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.

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.

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).

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.

FIRST REPORT OF ENTEROCYTOZOON BIENEUSI FROM AN AFRICAN LION (PANTHERA LEO) IN A ZOO IN THE REPUBLIC OF KOREA.

Enterocytozoon bieneusi is the most common species of microsporidia that infects humans and animals worldwide. However, no information is available on E. bieneusi infection among zoo animals in the Republic of Korea (ROK). Here, we investigated the prevalence of E. bieneusi among animals kept in zoos and the zoonotic potential of the E. bieneusi identified. E. bieneusi was detected only in one African lion (Panthera leo) with diarrhea, using PCR and sequencing analysis of the internal transcribed spacer (ITS) of the rRNA gene. A phylogenetic analysis based on the ITS gene showed that the lion isolate was classified into a novel genotype KPL belonging to Group 2. The KPL genotype identified in this study differed from genotype I in 6 nucleotides and from genotype I-like in 3 nucleotides, respectively, indicating that Group 2 has the capacity to infect a wide range of hosts. This is the first report of the presence of E. bieneusi in an African lion housed in a zoo in the ROK. Further investigation is necessary to study E. bieneusi infection among zoo animals in various regions and to determine the transmission route, in order to control E. bieneusi infection.

Prevalence and Molecular Characterization of Coxiella burnetii in Cattle, Goats, and Horses in the Republic of Korea.

Coxiella burnetii is an obligate intracellular zoonotic bacterium with a global distribution. This study was conducted to investigate the prevalence of C. burnetii in different animals and to assess the potential role of these species as reservoirs of infection and transmission to humans. A total of 592 blood samples (105 beef cattle, 61 dairy cattle, 110 Korean native goats, 83 Boer goats, and 233 horses) were collected in the Republic of Korea (ROK). The C. burnetii DNA was detected from blood samples using the transposon-like repetitive region (IS1111) by PCR method. The results showed that 22.7% of the Korean-native goats, 16.4% of the dairy cattle, 15.2% of the beef cattle, 6.0% of the Boer goats, and 5.2% of the horses were positive for C. burnetii. Significant differences were found between the animal species. The univariable binary logistic regression analysis revealed that the risk of contracting C. burnetii was significantly high by 5.4-fold in Korean-native goats (95% confidence interval [CI]: 2.60%-11.27%, p = 0.000), 3.6-fold in dairy cattle (95% CI: 1.48%-8.82%, p = 0.005), and 3.3-fold in beef cattle (95% CI: 1.51%-7.28%, p = 0.003) compared with horses. A phylogenetic tree based on the IS1111 gene revealed that our sequences had 92.2%-99.9% similarity and were clustered with those detected in humans, cattle, goats, dogs, rodents, and ticks. C. burnetii circulating in the ROK exhibits genetic variation. To the best of our knowledge, this is the first study to identify C. burnetii DNA in a horse in the ROK. These results suggest that cattle, goats, and horses can be potential reservoirs for C. burnetii and play an important role in the transmission of infection. Further studies should assess the pathogenicity of C. burnetii circulating in the ROK.

Seroprevalence and Molecular Characterization of Coxiella burnetii in Cattle in the Republic of Korea.

This study was conducted to determine the prevalence of Coxiella burnetii in cattle and how that prevalence is influenced by cattle breed and growth type. A total of 491 cattle [cattle breed: 216 dairy cattle and 275 beef cattle; growth type: indoor housed (n = 294) and grazing (n = 197)] were used. The presence of C. burnetii DNA and antibodies was detected from blood and serum samples using polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The overall prevalence of C. burnetii was: 10.8% (95% CI: 8.0-13.5%) using PCR and 8.8% (95% CI: 6.3-11.3%) using ELISA. The prevalence of C. burnetii was significantly higher in beef cattle than in dairy cattle using both PCR (13.5% vs. 7.4%; P = 0.032) and ELISA (14.5% vs. 1.4%; P = 0.000), respectively. Comparison by growth type revealed that C. burnetii infection was significantly higher in grazing cattle than in housed cattle when using both PCR (24.9% vs. 1.4%; P = 0.000) and ELISA (21.3% vs. 0.3%; P = 0.000). Beef cattle were at a significantly higher risk of contracting C. burnetii compared with dairy cattle (odds ratio = 3.20, 95% CI: 1.80-5.67; P = 0.000). The risk of contracting C. burnetii in grazing cattle was increased by 32.57-fold (95% CI: 12.84-82.61; P = 0.000) compared with indoor housed cattle. The phylogenetic analysis based on the IS1111 gene revealed that our sequences grouped with human, tick, goat, and cattle isolates/strains found in several countries. C. burnetii sequences circulating in the Republic of Korea exhibit genetic variations. Thus, grazing is a high risk factor for the prevalence and transmission of C. burnetii.

Identification of Zoonotic Tick-Borne Pathogens from Korean Water Deer (Hydropotes inermis argyropus).

