Evaluation of haematological parameters in haemolytic anaemia caused by tick-borne pathogens in grazing cattle.

BACKGROUND: No tick-borne pathogens (TBPs) causing haemolytic anaemia in cattle have been reported, except Theileria orientalis and complete blood count (CBC) profile is the only haematological parameter to determine the severity of regenerative haemolytic anaemia. OBJECTIVES: To identify the causative agents of TBP-induced haemolytic anaemia and determine haematological parameters that indicate haemolytic anaemia in grazing cattle. METHODS: Eighty-two Korean indigenous cattle (Hanwoo) were divided into two groups: grazing (n = 67) and indoor (n = 15) groups. CBC and serum biochemistry were performed. PCR was conducted using whole blood-extracted DNA to investigate the prevalence of TBPs. RESULTS: TBP-induced haemolytic anaemia was observed in the grazing group. In grazing cattle, co-infection (43.3%, 29/67) was most frequently detected, followed by T. orientalis (37.6%, 25/67) and Anaplasma phagocytophilum infections (1.5%, 1/67). In indoor cattle, only co-infection (20%, 3/15) was identified. Grazing cattle exhibited regenerative haemolytic anaemia with marked monocytosis, mild neutropenia, and thrombocytopenia. According to grazing frequency, the 1st-time grazing group had more severe anaemia than the 2nd-time grazing group. Elevations in indirect bilirubin and L-lactate due to haemolytic anaemia were identified, and correlations with the respective markers were determined in co-infected grazing cattle. CONCLUSIONS: Quantitative evaluation of haematocrit, mean corpuscular volume, and reticulocytes (markers of regenerative haemolytic anaemia in cattle) was performed for the first time. Our results show that, in addition to T. orientalis, A. phagocytophilum is strongly associated with anaemia. The correlation between haemolytic anaemia severity and haematological parameters (indirect bilirubin, reticulocytes, and L-lactate) was confirmed.

Comparison of blood profiles between housed and grazing Korean indigenous cattle (Hanwoo).

The objective of this study was to compare the hematology profiles of Korean indigenous cattle (Hanwoo) raised in a barn (housed) or on pasture (grazing). Our findings showed significant differences in the red blood cell (RBC) profiles of these 2 groups. When compared to cattle raised in a barn, a significant decrease in hematocrit (P = 0.000), hemoglobin (P = 0.000), and red blood cells (RBCs) (P = 0.000) and a significant increase in mean cell volume (P = 0.015) and reticulocytes (P = 0.000) were observed in grazing cattle, which indicate regenerative anemia. Furthermore, indirect bilirubin was significantly higher in grazing cattle, which indicates intravascular hemolysis and neutropenia (P = 0.000), and monocytosis (P = 0.000) was also identified. To the best of our knowledge, this is the first study that demonstrates changes in reticulocyte count and indirect bilirubin levels secondary to regenerative intravascular hemolysis in grazing cattle.

L'objectif de cette étude était de comparer les profils hématologiques de bovins coréens indigènes (Hanwoo) gardés dans une étable ou au pâturage. Nos résultats ont montré des différences significatives dans les profils des globules rouges de ces 2 groupes. Lorsque comparé aux bovins gardés dans l'étable, une réduction significative de l'hématocrite (P = 0,000), de l'hémoglobine (P = 0,000), et des globules rouges (P = 0,000) et une augmentation significative du volume cellulaire moyen (P = 0,015) et des réticulocytes (P = 0,000) ont été observées chez les bovins au pâturage, ce qui indique une anémie régénératrice. Également, la bilirubine indirecte était significativement plus élevée chez les bovins au pâturage, indicatif d'une hémolyse intravasculaire, et une neutropénie (P = 0,000) et une monocytose (P = 0,000) furent également identifiées. Au meilleur de nos connaissances, ceci est la première étude qui démontre des changements dans le dénombrement des réticulocytes et les niveaux de bilirubine indirecte secondaires à une hémolyse intravasculaire régénérative chez des bovins au pâturage.(Traduit par Docteur Serge Messier).

Preliminary report of Mycoplasma Wenoynii and Candidatus Mycoplasma haemobos infection in Korean native cattle.

