Canine Infectious Respiratory Disease Complex (CIRDC), commonly known as kennel cough, is a highly prevalent and contagious respiratory condition in the dog population worldwide (1, 2). Outbreaks most commonly occur in settings where dogs are housed or gathered nearby, such as boarding facilities, animal shelters, and dog daycares (3, 4). CIRDC manifests as a multifactorial syndrome characterized by acute respiratory signs, including coughing, nasal or ocular discharge, sneezing, fever, and respiratory distress, that generally lasts up to 2 weeks (1). The disease is primarily transmitted through direct dog-to-dog contact and airborne transmission via respiratory secretions (2). Most infections are self-limiting, with affected dogs typically recovering within a few days to a few weeks. CIRDC is a complex disease involving a wide array of viral and bacterial agents, including canine adenovirus 2 (CAdV-2) (5), canine distemper virus (CDV; Morbilivirus canis) (6), canine herpesvirus 1 (CaHV-1; Varicellovirus canidalpha1) (7), canine influenza virus (CIV) (8), canine pneumovirus (CnPnV) (9, 10), canine parainfluenza virus (CPiV) (11), canine respiratory coronavirus (CRCoV) (12, 13), Bordetella bronchiseptica (14), Mycoplasma canis (15), M. cynos (16), and Streptococcus equi zooepidemicus (17, 18). Additionally, sporadic infections with other canine viruses were also reported, including canine calicivirus, canine circovirus, and canine hepacivirus (19–22). Co-infections and certain host factors can contribute to more severe clinical outcomes (23–26). Vaccines targeting specific pathogens associated with CIRD, such as B. bronchiseptica, CAdV-2, CDV, and CPiV, are available in the United States and are commonly administered to puppies (27). Nevertheless, outbreaks continue to be frequently reported (28, 29).

Accurate aetiologic diagnosis of CIRDC is challenging due to the overlapping clinical presentations among pathogens and the frequent occurrence of asymptomatic carriers. For example, B. bronchiseptica, M. cynos, CaHV-1, and CRCoV are often found in both healthy and clinically ill dogs (12, 30). As such, their contribution to the clinical signs observed in dogs remains uncertain. Asymptomatic dogs significantly contribute to the spread of infections and illnesses among susceptible dogs and may also serve as a reservoir for pathogens between disease outbreaks. Most pathogens have a short incubation period, typically from a few days up to 2 weeks (2). However, given the similarity in clinical signs induced by these pathogens, diagnosis cannot rely solely on history and clinical signs. Thus, laboratory confirmation is essential to confirm clinical diagnosis by accurate pathogen identification. Molecular diagnostic methods such as polymerase chain reaction (PCR) and quantitative PCR (qPCR or reverse transcriptase-qPCR [RT-qPCR]) are widely used for detecting CIRDC agents (31–33). These assays offer high sensitivity and specificity, enabling the detection of active infections. Multiplex qPCR/RT-qPCR platforms, in particular, allow simultaneous detection of multiple pathogens in clinical specimens, making them well-suited for investigating the complex etiology of CIRDC (26, 34). Furthermore, next-generation sequencing (NGS) has become a rapid and comprehensive technique for identifying and characterizing multiple pathogens, including the detection of novel agents in clinical specimens (35). Therefore, these novel molecular techniques (multiplex qPCR/RT-qPCR and NGS) offer high sensitivity, specificity, and quick turnaround time compared to classical virology and bacterial isolation and characterization methods.

In late 2023, veterinarians in several states across the US reported cases of an unusual respiratory illness in dogs, referred to as atypical canine infectious respiratory disease complex (aCIRDC). These cases raised alarms about the possible emergence of a new pathogen (36–39). Unlike typical infections seen in previous years, affected dogs experienced prolonged and severe respiratory clinical signs. Notably, the animals often tested negative for common pathogens associated with canine infectious respiratory disease (CIRD). The atypical nature of CIRDC was further underscored by its poor response to standard antibiotic treatments and a noticeable rise in mortality rates (40). This study aimed to investigate a potential emerging pathogen associated with CIRDC cases during the winter of 2023–2024. A comprehensive one-step multiplex qPCR/RT-qPCR assay was employed to detect the most common CIRDC pathogens, including CAdV-2, CDV, CaHV-1, CIV, CnPnV, CPiV, CRCoV, B. bronchiseptica, M. canis, M. cynos, and S. equi subsp. zooepidemicus, and SARS-CoV-2. Furthermore, specimens were tested for the possible presence of novel or emerging pathogens by NGS.

