingxinensis”. Conclusions The UR-qPCR system was demonstrated to be useful tool for accurate and rapid detection of Rickettsia from its vector, ixodid ticks, within 20 min. The data on Rickettsia spp. in ticks detected in this study provide useful information on the distribution of Rickettsia in previously unstudied Korean provinces, which are important for the prevention and control of the spread of rickettsioses in both animals and humans in the country.

Honey bees play an important role in the pollination of crops and wild plants and provide important products to humans. Pathogens and parasites are the main factors that threaten beekeeping in South Korea. Therefore, a nationwide detection of 14 honey bee pathogens, including parasites (phorid flies, Nosema ceranae, and Acarapis woodi mites), viruses, bacteria, and fungal pathogens, was conducted from 2017 to 2021 in the country. The infection rate and the trend of detection of each pathogenic agent were determined. A total of 830 honey bee samples from Apis cerana (n = 357) and A. mellifera (n = 473) were examined. N. ceranae (35.53%), deformed wing virus (52.63%), sacbrood virus (SBV) (52.63%), and black queen cell virus (55.26%) were the most prevalent honey bee pathogens, and their prevalence rapidly increased from 2017 to 2021. The prevalence of Paenibacillus larvae, Israeli acute paralysis virus, Ascosphaera apis, A. woodi, Melissococcus plutonius, and chronic bee paralysis virus remained stable during the surveillance period, with infection rates ranging from 5.26% to 16.45% in 2021. Other pathogens, including acute bee paralysis virus, phorid flies, Kashmir bee virus, and Aspergillus flavus, had low infection rates that gradually declined during the detection period. The occurrence of honeybee pathogens peaked in July. SBV was the most common pathogen in A. cerana, whereas N. ceranae was predominant in A. mellifera. This study provides information regarding the current status of honey bee pathogens and presents the trend of the occurrence of each pathogen in South Korea. These data are important for predicting outbreaks of honey bee diseases in the country.

Abstract Background Q fever, a zoonosis caused by Coxiella burnetii, has adverse effects on public health. Ticks are vectors of C. burnetii and they contribute to the transmission of the pathogen. A tool for rapid, sensitive, and accurate detection of C. burnetii from ticks is important for the prevention of Q fever. Methods Ultra-rapid real-time PCR (UR-qPCR) as a chip-based real-time PCR system was developed for the detection of C. burnetii from ticks. The UR-qPCR system was established and evaluated for the rapidity, sensitivity, and specificity of C. burnetii detection. Results C. burnetii was detected using UR-qPCR from 5644 larval, nymphal, and adult ticks from 408 pools collected from livestock and epidemiologically linked environments in two provinces, Gangwon and Jeju, in Korea. Ticks from three species were identified; Haemaphysalis longicornis accounted for the highest number, present in 333 of 408 pools (81.62%), followed by Haemaphysalis flava in 62 pools (15.19%) and Ixodes nipponensis in 13 pools (3.19%). The rapidity and sensitivity of PCR detection was demonstrated with the sufficient amplification and detection of approximately 56 copies of C. burnetii DNA with only 20 min of PCR amplification. The kappa value for the diagnostic agreement between UR-qPCR and stationary qPCR was in perfect agreement (κ = 1). PCR detection and sequencing indicated that C. burnetii was present in 5 of the 408 pools (1.23%), in which four pools contained H. longicornis and one pool contained H. flava. The infection rates of C. burnetii in the tick pools collected from Gangwon and Jeju Provinces were 1.70% and 0.58%, respectively. Phylogenetic analysis indicated a close relationship between the detected C. burnetii and those originating from goats, humans, and ticks in different countries, such as the USA, France, Germany, and Serbia. Conclusions The methods described in this study could be important for the prevention and control of Q fever in the two provinces. The UR-qPCR, with its features of mobility, sensitivity, and rapidity, is helpful for constructing early alert systems in the field for C. burnetii in ticks and could help alleviate the transmission of and economic damage due to Q fever. Graphic abstract

