The First Identification of Cryptosporidium parvum Virus-1 (CSpV1) in Hanwoo (Bos taurus coreanae) Calves in Korea.

Cryptosporidium is an obligate coccidian parasite that causes enteric diseases in bovine species. A double-stranded RNA virus associated with C. parvum oocysts, Cryptosporidium parvum virus-1 (CSpV1), has been characterized. However, the relationship between the abovementioned coccidian parasite and the virus has not been studied in the context of the known clinical outcomes. This study aimed to characterize the prevalence and molecular traits of CSpV1 in diarrheal feces of Hanwoo (Korean indigenous cattle) calves. Of the 140 fecal samples previously tested for C. parvum, which were obtained from Hanwoo calves aged 60 days, 70 tested positive and 70 tested negative. These samples were included in this study. By using the polymerase chain reaction (PCR) analysis targeting the RdRp gene of CSpV1, we detected CSpV1 in 28 samples (20.0%), with infection rates of 31.4% (22/70) in C. parvum-positive and 8.6% (6/70) in C. parvum-negative samples. CSpV1 samples detected in the same farm were clustered together. To the best of our knowledge, this is the first study to report the prevalence and molecular characteristics of CSpV1 in Hanwoo calves in the Republic of Korea, providing important insights into the relationship between C. parvum and CSpV1 in bovine hosts.

Development of a one-step reverse transcription-quantitative polymerase chain reaction assay for the detection of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is an important disease that severely affects the swine industry and, therefore, warrants rapid and accurate diagnosis for its control. Despite the progress in developing diagnostic tools, including polymerase chain reaction (PCR)-based methods such as reverse transcription quantitative PCR (RT-qPCR) to diagnose PRRSV infection, its diagnosis at the genetic level is challenging because of its high genetic variability. Nevertheless, RT-qPCR is the easiest and fastest method for diagnosing PRRSV. Therefore, this study aimed to develop an RT-qPCR assay for rapid and accurate diagnosis of PRRSV by encompassing all publicly available PRRSV sequences. The developed assay using highly specific primers and probes could detect up to 10 copies of PRRSV-1 and -2 subtypes. Furthermore, a comparison of the performance of the developed assay with those of two commercial kits widely used in South Korea demonstrated the higher efficiency of the developed assay in detecting PRRSV infections in field samples. For PRRSV-1 detection, the developed assay showed a diagnostic agreement of 97.7% with the results of ORF5 sequencing, while for commercial kits, it showed 95.3% and 72.1% agreement. For PRRSV-2, the developed assay showed a diagnostic agreement of 97.7%, whereas the commercial kits showed 93% and 90.7% agreement. In conclusion, we developed an assay with higher accuracy than those of the tested commercial kits, which will contribute markedly to global PRRSV control.

Severe Fever with Thrombocytopenia Syndrome Virus in Ticks in the Republic of Korea.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a zoonotic, tick-borne RNA virus of the genus Bandavirus (Family Phenuiviridae), mainly reported in China, Japan, and the Republic of Korea (Korea). For the purpose of this study, a total of 3,898 adult and nymphal ticks of species Haemaphysalis longicornis (94.2%), Haemaphysalis flava (5.0%), Ixodes nipponensis (0.8%), and 1 specimen of Ixodes ovatus, were collected from the Deogyusan National Park, Korea, between April 2016 and June 2018. A single-step reverse transcriptase-nested PCR was performed, targeting the S segment of the SFTSV RNA. Total infection rate (IR) of SFTSV in individual ticks was found to be 6.0%. Based on developmental stages, IR was 5.3% in adults and 6.0% in nymphs. The S segment sequences obtained from PCR were divided into 17 haplotypes. All haplotypes were phylogenetically clustered into clades B-2 and B-3, with 92.7% sequences in B-2 and 7.3% in B-3. These observations indicate that the Korean SFTSV strains were closer to the Japanese than the Chinese strains. Further epidemiological studies are necessary to better understand the characteristics of the Korean SFTSV and its transmission cycle in the ecosystem.

The prevalence of causative agents of calf diarrhea in Korean native calves.

