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.

Post-mortem examination revealed epistaxis and haemorrhagic frothy fluids in the trachea and bronchial airways on cut sections. Haemorrhages were present in the thymus, epicardium, intercostally, and in the pleural space 200 mL of uncoagulated blood were present. The lungs were congested, wet, consolidated and diffusely to cavernous haemorrhagic, these changes being more severe in the left lung lobes (Figure 1).

Histopathology of the lungs revealed a subacute necrotising suppurative pneumonia, with haemorrhagic, often cavernous areas in the lungs and intra-lesional gram-positive cocci. A large number of macrophages with phagocytosed erythrocytes were present (Figures 2 and 3). A subacute pleuritis was also seen.

Streptococcus equi subsp. zooepidemicus was isolated in pure culture from the lung tissue with identification based on morphology, microscopy, Lancefield grouping (Streptococcal grouping kit, Oxoid, Basingstoke, Hampshire, England) and biochemical testing. Properties included β-haemolytic colonies on bovine blood agar, gram-positive, katalase negative cocci belonging to Lancefield group C. Glucose, lactose, and sorbitol were fermented, trehalose was not. The isolate was also tested by using API20STREP (bioMérieux®, Lyon, France) with the same conclusion.

Toxicological diagnostic screening of the liver tissue showed no evidence of anticoagulant poisons.

Acute respiratory infections (ARI) result in the death of an estimated 4 to 5 million children each year in developing countries–[4]. Most of these deaths are among children with pneumonia. Respiratory tract infection etiology is complex and diverse. In developed counties, the major causes of ARI in children and adults are influenza A and B viruses (infA, infB), parainfluenza virus type 1 (PIV1), PIV2, PIV3, respiratory syncytial virus (RSV), adenovirus (ADV) and rhinovirus–[6]. However, there is a lack of data on the characteristics of ARI in developing nations. Over the past decade, a number of new pathogens have been reported, including human metapneumovirus (HMPV) and human bocaviurs (HBoV), thus increasing the urgency for the study of epidemiology of respiratory tract pathogen infections in developing countries.

Over the past two decades, virus isolation and serology have been the mainstay of clinical laboratory diagnosis for respiratory virus infections. The introduction of molecular-based detection methods has made diagnosis quicker and cheaper and increased the ability to detect more than one virus simultaneously. In this work, the epidemiological features of 17 respiratory pathogens in children with ARI were studied in Guangzhou, southern China. This study should serve as a valuable resource for information on ARI and provide useful data for future research and development of vaccines.

Laryngectomees run a high risk of developing severe respiratory tract infections. Following laryngectomy the tracheal epithelium becomes directly exposed to the relatively cold and dry ambient air entering the tracheostoma [1, 2]. This can cause: drying of the mucus, which makes it more viscous; reduction of ciliary activity that causes impaired mucociliary clearance [2–4]; and tracheal epithelium damage (loss of ciliated cells, goblet cell hyperplasia, and excessive mucus production and metaplasia).

Severe pulmonary infections (tracheobronchitis and pneumonia) in laryngectomees are more frequent in the wintertime and the accompanying tracheal crusting often requires antibiotic treatment or even hospitalization. Tracheobronchitis in laryngectomees was described as a “suffocating” respiratory infection because of the difficulties in maintaining a patent airway in these patients [6, 7].

A case of severe tracheobronchitis in a laryngectomee is presented that illustrates the risks and difficulties encountered in managing this infection in a neck breather.

Canine infectious respiratory disease complex (CIRDC) is a major cause of respiratory illness and morbidity. It is a complex condition for disease occurrence, involving multifactorial etiologies. Overcrowding, stress, age and other underlying factors result in interference with the dog's immune response and play a role in disease predisposition [2, 3, 4, 5, 6]. Infectious agents serve as a key factor in promoting the severity of a clinical symptom. The CIRD viruses (CIRDVs) that are commonly associated with CIRDC are canine parainfluenza (CPIV), canine distemper (CDV), canine adenovirus type 2 (CAdV-2), canine herpesvirus 1 (CaHV-1), canine respiratory coronavirus (CRCoV), and canine influenza virus (CIV) [1, 7, 8, 9].

