The CDV-infected Dogs 1, 3 and 4 were hospitalized for two to three days and received intravenous infusions of crystalloids, intravenous antibiotic therapy and inhalation (Table 1). Dog 3, which was co-infected with Babesia spp., was treated with two imidocarb injections two weeks apart. Dog 4, which was co-infected with L. infantum was treated using allopurinol. All three dogs showed rapid clinical improvement with treatment and were discharged with oral antibiotic therapy. Repeated haematological examination in Dog 3 two weeks later revealed that the pancytopenia had resolved and had returned to moderate neutrophilia, eosinophilia and slight monocytosis. Mild anaemia was still present in Dog 3 at that time (data not shown). Repeated haematology in Dog 4 two weeks after the initial presentation showed normal platelets counts and nearly normal PCV values (data not shown). All three dogs were clinically asymptomatic in the 6-month follow-up period.

Dog 2 was ambulatory and was treated with oral antibiotics and antibiotic eye drops (Table 1). The dog showed several relapses with purulent nasal and ocular discharge after antibiotic therapy ceased and was repeatedly treated with antibiotics for two months. Thereafter, there was no relapse, and the dog was clinically asymptomatic in the remaining 4-month follow-up period.

Five CDV PCR-positive dogs (Dogs 5 to 8 and 10) received oral antibiotic therapy (amoxicillin clavulanic acid or doxycycline) for seven to ten days after their arrival in Switzerland. Dog 5 developed watery diarrhoea two weeks after arrival and was additionally treated with metronidazole, deworming and a highly digestible diet. Dog 8, which was co-infected with Babesia spp., received two injections of imidocarb diproprionate two weeks apart and antibiotic ear drops because of otitis externa. At the end of the 6-month follow-up period, all of the CDV PCR-positive dogs had recovered and none had developed neurological signs.

Fluid replacement, systemic antibiotic administration, antinausea medicines, antidiarrhea medicines, and a rigorous diet combined with monoclonal antibodies are the main treatment methods for CPV-2-infected dogs; however, the recovery rates vary from 27.8 to 93.5% (39, 53, 55, 56, 64, 65). Both disease and pathology of the infected animal differ depending on the age. CPV-2 infection in adult dogs results in temporary panleukopenia or lymphopenia; CPV-2 infection in neonatal animals causes myocarditis (12, 66). CPV-2 monoclonal antibodies are highly therapeutic in a short period of time and have treatment well effect (53). The cure rate for a CPV-2 single infection is higher than that of a coinfection with other viruses (41). With improvements in medical treatments, CPV-2 cure rates have been improved using specific drugs or other treatments.

All of the owners of the CDV PCR-positive dogs were instructed by the first author (BW) to quarantine the dogs until they tested CDV PCR-negative. The owners of the CDV-positive dogs in multidog households (Dogs 3, 6, 7, 8 and 9) were instructed to separate the infected dog from the other dogs in the household. However, several dogs (Dogs 6, 7, 8 and 9) had already had contact with adult dogs within the household at the time when the CDV diagnosis was made. All of the contact dogs had been vaccinated against CDV, although several dogs had only received the initial vaccination series as puppies and had received no booster vaccinations (data not shown). Two dogs that were in close contact with Dogs 7 and 9 were tested for the CDV infection with PCR one month and two months after the initial CDV diagnosis in Dogs 7 and 9. One contact dog exhibited a single, very weak CDV-positive result in the conjunctival swab in the first sampling but was negative in all of the swabs collected one month later (data not shown). The other contact dog tested PCR-negative in all of the collected samples (data not shown). In all of the other multidog households, no samples were collected for CDV PCR, but no clinical signs of the disease were noted in the 6-month follow-up period.