BACKGROUND: Hemotropic mycoplasmas or hemoplasmas are bacteria that attach to the erythrocyte surface and cause bovine hemoplasmosis. Two species, Mycoplasma wenyonii and Candidatus Mycoplasma haemobos, have been identified and shown to be distributed worldwide. However, there is currently no information available on hemoplasmas in cattle in the Republic of Korea. The aim of this study was to investigate the presence of hemoplasmas in Korean native cattle and to evaluate the association between hemoplasma infection and anemia. METHODS: One farm was selected, at which blood samples were collected from 104 Korean native cattle [grazing cattle (n = 89) and housed cattle (n = 15)]. Hemoplasmas were detected via polymerase chain reaction analysis and complete blood counts were also performed. RESULTS: The overall prevalence of hemoplasmas was 34% (35/104); 20.2% (21/104) for M. wenyonii, 3.8% (4/104) for C. M. haemobos, and 9.6% (10/104) for co-infection. Candidatus Mycoplasma haemobos was detected only in grazing cattle. Of red blood cell (RBC) parameters, C. M. haemobos-infected cattle had lower RBC and hematocrit, and higher mean cell volume than hemoplasma-negative cattle, although none of these differences were statistically significant. This is the first study to report the occurrence of M. wenyonii and C. M. haemobos. Mycoplasma wenyonii is more prevalent than C. M. haemobos in Korean native cattle. The results did not show an association between hemoplasma infection and anemia. CONCLUSIONS: Considering the infection rate of hemoplasmas shown in this study, further studies, such as on the pathogenicity and clinical significance of hemoplasmas are necessary.

Serologic and Molecular Prevalence of Severe Fever with Thrombocytopenia Syndrome Virus Among Poultry in the Republic of Korea.

Background: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by Dabie bandavirus, which belongs to the genus Bandavirus, family Phenuiviridae, and order Bunyavirales. It has been found in tick species, various animals, and humans. The aim of this study was to detect RNA of antigens and antibodies against SFTS virus (SFTSV) among poultry such as chickens, ducks, and wild geese from five provinces in the Republic of Korea (ROK). Materials and Methods: A one-step reverse transcriptase (RT)-PCR and nested PCR were performed after viral RNA extraction. The phylogenetic tree was constructed after sequencing data were analyzed and aligned. An indirect enzyme-linked immunosorbent assay (ELISA) and a neutralization test (NT) were performed to test for IgG antibodies of SFTSV. Results: Of a total of 606 poultry serum samples collected, 568 and 539 serum samples were used to perform ELISA and NT, respectively. Of a total of 606 serum samples tested by RT-PCR targeting the S segment, 15 (2.5%) were positive for SFTSV. From the 15 positive serum samples for the SFTSV antigen, three from chickens, three from ducks, and one from wild geese were classified as genotype B-2; one from chickens was classified as genotype B-3; and three from chickens and four from wild geese were classified as genotype D. Of the 568 serum samples tested by ELISA, 83 (28.0%) from chickens, 81 (32.9%) from ducks, and 8 (30.8%) from wild geese were seropositive. Of the 539 serum samples for which an NT was performed, 113 (38.6%) from chickens and 75 (30.5%) from ducks were positive for SFTSV antibodies. Conclusions: The results of this study provide useful information regarding detection of SFTSV RNA and antibodies among poultry and the possibility of SFTSV transmission in various types of poultry, including chickens, ducks, and wild geese, in the ROK.

Molecular and Serological Survey of Severe Fever with Thrombocytopenia Syndrome Virus in Horses from the Republic of Korea.

Background: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic tick-borne disease in East Asia caused by the SFTS virus (SFTSV). It is to investigate the presence of SFTSV RNA and antibodies in horses from a slaughterhouse and equestrian centers in the Republic of Korea (ROK). A prevalence study of SFTSV-specific RNA and antibodies was designed from 889 horses in the ROK. Materials and Methods: Serum samples were collected from horses at a slaughterhouse and equestrian centers from 2018 to 2020. To detect the presence of SFTSV, RNA was extracted from the serum samples, and a nested reverse transcription-polymerase chain reaction (RT-PCR) was conducted. Sequencing data were analyzed, and a phylogenetic tree was constructed using the maximum-likelihood method with Molecular Evolutionary Genetics Analysis Version 7.0 software. The horse sera were also tested for SFTSV-specific immunoglobulin G antibodies using enzyme-linked immunosorbent assay (ELISA). Results: Twelve of 889 (1.3%) horse sera were positive for SFTSV RNA, and 452 of 887 (51.0%) horse sera were seropositive by ELISA. Among the RT-PCR-positive samples, 12 of the SFTSV S-segment sequences were classified as sub-genotypes B-2 (n = 6) and B-3 (n = 6). ELISA analysis was evaluated by comparison with neutralization test. We investigated SFTSV infection in horses over a 3-year period, but sampling was not performed evenly by season; continuous surveillance of SFTSV in horses is needed. Conclusions: We report the detection of SFTSV RNA and provide serological data on SFTSV prevalence in horses in the ROK. The detection of SFTSV-specific RNA and antibodies in horses, which are in close proximity to humans, suggests that SFTS is an emerging and important health issue, indicating that more attention to its relevance for equestrian workers is needed.