The present study was performed to survey the dominant tick populations and molecularly determine the pathogenic agents of anaplasmosis in ticks from Gyeongsang, Republic of Korea. A total of 3825 questing ticks were collected by the flagging method from 12 sites near animal farms in Gyeongsang from March to October 2021. A molecular genomic study was performed with ticks stored in 70% ethanol to detect Anaplasma genes by the previously described method. The monthly incidence of ticks varied by developmental stages, i.e., nymphs, adults, and larvae, and each of their populations peaked in May, March, and October, respectively. The predominant tick species were Haemaphysalis longicornis, Haemaphysalis sp., Haemaphysalis flava, Ixodes nipponensis, and Amblyomma testudinarium in order. To determine the Anaplasma infection rate, collected ticks were pooled into 395 groups. The minimum infection rate (MIR) of Anaplasma was 0.7% (27 pools). That of A. phagocytophilum was highest (23 pools, MIR 0.6%), followed by A. phagocytophilum-like Anaplasma spp. clade B (2 pools, MIR 0.1%), A. bovis (1 pool, MIR 0.1%), and A. capra (1 pool, MIR 0.1%), respectively. In this study, five species of ticks, including unidentified Haemaphysalis species, were collected in 12 survey sites in Gyeongsang, but their prevalence was somewhat different according to the tick species and survey sites. Further, the incidence rate (6.8%) of 4 Anaplasma spp. was not as high in tick pools. However, the results of this study may offer a basis for future epidemiological research and risk assessment of tick-borne diseases.

Clostridium botulinum produces neurotoxic substrates that can cause fatal flaccid paralysis called botulism. These neurotoxins are classified into types A–G. Several botulism cases were recorded in 2012–2013 in the Gyeonggi province, South Korea. We assessed the distribution of C. botulinum types B, C, and D in several South Korean farms. A total of 184 samples collected in 2012–2013, including feces (n = 72), hay and silage (n = 50), soil (n = 26), water trough (n = 21), and stomach contents (n = 15), were subjected to multiplex polymerase chain reaction (PCR) to screen for types B, C, and D. Twenty-four samples tested PCR-positive as follows: type B (n = 11), type C/D (n = 4), and type D (n = 18). Eight of the 11 type B samples were detected in hay and silage. Sixteen of the 18 type D samples were detected in fecal and stomach content samples. PCR-positivity was observed in fecal (n = 9, 12.5%), hay and silage (n = 10, 20.0%), water trough (n = 2, 9.5%), and stomach content (n = 12, 80.0%) samples. Fourteen (42.4%) C. botulinum-positive samples were isolated from the PCR-positive samples (type B [n = 8], type C/D [n = 1], and type D [n = 5]). Our findings demonstrate that C. botulinum types B, C/D, and D were prevalent in South Korean cattle farms between 2012 and 2013.

Honeybees play a vital role in the ecological environment and agricultural economy. Increasing evidence shows that the gut microbiome greatly influences the host’s health. Therefore, a thorough understanding of gut bacteria composition can lead to the development of probiotics specific for each development stage of honeybees. In this study, the gut microbiota at different developmental stages (larvae, pupae, and adults) of the honeybees Apis cerana in Hanoi, Vietnam, was assessed by sequencing the V3–V4 region in the 16S rRNA gene using the Illumina Miseq platform. The results indicated that the richness and diversity of the gut microbiota varied over the investigated stages of A. cenara. All three bee groups showed relative abundance at both phylum and family levels. In larvae, Firmicutes were the most predominant (81.55%); however, they decreased significantly along with the bee development (33.7% in pupae and 10.3% in adults) in favor of Proteobacteria. In the gut of adult bees, four of five core bacteria were found, including Gilliamella apicola group (34.01%) Bifidobacterium asteroides group (10.3%), Lactobacillus Firm-4 (2%), and Lactobacillus Firm-5 (1%). In contrast, pupae and larvae lacked almost all core bacteria except G. apicola (4.13%) in pupae and Lactobacillus Firm-5 (4.04%) in larvae. This is the first report on the gut microbiota community at different developmental stages of A. cerana in Vietnam and provides potential probiotic species for beekeeping.

