ARI is one of the most common human diseases, and the heaviest burden of viral respiratory illness is carried by children. Studies using viral culture diagnosis have estimated that, in developed countries, infants and preschool children experience a mean of 6–10 viral infections annually and school-age children and adolescents experience 3–5 illnesses annually–[12]. RSV, infA and infB, PIV1, 2, and 3, and ADV are considered to be the most common causative viruses for ARI, especially for lower respiratory tract illness–[6],. MP and CP are the causative agent of atypical pneumonia and have been studied widely in developed countries. However, the characteristics of respiratory pathogens in the etiology of ARI in developing countries are not well studied. In this work, we analyzed the characteristics of 17 common respiratory pathogens from throat swab samples which were collected from children with ARI in Guangzhou, southern China over a 3-year period. Although the best recovered samples should be bronchial alveolar lavage fluid (BALF) in the location of pulmonary infection. However, it is hardly carried out in clinic for harvesting BALF in illness kids. Throat swab is practically clinical behavior for taking the specimen in patients with ARI so far.

Among pediatric patients with ARI in this study, 55.7% (2361/4242) patients were positive for one or more of the 17 pathogens studied, and the positivity rate would have been higher if rhinovirus and bacteria had been included. 10 of the 17 pathogens showed positivity rates greater than 5%, demonstrating the wide diversity of pathogens contributing to ARI (Figure 1; Table 1). In general, the distributions of pathogens vary among different countries and regions because of climatic and other variables. In southern China, RSV, infA, EV, MP, ADV were the predominant pathogens in this study and thus were the major influence on the structure of our analysis (Figure 3). Seasonal distributions of most of the pathogens rose during the change of seasons (Figure 4).

RSV (32.5%, 768/2361) was the most frequently isolated virus occurring mainly among children less than 2 years old. This distribution is consistent with previous reports of RSV in both developed and developing countries–[18] (Table 1, Table 2). RSV is known to occur in well-defined recurrent epidemics during the cold season in temperate climates,. In tropical and subtropical areas, RSV infections have been reported to peak more often in the wet season, but locations close to the equator show a less consistent pattern, some with almost continuous RSV activity and with varying seasonal peaks,. In this study, two seasonal peaks of RSV were found at the changes of season from winter to spring and from summer to autumn. This pattern is similar to that reported in Nepal.

Influenza virus is one of the major causative agents of respiratory disease in humans, and leads to a more severe disease than the common cold which is caused by a different type of virus. Influenza occurs worldwide with outbreaks during the winter season in temperate countries–[25]. In this study, infA (428/2361, 18.1%) was the second most frequent pathogen-isolated, and showed a seasonal distribution that was similar to RSV, except for the period of the pandemic of H1N1 in 2009 and 2010–[28] (Table 1, Figure 4). infB occurred less often than infA and was the sixth most frequent pathogen found.

EV, MP and ADV were also important pathogens in our study. EV is frequently isolated from the throats of people with respiratory infection and many studies have supported the role of EV in ARI–[30]. In a seven-year study in Rio de Janeiro EV was found less frequently than RSV, ADV and influenza viruses in patients with ARI.

MP is the causative agent of atypical pneumonia and is also responsible for other respiratory tract infections such as tracheobronchitis, bronchiolitis, croup, and less severe upper respiratory tract infections in older children and young adults. Epidemics generally occur at intervals of 4–7 years. In our study, MP was the fourth most frequent pathogen isolated and tended to increase when RSV was declining (Figure 4), in contrast with a previous report. However, both pathogens could be found all year long.

ADV infection usually causes symptoms of low respiratory illness (LRI) in children, and can occur all year round. In Guangzhou, ADV was mainly found in summer and autumn, but was also present all year long (Figure 4).

PIVs are important causes of ARI, especially in children–[36]. An estimated five million LRI occur each year in the United States among children under 5 years old, and PIVs have been isolated in up to one third of these infections–[39]. However, a total of 11.4% (269/2361) of patients in our study were PIV positive, and PIV3 which is the most frequent PIV isolated was found in only 5.9% of patients, which is lower than that found in the United States study.

In the past decade, at least six new viruses associated with respiratory infection have been identified, including HMPV, severe acute respiratory syndrome coronavirus, human coronavirus NL63 and HKU1, PIV4, and HBoV,, demonstrating the diversity of ARI pathogens. Many viruses like rhinovirus and human coronavirus have been largely ignored by the medical community because their clinical impact was considered to be minor. It is now clear that these viruses, once thought to cause only a common cold, can also cause pneumonia in adults. All these viruses are common causes of sporadic cases or outbreaks of community-acquired infections and can be fatal in immunosuppressed patients and the elderly. Thus, there is still much work that needs to be done in characterizing the effects of these new pathogens.

Over the past two decades, virus isolation and serology have been the mainstay of clinical laboratory diagnosis for respiratory virus infections. Because of the limited sensitivity of these methods, co-pathogen rates have generally been low. As expected, with the development of new diagnostic methods based on real-time PCR many more co-pathogens are being detected. Co-pathogen infections were found in 503 of the 2361 (21.3%) positive patients, and the frequency of the causative pathogens was similar to that among the primary pathogens with the exception of HBoV, HCoV-OC43 and HMPV (Figure 1), with HMPV occurring less as a co-infection than HBoV and HCoV-OC43. The clinical relevance of detection of co-pathogens in pneumonia, and the association with severe illness, is uncertain–[47]. Viral-viral interaction in vivo is poorly understood. Viruses might interact indirectly or directly with each other, resulting in complementation or inhibition. In one study, children with pneumonia caused by co-pathogenic infection with HBoV and other viruses suffered more wheezing than those with viral pneumonia associated with only one pathogen.

In general, ARI occurs mostly in children under the age of 5,. In our study, 87.5% (3713/4242) of the patients were less than 5 years old. No significant difference was found in positivity rate (p = 0.338) and co-pathogen rate (p = 0.117) among different age groups (Table 2). However, most pathogens showed significant differences in age prevalence with the exception of PIV1 (p = 0.239), HCoV-OC43 (p = 0.077), and pathogens for which there were too few positive samples to analyze such as PIV4, 229E, NL63, HKU1 and CP. Four age group distribution patterns could be observed as shown in Figure 2. In general, influenza virus (infA and infB) increased with age, while RSV declined as age increased (Figure 2A and 2B). The attack rate of influenza virus was typically highest in school-age children and daycare populations as has been shown in previous reports–[52]. This probably represents the higher intensity of transmission behavior in this population coupled with a relatively low rate of immunity. RSV is a cause of pediatric hospital admissions for LRI in most areas of the world. Most severe disease occurs in children aged under one year, with a peak occurring between 2 and 5 months of age, as indicated previously–[54]. In addition, evidence suggests that RSV LRIs are associated with persistent diminished airway size in part owing to altered regulation of airway tone.

Significant differences in age distribution were seen in influenza and RSV (p<0.001), also in seven other pathogens EV, ADV, PIV2, PIV3, HBoV, HMPV and MP (p≤0.009) (Table 2), and different age groups showed different distribution peaks (Figure 2). This was consistent with previous serologic and epidemiologic studies of these pathogens,,,,,–[57]. For the seven remaining pathogens no statistical differences in age distribution or few positive samples were found to analyze.

ARI is a complex and diverse group of diseases; it is difficult to identify the pathogen from clinical symptoms. Despite many advances, further studies are still needed to better understand the role of different pathogens in the cause and pathogenesis of ARI, especially in developing countries. The data from this work might be useful for advance warning of ARI and for development of effective vaccines.

Analysis by multiplex PCR assays revealed that most dogs in both groups were positive for at least one CIRDV (96.9% in CAI and 94.7% in HAI groups). Among the six common CIRDVs, CIV and CRCoV were commonly found in both groups (CIV; 57.9% for CAI, 60.5% for HAI and CRCoV; 62.4% for CAI, 59.2% for HAI), while CAdV-2 was the least frequently detected (8.3% for CAI and 11.8% for HAI). The populations of other viruses in the CAI and HAI groups were CPIV, CaHV-1, and CDV, respectively. The CAI dogs had no statistical significance to the HAI dogs in term of virus detections (Table 1).

Multiple virus infections were detected at similar levels in both groups, at 81.2% (108/133) of CAI dogs and 78.9% (60/76) of HAI dogs, where double viral detections were the most frequently found (Table 2). The frequency of multiple virus detections in both groups decreased with increasing numbers of viruses, with no infection with all six viruses being found in either the CAI or HAI dogs. There was no significant difference between the CAI and HAI dogs in terms of multiple virus infections.