Briefly, FK-81 cell monolayers (from Toronto University of Canada and stored in this lab) in Costar flasks were inoculated with 1 mL supernatant (obtained in Section 2.1) treated with antibiotics at final concentration 100 U/mL. After gentle rotation, the flasks were incubated for 1 h at 37 °C in 5% CO2 to allow for attachment; then, the supernatants were removed, and the monolayers were washed three times with MEM without FBS. After washing, 7 mL DMEM with 2% FBS was added, and the flasks were then incubated for 3 days at 37 °C in 5% CO2. If the cells did not exhibit a cytopathic effect (CPE) by the fourth day, the incubated monolayers were subjected to another two passages, and the culture supernatant (500 μL) was collected for PCR/RT-PCR. The monolayers were not discarded, unless the PCR/RT-PCR results were negative and no CPE was observed in the third FK-81 passage. If 80% of cells exhibited CPE and the PCR/RT-PCR results were positive, the cells were frozen for further analysis.

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.

Five healthy domestic cats free of FHV-1 and FHV-1 antibodies, aged 3 months old and weighing from 1.8 kg to 2.0 kg, were divided into three groups. Because the transmission of FHV-1 is largely by direct contact with an infected animal, the cage for cats No. 1 and No. 2, which made up the IG (inoculated group), was placed at the bottom, the cage for cats No. 3 and No. 4, which made up the EG (exposure group), was placed in the middle, and the cage for cat No. 5, the control cat, was placed at the top. Under anesthesia, the two cats of the IG were inoculated by intranasal and ocular routes with 0.5 mL sample/cat containing 106 TCID50 of the SZ12 strain, and the other cats were mock-inoculated with 0.5 mL PBS/cat. Each cat’s temperature was taken, and clinical signs were observed daily. From the fourth day post-inoculation, nose, eye and throat secretions were collected for PCR to detect FHV-1 infection.

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.

There are currently five other known canine parvovirus species belonging to two genera of the Parvoviridae family. Canine parvovirus 2 (CPV2) in the Carnivore protoparvovirus 1 species is a highly pathogenic virus that is closely related to feline parvovirus (FPV), the cause of feline panleukopenia, and can infect other carnivores such as coyotes, wolfs, raccoons and pumas. Canine bufavirus, a second protoparvovirus (in the species Carnivore protoparvovirus 2) was reported in 2018 in fecal and respiratory samples from both healthy and dogs with signs of respiratory illness. That same protoparvovirus was recently reported as a frequent component of juvenile cats fecal and respiratory samples. The canine minute virus (CnMV) in the Carnivore bocaparvovirus 1 species is less pathogenic than CPV2 but can cause diarrhea in young pups and is frequently found in the context of co-infections. Distantly related to CnMV, a second canine bocavirus in the Carnivore bocaparvovirus 2 species was sequenced in dogs with respiratory diseases. A third bocavirus was then characterized from the liver of a dog with severe hemorrhagic gastroenteritis.

Here, we describe the near complete genomes of two closely related cachaviruses, members of a new tentative species (Carnivore chapparvovirus 1) in a proposed genus Chapparvovirus, the third genera of viruses from the Parvoviridae family now reported in canine samples. The chapparvovirus was found in only two animals of the initial nine sampled. Many of the dogs in the outbreak analyzed were sampled more than 10 days after onset of clinical signs, increasing the possibility that they were no longer shedding viruses. Additionally, diarrhea is one of the top reasons for veterinary visits and some patients may have coincidentally presented with diarrhea from some other cause.

The two samples positive for CachaV-1 presented in the same week and were in the group of patients with the most severe clinical signs, requiring plasma transfusion and more aggressive supportive care. One of the two dogs, sampled at nine days after onset, died two days later. Because of the variable and often delayed feces sampling, it was therefore not possible to determine a clear disease association in this small group of diarrheic dogs (i.e., not all affected animals were shedding cachavirus).