Monofloral honey is produced from the nectar of a single predominant botanical species in a particular season and has certain unique properties. Valuable monofloral honey produced in a particular season with unique properties is often targeted for adulteration. Herein, a method for the identification of monofloral honey and determination of its production season was developed. Major nectar plants, including Prunus sp., Robinia pseudoacacia, Castanea sp., and Kalopanax sp., were selected to evaluate the honey produced between April and July in South Korea. Results showed that the highest amount of DNA from each plant was detected in the corresponding flowering season. The pollens tended to accumulate in the honeycomb after the flowering season. The accumulations result in an increase in the diversity of pollen detected in honey. Additionally, DNA quantity of each plant decreased in the samples as the number of plant DNA types increased from May to July. Moreover, the authenticity of the commercial monofloral honey samples showed only cherry blossom honey was found authentic, which exhibited the expected high amount of Prunus sp. DNA. This molecular tool is expected to be useful in verifying the origin of monofloral honey and its production season.

The horse industry has grown rapidly as a leisure industry in the Republic of Korea (ROK) in parallel with an increased demand for equestrian activities. As a result, there has been an increase in horse breeding and equestrian population and potential exposure to ticks and their associated pathogens. To provide a better understanding of the potential disease risks of veterinary and medical importance, a study was conducted to determine the geographical distribution and diversity of ticks collected from horses and vegetation associated with horse racetracks/ranches throughout the ROK. This included a survey of five associated common pathogens, Anaplasma phagocytophilum, Ehrlichia chaffeensis, Borrelia spp., Babesia caballi, and Theileria equi. A total 9220 ticks were collected from horses and associated pastures. Ticks were identified to species, stage of development, and sex. Two species of ticks, Haemaphysalis longicornis (99.9%) and Ixodes nipponensis (0.1%) were identified. Two of the target pathogens, A. phagocytophilum and Borrelia spp., were detected in 5/1409 tick pools (0.35%) and 4/1409 pools (0.28%) of H. longicornis, respectively, both of which are zoonotic pathogens of medical importance. The results of 16S rRNA phylogenetic analysis of A. phagocytophilum showed a close relationship to strains distributed in China, USA, Germany, Italy, Turkey, and Poland. Borrelia spp. showed a close relationship, based on 16S rRNA gene, to the strains reported from the USA (B. burgdorferi and B. americana) and Japan (B. tanukii and B. garinii). These results provide information about the potential risks of veterinary and medical importance and the development of mitigation strategies for disease prevention.

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.

Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is one of the major causes of viral encephalitis. To reduce the impact of Japanese encephalitis among children in the Republic of Korea (ROK), the government established a mandatory vaccination program in 1967. Through the efforts of this program only 0-7 (mean 2.1) cases of Japanese encephalitis were reported annually in the ROK during the period of 1984-2009. However, in 2010 there was an outbreak of 26 confirmed cases of Japanese encephalitis, including 7 deaths. This represented a >12-fold increase in the number of confirmed cases of Japanese encephalitis in the ROK as compared to the mean number reported over the last 26 years and a 3.7-fold increase over the highest annual number of cases during this same period (7 cases). Surveillance of adult mosquitoes was conducted during the 2010 outbreak of Japanese encephalitis in the ROK. A total of 6,328 culicine mosquitoes belonging to 12 species from 5 genera were collected at 6 survey sites from June through October 2010 and assayed by reverse-transcription polymerase chain reaction (RT-PCR) for the presence of JEV. A total of 34/371 pooled samples tested positive for JEV (29/121 Culex tritaeniorhynchus, 4/64 Cx. pipiens, and 1/26 Cx. bitaeniorhynchus) as confirmed by sequencing of the pre-membrane and envelope protein coding genes. The maximum likelihood estimates of JEV positive individuals per 1,000 culicine vectors for Cx. tritaeniorhynchus, Cx. pipiens, and Cx. bitaeniorhynchus were 11.8, 5.6, and 2.8, respectively. Sequences of the JEV pre-membrane and envelope protein coding genes amplified from the culicine mosquitoes by RT-PCR were compared with those of JEV genotypes I-V. Phylogenetic analyses support the detection of a single genotype (I) among samples collected from the ROK in 2010.