When the variable demographic factors and single or multiple CIRDV detections were analyzed, the main population was male and puppy in both groups. The common respiratory problems in both groups were nasal discharge, cough and depression. However, there was no association between sex, age, vaccination status and respiratory signs for single or multiple CIRDV detections (Table 3). The variable demographic factors were also compared with the clinical severity level, revealing that most CIRDV-infected puppies had a greater severity compared with the other age groups. There was a statistically significant association between the age of dogs and clinical severity level (P = 0.012), with the exception of sex, vaccination status, type of affected dog and number of different CIRDVs detected (Table 4). The respiratory score was compared with CIRD agents detected in both the CAI and HAI groups (Table 5). Most CIRDV-positive dogs expressed a respiratory score of 3 or 4 in both the CAI and HAI groups, which accounted for 60.2% (80/133) and 61.8% (47/76) respectively. Moreover, double infection with CIV and CRCoV was predominantly detected in both groups with a statistical association (P = 0.009, Table 5).

The CIRDC, a common respiratory disease complex in dogs, is associated with environmental factors, individual host susceptibility and infectious pathogens, which are primarily viruses [1, 31, 32, 33]. The CIRDVs are commonly detected in dogs with respiratory problems and are endemic in poor conditioned dogs, overcrowded shelters, hospitals and pet grooming centers [2, 3, 8, 19, 34]. Currently, the prevalence and types of HAIs are not well established compared to CAIs. The epidemiology of CIRDV-infected dogs has been reporting over many years but with contrasting results [3, 8, 24]. In this study, we focused on the incidence of CIRDV detections in terms of CAI and HAI, which are supposed to be an important factor of CIRDC infections. The HAI-diseases, especially respiratory tract infections, have been considered as a risk for nosocomial transmission, but they have not been investigated in clinically-infected dogs [19, 34]. In this study, all six common CIRDVs were detected in both sample groups, suggesting that these viruses might either be present or disseminated in both environments. Moreover, we found that CRCoV and CIV had a higher detection rate in both the CAI and HAI dogs, while CPIV had a lower rate. This finding is in contrast to previous observations that found CPIV was the most commonly detected virus in CIRDC dogs [3, 7, 24]. Furthermore, it is surprising that 96.9% in CAI and 94.7% in HAI groups displayed at least one virus detection, which is unusual in previous observations for canine respiratory disease that revealed the low occurrence of CIRD viruses [4, 5, 7, 35], although this might be influenced by various factors, including the different geography, sample size, sample population, sampling protocols and also validated detection method.

The evidence of CDV, CPIV, CAdV-2, CaHV-1, and CIV circulation in Thailand has been documented previously [17, 18, 26, 36, 37], but not for CRCoV. This study, therefore, is the first report of CRCoV-infected dogs in Thailand. The most commonly detected viruses were CIV and CRCoV (>50%) in both the CAI and HAI groups, which is in contrast to previous reports in Asia and Europe [3, 7, 24, 39].

Meanwhile, CIRDVs were detected in both the CAI and HAI dogs without any significant difference in the frequency of occurrence among the two groups, potentially suggesting that these viruses are already circulating in both sample groups. This is in accordance with a recent study that reported that there was no significant difference in CPIV, CAdV-2 and CRCoV infection levels between dogs from private households and those from shelters or kennels [3, 40].

In the present study, 81.2% (108/133) of CAI and 78.9% (60/76) of HAI dogs had multiple CIRDV infections, supporting the complex condition of the disease, which is often manifested as a co-infection rather than a single pathogen [2, 3, 24, 26, 38]. We also observed that the majority of multiple CIRDV infections were co-detected with either CIV, CRCoV, or both. However, this observation does not allow interpretation as to whether they are primary and/or secondary pathogens. Infection with CIV and CRCoV usually induces mild clinical symptoms by interfering with the respiratory defense mechanism and so leads to super-infections with other pathogens [1, 41, 42]. Moreover, co-infection of CIV and CRCoV may be synergistic and lead to severe tracheobronchitis.

There were no significant differences in sex, age and vaccination status of the dogs between single and multiple virus detections. However, the vaccinated dogs had a lower proportion of both single and multiple detections compared with the unvaccinated dogs. Interestingly, there was a significant difference in the CIRD-affected age groups when compared with the clinical severity level. CIRD-affected puppies had a more severe clinical level compared with the dogs in the other age groups. This could reflect their increased host susceptibility, such as from a premature immune response and being unvaccinated. Further study with large-scale sample populations and meta-analysis of clinical information is warranted.

Significant association between CIRD agents and clinical respiratory scores was not observed in co-detection with CIV and CRCoV. This co-detection represented the highest proportion and was most often found with other CIRDVs. Thus, CIV and/or CRCoV could be either primary or secondary agents that are commonly found as co-infections with the other pathogens. However, bacterial infections could not be ruled out in this study. Many investigations have revealed that either viral or bacterial co-infection leads to an increased severity, but some studies have revealed no significant differences in the clinical severity between single and multiple infections [24, 39, 43, 44].

Since healthy or control dogs were not included in this study, then the prevalence of the CIRDV infections could not be assessed. Furthermore, this study used upper airway swabs as the sampling method for virus detection with PCR. It has been widely shown that healthy dogs may also have positive PCR results for CIRDVs in upper airway samples. Thus, a positive viral PCR in an upper airway sample is not necessarily proof of a symptomatic infection but could represent only exposure to the pathogen. Thus, the result of CIRDV detection by PCR from respiratory swabs can only imply that they represented the CIRD pathogens that might be associated with respiratory problem. Moreover, since we only focused on six viral pathogens associated with CIRDC, then the role of other pathogens, including known bacterial pathogens, such as Bordetella bronchoseptica and Mycoplasma sp., and other novel viruses, such as CnPnV, that might be implicated remains unknown.

Samples from a total of 4242 pediatric patients were analyzed over three years from July 2009 to June 2012 in Guangzhou, southern China. Male to female ratio of the patients was 1.92∶1. Median age was 1.5 years (interquartile range, 0.67 to 3.00), with a range from one day to 14 years old, and with 3713/4242 (87.5%) patients being less than 5 years old. Pathogens were detected in 2361/4242 (55.7%) patients with a median age of 1.42 years (interquartile range, 0.67 to 3.0). Male to female ratio was 2.04∶1 (p = 0.076) in the positive patients and 1.82∶1 in the negative patients.

The most frequently detected pathogens in this study were RSV (768/2361, 32.5%), infA (428/2361, 18.1%), EV (138/2361, 13.3%), MP (267/2361, 11.3%) and ADV (213/2361, 9.0%), followed by HMPV, infB, PIV3, HCoV-OC43, HBoV all with the detection rates higher than 5.0%. The positivity rates of the remaining seven pathogens were lower than 5.0% (Table 1). Co-pathogens were common and found in 503 of 2361 (21.3%) positive-samples (Table 1). The frequency of detection of the co-pathogens followed almost the same ranking order as that of the primary pathogens with the exception of HBoV, HCoV-OC43 and HMPV (Figure 1).

In this study, patients were divided by age into seven groups. There was no difference in pathogen positivity rate (p = 0.338) and co-pathogen detection rate (p = 0.117) among the age groups. However, significant differences were found in age prevalence between infA, infB, RSV, EV, ADV, PIV2, PIV3, HBoV, HMPV, MP infections (p≤0.009, Table 2). In general, four age/prevalence patterns could be distinguished among children in this study; A: Detection rates increased with age as illustrated in infA and infB (Figure 2A); B: Detection rates declined with age, as shown in RSV (Figure 2B); C: The detection rates peaked at different ages as in EV (peak age 1–2 years, 10.9%), ADV (peak age 3–5 years, 9.0%), PIV2 (peak age 4–6 months, 2.8%), PIV3 (peak age 4–6 months, 6.1%), HBoV (peak age 7–12 months, 5.1%), HMPV (peak age 3–5 years, 5.5%) and MP (peak age 6–10 years, 20.1%) (Figure 2C and 2D) and D: the number of samples was too small to analyze or no significant pattern was present (p>0.05), as in PIV1, PIV4, HCoV-OC43, −229E, -NL63, -HKU1 and CP.

In general, sample positivity rates increased when seasons changed as is shown in Figure 3. In this study, RSV, infA, EV, MP and ADV formed the bulk of the positive sample (Figure 3). In seasonal distribution, RSV and infA, PIV, and human coronavirus infection mainly occurred at the change from winter to spring and summer to autumn; MP and infB increased as RSV and infA declined, respectively. ADV mainly occurred in summer and autumn, although it was generally distributed all year round; HMPV occurred at the change of season from spring to summer and from winter to spring; EV and HBoV occurred mostly in summer and HBoV was prevalent in winter (Figure 4). Few CP positive samples were found, and so a seasonal pattern could not be determined.

This case report described the occurrence of a serious bacterial tracheobronchitis due to H. influenzae in a laryngectomee. It illustrates the difficulties in managing the airway access in laryngectomees because of the increased mucus production that can block them. It also highlights the need to recognize the potential of bacterial infection in this population.