Infectious calf diarrhea is one of the most significant diseases of neonatal calves. This study is conducted to identify the prevalence of pathogens in calf diarrhea for 2 years. A total of 544 feces samples from Korean native beef calves were obtained to investigate selected seven pathogens causing calf diarrhea: bovine rotavirus, bovine coronavirus, Cryptosporidium parvum, bovine viral diarrhea virus, Eimeria species, Escherichia coli K99, and Salmonella species. The presence of diarrhea, the number and species of detected pathogens, and the calves' ages were analyzed using various statistical methods depending on the case. Of the 544 calves, 340 calves (62.5%) had normal feces and 204 calves (37.5%) had diarrhea. The presence of pathogens was significantly associated with diarrhea (p < 0.01) and fecal scores and the number of detected pathogens showed a significant linear trend (p < 0.001). Of the 7 target pathogens, 6 were detected in samples, but only C. parvum (p = 0.001) and bovine rotavirus (p < 0.001) were found at significantly higher rates in diarrheic calves than in non-diarrheic calves. Only Eimeria spp. showed a significant linear trend between the detection rate of the pathogen and the age groups (p < 0.05).

First detection of Borrelia and Rickettsia species from Ornithodoros ticks in the Republic of Korea.

Ticks are considered important vectors among arthropods and are linked to serious medical and veterinary health problems. In this study, we investigated tick-borne pathogens (TBPs) of Ornithodoros (Carios) sawaii and a newly identified Ornithodoros species from migratory bird nests in the uninhabited islands of the Republic of Korea (ROK). Ticks were collected from seabird nests with soil using a Tullgren funnel. Polymerase chain reaction (PCR) was performed using specific primer sets targeting genes of Borrelia spp., Rickettsia sp., Anaplasma phagocytophilum, Anaplasma bovis, and Bartonella spp. for molecular identification of TBPs, and two pathogens, Borrelia sp. and Rickettsia sp. were detected via PCR. Sequence data were analyzed and a phylogenetic analysis was conducted using the maximum-likelihood method in MEGA v.7. The detection rate of Borrelia sp. in O.(C.) sawaii was 6.8 % (5/74), and that of Rickettsia sp. in O. sawaii and the newly identified Ornithodoros species. was 36.5 % (27/74). Sequencing analysis revealed that the 16S ribosomal (r) RNA and flagellin genes of Borrelia sp., and the citrate synthase (gltA) and 17-kDa antigen gene of Rickettsia sp. were closely phylogenetically related to those of Borrelia turicatae and Rickettsia asembonensis. This is the first report identifying Borrelia sp. and Rickettsia sp. from O. sawaii, and Rickettsia sp. from the newly identified Ornithodoros species in the ROK, and these results imply that soft ticks (O. sawaii, and the newly identified Ornithodoros species) may function as pathogen carriers with important implications for public health throughout their distribution areas in Asia.

First report of Newly Identified Ornithodoros Species in the Republic of Korea.

Ticks and tick-borne diseases are important issues worldwide because of their effects on animal and human health. The genus Ornithodoros, which is included in the family Argasidae, is typically associated with wild animals, including seabirds. In this study, samples from the nests of seabirds and surrounding soil were collected to investigate Ornithodoros spp. from 9 uninhabited islands in the western, eastern, and southern parts of Korea from April 2017 to October 2018. The islands are known as the breeding places of migratory and resident birds. Ticks were collected from soil and nest material of seabirds using a Tullgren funnel and identified using 16S rRNA and the cytochrome c oxidase 1 gene (COI), and host animals of soft ticks were identified using the mitochondrial DNA cytochrome b gene by a polymerase chain reaction. In the sequence identity of the 16S rRNA gene fragment of Ornithodoros sp., Ornithodoros sawaii was identified as the closest homologous sequence, and the new Ornithodoros sp. was newly identified. We found that the newly identified Ornithodoros sp. in the Republic of Korea was located in uninhabited islands used as breeding places by the black-tailed gull, Larus crassirostris.

Evaluation of the efficacy of ivermectin against Theileria orientalis infection in grazing cattle.

