Clinical data for cases 2 and 3 are present in Table 1.

On presentation, the dog was depressed, dehydrated, shivering, hypersalivating with blood stained saliva, and coughed spontaneously with haemorrhagic expectorate. The neck was slightly stretched and auscultation of thorax revealed increased vesicular sounds.

Thoracic radiographs showed moderately increased attenuation of the ventral part of the right middle lung lobe, moderately to severely increased attenuation of the ventrocaudal part of the right caudal lung lobe as well as air bronchograms (Figures 4 and 5). These changes are consistent with acute pneumonia. A faint soft-tissue opacity was seen in the lung fissures, interpreted as a possible low amount of free pleural fluid.

The dog was treated with intravenous (IV) ringer acetate 100 ml/kg/hr for eight hours, thereafter 50 ml/kg/hr, enrofloxacin (Baytril, vet. Bayer; 5 mg/kg bodyweight (BW) IV once daily), ampicillin (Pentrexyl, Bristol-Myers Squibb; 35 mg/kg BW IV three times daily) and buprenorfin (Temgesic, Schering-Plough; 0.02 mg/kg BW IV three times daily). All medications were given for four days. The dog was hospitalised in an oxygen cage. Simultaneously as the treatment was initiated an expectorate sample was sent for routine bacteriological cultivation. S. equi subsp. zooepidemicus was isolated in pure culture and directly demonstrated to belong to the Lancefield Group C of streptococci. Complete blood count with serum biochemistry analysis was normal except for a mild leucopenia (Table 2). The coagulation profile was normal.

On the second day of hospitalisation the dog showed substantial clinical improvement and was normothermic (38.1°C), less dyspneic, less tachypneic (respiration rate (RR) = 44/min), and had reduced salivation and coughing. There was no longer blood in the saliva nor epistaxis. The result from the bacteriological investigation of the expectorate demonstrated sensitivity against penicillin, tetracyclin, cefalexin, ampicillin, amoxicillin/clavulanate, enrofloxacin and linkomycin. The antibiotic regimen was switched to phenoxymethylpenicillin (Apocillin; Actavis) 660 mg per os (PO) three times daily for 14 days. No diagnostic tests for respiratory viruses were performed. Clinical signs gradually resolved over the next few days and the dog was sent home seven days after hospitalisation. Control radiographs before departure from the clinic revealed absence of air bronchograms, though a mild to moderate increased attenuation with an interstitial pattern was still present.

Descriptive analysis was used as the median for age. Data were evaluated using the SPSS statistical analyser version 22.0 software (IBM Corp, New York, U.S.A). Differentiation of the CAI and HAI groups was compared using Fisher's exact and χ2 tests. Clinical severity level against the demographic of infected dogs was performed using χ2 tests. Incidence of CIRDC viruses was calculated with a confidence interval (CI) of 95%. In these cases, a P-value of <0.05 was considered statistically significant. The associated respiratory score with respect to the CIRDCV detections was evaluated using χ2 tests for individual infection, while logistic regression was evaluated in multiple infections. Multiple comparisons with a Bonferroni correction a significant P-value at 0.001 was considered statistically significant.

Nasal and tonsillar swabs (sterile cotton swabs) were collected from all 264 dogs (groups A, B, C) in this study. In 31 animals with progressive respiratory disorders that had been resistant to previous treatment, bronchoalveolar lavage fluid (BALF) samples were additionally collected during tracheobronchoscopy. From 30 acutely diseased dogs, paired blood samples were retrieved for serological examinations.

Swab samples were transferred into tubes containing 1 mL of diethyl pyrocarbonate‐treated water and vortexed for 10 seconds. BALF obtained from right and left lung was pooled in equivalent amounts. Swab and BALF samples were frozen at −80°C until further analysis.

Serum was prepared by centrifugation (10 minutes at 3000g), was subsequently lifted from the blood samples, and frozen at −20°C.

From 30 available paired serum samples 17 (56.7%) were obtained from privately‐owned dogs (group A). Five of these 17 samples (29.4%) showed a significant increase in anti‐CRCoV antibody titres. The antibody titres of two dogs increased more than 128‐fold. CRCoV‐specific nucleic acid from nasal or tonsillar swabs was detected in these two dogs.

Another 13 paired serum samples (43.3%) were collected from a population of 15 kennelled working dogs with an acute episode of CIRD (group B). As two of the 15 dogs were non‐compliant with blood sampling, in these cases no serum samples were obtained. In 10 of the 13 paired serum samples (76.9%), a significant increase in anti‐CRCoV antibodies was found. Six dogs revealed a 16‐ to 128‐fold antibody titre increase and concurrent evidence of CRCoV RNA in sample material from the nose or tonsils. No further causative viral agent was detected in these cases (Table 4).

Out of 885 patients tested for M. pneumoniae in the study period, 139 were excluded due to age (older than 15 years of age), double sampling or repeated samples within three months. A total of 746 children with respiratory tract infection (RTI) were enrolled in the study and 134 had a positive test for M. pneumoniae. Out of 740 M. pneumoniae PCR analyses included, 132 were positive and out of 20 serological tests, 3 were positive. Fourteen children were tested with both real-time PCR and serology.

Fig 1 shows the number of children tested for M. pneumoniae and the numbers of positive cases by month. The epidemic peaked in October 2010 and again in October to December 2011. The number of M. pneumoniae positive cases was 50 (of 386) in the first epidemic season and 84 (of 360) in the second period of the epidemic, with a significantly higher rate (0,13 vs 0,23) of positive cases in the later (p = 0,0002, chi2-test). Overall 18% of the children tested for M. pneumonia had a positive sample.

The highest rate of M. pneumoniae positive samples was in school-aged children (65%) but notably also pre-school children (30%), and even six children under the age of two (4%) had M. pneumoniae positive tests.

