Respiratory Disease In Dogs Research Articles

Efficacy and Safety in Dogs Following Administration of an Alphavirus RNA Particle Canine Influenza H3N2 Vaccine.

Canine influenza virus (CIV) H3N2 causes a highly contagious respiratory disease in dogs and has been the source of outbreaks across North America since 2015. An injectable RNA Particle (RP)-CIV H3N2 vaccine has been developed to protect dogs against this disease. To demonstrate efficacy, dogs were randomized into two treatment groups, then vaccinated subcutaneously twice, 21 days apart, with a placebo vaccine (n = 20) or an RP-CIV H3N2 vaccine (n = 20). Three weeks later, dogs were challenged intranasally with virulent CIV H3N2 and observed daily for 10 days for clinical signs of disease. Nasal swabs were also collected daily to evaluate the shedding of the challenge virus. Ten days post-challenge, the dogs were euthanized, and the lungs were examined for consolidation. RP-CIV H3N2 vaccination demonstrated a significant reduction in the duration of clinical signs, duration and amount of virus shed, lung consolidation, and the incidence of suppurative pneumonia. To evaluate safety, dogs from multiple geographic regions were vaccinated subcutaneously, 3-4 weeks apart, with an RP-CIV H3N2 vaccine and observed for adverse events for 14 days after each administration. The RP-CIV H3N2 vaccine was deemed safe, with lethargy being the most reported adverse event at a rate of 1.6%.

Investigation of the epidemiology, pathogenicity and immunogenicity of Bordetella bronchiseptica isolated from cats and dogs in China from 2021 to 2023

Bordetella bronchiseptica (Bb) is recognized as a leading cause of respiratory diseases in dogs and cats. However, epidemiological data on Bb in dogs and cats in China are still limited, and there is no commercially available vaccine. Live vaccines containing Bb that are widely used abroad are generally effective but can establish latency and potentially reactivate to cause illness in some immunodeficient vaccinated recipients, raising safety concerns. In this study, 34 canine-derived and two feline-derived Bb strains were isolated from 1809 canine and 113 feline nasopharyngeal swab samples collected from eight provinces in China from 2021 to 2023. The PCR results showed that the percentage of positive Bb was 22.94% (441/1922), and more than 90% of the Bb isolates had four virulence factor-encoding genes (VFGs), namely, fhaB, prn, betA and dnt. All the isolated strains displayed a multidrug-resistant phenotype. The virulence of 10 Bb strains isolated from dogs with respiratory symptoms was tested in mice, and we found that eight isolates were highly virulent. Furthermore, the eight Bb isolates with high virulence were inactivated and intramuscularly injected into mice, and three Bb strains (WH1218, WH1203 and WH1224) with the best protective efficacy were selected. Dogs immunized with these three strains exhibited strong protection against challenge with the Bb field strain WH1218. Ultimately, the WH1218 strain with the greatest protection in dogs was selected as the vaccine candidate. Dogs and cats that received a vaccine containing 109 CFU of the inactivated WH1218 strain showed complete protection against challenge with the Bb field strain WH1218. This study revealed that Bb is an important pathogen that causes respiratory diseases in domestic dogs and cats in China, and all the isolates exhibited multidrug resistance. The present work contributes to the current understanding of the prevalence, antimicrobial resistance, and virulence genes of Bb in domestic dogs and cats. Additionally, our results suggest that the WH1218 strain is a promising candidate safe and efficacious inactivated Bb vaccine.

Molecular determination of virulence factors of Mycoplasma cynos isolated from household dogs

Mycoplasma cynos is a member of the class Mollicutes. It is more pathogenic, more host specific than other canine Mycoplasma, and only associated with respiratory diseases in dogs due to producing many virulence factors, recently neuraminidase and hemagglutinin. The study was done to detect the neuraminidase and hemagglutinin of Mycoplasma cynos isolated from the upper respiratory tract and conjunctival infections in household dogs. Mycoplasma isolates obtained from nasal, oropharyngeal, and conjunctival swabs from household dogs suffering from respiratory diseases in different veterinary clinics in Mosul city. DNA extraction was prepared , PCR and sequencing of 16S rRNA, then Mycoplasma cynos was examined for neuraminidase and hemagglutinin using specific primers.The results showed the identification of Mycoplasma cynos strain "SM-MY-M23" under accession number“OQ446513” in percentage (67.6%), and detection of neuraminidase only. Results obtained are considered the first record of M. cynos and the important virulence factor (Neuraminidase) from household dogs in Iraq.