BACKGROUND: Raising cattle on pastures is known to be beneficial for animal welfare and cost reduction. However, grazing is associated with the risk of contracting tick-borne diseases, such as theileriosis. Here, the efficacy of ivermectin against these diseases and associated clinical symptoms were evaluated. RESULTS: A total of 68 cattle from a grazing cattle farm were selected and divided into two groups: the control group (17 cattle) with no preventive treatment and the ivermectin-treated group (51 cattle) in which cattle were treated with pour-on ivermectin prior to grazing. The infection rates of Theileria orientalis and the red blood cell (RBC) profile (e.g., RBC count, hematocrit value, and hemoglobin concentration) were compared in the spring (before grazing) and summer (during grazing) between the two groups. Based on PCR amplification of the major piroplasm surface protein (MPSP) gene, 12 cattle were positive for T. orientalis infection. Phylogenetic analysis revealed that the isolates identified in this study consisted of three MPSP types (1, 2, and 7). The T. orientalis infection rate in the control group during grazing was 3-fold higher than that in the ivermectin-treated group. Moreover, differences in RBC parameters during grazing were greater in the control group than in the ivermectin-treated group. In particular, the hematocrit value was significantly reduced in the control group. CONCLUSIONS: The results of this study demonstrated that ivermectin had protective effects against T. orientalis infection and RBC hemolysis in grazing cattle.

Epidemiological Investigation of Tick Species from Near Domestic Animal Farms and Cattle, Goat, and Wild Boar in Korea.

This study aimed to investigate the tick species and give background for tick-borne investigations in Korea. Ticks were collected from the area within 2 km radius of the 4 domestic animal farms, where they were located in mountainous areas and raising animals on pasture, and from animal bodies in 2014 and 2015. In total, 7,973 nymphal and adult ticks were collected from the farms - 7,758 Haemaphysalis longicornis, 198 Haemaphysalis flava, and 17 Ixodes nipponensis, and 1,763 were collected from animals - 729 H. longicornis from cattle; 569 H. longicornis from goats; and 297 H. longicornis, 118 H. flava, 1 I. nipponensis, and 49 Amblyomma testudinarium from wild boars. As more species of ticks were collected from wild boars than domesticated animals and their habitats, various animal hosts should be considered while investigating tick species.

Acute phase response in bovine coronavirus positive post-weaned calves with diarrhea.

Bovine coronavirus (BCoV) is associated with severe diarrhea in calves, winter dysentery in adult cattle, and respiratory diseases in cattle of all ages. This study aimed to investigate the relationship between white blood cell counts and haptoglobin (Hp) and serum amyloid A (SAA) levels in post-weaned calves with diarrhea caused by BCoV and those that recovered from diarrhea. Blood and fecal samples were collected twice from the same animals; 17 post-weaned calves with diarrhea (first) and 15 post-weaned calves that recovered from diarrhea (second). Real-time polymerase chain reaction revealed that all 17 fecal samples from post-weaned calves with diarrhea and one out of 15 from diarrhea-recovered calves were positive for BCoV and negative for Cryptosporidium spp., Escherichia coli K99, Salmonella spp., bovine rotavirus, and bovine viral diarrhea virus. No Eimeria oocysts were detected using the flotation method. In comparison with post-weaned calves with diarrhea, in diarrhea-recovered calves, the lymphocyte count was significantly higher (P = 0.018), and the monocyte count was significantly lower (P = 0.001); however, the number of monocytes was still high. Post-weaned calves with diarrhea had a significantly higher Hp concentration (P < 0.001) compared with diarrhea-recovered calves. The results indicated that increased Hp concentration and monocytosis but not SAA may be associated with diarrhea caused by BCoV. The present study suggests that the monitoring of Hp concentration and monocyte count is useful in the diagnosis of post-weaned calves with diarrhea caused by BCoV in this field.

Severe Fever with Thrombocytopenia Syndrome Virus in Dogs, South Korea.

Of 103 serum samples collected from dogs in South Korea, 3 (2.9%) were positive for severe fever with thrombocytopenia syndrome virus (SFTSV) and 22 (21.4%) were positive for antibodies against SFTSV. A dog-derived isolate of SFTSV clustered with many South Korea SFTSV strains in the Japanese clade.