A total of 209 dogs with respiratory illness were included in the study. A nasal swab (NS) and an oropharyngeal swab (OS) were obtained from each dog. These dogs with respiratory illness were obtained from the animal hospitals and private clinics located at Central (n = 127), Eastern (n = 21), Southern (n = 49), and Western (n = 12) regions of Thailand. Any dog with respiratory problems associated with underlying cardio-pulmonary disease, functional or anatomical airway disease and neoplasia, based on physical examination and radiographic investigation, or any dog with a history of vaccination within 2 weeks before showing respiratory clinical signs, was excluded from the study. The history of respiratory clinical presentation at hospital or clinic for at least 72 h after being admitted was recorded and used to categorize the dogs into the CAI and HAI dog groups. The CAI group was comprised of dogs with respiratory illness at first presentation at a hospital or a clinic and with no history of hospital or clinic admission within last 3 months, while the HAI group was comprised of likewise symptomatic dogs that showed respiratory problems within 72 h after admission to a hospital or clinic.

Respiratory signs of the dogs were recorded by the attending veterinarian on the date of sampling, as follows: 1, mild cough; 2, nasal discharge and cough; 3, nasal discharge and cough with depression; 4, nasal discharge and cough with depression and evidence of bronchopneumonia based on radiographs. Due to the fact that the different viral infections may show in either local and systemic diseases and result in difference of clinical severity, and the respiratory scores were further graded to clinical severity level for clinical screening [28, 29, 30]. The record was further graded for the clinical severity level as mild (score 1 and 2), moderate (score 3) and severe (score 4), as described previously [6, 7]. General signalment of age, breed, sex and vaccination history for CPIV, CAdV-2 and CDV was systemically recorded for further interpretation.

This animal was presented to NSVS one day after Case 1. The dog had been coughing for several days and gradually worsened with reduced appetite and depression developing on the day of presentation. On physical exam there was moderate dyspnoea with abdominal respiration and increased vesicular sounds, slight neck extension, blood stained saliva as well as fever (Table 1). Haematology showed moderate leucocytosis due to neutrophilia and monocytosis (Table 2). Radiography of the thorax showed the same changes as described for Case 2.

The dog was hospitalised and medically treated in the same way as for Case 2. Clinical progression was similar to Case 2, with normalisation of temperature (38.8°C), respiration rate (28 breaths/min), heart rate (100 beats/minute) and appetite on the second day of hospitalisation. No salivation or spontaneous coughing was observed unless whilst excited after visiting the exercise pen. Repeat thoracic radiographs on the seventh day of hospitalisation revealed air bronchograms in the right middle lung lobe, but reduced consolidations. The dog was sent home on phenoxymethylpenicillin (Apocillin; Actavis) 660 mg PO three times daily for another 14 days.

Follow-up hospital care of both dogs included radiographs of the thorax after one, three, five and eight weeks (Figures 6 and 7) together with complete blood counts (Table 2). After thoroughly scrutinising the last taken radiographs together with assessing their clinical condition they started a step-wise training program.

The radiographs taken during recovery revealed a very mild interstitial attenuation of the lung lobes that had been most severely affected and mild to faint visualisation of fissure lines which was interpreted as either mild amount of free fluid or mild fibrosis. These minor findings were gradually reduced, but faint fissure lines could still be seen after five weeks for Case 3 and after eight weeks for Case 2 (Figures 6 and 7).

The dogs were kept confined for one week after they were released from the hospital, and did run free on a large (2–3 acres) fenced yard for two weeks. Case 2 was in full training eight weeks post infection and Case 3 was slightly behind.

Early in January the following year both dogs participated in a sled race resulting in a time track record, and medal placements in various championships were achieved the following season.

We designed a retrospective analysis of all children younger than 16 years of age admitted to the Department of Paediatrics, Nordsjællands Hospital, Hillerød, Denmark, that were investigated for M. pneumoniae either by polymerase chain reaction (PCR) or by serology (M. pneumoniae antibody test and cold-agglutinin test). The study period was 01.02.2010 to 31.01.2012. We collected PCR results and blood sample results from both our local microbiology laboratory (PCR) and from SSI (PCR and serology). Data were matched on social security number to avoid double sampling. PCR was performed on oropharyngeal-swabs that were stored and transported cooled to the laboratory. Diagnosis of M. pneumoniae was based on a commercially available real-time-PCR (RT-PCR) kit (Minerva Biolabs VenorR Mp Mycoplasma pneumoniae -Diagnostic Kit for qPCR type I) targeting the P1 cytoadhesion gene. The PCR was a multiplex analysis also targeting Chlamydophila pneumoniae/psittaci. The assay was performed in accordance with the manufactures description. In short 10μl sample was added to 14.4μl mastermix containing 14μl buffer, 0.2μl Taq-polymerase and 0.2μl Uracil-N-Glycosylase. RT-PCR was performed under the following conditions on a Stratagene Mx 3005P RT-PCR machine: 10 min at 95°C followed by 45 cycles of 30 sec at 95°C, 30 sec at 55°C and 30 sec at 60°C. M. pneumoniae was detected in the FAM filter.

The serological tests were performed by an external commercial provider (SSI) and were based on M. pneumoniae specific IgM antibodies (MPT) together with cold-agglutinins (KAT). A titre of both MPT and KAT above or equal to 64 was considered as a positive test. Since the KAT titres fall more rapidly after a passed infection, it was used to strengthen the likelihood of an ongoing infection. It is estimated that 95% of persons under the age of 20 have positive KAT titres in response to an M. pneumoniae infection (SSI test-information).

Children who presented with two or more positive PCR-tests within three months were considered as the same infective episode and only included once.

The children were referred by general practitioners or the hospital's emergency department. The doctor on call ordered the M. pneumoniae sampling according to the clinical evaluation.

Demographic and clinical data were collected for the 134 children, who tested positive for M. pneumoniae, by medical chart review. The charts of 612 children with negative tests have not been audited. Medical history was systematically reviewed using computerised medical records, radiological reports and laboratory reports. Characteristics analysed included demographics (age, gender, medical history prior to admission), clinical presentation (pulmonary and extra-pulmonary symptoms, clinical examination; respiratory rate, auscultation, temperature, oxygen saturation), radiological findings, biochemistry (C-reactive protein (CRP), leucocyte count at admission) co-infections, complications and medical treatment. Children were divided in age-groups for data analysis, less than two years old (referred to in the text as infants), 2-6-year-olds and 7-15-year-olds (school-aged children).