NTHi is a known cause of community-acquired pneumonia in adults and may be associated with severe disease and high mortality in higher risk populations. Although bacteremia that complicates H. influenzae pneumonia occurs occasionally, disseminated infections following NTHi bacteremia are uncommon except in newborns and immunocompromised patients. H. influenzae was recovered from 6 of 27 (22%) tracheal aspirates of children with tracheostomy who had pneumonia, and in 4 of 14 (29%) children with bacterial tracheitis.

The stoma in neck breathers allows the inhaled air to bypass the natural defenses (nasal hair and mucus membranes) of the upper airway that filter out dust and bacteria. Wearing an HME that covers the stoma can provide important benefits [1, 2]. The HME filter serves as a stoma cover and creates a tight seal around the stoma. In addition to filtering dust and other larger airborne particles, HMEs preserve some of the moisture and heat inside the respiratory tract and prevent their loss and adds resistance to the airflow [1, 2]. HME filters assist in restoring the temperature, moisture, and cleanliness of the inhaled air to the same condition as before laryngectomy.

The number of bronchitis, tracheobronchitis, and pneumonia episodes as well as mortality due to these infections in non-HME users was found to be three times higher than in HME users. Laryngectomees especially those who do not wear an HME or have uncovered stoma are therefore at a higher risk for lower respiratory infections.

The exposure of our patient to cold air without an HME most likely compromised his airway and led to the development of the infection.

Paroxysmal hypertension is a known complication following head and neck radiation, and can be attributed to damage to the carotid artery baroreceptors. Aggravation of this condition during our patient’s illness further complicated his condition.

Treatment of tracheobronchitis in laryngectomees is more challenging and requires keeping the stoma open by manually removing accumulated mucus that can dry out and clog it, keeping the excessive sputum moist by breathing humidified air and inserting saline as needed, coughing out or suctioning accumulated sputum, keeping the patient well hydrated, and removing the HME during the illness or prior to coughing to prevent blocking it with the coughed out sputum.

Contagious upper airway infections in dogs occur regularly and are most commonly caused by canine parainfluenza virus (CPIV) or Bordetella bronchiseptica, amongst other agents. This clinical syndrome has also been named infectious tracheobronchitis (ITB), canine infectious respiratory disease (CIRD), “kennel cough” or “kennel croup”, so named due to its occurrence in environments where many dogs live or stay close together for shorter periods of time. Characteristic clinical signs include a self-limiting paroxysmal cough lasting for up to two weeks, which usually resolves without treatment. In Norway, immunisation against CIRD is performed using live attenuated viruses, annually with CPIV and every third year against canine adenovirus type 2 (CAV-2). However, in spite of vaccination, outbreaks of CIRD remain common. Some dogs with CIRD will develop serious pneumonia due to an immature immune system or other causes of immunodeficiency. Occasionally, bacteria such as Streptococcus equi subsp. zooepidemicus can cause fatal pneumonia.

This case-report describes the first canine outbreak of haemorrhagic pneumonia in the Nordic countries caused by S. equi subsp. zooepidemicus. Most of the animals in the pack of athletic sled dogs showed symptoms of CIRD with three dogs demonstrating symptoms of severe peracute infection. One sled dog died while two were successfully treated, rehabilitated and returned to competition. To the author’s knowledge, this is the first report documenting the chronology from onset of clinical signs, through convalescence to complete recovery for peracute haemorrhagic pneumonia in dogs. The vaccination regimen related to season and extreme training will also be discussed.

We present data from a large cohort of children with M. pneumoniae infection. All children enrolled were referred from the primary healthcare system to hospitalisation due to character and severity of symptoms. The majority of M. pneumoniae PCR test-positive children had LRTI, which we confirmed by a high rate of radiological findings (94%).

We found a higher rate of positive samples in the later wave of the epidemic in 2010 and 2011. School-aged children were more often M. pneumoniae positive (65%) than younger children, but even amongst the 2 to 6-year-old children 30% were M. pneumoniae positive substantiating our initial suspicion that M. pneumoniae also affects small children. Even a small number of infants; 6 out of 276 were diagnosed.

This study was conducted as a retrospective chart study. The doctor on call decided whom to test for M. pneumoniae. Children with M. pneumoniae infection might have been under-diagnosed if they had minor respiratory symptoms especially during the first wave of the epidemic period. Due to commonly held concepts of CAP epidemiology, originally based on a long-term study conducted in primary care from 1963–1975, we expect infants and young children to be under-diagnosed due to selection-bias.

Even very young children can become ill from M. pneumoniae even though it is less common. The differential diagnosis of respiratory viral infections and exacerbation of asthma-like symptoms must be considered. The clinical presentation with a cough, wheezing, low-grade-fever, CRP below 50 mg/L and rhonchi on auscultation in 33% of the youngest children can also be considered as a childhood asthma-like exacerbation, primarily due to viral infection in pre-school children. Indeed, we also had a minor degree of mixed viral co-infections discovered in our post-hoc analysis. It can only be speculated in what pathogen was the primary cause of disease in these cases. Due to our post-hoc findings, we would advise that small children with wheezing and rhinorrhoea should be tested for both M. pneumoniae and respiratory viral infections simultaneously. During the Norwegian M. pneumoniae epidemic, Inchly et al. described a similar relative number of viral co-infections.

In a Dutch childhood study of carriage of M. pneumoniae in the upper respiratory tract (URT), season and year of enrolment affected the prevalence of asymptomatic carries ranging from 3% to 52%. In our study, some of the children discharged from the ward on the same day as admitted to the hospital could have been carriers of M. pneumoniae. However, several of these children were treated with first-line antibiotics, prior to admission, and referred to our department because of insufficient response to beta-lactam antibiotic management.

Kroppi et al. found that 50% of children with LRTI caused by M. pneumoniae were co-infected with primarily S. pneumoniae or Chlamydiae spp. Only a small part of this cohort was tested for bacterial co-infection, but we did not regard this as a major problem. Again, it is noteworthy that 59% of the children had been treated with a beta-lactam antibiotic before examination for M. pneumoniae without improvement of the infection.

In parts of the same epidemic period in Denmark (2010–2011), Stockholm et al. identified an effect of azithromycin (a macrolide antibiotic) on episode duration of asthma-like symptoms in young children. No investigations for M. pneumoniae were done, and exclusion criteria of respiratory rate over 50/min, temperature over 39°C and CRP over 50 mg/L would not exclude all children with a possible M. pneumoniae infection [19–21]. Two recently published Norwegian studies described the discrepancy of the incidence of clinical symptomatic M. pneumoniae infections in preschool children between epidemic and endemic periods. Randomised Controlled Trials concerning the efficacy of macrolides on asthma-like symptoms should be conducted in endemic periods or better controlled for M. pneumoniae infections, especially in young children born after an epidemic period. The anti-inflammatory effect of macrolides still has to be further addressed.

10% of this cohort was affected by chronic illness, mainly respiratory severe illness. Severe asthma exacerbations were diagnosed in the current asthmatics.

Older children tended to be seen later after onset of symptoms and were accountable for the longer hospitalisations. This might indicate that older children had more severe infections, or that the delay in admission to the hospital resulted in more severe disease and thereby a prolonged period of rehabilitation. Despite that, we also identified severe pneumonia based on the radiological findings (atelectasis, pleural effusions) in the 2-6-year-olds. If adjusted for population size these preschool children had an increased risk of developing severe pneumonia compared with school children during this epidemic. Inchley et al. showed the same pattern even if their definition of severe pneumonia differed. Treating the infections earlier might reduce severe morbidity and length of hospital stays.

Treatment of M. pneumoniae infections with macrolide antibiotics is controversial since a Cochrane review, concluded that there is insufficient evidence to draw any specific conclusions about the efficacy of antibiotics in M. pneumoniae infections in children. The efficacy of antibiotic treatment should be discussed in light of a correct diagnostic test. Asymptomatic carriers of M. pneumoniae have to be differentiated from children suffering from symptomatic infections, LRTI, caused by M. pneumoniae. Gardiner et al. underline the need for RCT on this topic. In Denmark, SSI still recommends treatment of M. pneumoniae positive LRTI in children. Macrolide resistance is a growing problem worldwide. In Denmark, the occurrence is estimated to be 2%. No macrolide resistance was identified in our childhood cohort.

We found radiological changes in 94% of the chest x-rays taken in this study. The radiological findings were quite diverse, but notably, over 80% of children older than two years had a lobar infiltration while the younger children had significantly more subtle findings. This was in accordance with an Italian prospective childhood study.

Even in older children, symptoms could not be distinguished from CAP caused by other pathogens. Radiological findings in M. pneumoniae pneumonia were not distinguishable from CAP in general.

Almost 25% of all children had some kind of rash (erythema/hives) during the illness, and 33% had gastrointestinal symptoms like nausea and or vomiting. Severe extra-pulmonary manifestations accompanying respiratory infections caused by M. pneumoniae are expected to occur. Two children in our cohort were diagnosed with SJS, which is a known complication of M. pneumoniae. Outbreaks of M. pneumoniae–associated SJS in children has recently been reported. We did not see any children with neurological symptoms in this cohort which would be expected.