Japanese encephalitis virus (JEV) is a mosquito-borne virus that can infect pigs, horses, and other mammals, including humans. Sero-epidemiological investigations of JEV have been performed using hemagglutination inhibition (HI), virus neutralization (VN) tests and enzyme-linked immunosorbent assay (ELISA). A need exists for a new ELISA that can detect JEV antibodies in the sera of several animal species. We aimed to develop a blocking ELISA (B-ELISA) for detecting JEV antibodies in pig and horse serum samples. JEV antibodies in 218 pig and 315 horse serum samples were measured using HI and VN tests. The purified KV1899-306 strain was used as an antigen for B-ELISA. The purified antibody (7A13) was conjugated with horseradish peroxidase and used as a detector antibody. The sera of pigs and horses to measure antibody against JEV were subjected to B-ELISA and analyzed. The B-ELISA had a diagnostic sensitivity of 94.6% to 100%, a specificity of 91.2 to 100%, and an accuracy of 94.9 to 98.6% compared with those of the HI and VN tests in pig and horse sera. The B-ELISA had a higher correlation with pig sera (r = 0.89 and 0.90 for VN and HI) than with horse sera (r = 0.75 and to 0.79). The new B-ELISA could be useful in the sero-surveillance of JEV in pig and horse sera and replace indirect ELISA. [ABSTRACT FROM AUTHOR]

Canine adenovirus type 2 (CAV-2) is a common causative agent of respiratory disease in canines. There have been no reports of CAV-2 variants isolated from raccoon dogs. This study aims to investigate the biological and genetic characteristics of a novel Korean CAV-2 variant. Madin-Darby canine kidney cells were used to isolate the CAV-2 variant from 45 fecal swab samples. Diagnostic tools such as the cytopathic effect (CPE) assay, electron microscopy, polymerase chain reaction, and immunofluorescence and hemagglutination assays were used to confirm the presence of the CAV-2 isolate. A cross-virus neutralization assay was performed to verify the novelty of this CAV variant. Genetic analysis was performed using nucleotide sequences obtained through next-generation sequencing. The isolate was confirmed to be a CAV-2 variant based on the aforementioned methods and designated CAV2232. The number of bases in the fiber and E3 genes of CAV2232 were 1,626 and 414, respectively. Phylogenetic analysis of the fiber and E3 genes confirmed that CAV2232 was classified into a different clade from the known CAV-1 and CAV-2 strains. Mice inoculated with the CAV2232 vaccine developed high virus neutralization antibody titers of 1,024 (210) against CAV2232, while mice inoculated with CAV-1 and CAV-2 vaccines had low virus neutralization antibody titers of 12.9 (23.7) and 6.5 (22.7), respectively, against CAV2232. CAV2232 isolated from wild raccoon dog feces was classified as a novel CAV-2 variant. CAV2232 may therefore be used as an antigen for new vaccine development and serological investigations. [ABSTRACT FROM AUTHOR]

Background: The microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen. Objectives: The present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees. Methods: A procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration. Results: UR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 104 spores/bee, and the stable detection level was = 2.40 × 105 spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 104 copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min. Conclusions: UR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection. [ABSTRACT FROM AUTHOR]