To further meet the question of viral co-infections, a post-hoc analysis of the original, frozen, oropharyngeal swab specimen, from 49 of the M. pneumoniae positive cohort, were tested by PCR using a commercially available kit (Biomerieux Respiratory MWS R-Gene range) for a wide range of respiratory viruses (including; respiratory syncytial virus (RSV), influenza A and B, human metapneumovirus, rhinovirus, parainfluenza virus, coronavirus, bocavirus and adenovirus). In short, PCR was performed in four dual-plexes; a 10μl sample was added to 15μl mastermix containing a buffer, Reverse transcriptase and primer/probe-mix. RT-PCR was performed, under the following conditions, on a Stratagene Mx 3005P RT-PCR machine: 5 min at 50°C, 15 min at 95°C followed by 45 cycles of 10 sec at 95°C, 40 sec at 60°C and 25 sec at 72°C. Al targets were detected in the either the FAM or HEX filter.

Samples positive after more than 35 Ct-cycles were re-evaluated to confirm a true positive result.

The Ethical Committee of Region Hovedstaden (protocolnumber: H-2-2012-132) as well as the Danish Data Protection Agency approved the study. This approval included acceptance of reviewing the medical records without informed consent of the patients.

Statistical analyses were performed in STATA 10.0, software packages SPSS version 22 and Microsoft Excell. Chi2-test, Fishers Exact test or Z-score test was used for categorical data analysis and two-sample T-test for numerical data. A p-value of < 0,05 was considered significant.

Infectious causes of respiratory disease are common in dogs; canine distemper virus, adenovirus 2, parainfluenza, influenza, herpesvirus, pneumovirus, respiratory coronavirus, Bordetella bronchiseptica, various Mycoplasma spp., and Streptococcus equi var. zooepidemicus are documented causes.1 Molecular diagnostic assays to detect viral and bacterial pathogens are available for these agents. In the United States, modified live vaccines (MLVx) for intranasal (IN) administration are currently available for adenovirus 2, B. bronchiseptica, and parainfluenza. These vaccines do not induce sterilizing immunity, and vaccinated dogs can still develop clinical signs of disease if exposed to virulent strains of the organisms.2 It is currently unknown if IN administration of MLVx against these agents results in positive molecular diagnostic assay results in dogs without previous vaccination. If transient positive molecular diagnostic assay results are common after vaccination, the positive predictive value of the diagnostic assays to predict disease caused by these agents in dogs would be decreased.

The purpose of this study was to determine the impact of administration of a single IN dose of a commercially available MLVx adenovirus 2, B. bronchiseptica, and parainfluenza containing vaccine,1 included as part of a facility standard initial vaccination series with a parenteral administration of MLVx containing adenovirus 2, canine distemper virus, and parvovirus, on the results of a commercially available polymerase chain reaction (PCR) panel that amplifies the RNA or DNA of the agents.2

The study was completed with Institutional Animal Care and Use approval. Beagle puppies housed at a commercial breeding facility were used.3 The puppies were housed in a closed facility without contact with other dogs and staff members followed facility barrier precautions over the course of the study. A sterile cotton swab was gently rubbed at the entrance to the external nares, and a second swab gently rubbed against the mucosa of the oropharynx in nonsedated puppies. The swabs were stored separately at 4°C in sterile plastic tubes and stored until shipped by overnight express on cold packs for performance of the molecular assays.2

A total of 12 puppies were screened twice as described, 1 week apart, and all were negative for nucleic acids of the target organisms. Eight puppies were randomly selected for the study and housed in a separate room at the breeding facility for the duration of the study. The puppies were approximately 9 weeks of age when samples were collected on Day 0 before the SQ administration of a MLVx containing adenovirus 2, canine distemper virus, and parvovirus4 and the IN administration of a MLVx1 containing adenovirus 2, B. bronchiseptica, and parainfluenza following manufacturer's instructions (approximately ½ mL per nares). Nasal and pharyngeal swabs were then collected on days 1, 2, 3, 4, 5, 6, 7, 10, 14, 17, 21, 24, and 28 for molecular analysis.2

Sneezing or coughing which have been associated with IN MLVx administration was not noted over the course of the study. Adverse effects associated with the collection of the nasal and oropharyngeal swabs were not noted. At the time the study was performed, the PCR panel utilized also included primers for canine distemper virus RNA; and none of the samples collected over the course of the study were positive. In contrast, nucleic acids of adenovirus 2, B. bronchiseptica, and parainfluenza were amplified from both sampling sites, from all 8 puppies, on multiple days after vaccine administration (Table 1). Because adenovirus 2 was administered in both vaccine types, source of that virus cannot be determined. Increasing numbers of positive samples after vaccination suggest local replication of the vaccinal strains. Decreasing numbers of positive samples over time suggest immune responses inhibiting organism replication. However, quantitative PCR assays normalized to total DNA/RNA on the swab would be needed to confirm or deny these hypotheses. The PCR laboratory adheres to standard operating procedures including use of positive and negative controls thus erroneous results are unlikely.

Agents considered most common for kennel cough syndrome include canine distemper virus, adenovirus 2, parainfluenza, and B. bronchiseptica. However, emerging pathogens include influenza, herpesvirus, respiratory coronavirus, pantropic coronavirus, pneumovirus, and others.1 All of these agents, as well as S. equi var. zooepidemicus and Mycoplasma spp., have been identified as causes of canine infectious respiratory disease. Determination of the agent is important for targeting treatment, particularly for dogs who fail to respond to standard treatment recommendations.2 In animal shelter environments, agent identification is critical for outbreak control and individual case management.3

Bacterial and viral shedding postvaccine administration complicates diagnostic testing and treatment. This is especially problematic in shelter environments as dogs are routinely vaccinated on intake. Viral shedding after vaccination has been detected in cats,4 people,5 cattle,6 pigs,7 and dogs.8 A vaccine strain of B. bronchiseptica was detected via nasal culture up to 4 weeks after IN vaccination of 2–week‐old puppies.9

Commercially available respiratory PCR panels are a relatively cost and time effective diagnostic method for identifying multiple respiratory pathogens. However, amplification of nucleic acids may inherently lead to inaccurate clinical diagnosis because small amounts can be amplified from some animals even though the agent may not be present in sufficient quantity to cause disease. In this study, nucleic acids of all 3 organisms contained in the IN vaccine were amplified from both sites on multiple days via PCR, although no clinical signs of respiratory disease were observed. Thus, interpretation of PCR panel results for diagnoses should include consideration of recent vaccination status and clinical signs of disease. Use of quantitative PCR and wild‐type sequence differences may be able to differentiate between vaccine and pathogenic agent shedding and may be used diagnostically in the future.