CIRDC is a complex infectious disease in dogs, attributable to single or synergistic actions of diverse viral and bacterial agents (1, 23). A rapid detection of the implicated pathogens is important to provide the most appropriate treatment strategy and implement proper biosecurity measures to prevent the spread of respiratory infections. Moreover, the detection of emerging pathogens is essential to understand their epidemiology and to inform owners and public health officials. An increase in the number of CIRDC cases in the US was perceived in late 2023 and was relayed by several public news outlets (36–39). Between December 2023 and February 2024, 50 nasal and nasopharyngeal swab specimens were collected from dogs experiencing respiratory disease and submitted for diagnostic testing to LSU Diagnostics. These samples were tested for common canine respiratory pathogens by multiplex qPCR/RT-qPCR and by NGS to attempt the identification of potential emerging pathogens.

When considering both qPCR/RT-qPCR and NGS results, 32 samples (64%) were positive for one pathogen known or suspected to induce respiratory infections in dogs. M. canis and M. cynos are the most common pathogens identified from CIRDC dogs, which is consistent with previous findings (24, 26, 34). Additionally, cases of CRCoV, CPiV, and B. bronchiseptica were reported, aligning with earlier reports (23, 48). The use of NGS has the advantage of allowing the identification of pathogens not included in the qPCR/RT-qPCR diagnostic panels. Here, canine calicivirus, canine circovirus and, for the first time, vientovirus (family: Redondoviridae) were detected in dogs with CIRDC. Canine calicivirus (CaCV) is an unclassified virus in the family Caliciviridae. CaCV was isolated and characterized for the first time in 1985 from the feces of a diarrheic dog in the US (49). Clinical signs induced by CaCV are diverse and range from the absence of clinical signs to severe diseases characterized by bloody diarrhea, hemorrhagic gastroenteritis, vomiting, and depression (20). However, CaCV has not been detected in respiratory tract specimens before. Additionally, experimentally infected dogs with different strains of CaCV failed to induce respiratory clinical signs (20, 49). Canine circovirus (species: Circovirus canine; genus: Circovirus; family: Circoviridae) was detected for the first time in 2011 in the serum of healthy dogs (50). The pathogenicity of canine circovirus is currently poorly understood, with the virus being mainly detected in fecal samples. Canine circovirus infection has been associated with clinical profiles of acute gastroenteritis, hemorrhagic diarrhea, signs of vasculitis, lymphadenitis, thrombocytopenia, neutropenia, and lymphopenia (51). Canine circovirus has also been associated with respiratory signs in dogs such as dyspnea, nasal discharge, cough, or rales (52). However, the virus has also been detected in samples of asymptomatic dogs, so its role in clinical disease and its pathogenesis is not clearly defined. It is interesting to note that M. canis, which is commonly found in the respiratory tract of healthy dogs, was detected in all samples that tested positive for CaCV and canine circovirus. Although it is not yet clear whether M. canis contributes to secondary infections in the upper airways, it is generally considered a commensal or opportunistic cofactor in respiratory and urogenital tract diseases in dogs (15). In this study, vientovirus DNA was found in a nasal/pharyngeal swab from one dog with prolonged respiratory illness (30 days) with no other detectable pathogens. Vientovirus (Torbevirus viento), a member of the Redondoviridae family (53), has so far been identified only in human respiratory samples through metagenomics, with no confirmed detection in animals (such as pigs, chickens, ducks, and dogs) or the environment (freshwater, marine environments, or soil) (54, 55). The transmission of this virus from humans to animals or animals to humans has not yet been demonstrated. Although prior infection with common respiratory agents cannot be ruled out for the dog positive for vientovirus DNA, the relevance of this virus to the clinical signs remains uncertain. Both NGS and PCR confirmed its detection. However, sample collection occurred late in the illness, and no other samples were found to be positive. While human DNA contamination was considered, it is unlikely due to the low human read counts and the inability to detect this virus in other specimens, simultaneously processed and tested by the same technicians. However, the detected viral sequence showed greater than 90% similarity to human-associated strains. Without supporting pathological or serological data, the role of the vientovirus virus in canine disease remains unclear.

