Canine Infectious Respiratory Disease 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.

Genomic analysis of canine pneumoviruses and canine respiratory coronavirus from New Zealand

ABSTRACT Aims To isolate canine respiratory coronavirus (CRCoV) and canine pneumovirus (CnPnV) in cell culture and to compare partial genomic sequences of CRCoV and CnPnV from New Zealand with those from other countries. Methods Oropharyngeal swab samples from dogs affected by canine infectious respiratory disease syndrome that were positive for CnPnV (n = 15) or CRCoV (n = 1) by virus-specific reverse transcriptase quantitative PCR (RT-qPCR) in a previous study comprised the starting material. Virus isolation was performed in HRT-18 cells for CRCoV and RAW 264.7 and Vero cells for CnPnV. The entire sequence of CnPnV G protein (1,266 nucleotides) and most (8,063/9,707 nucleotides) of the 3’ region of CRCoV that codes for 10 structural and accessory proteins were amplified and sequenced. The sequences were analysed and compared with other sequences available in GenBank using standard molecular tools including phylogenetic analysis. Results Virus isolation was unsuccessful for both CRCoV and CnPnV. Pneumovirus G protein was amplified from 3/15 (20%) samples that were positive for CnPnV RNA by RT-qPCR. Two of these (NZ-048 and NZ-049) were 100% identical to each other, and 90.9% identical to the third one (NZ-007). Based on phylogenetic analysis of the G protein gene, CnPnV NZ-048 and NZ-049 clustered with sequences from the USA, Thailand and Italy in group A, and CnPnV NZ-007 clustered with sequences from the USA in group B. The characteristics of the predicted genes (length, position) and their putative protein products (size, predicted structure, presence of N- and O-glycosylation sites) of the New Zealand CRCoV sequence were consistent with those reported previously, except for the region located between open reading frame (ORF)3 (coding for S protein) and ORF6 (coding for E protein). The New Zealand virus was predicted to encode 5.9 kDa, 27 kDa and 12.7 kDa proteins, which differed from the putative coding capacity of this region reported for CRCoV from other countries. Conclusions This report represents the first characterisation of partial genomic sequences of CRCoV and CnPnV from New Zealand. Our results suggest that the population of CnPnV circulating in New Zealand is not homogeneous, and that the viruses from two clades described overseas are also present here. Limited conclusions can be made based on only one CRCoV sequence, but the putative differences in the coding capacity of New Zealand CRCoV support the previously reported variability of this region. The reasons for such variability and its biological implications need to be further elucidated.

Challenges in Developing Effective Canine Infectious Respiratory Disease Vaccines

Challenges in Developing Effective Canine Infectious Respiratory Disease Vaccines

Evaluation of hemagglutination inhibition test for canine respiratory coronavirus antibodies and seroprevalence in Korean dogs

Canine respiratory coronavirus (CRCoV) is a significant pathogen that causes respiratory diseases in dogs, collectively known as a canine infectious respiratory disease. The virus is highly contagious and exhibits high seroprevalence worldwide. Currently, bovine coronavirus (BCoV) enzyme-linked immunosorbent assay (ELISA) kits are used to detect CRCoV antibodies. However, BCoV-ELISA kits cannot differentiate between infections caused by BCoV and those caused by CRCoV. In this study, we evaluated the hemagglutination inhibition (HI) test for CRCoV by comparing it with the virus neutralization (VN) test. Subsequently, we evaluated the seroprevalence of CRCoV in 383 dog serum samples collected from South Korea utilizing the HI test. The HI test for CRCoV showed a strong correlation with the VN test (R = 0.83, p < 0.001). The analysis of seroprevalence revealed that 52.2% (95% confidence interval [CI], 47.2%–57.1%) of the Korean dog serum samples were positive. The seroprevalence exhibited varied with age, with a positivity rate of 43.9% in dogs under 1 year of age and 66.7% in dogs aged 3 to 5 years (odds ratio [OR], 2.54; 95% CI, 1.43–4.59). In conclusion, the HI test to monitor CRCoV antibody proved to be closely related to the VN test. Furthermore, over half of the dogs in Korea tested positive for CRCoV antibodies. These findings contribute to a better understanding of the sero-epidemiology of CRCoV.

