Coinfection with porcine epidemic diarrhea virus and Clostridium perfringens type A enhances disease severity in weaned pigs.

Clostridium perfringens is a constituent of the normal gut microbiome in pigs; however, it can potentially cause pre- and post-weaning diarrhea. Nevertheless, the importance of this bacterium as a primary pathogen of diarrhea in piglets needs to be better understood, and the epidemiology of C. perfringens in Korean pig populations is unknown. To study the prevalence and typing of C. perfringens, 203 fecal samples were collected from diarrheal piglets on 61 swine farms during 2021-2022 and examined for the presence of C. perfringens and enteric viruses, including porcine epidemic diarrhea virus (PEDV). We determined that the most frequently identified type of C. perfringens was C. perfringens type A (CPA; 64/203, 31.5%). Among the CPA infections, single infections with CPA (30/64, 46.9%) and coinfections with CPA and PEDV (29/64, 45.3%) were the most common in diarrheal samples. Furthermore, we conducted animal experiments to investigate the clinical outcome of single infections and coinfections with highly pathogenic (HP)-PEDV and CPA in weaned piglets. The pigs infected with HP-PEDV or CPA alone showed mild or no diarrhea, and none of them died. However, animals that were co-inoculated with HP-PEDV and CPA showed more-severe diarrheal signs than those of the singly infected pigs. Additionally, CPA promoted PEDV replication in coinfected piglets, with high viral titers in the feces. A histopathological examination revealed more-severe villous atrophy in the small intestine of coinfected pigs than in singly infected pigs. This indicates a synergistic effect of PEDV and CPA coinfection on clinical disease in weaned piglets.

Rate of Multiple Viral and Bacterial CoInfection(s) in Influenza A/H9N2-Infected Broiler Flocks.

Repeated cases of low pathogenic influenza A/H9N2 virus (IAV/H9N2) have been reported in commercial chickens since its emergence in 1998 in Pakistan. However, recently increased mortality and severe respiratory complications under field conditions have been noticed, suggesting concomitant influenza infections with respiratory viral and/or bacterial pathogens. Therefore, the present study aimed to investigate the presence of IAV/H9N2 coinfecting with multiple viral and bacterial pathogens in broiler chicken flocks. We surveyed 60 broiler flocks with respiratory signs from March through July 2019 in Punjab, Pakistan. Suspected flocks were screened for the presence of IAV using a lateral-flow device. Tracheal, cloacal, and bone marrow samples were collected and further tested for seven viral agents (chicken anemia; Newcastle disease; infectious bronchitis; infectious laryngeotracheitis [ILT]; and IAV subtypes H9, H7, and H5) and three bacterial agents (Mycoplasma gallisepticum; Mycoplasma synovae; Ornithobacterium rhinotracheale [ORT]) using PCR assays. Upon initial screening for IAV, 35/60 (58.3%) flocks tested positive. The coinfection of IAV/H9N2 with other pathogens was detected in 25 (71.4%) flocks and only IAV/H9N2 was detected in 10 (28.6%) flocks out of total positive IAV flocks (n = 35). IAV subtypes H5 and H7, ILT, and ORT were not detected throughout the study period. The detection rate of double, triple, and quadruple combinations of coinfections with IAV/H9N2 were 37% (13 flocks), 26% (9 flocks), 9% (3 flocks), respectively. Higher average mortality (28.5%) was found in broiler chicken flocks coinfected with viral and/or bacterial pathogens than in flocks where only H9 low pathogenic IAV/H9N2 was detected (20.8%). In conclusion, higher circulation of IAV/H9N2 with other viral and bacterial pathogens may contribute to higher production and economic losses at the farm level.

