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Simple Summary: Fleas are blood-sucking insects that are not only a nuisance but can also act as vectors for various diseases in animals and humans, including dangerous ailments such as the bubonic plague. Identifying and classifying these insects accurately is crucial for understanding how they spread and how to control them. Geometric morphometrics, a cutting-edge technique, is proving to be an invaluable tool in this regard, alongside traditional methods and molecular biology. In the present study conducted in Andalusia, Spain, this technique successfully differentiated between three populations of fleas, providing insights into their distribution, size, and characteristics. Image processing software was employed to obtain measurements, such as perimeters and areas, of the fleas under study. These findings underscore the importance of geometric morphometrics in studying and managing arthropod populations, particularly in cases where other methods fall short or are not available. Fleas (Siphonaptera) are ectoparasitic hematophagous insects responsible for causing bites and itchy skin conditions in both humans and animals. Furthermore, they can act as vectors of different pathogens of a wide variety of diseases worldwide, including bartonellosis, rickettsiosis, and bubonic plague. Accurate identification of fleas is necessary for the study of their epidemiology, prevention, and control. In addition to traditional morphological classification approaches and molecular biology techniques, geometric morphometrics is increasingly proving to be a useful complementary tool for discriminating between Siphonaptera taxa. With the objective of determining the capacity of this technique to identify and differentiate synanthropic fleas, a principal component analysis was carried out on populations of Ctenocephalides felis, Pulex irritans, and Archaeopsylla erinacei collected in distinct regions of Andalusia (Spain). The analysis carried out on 81 male and female specimens revealed factorial maps that allowed the differentiation of the populations under study, with only partial overlaps that did not prevent their correct identification. Global size differences were also detected, with a slightly larger size in P. irritans males and a bigger size in A. erinacei females. Therefore, the present study emphasizes the role of geometric morphometrics as a useful complementary technique in taxonomic studies of arthropods, especially in the case of flea specimens lacking representative morphological features. [ABSTRACT FROM AUTHOR]

Simple Summary: In order to understand the prevalence of respiratory diseases in urban stray cats in Shanghai and provide scientific evidence for the development of targeted prevention and control strategies for respiratory diseases in stray cats, we performed a molecular survey in Shanghai in 2022. A total of 374 stray cats were screened against six respiratory pathogens. Out of the 374 samples, 146 tested positive. Mycoplasma felis exhibited the highest positivity rate at 18.72% (70/374), followed by Chlamydia felis at 11.76% (44/374), feline calicivirus at 3.74% (14/374), feline herpesvirus type-1 at 3.48% (13/374), and Bordetella bronchiseptica at 1.34% (5/374); influenza A virus was not detected. Mycoplasma felis and Chlamydia felis were the primary causative agents of respiratory infections in urban stray cats, with Mycoplasma felis demonstrating a notably higher positivity rate compared to other respiratory pathogens and frequently co-infecting with Chlamydia felis and feline calicivirus. The positivity rate of Mycoplasma felis was elevated during the summer, autumn, and winter seasons, with no significant variance between seasons. These results highlight a substantial overall prevalence of respiratory pathogens in urban stray cats in the Shanghai region, characterized by seasonal patterns and concurrent infections with other pathogens. These findings suggest the need for comprehensive prevention and control measures to address respiratory pathogen infections in urban stray cats in the Shanghai area. Urban stray cats are cats without owners that survive in the wild for extended periods of time. They are one of the most common stray animals in cities, and as such, monitoring the pathogens carried by urban stray cats is an important component of urban epidemiological surveillance. In order to understand the prevalence of respiratory diseases in urban stray cats in Shanghai and