Real‐time reverse transcriptase PCR has been used to amplify canine distemper virus RNA in blood, urine, and conjunctival swabs after administration of SQ MLVx.10 In this study, the PCR panel did not amplify distemper virus RNA from nasal or pharyngeal swabs. Further studies are needed to determine whether the negative result is because this strain of vaccine virus does not reach the nasal or pharyngeal tissues or was present at levels below the detectable limit of the assay used.

Statistical analysis was performed using SPSS statistical software (version 19.0; SPSS Inc., Chicago, IL, USA). For comparisons of categorical data, the χ2 test and Fisher’s exact test were used as appropriate. All tests were two-tailed and p<0.05 was considered statistically significant.

Pediatric patients (≤14 years old) who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, snivel, sneeze, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week, were enrolled in this study. Chest radiography was conducted according to the clinical situation of the patient, and pneumonia was defined as an acute illness with radiographic pulmonary shadowing (at least segmental or in one lobe) by chest radiography.

Throat swab samples were collected from the enrolled patients at three hospitals in Guangzhou, southern China between July 2009 and June 2012. The samples were refrigerated at 2 to 8°C in viral transport medium, transported on ice to State Key Laboratory of Respiratory Diseases and analyzed immediately or stored at −80°C before testing.

All RNA extracts were subjected to a previously-established RT-PCR assay for detection of CnPnV RNA, with minor modifications. Briefly, a one-step method was adopted using SuperScript™ One-Step RT-PCR for Long Templates (Invitrogen srl, Milan, Italy), according to the manufacturer’s instructions, and primers SH1F/SH187R that amplify a 208-bp of the small hydrophobic (SH) protein gene (Table 1). The following thermal protocol was used: reverse transcription at 50°C for 30 min, inactivation of Superscript II RT at 94°C for 2 min, 40 cycles of 94°C for 30 s, 54°C for 30 s, 68°C for 60 s, with a final extension at 68°C for 10 min. The PCR products were detected by electrophoresis through a 1.5% agarose gel and visualisation under UV light after ethidium bromide staining.

In addition to the gel-based RT-PCR, a real-time RT-PCR assay based on the TaqMan technology was developed for the rapid detection and quantification of the CnPnV RNA in all clinical samples. Reactions were carried out using Platinum® Quantitative PCR SuperMix-UDG (Invitrogen srl) in a 50-µl mixture containing 25 µl of master mix, 300 nM of primers CnPnV-For and CnPnV-Rev, 200 nM of probe CnPnV-Pb (Table 1) and 10 µl of template RNA. Duplicates of log10 dilutions of standard RNA were analyzed simultaneously in order to obtain a standard curve for absolute quantification. The thermal profile consisted of incubation with UDG at 50°C for 2 min and activation of Platinum Taq DNA polymerase at 95°C for 2 min, followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 48°C for 30 s and extension at 60°C for 30 s.

CnPnV positive samples were inoculated into semiconfluent canine fibroma (A-72) cells, as previously described. Inoculated cells were maintained in D-MEM supplemented with 5% FCS and monitored daily for the occurrence of cytopathic effect (CPE). After 6 days of incubation, the monolayers were tested for CnPnV antigen by an immunofluorescence (IF) assay using a monoclonal antibody targeting HRSV (Monosan®, Sanbio BV, Uden, The Netherlands). The cells were sub-cultured every 6–8 days for 5 consecutive passages.

Nasal swab, urine, fecal and blood samples from each affected panda were collected at the time of clinical disease onset. Viral DNA and RNA were isolated from samples using the AxyPrep Multisource Genomic DNA Miniprep kit (AXYGEN, Union City, USA) and RNeasy Mini kit (QIAGEN, Germantown, MD) according to manufacturer’s protocols. Extracted nucleic acids were tested by RT-PCR for CDV using primers specific for CDV H gene (P1:5′-CGAGTCTTTGAGATAGGGTT-3′ and P2: 5′-CCTCCAAAGGGTTCCCATGA-3′). RT-PCR and PCR testing for other viruses threatening giant pandas (canine adenovirus, canine herpesvirus, canine coronavirus, and canine parainfluenza virus) were performed using previously reported methods1730. RT-PCR testing for CDV was also performed on samples collected from the heart, liver, spleen, lungs, kidneys, intestines, and brain of each deceased giant panda, with the exception of Chengcheng for whom tissue samples were not available. Serum samples were collected from the giant pandas during the outbreak to measure SN antibody titers against CDV.