A possible role for the cachavirus infection in canine diarrhea was further tested by comparing cachavirus DNA PCR detection in larger groups of healthy and diarrheic animals including a group of animals with bloody diarrhea. A statistically significant difference (p = 0.037) was seen when diarrhea samples from 2018 were compared to the feces from healthy animals collected the same year. When 2017 diarrheic samples were compared to e 2018 healthy samples, the p-value was 0.08. When 2017 and 2018 diarrhea samples were combined and compared to the healthy samples, the p-value was 0.05. The association of cachavirus with diarrhea is therefore borderline and the detection of viral DNA remains limited to ~4% of cases of diarrhea. The limited number of healthy samples available for PCR limited the statistical power of this analysis and a larger sample size will be required for further testing of disease association. The absence of detectable cachavirus DNA in 83 other cases of bloody diarrhea was unexpected given the similar signs that developed in the initial outbreak. Detection of viral DNA in feces may be related to timing of sample collection as shedding of the intestinal lining during hemorrhagic diarrhea may preclude viral replication and fecal shedding.

The detection of this virus in multiple fecal samples, the absence of prior cachavirus reports from tissues or fecal samples from other animals, and the confirmed vertebrate (murine) tropism of another chapparvovirus (mouse kidney parvovirus), support the tentative conclusion that cachavirus infects dogs. Given its relatively low viral load and only borderline association with diarrhea, this virus’ possible role in canine diarrhea or other diseases will require further epidemiological studies. Because viral nucleic acids in fecal samples may also originate from ingestion of contaminated food (rather than replication in gut tissues), the tropism of cachavirus for dogs will require further confirmation such as specific antibody detection, viral culture in canine cells, and/or evidence of replication in vivo such as RNA expression in enteric tissues of dogs shedding cachavirus DNA.

Canine distemper (CD) is a highly contagious and fatal disease of dogs caused by the canine distemper virus (CDV), which is a single-stranded negative RNA virus belonging to the Morbillivirus genus within the Paramyxoviridae family. Other members of the genus include measles virus (MV) and rinderpest virus (RPV). The genome of CDV is approximately 15,690 nucleotides (nt) in length, containing several genes encoding N, P, M, F, H, and L proteins. Only one serotype has been characterized.

A large number of dogs, minks, foxes die from CDV infections every year, causing significant economic losses[2]. Previous studies[3,4] have reported that vaccinated dogs were infected with CDV in Europe and Japan. Harder et al. also reported that there are marked differences between wild-type and vaccine strains of CDV[5], thus whether CDV vaccine strains are able to provide protection from the current strains of CDV remains a question. It is difficult and necessary to discriminate between wild-type and vaccine strains because the attenuated CDV vaccine is used widely in China. So a method to specifically detect the wild-type CDV strains is necessary. The multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) method could be used to effectively detect and differentiate between wild-type CDV-infected dogs from dogs which were vaccinated with CDV vaccine, which would make it useful in clinical diagnosis and epidemiological monitoring.

For detection of antibodies against betacoronaviruses, 30 acute serum samples as well as the corresponding sera (obtained 2 to 3 weeks later) of the convalescent dogs were examined by an indirect immunofluorescence test. Madin‐Darby bovine kidney cells were disseminated on 96‐well microtitre plates (100 μL/well) and then incubated at 37°C in a humid 5% CO2‐atmosphere overnight. After washing the plates with phosphate‐buffered saline (PBS) solution, the adherent cells were infected with BCoV strain 15317/82 and incubated at 37°C for 48 hours. Subsequently cells were washed with PBS again and fixed with 100 mL of 96% ethanol.

The sera underwent twofold serial dilutions from 1:20 to 1:5120 with PBS and immunofluorescence test was performed as follows:

Ethanol was discharged, and the 96‐well microtitre plates were washed three times with PBS; 50 μL of the previously diluted sera per well were added and incubated at 37°C for 30 minutes. Thereafter, the plates were washed three times with PBS and 50 μL of 1:40 diluted fluorescein isothiocyanate (FITC)‐conjugate (anti‐dog IgG, Jackson) was added to each well. After incubation at 37°C for another 30 minutes and three washing cycles with PBS, 50 μL/well Eriochrome black T indicator (diluted 1:200 with PBS) was filled in each well of the 96‐well microtitre plate to reduce background fluorescence. Plates incubated for 5 minutes at room temperature before cells were washed three times with PBS once more. Finally, wells were filled with 50 μL/well of glycerine buffer solution to prevent the cells from drying.