Nota de investigación- Tasa de coinfecciones virales y bacterianas múltiples en parvadas de pollos de engorde infectadas con virus influenza A/H9N2. Se han reportado varios casos del virus de influenza A de baja patogenicidad H9N2 (IAV/H9N2) en pollos comerciales desde su aparición en 1998 en Pakistán. Sin embargo, recientemente se ha observado un aumento de la mortalidad y complicaciones respiratorias graves en condiciones de campo, lo que sugiere infecciones concomitantes de influenza con patógenos respiratorios virales y/o bacterianos. Por lo tanto, el presente estudio tuvo como objetivo investigar la presencia del virus de influenza aviar H9N2 coinfectando con múltiples patógenos virales y bacterianos en parvadas de pollos de engorde. Se evaluaron 60 parvadas de pollos de engorde con signos respiratorios desde marzo hasta julio del año 2019 en Punjab, Pakistán. Las parvadas sospechosas fueron analizadas para detectar la presencia del virus de influenza aviar utilizando un dispositivo de flujo lateral. Se recolectaron muestras traqueales, cloacales y de médula ósea y se analizaron para detectar siete agentes virales (anemia infecciosa aviar, enfermedad de Newcastle, bronquitis infecciosa, laringeotraqueítis infecciosa [ILT] y subtipos H9, H7 y H5 de influenza aviar) y tres agentes bacterianos (Mycoplasma gallisepticum ; Mycoplasma sinovae; Ornithobacterium rhinotracheale [ORT]) utilizando ensayos de PCR. Tras la detección inicial del virus de la influenza aviar, 35/60 (58.3 %) parvadas resultaron positivas. La coinfección del virus de la influenza H9N2 con otros patógenos se detectó en 25 (71.4 %) parvadas y el virus de influenza aviar H9N2 fue detectado solo en 10 (28.6 %) parvadas del total de parvadas positivas (n = 35). Los subtipos H5 y H7 del virus de influenza, ILT y ORT no se detectaron durante el período de estudio. La tasa de detección de combinaciones dobles, triples y cuádruples de coinfecciones con el virus de influenza H9N2 fue del 37 % (13 parvadas), del 26% (9 parvadas), del 9 % (3 parvadas), respectivamente. Se encontró una mortalidad promedio más alta (28.5 %) en lotes de pollos de engorde coinfectados con patógenos virales y/o bacterianos que en lotes donde solo se detectó al virus de influenza H9 de baja patogenicidad (20.8%). En conclusión, una mayor circulación del virus de influenza aviar H9N2 con otros patógenos virales y bacterianos puede contribuir a mayores pérdidas en la producción y económicas a nivel de granja.

Understanding the mechanisms of viral and bacterial coinfections in bovine respiratory disease: a comprehensive literature review of experimental evidence.

Bovine respiratory disease (BRD) is one of the most important diseases impacting the global cattle industry, resulting in significant economic loss. Commonly referred to as shipping fever, BRD is especially concerning for young calves during transport when they are most susceptible to developing disease. Despite years of extensive study, managing BRD remains challenging as its aetiology involves complex interactions between pathogens, environmental and host factors. While at the beginning of the twentieth century, scientists believed that BRD was only caused by bacterial infections ("bovine pasteurellosis"), we now know that viruses play a key role in BRD induction. Mixtures of pathogenic bacteria and viruses are frequently isolated from respiratory secretions of animals with respiratory illness. The increased diagnostic screening data has changed our understanding of pathogens contributing to BRD development. In this review, we aim to comprehensively examine experimental evidence from all existing studies performed to understand coinfections between respiratory pathogens in cattle. Despite the fact that pneumonia has not always been successfully reproduced by in vivo calf modelling, several studies attempted to investigate the clinical significance of interactions between different pathogens. The most studied model of pneumonia induction has been reproduced by a primary viral infection followed by a secondary bacterial superinfection, with strong evidence suggesting this could potentially be one of the most common scenarios during BRD onset. Different in vitro studies indicated that viral priming may increase bacterial adherence and colonization of the respiratory tract, suggesting a possible mechanism underpinning bronchopneumonia onset in cattle. In addition, a few in vivo studies on viral coinfections and bacterial coinfections demonstrated that a primary viral infection could also increase the pathogenicity of a secondary viral infection and, similarly, dual infections with two bacterial pathogens could increase the severity of BRD lesions. Therefore, different scenarios of pathogen dynamics could be hypothesized for BRD onset which are not limited to a primary viral infection followed by a secondary bacterial superinfection.

Bagaza virus and Plasmodium spp. coinfection in red-legged partridges (Alectoris rufa), in Southern Spain 2019.