Equine piroplasmosis (EP) is caused by Babesia caballi and Theileria equi infection. We investigated antigen and antibody of EP in horses in the Republic of Korea during 2016- 2017. Antigen and antibody of T. equi was detected 0.06% (1/1,650). Phylogenetic analysis of 18S rRNA revealed that the T. equi was highly homologous with the strains from China, Mongolia, and Spain. Two Theileria spp. were also detected and highly homologous with T. buffeli, T. luwenshuni, and T. orientalis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To investigate the prevalence of Toxoplasma gondii in pork on the market in Korea, an in-house enzyme-linked immunosorbent assay for tissue fluid (CAU-tf-ELISA) was developed using a soluble extract of T. gondii RH strain tachyzoites. As the standard positive controls, the piglets were experimentally infected with T. gondii: Group A (1,000 cysts-containing bradyzoites), Group B (500 cysts-containing bradyzoites) and Group C (1.0×103 or 1.0×104 tachyzoites). The CAU-tf-ELISA demonstrated infection intensity-dependent positivity toward tissue fluids with average cut-off value 0.15: 100% for Group A, 93.8% for Group B and 40.6% for Group C. When tissue-specific cut-off values 0.066- 0.199 were applied, CAU-tf-ELISA showed 96.7% sensitivity, 100% specificity, 100% positive and 90.0% negative predictive values. When compared with the same tissue fluids, performance of CAU-tf-ELISA was better than that of a commercial ELISA kit. Of the 583 Korea domestic pork samples tested, anti-T. gondii antibodies were detected from 9.1% of whole samples and 37.9% from skirt meat highest among pork parts. In the 386 imported frozen pork samples, 1.8% (skirt meat and shoulder blade) were positive for anti-T. gondii antibodies. In Korea, prevalence of anti-T. gondii antibodies in the pork on retail markets appeared high, suggesting that regulations on pig farming and facilities are necessary to supply safe pork on the tables. [ABSTRACT FROM AUTHOR]

An 8-year-old female Eurasian otter (Lutra lutra) reared in a wetland center, died 2 h after sudden onset of astasia and dyspnea despite medical treatment. Gross examination of internal organs revealed 10 adult filarioid nematodes in the right ventricle of the heart and three between the left and right cerebral hemispheres. All nematodes were identified as Dirofilaria immitis by direct microscopy and polymerase chain reaction assay. Histopathological observation revealed multifocal hemorrhage in the cerebral subarachnoid space and focal necrosis with hemorrhage in the cerebellar parenchyma. Although rare, veterinarians should consider cerebral dirofilariasis as a differential diagnosis in unexplained neurological cases. [ABSTRACT FROM AUTHOR]

Between 2011 and 2012, a total of 896 pig fecal samples were collected from nine provinces in Korea, and 50 salmonella enterica susp. enterica serovar Typhimurium (S. Typhimurium) was isolated. The characteristics of the 50 strains were analyzed, and 4 strains were identified as Salmonella enterica subsp. enterica serovar 4,[5],12:i:-. Salmonella 4,[5],12:i:- could not be distinguished from S. Typhimurium through phage typing, antimicrobial resistance testing or multiple-locus variable-number tandem repeat analysis (MLVA). However, among the four Salmonella 4,[5],12:i:- strains, one (KVCC-BA1400078) was identified as a Salmonella 4,[5],12:i:- clone isolated from humans in the United States, and another (KVCC-BA1400080) was identified as DT193, which has been primarily isolated from humans and animals in European countries. The presence of Salmonella 4,[5],12:i:- in Korea poses a significant threat of horizontal transfer between pigs and humans. [ABSTRACT FROM AUTHOR]

Background: Schmallenberg virus (SBV), Akabane virus (AKAV) and Aino virus (AINV) are members of the Simbu serogroup within the genus Orthobunyavirus, family Bunyaviridae, which can cause reproductive disorders including abortion, stillbirth and congenital malformation in ruminants. Because, the clinical signs are similar, confirmatory diagnosis requires viral detection to differentiate infection between these three viruses. Methods: In this study, a one-step multiplex reverse-transcriptase quantitative PCR (one-step mRT-qPCR) was developed for the simultaneous detection and differentiation of SBV, AKAV and AINV. Results: The detection limit of the one-step mRT-qPCR for SBV, AKAV and AINV were 2.4 copies (10 0.6 TCID 50/ml), 96.2 copies (10 1.5 TCID 50/ml) and 52.3 copies (10 1.2 TCID 50/ml), respectively. Various field samples such as bovine serum, bovine whole blood, bovine brain, goat serum and Culicoides were analyzed using the one-step mRT-qPCR and compared with previously published RT-qPCRs. The test results of the field samples were identical for the one-step mRT-qPCR and RT-qPCRs, which showed all samples to be negative for SBV, AKAV and AINV, except for one bovine brain sample (1/123) that was positive for AKAV. Conclusion: The one-step mRT-qPCR allows for the simultaneous detection of three viral pathogens (SBV, AKAV and AINV) that cause reproductive failure. [ABSTRACT FROM AUTHOR]