provide scientific evidence for the development of targeted prevention and control strategies for respiratory diseases in stray cats, we collected 374 ocular, nasal, and oropharyngeal swabs from urban stray cats in Shanghai from January 2022 to December 2022. After RNA extraction, we used real-time PCR to detect six respiratory pathogens, including influenza A virus, feline calicivirus, feline herpesvirus type 1, Mycoplasma, Chlamydia, and Bordetella bronchiseptica. The results showed that among the 374 samples, 146 tested positive, with a positivity rate of 39.04%. The highest positivity rate was observed for Mycoplasma felis at 18.72% (70/374), followed by Chlamydia felis at 11.76% (44/374), feline calicivirus at 3.74% (14/374), feline herpesvirus 1 at 3.48% (13/374), Bordetella bronchiseptica at 1.34% (5/374), and influenza A virus was not detected. The highest positivity rate for Mycoplasma felis was in Minhang District at 31.94% (23/72), while Chlamydia felis and Bordetella bronchiseptica had the highest positivity rates in Jiading District at 23.53% (8/34) and 5.88% (2/34), respectively. The highest positivity rates for feline calicivirus and feline herpesvirus 1 were both observed in Qingpu District, at 14.46% (12/83) and 9.64% (8/83), respectively. A total of 36 samples showed mixed infections with two or more pathogens, with Mycoplasma felis being involved in 32 of these mixed infections, with the highest number of mixed infections being with Chlamydia felis at 25 samples. Respiratory pathogen positivity was detected throughout the year, with peak detection rates in summer and winter. The positivity rates of cat respiratory pathogens in different seasons showed statistical differences (χ2 = 27.73, p < 0.01). There was no statistical difference in the positivity rates of respiratory pathogens between cats of different genders (χ2 = 0.92, p > 0.05). The positivity rates of respiratory pathogens in cats of different age groups showed statistical differences (χ2 = 44.41, p < 0.01). Mycoplasma felis and Chlamydia felis were the main pathogens causing respiratory infections in stray cats, with Mycoplasma felis showing a much higher positivity rate than other respiratory pathogens and often co-infecting with Chlamydia felis and feline calicivirus. The positivity rate of Mycoplasma felis was high in summer, autumn, and winter, with no statistical difference between seasons. These results indicate a serious overall prevalence of respiratory pathogens in urban stray cats in the Shanghai area, showing seasonal trends and mixed infections with other pathogens. These findings suggest the need for comprehensive prevention and control measures to address respiratory pathogen infections in urban stray cats in the Shanghai area. [ABSTRACT FROM AUTHOR]

Simple Summary: Vector-borne infectious zoonotic diseases are a relevant problem, not only in veterinary medicine, but also in human medicine. Dogs are considered the main reservoir of these zoonoses and sentinels for the control of these diseases that, due to the increase in temperatures due to climate change, are becoming more and more frequent. Most of these zoonotic diseases are endemic to the Mediterranean Basin, and knowing their prevalence in different canine breeds and other related factors could contribute to controlling these diseases. This paper analyzes the prevalence of different infections and coinfections of zoonotic disease in four different canine breeds, and the results obtained indicate that the most frequent infections was Leishmania infantum, followed by Ricketsia rickesii. Changes in the hematological and biochemical values in infected dogs of different breeds could suggest a certain resistance against L. infantum infection in the Cirneco dell'Etna canine breed, autochthonous of Sicily. Dogs are considered the main reservoir of several zoonoses endemic to the Mediterranean Basin. In this study, a prevalence of infections and coinfections of canine vector-borne diseases was analyzed in apparently healthy dogs of different canine pure breeds in Sicily (Italy), where these diseases are endemic. The seroprevalence of Leishmania infantum, Ricketsia ricketsii, Anaplasma phagocytophilum, and Erlichia canis was assessed, as single and coinfections. Biochemical and hematological parameters were evaluated, and epidemiological factors, including sex, age, and canine breed, were recovered. The most frequent infection was L. infantum (45.61%), following R. ricketsii (36.84%), both as