Detection and Genotypic Analysis of Anaplasma bovis and A. phagocytophilum in Horse Blood and Lung Tissue.

A clinical case of Anaplasma bovis was reported for the first time in our previous study (2019) in a horse, a nondefinitive host. Although A. bovis is a ruminant and not a zoonotic pathogen, it is responsible for persistent infections in horses. In this follow-up study, the prevalence of Anaplasma spp., including A. bovis, was assessed in horse blood and lung tissue samples to fully understand Anaplasma spp. pathogen distribution and the potential risk factors of infection. Among 1696 samples, including 1433 blood samples from farms nationwide and 263 lung tissue samples from horse abattoirs on Jeju Island, a total of 29 samples (1.7%) tested positive for A. bovis and 31 (1.8%) samples tested positive for A. phagocytophilum, as determined by 16S rRNA nucleotide sequencing and restriction fragment length polymorphism. This study is the first to detect A. bovis infection in horse lung tissue samples. Further studies are needed to clarify the comparison of sample types within cohorts. Although the clinical significance of Anaplasma infection was not evaluated in this study, our results emphasize the need to clarify the host tropism and genetic divergence of Anaplasma to enable the development of effective prevention and control measures through broad epidemiological studies.

Molecular Detection and Genetic Diversity of Blastocystis in Korean Dogs.

Blastocystis is a genus of unicellular heterokont parasites belonging to a group of organisms known as Stramenopiles, which includes algae, diatoms, and water molds. Blastocystis includes several species that habitat in the gastrointestinal tracts of organisms as diverse as humans, farm animals, birds, rodents, reptiles, amphibians, fish, and cockroaches. It is important to public health and distributed globally, but its prevalence in dogs in Korea has not been reported to date. Here, we collected 787 canine fecal samples and assessed Blastocystis infection by age, sex, region, season, and diarrhea symptoms. We determined Blastocystis subtypes using phylogenetic analyses based on 18S rRNA gene sequences. We identified, 10 Blastocystis positive samples (1.3%). A higher proportion of infected dogs was asymptomatic; however, infection rates did not significantly differ according to region, age, sex, and season. Phylogenetic analysis showed that the Blastocystis sp. identified belonged to 4 subtypes (STs), ST1, ST5, ST10, and ST14, thus revealed the genetic diversity of Blastocystis sp. in dogs Korean. This is first report on the presence of Blastocystis sp. in dogs Korean. This study revealed a lower infection rate than expected and differed from previous studies in STs. Further studies are warranted to observe the national infection status of Blastocystis in dogs and the genetic characteristics of this genus.

Distribution and genotypic analysis of Enterocytozoon bieneusi from wild boars in Korea.

Enterocytozoon bieneusi, an important microsporidian fungus, causes chronic diarrhea in humans and animals worldwide. Out of the 502 fecal samples from wild boars, 13 were positive for the E. bieneusi internal transcribed spacer region, with a prevalence of 2.6%. Six E. bieneusi genotypes, D, EbpC, and four novel KWB1-KWB4, were identified with zoonotic potential. Genotypes D (subgroup 1a) and EbpC (subgroup 1d) were first reported in Korean swine and Korea, respectively; KWB1-KWB4 (subgroup 1e) were most prevalent in this study. Because zoonotic genotypes have been identified, E. bieneusi transmission through wild boars must be closely monitored for proper prevention and treatment, despite their low prevalence. LAY SUMMARY: Enterocytozoon bieneusi is an important microsporidian fungus. Its sequences from wild boars were identified with zoonotic potential. Genotypes D and EbpC were first reported in Korean swine and Korea, respectively. E. bieneusi should be closely monitored to properly prevent and treat animals.

High Prevalence of Rickettsia raoultii and Associated Pathogens in Canine Ticks, South Korea.

We studied the prevalence of tickborne pathogens in canine ticks, South Korea, during 2010-2015. Results revealed a high prevalence of the emerging pathogen Rickettsia raoultii. Dog ticks may be maintenance hosts for tickborne pathogens, suggesting the need to continually evaluate the potential public health threat posed by R. raoultii-infected ticks.