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.

Patient characteristics stratified by age group are presented in Table 1.

Children under two years of age were admitted to the hospital significantly earlier after the onset of symptoms than older children, (p = 0.01, two-sample t-test). 61% of the children were discharged from hospital on the day of admission.

The most common respiratory manifestation was a cough (100%) together with an age-depending degree of wheezing (Table 1). At admission to the hospital, 92% of infants and children were diagnosed with an episode of troublesome lung symptoms lasting at least three days. Significantly more young children (less than three years of age) had objective wheezing and cough (asthma-like symptoms) than older children (p = 0.01, Z-score test). Previous and clinically significant chronic disease was diagnosed in 10% of the children. Of these 4/13 (31%) had previously diagnosed and current asthma, and had a severe exacerbation during the M. pneumoniae infection.

The majority of the patients (84%) had a chest x-ray taken, and 96% of these had positive radiological findings (Table 1). Among infants and young children, exclusive hilar adenopathy was more frequent, while older children usually had significant peripheral infiltration on the chest X-ray (Table 1). Children with atelectasis had a significantly longer duration of hospital stay; more than three days (35% versus 25%; p = 0.05). The rate of pulmonary complications was the same for children with CRP over 50 mg/l (POCT(Point of Care Testing), part of an adult definition of significant pneumonia) as below 50 mg/l (18% versus 19%, p > 0.05). No pulmonary complications were reported in the under two age-group. The number of severe manifestations of pneumonia was equal in the age-groups; 2–6 years and 7–15 years (Tabel 1). Overall 20% of the children had an increased CRP level of more than 50 mg/l, and they were all older than three years of age.

A total of 120 children received antibiotic treatment. The majority were treated with clarithromycin according to the local Guideline. Sixty-four patients, or 46%, were treated upon suspicion. Out of these, 53% had received other antibiotics (beta-lactam) prior to M. pneumoniae testing. Fifty-six patients (42%) were started on treatment upon receiving the positive test result. Only six children were treated with macrolide antibiotics twice due to suspicion of recurrence or treatment failure. One sample was tested for macrolide resistance and found negative.

The most common extra-pulmonary manifestation was nausea, with or without vomiting, reported by a third of all children. 23% of all children had some type of rash, and 9% had hives. In infants, there were skin manifestations in 33% of the cases. Two children developed Steven Johnson syndrome (SJS) with mucosal symptoms arising prior to or at the same time as the antibiotic treatment was started.

A total of 37 children had simultaneously been tested with sputum-culture for other bacterial pathogens. In 41% of these, a co-infection was diagnosed. The most common bacteria were Moraxella catarrhalis, Haemophilus influenzae and S. pneumonia. Due to methodological setup, all children were tested for Chlamydophila pneumoniae none were found positive.

Only two children were tested for viral infections during the clinical setup. The post-hoc analyses of 49 oropharyngeal-swabs showed that 27% of these had a single or mixed viral co-infection (RSV (1 child), influenza A (2), human metapneumovirus (1), rhinovirus (2), coronavirus (3), bocavirus (2) and adenovirus (5)). Four children were PCR positive for two viruses as well as for M. pneumoniae. Table 2 shows, in a similar matter as Table 1, the clinical characteristics of children with, without and of unknown viral infection. The data suggest that significantly more children with mixed infection of M. pneumoniae and a respiratory virus had rhinorrhoea (p = 0.02), and were wheezing (tendency, p = 0.07), compared to those who were only positive for M. pneumoniae. We could not identify other differences between the two groups, including no radiological discrepancies.

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.

Laryngectomees are at a higher risk of developing lower respiratory tract infections especially in the winter and when not wearing an HME. Maintaining the patency of the airway is of utmost importance as the mucus can be very dry and viscous and can stick to the walls of the trachea and the stoma. The risk of acquiring these infections can be reduced by: getting vaccinated for respiratory pathogens that include Streptococcus pneumoniae, H. influenzae, and the influenza viruses; washing hands before any stoma care; wearing an HME at all times; maintaining adequate respiratory tract humidification; and avoiding hypothermia or inhaling cold air.

RSV was detected in 243 (11%) of 2257 encounters (representing 241 of 1832 individuals) for acute respiratory illness. RSV cases were equally represented by RSV A (n = 121) and RSV B (n = 122). Of the 243 RSV cases, 23 (9%) had an additional viral target identified. Influenza was detected in 519 (23%), and 5 (<1%) were positive for both RSV and influenza. There were 2 individuals with an episode of RSV during 2 different seasons; none of the study participants had 2 RSV episodes in the same season. Among 1495 encounters negative for RSV and influenza by RT-PCR, the most common viral pathogens were coronavirus OC43, NL63, HKU1, 229E (n = 210; 14%), human metapneumovirus (n = 184; 12%), human rhinovirus (n = 135; 9%), and parainfluenza virus types 1–4 (n = 74; 5%); 873 (39%) encounters were negative for all viral targets in the multiplex RT-PCR assay.

Patients with RT-PCR-confirmed RSV were similar to those with non-RSV respiratory illness in terms of age, gender, number of ED visits, and hospital admissions in the prior 12 months (Table 1). The mean (median) interval from symptom onset to study enrollment and swab collection was 4.0 (4.0) days for RSV-negative patients and 4.4 (4.0) days for RSV-positive patients (P = .006). Chronic obstructive pulmonary disease (COPD) was present in 15% of those with RSV illness, 18% of those with other viral respiratory infection, and 27% of those with no virus detected. RSV prevalence among adults with acute respiratory illness varied from a low of 5% in 2004–2005 to a high of near 18% in 2005–2006 and 2011–2012. Both RSV A and B were detected in every season. Type A accounted for the majority of RSV-positive detections in 5 seasons, and type B in 4 seasons. In 3 seasons (2007–2008, 2010–2011, 2012–2013), the numbers of RSV A and B cases were approximately equal.

Common symptoms of RSV illness included sore throat, sputum production, cough, fever/feverishness, dyspnea, myalgia, and wheezing (Table 2). The most common diagnosis codes on the date of enrollment for RSV-positive patients were cough (45%), acute upper respiratory infection (21%), acute bronchitis (13%), acute sinusitis (12%), and bronchitis, unspecified (12%). Fifty-nine patients (24%) met the criteria for RSV-msLRTD during the enrollment encounter. The presence of RSV-msLRTD on enrollment was associated with a 3-fold higher risk of serious clinical outcome (relative risk, 2.99; 95% confidence interval [CI], 1.83–4.88) (Table 3). RSV-positive participants with msLRTD during the enrollment encounter had a higher prevalence of COPD and congestive heart failure (CHF) and a greater number of medical encounters in the 28 days after enrollment (median, 1 vs 2; P = .05).

The clinical outcome for RSV illness was serious in 47 (19%), moderate in 155 (64%), and mild in 41 (17%). Nearly half of serious outcomes occurred in patients ≥75 years of age with RSV infection (Table 3). Serious outcomes were not mutually exclusive and included hospital admission (n = 29), ED visit (n = 13), and pneumonia (n = 23). The 13 ED visits included 5 patients who were admitted and 8 who were discharged. Among 155 patients with a moderate outcome, 144 (93%) received a new antibiotic prescription, 45 (29%) received bronchodilator or nebulizer treatment, and 28 (18%) received new systemic corticosteroid therapy; 5 (3%) were treated with an antiviral drug. The risk of a serious outcome was approximately double for patients with COPD or CHF compared with patients without these conditions (Table 3).

Thirty-two participants with RSV infection were admitted to a hospital within 28 days. Three of these hospitalizations were for noncardiopulmonary conditions that were unlikely to be related to RSV infection, including incarcerated hernia, small bowel obstruction, and acute gastrointestinal illness. These 3 hospital admissions were excluded from the serious outcome group. Fifteen of the remaining 29 hospitalized patients were enrolled in the inpatient setting. Four of those were directly admitted from an outpatient clinic, whereas the remaining 11 were admitted from the emergency department. Seven patients were enrolled during an outpatient encounter on the same day as their hospital admission, leaving 7 with hospital admissions 1 or more days after enrollment. The median interval from symptom onset to admission for RSV-positive hospitalized individuals was 4 days.

Preexisting chronic diseases were common among hospitalized patients with RSV, including COPD (n = 9; 31%), CHF (n = 8; 28%), asthma (n = 8; 28%), and diabetes (n = 9; 31%) (Table 4). Twenty-one (72%) received a discharge diagnosis of respiratory tract infection (eg, pneumonia, bronchitis, exacerbation of COPD). Only 1 patient was recognized to have RSV at the time of hospital discharge. The mean (SD) duration of hospital admission was 3.5 (2.5) days. Twenty-seven (93%) were discharged to home, and 2 (7%) were transferred to a rehabilitation or long-term care facility; there were no deaths within 28 days. The hospital course was uncomplicated for most patients. None required mechanical ventilation or ICU admission. Twenty-five (86%) received antimicrobials; 4 (16%) were treated with antiviral drugs.