First report of Cryptosporidium andersoni and risk factors associated with the occurrence of Cryptosporidium spp. in pre-weaned native Korean calves with diarrhea.

Cryptosporidium spp. are important enteric protozoan parasites that infect humans and other animals throughout the world. Cryptosporidium infection in cattle industry leads to substantial economic losses due to diarrhea, growth retardation, weight loss, and possibly death. Most studies have focused on C. parvum, and studies on other Cryptosporidium spp. and calf diarrhea are limited. Therefore, this study aimed to investigate the occurrence of Cryptosporidium spp. in pre-weaned calves, to determine the risk factors for Cryptosporidium spp. infection such as age and season, and to identify subtypes of C. parvum circulating in the Republic of Korea (ROK). A total of 510 fecal samples were collected from calves with diarrhea and divided by age and season. Cryptosporidium spp. were first screened using PCR targeting the small subunit (SSU) rRNA gene and further the 60-kDa glycoprotein gene for subtyping of C. parvum. Out of 510 fecal samples, 71 (13.9%) were positive for Cryptosporidium spp. in pre-weaned calves with diarrhea. C. andersoni (2.8%), C. bovis (30.9%), C. parvum (29.6%), and C. ryanae (36.6%) were identified. C. ryanae was the most predominant in calves in the ROK. Calf age was a significant risk factor for C. bovis (χ2 = 13.83, P = 0.001), C. parvum (χ2 = 7.57, P = 0.023), and C. ryanae (χ2 = 20.18, P = 0.000) occurrence. Additionally, C. parvum was detected 3.1-fold more frequently in pre-weaned calves with diarrhea in fall (95% CI: 1.23-7.81; P = 0.016) than in spring, whereas C. ryanae was 8.9-fold more frequently detected in summer (95% CI: 1.65-48.68; P = 0.011) than in spring. Three subtypes (IIaA17G4R1, IIaA18G3R1, and IIaA20G3R1) of C. parvum were identified. Of them, IIaA17G4R1 was the most common, whereas IIaA20G3R1 was not previously detected in calves in the ROK. To our knowledge, this is the first report of C. andersoni in pre-weaned calves in the ROK. The occurrence of Cryptosporidium spp. appears to be age-dependent in calves. Season had a significant effect on the occurrence of C. parvum and C. ryanae. Taken together, C. bovis and C. ryanae along with C. parvum are detected in pre-weaned calves with diarrhea and these two pathogens should not be overlooked in the diagnosis of calf diarrhea.

Successful treatment of idiopathic tetanus using metronidazole, magnesium, and acepromazine in Hanwoo (Korean indigenous cattle) yearling bull.

Bovine tetanus is a serious infectious disease of the central nervous system caused by the exotoxin produced by Clostridium tetani and is characterized by persistent tension and spasm of the rhabdomyocytes. Currently, many studies have focused on diagnosing tetanus; however, only a few studies on treatment methods have been conducted. Therefore, cattle with tetanus have been treated using symptomatic therapy. In this case, severe muscle spasticity and spasms were observed in a 9-month-old Hanwoo (Korean indigenous cattle) bull, and aspartate aminotransferase and creatine kinase levels were increased in serum biochemical tests. Clinically, bovine tetanus was strongly suspected, and metronidazole was administered orally for 5 days. To treat the intensifying bloat, a temporary rumenostomy was performed on the third day of onset, and the toxin gene (tetanospasmin) of C. tetani was amplified by polymerase chain reaction analysis from the collected ruminal fluid. Magnesium and sedatives (acepromazine) were administered for 7 days to treat muscle spasticity and spasms. Muscle spasticity and spasm markedly improved, and the bull stood up from the lateral recumbent position. On the 17th day after onset, all tetanus-related symptoms resolved and a normal diet was started. Our findings demonstrated that treatment with metronidazole, magnesium, and acepromazine was effective in the bull with tetanus.

Molecular Detection of Toxoplasma gondii in Blood Samples of Domestic Livestock in the Republic of Korea.