single, double, or triple coinfections. Coinfections change the biochemical and hematological parameters of the host, and canine breeds are related to the infection frequency and the parameters observed during infections. Changes in the complete blood count (CBC) and biochemical values also differ between canine breeds, with the Cirneco dell'Etna dogs infected with L. infantum being the animals presenting the most interesting results in our study. High values of RBC, hemoglobin, hematocrit, mean corpuscular hemoglobin (MCH), the albumin/globulin (A/G) ratio, and albumin and low levels of β-2 globulin and γ-globulin were found only in this canine breed, suggesting some resistance to infection in these dogs. Future studies about the immune response of this canine breed could be interesting to determine their possible resistance to zoonotic pathogens, such as L. infantum. [ABSTRACT FROM AUTHOR]

Simple Summary: The programmed grooming model is an evolutionary hypothesis for the regulation of ectoparasite-defense grooming behavior. It proposes that grooming to remove ectoparasites such as ticks is regulated by a type of internal biological clock that has evolved to pre-emptively remove parasites before they can blood feed. Programmed grooming contrasts with the stimulus-driven model, in which grooming is stimulated by direct peripheral irritation from ectoparasites. Since programmed grooming was first proposed in 1992, 26 studies have provided robust support for the model with ungulate hosts and ticks. Additional studies from unaffiliated investigators have evaluated the predictions of the model in different host systems (including rodents and primates) and in a variety of ectoparasites (fleas, lice, and keds). After reviewing the current evidence, I conclude that (1) tests of the programmed grooming model should utilize the established protocol, so that the results can be compared and assessed in light of previous studies; (2) the model predictions should be tailored to the host biology under investigation; and (3) the model predictions should be tailored to the ectoparasite biology, since the efficacy of grooming depends on the parasite. It is hoped that future studies will reveal much more about how grooming helps wild animals to defend themselves against the threat of parasites. In 1992, an evolutionary model for the endogenous regulation of parasite-defense grooming was first proposed for African antelope by Ben and Lynette Hart. Known as the programmed grooming model, it hypothesized that a central control mechanism periodically evokes grooming so as to remove ectoparasites before they blood feed. The programmed grooming model contrasts with a stimulus-driven mechanism, in which grooming is stimulated by direct peripheral irritation from ectoparasite bites. In the 30+ years since the seminal 1992 paper, 26 studies have provided robust support for the programmed grooming model in ungulate hosts and ticks. In addition, multiple studies from unaffiliated investigators have evaluated the predictions of the model in different host systems (including rodents and primates) and in a variety of other ectoparasites (fleas, lice, and keds). I conducted a tricennial review of these studies to assess the current evidence and arrived at the following three conclusions: (1) tests of the programmed grooming predictions should use a similar methodology to the well-established protocol, so that the results are comparable and can be properly assessed; (2) the predictions used to test the model should be tailored to the biology of the host taxa under investigation; and (3) the predictions should likewise be tailored to the biology of the ectoparasites involved, bearing in mind that grooming has varying degrees of effectiveness, depending on the parasite. Further research is warranted to enhance our understanding of the role of grooming in maintaining the health of wild animals in the face of parasite attacks. [ABSTRACT FROM AUTHOR]