Molecular Detection of Coxiella burnetii in Cattle on Ulleung Island, Korea: A Population-based Study with Four Years of Follow Up.

In a population-based study with 4 years of follow up, we evaluated the prevalence of Coxiella burnetii in cattle on Ulleung Island, Korea. In this study, the rates of C. burnetii infection in cattle on Ulleung Island were determined by PCR and were found to be 0.3-1.0% in the period 2011-2014. All 17 C. burnetii partial 16S rRNA gene sequences from PCR-positive cattle were identical and 2 geographic representatives were included in our analysis. The nucleotide sequences of the 2 samples showed high (98.4-100%) identity with C. burnetii sequences obtained from the GenBank. In this long-term tracking study, the number of cattle positive for C. burnetii on Ulleung Island was low. To prevent the transmission of C. burnetii on Ulleung Island, control strategy should include biosecurity improvement in surveillance, livestock management, administering suitable tests before purchasing animals to detect C. burnetii shedders, and restricting movements between herds.

Rapid Engineering of Foot-and-Mouth Disease Vaccine and Challenge Viruses.

There are seven antigenically distinct serotypes of foot-and-mouth disease virus (FMDV), each of which has intratypic variants. In the present study, we have developed methods to efficiently generate promising vaccines against seven serotypes or subtypes. The capsid-encoding gene (P1) of the vaccine strain O1/Manisa/Turkey/69 was replaced with the amplified or synthetic genes from the O, A, Asia1, C, SAT1, SAT2, and SAT3 serotypes. Viruses of the seven serotype were rescued successfully. Each chimeric FMDV with a replacement of P1 showed serotype-specific antigenicity and varied in terms of pathogenesis in pigs and mice. Vaccination of pigs with an experimental trivalent vaccine containing the inactivated recombinants based on the main serotypes O, A, and Asia1 effectively protected them from virus challenge. This technology could be a potential strategy for a customized vaccine with challenge tools to protect against epizootic disease caused by specific serotypes or subtypes of FMDV.IMPORTANCE Foot-and-mouth disease (FMD) virus (FMDV) causes significant economic losses. For vaccine preparation, the selection of vaccine strains was complicated by high antigenic variation. In the present study, we suggested an effective strategy to rapidly prepare and evaluate mass-produced customized vaccines against epidemic strains. The P1 gene encoding the structural proteins of the well-known vaccine virus was replaced by the synthetic or amplified genes of viruses of seven representative serotypes. These chimeric viruses generally replicated readily in cell culture and had a particle size similar to that of the original vaccine strain. Their antigenicity mirrored that of the original serotype from which their P1 gene was derived. Animal infection experiments revealed that the recombinants varied in terms of pathogenicity. This strategy will be a useful tool for rapidly generating customized FMD vaccines or challenge viruses for all serotypes, especially for FMD-free countries, which have prohibited the import of FMDVs.

Molecular detection of Anaplasma phagocytophilum-like Anaplasma spp. and pathogenic A. Phagocytophilum in cattle from South Korea.

Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis and tick-borne fever in domestic ruminants. Differential diagnosis of zoonotic and pathogenic tick-borne diseases like granulocytic anaplasmosis is important for the efficient implementation of control programs. Thus, the differentiation of pathogenic A. phagocytophilum from non-pathogenic A. phagocytophilum-like (APL) Anaplasma spp. is essential. Recent molecular analyses of APL revealed its distinct phylogenetic position from A. phagocytophilum. This study was conducted to detect A. phagocytophilum and genetically related strains in 764 cattle in South Korea using PCR and restriction fragment length polymorphism assays. APL clade A and A. phagocytophilum were identified in 20 (2.6%) and 16 (2.1%) cattle, respectively, with 16 cattle (2.1%) displaying co-infection. The 16S rRNA sequences of APL clade A were similar (98.3-99.9%) to those clustered in the APL clade A from eastern Asia. The A. phagocytophilum 16S rRNA sequence shared 98.6-100% identity to those of the A. phagocytophilum group. We used PCR to amplify the groEL and msp2 genes from the 20 samples positive for the 16S rRNA gene and found that 16 were positive for the groEL sequences in the APL clade A, which showed identity (82.8-84.4%) to those clustered in the APL clade A from Japan. Amplification of msp2 was unsuccessful. The co-infection results suggested sequence diversity in Anaplasma spp. Till date, both A. phagocytophilum and APL have been reported to be distributed separately in several animals throughout South Korea. This report is the first co-detection of A. phagocytophilum and APL in Korean cattle using molecular methods. Further studies are needed to provide additional molecular background and trace the evolutionary tree of Anaplasma species in animals and ticks.