Korean water deer (Hydropotes inermis argyropus) are widespread in the Republic of Korea (ROK). Mostly, Korean water deer are essential hosts for maintaining ticks and tick-borne diseases (TBDs). Here, we investigated the prevalence of tick-borne pathogens (TBPs) among rescued Korean water deer. Anaplasma phagocytophilum (21.4%, 6/28), Anaplasma capra (14.3%, 4/28), Babesia capreoli (3.6%, 1/28), and Coxiella burnetii (3.6%, 1/28) were detected, but Borrelia burgdorferi, Ehrlichia, Rickettsia, and Theileria infections were not found. A. phagocytophilum was the most commonly detected pathogen, and co-infection with A. capra and B. capreoli was also noted in one Korean water deer. To our knowledge, this is the first article of B. capreoli infection in Korean water deer in the ROK. The infecting isolate of A. phagocytophilum was genetically characterized by 16S ribosomal RNA (rRNA) gene and ankyrin-related protein (ankA) gene. Although the 16S rRNA gene alone may not be informative enough to delineate distinct host species, ankA-based phylogeny revealed a high identity of Korean water deer sequences with those of the causative agent of human granulocytic anaplasmosis. A. capra was detected by using citrate synthase gene (gltA), heat-shock protein (groEL), and major surface protein 4 (msp4) genes. Phylogenetic tree based on these gene markers revealed that there were at least two distinct variants within A. capra circulating in the ROK. One variant originated from different hosts including humans, ticks, goats, and sheep, whereas the other variant was reported recently in Korean water deer in the ROK. Consequently, these sequences were identified to belong to a zoonotic species. Sequencing analysis of the 18S rRNA gene revealed that our isolate belonged to B. capreoli and was distinct from Babesia divergens and Babesia venatorum. Moreover, our isolate showed 92.2% homology with B. capreoli sequences, indicating that these differences may be attributed to the different tick species that transmit B. capreoli or to different host species. Genotyping and phylogenetic analysis of C. burnetii based on 16S rRNA and IS1111 genes revealed that our isolate was grouped with several strains of C. burnetii and was genetically distant from Coxiella-like bacteria isolates. The present results highlight that Korean water deer act as potential reservoir hosts for zoonotic TBPs, and thus play an important role in the transmission of TBDs in humans, animals, and livestock.

Zoonotic potential of Enterocytozoon bieneusi in pre-weaned Korean native calves.

BACKGROUND: Enterocytozoon bieneusi is the most common microsporidian species infecting humans and various animals worldwide. To date, there has been limited information on the prevalence of infection and genotypes of E. bieneusi in cattle in the Republic of Korea. Therefore, this study investigated the prevalence and genotypes of E. bieneusi circulating in pre-weaned Korean native calves and determined the age pattern of E. bieneusi infection and the relationship between E. bieneusi infection and diarrhea. METHODS: The prevalence of E. bieneusi infection in pre-weaned Korean native calves was screened by polymerase chain reaction. PCR-positive products were sequenced to determine the genotype of E. bieneusi. A Chi-square analysis was used to compare the association between diarrhea and the infection rate of E. bieneusi in each age range or for all ages. RESULTS: PCR and sequencing analysis revealed an overall prevalence (16.9%, 53/314) of E. bieneusi in pre-weaned calves. The prevalence of E. bieneusi was highest in September (36.2%), followed by March (28.3%). Enterocytozoon bieneusi infection was associated with diarrhea in calves (χ2 = 5.82, P = 0.016). Our results also indicated that E. bieneusi infection was significantly associated with calf age (χ2 = 11.61, P = 0.003), and the prevalence of E. bieneusi infection was significantly higher in calves aged 21-40 days-old (odds ratio: 2.90, 95% confidence interval: 1.54-5.45; P = 0.001) than in those aged 1-20 days-old. Interestingly, the association between E. bieneusi infection and diarrhea was observed only in calves aged 1-20 days-old (χ2 = 5.82, P = 0.010). We identified three known genotypes, BEB4 (n = 12), BEB8 (n = 21) and J (n = 16), and three novel genotypes, BEB8-like (n = 21), KCALF1 (n = 1) and KCALF2 (n = 1). The genotype BEB8 was the most prevalent among all age groups. All genotypes identified in this study exhibited zoonotic potential. CONCLUSIONS: To our knowledge, this is the first report of the genotype BEB4 in pre-weaned Korean native calves. Zoonotic E. bieneusi infection was prevalent in pre-weaned calves, indicating that cattle may play an important role as a reservoir host for E. bieneusi transmission to humans.

Molecular surveillance of viral pathogens associated with diarrhea in pre-weaned Korean native calves.