Simple Summary: While it is widely believed that feline infectious peritonitis (FIP) is a dangerous epidemic disease, the prevalence of FIP in Chinese cats is currently unknown. Our findings indicate that the prevalence of FIP in Chinese cats is influenced by age, gender, and breed. We recommend controlling the scale of cat breeding, enhancing cat immunity, and particularly focusing on health management in kittens and continuous monitoring of FIP in cats. To evaluate the overall prevalence of FIP infection in cats in mainland China and associated risk factors, studies on the prevalence of FIP conducted from 1 January 2008 to 20 December 2023 were retrieved from five databases—CNKI, Wanfang, PubMed, Web of Science, and ScienceDirect—and comprehensively reviewed. The 21 studies selected, with a total of 181,014 samples, underwent a rigorous meta-analysis after quality assessment. The results revealed a 2% prevalence of FIP (95% CI: 1–2%) through the random-effects model, showing considerable heterogeneity (I2 = 95.2%). The subsequent subgroup analysis revealed that the age and gender of cats are significant risk factors for FIP infection in mainland China. In order to effectively reduce and control the prevalence of FIP on the Chinese mainland, we suggest improving the immunity of cats, with special attention given to health management in kittens and intact cats, and continuously monitoring FIPV. [ABSTRACT FROM AUTHOR]

Simple Summary: Inflammatory bowel disease (IBD) is a common chronic gastrointestinal disorder in dogs. Hematological ratios, in particular red blood cell distribution width (RDW), neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), and platelet-to-lymphocyte ratio (PLR), are considered cost-effective predictors of clinical disease activity in human patients with IBD and Crohn's disease. This study evaluated the diagnostic value of hematological parameters and ratios in 85 client-owned dogs, comparing 60 dogs with IBD to 25 clinically healthy dogs. Monocytes count and the hematological ratios MLR, NLR, and PLR can be useful in the diagnostic work-up of dogs with IBD. This is an observational retrospective study on 85 client-owned dogs, 60 with IBD and 25 clinically healthy dogs. This study aims to assess the clinical relevance of some easy to obtain and cost-effective hematological parameters including red blood cell distribution width (RDW), neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), and platelet-to-lymphocyte ratio (PLR) in dogs with IBD. Comparison of clinical and laboratory parameters between dogs with IBD and control dogs was carried out and the ability to distinguish between these two groups of dogs was evaluated by calculating the area under the receiver-operating characteristic curve (AUCROC). Univariate and multivariable logistic regression analysis estimated the odds ratio (OR) of developing IBD with a 95% confidence interval (CI). MLR and monocytes count had the highest accuracy in facilitating the discrimination of dogs with IBD from control dogs with an AUCROC of 0.839 and 0.787 at the cut-off of >0.14% and >3.7 cells*102/µL, respectively. According to two multivariable models, monocytes count (OR = 1.29; p = 0.016), NLR (OR = 1.80; p = 0.016), and MLR > 0.14 (OR = 8.07; p < 0.001) and PLR > 131.6 (OR = 4.35; p = 0.024) were significant and independent predictors of IBD for models one and two, respectively. Monocytes count and the hematological ratios MLR, NLR, and PLR can be useful in the diagnostic work-up of dogs with IBD. [ABSTRACT FROM AUTHOR]

Simple Summary: Amantadine, a drug initially used as an antiviral agent to treat the influenza A virus (though currently, its main application is in the treatment of Parkinson's disease) has also been used to control pain due to its mechanism of action: exerting non-competitive antagonism N-methyl-D-aspartate (NMDA) glutamatergic receptors, which participate in the neurophysiological project of pain via the inhibition of central sensitization. We hope that our study makes a significant contribution to the literature because the preemptive oral administration of amantadine at a dose of 5 mg/kg resulted in superior postsurgical pain control. In addition, the administration of amantadine did not result in cardiovascular or respiratory alterations or adverse effects during the intraoperative period of OVH in the evaluated cats. This study aimed to evaluate the effect of the preemptive administration of amantadine on postoperative analgesia in cats undergoing ovariohysterectomy and its influence on the physiological parameters. Twenty healthy domestic cats scheduled to undergo ovariohysterectomy at the Santa Cruz State University, Ilhéus, were divided into two groups: the control group (Group C; n = 10) and the amantadine group (Group A; n = 10). The cats in Group C received placebo capsules 30 min prior to the standard anesthetic protocol, whereas those in Group A received 5 mg/kg of amantadine orally 30 min prior to the standard anesthetic protocol. Postoperative pain was assessed using the visual analog scale and the UNESP-Botucatu multidimensional scale for the evaluation of postoperative pain in cats. The administration of amantadine had no effect on the physiological parameters evaluated. The pain scores in Group A were lower than those in Group C, indicating that the frequency of rescue analgesic administration cats in Group A was lower. That way, preemptive oral administration of amantadine at a dose of 5 mg/kg was effective at controlling postoperative pain in cats undergoing ovariohysterectomy. Moreover, no adverse effects or alterations in the physiological patterns were observed in the treated animals. [ABSTRACT FROM AUTHOR]