Molecular Detection and Phylogenetic Analysis of Anaplasma phagocytophilum in Horses in Korea.

The identification and characterization of pathogenic and zoonotic tick-borne diseases like granulocytic anaplasmosis are essential for developing effective control programs. The differential diagnosis of pathogenic Anaplasma phagocytophilum and non-pathogenic A. phagocytophilum-like Anaplasma spp. is important for implementing effective treatment from control programs. The objective of the present study was to investigate the prevalence of Anaplasma spp. in horses in Korea by nucleotide sequencing and restriction enzyme fragment length polymorphism assay. Of the 627 horses included in the study, only 1 (0.2%) was infected with A. phagocytophilum. Co-infection with A. phagocytophilum- like Anaplasma spp. was not detected in the study. The 16S rRNA sequence of A. phagocytophilum was similar (99.5- 100%) to A. phagocytophilum 16S rRNA isolated from horses in other countries. PCR adapted to amplify A. phagocytophilum groEL and msp2 genes failed to generate amplicons, suggesting genetic diversity in these genes. This study is the first molecular detection of A. phagocytophilum in horses in Korea. Human granulocytic anaplasmosis and animal infection of A. phagocytophilum have been reported in Korea recently. Because of vector tick distribution, global warming, and the increase of the horse industry, horses should be considered as a potential reservoir for A. phagocytophilum, and cross infectivity should be evaluated even though a low prevalence of infection was detected in this study. Furthermore, continuous surveillance and effective control measures for A. phagocytophilum should be established to prevent disease distribution and possible transmission to humans.

Serological Detection of Antibodies against Anaplasma spp. in Cattle Reared in the Gyeongsangbuk-do, Korea.

Anaplasmosis is a tick-borne, non-contagious, zoonotic disease caused by Anaplasma spp., which include Anaplasma marginale, A. centrale, A. phagocytophilum, A. platys, A. ovis, and A. bovis. Recently, in Korea, the prevalence of Anaplasma spp. has been investigated in some animals, such as dogs, horses, goats, cats, and Korean water deer. In cattle, A. marginale is the most virulent species and regarded as the typical type of species. However, data on the seroprevalence of Anaplasma spp. in cattle in Korea during the last decade is limited. This study was designed to investigate the seroprevalence of bovine anaplasmosis in Korea. From 2010 to 2013, blood samples were collected from 568 cattle. Forty animals (7.0%) tested seropositive for Anaplasma spp. by cELISA. Despite that current bovine anaplasmosis seropositivity rate in the Gyeongsangbuk-do is lower than those in tropical countries, anaplasmosis needs to be regarded as a concerning disease. The identification of the specific Anaplasma species infecting cattle in this province requires additional molecular studies. Moreover, further monitoring and control programs for bovine anaplasmosis is required, and the information from this study will be beneficial to develop these programs.

Herd prevalence and genotypes of Coxiella burnetii in dairy cattle bulk tank milk in Gyeongsang provinces of South Korea.

Coxiella burnetii is the causative agent of the zoonotic Q fever, and its reservoirs include ticks and livestock, which are key sources of transmission to humans. Although there have been several studies on the prevalence of C. burnetii antibodies in dairy cattle bulk tank milk (BTM), there is a lack of information on the molecular detection of C. burnetii in BTM in South Korea. Thus, this study was designed to assess milk shedding of C. burnetii in BTM from dairy cattle herds. Among the 607 BTM samples collected from 41 counties in Gyeongsang provinces in 2015, 108 (17.8%) from 23 (56.1%) counties tested positive for C. burnetii by PCR. Because the 16S rRNA sequences of C. burnetii from all 108 PCR-positive samples were identical, two representative samples (BTM-GB-10 and BTM-GN-63) are described in this paper. These sequences showed high identity (96.9-100%) to other C. burnetii sequences deposited in GenBank. Phylogenetic analysis showed that these two sequences were clustered with existing C. burnetii strains. The relatively high prevalence rates of C. burnetii in BTM detected in this study suggest that C. burnetii is prevalent among dairy cattle herds in South Korea. Thus, implementation of continuous monitoring and control strategies for domestic animals is needed to prevent disease transmission and protect public health.