Prevalence of Severe Fever with Thrombocytopenia Syndrome Virus in Ticks Collected from National Parks in Korea.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral disease in East Asian countries, including China, Japan, and the Republic of Korea (ROK). The causative agent of SFTS is the SFTS virus (SFTSV), which is transmitted by ticks. To investigate the prevalence of SFTSV in the ROK, a total of 9744 ticks were collected from vegetation in five national parks between July and November 2015. Of the collected adult and nymph ticks, Haemaphysalis longicornis (68.44%) was the most abundant, followed by Haemaphysalis flava (29.66%), Ixodes nipponensis (1.56%), and Amblyomma testudinarium (0.34%). Collected larval ticks were of the genera Haemaphysalis (99.61%) and Ixodes (0.39%). One-step RT-PCR and nested PCR were used to detect SFTSV-specific genes from each individual adult and nymph tick and pooled larval ticks. SFTSV was detected in 4.77% (48/1006) in H. longicornis, 1.15% (5/436) in H. flava, 0% (0/23) in I. nipponensis, and 20% (1/5) in A. testudinarium. The infection rate of SFTSV in adult and nymph ticks was 3.61%. The prevalence of SFTSV in adult and nymph ticks was relatively high, compared with previous reports. In larval ticks, the minimum infection rate was 0.31%. SFTSV was detected in ticks collected from both trail and nontrail areas in the national parks, and up to 800 meters above sea level. The sequences obtained showed 99.4-99.7% homology with SFTS virus S segment sequences from Chinese and Japanese ticks.

Molecular Detection of Severe Fever with Thrombocytopenia Syndrome Virus in Korean Domesticated Pigs.

BACKGROUND: Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus in China, Japan, and Korea. Although the incidence of SFTS has been increasing annually since the first SFTSV case was reported in Korea, the prevalence of SFTSV in Korean livestock has not conducted. The purpose of this study was to investigate the prevalence of SFTSV in domesticated pigs (Sus scrofa domesticus) throughout Korea. METHODS: A total of 240 sera were collected from 30 domesticated pigs in eight provinces. To detect SFTSV, one-step reverse transcription RT nested PCR was conducted using SFTSV genome-specific primer sets. The amplified PCR products were cloned and then sequenced. RESULTS: Four of 240 (1.7%) pig sera tested positive for SFTSV through one-step RT nested PCR. Two of the four obtained sequences were included in the Japanese clade, whereas the other two sequences were related to the Chinese clade based on phylogenetic analysis. Moreover, the obtained SFTSV sequences from this study were distinct from human SFTSV strains. CONCLUSIONS: This study is the first to attempt the identification of SFTSV sequences from domesticated pigs and the first molecular detection of SFTSV in Korean livestock. Our findings indicate that a new subclade of SFTSV-different from that in humans-may be present in domesticated pigs; the surveillance of SFTSV in livestock is required to better understand the life cycle of SFTSV.

Prevalence of severe fever with thrombocytopenia syndrome virus in black goats (Capra hircus coreanae) in the Republic of Korea.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen in China, Japan, and the Republic of Korea (ROK). The aim of this study was to investigate the prevalence of SFTSV antigens and anti-SFTSV antibodies in black goats (Capra hircus coreanae) throughout the ROK. Sera were collected from 737 black goats in nine provinces in the ROK. Eighteen of 737 (2.4%) goat sera were positive for SFTSV on one-step reverse transcription nested polymerase chain reaction. The amplified 346-bp S segments of SFTSV sequences were classified into three genotypes (BG1, BG2, and BG3), and were included in the Japanese clade rather than the Chinese clade, based on phylogenetic analysis. Forty-three of 624 (6.9%) serum samples were seropositive for anti-SFTSV antibodies on enzyme-linked immunosorbent assay analysis. This study is the first to examine the molecular prevalence of SFTSV in goats and the first to perform serological detection of anti-SFTSV antibodies in livestock in the ROK. Moreover, the results indicate that SFTSV is widely distributed in goats and that additional monitoring for SFTSV is needed in livestock in the ROK.

Identification of zoonotic Giardia duodenalis in Korean native calves with normal feces.

