Detection Of Bovine Coronavirus Research Articles

Specific and sensitive detection of bovine coronavirus using CRISPR-Cas13a combined with RT-RAA technology.

Bovine coronavirus (BCoV) is an important pathogen of enteric and respiratory disease in cattle, resulting in huge economic losses to the beef and dairy industries worldwide. A specific and sensitive detection assay for BCoV is critical to the early-stage disease prevention and control. We established a specific, sensitive, and stable assay for BCoV nucleic acid detection based on CRISPR/Cas13a combined with reverse transcription recombinase-aided amplification (RT-RAA) technology. The specific primers for RT-RAA and CRISPR RNA (crRNA) were designed in the conserved region of the BCoV nucleocapsid (N) gene. The detection limit of the RT-RAA CRISPR/Cas13a assays for BCoV detection was 1.72 copies/μl, and there were no cross-reactions with the other 10 common bovine enteric and respiratory disease-associated pathogens. The coefficient of variations (CVs) of within and between batches were less than 4.98 and 4.58%, respectively. The RT-RAA-CRISPR/Cas13a assays work well in clinical samples of cattle and yak, the BCoV positive rate of 84 clinical samples detected by RT-RAA-CRISPR/Cas13a assays was 58.3% (49/84), it was notably higher than that of RT-qPCR (2.4%, 2/84; p < 0.001). The 49 positive samples detected by RT-RAA-CRISPR/Cas13a assays were further confirmed as BCoV by Sanger sequencing. A specific, sensitive, and stable assay based on RT-RAA-CRISPR/Cas13a assays for BCoV was developed, providing new technical support for the clinical detection and epidemiological monitoring of BCoV.

Novel CRISPR/Cas13-based assay for detection of bovine coronavirus associated with severe diarrhea in calves.

Bovine coronavirus (BCoV) is one of the important causes of diarrhoea in cattle. The virus is responsible for the high fatality rate associated with acute diarrhoea in calves. Rapid and accurate tests need to be conducted to detect the virus and minimise economic losses associated with the disease. Nucleic acid-based detection assays including PCR is an accurate test for detecting pathogens. However, these tests need skilled personnel, time and expensive devices. In this study, we developed a novel assay for the detection of BCoV in clinical cases. This novel assay combined reverse transcription-recombinase polymerase amplification with CRISPR/Cas13 and conducted a rapid visualisation of cleavage activity using a Lateral Flow Device. A conserved sequence of the BCV M gene was used as a target gene and the assays were tested in terms of specificity, sensitivity and time consumption. The result showed the specificity of the assay as 100% with no false positives being detected. Ten copies of the input RNA were enough to detect the virus and perform the assay. It took up to forty minutes for reading the results. Conducted together, the assay should be used as a rapid test to clinically diagnose infectious pathogens including bovine coronavirus. However, the assay needed the RNA to be extracted from the clinical sample in order to detect the virus. Therefore, more studies are needed to optimise the assay to be able to detect the virus in the clinical sample without extracting the RNA.

Advances in Laboratory Diagnosis of Coronavirus Infections in Cattle.

Coronaviruses cause infections in humans and diverse species of animals and birds with a global distribution. Bovine coronavirus (BCoV) produces predominantly two forms of disease in cattle: a respiratory form and a gastrointestinal form. All age groups of cattle are affected by the respiratory form of coronavirus, whereas the gastroenteric form causes neonatal diarrhea or calf scours in young cattle and winter dysentery in adult cattle. The tremendous impacts of bovine respiratory disease and the associated losses are well-documented and underscore the importance of this pathogen. Beyond this, studies have demonstrated significant impacts on milk production associated with outbreaks of winter dysentery, with up to a 30% decrease in milk yield. In North America, BCoV was identified for the first time in 1972, and it continues to be a significant economic concern for the cattle industry. A number of conventional and molecular diagnostic assays are available for the detection of BCoV from clinical samples. Conventional assays for BCoV detection include virus isolation, which is challenging from clinical samples, electron microscopy, fluorescent antibody assays, and various immunoassays. Molecular tests are mainly based on nucleic acid detection and predominantly include conventional and real-time polymerase chain reaction (PCR) assays. Isothermal amplification assays and genome sequencing have gained increased interest in recent years for the detection, characterization, and identification of BCoV. It is believed that isothermal amplification assays, such as loop-mediated isothermal amplification and recombinase polymerase amplification, among others, could aid the development of barn-side point-of-care tests for BCoV. The present study reviewed the literature on coronavirus infections in cattle from the last three and a half decades and presents information mainly on the current and advancing diagnostics in addition to epidemiology, clinical presentations, and the impact of the disease on the cattle industry.