Pathogenic Characteristics of an Infection with Canine Influenza Virus and Streptococcus equi subsp. zooepidemicus Alone or in Combination in Mice

Both Streptococcus equi subsp. zooepidemicus (SEZ) and canine influenza virus (CIV) are two important pathogens causing infectious respiratory disease in dogs and are frequently codetected in respiratory secretions. However, the clinical significance of viral/bacterial coinfection remains unknown. This study investigated the pathogenic characteristics of infection with CIV and SEZ alone or in combination in mice. Our data indicated that the severity of the disease is related to the challenge order of CIV and SEZ. Coinfection of CIV and SEZ induced higher weight loss in mice than single infection, except for the VB group (viral followed by secondary bacterial infection). Compared with the concurrent or sequential infection groups of CIV and SEZ, mice in the CIV–SEZ preincubation group exhibited more obvious weight loss, higher mortality, and significantly enhanced burden of SEZ and CIV in tissues. Interestingly, viral and bacterial preincubation before coinfection caused typical pulmonary fibrosis in mice. Correspondingly, transforming growth factor (TGF)-β was upregulated, and its canonical small mother against decapentaplegic (Smad) 2/3 signaling was noticeably induced. Further investigation indicated that the activity of the viral neuraminidase (NA) enzyme upon sialic acid was considerably increased due to the direct interaction of CIV with SEZ, which may be related to the activation of the TGF-β signaling pathway. These findings implicate an unexpected contribution of the direct interaction between CIV and SEZ to synergistic pathogenicity.

Relationship between Respiratory Rate, Oxygen Saturation, and Blood Test Results in Dogs with Chronic or Acute Respiratory Disease: A Retrospective Study.

This study aimed to investigate the association of respiratory rate (RR), oxygen saturation (SpO2), and blood findings with respiratory disease in dogs and to compare the examination findings in the chronic and acute phases. Dogs that visited a veterinary referral hospital with respiratory symptoms were classified into the chronic disease group (GC), and those that visited the emergency veterinary hospital were classified into the acute disease group (GA). In total, 704 and 682 dogs were included in GC and GA, respectively. The RR and SpO2 were significantly higher and lower, respectively, in patients with lung disease compared to other disease sites in both groups. White blood cell counts were significantly increased in patients with lung and pleural diseases in both groups. Respiratory alkalosis and respiratory acidosis were most common in GC and GA, respectively. The C-reactive protein levels were elevated in both groups, primarily in patients with lung disease. Associations between the results of several tests for understanding and diagnosing respiratory conditions and diseases were recognized, and differences in the trends of the chronic and acute phases were clarified. These tools may be used as adjuncts to other tests for the diagnosis and monitoring of treatment responses.

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.

Bronkopnömonili Köpeklerde Mycoplasma spp., Streptococcus spp., Bordetella bronchiseptica, Klebsiella spp. ’nin Real-time PCR ve Patolojik Yöntemler ile Teşhisi