Simple Summary: This study intended to show the use of external fixation to resolve bilateral acetabular physeal fractures in an immature cat, obtaining a good outcome with a low rate of complications. A great part of this success was because external fixation allows fractures to be stabilised through a minimally invasive procedure. In addition to its relevance as a fixation method, it was possible to show that external fixation provided comfort and a high level of tolerance for the patient during the whole healing period. This study aimed to assess the outcome of a bilateral acetabular physeal fracture treated with external fixation in an immature cat, a surgical technique not usually employed in immature patients. The fixator took 40 days, and it was removed after radiographic bone healing was achieved. No significant complications related to the technique were identified, and the outcome was classified as good based on the functional assessment and pain scales employed. The use of external fixation for stabilising acetabular fractures in immature cats should be considered a viable technical option, especially for minimally invasive stabilisation. [ABSTRACT FROM AUTHOR]

Simple Summary: Post-operative pain after fracture fixation in dogs can be severe. We already know that intra-operative bupivacaine hematoma block (HB, the infusion of local anaesthetic–bupivacaineto the fracture site during the operation) is effective in reducing post-operative pain in dogs undergoing long-bone fracture fixation (Dimopoulou et al. 2017). The aim of the present study was to compare the efficacy of other local anaesthetics, such as lidocaine or ropivacaine, for post-operative pain relief when administered via an HB in the same clinical scenario and also to investigate which is the best intra-operative time point to perform the HB in order to achieve better post-operative pain relief. For this aim, we infused two different local anaesthetics (lidocaine or ropivacaine) at the same surgical time point to compare their analgesic efficacy but also the same local anaesthetic (ropivacaine) at three different distinct surgical time points. Post-operative pain was estimated with the use of the University of Melbourne Pain Scale (a multimodal pain scale) and an algometer (a device that measures the mechanical pain threshold). The results of the present study indicate that the dogs that received a lidocaine HB experienced better post-operative pain relief than those who received a ropivacaine HB. Furthermore, the dogs that received the HB (ropivacaine) right before surgical closure had better post-operative pain relief. Objective: We aimed to compare the efficacy of intra-operative lidocaine hematoma block (HB) to ropivacaine HB and to compare the efficacy of different timings of ropivacaine HB in controlling post-operative pain in dogs undergoing the osteosynthesis of long-bone fractures. Study Design: We conducted a randomized, blinded, prospective clinical study. Animals: Forty-eight dogs with long-bone fractures were included and were randomly allocated to four groups: lidocaine (L), ropivacaine (Rmid), ropivacaine pre- (Rpre) and ropivacaine post- (Rpost) groups. Methods: The dogs in group L (n = 14) and in group Rmid (n = 11) received a lidocaine or ropivacaine HB, respectively, after fracture reduction and before osteosynthesis material placement. Rpre dogs (n = 11) received ropivacaine HB before fracture reduction, and Rpost dogs (n = 12) received ropivacaine HB after osteosynthesis material placement. Eight post-operative pain assessments were performed using the University of Melbourne Pain Scale (UMPS) and an algometer. Rescue analgesia was administered based on UMPS scoring. For data analysis, the Shapiro–Wilk test of normality, chi-square, Student t test and Split Plot analysis were used. The level of