During the last decade, novel viruses, such as canine pneumovirus (CnPnV) [10, 11] and pantropic canine coronavirus, which induce respiratory problems in dogs, have also been discovered. In addition, canine bocavirus [13, 14], canine hepacivirus and canine circovirus have been detected in respiratory tract samples of dogs showing respiratory illness, but the pathogenic roles of those viruses are poorly determined. Although new viruses have emerged, the common CIRDVs are still important contributors to respiratory disease [17, 18]. Additionally, current core vaccines that are routinely used in dogs prevent some CIRDVs, such as CPIV, CDV, and CAdV-2, but not CaHV-1, CRCoV and CIV. This lack of a vaccine for CaHV-1, CRCoV, and CIV is likely to allow the spread of infection.

Hospital-associated infection (HAI) has recently been described as an “ever-present risk”. The current literature includes reported outbreaks of CPIV and CaHV-1 in animal healthcare facilities, where these nosocomial infections worsened any ongoing disease and enhanced the morbidity and mortality rates. In addition, infections among communities are documented and serve as a source for disease dissemination. Community-acquired infections (CAIs) are those that are acquired without exposure to hospitals or with limited regular exposure with a health care center or both. Currently, CAIs with CIRDVs are believed to have a concomitant effect in respiratory disease [1, 21, 22, 23, 24].

The identification of multiple-viral-induced CIRDC involves the simultaneous rapid nucleic acid-based detection, typically by the reverse transcription polymerase chain reaction (RT-PCR) for RNA viruses and PCR for DNA viruses. These detection methods have led to an increased and better realization of the prevalence of CIRDC worldwide [3, 25, 26]. However, whether the severity of the respiratory disease depends on the number of viral infections is equivocal. Although epidemiological evidence of CIRDC has been reported periodically, the classification among HAIs and CAIs has largely not been evaluated [7, 24].

A comprehensive understanding of the source of infection (HA or CA) and other associated risk factors may help guide the successful implementation of preventive strategies. To emphasize this point, this study focused on the associations between the incidence of all six common CIRDVs, source of infection and the possible related risk factors of the dog's age, sex and vaccine status. Moreover, the relationship between clinical severity and number of viral infections was also evaluated in respiratory-ill dogs during 2013–2016 in Thailand.

Canine infectious respiratory disease (CIRD), synonymous for infectious tracheobronchitis or “kennel cough,” is a disease caused by single or multiple infectious agents with a high worldwide prevalence. Apart from several viral and bacterial agents, the individual health and constitution, vaccination status and environmental influences including husbandry conditions (e.g. crowding of animals) may have an impact on the manifestation of clinical signs. Non‐complicated forms of typically self‐limiting character may be distinguished from complicated forms associated with, possibly fatal, pneumonia. A severe course of disease typically develops as a consequence of coinfections (Chalker et al. 2003, Chvala et al. 2007, Schulz et al. 2014a). However, even isolated viral infections [e.g. canine influenza virus (CIV)] may lead to clinically relevant and sometimes lethal respiratory disease (Crawford et al. 2005). Commonly recognised viral causes of CIRD are canine parainfluenza virus (CPiV), canine adenovirus type 2 (CAV‐2) and canine distemper virus (CDV) (Ford 2012).

However, according to more recent studies, the understanding of this disease complex has changed. New viral pathogens have been detected within the past two decades. In 2003, canine respiratory coronavirus (CRCoV) emerged as a cause of CIRD in a rehoming centre in the UK (Erles et al. 2003). Further studies from several countries detected CRCoV‐specific nucleic acid in dogs suffering from respiratory disease (Yachi & Mochizuki 2006, Decaro et al. 2007, Spiss et al. 2012, Schulz et al. 2014a, Viitanen et al. 2015).