Samples used in this study were regular submissions to the Athens Veterinary Diagnostic Laboratory (AVDL, Athens, GA, USA) by licensed veterinarians from client owned dogs for the diagnosis of respiratory disease using molecular methods. Clinical samples submitted to the AVDL are accompanied by a paper submission form, which asks questions regarding clinical history, vaccination records and clinical signs. These submission forms are then scanned and partially transcribed to an electronic database. This database was queried to retrieve all submission forms between 2011 and 2017 that requested a canine respiratory PCR panel. Signalment, clinical presentation and history of vaccination were retrieved from 559 of these electronically stored paper-based forms. The majority of these samples were received from the southeastern region of the United States. Submission forms provided no information as to whether the animals had been previously kenneled. From 2011 to 2016, the AVDL canine respiratory PCR panel included Mycoplasma spp., B. bronchiseptica, CAV, CDV, coronavirus (CoV) and influenza A Matrix (H3N2 and H3N8). In July 2017, PCR tests for identification of M. canis, M. cynos and S. equi subsp. zooepidemicus were added to the panel. Since our laboratory stores DNA for 6 months (-20°C), we were able to perform the new diagnostic tests included in the panel on all clinical samples received in 2017, by reanalyzing stored DNA. In order to investigate the presence of canine respiratory pathogens in dogs without clinical signs of respiratory disease (controls), nasal swabs were prospectively collected 4h to 24h postmortem from carcasses of asymptomatic dogs (n = 52) that were submitted to the AVDL for necropsy. These animals were selected to represent a similar age and sex distribution as the animals with clinical signs of CIRD (~ 50% males, 50% females; age mean ± SD = 2.1 ± 1.08 years). These control animals had no history of respiratory clinical disease according to the referring veterinarians and available medical records, which covered the entire life span of young animals (< 2-year-old) and at least the last year of life in older (> 2-year-old) dogs. Board-certified pathologists examined the clinical records from the asymptomatic control group, and performed complete postmortem and histological examinations to confirm that the animals were not affected by respiratory disease at the time of euthanasia. All the data collected in this study was part of routine diagnostic work-up in client-owned animals, and no additional testing or diagnostic procedures were performed for the purpose of this study. Therefore, The University of Georgia does not require Institutional Animal Care and Use Committee review and approval of such studies as long as the retrospective records review does not contain animal ID or client information. The animals investigated in this study were not identifiable in the retrospective records (e.g. only sample ID barcoding was used to identify samples).

Nucleic acids were extracted using QIAamp cador Pathogen Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions using QIAcube automated nucleic acid extraction system (Qiagen). DNA samples were stored at -20°C until molecular analysis. Primers and probes are described in supplementary table 1 (S1 Table). B. bronchiseptica, CAV type 1 and type 2, CDV, influenza type A, CoV, CPIV, and S. equi subsp. zooepidemicus were performed as previously published. For CoV, the primers targeted the replicase gene, which is well conserved among coronaviruses, and therefore, do not allow distinction between respiratory (CRCoV) and enteric (CCoV) canine coronaviruses. Further details of the PCR assays are described in supplementary table 2 (S2 Table). All assays were performed with a positive amplification control, negative amplification control, and an exogenous internal control (Qiagen Quantifast pathogen PCR and RT-PCR internal control kit). Assays were performed and results were interpreted taking into consideration the MIQE guidelines [30, 31].

Necropsies were performed on all deceased giant pandas at which time tissue samples were collected for histologic examination. Lung samples from the giant panda named Fengfeng, who experienced a long illness duration, were selected for histologic review using routine methods. Tissue samples were fixed in 10% phosphate-buffered formalin, embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin prior to analysis.

Seropositivity ratios were evaluated by χ

2 test (Minitab 12.0). P < 0.05 value was taken to indicate statistical significance.

In a longitudinal assessment over 12 seasons in a single community, we found that RSV was a common cause of outpatient respiratory illness in adults ≥60 years of age. RSV was detected in 11% of those with medically attended acute respiratory illness, and it was the second most common viral pathogen in this age group. The number of RSV A and RSV B cases was similar overall, but 1 subtype was usually dominant during any given season. The seasonal incidence of medically attended RSV was variable but was consistently higher in persons with preexisting cardiopulmonary disease.

Moderate or serious outcomes, including change in therapy, hospital admission, and pneumonia, occurred in >80% of patients with laboratory-confirmed RSV infection. Serious outcomes (hospital admission, ED visit, or pneumonia) occurred in nearly 1 of every 5 patients with RSV infection. Patients with serious outcomes were significantly more likely to present with dyspnea and objective signs of lower respiratory tract involvement, including wheeze, rales, and rhonchi. Hospital admission was the most common serious outcome, and the risk of a serious outcome increased with age. Serious RSV illness was significantly associated with chronic obstructive pulmonary disease and congestive heart failure.

The majority of outpatient RSV cases (64%) resulted in a moderate outcome, including a new prescription for antibiotics, antivirals, bronchodilators, or systemic corticosteroids. Nearly half of individuals with RSV required a chest x-ray and measurement of oxygen saturation during the enrollment visit or follow-up period. Overall, the most common therapeutic interventions included new antibiotic prescription and bronchodilator/nebulizer treatment. More than three-quarters of patients with RSV were treated with antibiotics. Although this study did not evaluate bacterial coinfections, it is possible that many of these antibiotic courses were unnecessary.

Few studies have assessed the occurrence, clinical spectrum, and outcomes for RSV illness among older adults in the outpatient setting. A sentinel system in the United Kingdom identified RSV in 15% of adults ≥65 years old with medically attended acute respiratory illness. This is similar to the percent positive (11%) that we observed over 12 seasons in adults ≥60 years old. In a previous study over a shorter time period, we found that cough, nasal congestion, and wheezing were more common in adults ≥50 years old with RSV compared with those with other causes of acute respiratory illness. Prospective RSV illness surveillance in nearly 3000 healthy, working-age adults from 1975 to 1995 demonstrated that 84% of RSV infections were symptomatic. Of the latter, 22% involved the lower respiratory tract (tracheobronchitis or wheezing). Additional studies are needed in diverse populations to estimate the burden of adult RSV illness and the potential impact of future licensed vaccines.

RSV-associated moderate to severe lower respiratory tract illness is a composite measure of lower respiratory tract illness that has been used as a proxy for serious respiratory disease outcomes in RSV vaccine clinical trials (ClinicalTrials.gov identifiers: NCT02608502 and NCT02266628). In this study, we found that RSV-msLRTD at the time of enrollment was significantly associated with a serious clinical outcome. In particular, patients with RSV-msLRTD at enrollment were significantly more likely to require hospital admission and were also more likely to develop pneumonia during the follow-up period. The incremental risk (absolute risk difference) for each of these outcomes exceeded 20% when RSV-msLRTD was present at enrollment. These findings suggest that RSV-msLRTD may be a useful surrogate measure to identify individuals at risk for more serious clinical end points in trials of vaccines and antivirals.