significance was set at α = 0.05. Results: Rescue analgesia was administered to one dog in group L, one in group Rmid and one in group Rpost, with no significant differences detected. Compared to group Rmid, group L dogs exhibited significantly higher mean mechanical pain thresholds (p = 0.049) and lower mean UMPS scores (p = 0.001). Group Rpost dogs had statistically significantly higher mean pain thresholds compared to group Rmid (p = 0.009). Clinical Implications: When performed after fracture reduction and before osteosynthesis material placement, lidocaine HB seems to be more effective than ropivacaine HB in controlling post-operative pain in dogs undergoing osteosynthesis of long-bone fractures. The administration of ropivacaine HB after osteosynthesis material placement seems to be more effective than administration after fracture reduction and before osteosynthesis material placement or administration before fracture reduction in controlling post-operative pain in dogs undergoing osteosynthesis of long-bone fractures. [ABSTRACT FROM AUTHOR]

Simple Summary: Recent data suggest that feline lymphoma may no longer be strongly associated with viruses like feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV), possibly causing a change in the occurrence of specific anatomical subtypes and treatment outcomes. This study aimed to analyze this shift in disease presentation in a country with a low appearance of FIV/FeLV, such as the Netherlands, to investigate if these changes can be solely attributed to the prevalence of these viruses or if other risk factors should be considered. In total, 174 cats with large cell lymphoma (110 treated) during the last 10 years were included and compared to previous data from the Netherlands. This study demonstrates a shift in patient characteristics of cats with malignant lymphoma in the Netherlands, which could not be ascribed to differences in FIV/FeLV occurrence. While treatment response remained consistent, some anatomical subtypes showed longer disease-free periods. Feline lymphoma is currently less commonly associated with retrovirus infections as the feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV). This is thought to have caused a shift in the distribution of anatomical subtypes and eventually have led to poorer treatment outcomes. The aim of this study was to evaluate whether this change was also notable in the Netherlands, a country historically known for its low prevalence of FeLV and FIV, and to determine its consequences on treatment response. A 10-year cohort of 174 cats with large cell lymphoma (110 treated) were included and compared to historical data from previously published reports in the Netherlands. Of the 90 cats screened, only one tested positive for FeLV and three for FIV. The most current cohort had an increased age (median 8.7 years) and fever Siamese cats (6.3%) compared to previous reports, with alimentary (24.5%) and nasopharyngeal lymphoma (22.7%) being the most common subtypes. Sixty-six of the one hundred and ten cats (60%) went into complete remission, (CR) resulting in a median disease-free period (DFP) of 763 days, with nasopharyngeal and mediastinal having the longest DFP. The median overall survival time was 274 days with an estimated 1-year survival of 41.3% and a 2-year survival of 34.6%, respectively. Patient characteristics of cats with malignant lymphoma in the Netherlands have changed over the years, but this cannot be explained by differences in FeLV/FIV prevalence. Although the overall response rate to therapy did not change over time, for some lymphoma subtypes, longer DFPs were observed compared to 30 years ago. [ABSTRACT FROM AUTHOR]

Simple Summary: Prebiotics and essential oils are some of the feed additives that can be added to pet food, individually or in combination. These additives can exert important properties on the animal, such as antioxidant, anti-inflammatory, and immunomodulatory effects, as well as modulation of gut microbiota. Therefore, the aim of this study is to evaluate the combined effects of yeast cell wall and oregano essential oil on nutrient digestibility, diet palatability, intestinal fermentation products, and fecal microbiota in dogs. The addition of the blend with yeast cell wall and oregano essential oil to the dog's diet reduced the apparent total tract digestibility of dry matter and the intake ratio compared to the control diet, modulated fecal microorganisms considered beneficial, caused greater bacterial diversity, and