Detection of bovine coronavirus (BCoV) infection in cattle with clinical respiratory signs by PCR and investigation of the serological status of these animals

Aim: It was aimed to reveal the antigenic and serological prevalence of bovine coronavirus (BCoV) in cattle with clinically respiratory infection and to evaluate the relationship between virus shedding and serological status of animals in this study. Materials and Methods: In the study, nasal swab samples were collected from a total of 92 cattle of different gender between 0 and 2 years of age with clinical respiratory symptoms. These samples were subjected to RT-PCR using specific primer pairs. Furthermore, serum samples were also collected from the same animals and evaluated for BCoV-specific antibodies through an indirect ELISA method. Results: Ten (10.87%) swap samples were defined as positive for BCoV using RT-PCR. The seropositivity rate was 93.48% (86/92) in cattle by ELISA and 90% (9/10) in virus-shedding animals with nasal swab samples. Conclusion: The fact that 93.47% seropositivity was detected in unvaccinated animals against coronavirus in this study indicates that the animals were exposed to BCoV through colostrum or at a certain period of their lives. However the fact that 9 out of 10 animals with BCoV detected in swab samples also have BCoV-specific antibodies casts doubt on the efficacy of these antibodies in protecting against respiratory BCoV infections. We concluded that more detailed studies are needed to reveal the role of antibodies in respiratory coronavirus infections.

Prevalence of bovine coronavirus in cattle in China: A systematic review and meta-analysis

Bovine coronavirus (BCoV) is one of the important pathogens that cause calf diarrhea (CD), winter dysentery (WD), and the bovine respiratory disease complex (BRDC), and spreads worldwide. An infection of BCoV in cattle can lead to death of young animals, stunted growth, reduced milk production, and milk quality, thus bringing serious economic losses to the bovine industry. Therefore, it is necessary to prevent and control the spread of BCoV. Here, a systematic review and meta-analysis was conducted to assess the prevalence of BCoV in cattle in China before 2022. A total of 57 articles regarding the prevalence of BCoV in cattle in China were collected from five databases (PubMed, ScienceDirect, CNKI, VIP, and Wan Fang). Based on the inclusion criteria, a total of 15,838 samples were included, and 6,136 were positive cases. The overall prevalence of BCoV was 30.8%, with the highest prevalence rate (60.5%) identified in South China and the lowest prevalence (15.6%) identified in Central China. We also analyzed other subgroup information, included sampling years, sample sources, detection methods, breeding methods, age, type of cattle, presence of diarrhea, and geographic and climatic factors. The results indicated that BCoV was widely prevalent in China. Among all subgroups, the sample sources, detection methods, breeding methods, and presence or absence of diarrheal might be potential risk factors responsible for BCoV prevalence. It is recommended to strengthen the detection of BCoV in cattle, in order to effectively control the spread of BCoV.

Development of a multienzyme isothermal rapid amplification and lateral flow dipstick combination assay for bovine coronavirus detection.

Bovine coronavirus (BCoV) is a major cause of infectious disease in cattle, causing huge economic losses to the beef and dairy industries worldwide. BCoV can infect humans and multiple other species of animals. A rapid, reliable, and simple test is needed to detect BCoV infection in suspected farms. In this study, we developed a novel multienzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) combination assay, targeting a highly conserved region of the viral nucleocapsid (N) gene for BCoV detection. The MIRA-LFD assay was highly specific and sensitive, comparable to a published reverse transcription quantitative PCR (RT-qPCR) assay for BCoV detection. Compared with the published RT-qPCR assay, the κ value of the MIRA-LFD assay in the detection of 192 cattle clinical samples was 0.982. The MIRA-LFD assay did not require sophisticated instruments and the results could be observed with eyes. Our results showed that the MIRA-LFD assay was a useful diagnostic tool for rapid on-site detection of BCoV.

Bovine coronavirus in the lower respiratory tract of cattle with respiratory disease.