To estimate the number of hospitalized RSV cases that were not included in this analysis, we extracted hospital diagnosis codes for community cohort members during periods of study enrollment. We identified an additional 20 individuals ≥60 years of age who were hospitalized during study enrollment periods with a diagnosis code for RSV but were not enrolled in the influenza vaccine effectiveness study.

The median community cohort size for adults aged ≥60 years was 13 807 (range, 12 142–13 807) for the seasons from 2006–2007 through 2015–2016. The overall seasonal incidence of medically attended RSV illness was 139 cases per 10 000 (95% CI, 122–160). The RSV incidence was 196 cases per 10 000 (95% CI, 162–236) among persons with chronic cardiopulmonary disease and 103 (95% CI, 85–125) among those without cardiopulmonary disease (incidence rate ratio [IRR], 1.89; 95% CI, 1.44–2.48). There was a significant decline in the incidence of medically attended RSV from 2006–2007 through 2015–2016 for the entire cohort (P = .003, chi-square test for trend) and for those with cardiopulmonary disease (P = .014, chi-square test for trend). The incidence of medically attended RSV was also higher in women compared with men (IRR, 1.46; 95% CI, 1.09–1.95). The overall incidence rates of medically attended illness caused by RSV A and RSV B were nearly identical (IRR, 1.01; 95% CI, 0.75–1.35), although 1 or the other subtype was often dominant during a single season. The temporal trend in RSV incidence suggests an overall reduction among adults ≥60 years of age after the 2011–2012 season (Figure 1).

We compared the peak month for RSV and influenza positives during each season among study participants ≥60 years old. In 7 seasons, the RSV peak and the influenza peak occurred in the same calendar month. In 2 seasons, the RSV peak occurred before the influenza peak, and in 3 seasons, the RSV peak occurred after the influenza peak. We also compared the peak month for RSV detection among study participants with the peak month based on local clinical testing of children <24 months. In 5 seasons, the peak calendar month was the same for enrolled adults ≥60 years old and children; the pediatric peak occurred 1–2 months earlier in 5 seasons and 1 month later in 2 seasons.

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.

No conflicts of interest have been declared.

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.

Focussing on upper respiratory tract samples (nasal and tonsillar swabs), viral nucleic acids were detected in 31 of 214 diseased dogs (14.5%). Sixteen dogs tested positive for CRCoV (7.5%), 14 dogs for CPiV (6.5%) and one of these dogs additionally for CAV‐2‐specific nucleic acid (0.5%). One single dog tested positive for CDV‐specific nucleic acid (0.5%). In none of the obtained samples from the upper respiratory tract was CIV‐specific nucleic acid detected. Of those 31 positive dogs, 21 were privately owned (group A), and 10 kept in shelters (group B). They consisted of five puppies, 12 adolescent dogs and 14 adult dogs. Twenty‐seven of the 31 positive dogs (87.1%) showed acute onset of signs, three suffered from chronic disease (9.7%) and for one diseased dog this information was not available (Table 2).

Furthermore, upper respiratory tract samples from two dogs (4.0%) of the clinically healthy control group C tested positive for CRCoV‐specific nucleic acid (Table 2).

Nine dogs from group A (5.2%) and seven dogs out of group B (17.0%) tested positive for CRCoV in either nasal, tonsillar or both samples at one time. One of these dogs belonged to the subgroup of puppies; nine dogs were from the adolescent subgroup and six animals from the subgroup of adult dogs. With one exception, all these animals showed acute onset of CIRD (93.7%).

Fourteen diseased dogs (6.5%) tested positive for CPiV. From those, 11 belonged to group A and three to group B. They all harboured CPiV‐specific nucleic acid in sample material from the nose and one dog concurrently from the tonsils. Four of these dogs were classified as puppies; three dogs were from the adolescent subgroup and seven dogs were adults. Twelve out of these 14 animals showed acute onset of clinical signs (85.7%), one dog was chronically ill, and for another dog this information was not available. Seven dogs (50.0%) were regularly vaccinated‐including against CPiV.

In one of these 14 CPiV‐positive dogs, CAV‐specific nucleic acid was detected concurrently. This dog was privately owned (group A) and tested positive for CAV in both nasal and tonsillar swabs and CAV‐2 strain (Toronto) was confirmed by DNA sequencing. Belonging to the subgroup of adults this dog had been irregularly vaccinated and received its latest vaccine 45 days before sample collection. It presented with a several week history of clinical signs including severe coughing, nasal and ocular discharge, dyspnoea and fever. Apart from that case, in no other dog was viral nucleic acid of two or more different viruses detected. In addition, no other proband of the study tested positive for CAV.

One dog from group A tested positive for CDV‐specific nucleic acid in a sample retrieved from the tonsils. RNA sequencing enabled the identification of a CDV vaccine strain (Onderstepoort). The dog was an adult and presented with chronic respiratory disease but no other signs consistent with CDV infection. The vaccination status of this dog was unknown.

Additional information regarding all PCR‐positive dogs is summarised in Table 3.

All BALF samples collected from 31 chronically ill dogs revealed negative PCR results.

Canine infectious respiratory disease (CIRD), also known as “Kennel cough”, is an endemic syndrome with multiple viral and bacterial pathogens being involved in disease causation. CIRD is most common when dogs are kept in large groups with continuous intake of new animals, particularly in kennels, but also occurs in singly housed pets. Clusters of infection have also been documented in veterinary hospitals. Common clinical signs include nasal discharge, coughing, respiratory distress, fever, lethargy and lower respiratory tract infections [1, 3–5]. The clinical signs caused by the different pathogens associated with this syndrome are similar, which makes differential diagnosis challenging. Vaccination plays an important role in managing CIRD, and as such, several mono and multivalent vaccines are available; however, despite the widespread use of vaccines to prevent CIRD, clinical disease is still common in vaccinated dogs [2, 6]. Vaccines are commercially available for some, but not all pathogens, which may explain the occasional lack of protection.

The complex multifactorial etiology of this disease involves the traditional CIRD viral and bacterial agents, canine parainfluenza virus (CPIV), canine adenovirus (CAV), canine distemper virus (CDV), canine herpesvirus (CHV), and Bordetella bronchiseptica. New or emerging microorganisms associated with CIRD include canine influenza virus (CIV), canine respiratory coronavirus (CRCov), Mycoplasma cynos and Streptococcus equi subsp. zooepidemicus (S. zooepidemicus). Other novel canine respiratory agents include canine pneumovirus, canine bocavirus, canine hepacivirus [17, 18] and canine picornavirus. There is debate on whether these are truly new emerging pathogens or pre-existing pathogens that are now easier to detect due to the advent of sophisticated molecular diagnostic tools and more frequent diagnostic testing. In recent years, the role of other bacterial agents such as Mycoplasma canis has been questioned [13, 20]. It is unknown whether certain Mycoplasma species such as M. canis act as a commensal, primary or secondary agent.

The detection of co-infections of CIRD pathogens in a single dog has been previously documented [2, 12, 20]. It is most likely that a single pathogen alters the protective defense mechanisms of the respiratory tract, thereby allowing additional pathogens to infect the respiratory tissues. The presence of co-infections may increase disease severity compared with single pathogen infections [2, 5, 20]; however, the prevalence and role of co-infections in CIRD causation remain unclear.

Previous epidemiologic studies of CIRD pathogens in the United States have focused on asymptomatic dogs or on specific pathogens implicated in clinical cases [11, 22, 23]; therefore, a comprehensive etiologic and epidemiologic study involving multiple CIRD agents in a diverse population of dogs has not yet been reported. Understanding disease prevalence facilitates the improvement or establishment of new vaccination programs and alternative treatments. To aid in addressing this question, we conducted a disease surveillance study using molecular methods to detect nine pathogens currently known to be involved in CIRD using samples from symptomatic and asymptomatic dogs that were received at a veterinary diagnostic laboratory. The aim was to attain information regarding pathogen occurrence according to age, seasonality, sex, clinical signs, and vaccination history. This study also aimed to evaluate the role of co-infections in disease severity, and to develop a novel probe-based multiplex real-time PCR assay to simultaneously detect and differentiate M. cynos and M. canis.

From 2003 to December 2007, medical data of 1980 patients discharged from the hospital ICU were colleted. The average length of stay was 9.95 days, giving 19700 patient-days. Among these patients, 531 patients acquired a total of 1005 nosocomial infections, including 125 patients with two infections, 33 patients with three infections, 12 patients with four infections and one patient with five infections (Table 1).