The strengths of this study include consistent prospective recruitment of patients with acute respiratory illness from a defined community cohort, inclusion of 12 consecutive winter seasons, collection of standardized clinical data during the enrollment encounter, and detailed abstraction of outpatient and inpatient medical records for the 28-day follow-up period.

This observational study also has several limitations. Recruitment was restricted to individuals who sought medical care for respiratory illness during periods of influenza transmission, and cough was required for enrollment during seasons after the 2009 pandemic. This study underestimated the occurrence of RSV hospital admissions as enrollment was restricted to primary care and urgent care outpatient clinics after 2010. Based on diagnosis codes, we identified an additional 20 individuals ≥60 years old in our community cohort who were hospitalized with a diagnosis of RSV but not enrolled in the vaccine effectiveness study. These patients were most likely admitted through the emergency department or subspecialty clinics where study recruitment did not occur. In addition, some RSV cases may have been missed by RT-PCR testing, as serology has been shown to improve the detection of RSV infection in adults with community-acquired pneumonia. Finally, this study was conducted in a largely rural and racially homogenous population, and results may not reflect outpatient RSV occurrence and outcomes in urban and racially diverse settings.

As new vaccines and antivirals are licensed for RSV prevention and treatment in adults, there will be a great need for data to estimate the population burden and the potential reduction in cases and serious outcomes. The potential impact on reducing antimicrobial use is another potential benefit that requires further evaluation. These data will be needed to increase awareness of RSV among adult health care providers and to inform cost-effectiveness analyses for public health planning and policy deliberations. In preparation for these decisions, additional research is urgently needed to estimate disease burden and outcomes in larger and more diverse populations.

Blood serum samples belonging to 188 dogs, which had either been admitted to the Internal Medicine Clinic of Selcuk University, Faculty of Veterinary Medicine, with clinical symptoms or had been sampled at the dog shelters they were cared after in Isparta and Burdur provinces, were examined using the ELISA method. Of these samples, 103 (54.7%) were found to be positive for antibodies against CAV infection (Table 3).

Of the 108 female animals sampled in the study, 55 (50.9%) were determined to be positive for CAV antibodies, while 48 (60%) of the sampled 80 male animals were confirmed to be positive (Table 3). Of the 7 animals below 1 year of age, only 1 (14.2%; 2-month-old female puppy) was positive, and the remaining ones were found to be negative for CAV antibodies. Of the 53 animals aged 1-2 years, 22 (41.5%); of the 58 animals aged 2 years, 31 (53.4%); of the 64 animals aged 3 years, 44 (68.7%); and of the 6 animals aged >4 years, 5 (83.33%) were found to be positive (Table 2).

Blood leukocyte samples from dogs were processed and inoculated onto confluent monolayers of MDCK cells using standard virological techniques. The inoculated cells were incubated at 37°C and observed daily for the appearance of cytopathic effect (CPE). After third passage, cells were examined by immunofluorescence test for virus isolation. No morphological changes were observed in cell cultures, and a positive result was not detected by immunofluorescence test.

Clinical Findings. Blood samples were taken from 111 dogs showing clinical symptoms which were brought to the Internal Medicine Clinic of Selcuk University, Faculty of Veterinary Medicine. Seventy-seven dogs were sampled from Isparta and Burdur dog shelters by random sampling, regardless of the clinical findings. Dogs showed a systemic disease, characterized by fever, diarrhea, vomiting, mucopurulent oculonasal discharge, mucopurulent conjunctivitis, severe moist cough, signs of pulmonary disease, and dehydration. Corneal opacity and photophobia were determined for two dogs.

Electronic diagnosis codes, procedure codes, laboratory tests, and prescribing data were extracted from the medical record for all RSV-positive patients. Trained research coordinators abstracted detailed clinical data for the initial respiratory illness visit, subsequent outpatient or ED visits, and hospital admissions within 28 days for all participants with PCR-confirmed RSV illness. Symptoms of acute respiratory illness and onset date were available from the original study enrollment interview. Abstracted clinical data included additional symptoms not assessed during study enrollment, functional status, smoking status, physical exam findings, treatment, pulmonary function, and presence of specific comorbid chronic diseases. For each clinical encounter in the 28-day window, information was abstracted regarding diagnosis or suspicion of pneumonia, acute sinusitis, and acute otitis media.

Abstraction of laboratory results included hemoglobin, total leukocyte count, BUN, creatinine, glomerular filtration rate, hemoglobin A1c, procalcitonin, and brain natriuretic peptide (BNP). Measures of cardiac and pulmonary function were abstracted, including peak flow, FEV1/FVC, oxygen saturation, and cardiac ejection fraction (from echocardiogram). Additional abstractions were performed for participants who were hospitalized within 28 days after PCR-confirmed RSV illness. Abstracted data included admission and discharge diagnoses, hospital course, use of supplemental oxygen or ventilation support, intensive care unit (ICU) admission, antibiotic or antiviral treatment, and disposition at discharge.

All chest x-ray and chest computed tomography (CT) reports for participants with RT-PCR-confirmed RSV illness were independently reviewed by 2 physicians to identify a new radiographic opacity or infiltrate within 28 days after enrollment. Chest x-rays obtained >48 hours after hospital admission were excluded as they may represent nosocomial rather than community-acquired illness. Discrepancies in x-ray interpretation were adjudicated by consensus. For the purposes of this analysis, pneumonia was defined as an illness meeting all of the following criteria: (1) clinical diagnosis or suspicion of pneumonia mentioned in the medical record; (2) new opacity or infiltrate identified by chest x-ray or CT scan; and (3) antimicrobial treatment.

The CAV-2-, CDV-, CPIV- and CPV-positive samples were obtained from the live vaccine Nobivac DHPPi (MSD Animal Health Trading Co., Ltd., Shanghai, China), which contains CAV-2 (104.0 TCID50/ml), CDV (104.0 TCID50/ml), CPIV (105.5 TCID50/ml) and CPV (107.0 TCID50/ml). The rabies virus (RABV) was obtained from the inactivated vaccine Nobivac Rabies. The positive CanineCV, CCoV, and H3N2 CIV strains used in this study were maintained in our laboratory. The Escherichia coli (E. coli, ATCC-29522) and Salmonella enterica samples were kindly provided by Associate Prof. Zhang, College of Veterinary Medicine, South China Agricultural University.