Flaviviruses such as West Nile (WNV), Usutu (USUV) and Bagaza (BAGV) virus and avian malaria parasites are vector borne pathogens that circulate naturally between avian and mosquito hosts. WNV and USUV and potentially also BAGV constitute zoonoses. Temporal and spatial cocirculation and coinfection with Plasmodium spp., and West Nile virus has been documented in birds and mosquito vectors, and fatally USUV-infected passerines coinfected with Plasmodium spp. had more severe lesions. Also, WNV, USUV and BAGV have been found to cocirculate. Yet little is known about the interaction of BAGV and malaria parasites during consecutive or coinfections of avian hosts. Here we report mortality of free-living red-legged partridges in a hunting estate in Southern Spain that were coinfected with BAGV and Plasmodium spp. The outbreak occurred in the area where BAGV first emerged in Europe in 2010 and where cocirculation of BAGV, USUV and WNV was confirmed in 2011 and 2013. Partridges were found dead in early October 2019. Birds had mottled locally pale pectoral muscles, enlarged, congestive greenish-black tinged livers and enlarged kidneys. Microscopically congestion and predominantly mononuclear inflammatory infiltrates were evident and Plasmodium phanerozoites were present in the liver, spleen, kidneys, muscle and skin. Molecular testing and sequencing detected Plasmodium spp. and BAGV in different tissues of the partridges, and immunohistochemistry confirmed the presence and colocalization of both pathogens in the liver and spleen. Due to the importance of the red-legged partridge in the ecosystem of the Iberian Peninsula and as driver of regional economy such mortalities are of concern. Such outbreaks may reflect climate change related shifts in host, vector and pathogen ecology and interactions that could emerge similarly for other pathogens.

Co-infection of porcine deltacoronavirus and porcine epidemic diarrhoea virus alters gut microbiota diversity and composition in the colon of piglets.

Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhoea virus (PEDV) are the main porcine enteric coronaviruses that cause severe diarrhoea in piglets, posing huge threat to the swine industry. Our previous study verified that the co-infection of PDCoV and PEDV is common in natural swine infections and obviously enhances the disease severity in piglets. However, the effects of co-infection of PDCoV and PEDV on intestinal microbial community are unknown. In current study, the microbial composition and diversity in the colon of piglets were analyzed. Our results showed that both of PDCoV and PEDV were mainly distributed in the small intestines and caused severe damage of ileum but not colon in the co-inoculated piglets. Furthermore, we observed that PDCoV and PEDV co-infection alters the gut microbiota composition at the phylum, family and genus levels. The abundance of Mitsuokella and Collinsella at genus level were significantly increased in PDCoV-PEDV co-infection piglets. Spearman's correlation analysis further suggested that there existed strong positive correlation between Mitsuokella and TNF-α, IL-6 and IL-8 secretion, these two factors may together aggravating the small intestine pathological lesions. These results proved there existed obvious correlation between the disease severity caused by PDCoV-PEDV co-infection and intestinal microbial community.

Interference between avian corona and influenza viruses: The role of the epithelial architecture of the chicken trachea.

Respiratory viral infections are among the major causes of disease in poultry. While viral dual infections are known to occur, viral interference in chicken airways is mechanistically hardly understood. The effects of infectious bronchitis virus (IBV) infection on tissue morphology, sialic acid (sia) expression and susceptibility of the chicken trachea for superinfection with IBV or avian influenza virus (AIV) were studied. In vivo, tracheal epithelium of chickens infected with IBV QX showed marked inflammatory cell infiltration and loss of cilia and goblet cells five days post inoculation. Plant lectin staining indicated that sialic acids redistributed from the apical membrane of the ciliated epithelium and the goblet cell cytoplasm to the basement membrane region of the epithelium. After administration of recombinant viral attachment proteins to slides of infected tissue, retained binding of AIV hemagglutinin, absence of binding of the receptor binding domain (RBD) of IBV M41 and partial reduction of IBV QX RBD were observed. Adult chicken trachea rings were used as ex vivo model to study the effects of IBV QX-induced pathological changes and receptor redistribution on secondary viral infection. AIV H9N2 infection after primary IBV infection was delayed; however, final viral loads reached similar levels as in previously uninfected trachea rings. In contrast, IBV M41 superinfection resulted in 1000-fold lower viral titers over the course of 48 h. In conclusion, epithelial changes in the chicken trachea after viral infection coincide with redistribution and likely specific downregulation of viral receptors, with the extend of subsequent viral interference dependent on viral species.

Severe acute respiratory syndrome coronavirus 2 and influenza A virus co-infection alters viral tropism and haematological composition in Syrian hamsters.