Detection and Genotyping of Coxiella burnetii in Pigs, South Korea, 2014-2015.

We assessed Coxiella burnetii prevalence and genotypes in pigs in South Korea during 2014-2015. Prevalence was low among 1,030 samples tested by ELISA and immunofluorescent assay and 1,124 samples tested by PCR. Despite this finding, possible transmission of C. burnetii from pigs to humans cannot be excluded.

Robust Protection against Highly Virulent Foot-and-Mouth Disease Virus in Swine by Combination Treatment with Recombinant Adenoviruses Expressing Porcine Alpha and Gamma Interferons and Multiple Small Interfering RNAs.

UNLABELLED: Because the currently available vaccines against foot-and-mouth disease (FMD) provide no protection until 4 to 7 days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is the application of antiviral agents. Combination treatment strategies have been used to enhance the efficacy of antiviral agents, and such strategies may be advantageous in overcoming viral mechanisms of resistance to antiviral treatments. We have developed recombinant adenoviruses (Ads) for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-αγ) as well as 3 small interfering RNAs (Ad-3siRNA) targeting FMDV mRNAs encoding nonstructural proteins. The antiviral effects of Ad-porcine IFN-αγ and Ad-3siRNA expression were tested in combination in porcine cells, suckling mice, and swine. We observed enhanced antiviral effects in porcine cells and mice as well as robust protection against the highly pathogenic strain O/Andong/SKR/2010 and increased expression of cytokines in swine following combination treatment. In addition, we showed that combination treatment was effective against all serotypes of FMDV. Therefore, we suggest that the combined treatment with Ad-porcine IFN-αγ and Ad-3siRNA may offer fast-acting antiviral protection and be used with a vaccine during the period that the vaccine does not provide protection against FMD. IMPORTANCE: The use of current foot-and-mouth disease (FMD) vaccines to induce rapid protection provides limited effectiveness because the protection does not become effective until a minimum of 4 days after vaccination. Therefore, during outbreaks antiviral agents remain the only available treatment to confer rapid protection and reduce the spread of foot-and-mouth disease virus (FMDV) in livestock until vaccine-induced protective immunity can become effective. Interferons (IFNs) and small interfering RNAs (siRNAs) have been reported to be effective antiviral agents against FMDV, although the virus has associated mechanisms of resistance to type I interferons and siRNAs. We have developed recombinant adenoviruses for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-αγ) as well as 3 small interfering RNAs (Ad-3siRNA) to enhance the inhibitory effects of these antiviral agents observed in previous studies. Here, we show enhanced antiviral effects against FMDV by combination treatment with Ad-porcine IFN-αγ and Ad-3siRNA to overcome the mechanisms of resistance of FMDV in swine.

Inactivation of foot-and-mouth disease virus by citric acid and sodium carbonate with deicers.

Three out of five outbreaks of foot-and-mouth disease (FMD) since 2010 in the Republic of Korea have occurred in the winter. At the freezing temperatures, it was impossible to spray disinfectant on the surfaces of vehicles, roads, and farm premises because the disinfectant would be frozen shortly after discharge and the surfaces of the roads or machines would become slippery in cold weather. In this study, we added chemical deicers (ethylene glycol, propylene glycol, sodium chloride, calcium chloride, ethyl alcohol, and commercial windshield washer fluid) to keep disinfectants (0.2% citric acid and 4% sodium carbonate) from freezing, and we tested their virucidal efficacies under simulated cold temperatures in a tube. The 0.2% citric acid could reduce the virus titer 4 logs at -20°C with all the deicers. On the other hand, 4% sodium carbonate showed little virucidal activity at -20°C within 30 min, although it resisted being frozen with the function of the deicers. In conclusion, for the winter season, we may recommend the use of citric acid (>0.2%) diluted in 30% ethyl alcohol or 25% sodium chloride solvent, depending on its purpose.