The overall patient nosocomial infection rate was 26.8%, ranging from 22.6% to 33.2% among the 5 years. There was a significant difference in the infection rates among the 5 years (χ2 = 10.395, P = 0.035). The incidence density rate of nosocomial infections was 51.0 per 1000 patient days, ranging from 38.5 to 59.2 among the 5 years. The excess length of stay was 9.4 days, ranging from 3.3 days in 2003 to 11.5 days in 2004 [see Additional file 1].

Lower respiratory tract infections (LRTIs) including bronchitis, tracheobronchitis and pneumonia, were the most common infections, occurring in 34.7% of the 1980 patients, followed by urine tract infections (UTIs) (8.1%), and bloodstream infections (BSIs) (3.0%). Among the 1005 nosocomial infections, LRTIs accounted for 68.4%, followed by UTIs (15.9%), BSIs (5.9%) and gastrointestinal tract infections (2.5%). Most (76.0%) patients with nosocomial LRTIs had received mechanical ventilation or tracheotomy before the infections, whereas 50.0% of nosocomial UTIs and 54.2% of nosocomial BSI were catheter associated (Table 1).

There is no significant change in LRTI, UTI and BSI rates during the 5 years. The GI infection rate was significantly decreased from 5.5% in 2003 to 0.4% in 2007 (χ2 = 12.603, P = 0.012), whereas nosocomial infections in other sites was increased significantly (χ2 = 12.858, P = 0.012). The nosocomial infection rates at the surgical sites and skin and soft tissues remained under 2% (Table 1).

Pathogens were isolated and identified from 530 (52.7%) of 1005 nosocomial infections, or, in 338 (63.7%) of the 531 patients. The isolated pathogens responsible for nosocomial infections differed among the infection sites (Table 2). In patients with LRTIs, Acinetobacter baumannii and Klebsiella pneumoniae were the most frequently isolated pathogens, followed by Pseudomonas aeruginosa and Staphylococcus aureus, accounting for more than half of the LRTI related pathogen population. In patients with UTIs, the fungi, especially Candida albicans, were the most common pathogens, followed by Escherichia coli. Staphylococcus epidermidis, E. coli, and S. aureus were the first three most common pathogens for BSIs. In addition, A. baumannii was commonly isolated in UTIs and BSIs (Table 2).

Data on susceptibility testing were available for 3328 isolates, including 195 isolates of E. coli, 359 isolates of S. aureus, and 549 isolates of P. aeruginosa. Overall, 79.3%, 80.0%, 82.3% and 77.8% of E. coli isolates were resistant to trimethoprim/sulfamethoxazole (TMP/SMX) and ciprofloxacin, cefotaxime, and amoxicillin/clavulanic acid, respectively. All S. aureus isolates were sensitive to vancomycin, but 24.0%, 37.9%, 68.1% and 89.6% of isolates were resistant to nitrofurantoin, TMP/SMX, rifampin and ciprofloxacin, respectively. In addition, 93.1%, 41.3% and 66.9% of P. aeruginosa were resistant to TMP/SMX, ciprofloxacin and levofloxacin, respectively [see Additional file 2].

All patients with nosocomial infections were treated with empirical antimicrobial therapies or according to the antimicrobial susceptibility test results, when available. The mortality rates in the patients with nosocomial infections were 20.2% (17/84), 12.2% (12/98), 15.1% (18/119), 15.1% (18/119) and 17.1% (19/111), respectively, in 2003, 2004, 2005, 2006 and 2007. However, based on the medical records, none of the mortalities were directly related to nosocomial infections.

Canine infectious respiratory disease (CIRD) is a multifactorial disease affecting dogs of all ages, which is typically induced by simultaneous viral and bacterial infections. Apart from well-known canine respiratory pathogens, such as canine adenovirus type 2, canine herpesvirus, canine distemper virus, and canine parainfluenza virus, novel viruses are being continuously associated with CIRD occurrence in dogs. These include canine influenza virus, canine respiratory coronavirus, canine pantropic coronavirus–[8], canine bocaviruses, and canine hepacivirus.

Pneumoviruses (family Paramyxoviridae, subfamily Pneumovirinae, genus Pneumovirus) are enveloped, single-strand negative-sense RNA viruses that are associated with respiratory disease in mammals and birds. Apart from the prototype species human respiratory syncytial virus (HRSV) and its ruminant relative bovine respiratory syncytial virus (BRSV), a murine pneumovirus (MPV), also known as pneumonia virus of mice, is included in the genus Pneumovirus

[11]. This virus, which is only distantly related to human and ruminant RSVs, is a natural rodent pathogen circulating among research and commercial rodent colonies.

Recently, a pneumovirus was associated to respiratory disease in canine breeding colonies in the United States–[14]. The virus, designated as canine pneumovirus (CnPnV), was found to be very closely related to MPV, displaying 95% nucleotide identity with the MPV prototype isolate J3666. Experimental infection of mice with the canine isolate demonstrated that CnPnV is able to replicate in the mouse lung tissue inducing pneumonia. Although the virus was discovered more than 4 years ago, to date there is no complete genomic sequence, which prevents a comprehensive comparative study with other members of the Pneumovirinae subfamily.

The aim of the present manuscript is to report the detection and molecular characterisation of this emerging virus in dogs with respiratory disease in Italy. The full-length genome of a prototype strain was determined and analysed in comparison with American strains and other pneumoviruses.

This study provided new insights into the etiology and epidemiology of CIRD associated pathogens using a molecular surveillance approach in a veterinary diagnostic laboratory. We explored two main aspects: (i) the rate of detection of nine CIRD associated pathogens by age, season, sex, clinical signs, and vaccination status; and (ii) the effect of co-infections on the severity of clinical disease. Our results indicated that the presence of co-infections and young age were associated with the severity of clinical signs. Additionally, we found a low occurrence of classical CIRD pathogens such as B. bronchiseptica, CAV and CDV, while identifying a higher than expected detection of bacterial agents such as M. canis and M. cynos.

The low detection rate of traditional CIRD agents such as B. bronchiseptica, CAV and CDV might be associated with the extensive vaccination programs adopted in the United States, which may have reduced the circulation of these pathogens in the canine population. In fact, our data show that very few clinically ill animals (n = 17), previously vaccinated against B. bronchiseptica, CDV, CPIV and influenza, were PCR positive for these agents. This result emphasizes the important role of CIRD vaccination regimes and suggests that currently available vaccines against B. bronchiseptica, CAV and CDV are effective. Similar findings were reported in a large surveillance study across Europe, where disease occurrence was significantly reduced in dogs vaccinated against classical CIRD pathogens (CDV, CAV-2 and CPIV).

It is noteworthy that CPIV, traditionally considered one of the most important viral agents of CIRD, was the most commonly detected microorganism in our study (29%, 33/114), and it was significantly associated with mild and moderate/severe clinical signs. Previous investigations on canine respiratory viruses have found high prevalence of CPIV in clinically ill dogs [5, 20, 37], and a Canadian study reported CPIV as the cause of a respiratory disease outbreak in a veterinary hospital. Influenza is a newly described CIRD respiratory virus [11, 12]. In the United States, canine influenza is caused by two subtypes: H3N8 and H3N2. Canine H3N8 was first identified in 2004 in racing greyhounds in Florida, United States, and it has been reported that this strain developed from an equine H3N8 influenza strain that was transmitted from horses to dogs. Canine H3N2 was first identified in the United States in March 2015, following an outbreak of respiratory illness in dogs from Chicago. Between 2011 and 2017, we found 57 PCR positive samples for influenza A (including H3N8 and H3N2); interestingly, 51/57 of these positive samples were processed in 2015, which coincides with the H3N2 canine influenza outbreak in the United States. To date, there is no evidence of spread of canine influenza viruses from dogs to people; however, special attention is warranted due to the risk of viral genetic reassortment. Therefore, continued monitoring of canine influenza by veterinary and human health agencies is of critical importance.

M. cynos is an emerging bacteria implicated in canine respiratory disease, which was shown to cause pneumonia in dogs after experimental endobronchial inoculation. Despite the clear involvement of M. cynos in lower respiratory tract infections [4, 13, 41], there is limited information on the prevalence and pathogenesis of this agent. In this study, M. cynos was one of the most prevalent pathogens (24.5%), and it was more commonly identified in symptomatic than asymptomatic dogs. In the first group, there was a significant association between the presence of this pathogen and the development of moderate and severe clinical signs. Similar findings were observed in a large rehoming kennel where M. cynos was most common in dogs with moderate signs of CIRD. The role of other Mycoplasma species in canine respiratory infections, including M. canis, has been a recent topic of research and debate. In this study, we found a significant association between the presence of M. canis and the severity of clinical signs. M. canis is considered part of the microbiota of the upper respiratory tract of dogs [13, 42], and was previously found not to be significantly associated with respiratory disease. Our interest in this microorganism was triggered by a recent study where M. canis was associated with acute respiratory disease in dogs, which along with our findings provide further insights into a potential role of M. canis in CIRD. In addition, M. canis appears to be a common co-infection and warrants further consideration by veterinarians and diagnostic laboratories.