Giardia duodenalis is an important zoonotic protozoan parasite that infects humans and other animals. This study was conducted to investigate the prevalence and molecular characterization of G. duodenalis infection in the feces of normal and diarrheic pre-weaned calves aged 1-60 days. Fecal samples were collected from 90 Korean native calves in 7 different regions in the central region of the Republic of Korea (ROK). Of the 90 fecal samples, 9 (10%, 9/90) were positive for G. duodenalis by PCR using the ß-giardin gene. Phylogenetic analysis identified G. duodenalis assemblages A and E in the farms examined and G. duodenalis with both assemblage A and E was detected in one farm. Assemblage A was detected in calves with normal feces, while assemblage E was frequently identified in calves with diarrhea. This is the first report of the identification of assemblage A in pre-weaned calves with normal feces in the ROK. These results suggest that G. duodenalis infection is closely associated with calf age and management systems including overcrowding, age-mixed grouping, and hygiene. Thus, giardiasis in calves is an important disease because of its zoonotic significance and should be screened in young animals. Further studies are necessary to elucidate the transmission routes of zoonotic G. duodenalis assemblages.

First report of Anaplasma phagocytophilum infection in Holstein cattle in the Republic of Korea.

Global warming has increased the incidence and risk of tick-borne diseases in domestic animals and humans in the Republic of Korea (ROK). In this study, we investigated the prevalence of Anaplasma phagocytophilum in Holstein cattle (n = 214) in the ROK using specific PCR assays. A. phagocytophilum infection was detected in only two animals (0.93%, 2/214). Our findings showed that PCR assay using the 16S rRNA gene, but not groEL, was suitable for detection of A. phagocytophilum in cattle. Phylogenetic analysis based on the16S rRNA gene showed that A. phagocytophilum was divided into two clades. Clade 1 included Korean isolates, such as those from dogs, cats, Korean water deer, and ticks, while A. phagocytophilum identified in Holstein cattle formed clade 2. Our results suggest that there is genetic variability among isolates of A. phagocytophilum circulating in the ROK. This is the first study to report A. phagocytophilum infection in Holstein cattle in the ROK. As A. phagocytophilum has zoonotic potential, additional epidemiological studies are needed to investigate the prevalence and genetic characterization of A. phagocytophilum from different regions and hosts.

Molecular detection of Anaplasma bovis in Holstein cattle in the Republic of Korea.

Anaplasmosis is a tick-borne infectious disease that affects both human and animal health. This study was performed to characterize and investigate the prevalence of infection with Anaplasma bovis in Holstein cattle originating from two regions in the Republic of Korea (ROK). Blood samples (n = 151; 80 from Namwon and 71 from Jeju Island) were analyzed by polymerase chain reaction, and the prevalence of A. bovis infection was compared before and after grazing. In Namwon, A. bovis infection was not detected, while in the Jeju Island, A. bovis infection was detected in three of 13 animals after grazing. Phylogenetic analysis revealed that the A. bovis isolates had homology (97.1-99.7%) with a Korean spotted deer (Cervus nippon) isolate and Haemaphysalis longicornis tick isolates identified in the ROK. A. bovis infection has not previously been diagnosed in cattle in the ROK. This study shows that A. bovis infection in the Jeju Island is closely related to grazing.

Molecular Detection of Anaplasma, Bartonella, and Borrelia theileri in Raccoon Dogs (Nyctereutes procyonoides) in Korea.

Anaplasmosis, cat-scratch disease, and Lyme disease are emerging vector-borne infectious diseases in Korea. Although the prevalence of vector-borne pathogens (VBPs) in domestic animals and vector arthropods has been documented, there is limited information on the presence of VBPs in wild animals. The raccoon dog (Nyctereutes procyonoides), a wild canid found in East Asia and Europe, represents a potential wildlife reservoir for zoonotic diseases. To investigate the prevalence of VBPs in raccoon dogs, 142 carcasses and 51 blood samples from captured raccoon dogs were collected from 2003 to 2010 and from 2008 to 2009, respectively, in Korea. In addition, 105 Haemaphysalis flava (14 larvae, 43 nymphs, 32 males, and 16 females) and nine Haemaphysalis longicornis (all female) were collected from three raccoon dogs. Samples of the spleen and blood were tested for the presence of VBPs by using nested polymerase chain reaction. Among the samples collected from 193 raccoon dogs and 114 ticks, two samples were positive for Anaplasma phagocytophilum, four for Anaplasma bovis, two for Borrelia theileri, and two for Bartonella henselae. To the best of our knowledge, this study is the largest survey of raccoon dogs aimed at the analysis of VBPs in this species. Moreover, the present study represents the first identification of A. phagocytophilum, B. henselae, and B. theileri in raccoon dogs in their native habitat (East Asia).