In association with an outbreak of respiratory disease in racing greyhounds in Florida, CIV types closely related to influenza subtype H3N8, originally detected in horses were isolated (Crawford et al. 2005). Subsequently, several studies from different countries detected CIV isolates in respiratory samples and concurrent anti‐CIV antibodies in dogs with mild respiratory signs as well as cases of fatal respiratory disease (Yoon et al. 2005, Daly et al. 2008, Payungporn et al. 2008, Song et al. 2008, Kirkland et al. 2010, Li et al. 2010, Song et al. 2013). Furthermore, isolation of human‐related influenza strains from dogs was successful (Lin et al. 2012). To date, at least seven influenza virus subtypes showing different ability of interspecies and intraspecies transmission have been isolated from dogs. These subtypes are mainly prevalent in the USA (H3N8), Eastern China and South Korea (e.g. H3N2), but some of them have also been reported from European countries (Sun et al. 2013, Xie et al. 2016) supporting the hypothesis that dogs may play a role in transmission and spread of influenza virus among animal species and even humans.

Detection of further viral pathogens (e.g. canine herpesvirus, canine reovirus, canine pneumovirus (CnPnV), pantropic canine coronavirus, canine hepacivirus and canine bocavirus) has been associated with respiratory disease in dogs (Buonavoglia & Martella 2007, Decaro & Buonavoglia 2008, Kawakami et al. 2010, Renshaw et al. 2010, Decaro & Buonavoglia 2011, Kapoor et al. 2011, Kapoor et al. 2012, Mitchell et al. 2013b, Priestnall et al. 2014). However, these viruses are uncommonly detected in dogs with CIRD or their possible role as causative agents is not yet completely determined.

The aim of this study was to assess the prevalence of common CIRD‐associated viruses (CPiV, CAV‐2, CDV) in dogs in and around Vienna, Austria. Although there may be environmental factors specific to this location, our findings are likely to generalise to other locations within Western Europe and possibly further afield. It was further investigated whether emerging viruses (CRCoV and CIV) have a significantly higher prevalence in dogs with CIRD compared to dogs without respiratory disease.

The study was approved by The First Affiliated Hospital of Guangzhou Medical University Ethics Committee for research on human beings, and all participants or their guardians gave signed informed consent for participation in the study.

Mycoplasma pneumoniae (M. pneumoniae) is an important cause of respiratory tract infections, especially in children and younger adults and are estimated to be accountable for up to 30–40% of community-acquired pneumonia (CAP) [1–4]. Pulmonary manifestations are typically tracheobronchitis and pneumonia accompanied by a cough but also wheezing. Young children are considered not to be as susceptible to M. pneumoniae as school-aged children. In Denmark, M. pneumoniae is mainly epidemic with a recurrence rate every 4-7th year.

Denmark experienced an M. pneumoniae epidemic, peaking in the autumn of 2010 and again in the autumn of 2011. Statens Serum Institut (SSI), responsible for the national surveillance system in Denmark, has described it as two waves of the same epidemic.

The realisation that also pre-school children and even infants can be susceptible and have clinical symptoms from M. pneumoniae infection has evolved during recent years and has been reported in studies from Europe and Australia [8–14]. From a clinical view, it is essential to establish if M. pneumoniae plays a significant differential role in lower respiratory tract infections and asthma-like exacerbations in young children and infants. In recent years the issue of co-infections and non-symptomatic carriage of M. pneumoniae and hence over-diagnosing of M. pneumoniae by PCR has been debated.

The purpose of this study was to describe and characterise the M. pneumoniae epidemic in a hospital setting, and to evaluate possible age-dependent clinical features in infants, young children and older children hospitalised and diagnosed with an M. pneumoniae infection. The study was set up since we experienced a shift in the clinical picture of our M. pneumoniae patients during the epidemic. The literature on this area is extensive with varying opinions, as mentioned, on the pathogenicity of the bacteria and the indication for treatment as well as on the significance of infections in pre-school children. Our report is another brick in understanding this pathogen.

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.

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 76-year-old Caucasian man who underwent laryngectomy 10 years earlier, presented with fever (38.9 °C; 102.0 °F), increased sputum production, and purulent conjunctivitis. These symptoms emerged gradually over a period of 48 hours. He noted increasing difficulty in coughing out his sputum that became brownish and viscous. He had been wearing a heat and moisture exchanger (HME) filter that covered his stoma and spoke through a tracheoesophageal voice prosthesis. The symptoms started a day after a very cold weather spell with temperatures of −7 to −1 °C (19–31 °F). He had to remove his HME on several occasions for extended periods of time to enable him to breathe when he walked outside his home.