There are many bacterial factors that cause respiratory diseases in dogs. Bacterial bronchopneumonia is a lung disease caused by bacteria in the lower respiratory tract and lung parenchyma. In this study, it was aimed to determine Mycoplasma spp., Streptococcus spp., Bordetella bronchiseptica, Klebsiella spp., in dogs with bronchopneumonia by Real-time PCR and to compare the histopathological findings determined according to the agents. The material of the study consisted of lung paraffin blocks of 37 dogs that died due to different reasons and were found to have bronchopneumonia in microscopic examination. Microscopically, edema in the alveolar lumens, shedding of the alveolar epithelium, shedding of the bronch and bronchial epithelium and bronchiectasis, mononuclear cell infiltration (MCI) and Polymorphonuclear leukocytes (PMNL) infiltration in the bronchial lumens, peribronchiolar MCI infiltration, Bacterial clusters localized to the bronch epithelium, PMNL infiltration in the interstitium, multifocal necrosis areas, bleeding and pleuritis were observed. Real-time PCR analysis revealed Bordetella bronchiseptica in 18 (48.64%) cases, Mycoplasma spp. in 9 (24.32%) cases, Streptococcus spp. in 10 (27.02%) cases, and Klebsiella spp. in 2 (5.4%) cases. In conclusion, this study showed that the causative agent can be determined in bacterial bronchopneumonias of dogs with Real-time PCR even in tissues without culture opportunity. In addition, this study indicates that polymicrobial lower respiratory tract infections can also be seen in dogs and reveals that more than one bacterial species should be investigated for diagnosis.

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 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.

Bacterial Contamination of Inhalation Chambers Used for Cats and Dogs with Chronic Airway Diseases.

Inhalation chambers (ICs) are regularly used in veterinary medicine for the inhalative treatment of chronic respiratory diseases in dogs and cats. Since therapy is usually required lifelong and daily, devices are frequently in use. The aim of this study was to identify bacterial contamination of ICs used for cats and dogs in relation to the applied cleaning measures. Swabs from ICs of 66 cats and 19 dogs with chronic airway diseases were obtained using a standardized protocol and subsequently cultured. A questionnaire was completed by the pet owners regarding the history of their pet's illness and applied device cleaning measures. Overall, 64% (54/86) of the ICs were found to be contaminated; the mask was significantly (p < 0.001) more often contaminated than other device parts. Most cultured bacteria were environmental contaminants; however, some harbored pathogenic potential. Cleaning frequency and method did not significantly influence the presence of contamination. Bacterial contamination of ICs, used for cats and dogs, is common but is not significantly influenced by the type or frequency of cleaning. To avoid potential infection by opportunistic bacteria, the instruction of pet owners regarding the maintenance of the ICs is recommended.

Extraesophageal reflux and reflux aspiration in dogs with respiratory diseases and in healthy dogs.

Salivary bile acids are used to diagnose extraesophageal reflux (EER) and to evaluate the risk of reflux aspiration that is associated with respiratory diseases in dogs. To study total bile acid (TBA) concentrations in saliva and in bronchoalveolar lavage fluid (BALF) to investigate EER and reflux aspiration in dogs with respiratory diseases and in healthy dogs. Thirty-one West Highland White Terriers (WHWTs) with idiopathic pulmonary fibrosis (IPF), 12 dogs with inflammatory airway disease (IAD), 6 dogs with recurrent pneumonia (RP), 26 brachycephalic dogs (BD), 27 healthy WHWTs (HW), 52 healthy dogs (HD). All privately-owned dogs. Saliva and BALF were collected from dogs in each group. Salivary TBA concentrations were higher in IPF (median 0.1692 μM, interquartile range [IQR] 0.1115-0.2925 μM, Cohen's d 3.4, 95% confidence interval [CI] 2.2-4.0, P < .001) and BD (0.0256 μM, IQR 0.0086-0.0417 μM, d 0.5, CI -0.1 to 1.1, P=.003) compared to HD (0μM, IQR not quantifiable [n.q.]-0.0131 μM). Bronchoalveolar lavage fluid TBA concentrations were higher in IPF (0.0117 μM, IQR 0.0048-0.0361 μM, d 0.5, CI 0-1.1, P < .001) compared to HD (0μM, IQR n.q.-0.0074 μM). Extraesophageal reflux and reflux aspiration occur in healthy dogs and those with respiratory diseases.

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.

Retrospective study of 1050 dogs with respiratory symptoms in Japan (2005-2020).