lowered histamine, phenol, and ammonia concentrations. Feed additives, such as prebiotics and essential oils, are used in pet foods and can affect digestibility, palatability, and intestinal functionality of dogs. The combined effects of yeast cell wall and oregano essential oil on apparent total tract digestibility (ATTD) and palatability of diet, intestinal fermentation products, and fecal microbiota in dogs were analyzed. Eighteen adult dogs were fed for 20 days with three dry extruded diets for adult dogs: control (without the additive), a diet containing 1.5 kg/ton of yeast cell wall and oregano essential oil (1.5YCO), and a diet containing 3.0 kg/ton of yeast cell wall and oregano essential oil (3.0YCO). The inclusion of both levels of YCO reduced the intake ratio. The addition of 3.0YCO reduced the ATTD of dry matter, compared to the control group (p < 0.05). There were greater putrescine and cadaverine concentrations and lower histamine and ammonia (p < 0.05) in the feces of dogs fed 3.0YCO. In addition, fecal odor of dogs fed YCO was less fetid than the control group (p < 0.05). There was greater fecal bacterial diversity in dogs fed with both dietary concentrations of YCO evaluated (p < 0.05). Dogs fed 1.5YCO and 3.0YCO showed higher relative abundance of Blautia and Faecalibacterium and lower abundance of Streptococcus (p < 0.05) in the feces, in comparison to the control group. Given the modulation of microorganisms considered beneficial and the lower fecal concentrations of histamine, phenols, and ammonia, the YCO blend resulted in indicators of improvement of intestinal functionality in dogs. [ABSTRACT FROM AUTHOR]

Simple Summary: Given that SARS-CoV-2 infections in companion dogs and cats have been frequently reported worldwide during the ongoing COVID-19 pandemic, a multiplex real-time RT-PCR assay is urgently required to reliably detect SARS-CoV-2 infection in companion animals. In this study, we developed a tailored multiplex real-time RT-PCR assay to simultaneously detect RdRp and N genes of currently circulating SARS-CoV-2 variants and canine or feline 16S rRNA as an endogenous internal positive control. The developed assay had high sensitivity, specificity, and accuracy and could detect all tested SARS-CoV-2 variants, including Omicron subvariants. Clinical evaluation of canine and feline specimens revealed that the diagnostic sensitivity of the assay was equivalent to that of a commercial SARS-CoV-2 multiplex real-time RT-PCR kit. Furthermore, canine or feline endogenous internal positive control was amplified using the developed assay while avoiding false-negative results. Considering the high sensitivity, specificity, accuracy, and reliability, the developed assay can help diagnose COVID-19 in dogs and cats and potentially play a vital role in the rapid diagnosis and control of SARS-CoV-2 infections in companion animals. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been frequently reported in companion dogs and cats worldwide during the ongoing coronavirus disease. However, RT-qPCR methods developed for humans have been used for the diagnosis of SARS-CoV-2 infections in suspected companion dogs and cats owing to the lack of the companion animal-tailored methods. Therefore, we developed a multiplex RT-qPCR (mRT-qPCR) using newly designed primers and probes targeting RdRp and N genes of all currently circulating SARS-CoV-2 variants as well as the canine or feline 16S rRNA gene as an endogenous internal positive control (EIPC) for reliable diagnosis of SARS-CoV-2 infection from suspected dogs and cats. The developed mRT-qPCR assay specifically detected the target genes of SARS-CoV-2 but no other canine or feline pathogens. Furthermore, canine and feline EIPCs were stably amplified by mRT-qPCR in samples containing canine- or feline-origin cellular materials. This assay has high repeatability and reproducibility, with an optimal limit of detection (<10 RNA copies per reaction) and coefficients of variation (<1.0%). The detection rate of SARS-CoV-2 of the developed mRT-qPCR was 6.6% for canine and feline nasopharyngeal samples, which was consistent with that of a commercial mRT-qPCR kit for humans. Collectively, the newly developed mRT-qPCR with canine and feline EIPC can efficiently diagnose and evaluate the viral load in field specimens and will be a valuable tool for etiological diagnosis, epidemiological study, and controlling SARS-CoV-2 infections in canine and feline populations. [ABSTRACT FROM AUTHOR]