At present, the emergence of new pathogens and the continuous circulation of common etiological agents in dogs have made canine diseases more complex and difficult to diagnose. Dog infectious diseases mainly include respiratory and intestinal viral diseases, including CRV (CAV-2, CDV, CIV and CPIV) and CEV (CAV-2, CanineCV, CCoV and CPV). However, the traditional methods of virus identification and isolation are time consuming, causing delays in treatment initiation. A few methods for detecting virus-induced respiratory or enteric disease have been developed [4, 27, 28, 34], but no previous study had developed a systematic way to detect both CRV and CEV in dogs. Here, we developed two mPCR methods for detection of the most frequently coinfected viruses; these methods could be performed to diagnose dogs according to their clinical symptoms.

Primer design is the first and most important step in the process of establishing a detection method, and the following conditions must be satisfied: primers were designed to bind to conserved sequence regions, to have similar annealing temperatures, and to lack dimers or hairpin structures. In these novel mPCR methods, the primer combination produced amplicons that were easy to distinguish from each other, the primer annealing temperatures were similar, and degenerate bases were required only infrequently. The specificity, sensitivity and reproducibility tests all showed good results.

The mPCR methods were tested on 20 NS and 20 AS samples collected from dogs with symptoms of respiratory disease or enteric disease. The ratio of positive samples to total samples was 80% (16/20) for CRV detection and 85% (17/20) for CEV detection. Because the sample number was insufficient, these results were not statistically significant. However, CPV and CDV clearly remain two of the more serious and epidemic diseases in dogs in worldwide at present [35–38]. Epidemiological monitoring of CPV is particularly important because CPV evolves at a rapid rate, similar to that of Porcine Circovirus 3 [39, 40]. Because a small number of dogs were negative for the viruses tested by the CRV or CEV detection assays, although they suffered respiratory illness or intestinal problems, we suggest that some viruses with low prevalence and pathogenic bacteria may also cause disease in dogs [2, 41]. A variety of pathogenic bacteria are often present along with viruses in canine infections [42, 43], and thus, it is essential to expand the coverage of mPCR detection in the future. For example, CIRD also include CHV-1, canine reovirus, and Bordetella bronchiseptica and so on. At the same time, other pathogens causing serious zoonotic diseases, such as pseudorabies virus, should also be monitored in future [44, 45].

In this study, the detection of CanineCV was added to an mPCR method for the first time, because coinfection of this pathogen with other pathogens is common. Though the pathogenic mechanism of CanineCV is unclear, epidemiological testing is important for future research. CanineCV was not detected from the AS clinical samples; perhaps the limited source of these clinical samples was responsible for this result. We didn’t get a lot of clinical samples because it was not easy to get disease samples. CAV-2 mostly replicates in the lower respiratory tract and was detected in the NS samples; however, the CAV-2 primer pair used in this study was probably able to amplify the CAV-1 DNA virus despite the optimization performed. Notably, the live vaccine strains used may have an unavoidable impact on disease detection using the methods developed in this study. Additionally, discriminating between wild-type infections and vaccines is important, and therefore, a trend exists toward later development of broad-spectrum and accurate mPCR detection methods. Sometimes, cross contamination may lead to experimental failure. It is worth noting that PCR pretreatment and post-treatmen performed in different isolation zones can effectively avoid pollution. Besides, regular air spray cleaning will also play a role.

In conclusion, these newly established mPCR methods provide an efficient, sensitive, specific and low-cost testing tool for the detection of CRV (CAV-2, CDV, CIV and CPIV) and CEV (CAV-2, CanineCV, CCoV and CPV). The use of Taq Master Mix makes the detection process more convenient and reduces the chance of contamination during the process of sample addition; PCRs can be initiated by simply adding enzyme, ddH2O, premixed primers, and template, and thus, this method is superior to other mPCR detection methods. Here, detection of CanineCV was added to mPCR for the first time, making this method suitable for the further study of coinfection by CanineCV and other pathogens. This study provides a novel tool for systematic clinical diagnosis and laboratory epidemiological surveillance of CRV and CEV among dogs.

Bacterial pneumonia is a serious lower respiratory tract infection in dogs with substantial morbidity and risk of mortality. Although BP was described in Dogs decades ago, information on the mechanisms leading to the development of the disease still is limited. Factors such as diseases predisposing to aspiration, immunodeficiency, or ciliary dysfunction that lead to impairment of pulmonary defense mechanisms and thereby predispose to the development of BP have been described.3 However, the role of preceding or concurrent infections with CIRD viruses has not been fully evaluated in dogs with BP, although it has been suspected to play a role in the etiology, as reported in humans with CAP.34, 35, 36, 37, 38, 39 Previously, respiratory viral‐bacterial co‐infections mostly have been reported in dogs housed in dense populations, such as kennels and rescue shelters, and bacteria accompanying viruses have been primary CIRD bacteria (B. bronchiseptica, S. equi sp. zooepidemicus, and Mycoplasma spp.).6, 7, 19, 44

Our study indicates that respiratory viruses, primarily CPIV, frequently are also found in dogs with BP, which is caused by opportunistic bacteria. Therefore, it is likely that CIRD viruses can predispose dogs to opportunistic bacterial lung infections by increasing bacterial adhesion, as has been reported in humans.35

In this study, CPIV was the most common viral pathogen detected, which is in accordance with previous reports describing viruses responsible for CIRD in different countries.6, 13, 19 Novel CRCoV was detected in 1 dog with BP, further demonstrating that CRCoV has a worldwide distribution and also may be detected in Northern Europe.