Molecular biology of canine parainfluenza virus V protein and its potential applications in tumor immunotherapy.

Canine parainfluenza virus (CPIV) is a zoonotic virus that is widely distributed and is the main pathogen causing canine infectious respiratory disease (CIRD), also known as "kennel cough," in dogs. The CPIV-V protein is the only nonstructural protein of the virus and plays an important role in multiple stages of the virus life cycle by inhibiting apoptosis, altering the host cell cycle and interfering with the interferon response. In addition, studies have shown that the V protein has potential applications in the field of immunotherapy in oncolytic virus therapy or self-amplifying RNA vaccines. In this review, the biosynthesis, structural characteristics and functions of the CPIV-V protein are reviewed with an emphasis on how it facilitates viral immune escape and its potential applications in the field of immunotherapy. Therefore, this review provides a scientific basis for research into the CPIV-V protein and its potential applications.

Development of a one-step multiplex (RT)-PCR for simultaneous detection of virus-induced respiratory disease complex in dogs in Vietnam

Canine infectious respiratory disease (CIRD) viruses, particularly canine distemper virus (CDV), canine adenovirus type 2 (CAV-2), and canine parainfluenza virus (CPIV), are the primary agents responsible for respiratory diseases in dogs. A dog can be infected with a single or multiple viruses with similar clinical signs. Although CIRD is globally prevalent in dogs, investigation into the causative viruses in Vietnam remains limited. This study aims to identify the presence of CIRD virus(es) in dogs via molecular detection. Three primer sets were newly designed and applied into (RT)-PCR reaction to detect viral genes of CDV, CAV-2, and CPIV in a commercial vaccine (Vanguard® Plus 5/L, Zoetis, USA). Both simplex and multiplex (RT)-PCR reactions using the three primer sets could detect the presence of CAV-2, CDV, and CPIV in the vaccine sample. Subsequently, the applicability of the one-step multiplex (RT)-PCR was demonstrated to test for 27 clinical nasal swab samples collected from dogs suspected of having CIRD. The results showed that our (RT)-PCR could detect CIRD virus(es) in all tested clinical samples. PCR amplicons for each representative CAV-2, CDV, and CPIV were selected for sequencing and showed high genetic similarity to respective field viruses. In conclusion, this study successfully developed a one-step multiplex (RT)-PCR reaction to detect and differentiate causative viruses of CIRD in dogs.

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.

Epidemiologic investigation and genetic characterization of canine respiratory coronavirus in the Southeastern United States.

Canine respiratory coronavirus (CRCoV) is one of the main causative agents of canine infectious respiratory disease (CIRD), an illness whose epidemiology is poorly understood. We assessed the prevalence, risk factors, and genetic characterization of CRCoV in privately owned dogs in the Southeastern United States. We PCR-screened 189 nasal swabs from dogs with and without CIRD clinical signs for 9 CIRD-related pathogens, including CRCoV; 14% of dogs, all diagnosed with CIRD, were positive for CRCoV, with a significantly higher rate of cases in younger dogs and during warmer weather. Notably, the presence of CRCoV, alone or in coinfection with other CIRD pathogens, was statistically associated with a worse prognosis. We estimated a CRCoV seroprevalence of 23.7% retrospectively from 540 serum samples, with no statistical association to dog age, sex, or season, but with a significantly higher presence in urban counties. Additionally, the genomes of 6 CRCoVs were obtained from positive samples using an in-house developed targeted amplicon-based approach specific to CRCoV. Subsequent phylogeny clustered their genomes in 2 distinct genomic groups, with most isolates sharing a higher similarity with CRCoVs from Sweden and only 1 more closely related to CRCoVs from Asia. We provide new insights into CIRD and CRCoV epidemiology in the Southeastern United States and further support the association of CRCoV with more severe cases of CIRD. Additionally, we developed and successfully tested a new amplicon-based approach for whole-genome sequencing of CRCoV that can be used to further investigate the genetic diversity within CRCoVs.