Toxoplasma gondii, a major zoonotic pathogen distributed worldwide, causes severe infections in humans, animals, and birds. However, limited information is available regarding T. gondii infection in livestock in the Republic of Korea (ROK). Herein, we determined the prevalence of T. gondii infection in livestock in the ROK and identified animal species that can potentially transmit T. gondii to humans. B1 gene-targeting nested polymerase chain reaction detected T. gondii DNA in 3.3% (2/61), 2.9% (3/105), 14.1% (11/78), and 15.4% (14/91) of dairy cattle, beef cattle, Boer goats, and Korean native goats, respectively. The prevalence of T. gondii was significantly higher (p = 0.002) in goats than in cattle. The risk of contracting T. gondii infection was significantly higher by 6.18-fold in Korean native goats (95% confidence interval [CI]: 1.72-22.27%, p = 0.005) and by 5.58-fold in Boer goats (95% CI: 1.50-20.76%, p = 0.010) than in beef cattle. Our T. gondii DNA sequences exhibited 97.1-100% homology with those obtained from various hosts in other countries. To the best of our knowledge, this is the first study to report T. gondii infection using the blood samples of domestic ruminants in the ROK. The results revealed that the prevalence of T. gondii infection is higher in goats than in cattle as determined by molecular detection. Thus, these findings suggest that T. gondii can be transmitted from ruminants to humans via meat consumption.

Multilocus genotyping of Giardia duodenalis in pre-weaned calves with diarrhea in the Republic of Korea.

Giardia duodenalis is a protozoan parasite that infects humans, companion animals, livestock, and wildlife. Infections in cattle caused by this parasite are often asymptomatic, but such infections can cause diarrhea, reduced weight gain, and ill-thrift in young calves. Although G. duodenalis causes diarrhea in calves, only a few studies have been conducted on calves in the Republic of Korea (ROK). Here, we aimed to determine the prevalence and distribution of G. duodenalis assemblages in pre-weaned calves with diarrhea in the ROK, identify the association between the occurrence of G. duodenalis and the age of calf, and perform molecular characterization of G. duodenalis. We collected 455 fecal samples from pre-weaned native Korean calves (≤60 days old) with diarrhea in four different regions. G. duodenalis was detected using nested PCR targeting the beta-giardin (bg) gene, and positive samples were further genotyped for the glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes. The overall prevalence of G. duodenalis in calves with diarrhea was 4.4% (20/455) based on the analysis of bg. The highest prevalence was observed in calves aged 11-30 days (7.5%; 95% confidence interval: 3.7%-11.3%), whereas the lowest prevalence was observed in neonatal calves. From the 20 samples that were positive for bg, 16, 5, and 6 sequences were obtained following genotyping of bg, gdh, and tpi, respectively. Sequencing analysis of the bg gene revealed the presence of assemblage E (n = 15) and sub-assemblage AⅠ (n = 1) in the samples. Moreover, we detected mixed infections with assemblages E and A in two calves for the first time. Among the sequences obtained herein, two new subtypes of assemblage E were detected in gdh and tpi sequences each. The results suggest that G. duodenalis is an infectious agent causing diarrhea in calves, and pre-weaned calves are at a higher risk of infection than neonatal calves. Multilocus genotyping should be performed to confirm the presence of potentially zoonotic genotypes. These results highlight the importance of cattle as a source of zoonotic transmission of G. duodenalis to humans.

Non-cytopathic bovine viral diarrhoea virus 2 induces autophagy to enhance its replication.

BACKGROUND: Bovine viral diarrhoea virus (BVDV) is an important viral pathogen that has an economic impact on the livestock industry worldwide. Autophagy is one of the earliest cell-autonomous defence mechanisms against microbial invasion, and many types of viruses can induce autophagy by infecting host cells. OBJECTIVES: The aim of this study was to identify the role of autophagy in the pathogenesis of non-cytopathic (ncp) BVDV2 infection. METHODS: Madin-Darby bovine kidney (MDBK) cells were treated with ncp BVDV2, rapamycin, or 3-methyladenine (MA) and ncp BVDV2 and then incubated at 37°C for 24 h. Cells were harvested, and the effects of autophagy were determined by transmission electron microscopy (TEM), confocal laser microscopy, western blotting and qRT-PCR. Apoptotic analysis was also performed using western blotting and flow cytometry. RESULTS: In ncp BVDV2-infected MDBK cells, more autophagosomes were observed by TEM, and the number of microtubule-associated protein 1 light chain 3B (LC3B) with green fluorescent protein puncta was also increased. The ncp BVDV2-infected cells showed significantly enhanced conversion of LC3-I to LC3-II, as well as upregulation of autophagy-related proteins, including ATG5 and Beclin 1, and substantial degradation of p62/SQSTM1. These results are similar to those induced by rapamycin, an autophagy inducer. E2 protein expression, which is associated with viral replication, increased over time in ncp BVDV2-infected cells. Inhibition of autophagy by 3-MA in ncp BVDV2-infected MDBK cells downregulated the expressions of LC3-II, ATG5 and Beclin 1 and prevented the degradation of p62/SQSTM1. Moreover, the expressions of phosphorylated Akt and procaspase-3 were significantly increased in ncp BVDV2-infected cells. In addition, the mRNA level of protein kinase R (PKR) was significantly reduced in ncp BVDV2-infected cells. CONCLUSIONS: Our results demonstrate that ncp BVDV2 infection induced autophagy in MDBK cells via anti-apoptosis and PKR suppression. Therefore, autophagy may play a role in establishing persistent infection caused by ncp BVDV.

