Canine Infectious Respiratory Disease Complex Research Articles

Rapid diagnosis of canine respiratory coronavirus, canine influenza virus, canine distemper virus and canine parainfluenza virus with a Taqman probe-based multiplex real-time PCR

Canine Infectious Respiratory Disease Complex (CIRDC) is a highly infectious diseases. Canine respiratory coronavirus (CRCoV), Canine influenza virus (CIV), Canine distemper virus (CDV), and Canine parainfluenza virus (CPiV) are crucial pathogens causing CIRDC. Due to the similar clinical symptoms induced by these viruses, differential diagnosis based solely on symptoms can be challenging. In this study, a multiplex real-time PCR assay was developed for detecting the four RNA viruses of CIRDC. Specific primers and probes were designed to target M gene of CRCoV, M gene of CIV, N gene of CDV and NP gene of CPiV. The detection limit is 10 copies/μL for CIV or CRCoV, while the detection limit of CDV or CPiV is 100 copies/μL. Intra-group and inter-group repeatability coefficient of variation (CV) were both less than 2 %. A total of 341 clinical canine samples were analyzed, and the results indicated that the method developed in our study owns a good consistency and better specificity compared with the conventional reverse transcription PCR. This study provides a new method to enable the simultaneous detection of all four pathogens in a single reaction, improving the efficiency for monitoring the prevalence of four viruses in CIRDC, which benefits the control of CIRDC.

Development of a Large-Scale Pathogen Screening Test for the Biosafety Evaluation of Canine Mesenchymal Stem Cells

BackgroundThe action of mesenchymal stem cells (MSCs) is the subject of intense research in the field of regenerative medicine, including their potential use in companion animals, such as dogs. To ensure the safety of canine MSC batches for their application in regenerative medicine, a quality control test must be conducted in accordance with Good Manufacturing Practices (GMP). Based on guidance provided by the European Medicines Agency, this study aimed to develop and validate a highly sensitive and robust, nucleic acid-based test panel for the detection of various canine pathogens. Analytical sensitivity, specificity, amplification efficiency, and linearity were evaluated to ensure robust assessment. Additionally, viable spike-in controls were used to control for optimal nucleic acid extraction. The conventional PCR-based and real-time PCR-based pathogen assays were evaluated in a real-life setting, by direct testing MSC batches.ResultsThe established nucleic acid-based assays displayed remarkable sensitivity, detecting 100–1 copies/reaction of template DNA. They also exhibited high specificity and efficiency. Moreover, highly effective nucleic acid isolation was confirmed by the sensitive detection of spike-in controls. The detection capacity of our optimized and validated methods was determined by direct pathogen testing of nine MSC batches that displayed unusual phenotypes, such as reduced cell division or other deviating characteristics. Among these MCS batches of uncertain purity, only one tested negative for all pathogens. The direct testing of these samples yielded positive results for important canine pathogens, including tick-borne disease-associated species and viral members of the canine infectious respiratory disease complex (CIRDC). Notably, samples positive for the etiological agents responsible for enteritis (CPV), leptospirosis (Leptospira interrogans), and neosporosis (Neospora caninum) were also identified. Furthermore, we conducted biosafety evaluation of 12 MSC batches intended for therapeutic application. Eleven MSC batches were found to be free of extraneous agents, and only one tested positive for a specific pathogen, namely, canine parvovirus.ConclusionIn this study, we established and validated reliable, highly sensitive, and accurate nucleic acid-based testing methods for a broad spectrum of canine pathogens.

Predominance of Canine Parainfluenza Virus and Mycoplasma in Canine Infectious Respiratory Disease Complex in Dogs.

