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.

The COVID-19 pandemic, caused by SARS-CoV-2, is still significantly impacting health care systems around the globe. Refined animal models are needed to study SARS-CoV-2 pathogenicity as well as efficacy of vaccines and therapeutics.

Equine Infectious Anemia Virus (EIAV), the causative agent of Equine Infectious Anemia (EIA), presents a significant threat to equine populations worldwide. While local EIAV prevalence has been estimated in several studies, no global prevalence has been determined. Thus, this study aimed to review the literature on EIAV prevalence in the 21st century. A comprehensive electronic search was conducted in PubMed, Web of Science, Scopus, Google Scholar, SciELO.org, African Journals Online, and NZresearch.org, yielding 105 articles published between January 1st, 2000, and December 31st, 2024 (spanning 24 years). EIAV prevalence was estimated in 42 countries and is particularly high in Mexico (North America; 27.14 % [95 % CI, 25.11 - 29.17]) and Guatemala (Central America; 15.9 % [95 % CI, 9.66 - 22.14]). While EIAV prevalence in the United States of America (USA) remains low and stable over time, the relatively elevated prevalence in the Southern states, along with their extensive border with Mexico, places these areas at a higher risk. EIAV prevalence estimated per province/state in the USA, Argentina, and Brazil, indicated higher rates in humid and warmer regions. EIAV has a low presence in the Middle East, while the scarcity of epidemiological studies in Africa and Oceania complicates the estimation of EIAV prevalence in these regions. These findings highlight the need for continued intensive surveillance in both high-risk regions and areas lacking data. Given that insect vectors are the primary natural transmission route, global warming may lead to increased vector movement into temperate areas, potentially contributing to a surge in EIAV infections.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was transmitted from humans to dogs and cats (reverse zoonosis) during the COVID-19 pandemic. SARS-CoV-2 has been detected in fecal samples of infected dogs and cats, indicating potential fecal–oral transmission, environmental contamination, and zoonotic transmission (i.e., spillback). Additionally, gastrointestinal viral infections are prevalent in dogs and cats. In this study, we developed and validated a panel of multiplex one-step reverse transcription–quantitative polymerase chain reaction (RT-qPCR) assays for the simultaneous detection of SARS-CoV-2 and common canine enteric viruses: Canine Enteric Assay_1 (CEA_1) for the detection of canine adenovirus-1, canine enteric coronavirus, canine distemper virus, and canine parvovirus, and CEA_2 for the detection of rotavirus A (RVA), and SARS-CoV-2); or common feline enteric viruses (Feline Enteric Assay_1 (FEA_1) for the detection of feline enteric coronavirus, feline panleukopenia virus, RVA, and SARS-CoV-2). All assays demonstrated high analytical sensitivity, detecting as few as 5–35 genome copies/µL in multiplex format. The repeatability and reproducibility of the multiplex assays were excellent, with coefficient of variation <4%. Among the 58 clinical samples tested, 34.5% were positive for at least one of these viruses, and SARS-CoV-2 was detected in two samples collected from one dog and one cat, respectively. In conclusion, these newly developed one-step multiplex RT-qPCR assays allow for rapid diagnosis of enteric viral infections, including SARS-CoV-2, in dogs and cats.

Feline respiratory disease complex (FRDC) is caused by a wide range of viral and bacterial pathogens. Both Influenza A virus (IAV) and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) also induce respiratory diseases in cats. Two one-step multiplex qPCR/RT-qPCR assays were developed and validated: FRA_1 (Feline respiratory assay 1) for the detection of four viral targets and FRA_2 for the detection of three bacteria associated with FRDC. Both multiplex assays demonstrated high specificity, efficiency (93.51%–107.8%), linearity (> 0.998), analytical sensitivity (≤ 15 genome copies/μl), repeatability (coefficient of variation [CV] < 5%), and reproducibility (CV < 6%). Among the 63 clinical specimens collected from FRDC-suspected cats, 92.1% were positive for at least one pathogen and co-infection was detected in 57.1% of samples. Mycoplasma felis (61.9%) was the most found pathogen, followed by feline herpesvirus-1 (30.2%), Chlamydia felis (28.7%) and feline calicivirus (27.0%). SARS-CoV-2 was detected in two specimens. In summary, this new panel of qPCR/RT-qPCR assays constitutes a useful and reliable tool for the rapid detection of SARS-CoV-2 and viral and bacterial pathogens associated with FRDC in cats.