Reemergence of foot-and-mouth disease, South Korea, 2000-2011.

Five outbreaks of foot-and-mouth disease have occurred in South Korea during 2000-2011. Macro-analysis of these outbreaks showed a correlation with outbreaks in countries in eastern Asia. Genetic analyses of food-and-mouth disease viruses in South Korea showed a correlation with viruses that are prevalent in neighboring countries.

Molecular survey of Toxoplasma gondii B1 gene in pigs from various localities in Korea.

Toxoplasma gondii, a common protozoan parasite, poses significant public health risks due to its potential to cause toxoplasmosis in humans and can be contracted from pigs, which are considered its critical intermediate host. The aim of this study is to evaluate the prevalence of T. gondii in slaughtered pigs for human consumption, emphasizing the zoonotic implications and the need for improved biosecurity and monitoring practices in pig farming. A total of 1,526 pig samples (1,051 whole blood samples and 384 lung tissue samples from the local slaughterhouse and 91 aborted fetus samples from local farms) were collected throughout the whole country of Korea in 2020. Among them, 6 (0.4%) were found to be infected with T. gondii by nested PCR. When compared by sample type, the prevalence of T. gondii was significantly higher in the aborted fetus samples (2.2%, 2/91) than in the blood (0.3%, 3/1,051) and lung tissue samples (0.3%, 1/384). The B1 gene sequence of T. gondii was similar (97.9-99.8%) to that of the other T. gondii isolates. This study represents the first molecular genotyping survey of T. gondii in the lung tissue of fattening pigs and aborted fetuses in Korea. Our findings indicated the importance of adopting preventive measures including the implementation of rigorous farm hygiene protocols and the promotion of public awareness about the risks of consuming undercooked pork. By addressing the gaps in current control strategies and encouraging the One Health approach, this study contributes to the development of more effective strategies to mitigate the transmission of T. gondii from pigs to humans, ultimately safeguarding public health.

Tick-borne pathogens in Mongolian ticks: The high prevalence of Rickettsia raoultii and its public health implications.

In Mongolia, a substantial segment of the population is engaged in pastoralism, leading to frequent interactions with livestock and heightened exposure to tick-infested environments. Consequently, ticks and the endemic tick-borne pathogens (TBPs) they harbor present significant health threats. In May 2021, we conducted a study to evaluate the prevalence of 9 TBPs in ticks collected from four regions in Mongolia: Uvurkhangai, Tuv, Bayan-Ulgii, and Khentii. The pathogens surveyed included Anaplasma, Rickettsia, Bartonella, Borrelia, Ehrlichia, Babesia, Toxoplasma, Theileria, and lumpy skin disease virus. Molecular analysis of 1142 ticks revealed that, although the majority of TBPs were not detected, 89.1 % of the ticks were positive for Rickettsia. Genetic characterization using the 16S rRNA and gltA genes identified the pathogen identified the pathogen Rickettsia raoultii. Species identification indicated a predominance of Dermacentor nuttalli (70.0 %) and Dermacentor silvarum (30.0 %). These findings highlight the extensive prevalence of R. raoultii in Mongolia and underscore the urgent need for heightened awareness and preventive measures due to the high level of interaction between livestock and humans. The genetic similarity of R. raoultii sequences to those found in neighboring countries suggests potential cross-border transmission, underscoring the importance of conducting similar research in surrounding regions. This study advances our understanding of the epidemiology of tick-borne diseases in Mongolia and can inform public health strategies aimed at mitigating the risks associated with these infections.

Red ginseng extract inhibits lipopolysaccharide-induced platelet-leukocyte aggregates in mice.