Few studies have investigated the incidence of respiratory diseases based on anatomical sites or the relationship between breed and these diseases. The objective of this study was to investigate the prevalence of canine respiratory diseases among dogs in Japan, with relationship to the breed. We retrospectively reviewed the medical records of dogs with respiratory symptoms and calculated the odds ratio (OR) to evaluate the relationship between breed and disease. A total of 1050 dogs with respiratory symptoms were included in this study. Miniature dachshunds were the most common breed affected by respiratory diseases. Among tracheobronchial diseases, there was a significant association between some small breeds and tracheobronchial collapse, miniature dachshunds (OR: 4.44, 8.43, 95% confidence interval [CI]: 3.17-6.22, 4.33-16.0) and chronic bronchitis and bronchiectasis. Among nasal diseases, miniature dachshunds (OR: 27.2, 95% CI: 16.8-44.8) and golden retrievers (OR: 21.0, 95% CI: 6.43-69.3) were the most affected by non-infectious rhinitis and nasal aspergillosis, respectively. Brachycephalic obstructive airway syndrome was the most common disease among pharyngeal and laryngeal diseases, with a relationship with breed being found in some brachycephalic breeds, and Pomeranians (OR: 2.7, 95% CI: 1.42-5.17). Respiratory diseases in dogs are strongly correlated with popular breeds in Japan. Miniature dachshunds, in particular, are associated with many respiratory diseases, which may differ from international reports. Thus, this result may help in the early detection, prevention, treatment, and elucidation of the pathophysiology of canine respiratory diseases.

Effects of Intranasal and Oral Bordetella bronchiseptica Vaccination on the Behavioral and Olfactory Capabilities of Detection Dogs.

The bacterium Bordetella bronchiseptica is responsible for serious respiratory disease in dogs, most often associated with ‘kennel cough’ (canine infectious tracheobronchitis). It is recommended that dogs are vaccinated against the bacterium every 6–12 months, either by oral or intranasal administration. Any impairment of dogs' olfactory capabilities due to medical treatments may impact their efficiency and accuracy in their jobs. This study examined (1) the effect of intranasal and oral vaccines on the olfactory capabilities of detection dogs; as well as (1) effects of the vaccines on canine behavior. Dogs that were vaccinated initially with the oral and 28 days later with intranasal B. bronchiseptica were generally slower to find the target odor than the dogs that were assigned intranasal then oral vaccine. This result prompted a second between-subjects study to further investigate any impact of intranasal administration of the B. bronchiseptica vaccine on the olfactory capabilities of dogs. The intranasal vaccine was of particular interest due to its prevalent use and potential for nasal inflammation leading to decreased olfactory capabilities. Neither odor threshold nor time spent searching for odor were affected by the intranasal vaccine. Behavioral analyses showed that behaviors associated with the dogs' positive and negative motivation affected their time spent finding the target odor; this suggests that behavior should be considered in future studies of olfactory performance.

Retrospective evaluation of carboxyhemoglobin and methemoglobin levels in dogs and cats with respiratory disease

To evaluate carboxyhemoglobin (COHb) and methemoglobin (MetHb) levels in dogs and cats with respiratory disease in the ICU. Retrospective study. University veterinary teaching hospital. The ICU census was searched for dogs (n=466) and cats (n=97) hospitalized within the ICU between January 2016 and January 2019 in whom blood gas with co-oximetry was performed. Dogs and cats were stratified into those with primary respiratory and nonrespiratory categories; the underlying cause of the disease was also noted. Venous blood gas, co-oximeter, PaO2 /FiO2 (PF ratio), physical examination findings, and outcome were recorded. The median COHb and MetHb in dogs hospitalized in the ICU were 2.6% (0.1%-5.6%) and 1.1% (0.1%-2.9%), respectively. The median COHb and MetHb in cats hospitalized in the ICU were 2.2% (0.1%-5.4%) and 1.0% (0%-2.1%), respectively. Dogs with respiratory disease had a higher COHb than dogs without respiratory disease (median, 2.7% [range, 0.3%-5.0%] vs. 2.5% [0.1%-5.6%]; P=0.0148). COHb was positively associated with survival in cats (median, 2.2% [range, 0.1%-5.4%] vs. 1.9% [0.1%-3.9%]; P=0.0433). Both COHb and MetHb were higher in septic dogs than in nonseptic dogs (median COHb, 2.8% [range 0.3%-4.5%] vs. 2.6% [0.1%-5.6%]; P=0.02 and median MetHb, 1.1% [0.1%-2.9%] vs. 1.1% [0.1%-2.4%]; P=0.01, respectively). There may be a positive association between COHb and respiratory disease in dogs; prospective studies are needed to evaluate this further. No association between COHb and respiratory disease in cats or MetHb and respiratory disease in either species was detected. Additional prospective studies are needed to determine whether COHb and MetHb are biomarkers for sepsis in dogs and whether COHb is an indicator of mortality in cats.