Bovine tuberculin purified protein derivative (bPPD) is used as an intradermal test (IT) reagent to detect bovine tuberculosis (bTB) in most countries. Identification of bPPD proteins is critical to understanding the immunological reaction of IT at the molecular level. While bPPD from the United Kingdom (UK) and Brazil (BR) have been recently defined at the proteomic level, bPPD from the Republic of Korea (KR) has not yet been analyzed. Here, bPPD KR proteome was examined for the first time. In total, 271 proteins were identified, including Mycobacterium bovis-specific proteins Mb0854c and Mb2898, and 42 known T cell antigens. On comparing with proteomes of bPPD UK and BR, 33 proteins were found to be common among all three bPPDs, of which 15 proteins were T cell antigens. M. bovis-specific antigens with T cell activity in bPPD may be novel candidates for use as alternatives to currently available bPPD in diagnostics. [ABSTRACT FROM AUTHOR]

Apis mellifera, especially weak ones, are highly vulnerable to Carpoglyphus lactis mites, which can rapidly infest and consume stored pollen, leading to weakened colonies and potential colony collapse. This study aimed to ascertain and investigate the prevalence of this mite in honeybee colonies across nine provinces in the Republic of Korea (ROK). A total of 615 honeybee colony samples were collected from 66 apiaries during the spring and 58 apiaries during the summer of 2023. A 1242 bp segment of the Cytochrome c oxidase subunit 1 (COI) gene was amplified using the polymerase chain reaction method. The detection levels of C. lactis in the honeybees were compared between winter and summer. Based on the COI sequence analysis, the nucleotide sequence similarity of C. lactis mites isolated in the ROK with those from China (NC048990.1) was found to be 99.5%, and with those from the United Kingdom (KY922482.1) was 99.3%. This study is the first report of C. lactis in Korean apiaries. The findings of this study demonstrate a significantly higher detection rate in winter, which is 4.1 times greater than that in summer (p < 0.001). Furthermore, the results underscore the usefulness of molecular diagnostic techniques for detecting C. lactis mites.

Sacbrood virus (SBV) infection has emerged as a remarkable threat to Apis cerana colonies in South Korea, necessitating prompt control measures. In this study, RNA interference (RNAi) targeting the VP3 gene was developed to assess its safety and efficacy in protecting and treating SBV in vitro and in infected colonies in South Korean apiaries. The efficacy of VP3 double-stranded RNA (dsRNA) was demonstrated in laboratory-based experiments, wherein infected larvae treated with VP3 dsRNA exhibited a 32.7% increase in survival rate compared to untreated larvae. Data from a large-scale field trial indicate the efficacy of dsRNA treatment since none of the treated colonies had symptomatic SBV infections, whereas disease was observed in 43% (3/7) of the control colonies. In the 102 colonies exhibiting symptoms of SBV disease, RNAi treatment provided partial protection with weekly treatment, prolonging the survival period of colonies to 8 months compared to 2 months in colonies treated at 2- and 4-week intervals. Therefore, this study demonstrated that RNAi is a valuable tool for preventing SBV disease outbreaks in healthy and low-level SBV-infected colonies.

Abstract Excessive alcohol consumption induces acute intoxication and various hepatic diseases. In this study, we investigated the effect of the CureZyme-ACE (CA), Acetobacter Pasteurianus (AP)-derived product, in acute intoxication rats. The ethanol and acetaldehyde levels of serum were lower in rats treated with CA than those who only treated ethanol. The activities of alcohol dehydrogenase and acetaldehyde dehydrogenase also recovered faster in the CA group than only-ethanol group. The transaminase levels (AST, ALT) in the CA group were significantly lower than only-ethanol group. In addition, Hepatic histological analyses and stomach wall were demonstrated that the CA-treated group recovered faster than only-ethanol group. With regard to most characteristics, we found that CA had dose-dependent effects. At high concentrations of CA, there were no differences in the tested parameters compared to those of normal rats. These findings indicate that CA reduces the serum alcohol concentration and some of the hepatic damage caused by alcohol intoxication.