Canine infectious respiratory disease complex (CIRDC) is caused by different viruses and bacteria. Viruses associated with CIRDC include canine adenovirus type 2 (CAV-2), canine distemper virus (CDV), canine influenza virus (CIV), canine herpesvirus type 1 (CHV-1), canine respiratory coronavirus (CRCoV), and canine parainfluenza virus (CPIV). Bacteria associated with CIRDC include Bordetella bronchiseptica, Streptococcus equi subspecies zooepidemicus (S. zooepidemicus), and Mycoplasma spp. The present study examined the prevalence of CIRDC pathogens in specimens received by a Veterinary Diagnostic Laboratory in Georgia, USA., from 2018 to 2022. Out of 459 cases, viral agents were detected in 34% of cases and bacterial agents were detected in 58% of cases. A single pathogen was detected in 31% of cases, while two or more pathogens were identified in 24% of cases. The percentages of viral agents identified were CAV-2 (4%), CDV (3%), CPIV (16%), CRCoV (7%), and CIV (2%). The percentages of bacterial agents were B. bronchiseptica (10%), Mycoplasma canis (24%), Mycoplasma cynos (21%), and S. zooepidemicus (2%). Over the five-year period, the positive cases ranged from 2-4% for CAV-2, 1-7% for CDV, 1-4% for CHV-1, 9-22% for CPIV, 4-13% for CRCoV, and 1-4% for CIV. Overall, the most prevalent pathogens associated with CIRDC were CPIV, M. canis, and M. cynos.

Canine respiratory disease outbreak involving canine pneumovirus at an American animal shelter

AbstractAn outbreak of canine infectious respiratory disease complex was attributed to canine pneumovirus in a municipal animal shelter in the northeastern United States. The outbreak comprised eight fatalities and over 100 cases in dogs of all signalments. Cases were characterised by mild to severe signs indicating upper airway, lower airway, and systemic involvement. Nasopharyngeal samples submitted to one commercial and one university laboratory for quantitative polymerase chain reaction testing revealed several canine infectious respiratory disease complex pathogens. However, only the university lab detected canine pneumovirus: multiple nasopharyngeal and lung samples tested positive. Vaccination status, lack of response to antimicrobials, and improvement after implementation of canine infectious respiratory disease outbreak management strategies tailored to canine pneumovirus characteristics indicate substantial involvement of this virus. The outbreak resolved within 1 month of adherence to clean break procedures, limitation of incoming dogs, and deep clean of the premises. This case highlights the difficulty in reliably detecting canine pneumovirus and how population management deficits permit canine infectious respiratory disease complex outbreaks.

Canine respiratory coronavirus in Thailand undergoes mutation and evidences a potential putative parent for genetic recombination.

Canine respiratory coronavirus (CRCoV) is associated with canine infectious respiratory disease complex. Although its detection has been reported worldwide, the genomic characteristics and evolutionary patterns of this virus remain poorly defined. In this study, 21 CRCoV sequences obtained from dogs in Thailand during two episodes (2013-2015, group A; 2021-2022, group B) were characterized and analyzed. The genomic characteristics of Thai CRCoVs changed from 2013 to 2022 and showed a distinct phylogenetic cluster. Phylogenetic analysis of the spike (S) genes divided the analyzed CRCoV strains into five clades. The full-length genome characterization revealed that all Thai CRCoVs possessed a nonsense mutation within the nonstructural gene located between the S and envelope genes, leading to a truncated putative nonstructural protein. Group B Thai CRCoV strains represented the signature nonsynonymous mutations in the S gene that was not identified in group A Thai CRCoVs, suggesting the ongoing evolutionary process of Thai CRCoVs. Although no evidence of recombination of Thai CRCoV strains was found, our analysis identified one Thai CRCoV strain as a potential parent virus for a CRCoV strain found in the United States. Selective pressure analysis of the hypervariable S region indicated that the CRCoV had undergone purifying selection during evolution. Evolutionary analysis suggested that the CRCoV was emerged in 1992 and was first introduced in Thailand in 2004, sharing a common ancestor with Korean CRCoV strains. These findings regarding the genetic characterization and evolutionary analysis of CRCoVs add to the understanding of CRCoVs. IMPORTANCE Knowledge of genomic characterization of the CRCoV is still limited and its evolution remains poorly investigated. We, therefore, investigated the full-length genome of CRCoV in Thailand for the first time and analyzed the evolutionary dynamic of CRCoV. Genomic characterization of Thai CRCoV strains revealed that they possess unique genome structures and have undergone nonsynonymous mutations, which have not been reported in previously described CRCoV strains. Our work suggests that the Thai CRCoVs were not undergone mutation through genetic recombination for their evolution. However, one Thai CRCoV strain PP158_THA_2015 was found to be a potential parent virus for the CRCoV strains found in the United States. This study provides an understanding of the genomic characterization and highlights the signature mutations and ongoing evolutionary process of CRCoV that could be crucial for monitoring in the future.

Development and Validation of a Panel of One-Step Four-Plex qPCR/RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Pathogens Associated with Canine Infectious Respiratory Disease Complex.