His past medical history included hypopharyngeal squamous cell carcinoma which was treated with intensity-modulated radiotherapy (IMRT) 12 years earlier. A recurrence of the cancer 2 years later required laryngectomy. He had no signs of tumor recurrence since then. He also suffered from paroxysmal hypertension, diverticulitis, and migraines.

He was vaccinated with the current Influenza virus vaccine 3 month earlier. He had also received a pneumococcal polysaccharide vaccine (PPSV23) 2 years earlier.

He was in mild respiratory distress especially when coughing. He had coughing spells and expectorated green-brown dry and viscous sputum. A physical examination revealed bilateral purulent conjunctivitis and auscultation of his lungs revealed coarse rhonchi and no crepitations. No lymphadenopathy was noted. The results of the rest of the physical and neurological examinations were within normal limits. A chest X-ray was normal.

Sputum and conjunctival culture grew heavy growth of beta-lactamase-producing nontypeable Haemophilus influenzae (NTHi) that was susceptible to levofloxacin and amoxicillin- clavulanate. A FilmArray® Respiratory Panel 2 (RP2) polymerase chain reaction (PCR) system test did not detect 14 viruses (adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus OC43, human rhinovirus/enterovirus, human metapneumovirus, influenza A, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus) and four bacteria (Bordetella pertussis, Bordetella parapertussis, Chlamydophila pneumoniae, Mycoplasma pneumoniae).

He was treated with orally administered levofloxacin 500 mg/day, ciprofloxacin eye drops, acetaminophen, and guaifenesin. Humidification of his trachea and the airway was maintained by repeated insertions of 3–5 cc respiratory saline into the stoma at least once an hour and by breathing humidified air.

The main challenge was to maintain a patent airway as the mucus was very dry and viscous and tended to stick to the walls of his trachea and the stoma. The mucus had to be repeatedly expectorated by vigorous coughing and by manual removal from the upper part of his trachea and stoma.

He experienced repeated episodes of sustained elevated blood pressure (up to 210/110) and tachycardia (112/minute). This was managed by administration of clonidine 0.1 mg as needed (1–2/day).

His fever started to decline 48 hours after antimicrobial therapy was started. The conjunctivitis improved within 36 hours. The sputum production declined and became less viscous over time, but persisted for 5 days.

Antimicrobial therapy was discontinued after 7 days.

His condition improved and he had a complete recovery in 7 days. He was seen in the clinic every 2 months and showed no recurrence of his infection for the following 8 months. He received vaccination for H. influenzae B and Prevnar 13® (pneumococcal conjugate vaccine; PCV13) 4 weeks after his recovery.

The NS samples were collected by gently inserting a sterile rayon tipped applicator (Puritan®, Puritan Medical Product, ME, U.S.A.) into the nostril to a one-third depth of the nasal passage, while the OS samples were collected by rolling the swab onto the soft palate. The swabs were immersed in 0.5 mL of 1% sterile phosphate buffered saline (PBS) and stored at -80 °C until assayed.

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.

M. pneumoniae is a significant cause of LRTI in children and can cause infections difficult to distinguish from CAP caused by other respiratory pathogens. M. pneumoniae also affect infants and young children in epidemic periods, and we believe that M. pneumoniae must be considered as a differential diagnosis to respiratory virus infections and as a cause of infant and childhood troublesome lung symptoms and pneumonia. Chest x-rays were without pathognomonic features to CAP. During an up-coming epidemic, assessment of extra-pulmonary manifestations, especially immunological based skin reactions, can be helpful when diagnosing M. pneumoniae infections.

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.