Comparison of blood electrolyte and biochemical parameters between single infections of rotavirus and Cryptosporidium parvum in diarrheic Hanwoo calves.

BACKGROUND: Neonatal calf diarrhea is a major problem in the cattle industry worldwide. Rotavirus and Cryptosporidium parvum are the primary causative agents, especially during the first three weeks of the calf's life. OBJECTIVES: This study investigated the differences in acid-base, electrolytes, and biochemical parameters of diarrheic calves with infection of either rotavirus or C. parvum. METHODS: A total of 61 Korean native calves (≤ 20 days old) were divided into two groups based on rotavirus or C. parvum infections: rotavirus infection (n = 44) and C. parvum infection (n = 17). The calves with at a specific blood pH range (pH 6.92-7.25) were chosen for comparison. The acid-base, electrolyte, chemistry, and serum proteins were analyzed, Further, fecal examinations were performed. RESULTS: Compared to C. parvum-infected calves, the rotavirus-infected calves showed lower levels of total carbon dioxide, bicarbonate (HCO3-), anion gap, total protein, and albumin/globulin ratio, and significantly lower levels of potassium, globulin, and α2-globulin (p < 0.05). The C. parvum-infected calves (r = 0.749) had stronger correlations between pH and HCO3- than the rotavirus-infected calves (r = 0.598). Compared to rotavirus-infected calves, strong correlations between globulin and α2-globulin, α2-globulin and haptoglobin were identified in C. parvum-infected calves. CONCLUSIONS: This study is the first to investigate acid-base, electrolyte, and biochemical parameters in calves in response to infections of rotavirus and C. parvum. Although rotavirus and C. parvum cause malabsorptive and secretory diarrhea in similar-aged calves, blood parameters were different. This would help establish the diagnostic and treatment strategies.

Clinical features and epidemiology of severe fever with thrombocytopenia syndrome in dogs in the Republic of Korea: an observational study (2019-2020).

Severe fever with thrombocytopenia syndrome (SFTS) is a zoonotic disease with a high mortality rate for humans and cats. The clinical course and prognosis of SFTS in dogs remains unclear. In the present study, we investigated the clinical and epidemiological characteristics of SFTS virus (SFTSV) infection in dogs. All evaluated dogs exhibited an acute course and symptoms including fever (57.1%), anorexia (57.1%), depression (42.9%), and vomiting (35.7%). Thrombocytopenia was present in 45.5% of dogs, while jaundice was not observed. C-reactive protein, alanine transaminase, and alkaline phosphatase were elevated in some cases. Viral clearance occurred within 6 to 26 days. Phylogenetic analysis revealed that the SFTSV sequences were consistent with viruses circulating in the Republic of Korea. As dogs often live in close contact with humans, awareness of the clinical and epidemiological features of SFTS in dogs is crucial. Further large-scale studies are necessary to investigate SFTSV infection in dogs.

Prevalence and genetic characterization of bovine coronavirus identified from diarrheic pre-weaned native Korean calves from 2019 to 2021.