Canine infectious respiratory disease complex (CIRDC) is the primary cause of respiratory disease in the canine population and is caused by a wide array of viruses and bacterial pathogens with coinfections being common. Since its recognition in late 2019, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been reported to cause respiratory disease in dogs. Therefore, the rapid detection and differentiation of SARS-CoV-2 from other common viral and bacterial agents is critical from a public health standpoint. Here, we developed and validated a panel of four one-step multiplex qPCR/RT-qPCR assays for the detection and identification of twelve pathogens associated with CIRDC (canine adenovirus-2, canine distemper virus, canine herpesvirus-1, canine influenza A virus, canine parainfluenza virus, canine pneumovirus, canine respiratory coronavirus, SARS-CoV-2, Bordetella bronchiseptica, Streptococcus equi subsp. zooepidemicus, Mycoplasma cynos, and M. canis), as well as the identification of three main CIV subtypes (i.e., H3N2, H3N8, and H1N1). All developed assays demonstrated high specificity and analytical sensitivity. This panel was used to test clinical specimens (n = 76) from CIRDC-suspected dogs. M. canis, M. cynos, and CRCoV were the most frequently identified pathogens (30.3%, 25.0%, and 19.7% of samples, respectively). The newly emerging pathogens CPnV and SARS-CoV-2 were detected in 5.3% of samples and coinfections were identified in 30.3%. This new multiplex qPCR/RT-qPCR panel is the most comprehensive panel developed thus far for identifying CIRDC pathogens, along with SARS-CoV-2.

Re-emergence of canine influenza in the United States of America; a call for One Health approach; a letter to the editor

Re-emergence of canine influenza in the United States of America; a call for One Health approach; a letter to the editor

Bordetella bronchiseptica and Bordetella pertussis: Similarities and Differences in Infection, Immuno-Modulation, and Vaccine Considerations.

Bordetella pertussis and Bordetella bronchiseptica belong to the genus Bordetella, which comprises 14 other species. B. pertussis is responsible for whooping cough in humans, a severe infection in children and less severe or chronic in adults. These infections are restricted to humans and currently increasing worldwide. B. bronchiseptica is involved in diverse respiratory infections in a wide range of mammals. For instance, the canine infectious respiratory disease complex (CIRDC), characterized by a chronic cough in dogs. At the same time, it is increasingly implicated in human infections, while remaining an important pathogen in the veterinary field. Both Bordetella can evade and modulate host immune responses to support their persistence, although it is more pronounced in B. bronchiseptica infection. The protective immune responses elicited by both pathogens are comparable, while there are important characteristics in the mechanisms that differ. However, B. pertussis pathogenesis is more difficult to decipher in animal models than those of B. bronchiseptica because of its restriction to humans. Nevertheless, the licensed vaccines for each Bordetella are different in terms of formulation, route of administration and immune responses induced, with no known cross-reaction between them. Moreover, the target of the mucosal tissues and the induction of long-lasting cellular and humoral responses are required to control and eliminate Bordetella. In addition, the interaction between both veterinary and human fields are essential for the control of this genus, by preventing the infections in animals and the subsequent zoonotic transmission to humans.

Mucosal Vaccination with Live Attenuated Bordetella bronchiseptica Protects against Challenge in Wistar Rats.

Bordetella bronchiseptica (Bb) is a Gram-negative bacterium responsible for canine infectious respiratory disease complex (CIRDC). Several vaccines targeting this pathogen are currently licensed for use in dogs, but their mechanism of action and the correlates of protection are not fully understood. To investigate this, we used a rat model to examine the immune responses induced and the protection conferred by a canine mucosal vaccine after challenge. Wistar rats were vaccinated orally or intranasally on D0 and D21 with a live attenuated Bb vaccine strain. At D35, the rats of all groups were inoculated with 103 CFU of a pathogenic strain of B. bronchiseptica. Animals vaccinated via either the intranasal or the oral route had Bb-specific IgG and IgM in their serum and Bb-specific IgA in nasal lavages. Bacterial load in the trachea, lung, and nasal lavages was lower in vaccinated animals than in non-vaccinated control animals. Interestingly, coughing improved in the group vaccinated intranasally, but not in the orally vaccinated or control group. These results suggest that mucosal vaccination can induce mucosal immune responses and provide protection against a Bb challenge. This study also highlights the advantages of a rat model as a tool for studying candidate vaccines and routes of administration for dogs.