Background: Platelet-leukocyte aggregates (PLAs) play important roles in cardiovascular disease and sepsis. Red ginseng extract (RGE) has been well-studied for its antiplatelet and anti-inflammatory activities. However, the potential inhibitory effects of RGE on PLA have not been investigated. Methods: Six-week-old ICR mice were given oral gavage of RGE for 7 days, followed by an intraperitoneal injection of 15 mg/kg of lipopolysaccharide. Mice were euthanized 24 h later, and blood samples were collected for further analysis. Flow cytometry was utilized to sort populations of PLAs and platelet-neutrophil aggregates (PNAs). By using confocal microscopy, PNAs were validated. Morphological changes in platelets and leukocytes were visualized with scanning electron microscopy. Expressions of tissue factor (TF) and platelet factor 4 (PF4) were investigated using enzyme-linked immunosorbent assay. Results: Populations of activated platelets, PLAs and PNAs, were significantly increased with LPS-induction. Treatment with 200 and 400 mg/kg of RGE decreased platelet activation. Moreover, the populations of PLAs and PNAs were reduced. PNAs were visible in the blood of septic mice, and this was attenuated by treatment with 400 mg/kg of RGE. Morphologically, sepsisinduced platelet activation and fibrin formation in the blood. This was reduced with RGE treatment. Sepsis-induced increase in the plasma levels of TF and PF4 was also reduced with RGE treatment. Conclusion: This study shows that RGE is a potential therapeutic that reduces the activation of platelets and targets PLA and PNA formation. Detailed inhibitory mechanisms of RGE should be studied.

Molecular analysis of Rickettsia spp. and related tick-borne pathogens detected in dogs in Korea.

Ticks are widespread in nature and serve as primary vectors for several tick-borne pathogens (TBPs). Ticks and TBPs cause considerable harm to humans and animals and have emerged as a major global public health concern. Domestic dogs are the major reservoirs of zoonotic agents owing to their constant interaction with humans. This study aimed to assess the prevalence and risk factors of canine TBPs, such as Rickettsiales, Coxiella burnetii, hepatozoa, and Borrelia spp., using molecular analyses. A total of 906 dogs were examined and 4 TBPs were identified: Anaplasma phagocytophilum (5; 0.6%), Hepatozoon canis (9; 1.0%), Candidatus Rickettsia longicornii (2; 0.2%), and Rickettsia tamurae (1; 0.1%). Ehrlichia spp., C. burnetii, and Borrelia spp. were not detected. To the best of our knowledge, this is the first study to perform a phylogenetic analysis of Candidatus R. longicornii and R. tamurae in dogs. These findings can help determine the potential public health risks by enhancing our understanding of the geographical and vector distributions of TBPs in Korea.

Molecular epidemiology of Toxoplasma gondii in cattle in Korea.

Toxoplasmosis is a major public health concern, with raw or undercooked meat being the primary source of human infection. Knowledge regarding the molecular epidemiology of Toxoplasma gondii in cattle destined for human consumption in Korea is lacking. The present study aimed to genetically characterize the infectious strains of T. gondii. Overall, 455 cattle blood samples from 84 farms in the Gyeongnam-do (Province) were randomly collected in 2017. Nested PCR analysis revealed that only 3 (0.7%) samples were infected with T. gondii. The B1 gene sequence of T. gondii was observed to be similar (97.3-99.6%) to that of other T. gondii isolates. This is the first study to perform the molecular detection of T. gondii in cattle in Korea. Although the prevalence of infection was low, our findings suggest that cattle present a potential public health issue. It may be crucial to recognize the importance of T. gondii infection in cattle meat.

Sudden death caused by Aeromonas hydrophila in a Crab-eating Macaque (Macaca fascicularis) associated with captivity stress.

Aeromonas hydrophila is a facultative anaerobic gram-negative bacterium regarded as an opportunistic pathogen in animals. A 17-year-old female crab-eating macaque (Macaca fascicularis) died after experiencing anorexia and depression for several days. The carcass was severely emaciated, and the sternum was exposed under subcutaneous lesions in the thorax. Many abnormal pathological lesions were found, including tracheal inflammation, pulmonary inflammatory emphysema, yellowish discoloration of the liver, enlargement of the gall bladder, necrosis of the heart, congested bilateral kidneys, and enlargement of the adrenal glands. The stomach was empty, mucosal ulcerations were found, and the duodenum was congested. Giemsa staining revealed rod-shaped organisms in the whole blood smear and major organs, which were identified as A. hydrophila. The animal had experienced stress, and decreased immune system function possibly contributed to the infection.