Generation and Characterization of Single-Cycle Infectious Canine Influenza A Virus (sciCIV) and Its Use as Vaccine Platform.

Influenza A viruses (IAVs) infect a broad range of hosts, including multiple avian and mammalian species. The frequent emergence of novel IAV strains in different hosts, including in humans, results in the need for vigilance and ongoing development of new approaches to fighting or prevent those infections. Canine influenza is a contagious respiratory disease in dogs caused by two subtypes of IAV, the equine-origin H3N8 canine influenza virus (CIV), and the avian-origin H3N2 CIV. A novel approach to influenza vaccination involves single-cycle infectious influenza A viruses (sciIAVs), which are defective for an essential viral gene. They are propagated in complementing cell lines which provide the missing gene in trans. As sciIAV cannot complete their replication cycle in regular cells they are limited to a single round of viral replication. Because of their safety profile and ability to express foreign antigens inside infected cells, sciIAVs have served both as live-attenuated vaccines and as vaccine vectors for the expression of heterologous antigens. Here, we describe experimental procedures for the generation of a single-cycle infectious CIV (sciCIV), where the viral hemagglutinin (HA) gene was exchanged for the gene for green fluorescent protein (GFP). Complementation of the viral HA protein is provided in trans by stable HA-expressing cell lines. Methods for the in vitro characterization of HA deficient but GFP-expressing sciCIV (sciCIV ΔHA/GFP) are described, as well as its use as a potential vaccine.

Induction of IFN-β through TLR-3- and RIG-I-Mediated Signaling Pathways in Canine Respiratory Epithelial Cells Infected with H3N2 Canine Influenza Virus.

Canine influenza virus (CIV) induces acute respiratory disease in dogs. In this study, we aimed to determine the signaling pathways leading to the induction of IFN-β in a canine respiratory epithelial cell line (KU-CBE) infected with the H3N2 subtype of CIV. Small interfering RNAs (siRNAs) specific to pattern recognition receptors (PRRs) and transcription factors were used to block the IFN-β induction signals in H3N2 CIV-infected KU-CBE cells. Among the PRRs, only the TLR3 and RIG-I expression levels significantly (p < 0.001) increased in CIV-infected cells. Following transfection with siRNA specific to TLR3 (siTLR3) or RIG-I (siRIG-I), the mRNA expression levels of IFN-β significantly (p < 0.001) decreased, and the protein expression of IFN-β also decreased in infected cells. In addition, co-transfection with both siTLR3 and siRIG-I significantly reduced IRF3 (p < 0.001) and IFN-β (p < 0.001) mRNA levels. Moreover, the protein concentration of IFN-β was significantly (p < 0.01) lower in cells co-transfected with both siTLR3 and siRIG-I than in cells transfected with either siTLR3 or siRIGI alone. Also, the antiviral protein MX1 was only expressed in KU-CBE cells infected with CIV or treated with IFN-β or IFN-α. Thus, we speculate that IFN-β further induces MX1 expression, which might suppress CIV replication. Taken together, these data indicate that TLR3 and RIG-I synergistically induce IFN-β expression via the activation of IRF3, and the produced IFN-β further induces the production of MX1, which would suppress CIV replication in CIV-infected cells.