Canine diarrhea is one of the most common illnesses treated by veterinarians with many possible causes of canine diarrhea, including bacteria, parasites, and viruses. One of the most important dog enteric viruses is canine parvovirus 2 (CPV-2) in the Carnivore protoparvovirus species 1. Parvoviruses are small, icosahedral, nonenveloped, single-stranded DNA viruses that are pathogenic to a variety of mammals. The vertebrate-infecting parvoviruses are classified in the subfamily Parvovirinae in the Parvoviridae family (which also includes the insect infecting subfamily Densovirinae). The Parvovirinae subfamily is currently subdivided into eight officially recognized genera (Dependoparvovirus, Copiparvovirus, Bocaparvovirus, Amdoparvovirus, Aveparvovirus, Protoparvovirus, Tetraparvovirus, and Erythroparvovirus). The recently proposed genus Chapparvovirus is currently comprised of a rat parvovirus 2 (KX272741), Eidolon helvum fruit bat parvovirus 1 (MG693107.1), and E. helvum bat parvovirus 2 (JX885610), Desmodus rotundus bat parvovirus (NC032097.1), simian parvo-like virus 3 (KT961660.1), Turkey parvovirus TP1-2012/Hun (KF925531), porcine parvovirus 7 (KU563733), murine chapparvovirus (MF175078), Tasmanian devil-associated chapparvovirus strains 1–6 (MK513528-MK53533), red-crowned crane-associated parvovirus (KY312548, KY312549, KY312550, KY312551), and chicken chapparvovirus 1 and 2 (MG846441 and MG846642). A close relative of murine chapparvovirus, initially reported in the feces of a wild Mus musculus from New York City, called murine kidney parvovirus (MH670588) was recently shown to cause nephropathy in immunocompromised laboratory mice. A recent survey of eukaryotic genomes for chapparvovirus sequences has also shown the presence of a likely exogeneous chapparvovirus genome in a fish (Gulf pipefish or Syngnathus scovelli) and of mostly defective germline sequences in another fish (Tiger tail seahorse or Hippocampus comes) as well as in multiple invertebrates, indicating an ancient origin for chapparvoviruses. A phylogenetic analysis of NS1 also indicated chapparvoviruses fall outside the traditional vertebrate-infecting Parvovirinae subfamily clade and closer to that of a subset of members of the subfamily Densovirinae.

Here an unexplained diarrhea outbreak among dogs was analyzed using viral metagenomics after diagnostic tests were negative for common canine enteric pathogens. The genome of a novel chapparvovirus was characterized and used to perform an epidemiological study to measure its prevalence and possible clinical significance.

Kobuvirus (KoV) is a single-strand positive-sense RNA virus. KoV belongs to the family Piconaviridae, genus Kobuvirus, which consists of four species Aichivirus A, B, C and D [1–3]. KoV has been reported in feces of several mammal species including humans, ruminants, pigs, dogs, cats, bats and rodents [3–10]. The Kobuvirus species Aichivirus A contains four types including Aichi virus 1, canine Kobuvirus 1 (CaKoV), Feline Kobuvirus 1 (FeKoV) and Murine Kobuvirus 1 (MuKoV). Canine Kobuvirus 1 (CaKoV) was first reported in dogs with acute gastroenteritis in the US in 2011 [5, 11]. CaKoV was subsequently reported in dogs in UK, Italy, Australia, Japan, Korea and China [4, 12–15]. The virus was reported in wild carnivores (Jackal and Hyena) and domestic dogs in Tanzania, Africa, in foxes in Spain and in foxes and wolves in Italy. Several studies have reported the detection of CaKoV infection in dogs with or without diarrhea and sometime systemic infection. To date, only 12 completed CaKoV genomes are available in the GenBank database.

During September 2016 to September 2018, the center of excellence for emerging and re-emerging infectious diseases in animals (CUEIDAs), Chulalongkorn University conducted a survey of canine Kobuvirus in domestic dogs at small animal hospitals in 5 provinces of Thailand. The survey was conducted under the Chulalongkorn University’s animal use and care protocol # 1731074. The result of this study provided the first detection and genetic characterization of CaKoV isolated from domestic dogs in Thailand.