Dogs with viral or bacterial co-infections present with moderate to severe clinical signs more often than dogs with single infection. The prevalence of these co-infections have been previously reported [2, 20, 37, 43]; however, to the best of our knowledge, none of the prior investigations have performed a detailed assessment on the effect of co-infections in the severity of clinical disease. The 3D network analysis showed that M. cynos, CPIV, M. canis and B. bronchiseptica were the most common pathogens seen in co-infections, with M. cynos and CPIV being the most frequent pathogen combination. Knowledge regarding the most common co-infections may help clinicians determine appropriate treatment strategies, improve patient outcomes, and facilitate antimicrobial stewardship.

In this study, the age of the animals was the most significant predictor of moderate to severe clinical signs. Other studies have reached similar conclusions [2, 13, 44], indicating that host factors, such as age, are important in CIRD severity. Young dogs may have lower levels of immunity against pathogens associated with CIRD, and may be subjected to crowded conditions more often, which could increase their susceptibility to infection and lead to more severe clinical signs.

It was challenging to find an ideal control group for a retrospective and diagnostic-based surveillance study. Our approach was to include necropsied dogs in order to ensure the absence of lesions in the respiratory tract. We carefully selected the necropsied dogs based on age, clinical history, macroscopic and histologic findings. The likelihood of detecting pathogens in post-mortem samples may differ; however, based on prior investigations on viral and bacterial persistence on inanimate surfaces and cadavers, we believe that the integrity of the nucleic acid material from the microorganisms was maintained. Our assumption is supported by other studies showing that Bordetella pertussis persisted on inanimate surfaces for 3–5 days, influenza virus for 1–2 days and adenovirus for 7 days. Tubercle bacilli was isolated from cadavers within 24 hours post-mortem, and viable human immunodeficiency virus was recovered from patients at autopsy 6 to 16 days after death [45, 46]. Furthermore, the pathogens surveyed in our study are routinely detected from nasal swabs collected during necropsy from dogs diagnosed with respiratory disease in our diagnostic laboratory.

This study has some limitations. First, the PCR-based assays could potentially yield false-positive results in dogs that recently received modified live vaccines containing the specific pathogens of interest. This assumption is based on findings from a previous study showing that PCR assays may detect vaccine content within 28 days after vaccination with modified live vaccines against CAV, B. bronchiseptica and CPIV. Since the time between vaccination and the onset of clinical CIRD was not documented in our submission forms, we cannot say with certainty whether positive PCR results corresponded to field or vaccine strains. Secondly, as with any pathogen detection test, the PCR assays may yield false-negative results if samples are collected at a point in the disease process when pathogens are not present at detectable levels.

The study did not involve any animal experiment. Only sample collection from naturally infected dogs was carried out, consisting of a single nasal swab per dog. This was needed for the laboratory analyses and did not involve any suffering of the sampled animals.

In the present study, we verified the frequency of five canine viral enteropathogens in a dog population with low vaccination coverage where CPV-2 was the most frequently detected virus (Table 2). The data presented herein agree with previous studies where CPV-2 is reported as the most common cause of severe diarrhea in puppies.7, 15, 16, 22 However, the frequency of CPV-2 found in our study (54.3%) was higher when compared with the ranges reported in other studies (16–48.7%).7, 15, 16, 22 The CPV-2 prevalence varies between studies, depending on the inclusion criteria for participation. However, since vaccination against canine pathogens other than rabies is not mandatory in Brazil, the vaccine coverage for these viruses must be drastically lower, which contributes to the high rate of detection observed in our study.

CCoV is generally the second most common viral agent detected in diarrheic dogs,7, 15, 16, 22 but in the present study, CDV had a higher frequency of detection (Table 2). However, the frequency of detection observed for CCoV was still higher than that reported other studies.7, 15, 16, 22 Some CCoV strains can cause severe diarrhea and intestinal damage indistinguishable from those caused by CPV-2.6

CDV is endemic to Brazil8, 23 but not to the regions sampled in previous studies.7, 16, 22 This could explain the high rates of CDV infection detected in the present study. Moreover, CDV causes a multisystemic disease with immunodepression that can favor infection by other pathogens including other diarrhea-associated viruses.24

CRV and CAdV were also identified in 8.2 and 4.9% of the dogs tested, respectively. Both viral agents are linked to diarrhea and vomiting13 and should not be excluded in the diagnosis of canine diarrhea. Moreover, CRV is a zoonotic pathogen, and its frequency in dogs needs to be determined to better assess the risk of infection in humans.

The samples analyzed in the present study were obtained by convenience from veterinary clinics which could bias information regarding sanity. Nevertheless, CPV-2 remained as the most frequent viral agent in the different dog groups (Table 1). The only exception was in dogs apparently healthy where CCoV and CDV were more frequent than CPV-2 but with no statistical significance.

More than one-third of the dogs tested for CPV-2, CCoV, CDV, CRV and CAdV were positive for co-infections (Table 2). No significant difference in co-infection rates were observed between the groups regarding clinical signs and vaccination status (Table 3). The occurrence of co-infection can increase the pathogenicity of the disease since these viral agents can act as immunosuppressants.12, 24, 25 There is a lack of studies searching for multiple viral pathogens in dogs, which could uncover the real etiology and interactions in the clinics. Moreover, a single search for the more common pathogens can reveal the real epidemiology of less common viral enteropathogens. Canine diarrhea can have other etiologies such as bacteria and parasites that are frequently reported.15, 16 Despite CPV-2 being the most frequently detected diarrhea-associated viral pathogen in the present study, a high degree of co-infections was observed. These co-infection rates reinforce that the search for more common individual pathogens could uncover the real epidemiology of less common viral enteropathogens.

In the present study, stool samples from dogs were evaluated for the presence of viral pathogens, and CPV-2 was found to be the most common. In contrast with previous studies, CDV was the second most common viral agent detected, probably since it is endemic to Brazil and not to the regions sampled in other studies. Moreover, the high frequency of co-infected dogs that was observed can increase the pathogenicity of the disease. The data presented herein can improve the clinical knowledge in regions with low vaccine coverage and highlight the need to improve the methods of controlling infectious diseases in domestic dogs.

In conclusion, there was a high and relatively stable rate of nosocomial infections in the ICU of a tertiary hospital in China through year 2003–2007, with some differences in the distribution of the infection sites, and pathogen and antibiotic susceptibility profiles from those reported in the Western countries. The Guidelines for Surveillance and Prevention of Nosocomial Infections must be implemented national wide in order to reduce the rate.

Pet dogs play an important role in humans’ daily lives. Recently, the emergence of new pathogens and the continuous circulation of common etiological agents in dog populations have complicated canine diseases. Among these diseases, canine infectious respiratory diseases (CIRD) and viral enteritis pose notable threats to dog health.

CIRD are complex and include canine adenovirus type 2 (CAV-2), canine distemper virus (CDV), canine influenza virus (CIV), canine parainfluenza virus (CPIV), canine herpesvirus (CHV), canine reovirus, Bordetella bronchiseptica and other pathogenic agents [2–4]. Among these, CAV-2, CDV or CPIV have frequently been detected in dogs with CIRD, according to previous studies [5, 6]. Avian-origin H3N2 CIV has been detected in domestic dogs in South Korea and China since 2007 [7, 8]. H3N2 CIV is now circulating in dog populations in China, South Korea, Thailand, and even the United States [9–11]. Distinguishing these pathogens can be challenging, because dogs often show similar clinical signs of infection with these viruses, such as low-grade fever, nasal discharge and cough. These respiratory symptoms are flu-like and difficult to diagnose.

Canine viral enteritis is common in dogs with acute vomiting and diarrhea. Canine parvovirus (CPV) is one of the major viruses leading to acute gastroenteritis in dogs; CPV infection is characterized by fever, severe diarrhea and vomiting, with high morbidity. Puppies tend to be intolerant of CPV infection and have higher mortality than adult dogs because of myocarditis and dehydration [14, 15]. Canine coronavirus (CCoV) is characterized by high morbidity and low mortality. Dogs infected with CCoV alone are likely to have mild diarrhea, whereas the disease may be fatal when coinfection by CCoV and CPV, CDV or canine adenovirus type 1 (CAV-1) occurs [16, 17]. CAV-2 is associated with mild respiratory infection and episodic enteritis [18, 19]. Canine circovirus (CanineCV), a newly discovered mammalian circovirus, was first reported by Kapoor et al. in 2012. CanineCV has been detected in dogs with severe hemorrhagic diarrhea, and it is more common in puppies than in adults [21, 22]. Coinfection of CanineCV with other intestinal pathogens (CPV or CCoV) is closely related to the occurrence of intestinal diseases [23, 24]. Dogs with intestinal diseases are often infected with one or more viruses, and their clinical symptoms are similar [17, 25, 26], making clinical differential diagnosis difficult. To date, no multiplex PCR (mPCR) method has been developed to detect CanineCV and other enteropathogens.