Hematological Changes Associated with Theileria orientalis Infection in Korean Indigenous Cattle.

Tick-borne pathogens can cause serious problems in grazing cattle. However, little information is available on tick-mediated diseases in cattle grazing on mountains. Thus, this study aimed to understand the potential problems related to tick-borne diseases in grazing cattle through the investigation of prevalent tick-transmitted infections, and their associated hematological changes, in terms of season and grazing type in Korean indigenous cattle (=Hanwoo). Hanwoo cattle from 3 regions of the Republic of Korea (=Korea) were either maintained indoors or placed on grassy mountains from spring to fall of 2014 and 2015. Cattle that grazed in mountainous areas showed a greater prevalence of tick-borne infections with an increased Theileria orientalis infection rate (54.7%) compared to that in non-grazing cattle (16.3%) (P<0.001). Accordingly, the red blood cell (RBC) count and hematocrit (HCT) values of grazing cattle were significantly lower than those of non-grazing cattle throughout the season (P<0.05). Moreover, RBC, hemoglobin (Hb), and HCT of T. orientalis-positive group were significantly lower than those of T. orientalis-negative group (P<0.05). T. orientalis is a widespread tick-borne pathogen in Korea. Grazing of cattle in mountainous areas is closely associated with an increase in T. orientalis infection (RR=3.4, P<0.001), and with consequent decreases in RBC count and HCT. Thus, these findings suggest that the Hanwoo cattle in mountainous areas of Korea are at a high risk of infection by T. orientalis, which can lead to hematological alterations. This study highlights the necessity of preventive strategies that target T. orientalis infection.

Pathogenic genotype of major piroplasm surface protein associated with anemia in Theileria orientalis infection in cattle.

Serious disease outbreaks in cattle caused by Theileria orientalis have emerged in the Asia-Pacific region. Genetic variables of the major piroplasm surface protein (MPSP) expressed on the surface of the piroplasm inside T. orientalis-infected erythrocytes are considered to be associated with variation in the pathogenicity of T. orientalis. Our study describes the clinically relevant MPSP types associated with anemia in Theileria-infected cattle. These results revealed that MPSP expression plays an important role in hematological alterations in Theileria-infected cattle, and that MPSP type 1 is strongly associated with bovine anemia, which can be a potential target for the prevention of bovine theileriosis.

Identification of Tick Species Collected from Wild Boars and Habitats of Wild Boars and Domestic Pigs in the Republic of Korea.

Tick is one of the most important arthropods in the transmission of vector-borne diseases. In this study, we investigated the abundance and species of ticks associated with swine and their habitats to assess the risk of spread of tick-borne diseases in host species, such as wild boars. Ticks were collected from 24 grazing or traditionally reared domestic pig farms and 8 habitats of wild boars in 8 provinces and 1 city in the Republic of Korea, by using the dragging and flagging methods. Ticks were also collected directly from 49 wild boars by using fine forceps. A total of 9,846 hard ticks were collected, including 4,977 Haemaphysalis longicornis, 4,313 Haemaphysalis flava, 508 Ixodes nipponensis, 1 Ixodes turdus, and 47 Amblyomma testudinarium. A total of 240 hard ticks were collected from 49 wild boars, including 109 H. flava, 84 H. longicornis, and 47 A. testudinarium. A total of 578 hard ticks were collected from areas around domestic pig farms. Only 2 hard tick species, 546 H. longicornis and 32 H. flava, were collected from these areas. A total of 9,028 hard ticks were collected from wild boars of 8 habitats, including 4,347 H. longicornis, 4,172 H. flava, 508 I. nipponensis, and 1 I. turdus. A. testudinarium was collected only from wild boars, and I. nipponensis and I. turdus were collected only from the habitats of wild boars.