Bovine coronavirus (BCoV) is a known cause of enteric disease in cattle; however, its role in bovine respiratory disease (BRD) is poorly understood, with a dearth of evidence of the detection of the virus in respiratory tract lesions. We coupled histologic evaluation of tracheal and lower airway tissues from 104 calves with BRD in which BCoV was detected in the lungs via PCR followed by direct detection of BCoV by immunohistochemistry and an RNA in situ hybridization assay (ISH; RNAscope technology). RNAscope ISH detected BCoV in respiratory epithelium in more cases than did IHC. Using both methods of direct detection, tracheal epithelial attenuation and identification of the virus within lesions were observed commonly. Our results confirm a role of BCoV in respiratory tract infection and pathology, and show that the virus likely plays a role in the development of BRD.

Molecular-based cross-species evaluation of bovine coronavirus infection in cattle, sheep and goats in Ghana

BackgroundApart from the huge worldwide economic losses often occasioned by bovine coronavirus (BCoV) to the livestock industry, particularly with respect to cattle rearing, continuous surveillance of the virus in cattle and small ruminants is essential in monitoring variations in the virus that could enhance host switching. In this study, we collected rectal swabs from a total of 1,498 cattle, sheep and goats. BCoV detection was based on reverse transcriptase polymerase chain reaction. Sanger sequencing of the partial RNA-dependent RNA polymerase (RdRp) region for postive samples were done and nucleotide sequences were compared with homologous sequences from the GenBank.ResultsThe study reports a BCoV prevalence of 0.3%, consisting of 4 positive cases; 3 goats and 1 cattle. Less than 10% of all the animals sampled showed clinical signs such as diarrhea and respiratory distress except for high temperature which occurred in > 1000 of the animals. However, none of the 4 BCoV positive animals manifested any clinical signs of the infection at the time of sample collection. Bayesian majority-rule cladogram comparing partial and full length BCoV RdRp genes obtained in the study to data from the GenBank revealed that the sequences obtained from this study formed one large monophyletic group with those from different species and countries. The goat sequences were similar to each other and clustered within the same clade. No major variations were thus observed between our isolates and those from elsewhere.ConclusionsGiven that Ghana predominantly practices the extensive and semi-intensive systems of animal rearing, our study highlights the potential for spillover of BCoV to small ruminants in settings with mixed husbandry and limited separation between species.

First report of group A rotavirus and bovine coronavirus associated with neonatal calf diarrhea in the northwest of Argentina.

Group A rotavirus (RVA) and bovine coronavirus (BCoV) are the two main viral enteropathogens associated with neonatal calf diarrhea. The aim of the present survey was to investigate the epidemiology and the role of RVA and BCoV in the presentation of dairy and beef calf diarrhea in Lerma Valley of Salta province, within the Northwest region of Argentina. Stool samples of calves with or without diarrhea younger than 2 months of age were collected from 19 dairy farms and 20 beef farms between the years 2014 and 2016. Stool samples were screened for RVA and BCoV detection by ELISA. Heminested multiplex RT-PCR was used for RVA typing and RT-PCR to confirm BCoV. Positive samples were submitted to sequencing analysis. Bovine RVA and BCoV were circulating in 63% (12/19) and 10.52% (2/19) of the dairy farms, respectively, where 9.5% (46/484) of the calves were positives to RVA and 0.4% (2/484) to BCoV. In beef herds, RVA was detected in 40% (8/20) of the farms and in 6.75% (21/311) of the calves, without positives cases of BCoV. Molecular analysis showed that in dairy farms, G6P[11] and G10P[11] were the prevalent RVA strains, while in beef farms, G10P[11] was the prevalent. The main finding was the detection for the first time of a G15P[11] causing diarrhea in beef calves of Argentina that represents a new alert to be consider for future vaccine updates. Analysis of detected BCoV showed that it is related to the other circulating strains of Argentina.