Bovine coronavirus (BCoV) is associated with severe diarrhea in calves, winter dysentery in adult cattle, and respiratory diseases in cattle. However, there is currently limited information regarding its molecular characterization in the Republic of Korea (KOR). Therefore, this study investigated the prevalence of BCoV in diarrheic pre-weaned calves (aged ≤60 days) and compared BCoV genome sequences identified globally. A total of 846 fecal samples were collected from calves with diarrhea across 100 beef farms in the KOR. The samples were divided into three groups based on age as follows: 1-10 days (n = 490), 11-30 days (n = 277), and 31-60 days (n = 79). BCoV infection was detected in 50 calves by real-time RT-PCR analysis. The results showed that the prevalence of BCoV was associated with calf age (P = 0.028) and was significantly higher in calves aged 31-60 days (odds ratio: 2.69, 95% confidence interval: 1.24-5.85; P = 0.012) than in those aged 1-10 days. Our findings show that BCoV is an important etiological agent of diarrhea in calves aged 31-60 days. Fifteen full genome sequences (2019-2021 variants) of the spike, hemagglutinin/esterase, and nucleocapsid were obtained from the 50 BCoV-positive samples. Phylogenetic analysis of each gene revealed that BCoVs circulating worldwide might have no boundary between enteric and respiratory tropisms, demonstrating the presence of three BCoVs groups: the classical, Asia/USA, and European. Initially, Korean BCoVs were originated from the USA, but diverged since the 1980s and rapidly evolved independently, unlike in other Asian countries. In this study, Korean BCoVs are more recent BCoVs and present relatively high nucleotide substitution rates in all genes compared with other BCoVs. Our results showed that the 2019-2021 variants undergo continuous genetic evolution and that there are genetic differences among globally distributed BCoVs.

Molecular surveillance of rotavirus A associated with diarrheic calves from the Republic of Korea and full genomic characterization of bovine-porcine reassortant G5P[7] strain.

Group A rotavirus (RVA) is the most common diarrhea-causing pathogen among humans and animals worldwide. Rotavirus infection in neonatal calves causes major problems in the livestock industry. This study aimed to determine the prevalence and genetic diversity of bovine rotavirus (BoRVA) infections in calves with diarrhea and to perform whole genome analysis of an unusual strain, designated as RVA/Calf-wt/KOR/KNU-GJ2/2020/G5P[7], that was detected in a 2-day-old diarrheic calf. From 459 diarrheic calves aged 1-40 days, fecal samples were collected and BoRVA infections were screened using real-time RT-PCR targeting VP6 gene. BoRVA was detected in 195 (42.4%) samples and was most prevalent in calves aged 1-10 days (47.2%). No significant difference in the BoRVA infection rate was observed between calves born in herds that were (42.1%) and were not (42.6%) vaccinated against BoRVA. A binomial regression analysis revealed that calves aged 1-10 days (95% confidence intervals [CI]:1.18-24.34; P = 0.000) and 11-20 days (95% CI: 0.76-16.83, P = 0.000) had a 5.37- and 3.58-fold higher BoRVA prevalence in comparison to those aged 31-40 days, respectively. The RVA-positive samples were subsequently subjected to amplification of the VP7 and VP4 genes for determining G and P genotypes. Overall, 45 (23.1%, 45/195) and 63 (32.3, 63/195) sequences for VP7 and VP4 were obtained. In this study, four G and three P genotypes were identified. G6 (86.7%) was the most prevalent genotype, followed by G8 (8.9%), G10 (2.2%), and G5 (2.2%). P[5] (92.1%) was the most frequently detected, followed by P[11] (6.3%), and P[7] (1.6%). The G6P[5] (82.2%) is the most common combination found in Korean native calves with diarrhea, whereas G6P[11] (4.4%) and G10P[11] (2.2%) had relatively low prevalence. G8P[5] (8.9%) was identified for the first time in diarrheic calves in the KOR. The uncommon strain KNU-GJ2 exhibited a G5-P[7]-I5-R1-C1-M2-A1-N1-T1-E1-H1 genotype constellation possessing a typical porcine RVA backbone, with the exception of the VP3 gene, which is derived from bovine. Phylogenetically, except for VP3, ten gene segments of KNU-GJ2 were closely related to porcine, porcine-like, and reassortant bovine strains. Interestingly, the VP3-M2 gene of KNU-GJ2 clustered with bovine-like strains as well as reassortant porcine and bovine strains. Comparison of the NSP4s within a species-specific region of amino acids 131-141 demonstrated that KNU-GJ2 belonged to genotype B with porcine RVAs; however, it differed from porcine RVAs by one to three amino acids. The present study is fundamental to understanding the epidemiology and genotypes of circulating RVAs throughout the KOR and underscoring the importance of continuous monitoring and molecular characterization of RVAs circulating within animal populations for future vaccine development.

Identification of Babesia capreoli from Korean Water Deer in the Republic of Korea.