Canine adenovirus (CAV) can be grouped into two distinct but related serotypes, CAV-1 and CAV-2, based on serological tests and molecular analyses [2–4]. Two types of Canine adenovirus (CAVs), Canine Adenovirus type 1 (CAV-1), the virus which causes infectious canine hepatitis, and Canine Adenovirus type 2 (CAV-2), which causes canine infectious laryngotracheitis, have been found in dogs. CAVs belong to the genus Mastadenovirus of the family Adenoviridae. Virus enters the host via direct contact with contaminated saliva, urine, and faeces. The incubation period is 4–7 days. CAV-1 replicates in vascular endothelial cells and causes a generalized infection characterized by hepatitis, whereas CAV-2 has an affinity for respiratory tract epithelium and is mainly associated with outbreaks of respiratory disease in kenneled dogs. CAV-1 causes fever, often above 40°C, apathy, anorexia, abdominal pain, blood in faeces, acute/chronic hepatitis and interstitial nephritis, tenderness, vomiting, and diarrhoea. Dogs may develop bronchopneumonia, conjunctivitis, photophobia, and a transient corneal opacity, “blue eye”, which may occur after clinical recovery as result of anterior uveitis and oedema [9, 10]. CAV-2 is characterized by respiratory disorders, with clinical signs that include tonsillitis, pharyngitis, tracheitis, and bronchitis [11–13]. Confirmation of diagnosis and identification of CAV-1 and CAV-2 infections are usually based on virus isolation, electron-microscopic observation and serological tests. There are distinct differences in structure, antigenicity, and pathogenicity between the two CAVs. Serological tests such as haemagglutination inhibition (HI), serum neutralization (SN), and enzyme-linked immunosorbent assay (ELISA) have been used detection of CAVs [15, 16]. The ELISA was found to be a highly efficient and rapid test to determine the immune status of dogs to infectious canine hepatitis virus and canine adenovirus type 2. The ELISA is a sensitive, reliable and fast method for the detection of anti-adenovirus antibody. When compared with SN test, the ELISA has several advantages. It does not require cell culture, the risk of contamination is less and the optimization is easier than other serological test methods. With the advent of molecular techniques, restriction endonuclease analysis (REA) of the viral genome has been said to differentiate between the two viruses [11, 14, 17].

The objective of this study was to determine the presence of antigen and the prevalence of CAV type 1 and 2 exposure in shelter-housed and household dogs in several regions of Turkey.

On December 3, 2014, an eight-year old giant panda named Chengcheng presented with jaw trembling and violent convulsions of the limbs. Over the ensuing fourteen weeks, four additional giant pandas housed in the same room or adjacent rooms began to display clinical signs including mucopurulent ocular discharge, nasal and footpad hyperkeratosis, and violent convulsions of the limbs (clinical onset dates are listed in Table 1). Nucleic acids isolated from nasal swabs, urine, feces and blood collected from affected pandas at the time of clinical presentation all tested positive for CDV by RT-PCR. PCR-based tests were negative for other virus previously isolated from giant pandas (canine coronavirus) or viruses regarded as a potential threat to giant pandas (canine adenovirus, canine herpesvirus, and canine parainfluenza virus)41617. CDV-positive giant pandas were monitored and treated with antiserum therapy.

Each of the five CDV-infected giant pandas that displayed clinical signs of infection died 7–34 days following disease onset (Table 1). No CDV serum neutralizing (SN) antibodies were detected in the five infected giant pandas showing clinical signs of CDV infection prior to death (Table 1). However, CDV RNA was detected by RT-PCR in heart, liver, spleen, lung, kidney, intestines and brain of four deceased giant pandas (Table 2). In addition, CDV RNA was detected by RT-PCR from blood and nasal swab samples collected from an asymptomatic giant panda named Zhuzhu, who was previously vaccinated against CDV in 2012 and had high-titer SN antibodies (Table 1). None of the additional sixteen giant pandas in the Shanxi Rare Wild Animal Rescue and Research Center tested positive for CDV by RT-PCR. Uninfected pandas within the Shanxi Rare Wild Animal Rescue and Research Center were placed in isolation on December 26, 2014 and vaccinated with a canarypox-vectored CDV vaccine.

Interest in the virome, or the entire population of viruses present in a biological sample, has increased recently due to improved availability of high throughput sequencing or next generation sequencing (NGS) technologies, and improved metagenomic analytical methods [1, 2]. The virome comprises all types of viruses, including those that infect prokaryotic and eukaryotic organisms, DNA or RNA viruses, and viruses that cause acute or chronic infections. Many of these viruses are difficult or impossible to propagate in cell culture, and molecular detection is difficult as no common gene such as the ribosomal 16S gene that is present in bacterial species exists in viruses. These limitations have hindered the identification and characterisation of uncultured viruses [3, 4]. Recently, due to the advent of molecular enrichment protocols, high throughput sequencing and new metagenomic analytical methods we are now able to explore, identify and characterise viruses from different biological and environmental samples with a greater capacity [2, 5–11]