The ongoing coronavirus disease 2019 pandemic and its overlap with the influenza season lead to concerns over severe disease caused by the influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) co-infections. Using a Syrian hamster co-infection model with SARS-CoV-2 and the pandemic influenza virus A/California/04/2009 (H1N1), we found (a) more severe disease in co-infected animals, compared to those infected with influenza virus alone but not SARS-CoV-2 infection alone; (b) altered haematological changes in only co-infected animals and (c) altered influenza virus tropism in the respiratory tracts of co-infected animals. Overall, our study revealed that co-infection with SARS-CoV-2 and influenza virus is associated with altered disease severity and tissue tropism, as well as haematological changes, compared to infection with either virus alone.

Role of Feline Coronavirus as Contributor to Diarrhea in Cats from Breeding Catteries.

(1) Background: Feline coronavirus infection (FCoV) is common in multi-cat environments. A role of FCoV in causing diarrhea is often assumed, but has not been proven. The aim of this study was to evaluate an association of FCoV infection with diarrhea in multi-cat environments. (2) Methods: The study included 234 cats from 37 catteries. Fecal samples were analyzed for FCoV RNA by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Potential co-infections were determined by applying a qPCR panel on different potential enteropathogens and fecal flotation. A fecal scoring system was used to categorize feces as diarrheic or non-diarrheic. (3) Results: Of the 234 cats included, 23 had diarrhea. The prevalence of FCoV infection was 87.0% in cats with and 58.8% in cats without diarrhea. FCoV infection was significantly associated with diarrhea (Odds Ratio (OR) 5.01; p = 0.008). In addition, presence of Clostridium perfringens α toxin (OR 6.93; p = 0.032) and feline panleukopenia virus (OR 13.74; p = 0.004) were associated with an increased risk of diarrhea. There was no correlation between FCoV load and fecal score. FCoV-positive cats with co-infections were not more likely to have diarrhea than FCoV-positive cats without co-infections (p = 0.455). (4) Conclusions: FCoV infection is common in cats from catteries and can be associated with diarrhea.

Diarrhoea outbreak caused by coinfections of Cryptosporidium parvum subtype IIdA20G1 and rotavirus in pre-weaned dairy calves.

Diarrhoea is one of the most important syndromes in neonatal calves. In industrialized nations with intensive animal farming, Cryptosporidium spp. and rotavirus are primary causes of calf diarrhoea, but the role of these and other enteric pathogens is not clear in China. In November and December 2018, a diarrhoea outbreak was identified in over 150 pre-weaned calves on a dairy farm in Heilongjiang Province, northeast China and approximately 60 calves died. To determine the cause of the outbreak, we analyzed 131 faecal samples collected from pre-weaned calves (0-2 months) during (n = 114) and after the outbreak (n = 17). Initially, 10 diarrheic samples during the outbreak and 10 non-diarrheic samples after the outbreak were screened for rotavirus, coronavirus, Escherichia coli K99 and Cryptosporidium parvum by using an enzymatic immunoassay (EIA). In addition, 81 other samples were tested specifically for rotavirus by EIA, and all 131 samples were analyzed for Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi by PCR. The initial EIA analysis identified C. parvum (8/10) and rotavirus (5/10) as the dominant pathogens in calves during the outbreak, while both pathogens were detected at lower frequency after the outbreak (2/10 and 1/10, respectively). Further PCR analyses indicated that the occurrence of C. parvum infections in calves was significantly higher during the outbreak (75.4%, 86/114) than after the outbreak (11.8%, 2/17; odds ratio [OR] = 23.0), and was significantly associated with the occurrence of watery diarrhoea (OR = 15.7) and high oocyst shedding intensity. All C. parvum isolates were identified as subtype IIdA20G1. Among other pathogens analyzed, the overall prevalence of rotavirus, G. duodenalis and E. bieneusi was 19.8% (20/101), 38.9% (51/131) and 42.0% (55/131) in calves, respectively, without significant differences during and after the outbreak. Among the three pathogens, only the rotavirus infection was associated with diarrhoea in calves. More importantly, coinfections of C. parvum and rotavirus were significantly associated with the occurrence of watery diarrhoea in calves and were seen only during the outbreak. Thus, C. parvum subtype IIdA20G1 and rotavirus appeared to be responsible for this diarrhoea outbreak. Control measures should be implemented to effectively prevent the concurrent transmission of these enteric pathogens in pre-weaned dairy calves in China.

