Molecular and phylogenetic analysis of transmissible gastroenteritis virus strain VET-16, isolated from piglets in Vietnam.

Porcine transmissible gastroenteritis virus (TGEV) is a major pathogen that causes viral enteritis and severe diarrhea in newborn piglets. TGEV strains have been isolated in the USA, Europe, and China, and their molecular characteristics are well known. However, there have been few reports of molecular analysis of TGEV strains isolated in Southeast Asia. In 2016, we isolated TGEV strain VET-16 from fecal samples collected from piglets in Vietnam and determined its complete genome sequence by Sanger sequencing. We found that, while the full genome of the VET-16 strain was 92.4-99.9% identical to those of other TGEV strains, the ORF3 gene showed very little sequence similarity. Phylogenetic analysis suggested that the VET-16 strain belongs to the Purdue subgroup. Comparison of the predicted amino acid (aa) sequence of the spike protein of strain VET-16 with those of other TGEV strains revealed three aa substitutions (V378L, S379T, and D380N) and a 3-aa insertion (F383_F387insWEK) in antigenic site D of the VET-16 strain. Also, a single aa deletion (∆F1413) was found in the transmembrane domain of the spike gene of VET-16. Like the ORF3 gene from the TGEV Miller M60 vaccine strain, the VET-16 strain has a large deletion (∆725 nt) in the ORF3 gene. Previous studies have suggested that these mutations in the spike and ORF3 genes might be associated with a reduction in pathogenicity. The data from this study will facilitate further genetic analysis and research into the evolution of TGEV in pigs in Vietnam.

Immunogenicity of a novel inactivated canine adenovirus type 2 variant vaccine for dogs.

Purpose: The immunogenicity of vaccines containing the canine adenovirus (CAdV) type 2 (CAdV-2) variant has not yet been reported. We prepared a novel inactivated CAdV-2 variant vaccine using the CAV2232-41 strain, and evaluated its safety and immunogenicity in raccoon dogs. Materials and Methods: The growth kinetics of CAV2232-41 were determined using Madin-Darby Canine Kidney (MDCK) cells. The nucleotide sequences of CAV2232 and CAV2232-41 were determined by next-generation sequencing. To generate the CAdV-2 variant vaccine, CAV2232-41 propagated in the MDCK cells was inactivated with 0.1% formaldehyde. Two vaccines were prepared by blending inactivated CAV2232-41 with Cabopol and Rehydragel adjuvants. Safety and immunogenicity of the CAV2232C and CAV2232R vaccines were evaluated in guinea pigs. Safety and immunogenicity of the CAV2232C vaccine were also evaluated in raccoon dogs. The virus neutralizing antibody (VNA) titer against CAV2232-41 was measured in sera collected from immunized guinea pigs and raccoon dogs. Results: CAV2232-41 showed the highest viral titer on days 4-6 post-inoculation and had a deletion in the E3 gene, which was confirmed as a CAdV-2 variant. Guinea pigs inoculated with CAV2232C showed slightly higher VNA titers than those inoculated with CAV2232R 2 weeks after booster vaccination. Raccoon dogs immunized with the CAV2232C vaccine developed high mean VNA titers, while non-vaccinated raccoon dogs were antibody-negative. Conclusion: The CAV2232C vaccine is safe and induces a protective VNA titer in raccoon dogs.

16S rRNA metabarcoding for the identification of tick-borne bacteria in ticks in the Republic of Korea.

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.

Evaluation of acaricidal activity in entomopathogenic fungi for poultry red mite (Dermanyssus gallinae) control.

The poultry red mite (PRM), Dermanyssus gallinae, significantly impacts the health of egg-laying hens. Mites feed on the blood of infested chickens and have a great economic impact on the poultry industry. Chemical treatment of mites raises concerns about their resistance to miticides and residues in eggs and poultry. Biocontrol using entomopathogenic fungi is expected to be a chemical-free strategy for reducing PRM infestations. Therefore, the present study aimed to investigate the effects of various entomopathogenic fungal species collected in South Korea on the inhibition of PRM. Seventeen strains of six fungal species collected from various sources were used to evaluate acaricidal activity against PRM. The results showed that 16/17 strains had acaricidal properties against PRM, of which strains of Metarhizium anisopliae had the highest acaricidal activity. Mites treated with M. anisopliae CBNU 4-2 showed 100 % mortality 5 d after inoculation, followed by M. flavoviride var. pemphigi. The M. flavoviride var. pemphigi CBNU 1-1-1 showed 97.78 % mortality after 10 d of exposure to fungi. The mortality rate of PRM treated with other strains slowly increased and reached its highest value on the 14th day of inoculation. The results of this study provide information on the acaricidal activity of different entomopathogenic fungi against PRM. This information is important for the selection of fungal species for developing biocontrol methods for PRM treatment. These strains could be used for further evaluation of PRM treatment on chicken farms, or in combination with other methods, to increase PRM treatment efficiency.

First detection and prevalence of Apis mellifera filamentous virus in Apis mellifera and Varroa destructor in the Republic of Korea.

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.