Proteomic Characterization of Canine Gastric Fluid by Liquid Chromatography–Mass Spectrometry for Development of Protein Biomarkers in Regurgitation, Vomiting, and Cough

Reflux and aspiration in people cause and exacerbate respiratory diseases in the absence of gastrointestinal signs. Protein biomarkers in humans detect extraesophageal reflux (EER) from oropharyngeal (OP) and bronchoalveloar lavage samples. Reflux likely contributes to respiratory disease in dogs. The objectives of this study were to analyze the canine gastric fluid (GF) proteome and compare this to the OP proteome in normal, vomiting/regurgitating, and coughing dogs to identify biomarkers for EER/aspiration. Twenty-three client-owned dogs were enrolled. Canine GF samples (n = 5) and OP swabs in normal (n = 6), vomiting/regurgitating (n = 7), and coughing (n = 5) dogs were within 2 weeks of sample collection. Protein digests were analyzed by liquid chromatography–mass spectrometry. Differential abundance (DA) of proteins between groups was evaluated by Fisher's exact test with p < 0.0004 significance level after correction for multiple comparisons. DA was found between all groups (p < 0.0001): GF vs. normal (n = 130 proteins), coughing vs. normal (n = 22 proteins), and vomiting/regurgitating vs. normal (n = 20 proteins). Protein abundance was highly variable between dogs. Gastrointestinal-specific proteins were found in OP swabs from vomiting/regurgitating and coughing dogs but not from healthy dogs. In conclusion, the proteomic composition of the OP varies between health and disease. The presence of gastrointestinal-specific proteins in OP of coughing dogs may suggest reflux and/or aspiration as contributing factors. The variable protein abundance warrants investigation into biomarker panels.

The inactivated vaccine of reassortant H3N2 canine influenza virus based on internal gene cassette from PR8 is safe and effective

Canine influenza (CI) is a contagious respiratory disease in dogs, which poses a threat to canine health. A safe, high-yield vaccine seed virus is critical for CI vaccine development. We developed a PR8-based reassortant H3N2 canine influenza virus (RT CIV) using the reverse genetic method and evaluated its yield in canine kidney epithelial (MDCK) cells, Vero cells, and specific pathogen-free (SPF) chicken embryos. Mice and dogs were infected with RT CIV, and the pathogenicity was evaluated. The viral titers of RT CIV increased in MDCK cells, Vero cells, and SPF chicken embryos; the HA yield in SPF chicken embryos increased 4-fold. However, RT CIV was not lethal to mice, and it showed similar virulence as wild-type CIV. RT CIV also showed minimal pathogenicity in dogs, which manifested as mild fever and rhinorrhea for the first two days post-infection. Thus, RT CIV carrying the internal gene cassette from PR8 showed almost no pathogenicity in dogs. And the reassortant virus inactivated vaccine could provide complete protection against H3N2 CIV. To our knowledge, this is the first report on the pathogenicity of PR8-based reassortant H3N2 CIV in dogs. These studies are relevant for developing a high-yield and safe CI vaccine.

Phosphoproteomics to Characterize Host Response During H3N2 Canine Influenza Virus Infection of Dog Lung.

Avian-origin H3N2 canine influenza viruses (CIVs) cause severe contagious respiratory disease in dogs, and quickly adapt to new environments. To further understand the mechanism of virus infection and host-virus interactions, we characterized the complete phosphoproteome of dogs infected with H3N2 CIV. Nine-week-old Beagle dogs were inoculated intranasally with 106 EID50 of A/canine/Guangdong/04/2014 (H3N2) virus. Lung sections were harvested at 5 days post-inoculation (dpi) and processed for global and quantitative analysis of differentially expressed phosphoproteins. A total of 1,235 differentially expressed phosphorylated proteins were identified in the dog lung after H3N2 CIV infection, and 3,016 modification sites were identified among all differentially expressed proteins. We then performed an enrichment analysis of functional annotations using Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) database analyses to predict the functions of the identified differential phosphoproteins. Our data indicate that H3N2 CIV infection causes dramatic changes in the host protein phosphorylation of dog lungs. To our knowledge, this is the first study to assess the effect of H3N2 CIV infection on the phosphoproteome of beagles. These data provide novel insights into H3N2-CIV-triggered regulatory phosphorylation circuits and signaling networks and may improve our understanding of the mechanisms underlying CIV pathogenesis in dogs.