Susceptibility of sheep to experimental co-infection with the ancestral lineage of SARS-CoV-2 and its alpha variant.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that has had significant impacts on human health and economies worldwide. SARS-CoV-2 is highly transmissible and the cause of coronavirus disease 2019 in humans. A wide range of animal species have also been shown to be susceptible to SARS-CoV-2 by experimental and/or natural infections. Sheep are a commonly farmed domestic ruminant that have not been thoroughly investigated for their susceptibility to SARS-CoV-2. Therefore, we performed in vitro and in vivo studies which consisted of infection of ruminant-derived cells and experimental challenge of sheep to investigate their susceptibility to SARS-CoV-2. Our results showed that sheep-derived kidney cells support SARS-CoV-2 replication. Furthermore, the experimental challenge of sheep demonstrated limited infection with viral RNA shed in nasal and oral swabs at 1 and 3-days post challenge (DPC); viral RNA was also detected in the respiratory tract and lymphoid tissues at 4 and 8 DPC. Sero-reactivity was observed in some of the principal infected sheep but not the contact sentinels, indicating that transmission to co-mingled naïve sheep was not highly efficient; however, viral RNA was detected in respiratory tract tissues of sentinel animals at 21 DPC. Furthermore, we used a challenge inoculum consisting of a mixture of two SARS-CoV-2 isolates, representatives of the ancestral lineage A and the B.1.1.7-like alpha variant of concern, to study competition of the two virus strains. Our results indicate that sheep show low susceptibility to SARS-CoV-2 infection and that the alpha variant outcompeted the lineage A strain.

Serological evidence of SARS-CoV-2 and co-infections in stray cats in Spain.

A new coronavirus known as SARS-CoV-2 emerged in Wuhan in 2019 and spread rapidly to the rest of the world causing the pandemic disease named coronavirus disease of 2019 (COVID-19). Little information is known about the impact this virus can cause upon domestic and stray animals. The potential impact of SARS-CoV-2 has become of great interest in cats due to transmission among domestic cats and the severe phenotypes described recently in a domestic cat. In this context, there is a public health warning that needs to be investigated in relation with the epidemiological role of this virus in stray cats. Consequently, in order to know the impact of the possible transmission chain, blood samples were obtained from 114 stray cats in the city of Zaragoza (Spain) and tested for SARS-CoV-2 and other selected pathogens susceptible to immunosuppression including Toxoplasma gondii, Leishmania infantum, feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) from January to October 2020. Four cats (3.51%), based on enzyme-linked immunosorbent assay (ELISA) using the receptor binding domain (RBD) of Spike antigen, were seroreactive to SARS-CoV-2. T. gondii, L. infantum, FeLV and FIV seroprevalence was 12.28%, 16.67%, 4.39% and 19.30%, respectively. Among seropositive cats to SARS-CoV-2, three cats were also seropositive to other pathogens including antibodies detected against T. gondii and FIV (n = 1); T. gondii (n = 1); and FIV and L. infantum (n = 1). The subjects giving positive for SARS-CoV-2 were captured in urban areas of the city in different months: January 2020 (2/4), February 2020 (1/4) and July 2020 (1/4). This study revealed, for the first time, the exposure of stray cats to SARS-CoV-2 in Spain and the existence of concomitant infections with other pathogens including T. gondii, L. infantum and FIV, suggesting that immunosuppressed animals might be especially susceptible to SARS-CoV-2 infection.

Coinfection of porcine deltacoronavirus and porcine epidemic diarrhea virus increases disease severity, cell trophism and earlier upregulation of IFN-α and IL12.

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) cause an enteric disease characterized by diarrhea clinically indistinguishable. Both viruses are simultaneously detected in clinical cases, but a study involving the co-infection has not been reported. The study was therefore conducted to investigate the disease severity following a co-infection with PEDV and PDCoV. In the study, 4-day-old pigs were orally inoculated with PEDV and PDCoV, either alone or in combination. Following challenge, fecal score was monitored on a daily basis. Fecal swabs were collected and assayed for the presence of viruses. Three pigs per group were necropsied at 3 and 5 days post inoculation (dpi). Microscopic lesions and villous height to crypt depth (VH:CD) ratio, together with the presence of PEDV and PDCoV antigens, were evaluated in small intestinal tissues. Expressions of interferon alpha (IFN-α) and interleukin 12 (IL12) were investigated in small intestinal mucosa. The findings indicated that coinoculation increased the disease severity, demonstrated by significantly prolonged fecal score and virus shedding and decreasing VH:CD ratio in the jejunum compared with pigs inoculated with either PEDV or PDCoV alone. Notably, in single-inoculated groups, PEDV and PDCoV antigens were detected only in villous enterocytes wile in the coinoculated group, PDCoV antigen was detected in both villous enterocytes and crypts. IFN-α and IL12 were significantly up-regulated in coinoculated groups in comparison with single-inoculated groups. In conclusion, co-infection with PEDV and PDCoV exacerbate clinical signs and have a synergetic on the regulatory effect inflammatory cytokines compared to a single infection with either virus.