N gene based detection and phylogenetic analysis of canine morbillivirus in dogs

Canine distemper, a highly fatal systemic disease in domestic dogs and wild carnivores, has the second highest mortality rate after rabies and is responsible for a large number of animal deaths around the world. It is considered a major pathogen in the canine infectious respiratory disease complex. This paper reports the finding of a study conducted to detect and characterise canine distemper virus (Canine Morbillivirus) based on N gene. A total of 59 samples collected from cases of respiratory infections in dogs were subjected to N gene based reverse transcriptase polymerase chain reaction (RT-PCR) and eleven of them (18.64 per cent) were found positive. Sequencing and phylogenetic analysis revealed that all the canine distemper viruses obtained in the present study were related to Indian strains that were previously reported. However, the viruses from the same district were similar among themselves.

Molecular identification and occurrence of bordetellosis among dogs in northern Kerala

Bordetella bronchiseptica is a gram–negative bacterium with a broad host range that is most commonly seen in animals kept in close quarters. It is considered to be one of the main causes of the canine infectious respiratory disease complex (CIRDC). In this study, 45 dogs with clinical signs suggestive of CIRDC were selected and nasal/ oropharyngeal swabs were collected aseptically. Total DNA was extracted from the swabs and subjected to polymerase chain reaction (PCR) targeting alc gene of Bordetella spp. for confirmation. Nine animals (20 per cent) tested positive for the presence of Bordetella antigen. All the dogs tested positive for Bordetellosis were less than one year of age. Most common clinical signs in positive dogs were cough and nasal discharge, and the majority of the bordetellosis positive dogs were from multiple dog houses.

Serum 25-hydroxyvitamin D concentration and infectious respiratory disease complex in shelter dogs.

BackgroundHypovitaminosis D is a risk factor for the development of respiratory infections in humans and repletion can be protective.ObjectivesDetermine if serum 25‐hydroxyvitamin (OH)D concentrations are lower in shelter dogs and if 25(OH)D concentrations are associated with clinical signs of canine infectious respiratory disease complex (CIRDC) or with time in the shelter.AnimalsOne hundred forty‐six shelter dogs (clinically ill n = 36, apparently healthy n = 110) and 23 nonshelter control dogs.MethodsProspective cohort study. Shelter dogs were grouped as clinically ill or apparently healthy based on the presence or absence, respectively, of clinical signs associated with CIRDC. Serum 25(OH)D concentrations were measured with a competitive chemiluminesence immunoassay. Nucleic acids of agents associated with the CIRDC were amplified by polymerase chain reaction assays.ResultsThe concentration of 25(OH)D was 7.3 ng/mL (4.5‐9.9, 95% confidence interval [CI]) lower in dogs with signs of CIRDC than apparently healthy shelter dogs (t(142) = 2.0, P = .04). Dogs positive for DNA of canine herpesvirus (CHV)‐1 had serum 25(OH)D concentrations 14.9 ng/mL (−3.7 to 29.6, 95% CI) lower than dogs that were negative (t(137) = 2.0, P = .04). Serum 25(OH)D concentrations in shelter dogs were not different from control dogs (t(45) = −1.4, P = .17). Serum 25(OH)D concentration was not associated with duration of time in the shelter (F(1, 140) = 1.7, P = .2, R 2 = 0.01).Conclusion and Clinical ImportanceVitamin D could have a role in acute respiratory tract infections in shelter dogs.

Canine Respiratory Coronavirus: A Naturally Occurring Model of COVID-19?

Discovered in 2003 at the Royal Veterinary College, London, canine respiratory coronavirus (CRCoV) is a betacoronavirus of dogs and major cause of canine infectious respiratory disease complex. Generally causing mild clinical signs of persistent cough and nasal discharge, the virus is highly infectious and is most prevalent in rehoming shelters worldwide where dogs are often closely housed and infections endemic. As the world grapples with the current COVID-19 pandemic, the scientific community is searching for a greater understanding of a novel virus infecting humans. Similar to other betacoronaviruses, SARS-CoV-2 appears to have crossed the species barrier, most likely from bats, clearly reinforcing the One Health concept. Veterinary pathologists are familiar with coronavirus infections in animals, and now more than ever this knowledge and understanding, based on many years of veterinary research, could provide valuable answers for our medical colleagues. Here I review the early research on CRCoV where seroprevalence, early immune response, and pathogenesis are some of the same key questions being asked by scientists globally during the current SARS-CoV-2 pandemic.