Our results indicate that dogs from different states were infected with several known canine pathogens during the winter of 2023–2024, originating from six states in the US. Previously, studies have focused on detecting the nasal virome in dogs, which identified similar pathogens, including a novel Taupapillomavirus (CPV21–23) (56). Similarly, studies have focused on identifying and comparing the canine microbiome, which has revealed numerous bacterial taxa (57). It is important to highlight that the NGS protocol employed in this study was specifically optimized for viral detection, showing concordant results with the multiplex qPCR/RT-qPCR. Although the NGS protocol was not optimized for bacterial identification, it was applied for this purpose, which may partly explain the differences observed between the two methods in detecting bacterial pathogens, particularly the reduced bacterial detection by NGS. The nucleic acid extraction method, which includes a step to deplete host nucleic acids, may have contributed to the failure to detect certain bacteria. While our findings highlight several pathogens detected during the outbreak, it is important to consider factors that may influence diagnostic test results and the interpretation of negative findings. The quality of test results depends on the timely collection of appropriate samples during the infection. A negative qPCR/RT-qPCR or NGS test result means that the target virus or bacteria were not detected in the specimen. This could be due to the sample being collected too early or too late in the course of infection, resulting in insufficient target DNA or RNA for detection, or because no infection was present. In this study, 38% of the samples yielded negative results. The absence of pathogen identification in these cases could be attributed to several factors, including, but not limited to, lower respiratory tract infections, nucleic acids depletion techniques used during samples preparation for NGS (i.e., specific for bacteria detection), pathogen clearance before sample collection, antibiotic treatment by veterinarians, or non-infectious causes that the attending veterinarians may have considered. Based on available information, antibiotic treatment had been initiated in eight dogs before sample collection, which may have influenced the results by reducing the detectability of primary bacterial pathogens at the time of sampling. Follow-up was not possible for these cases, which limited the ability to gather additional clinical data or repeat testing to clarify the etiology of illness. Further investigation into the other respiratory causes of these negative cases could provide valuable insights and may be an important area for future research. Given the limited number of samples available during the Winter 2023–2024 outbreak (n = 50), it is essential to note that the relatively small sample size does not represent the overall US canine population, and the findings of this study should be considered exploratory. Nevertheless, the results provide valuable preliminary insights that may inform future, larger-scale investigations into CIRDC-associated pathogens. Additionally, the vaccine status of these animals was unknown. For these reasons, the association of other known or new respiratory pathogens in CIRDC cases across the country cannot be ruled out. Finally, nasal and pharyngeal swabs were pooled together before performing the diagnostic assays; therefore, the comparison of the two collection methods was not possible in this study. The SISPA method is a valuable tool for viral and bacterial metagenomic studies (58–60); however, like other NGS methods, it has limitations that affect its sensitivity. A key challenge is the requirement for high viral loads for adequate amplification, as lower concentrations lead to non-specific products and poor genome recovery. SISPA is also prone to amplification biases, frequently resulting in incomplete viral genome assemblies, particularly when viral titers are low (61). Additionally, background contamination from host nucleic acids can reduce sensitivity, necessitating stringent preprocessing and enrichment strategies.

While Mycoplasma species were detected at a high rate in these samples, we believe it is unlikely that they were the primary pathogen responsible for the outbreak. Instead, it may have acted as a secondary pathogen, contributing to the overall disease burden. Future studies focusing on viral and bacterial co-infections, as well as the timing of pathogen detection relative to clinical signs, would be valuable in further elucidating the role of Mycoplasma in CIRDC.

In summary, our results suggest that the increase in CIRDC cases observed in the winter of 2023–2024 is unlikely to be attributed to a single pathogen or the emergence of a novel viral or bacterial pathogen. No conclusive evidence of a new causative agent was identified in this study.