Pathogen-specific risk factors in acute outbreaks of respiratory disease in calves

Respiratory tract infections (bovine respiratory disease) are a major concern in calf rearing. The objective of this study was to identify pathogen-specific risk factors associated with epidemic respiratory disease in calves. A cross-sectional study was conducted, involving 128 outbreaks (29 dairy, 58 dairy-mixed, and 41 beef) in Belgium (2016–2018). A semiquantitative PCR for 7 respiratory pathogens was done on a pooled nonendoscopic bronchoalveolar lavage sample for each herd. Potential risk factors were collected by questionnaire and derived from the national cattle registration databank. Most outbreaks occurred between October and March, and single and multiple viral infections were detected in 58.6% (75/128) and 13.3% (17/128), respectively. Bovine coronavirus (BCV) was the most frequently isolated virus (38.4%), followed by bovine respiratory syncytial virus (bRSV; 29.4%) and parainfluenzavirus type 3 (PI-3; 8.1%). Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni were detected in 33.3, 41.2, 89.1, and 36.4% of the herds, respectively. Specific risk factors for BCV detection were detection of M. haemolytica [odds ratio (OR) = 2.8 (95% confidence interval = 1.1–7.5)], increasing herd size [OR = 1.3 (1.0–1.8) for each increase with 100 animals] and detection of BCV by antigen ELISA on feces in calves in the last year [OR = 3.6 (1.2–11.1)]. A seasonal effect was shown for bRSV only {more in winter compared with autumn [OR = 10.3 (2.8–37.5)]}. Other factors associated with bRSV were PI-3 detection [OR = 13.4 (2.1–86.0)], prevalence of calves with respiratory disease [OR = 1.02 (1.00–1.04) per 1% increase], and number of days with respiratory signs before sampling [OR = 0.99 (0.98–0.99) per day increase]. Next to its association with BCV, M. haemolytica was more frequently detected in herds with 5 to 10 animals per pen [OR = 8.0 (1.4–46.9)] compared with <5 animals, and in herds with sawdust as bedding [OR = 18.3 (1.8–191.6)]. Also, for H. somni, housing on sawdust was a risk factor [OR = 5.2 (1.2–23.0)]. Purchase of cattle [OR = 2.9 (1.0–8.0)] and housing of recently purchased animals in the same airspace [OR = 5.0 (1.5–16.5)] were risk factors for M. bovis. This study identified pathogen-specific risk factors that might be useful for the development of customized control and prevention and for the design of decision support tools to justify antimicrobial use by predicting the most likely pathogen before sampling results are available.

Specific Detection of Bovine Coronavirus N Protein with TaqMan Probe qRT-PCR

Background: Bovine Coronavirus (BCoV) can cause acute diarrhea in newborn calves and adult cattle. BCoV infection may cause losses to production by reduced weight gain, reduced milk yield. Several methods have been applied to detect and diagnose BCoV. However, each assay has its deficiency. Currently, real-time quantitative PCR (qRT-PCR) has been utilized to identify and quantify many viral pathogens since it is a highly sensitive. However, the technical assay varies due to normalization control of the signal with an internal standard, typically a housekeeping gene. The main objective of the present study to establish a novel TaqMan probe real-time PCR (qRT-PCR) for detecting BCoV.Materials, Methods &amp; Results: The present study was aimed to establish a novel TaqMan probe real-time PCR (qRT-PCR) for detecting bovine coronaviruses (BCoV), and also to develop a diagnostic protocol which simplifies sample collection and processing. One pair of specific primers, one pair of universal primers and a TaqMan probe were designed from the known sequences of conserved nucleocapsid (N) protein of BCoV. Reaction systems of TaqMan qRT-PCR were optimized including concentrations of the primers and probe as well as annealing temperatures. Prior to optimizing the assay, the recombinant plasmids of pMD18-T-BCoV-N were successfully constructed to make standard curves. The sensitivity, specificity and reproducibility were evaluated on the TaqMan qRT-PCR, respectively. A total of 321 feces specimens collected from diarrheic calves were detected with this assay. The results showed the optimized reaction conditions for qRT-PCR were 14.5 μM/L primers, 19.5 μM/L probes and 45.0°C annealing temperatures. The established TaqMan qRT-PCR assay could specially detect BCoV without detecting any other viruses. Its minimum detection limit was 4.72 × 101 copies/μL. However, universal PCR could detect only 4.72 × 103 copies/μL. Its sensitivity was 100-fold stronger than universal PCR. In conclusion, this TaqMan qRT-PCR had excellent specificity, sensitivity and stability with a 100-fold sensitivity stronger than universal PCR. Minimum detection limit was 4.72 × 101 copies/μL. This method was a cost-effective method to diagnose diarrhea and distinguish pathogens in dairy farms.Discussion: In this study, the authors developed a quantitative real-time PCR (qRT-PCR) in this study based on the TaqMan probe of BCoV. This TaqMan qRT-PCR assay selected and used one pair of specific primers (BCoV-qF/BCoV-qR) and a specific TaqMan probe (BCoV-probe) targeting the conserved nucleocapsid (N) gene. The specificity of primers and probes was validated with Primer-BLAST. The specificity of the qRT-PCR was confirmed by the negative control and other six viruses. The findings demonstrated that TaqMan qRT-PCR could only detect BCoV. This verified the qRT-PCR had an excellent specificity. It is obvious that this TaqMan qRT-PCR assay can detect only BCoV with stronger sensitivity and reproducibility than other real-time PCR methods. The sensitivity test indicated the minimum detection limit of the TaqMan qRT-PCR was 4.72 × 101copies/μL, or 47.2 copies/μL. Sensitivity of the TaqMan qRT-PCR assay was increased by 100-fold as compared to universal PCR with a good inter-assay and intra-assay reproducibility. Thereby, based on the high sensitivity of the assay of this qRT-PCR assay it may be a cost-effective method to diagnose BCoV infections and indentify the etiologic agents of diarrhea syndrome in the dairy farms.