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.

Validation of a real-time PCR panel for detection and quantification of nine pathogens commonly associated with canine infectious respiratory disease

Canine infectious respiratory disease (CIRD) is a complicated respiratory syndrome in dogs [1–3]. A panel PCR was developed [4] to detect nine pathogens commonly associated with CIRD: Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica; canine adenovirus type 2, canine herpesvirus 1, canine parainfluenza virus, canine distemper virus, canine influenza virus and canine respiratory coronavirus [5–16]. To evaluate diagnostic performance of the assay, 740 nasal swab and lung tissue samples were collected and tested with the new assay, and compared to an older version of the assay detecting the same pathogens except that it does not differentiate the two Mycoplasma species. Results indicated that the new assay had the same level of specificity, but with higher diagnostic sensitivity and had identified additional samples with potential co-infections. To confirm the new assay is detecting the correct pathogens, samples with discrepant results between the two assays were sequence-confirmed. Spiking a high concertation target to samples carrying lower concentrations of other targets was carried out and the results demonstrated that there was no apparent interference among targets in the same PCR reaction. Another spike-in experiment was used to determine detection sensitivity between nasal swab and lung tissue samples, and similar results were obtained.•A nine-pathogen CIRD PCR panel assay had identified 139 positives from 740 clinical samples with 60 co-infections;•High-concentration target does not have apparent effect on detecting low-concentration targets;•Detection sensitivity were similar between nasal swab and lung tissue samples.

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.

Development of a long-read next generation sequencing workflow for improved characterization of fastidious respiratory mycoplasmas.

Mycoplasma cynos and Mycoplasma felis are often associated with canine and feline infectious respiratory disease in dogs and cats, respectively. Mycoplasmas have a reduced genome and dearth of many biosynthetic pathways, making them dependent on rich medium for growth. Due to this fastidious nature, mycoplasmas have been historically underdiagnosed. The aim of this study was to develop a cost-effective and accurate sequencing workflow for genotypic characterization of clinical isolates of M. cynos and M. felis using a rapid long-read sequencing platform. We explored the following critical aspects of bacterial whole genome sequencing, including: (i) five solid and liquid-based culture approaches based on a specialized media formulation for Mycoplasma culture, (ii) three DNA extraction methods modified for long-read sequencing purposes, and (iii) two de novo assembly platforms, Flye and Canu, as key components of a bioinformatics pipeline. DNA extraction method 1, a solid-phase and column-based kit with enzymatic lysis, provided the best DNA quality and concentration followed by high coverage and sequencing contiguity. This was obtained with a culture volume of 45 ml in modified Hayflick's broth incubated for 48 h. DNA extracted directly from colonies on agar or from small broth volumes (6 ml) did not meet the criteria required for long-read sequencing. Overall, Flye generated more contiguous assemblies than the Canu assembler and was more time efficient. This 4-5 day sample-to-sequence workflow provides the scientific and clinical communities with a more comprehensive tool than laborious conventional methods for complete genomic characterization of M. cynos and M. felis clinical isolates.

Development of a three-panel multiplex real-time PCR assay for simultaneous detection of nine canine respiratory pathogens