Co-infections of chickens with avian influenza virus H9N2 and Moroccan Italy 02 infectious bronchitis virus: effect on pathogenesis and protection conferred by different vaccination programmes.

Since the emergence of low pathogenic avian influenza (LPAI) H9N2 viruses in Morocco in 2016, severe respiratory problems have been encountered in the field. Infectious bronchitis virus (IBV) is often detected together with H9N2, suggesting disease exacerbation in cases of co-infections. This hypothesis was therefore tested and confirmed in laboratory conditions using specific-pathogen-free chickens. Most common field vaccine programmes were then tested to compare their efficacies against these two co-infecting agents. IBV γCoV/chicken/Morocco/I38/2014 (Mor-IT02) and LPAI virus A/chicken/Morocco/SF1/2016 (Mor-H9N2) were thus inoculated to commercial chickens. We showed that vaccination with two heterologous IBV vaccines (H120 at day one and 4/91 at day 14 of age) reduced the severity of clinical signs as well as macroscopic lesions after simultaneous experimental challenge. In addition, LPAI H9N2 vaccination was more efficient at day 7 than at day 1 in limiting disease post simultaneous challenge.RESEARCH HIGHLIGHTS Simultaneous challenge with IBV and AIV H9N2 induced higher pathogenicity in SPF birds than inoculation with IBV or AIV H9N2 alone.Recommended vaccination programme in commercial broilers to counter Mor-IT02 IBV and LPAIV H9N2 simultaneous infections: IB live vaccine H120 (d1), AIV H9N2 inactivated vaccine (d7), IB live vaccine 4-91 (d14).

Influenza C and D viral load in cattle correlates with bovine respiratory disease (BRD): Emerging role of orthomyxoviruses in the pathogenesis of BRD.

Bovine respiratory disease (BRD) is the costliest disease affecting the cattle industry globally. Orthomyxoviruses, influenza C virus (ICV) and influenza D virus (IDV) have recently been implicated to play a role in BRD. However, there are contradicting reports about the association of IDV and ICV to BRD. Using the largest cohort study (cattle, n = 599) to date we investigated the association of influenza viruses in cattle with BRD. Cattle were scored for respiratory symptoms and pooled nasal and pharyngeal swabs were tested for bovine viral diarrhea virus, bovine herpesvirus 1, bovine respiratory syncytial virus, bovine coronavirus, ICV and IDV by real-time PCR. Cattle that have higher viral loads of IDV and ICV also have greater numbers of co-infecting viruses than controls. More strikingly, 2 logs higher IDV viral RNA in BRD-symptomatic cattle that are co-infected animals than those infected with IDV alone. Our results strongly suggest that ICV and IDV may be significant contributors to BRD.

Development of a TaqMan-probe-based multiplex real-time PCR for the simultaneous detection of emerging and reemerging swine coronaviruses.

With the outbreak of the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, coronaviruses have become a global research hotspot in the field of virology. Coronaviruses mainly cause respiratory and digestive tract diseases, several coronaviruses are responsible for porcine diarrhea, such as porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and emerging swine acute diarrhea syndrome coronavirus (SADS-CoV). Those viruses have caused huge economic losses and are considered as potential public health threats. Porcine torovirus (PToV) and coronaviruses, sharing similar genomic structure and replication strategy, belong to the same order Nidovirales. Here, we developed a multiplex TaqMan-probe-based real-time PCR for the simultaneous detection of PEDV, PDCoV, PToV, and SADS-CoV for the first time. Specific primers and TaqMan fluorescent probes were designed targeting the ORF1a region of PDEV, PToV, and SADS-CoV and the ORF1b region of PDCoV. The method showed high sensitivity and specificity, with a detection limit of 1 × 102 copies/µL for each pathogen. A total of 101 clinical swine samples with signs of diarrhea were analyzed using this method, and the result showed good consistency with conventional reverse transcription PCR (RT-PCR). This method improves the efficiency for surveillance of these emerging and reemerging swine enteric viruses and can help reduce economic losses to the pig industry, which also benefits animal and public health.