Bovine coronavirus in Uruguay: genetic diversity, risk factors and transboundary introductions from neighboring countries.

Bovine coronavirus (BCoV) is a recognized cause of severe neonatal calf diarrhea, with a negative impact on animal welfare, leading to economic losses to the livestock industry. Cattle production is one of the most important economic sectors in Uruguay. The aim of this study was to determine the frequency of BCoV infections and their genetic diversity in Uruguayan calves and to describe the evolutionary history of the virus in South America. The overall detection rate of BCoV in Uruguay was 7.8% (64/824): 7.7% (60/782) in dairy cattle and 9.5% (4/42) in beef cattle. The detection rate of BCoV in samples from deceased and live calves was 10.0% (6/60) and 7.6% (58/763), respectively. Interestingly, there was a lower frequency of BCoV detection in calves born to vaccinated dams (3.3%, 8/240) than in calves born to unvaccinated dams (12.2%, 32/263) (OR: 4.02, 95%CI: 1.81–8.90; p = 0.00026). The frequency of BCoV detection was higher in colder months (11.8%, 44/373) than in warmer months (1.5%, 3/206) (OR: 9.05, 95%CI: 2.77–29.53, p = 0.000013). Uruguayan strains grouped together in two different lineages: one with Argentinean strains and the other with Brazilian strains. Both BCoV lineages were estimated to have entered Uruguay in 2013: one of them from Brazil (95%HPD interval: 2011–2014) and the other from Argentina (95%HPD interval: 2010–2014). The lineages differed by four amino acid changes, and both were divergent from the Mebus reference strain. Surveillance should be maintained to detect possible emerging strains that can clearly diverge at the antigenic level from vaccine strains.Electronic supplementary materialThe online version of this article (10.1007/s00705-019-04384-w) contains supplementary material, which is available to authorized users.

First detection of bovine noroviruses and detection of bovine coronavirus in Australian dairy cattle.

Background and objectiveNoroviruses have been recognised as a significant cause of neonatal enteritis in calves in many countries, but there has been no investigation of their occurrence in Australian cattle. This study aimed to establish whether bovine noroviruses could be detected in faecal samples from Australian dairy cattle. It also sought to determine whether bovine coronaviruses, also associated with neonatal enteritis in calves, could be detected in the same faecal samples.MethodsA selection of faecal samples that were negative for rotaviruses from dairy farms located in three geographically distinct regions of Victoria were pooled and tested by reverse transcription‐PCR for the presence of noroviruses (genogroup III), neboviruses and bovine coronaviruses.Results and conclusionGenetically distinct genogroup III noroviruses were detected in two sample pools from different geographic regions and bovine coronavirus was detected in a third pool of samples. This is the first report of bovine norovirus infection in Australian cattle and suggests that future work is required to determine the significance of these agents as a cause of bovine enteric disease in Australia.

Detection of bovine coronavirus in nasal swab of non-captive wild water deer, Korea.