Calf diarrhea causes severe economic losses in the cattle industry worldwide. This study investigated the prevalence of bovine coronavirus (BCoV), bovine norovirus (BNoV), bovine group A rotavirus (BoRVA), and bovine torovirus (BToV) in calves aged ≤ 60 days, regardless of diarrhea, across nine different regions in the Republic of Korea (ROK) and reported associations between these viruses and diarrhea. Fecal samples were collected rectally from 689 calves: normal (n = 360) and diarrheic (n = 329). BNoV (84/689, 12.2%) was the most prevalent regardless of diarrhea, followed by BCoV (37/689, 5.4%), BToV (15/689, 2.2%), and BoRVA (13/689, 1.9%). Although BCoV (P = 0.032) and BoRVA (P = 0.007) were significantly associated with diarrhea in pre-weaned calves, BNoV and BToV showed no association. Infection by the four pathogens had no relationship with calf age; BoRVA was detected only in calves aged < 30 days, but this finding was not statistically significant. Phylogenetic analysis revealed that BCoV isolates obtained in this study were distinct from the other known BCoVs, and all BNoV isolates belonged to GIII.2 genotype; genetic variations in BNoVs are present in the ROK. BoRVA isolates distributed in the ROK were assigned to G6P[5]. Within the P[5] genotype, our isolates were divided into two lineages: P[5]-III and P[5]-VIII. P[5]- VIII lineage was dominant in pre-weaned Korean native calves. Our BToV isolates were more closely related to a European isolate, B145, than to Japanese isolates. Here, BNoV was frequently identified in calves, suggesting its non-significant contribution to calf diarrhea, whereas BCoV and BoRVA were responsible for calf diarrhea in the ROK. Consequently, our results highlight the importance of rapid diagnosis against these viruses in calf diarrhea.

Prevalence and molecular characterization of Cryptosporidium and Giardia in pre-weaned native calves in the Republic of Korea.

Cryptosporidium spp. and Giardia duodenalis are protozoan parasites that cause diarrhea in humans and animals. Molecular data on Cryptosporidium spp. and G. duodenalis in calves in the Republic of Korea (ROK) is limited; thus, we investigated the prevalence of Cryptosporidium and Giardia in pre-weaned calves, analyzed the association between these parasites and diarrhea, and identified their zoonotic potential for human infection. Fecal samples were collected from 315 pre-weaned calves aged 1-60 days from 10 different regions in the ROK and screened for Cryptosporidium spp. and G. duodenalis using PCR. Overall prevalence of Cryptosporidium spp. and G. duodenalis was 4.4% (n = 14) and 12.7% (n = 40), respectively. Co-infection was not detected. All Cryptosporidium-positive samples were identified as C. parvum after sequence analysis of a small subunit rRNA fragment and further subtyped into zoonotic IIaA15G2R1 (n = 13) and IIaA18G3R1 (n = 1) by DNA sequencing of the 60-kDa glycoprotein gene. To our knowledge, this is the first report of C. parvum IIaA15G2R1 subtype in calves in the ROK. Based on ß-giardin (bg) gene, G. duodenalis-positive samples belonged to assemblages E (n = 36) and A (n = 4), with the latter belonging to subtype A1, the zoonotic genotype. Six subtypes of assemblage E were identified at the bg locus: E1 (n = 6), E2 (n = 3), E3 (n = 13), E5 (n = 1), E8 (n = 1), and E11 (n = 1). The occurrence of C. parvum and G. duodenalis was not associated with diarrhea in pre-weaned Korean native calves. The present results suggest that the prevalence of C. parvum is not related to calf age; in contrast, the prevalence of G. duodenalis was significantly higher in 41-50-day-old calves (odds ratio = 9.90, 95% confidence interval 2.37-41.34; P = 0.001) than in 1-10-day-old calves. Therefore, calves are a potential source of zoonotic transmission, which may have significant public health implications.

Noncytopathic bovine viral diarrhea virus 2 impairs virus control in a mouse model.

Bovine viral diarrhea virus (BVDV) is an economically important pathogen that causes development of mild to severe clinical signs in wild and domesticated ruminants. We previously showed that mice could be infected by BVDV. In the present study, we infected mice intraperitoneally with non-cytopathic (ncp) BVDV1 or ncp BVDV2, harvested the blood and organs of the infected mice at days 4, 7, 10 and 14 postinfection (pi), and performed immunohistochemical analyses to confirm BVDV infection. Viral antigens were detected in the spleens of all infected mice from days 4 through 14 and were also found in the mesenteric lymph nodes, gut-associated lymphoid tissue (GALT), heart, kidney, intestine, and bronchus-associated lymphoid tissue (BALT) of some infected mice. In ncp BVDV2-infected mice, flow cytometric analysis revealed markedly fewer CD4(+) and CD8(+) T lymphocytes and lower expression of costimulatory molecules CD80 (B7-1) and CD86 (B7-2) and major histocompatibility complex (MHC) class II (I-A/I-E) than those in ncp BVDV1-infected mice. Production of the cytokines interleukin (IL)-6 and monocyte chemotactic protein (MCP)-1 was higher in the plasma of ncp BVDV2-infected mice than that in that of ncp BVDV1-infected mice. Our results demonstrate that ncp BVDV1 and ncp BVDV2 interact differently with the host innate immune response in vivo. These findings highlight an important distinction between ncp BVDV1 and ncp BVDV2 and suggest that ncp BVDV2 impairs the host's ability to control the infection and enhances virus dissemination.