Analysis of the lung microbiota in dogs with Bordetella bronchiseptica infection and correlation with culture and quantitative polymerase chain reaction

Infection with Bordetella bronchiseptica (Bb), a pathogen involved in canine infectious respiratory disease complex, can be confirmed using culture or qPCR. Studies about the canine lung microbiota (LM) are recent, sparse, and only one paper has been published in canine lung infection. In this study, we aimed to compare the LM between Bb infected and healthy dogs, and to correlate sequencing with culture and qPCR results. Twenty Bb infected dogs diagnosed either by qPCR and/or culture and 4 healthy dogs were included. qPCR for Mycoplasma cynos (Mc) were also available in 18 diseased and all healthy dogs. Sequencing results, obtained from bronchoalveolar lavage fluid after DNA extraction, PCR targeting the V1–V3 region of the 16S rDNA and sequencing, showed the presence of Bb in all diseased dogs, about half being co-infected with Mc. In diseased compared with healthy dogs, the β-diversity changed (P = 0.0024); bacterial richness and α-diversity were lower (P = 0.012 and 0.0061), and bacterial load higher (P = 0.004). Bb qPCR classes and culture results correlated with the abundance of Bb (r = 0.71, P < 0.001 and r = 0.70, P = 0.0022). Mc qPCR classes also correlated with the abundance of Mc (r = 0.73, P < 0.001). Bb infection induced lung dysbiosis, characterized by high bacterial load, low richness and diversity and increased abundance of Bb, compared with healthy dogs. Sequencing results highly correlate with qPCR and culture results showing that sequencing can be reliable to identify microorganisms involved in lung infectious diseases.

Antimicrobial Prescribing Practices in Small Animal Emergency and Critical Care.

Background: Antimicrobial use contributes to emergence of antimicrobial resistance. It was hypothesized that antimicrobial prescribing behavior varies between the emergency (ER) and critical care (CC) services in a veterinary teaching hospital. This study aimed to: (i) describe antimicrobial prescribing patterns in the ER and CC services; (ii) assess adherence to stewardship principles; (iii) evaluate the prevalence of multidrug resistant (MDR) bacterial isolates.Methods: Institution electronic medical records were queried for all antimicrobial prescriptions from the ER and CC services between 1/1/2017 and 12/31/2017. Prescriptions were manually reviewed, and the following data recorded: drug, dosage, duration, diagnosis, outcome, hospitalization duration, culture submission, and susceptibility results.Results: There were 5,091 ER visits, of which 3,125 were not transferred to another service. Of these emergency visits, 516 (16.5%) resulted in 613 antimicrobial drug prescriptions. The most commonly prescribed drugs for the ER were amoxicillin/clavulanate (n = 243, 39.6%), metronidazole (n = 146, 23.8%), and ampicillin/sulbactam (n = 55, 9.0%). The most common reasons for antimicrobial prescriptions were skin disease (n = 227, 37.0%), gastrointestinal disease (n = 173, 28.2%), and respiratory disease (n = 50, 8.2%). For ER patients 18 cultures were submitted, equivalent to a 3.5% submission rate. The CC service managed 311 case visits for 822 patient days. Of these, 133 case visits (42.7%) resulted in 340 prescriptions. The most commonly prescribed drugs for the CC service were ampicillin/sulbactam (n = 103, 30.3%), enrofloxacin (n = 75, 22.1%), and metronidazole (n = 59, 17.4%). The most common reasons for antimicrobial prescriptions were gastrointestinal disease (n = 106, 31.2%), respiratory disease (n = 71, 20.9%), and sepsis (n = 61, 17.9%). On the CC service, 46 patients had ≥1 culture submitted, equivalent to a 34.6% submission rate. Of patients prescribed antimicrobials, 13/38 (34%) with urinary tract disease, 2/28 (7%) with pneumonia, 1/11 (9%) with canine infectious respiratory disease complex and 2/8 (25%) with feline upper respiratory infection were compliant with published guidelines.Conclusions: Antimicrobial prescription was common in both ER and CC services and followed similar patterns. Adherence to published guidelines for urinary and respiratory infections was poor.