SummaryBovine coronavirus (BCoV) is a causative agent of respiratory and enteric diseases in cattle and calves. BCoV infection was also evident in captive wild ruminants. Recently, water deer are recognized as the most common wildlife to approach farmhouses and livestock barns in Korea. Therefore, we investigated 77 nasal swab samples from non‐captive wild water deer (Hydropotes inermis) between November 2016 and September 2017 and identified three samples positive for coronavirus, indicating potential for respiratory shedding. The full genomic sequences of the water deer coronavirus were closely related to BCoV (>98%). Therefore, effective biosecurity system in bovine farms would be necessary to prevent contact between farm ruminants and free‐ranging wild water deer.

Detection of Bovine Coronavirus in Healthy and Diarrheic Dairy Calves.

Background BCoV is identified in both healthy and diarrheic calves, complicating its assessment as a primary pathogen.ObjectivesTo investigate the detection rates of bovine coronavirus (BCoV) in feces of healthy and diarrheic calves and to describe the usefulness of a pancoronavirus reverse transcriptase (RT) PCR (PanCoV‐RT‐PCR) assay to identify BCoV in samples of diarrheic calves.AnimalsTwo hundred and eighty‐six calves <21 days. Calves with liquid or semiliquid feces, temperature >39.5°C, and inappetence were considered as cases, and those that had pasty or firm feces and normal physical examination were designated as controls.MethodsProspective case–control study. A specific BCoV‐RT‐PCR assay was used to detect BCoV in fecal samples. Association between BCoV and health status was evaluated by exact and random effect logistic regression. Fecal (n = 28) and nasal (n = 8) samples from diarrheic calves were tested for the presence of BCoV by both the PanCoV‐RT‐PCR and a specific BCoV‐RT‐PCR assays. A Kappa coefficient test was used to assess the level of agreement of both assays.Results BCoV was detected in 55% (157/286) of calves; 46% (66/143), and 64% (91/143) of healthy and diarrheic calves, respectively. Diarrheic calves had higher odds of BCoV presence than healthy calves (OR: 2.16, 95% CI: 1.26 to 3.83, P = 0.004). A good agreement between PanCoV‐RT‐PCR and BCoV‐RT‐PCR to detect BCoV was identified (κ = 0.68, 95% CI: 0.392 to 0.967; P < 0.001).Conclusions and Clinical Importance BCoV was more likely to be detected in diarrheic than healthy calves. The PanCoV‐RT‐PCR assay can be a useful tool to detect CoV samples from diarrheic calves.

Molecular and phylogenetic characterization of bovine coronavirus virus isolated from dairy cattle in Central Region, Thailand.

Bovine coronavirus (BCoV) is involved mainly in enteric infections in cattle. This study reports the first molecular detection of BCoV in a diarrhea outbreak in dairy cows in the Central Region, Thailand. BCoV was molecularly detected from bloody diarrheic cattle feces by using nested PCR. Agarose gel electrophoresis of three diarrheic fecal samples yielded from the 25 samples desired amplicons that were 488 base pairs and sequencing substantiated that have BCoV. The sequence alignment indicated that nucleotide and amino acid sequences, the three TWD isolated in Thailand, were more quite homologous to each other (amino acid at position 39 of TWD1, TWD3 was proline, but TWD2 was serine) and closely related to OK-0514-3strain (virulent respiratory strain; RBCoV).The amino acid sequencing identities among TWD1, TWD2,TWD3, and OK-0514-3 strain were 96.0 to 96.6%, those at which T3I, H65N, D87G, H127Y, andQ136R were changed. In addition, the phylogenetic tree of the hypervariable region S1subunit spike glycoprotein BCoV gene was composed of three major clades by using the 54 sequences generated and showed that the evolutionally distance, TWD1, TWD2, and TWD3 were the isolated group together and most similar to OK-0514-3 strain (98.2 to 98.5% similarity). Further study will develop ELISA assay for serologic detection of winter dysentery disease.

Molecular detection of bovine coronavirus in a diarrhea outbreak in pasture-feeding Nellore steers in southern Brazil.