Identification and pathogen detection of a Neocypholaelaps species (Acari: Mesostigmata: Ameroseiidae) from beehives in the Republic of Korea.

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.

Molecular Identification and Prevalence of the Mite Carpoglyphus lactis (Acarina: Carpoglyphidae) in Apis mellifera in the Republic of Korea.

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.

Prevalence and genome features of lake sinai virus isolated from Apis mellifera in the Republic of Korea.

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.

Current Status of Q Fever and the Challenge of Outbreak Preparedness in Korea: One Health Approach to Zoonoses.

Human Q fever, a zoonosis caused by Coxiella burnetii, presents with diverse clinical manifestations ranging from mild self-limited febrile illnesses to life-threatening complications such as endocarditis or vascular infection. Although acute Q fever is a benign illness with a low mortality rate, a large-scale outbreak of Q fever in the Netherlands led to concerns about the possibility of blood transfusion-related transmission or obstetric complications in pregnant women. Furthermore, a small minority (< 5%) of patients with asymptomatic or symptomatic infection progress to chronic Q fever. Chronic Q fever is fatal in 5-50% of patients if left untreated. In South Korea, Q fever in humans was designated as a notifiable infectious disease in 2006, and the number of Q fever cases has increased sharply since 2015. Nonetheless, it is still considered a neglected and under-recognized infectious disease. In this review, recent trends of human and animal Q fever in South Korea, and public health concerns regarding Q fever outbreaks are reviewed, and we consider how a One Health approach could be applied as a preventive measure to prepare for zoonotic Q fever outbreaks.

First identification of Tyrophagus curvipenis (Acari: Acaridae) and pathogen detection in Apis mellifera colonies in the Republic of Korea.

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.

Probiotic candidates for controlling Paenibacillus larvae, a causative agent of American foulbrood disease in honey bee.

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.

Large-Scale Application of Double-Stranded RNA Shows Potential for Reduction of Sacbrood Virus Disease in Apis cerana Apiaries.

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.

Tick Populations and Molecular Analysis of Anaplasma Species in Ticks from the Republic of Korea.

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.

Prevalence of honey bee pathogens and parasites in South Korea: A five-year surveillance study from 2017 to 2021.

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.

Molecular epidemiology of Theileria species in ticks and its potential threat to livestock in the Republic of Korea.

The purpose of this study was to investigate molecular epidemiology of Theileria spp. in ticks in Korea and assess their potential threat from wildlife animals to domestic animals. A total of 21152 hard ticks were collected from Chungcheong and Jeolla provinces of Korea from March to October 2021. Tick species were identified by microscopy and Theileria spp. were screened by nested PCR targeting 18S rRNA. Haemaphysalis spp. were the most abundant tick species, followed by H. longicornis, H. flava, Amblyomma testudinarium, and Ixodes nipponensis. Of the collected ticks, 6914 ticks (541 pools) were screened, and PCR showed 211 positive pools (39.0%; MIR: 3.05). The PCR and phylogenetic analysis identified two Theileria species, T. luwenshuni and Theileria sp., with T. luwenshuni (162/211, 76.78%; MIR: 2.34) being more abundant than Theileria sp. (36/211, 17.06%; MIR: 0.52); co-infection of the two species were noted (13/211, 6.16%; MIR: 0.19). Among the tick species, H. longicornis, especially nymphs, showed the highest prevalence. Regarding season, the highest prevalence was observed in May. Considering the tick and Theileria species identified in this study, H. longicornis nymph and cervine play a critical role in maintaining Theileria spp. in Korea and could be a potential threat to domestic animals, including deer and goats. In addition, there are significant correlations among tick distribution, region, season, and prevalence of Theileria.

Genotyping of Coxiella burnetii from Cattle by Multispacer Sequence Typing and Multiple Locus Variable Number of Tandem Repeat Analysis in the Republic of Korea.

Genotyping of Coxiella burnetii using multispacer sequence typing (MST) and multiple locus variable number tandem repeat analysis (MLVA) was conducted from infected animals for the first time in the Republic of Korea. C. burnetii was detected by real-time PCR, and followed by MST and MLVA genotyping. The result showed that detected C. burnetii all had the same MLVA genotype, 6-13-2-7-9-10 for markers MS23-MS24-MS27-MS28-MS33-MS34, respectively, and genotype group 61 for MST. The same genotypes were previously identified in Poland. Importantly, this MLVA type was detected in humans in France, suggesting that the Korean strain can also potentially cause Q fever in humans. MST and MLVA were very useful tools for analyzing the molecular epidemiology of C. burnetii and helpful for interpreting the epidemiological relationship between isolates from domestic and international resources.

The Gut Microbiota at Different Developmental Stages of Apis cerana Reveals Potential Probiotic Bacteria for Improving Honeybee Health.

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.

Toxoplasma gondii and Rickettsia spp. in ticks collected from migratory birds in the Republic of Korea.

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.

Comparison of the gut microbiome of sacbrood virus-resistant and -susceptible Apis cerana from South Korea.

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.

Utility of ultra-rapid real-time PCR for detection and prevalence of Rickettsia spp. in ticks.

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.