In studies of human faeces, the virome has been shown to include viruses that infect eukaryotic organisms and viruses that infect prokaryotes (bacteriophages) [2, 5, 12–18]. Bacteriophages have been reported in many studies to be the most frequently detected viral constituent in the gut of humans [1, 2, 5, 8, 16, 19, 20]. The faecal virome has been characterised for several animal species including pigs, bats, cats, pigeons, horses and ferrets [2, 6, 7, 9–11, 21–31]. In dogs, the presence of enteric viral pathogens such as canine parvovirus, coronavirus, rotavirus and distemper virus (Paramyxoviridae) have been identified only through targeted studies [32–35]. To date, only one published study has used high throughput sequencing to investigate the faecal viral population in diarrhoeic dogs. These investigators analysed faeces from dogs with acute diarrhoea and detected two new virus species, canine sapovirus and canine kobuvirus; known canine enteric viruses such as canine coronavirus, canine parvovirus, canine rotavirus as well as plant and insect viruses were also reported.

The aim of this study was to describe the faecal virome of samples collected from healthy dogs, and compare these findings to the faecal virome of dogs with acute diarrhoea in Australia, using an Illumina MiSeq shotgun metagenomic sequencing approach.

Noroviruses are members of the RNA virus family Caliciviridae, and are a major cause of human infectious gastroenteritis worldwide. An estimated three million people each year in the UK suffer from ‘winter vomiting disease’ caused by human norovirus. Infection causes the classic symptoms of vomiting, diarrhea and malaise, and outbreaks are common in closed or semi-closed communities such as in hospitals, care homes, schools and cruise ships. In addition to the major burden of noroviruses on human health, noroviruses have also been found associated with intestinal disease in cows, pigs, mice, a lion, cats and dogs. The first canine norovirus (CNV) was reported from a single dog with enteritis in Italy in 2007. Subsequent studies have identified CNV in stools of dogs from Portugal,, Greece and the US. To date there have been no reports of CNV present in the UK.

Significant sequence variation has been found in different CNV strains identified to date. Noroviruses are assigned to six genogroups based on complete capsid sequences, with strains of norovirus assigned to the same genogroup if they share 55–85% amino acid identity. Human noroviruses are grouped together in genogroups I, II and IV whereas CNV strains have been assigned to genogroups IV and VI. Human and canine noroviruses in genogroup IV share <85% amino acid identity, thus are separated into different genotypes, IV.1 and IV.2 respectively. Genetic recombination is believed to occur between different norovirus strains, which may explain the significant heterogeneity between the CNV strains identified.

The prevalence of CNV in dogs with clinical signs of gastroenteritis across Europe has been estimated to be between 2.1% (Italy) and 40% (Portugal). A study in the US identified CNV at a prevalence of 11% in canine diarrhoea samples. CNV has also been detected in healthy dogs, and the association between infection and clinical signs is yet to be formally established. CNV has been identified in dogs also infected with other enteric viral pathogens such as canine parvovirus (CPV) and canine enteric coronavirus (CECoV), thus elucidating the role of CNV infection in disease is difficult.

Serological prevalence of CNV in countries where the virus has been detected has not been reported. A preliminary serological survey in Italy suggested less than 5% dogs were seropositive to a genogroup IV.2 lion norovirus (strain Pistoia/387/06/ITA) but the sample size was small. As with human norovirus, CNV has yet to be cultivated in cell culture thus obtaining sufficient quantities of virus for serological screening is not possible. However, the major capsid protein of noroviruses (VP1) has been shown to spontaneously assemble into virus-like particles (VLPs) when recombinant VP1 is expressed in an appropriate system. Production of lion and canine norovirus VLPs has previously been achieved and proven to be an efficient way of generating antigen for serological analysis,.

The aim of this study was to elucidate the importance of CNV in the UK dog population and evaluate any changes over time. No prior information on CNV prevalence in the UK has been described, so in order to address this our study has sought to determine the prevalence of CNV RNA in canine fecal samples, in conjunction with the prevalence of anti-CNV antibodies in different populations of dogs.