An effective diagnostic tool is important for the prevention, control and treatment of CIRD and viral-enteritis-related viral diseases. Although many methods exist to detect CIRD and canine viral enteritis, most can detect only 2 or 3 pathogens, and the current lack of systematic and comprehensive detection methods makes diagnosis impractical and time consuming [4, 27, 28]. Because mPCR can simultaneously detect multiple pathogens in a timely and inexpensive manner, this technique has become increasingly popular. Therefore, in this study, two new mPCR methods were developed for the detection of canine respiratory viruses (CRV, including CAV-2, CDV, CIV and CPIV) and canine enteric viruses (CEV, including CAV-2, CanineCV, CCoV and CPV), and we indicated that the mPCR methods established here are simple and effective tools for detecting the viruses of interest.

A high prevalence (68.3%) of diarrheic dogs, shown to harbor at least one pathogen by real-time PCR, was observed in the Brazilian samples, which exceeded those in the United States (54.5%), Australia (58.4%), Canada (52.0%), United Kingdom (51.7%), and Japan (49.6%), as shown in Table 4. The rate of co-infection observed here in diarrheic dogs (45.1%) was also higher than those in the other countries tested. Despite the higher prevalence of enteropathogens and co-infections in Brazil, the rates in the other countries are also relevant, indicating that infectious diarrhea may be a global phenomenon rather than a phenomenon specific to a particular country. Because all dogs with co-infections belonged to the diarrheic group and co-infections were observed in all age categories, this study highlights the importance of investigating multipathogen co-infections, especially in dogs aged 0–1 years, in which the rate of co-infection was 4-fold higher than in the other age groups.

Many enteric viruses, bacterial pathogens, and parasites probably contribute to disease both individually and in combination, and together, co-infecting pathogens may cause more severe diarrhea than infections with each pathogen alone. The pathogens involved in a co-infection can interact synergistically, for example via the host’s immune system, with the presence of one enhancing the abundance and/or virulence of the other, resulting in even greater pathogenesis and a greater contribution to the overall disease burden. Therefore, interspecific pathogen interactions can alter the pathogen dynamics, host health, and the success of control strategies.

In this study, co-infection did not increase the duration of diarrhea and there was no significant difference in the number of deaths in animals with or without co-infections. Because there was no reliable correlation between the interaction of enteropathogens in co-infections in this study, the cause–effect relationship between the presence of an organism and the occurrence of diarrhea is still unclear. Opportunistic or commensal organisms may be identified from an imbalance in the intestinal flora or dysbiosis, and not all the co-infecting agents present must be treated to produce a good outcome. However, because all the infectious agents evaluated here have been described as causing diarrhea in experimental studies, knowledge of their presence allows treatments and prevention strategies to be planned. In this study, even when diarrhea persisted for more than 10 days, the infectious diseases were still present in the differential diagnosis. Empirical treatments and the use of several antibiotics are common in routine veterinary practice and the use of a panel to detect multiples pathogens prevents the incorrect or excessive use of antimicrobial drugs, which could cause resistance. Furthermore, some of these pathogens are potential zoonotic agents, including hookworms, Giardia spp., Cryptosporidium spp., and Salmonella spp., and the identification of these organisms can reduce the risk of their transmission to humans and others animals.

Although this study focused on client-owned dogs, dogs received in animal shelters are also expected to carry pathogen co-infections, including zoonotic agents. However, differences have been observed in the prevalence of each agent, especially in terms of the co-infection rates, and dogs from shelters with diarrhea showed a higher prevalence of co-infection (96.0%) than was observed in this study (45.1%). That heterogeneous dog population had a higher rate of crowding, and the dogs may have been immunocompromised for clinical, nutritional, and/or psychological reasons, exposed to more environmental pathogens, and sometimes with inadequate health care, so their high co-infection rate cannot be compared validly with that of owned dogs in households.

The highest rate of co-infection in this study involved the association of viral and bacterial agents, in contrast to the highest co-infection in dogs in the United States, which was caused by viruses and protozoans. The highest co-infection rates were for CPV-2 and CPA, observed in 9/12 (75.0%) samples from dogs in Brazil, and for CCoV and Giardia spp., which occurred together in 35.4% of dogs from the United States. These co-infections may have clinical effects and may require more-intensive efforts to ensure the appropriate treatments to eliminate specific pathogens and to correct electrolyte, acid–base, and nutritional disturbances, potential sepsis, and other metabolic consequences.

The alpha toxin gene is present in all strains of Clostridium perfringens and may be found in asymptomatic dogs as part of the normal intestinal microflora, as in 14% of the control dogs in the present study. Data from some studies indicate that conventional PCR that targets only CPA will almost always be positive and of virtually no clinical use. However, a recent study demonstrated that the quantification of CPA may be used as a diagnostic marker for association of the agent in patients with diarrhea. Using the same methodology and cutoff value as a previous study, we observed a significant difference between the control group (4/43, 9.3%) and in the diarrheic group (37/104, 35.6%; P = 0.0025) in the proportion of animals positive for > 300,000 copies of CPA. The higher amount of CPA in the diarrheic dogs than in the control dogs suggests that the high concentrations of toxins produced by this organism exert a pathogenic effect on the gastrointestinal tract. In the present study, diarrheic dogs co-infected with CPA had 3-fold more copies than those that were infected with only CPA. We hypothesize that in these cases, C. perfringens overgrowth in the bacterial flora increases the toxin expressed, which contributes to the dog’s diarrhea.

All the control dogs were negative for CPV-2, which was strongly associated with diarrhea (P = 0.000004), with an overall occurrence of 36/104 (34.6%) in the diarrheic dogs. The same prevalence (18/51, 34.6%) was observed in a study also conducted in Brazil but performed only with puppies up to 6 months old and corroborated with previous surveys, which reported rates varying from 16% to 58%. Although CPV-2 has been considered to be primarily disease of puppies, the present study has shown the importance of also investigating adult dogs for CPV-2, since occurred in 11.1% of 1–8 year old dogs and in 12.0% of dogs older than 8 years. The CPV-2 was most prevalent agent involved in co-infections in this study, in which 58.3% of the diarrheic samples positive for CPV-2 were associated with others agents, contrasting with only 3.8% CPV-2 co-infection observed in the study with puppies. This variability may be related to the geographic regions examined, the populations studied, agents investigated and the diagnostic techniques used. Although the date of live-modified vaccination was not the focus of this study, the CPV-2 cutoff value used was able to differentiate vaccine strains from wild-type infections. Despite the use of vaccination, the CPV-2 was still spread among the dogs as observed in this study, in which only 13/36 (36.1%) of the positive animals for CPV-2 had no history of vaccination. Still remains unclear whether type-2 vaccines can provide protection against the new variants of the CPV, but a recent study observed that the cases of vaccine failure are most likely not associated to the mutations detected in the sequenced regions. Thus, further studies should elucidate whether local parvovirus strains are effectively controlled by the currently available vaccines and which factors may be associated with the vaccination efficacy. CPV-2 was the most prevalent agent associated with dual co-infections in the present study, which may be attributable to highly contaminated environments or low dog immunity.

Although the detection rate of CCoV was higher in the diarrheic dogs (11.54%) than in the control dogs (6.98%), the lack of a statistically significant difference in these rates may indicate a secondary role for CCoV as an intestinal pathogen in dogs. Although the shedding of CPV-2 seemed to be associated with clinical signs of gastroenteritis, CCoV was also detected in healthy dogs, as previously reported. Although CCoV infections are characterized by high morbidity and low mortality, with typically mild enteritis in dogs, 11/12 (91.7%) diarrheic dogs with CCoV were co-infected with other enteric pathogens, which may have aggravated their clinical signs and even caused higher mortality, as reported earlier for CPV-2, canine adenovirus type 1, and CDV. CDV tended to be significantly higher in the diarrheic dogs than in the control dogs; although not significant, the P value was very close to the limit of significance (P = 0.059).

Whereas some enteropathogens, such as Salmonella spp., Cryptosporidium spp., and Giardia spp., showed similar prevalences in the various countries tested (Table 4), CDV and CPV-2 were approximately 4-fold more prevalent in Brazil, indicating that the higher incidence of viral diseases may be related to differences in strain pathogenicity, vaccination status, and environmental factors.

Although the CPV-2 strain and vaccine status/response may play important roles in viral infection and host immunity, further studies are required to fully understand this specific pattern of CPV-2 infections in Brazil.

Although intestinal parasites contribute to diarrhea, 95.7% of positive and 49.2% negative samples on the parasitological tests were also positive on the real-time PCR diarrhea panel, indicating that fecal parasitological tests alone should not be used as a single diagnostic tool.