Babesiosis is a tick-borne disease caused by intraerythrocytic protozoa belonging to the genus Babesia that is primarily transmitted through Ixodid ticks and is highly pathogenic to ruminants, horses, pigs, dogs, cats, and in some cases, even humans. Babesia is considered an increasing threat to animal and human health. In this study we report Babesia capreoli infection in Korean water deer (Hydropotes inermis argyropus). Blood samples of 77 wild animals (62 Korean water deer and 15 Korean raccoon dogs [Nyctereutes procyonoides koreensis]) were analyzed to detect Babesia infection. First, PCR was performed to investigate Babesia infection using the partial 18S rRNA gene. Among the 77 wild animals examined, 11 Korean water deer were positive for infection as per the results of sequence analysis. Next, these 11 samples were reanalyzed by PCR using newly designed primers to differentiate between B. capreoli and Babesia divergens. Finally, only one Korean water deer sample was found to be positive for B. capreoli. The nucleotide sequence of this protozoan showed 99.6 - 99.7% identity to those of B. capreoli reported previously. Phylogenetic analysis revealed that the sequence belonged to the B. capreoli clade and diverged from the B. divergens clade. Three nucleotides at positions 631, 663, and 1637 were analyzed to differentiate between B. capreoli and B. divergens. The nucleotides at positions 631 and 1637 were identical to those of B. capreoli, whereas that at position 663 was different. The sequence detected in Korean water deer also exhibited differences at other four positions (669, 704, 711, and 1661) compared with other B. capreoli sequences. This study indicates that Korean water deer serve as a reservoir for B. capreoli. In addition, the primers designed in this study can be used to detect B. capreoli. Future studies should explore the tick vectors that transmit B. capreoli and its pathogenicity. This study was approved by the Institutional Committee of Graduate Studies and Research at Jeonbuk National University, ROK (IACUC decision no.: CBU 2014-00026).

Serological evidence of Anaplasma spp., Borrelia burgdorferi and Ehrlichia canis in dogs from the Republic of Korea by rapid diagnostic test kits.

BACKGROUND: Emergent and re-emergent canine tick-borne infections are attracting increasing attention worldwide. The rise in pet ownership and the close relationship between dogs and their owners are the most concerning factors because dogs may act as competent reservoirs for human tick-transmitted infectious agents. OBJECTIVES: This study contributes to the epidemiological surveillance of canine tick-transmitted infections with zoonotic risk in the Republic of Korea (ROK) by investigating the seroprevalence of the pathogens, Anaplasma spp., Borrelia burgdorferi, and Ehrlichia canis. METHODS: Four hundred and thirty whole blood samples from domestic dogs were collected in seven metropolitan cities and nine provinces in the ROK and tested using SensPERT Ab test kits (VetAll Laboratories®) to detect seroreactive animals. RESULTS: The seroprevalence rates identified were 9.8% (42/430) for Anaplasma spp., 2.8% (12/430) for B. burgdorferi, and 1.4% (6/430) for E. canis. The risk factors evaluated in this study that could be associated with the development of a humoral immune response, such as sex, age, and history of tick exposure, were similar. There was only one exception for dogs seroreactive to Anaplasma spp., where the risk factor "tick exposure" was statistically significant (p = 0.047). CONCLUSIONS: This serological survey exhibited the widespread presence of Anaplasma spp., B. burgdorferi, and E. canis throughout the ROK. Hence, dogs may play a key role as the sentinel animals of multiple zoonotic infectious agents in the country.

Prevalence and distribution pattern of Cryptosporidium spp. among pre-weaned diarrheic calves in the Republic of Korea.