Canine parainfluenza virus was prevalent despite recent vaccination, which can be considered indicative of poor vaccine‐induced antibody coverage against CPIV. In contrast, CAV and CDV were not encountered in dogs vaccinated against these viruses. This finding is in accordance with previous reports. In a longitudinal study on respiratory viruses in a rehoming center in England, CPIV was commonly detected despite regular vaccinations, but CDV and CAV‐2 were not encountered, most likely because of adequate vaccination coverage.13 It remains unknown whether more efficient CPIV vaccines and possible CRCoV vaccinations could decrease the incidence of BP, as has been shown in humans, in whom protection against influenza and respiratory syncytial virus decreased the incidence of secondary bacterial infections.50, 51, 52

Nosocomial infections with respiratory viruses also have been reported in dogs. An outbreak of CPIV was described in an animal hospital and an outbreak of CHV was reported in immunocompromised dogs.53, 54 Because co‐infections with CIRD viruses are shown to be common in dogs with BP, the infection risk needs to be taken into account when treating BP patients in the same premises (eg, intensive care units) with immunocompromised patients.

Dogs with viral co‐infections were significantly heavier than those without virus infection. This finding might be influenced by the structure of the virus‐negative group: All 4 dogs with another predisposing factor for the development of BP were <20 kg (West Highland White Terrier, Dachshund, Spanish Water Dog, and Schnauzer). Dogs with viral co‐infections also were younger than those without viral co‐infection, although this did not reach statistical significance. This finding is not unexpected, because young animals might have insufficient acquired immunity against CIRD viruses.2

Clinical findings, arterial blood gas analysis, and hematology, as well as respiratory sample cytology in both groups were in accordance with previously reported findings in BP and did not differ between virus‐negative and virus‐positive groups.42, 43, 48 On thoracic radiographs, an alveolar pattern in the cranial and the middle lobes was predominant in both groups without group predisposition. Radiographic findings in dogs with BP have been thoroughly reported previously for cases of aspiration etiology.55, 56 In our study, radiographic findings in dogs with BP caused by other etiologies were similar to those reported for aspiration pneumonia. Aspiration etiology was considered unlikely, because none of the dogs with BP had a history of vomiting, regurgitation, recent anesthesia or signs compatible with laryngeal paralysis. Our findings could indicate that an alveolar pattern in cranial and middle lung lobes may be typical for pneumonia, regardless of etiology. On the other hand, aspiration pneumonia might have played a role in some dogs but could not be confirmed or denied based on available history, examination findings, or imaging. We were unable to identify clinical variables to reliably distinguish dogs with BP and viral co‐infection, and PCR testing therefore appears to be required to identify viral respiratory infections in dogs with BP. A similar finding was reported in humans.57

There were no significant differences in the duration of hospitalization (P = .427) or partial pressures of arterial oxygen at presentation (P = .343) between BP dogs with and without viral co‐infection, indicating that viral co‐infections do not appear to cause a more severe course of BP. In dogs, limited information is available on the severity of BP of different etiological origins, and in humans the reports are contradictory. Some studies have shown that mixed infections with viruses and bacteria induce a more severe clinical disease, whereas others have been unable to demonstrate significant differences in disease severity.49, 58, 59, 60, 61

Previous studies reporting microbiological findings in dogs with pneumonia have found growth of a single species of bacteria in 40–74% of cases.42, 43 All of these studies used TTW as a sampling method. Factors that might have influenced the finding of primarily a single species of bacteria in our study may be the use of BAL as a sampling method in majority of cases, compared to previous studies where TTW was used and the widespread use of prior antimicrobial treatments in these dogs.

Novel molecular methods have allowed the rapid testing of several respiratory pathogens simultaneously. Lower respiratory tract samples are considered ideal when diagnosing viral respiratory infections in humans with CAP, and it has been shown that virus‐positive PCR in BALF is associated with respiratory symptoms in humans.62, 63 Naturally, the invasiveness of retrieving BALF, compared to upper respiratory sampling, limits the usefulness of this accurate sample. However, especially when using molecular methods, virus recovery from the upper respiratory tract may be suggestive of virus exposure rather than indicative of an active viral infection.6 We chose lower respiratory tract samples in order to decrease the number of false‐positive results, but a comparison of PCR findings between upper and lower respiratory tract samples would be useful. Underestimation of virus‐positive PCR results may have occurred in our dogs in cases in which viral infection preceded BP and sampling was performed outside of the viral shedding period.

Bordetella bronchiseptica and Mycoplasma spp. were tested using both PCR and conventional culture methods. Polymerase chain reaction was, as expected, able to reliably demonstrate both pathogens in dogs with positive culture results. Additionally, Mycoplasma PCR was positive in 3 dogs with negative culture results. The clinical relevance of these positive results is difficult to interpret, because Mycoplasma spp. are also encountered in the respiratory tract of healthy dogs.64 On the other hand, because Mycoplasma requires special culture methods (and in this study also shipping to an outside laboratory), there might have been dogs in which Mycoplasma culture was falsely negative. Quantitative PCR might have aided in assessing the clinical relevance of these PCR findings.

Respiratory viruses were not detected in control dogs with prolonged BBTB. Bordetella bronchiseptica commonly accompanies CIRD viruses in acute respiratory infections and signs usually are self‐limiting.6, 19, 28, 29 Dogs with prolonged BBTB were considered more likely than those of the general dog population to have been exposed to CIRD viruses previously. Infections with CIRD viruses are self‐limiting within the first weeks, and because all BBTB dogs had prolonged clinical signs, an active viral infection therefore was considered unlikely.7, 9, 11, 65 Consequently, the negative results in the BBTB group are considered to increase the reliability of positive virus PCR findings in dogs with BP.

The most important limitation in this study was the small number of dogs in each group. This decreases statistical power (ie, the possibility of detecting a true difference between groups or reporting a difference that does not truly exist). Additionally, because this study was performed in Northern Europe in household dogs with low infection pressure, the results may not be applicable in all situations.

In conclusion, respiratory viruses, primarily CPIV, were found frequently in lower respiratory samples of dogs with BP and may play an important role in the etiology and pathogenesis of BP. Additionally, clinical variables and disease severity did not differ between BP dogs with and without viral co‐infection.

The results were subjected to statistical analysis with the χ2 test or Fisher’s exact probability. Differences were considered significant when P < 0.05. The statistical analysis was performed at a website for statistical computation (VassarStats, Vassar College, Poughkeepsie, NY, USA).