Worldwide diarrhea outbreaks in cattle herds are more frequently detected in calves being that diarrhea outbreaks in adult cattle are not common. Winter dysentery (WD) is a bovine coronavirus (BCoV) enteric infection that is more reported in Northern hemisphere. Seasonal outbreaks of WD in adult cattle occur mainly in dairy cows. WD has not been described in beef cattle herds of tropical countries. This study describes the molecular detection of BCoV in a diarrhea outbreak in beef cattle steers (Nellore) raised on pasture in Parana, southern Brazil. During the outbreak, the farm had about 600 fattening steers. Watery and bloody diarrhea unresponsive to systemic broad-spectrum antibiotic therapy reveals a morbidity rate of approximately 15 %. The BCoV N gene was identified in 42.9 % (6/14) of the diarrheic fecal samples evaluated by semi-nested polymerase chain reaction (SN-PCR) technique. Other enteric microorganisms occasionally identified in adult cattle and evaluated in this study such as bovine groups A, B, and C rotavirus, bovine viral diarrhea virus, bovine torovirus, aichivirus B, and Eimeria sp. were not identified in the fecal samples. To the best knowledge of the authors, this is the first description of the BCoV diagnosis in fecal samples collected in a diarrhea outbreak in adult beef cattle grazing in the grass in a tropical country.

Seroprevalence of corona virus in dairy herds of Jabalpur region

Bovine Corona virus (BCV) is widely distributed in cattle population in many countries and specific antibodies can be found in a majority of adult animals. Enzyme linked immunosorbant assay (ELISA) is probably the most widely used diagnostic test for BCV serological surveys as it can be applied for mass screening and give reliable results quickly. Study was conducted to determine the seroprevalence of corona virus in dairy herds of Jabalpur region. The ELISA for detection of bovine corona virus was performed on total 90 serum samples collected from dairy animals of organised as well as unorganised dairy farms. 86.66% serum samples were found positive for corona virus antibody with more positive cases in non diarrhoeic animals. Maximal seroprevalence was observed in adult animals. However no correlation was observed in the clinical finding of diarrhoea and presence of bovine corona virus antibody in serum of dairy animals.

A new approach for diagnosis of bovine coronavirus using a reverse transcription recombinase polymerase amplification assay

Bovine coronavirus (BCoV) is an economically significant cause of calf scours and winter dysentery of adult cattle, and may induce respiratory tract infections in cattle of all ages. Early diagnosis of BCoV helps to diminish its burden on the dairy and beef industry. Real-time RT-PCR assay for the detection of BCoV has been described, but it is relatively expensive, requires well-equipped laboratories and is not suitable for on-site screening. A novel assay, using reverse transcription recombinase polymerase amplification (RT-RPA), for the detection of BCoV is developed. The BCoV RT-RPA was rapid (10–20min) and has an analytical sensitivity of 19 molecules. No cross-reactivity with other viruses causing bovine gastrointestinal and/or respiratory infections was observed. The assay performance on clinical samples was validated by testing 16 fecal and 14 nasal swab specimens and compared to real-time RT-PCR. Both assays provided comparable results. The RT-RPA assay was significantly more rapid than the real-time RT-PCR assay. The BCoV RT-RPA constitutes a suitable accurate, sensitive and rapid alternative to the common measures used for BCoV diagnosis. In addition, the use of a portable fluorescence reading device extends its application potential to use in the field and point-of-care diagnosis.

Multiplex semi-nested RT-PCR with exogenous internal control for simultaneous detection of bovine coronavirus and group A rotavirus

Neonatal calf diarrhea is a multi-etiology syndrome of cattle and direct detection of the two major agents of the syndrome, group A rotavirus and Bovine coronavirus (BCoV) is hampered by their fastidious growth in cell culture. This study aimed at developing a multiplex semi-nested RT-PCR for simultaneous detection of BCoV ( N gene) and group A rotavirus ( VP1 gene) with the addition of an internal control (mRNA ND5). The assay was tested in 75 bovine feces samples tested previously for rotavirus using PAGE and for BCoV using nested RT-PCR targeted to RdRp gene. Agreement with reference tests was optimal for BCoV (kappa = 0.833) and substantial for rotavirus detection (kappa = 0.648). the internal control, ND5 mRNA, was detected successfully in all reactions. Results demonstrated that this multiplex semi-nested RT-PCR was effective in the detection of BCoV and rotavirus, with high sensitivity and specificity for simultaneous detection of both viruses at a lower cost, providing an important tool for studies on the etiology of diarrhea in cattle.