Cross-sectional investigation and risk factor analysis of community-acquired and hospital-associated canine viral infectious respiratory disease complex

Canine infectious respiratory disease complex (CIRDC) is associated with multiple factors. The possible transmission source can be via community-acquired infection (CAI) or hospital-associated infection (HAI), but the variable factors within these two routes are not well described. This study aimed to (i) investigate a cross-sectional incidence of canine respiratory viruses, including influenza (CIV), parainfluenza, distemper (CDV), respiratory coronavirus (CRCoV), adenovirus-2, and herpesvirus, in respiratory-diseased dogs, and (ii) analyze the possibly related risk factors. In total 209 dogs with respiratory illness, consisting of 133 CAI and 76 HAI dogs, were studied. Both nasal and oropharyngeal swabs were sampled from each dog and subjected for CIRDC virus detection using multiplex PCRs. Common six viruses associated with CIRDC were detected in both groups with CIV and CRCoV being predominantly found. Only CDV was significantly more prevalent in CAI than HAI dogs. Multiple virus detections were found in 81.2% and 78.9% of CAI and HAI dogs, respectively. Co-detection of CIV and CRCoV was represented the highest proportion and most often found with other CIRD viruses. Moreover, the clinical severity level was notably related to the age of infected dogs, but not to the vaccination status, sex and transmission route. Since healthy or control dogs were not included in this study, the prevalence of the CIRD virus infections could not be assessed.

Viral molecular and pathological investigations of Canid herpesvirus 1 infection associated respiratory disease and acute death in dogs

Abstract Canid herpesvirus 1 (CaHV-1) is a member of the canine infectious respiratory disease complex (CIRDC). The outcome of CaHV-1 infection can be occasionally fatal. So far, no information on CaHV-1 circulation in Thailand has been reported resulting in a lack of preventive strategies. In this study, nasal (NS) and oropharyngeal (OS) swabs were collected from 100 live dogs with respiratory distress. Among them, 23 pleural effusions were aspirated. A panel of CIRDC-associated viruses was screened by (RT)-PCR, including CaHV-1, CIV, CPIV, CDV, CRCoV and CAdV-2, for all collected samples. The CaHV-1 was detected in 32 dogs. Additionally, CaHV-1 was consistently detected in six pleural effusions. Most CaHV-1 infected dogs were over 5 years of age (43.8%) and expressed a mild nasal discharge. Pathological results of four three-month-old puppies, naturally moribund from respiratory disease, revealed a severe multifocal necrotic-hemorrhagic disease in several organs without pathognomonic inclusion bodies. They were only found to be CaHV-1 positive by PCR. Phylogenetic analysis demonstrated concordant results of CaHV-1 circulation in Thailand. Although mostly found as a co-infection with other CIRDC viruses (68.8%) it also occurred alone. Therefore, rapid ante-mortem diagnosis might facilitate the investigation of unclassical CaHV-1 infection, which is fatal in neonates and causes illness in annually core-vaccinated adults.

Development and application of multiplex PCR assays for detection of virus-induced respiratory disease complex in dogs.

Canine infectious respiratory disease complex (CIRDC) viruses have been detected in dogs with respiratory illness. Canine influenza virus (CIV), canine parainfluenza virus (CPIV), canine distemper virus (CDV), canine respiratory coronavirus (CRCoV), canine adenovirus type 2 (CAdV-2) and canine herpesvirus 1 (CaHV-1), are all associated with the CIRDC. To allow diagnosis, two conventional multiplex polymerase chain reactions (PCR) were developed to simultaneously identify four RNA and two DNA viruses associated with CIRDC. The two multiplex PCR assays were then validated on 102 respiratory samples collected from 51 dogs with respiratory illness by sensitivity and specificity determination in comparison to conventional simplex PCR and a rapid three-antigen test kit. All six viruses were detected in either individual or multiple infections. The developed multiplex PCR assays had a >87% sensitivity and 100% specificity compared to their simplex counterpart. Compared to the three-antigen test kit, the multiplex PCR assays yielded 100% sensitivity and more than 83% specificity for detection of CAdV-2 and CDV, but not for CIV. Therefore, the developed multiplex PCR modalities were able to simultaneously diagnose a panel of CIRDC viruses and facilitated specimen collection through being suitable for use of nasal or oral samples.