Infectious respiratory disease is one of the most common diseases in dogs worldwide. Several bacterial and viral pathogens can serve as causative agents of canine infectious respiratory disease (CIRD), including Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica, canine adenovirus type 2 (CAdV-2), canine herpesvirus 1 (CHV-1), canine parainfluenza virus (CPIV), canine distemper virus (CDV), canine influenza virus (CIA) and canine respiratory coronavirus (CRCoV). Since these organisms cause similar clinical symptoms, disease diagnosis based on symptoms alone can be difficult. Therefore, a quick and accurate test is necessary to rapidly identify the presence and relative concentrations of causative CIRD agents. In this study, a multiplex real-time PCR panel assay was developed and composed of three subpanels for detection of the aforementioned pathogens. Correlation coefficients (R2) were >0.993 for all singleplex and multiplex real-time PCR assays with the exception of one that was 0.988; PCR amplification efficiencies (E) were between 92.1% and 107.8% for plasmid DNA, and 90.6–103.9% for RNA templates. In comparing singular and multiplex PCR assays, the three multiplex reactions generated similar R2 and E values to those by corresponding singular reactions, suggesting that multiplexing did not interfere with the detection sensitivities. The limit of detection (LOD) of the multiplex real-time PCR for DNA templates was 5, 2, 3, 1, 1, 1, 4, 24 and 10 copies per microliter for M. cynos, M. canis, B. brochiseptica, CAdV-2, CHV-1, CPIV, CDV, CIA and CRCoV, respectively; and 3, 2, 6, 17, 4 and 8 copies per microliter for CAdV-2, CHV-1, CPIV, CDV, CIA and CRCoV, respectively, when RNA templates were used for the four RNA viruses. No cross-detection was observed among the nine pathogens. For the 740 clinical samples tested, the newly designed PCR assay showed higher diagnostic sensitivity compared to an older panel assay; pathogen identities from selected samples positive by the new assay but undetected by the older assay were confirmed by Sanger sequencing. Our data showed that the new assay has higher diagnostic sensitivity while maintaining the assay's specificity, as compared to the older version of the panel assay.

Clinical and bacteriological analysis of respiratory tract infections in sheltered dogs and determination of antibacterial treatment options

After canine infectious respiratory disease complex-CIRDC, only bacterial pneumonia or accompanied with CIRDC has a higher proportion in respiratory disease in stray dogs. Management of these respiratory problems in terms of both treatment and prevention in shelters has a big importance for animal welfare. With this purpose, the present study evaluates bacterial pneumonia in terms of clinical, bacteriological and the antibacterial treatment options in 100 sheltered dogs with respiratory tract infection symptoms. In all dogs, status of respiratory disease and treatment efficacy were evaluated by haematological analysis and clinical scores. Haematological analyses showed that all of the dogs suffered leucocytosis before treatment. Health status of all animals before, during and after treatment were evaluated according to nine clinical scores include clinical condition, body temperature, respiratory and heart rates per minutes, nasal discharge, tracheal sensitivity, mucous membranes, coughing, auscultation. For bacteriological analysisand antimicrobial susceptibility tests, Bronchoalveolar lavage-BAL fluids were obtained from all dogs, twice. The bacterial agents isolated in the present study were Bordetella spp. (38.98%), Mycoplasma spp. (21.19%), Klebsiella spp. (16.10%), E. coli (5.93%), S. aureus (5.08%) and Pasteurella spp. (4.24%). Susceptibility tests were performed by using the disc diffusion method for Enrofloxacin (ENR), Trimethoprim/Sulpha (TS), Chloramphenicol (C), Amoxicillin clavulanate (AC), and Erythromycin (E) in all cases. Bordetella spp. isolated from 46 cases were found to be most susceptible to ENR (21/46 = 46%), TS (12/46 = 26%), and C (11/46 = 24%). Mycoplasma spp. were isolated from 25 cases and were found to be susceptible to C (14/25 = 56%), TS (8/25 = 32%), and ENR (3/25 = 12%). Klebsiella spp. were isolated from 19 cases and the antibiotics most effective were ascertained as C (9/19 = 47%), ENR (9/19 = 47%), and TS (1/19 = 5%).The results showed that clinical scores could be useful in the diagnosis and monitoring of respiratory tract diseases in sheltered dogs. Besides, in the light of the findings of presented study, enrofloxacin, chloramphenicol, and trimethoprim/sulpha were proven efficient against to bacterial isolates in sheltered dogs in the treatment of bacterial pneumonia. Antibacterial therapy should be conducted by antibiotic sensitivity test. But, in the cases this is not possible, antibiotic choice may contain enrofloxacin, trimethoprim/sulpha. If it is not forbidden to use for companion animals by administrations, chloramphenicol may also be thought as alternative.