Post-infectious bronchiolitis obliterans (PIBO), a syndrome in children most commonly caused by Mycoplasma pneumonia (1, 2) and adenovirus (1) and occasionally Bordetella pertussis (3, 4) is associated with chronic inflammatory and fibrotic lesions of small airways leading to chronic airflow obstruction (5). Treatment is generally supportive therapy and frequently includes glucocorticoids (4–6). In dogs, while many contagious respiratory pathogens cause tracheobronchitis and pneumonia, bronchiolar diseases including PIBO are not well recognized as spontaneous clinical syndromes. Importantly, severe damage to the lung can lead to end-stage and untreatable fibrosis, with most cases in dogs not having a recognizable trigger and thus being termed “idiopathic pulmonary fibrosis.” This report describes a puppy developing PIBO after Bordetella bronchiseptica pneumonia with histologic evidence of small airway changes strongly supporting development of pulmonary fibrosis. Recognizing addressable triggers of fibrotic lung disease could have important implications for delaying progression of end-stage lung lesions.

Late in the course of infection, giant pandas exhibited clinical signs of nasal hyperkeratosis (Fig. 1a) and footpad hyperkeratosis (Fig. 1b) which are characteristic of CDV infection in other animals18. Severe pneumonia with dark-red congestion was observed in affected lungs along with small white patches on the surface of the lungs of one CDV-infected giant panda named Fengfeng (Fig. 1c). Lung samples from Fengfeng were examined for histopathological analysis, whereas other samples (e.g ., brain, spleens) were not assessed due to cellular autolysis of tissues collected at necropsies performed >12 hours of after death. Histological analyses of lung tissue from Fengfeng showed interstitial pneumonia with congestion, multinuclear macrophage infiltration in the alveoli, and widening of alveolar septa (Fig. 1d). Histological observations were consistent with those previously reported in the lungs of cynomolgus monkeys and red fox (Vulpes vulpes) infected by CDV1419.

Because results of bacterial culture and antimicrobial susceptibility testing from specimens collected from the nasal cavity are difficult to interpret, monitoring the efficacy of treatment of cats with suspected chronic bacterial URI is usually based on clinical signs of disease.

The caliciviruses (family Caliciviridae) are non-enveloped, positive sense, single-stranded RNA viruses with diameters ranging from 27 to 40 nm. Caliciviruses cause a wide range of significant diseases in human and animals. At present, there are five recognized genera, i.e., Norovirus, Sapovirus, Lagovirus, Vesivirus, and Nebovirus with several additional candidate genera or species proposed and under evaluation by the International Committee on Taxonomy of Viruses (ICTV) [1, 2] (http://www.caliciviridae.com/unclassified/unclassified.htm). In the Vesivirus genus, Vesicular exanthema of swine virus (VESV) and Feline calicivirus (FCV) are two species currently approved by ICTV. Several canine caliciviruses (CaCV) isolates have been identified and shown to be phylogenetically related to vesiviruses with features distinct from both VESV and FCV in phylogeny, serology and cell culture specificities. CaCV is a probable species in the Vesivirus genus, as stated by ICTV. It is still unclassified to date and the evidence presented herein should facilitate the classification and acceptance of CaCV as a species of vesivirus.

Many viruses found in human and other animal species can also infect dogs asymptomatically or cause respiratory, digestive, neurologic and genital diseases with mild to severe symptoms. In response to the use of dogs in military services and laboratory studies, etiological studies of canine diseases were conducted in 1963–1978 at the Walter Reed Army Institute of Research (WRAIR) [3, 4]. In addition to several known canine viral pathogens [5, 6], four unidentified viruses were recovered in Walter Reed Canine Cells (WRCC) producing similar cytopathic effects (CPE). The isolates were not recognized by available human and dog reference virus antisera. Studies of their physicochemical properties and electron microscope observations identified the isolates as likely caliciviruses. Our recent whole genome sequencing of these canine isolates clearly identified them as vesiviruses and elucidated their genetic relationships to the other members of the Caliciviridae family. We herein report the viral isolation and characterization results, which were made in 1963–1978 canine diseases etiological study but were not published, and additional genomics analysis supporting the serological diversity of CaCV strongly suggesting that these isolates and similar CaCV are a unique species within Vesivirus genus [7–9].