Cryptosporidium spp. are protozoan parasites that belong to subphylum apicomplexa and cause diarrhea in humans and animals worldwide. Data on the prevalence of Cryptosporidium spp. and its subtypes among calves in the Republic of Korea (KOR) are sparse. Hence, our study aimed to investigate the prevalence and association between the age of calf and the identified Cryptosporidium spp. and to determine the genotypes/subtypes of Cryptosporidium spp. in pre-weaned calves with diarrhea in the KOR. A total of 460 diarrheic fecal samples were collected from calves aged 1-60 days and screened for Cryptosporidium spp. by the 18S rRNA gene. Species identification was determined using the sequencing analysis of the 18S rRNA gene, and C. parvum-positive samples were subtyped via the sequence analysis of the 60-kDa glycoprotein (gp60) gene. Sequence analysis based on the 18S rRNA gene revealed the presence of three Cryptosporidium spp., namely, C. parvum (n = 72), C. ryanae (n = 12), and C. bovis (n = 2). Co-infection by these species was not observed. The infection rate was the highest in calves aged 11-20 days (26.1%, 95% CI 17.1-35.1), whereas the lowest rate was observed in calves aged 21-30 days (7.7%, 95% CI 0.0-16.1). The prevalence of C. parvum was detected exclusively in calves aged ≤20 days, and the highest infection rate of C. ryanae was seen in calves ≥31 days of age. The occurrence of C. parvum (χ2 = 25.300, P = 0.000) and C. ryanae (χ2 = 18.020, P = 0.001) was significantly associated with the age of the calves. Eleven different subtypes of the IIa family that belonging to C. parvum were recognized via the sequence analyses of the gp60 gene. Except for two (IIaA18G3R1 and IIaA15G2R1) subtypes, nine subtypes were first identified in calves with diarrhea in the KOR. IIaA18G3R1 was the most frequently detected subtype (72.2% of calves), followed by IIaA17G3R1 (5.6%), IIaA15G2R1 (4.2%), IIaA19G4R1 (4.2%), IIaA16G4R1 (2.8%), IIaA17G4R1 (2.8%), IIaA19G3R (2.8%), IIaA14G1R1 (1.4%), IIaA14G3R1 (1.4%), IIaA15G1R1 (1.4%), and IIaA19G1R1 (1.4%) These results suggest that the prevalence of Cryptosporidium spp. is significantly associated with calf age. Furthermore, the findings demonstrate the high genetic diversity of C. parvum and the widespread occurrence of zoonotic C. parvum in pre-weaned calves. Hence, calves are a potential source of zoonotic transmission with considerable public health implications.

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).

Survey of severe fever with thrombocytopenia syndrome virus in wild boar in the Republic of Korea.

Severe fever with thrombocytopenia syndrome (SFTS) is caused by Dabie bandavirus that belongs to the genus Bandavirus in the family Phenuiviridae and order Bunyavirales and is transmitted by hard ticks. It has been detected in several tick species, various animals, and humans. The purpose of this study was to detect SFTS virus (SFTSV) antigen and antibody in wild boar in the Republic of Korea (ROK). A total of 768 sera samples were collected from wild boar in the ROK between January and December 2019. Viral RNA was extracted from sera using viral RNA extraction kit, and one-step RT-nested polymerase chain reaction (PCR) was performed to amplify the S segment of the SFTSV. The sequencing data were analyzed using Chromas and aligned using Clustal X. The phylogenetic tree was constructed using the maximum-likelihood method using MEGA7. In addition, wild boar sera were tested for IgG antibodies against SFTSV by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA). Of a total of 768 sera samples, 40 (5.2%) were positive for SFTSV by RT-PCR targeting the S segment. Two hundred twenty-one (28.8%) and 159 (20.7%) of 768 sera samples were seropositive by ELISA and IFA, respectively. Based on both ELISA and IFA tests of the same samples, 110 (14.3%) wild boar sera samples were positive for SFTSV antibodies. Of a total of 40 positive serum samples by RT-PCR, 33 (82.5%) and 7 (17.5%) sera were classified as the genotype B-3 and D, respectively, by sequence analysis,. These results provide useful information that demonstrates the detection of antigen and antibody in wild boar sera samples for every month of a certain year throughout the ROK.

Euryhelmis squamula (Digenea: Heterophyidae) Recovered from Korean Raccoon Dog, Nyctereutes procyonoides koreensis, in Korea.

In this study, we intended to describe an unrecorded species of heterophyid trematode recovered from the small intestine of a Korean raccoon dog, Nyctereutes procyonoides koreensis, in Korea. A total of 13 small flukes were collected from a deceased Korean raccoon dog which was found in Chuncheon-si, Gangwon-do, Korea in May 2017. The trematode body were covered with many small spines, rectangular, broader than long, 807-1,103 µm long and 1,270-1,550 µm wide. Oral sucker in the anterior end slightly smaller than acetabulum. Pharynx muscular and well developed. Esophagus relatively long and sigmoid. Acetabulum small and located at median in anterior 2/5 portion. Ceca bifurcated at the anterior of genital pore and acetabulum and terminated at testis level. Testes larger, deeply lobed and located at the near of posterior end of body. Ovary small, triangular and located at the slight left of median and the anterior of left testis. Vitelline follicles dendritic and extend from the middle level of esophagus to the posterior portion of body. Eggs embryonated, operculated, small and 33-35×15-16 µm in size. Based on the morphological characteristics, the small heterophyid flukes recovered from the small intestines of Korean raccoon dog, N. procyonoides koreensis, were identified as Euryhelmis squamula (Digenea: Heterophyidae). Accordingly, this species of heterophyid flukes is to be a new trematode fauna in Korea by this study.