For evaluation of the microtitre plates an inverse ultraviolet microscope was used. The highest dilution with a clear cytoplasmatic fluorescence was equivalent to the specific antibody titre of each serum sample. Samples that showed no fluorescence in dilution 1:20 were regarded as negative (no antibodies present). Each assay included a positive and a negative control serum.

In canine disease, viral diarrhea has a high incidence because of etiology complexity, which causes serious harm to the canine industry and dogs. At present, several viral pathogens are related to canine diarrhea in China, including canine parvovirus (CPV) (1, 2), canine coronavirus (CCoV), canine bocavirus, canine kobuviruses (CaKVs), and canine distemper virus (CDV). CPV is an important cause of mortality and morbidity in dogs, especially puppies, in China and the rest of the world (3–5). Variation, recombination, and coinfection have been shown to aggravate clinical symptoms and challenge the prevention and control of CPV infections (6–9). Dogs that become infected by CPV show illness within 3–7 days, presenting with severe gastroenteritis, lethargy, vomiting, fever, and diarrhea (usually bloody) (10–12).

CPV belongs to the genus Parvovirus, family Parvoviridae, and causes a highly contagious and fatal disease in dogs (1). The original viral strain, designated as CPV-2 to distinguish it from CPV-1 which is also known as canine minute virus and was believed to be non-pathogenic until 1992 (13). CPV-2 is a non-enveloped DNA virus with a linear single-stranded DNA genome (5.2 kb), containing two major open reading frames (ORFs). One ORF encodes the two non-structural proteins (NS1 and NS2), and the other encodes the two capsid proteins (VP1 and VP2) (14). The VP2 protein of CPV-2 is known to affect antigenic properties, playing important roles in controlling viral host ranges and tissue tropisms (15–17).

The main method for controlling the virus in domestic animals is by vaccination, antibody therapy, and traditional Chinese medicine therapy (18, 19). However, the virus is widely distributed in nature, and the morbidity and mortality of CPV-2-infected animals remain high (20). Furthermore, because of vaccine formulations, a dramatic increase in the number of dog or other factors potentially promote the spread of different CPV-2 antigenic variants, increasing disease complexity (21–23). In previous studies, researchers have investigated CPV-2 genetic evolution (5, 24–27), providing important reference information for the prevention and control of the CPV-2 infections. However, the molecular epidemiology and genetic diversity of CPV-2 need to be updated in China. In this review, we have summarized contemporary data on the progression of CPV-2 epidemiology in China, including the virus origin, prevalence, coinfection, and evolution. The aim is to unravel CPV-2 epidemiology and provide new information on virus infections, not only for Chinese dogs and their owners but also for all dog owners across the world.

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.

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

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

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

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

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

The protocol of the study was carried out in accordance with guidelines of animal welfare of World Organization for Animal Health. All experimental protocols were approved by the Review Board Military Veterinary Research Institute of the Academy of Military Medical Sciences.

Nine stool samples from dogs suffering from an infectious diarrhea outbreak in Colorado in October 2017 were submitted to IDEXX Reference Laboratories, Inc. (Sacramento, CA, USA) for pathogen testing. Fourteen dogs were involved in the initial outbreak which were identified by clinical signs that started with steatorrhea, progressed to hemorrhagic diarrhea with additional symptoms of lethargy, fever, and low lymphocyte counts pointing to a possible viral infection. At the time of feces collection, the nine sampled dogs were at various stages of the disease, with two of the dogs relapsing a month after initially experiencing parvo-like clinical signs. These stool samples were all negative for Giardia spp., Cryptosporidium spp., Salmonella spp., Clostridium perfringens enterotoxin gene (quantitative), Clostridium perfringens Alpha-toxin gene (quantitative), Canine enteric coronavirus (alphacoronavirus), Canine Parvovirus 2 and Canine Distemper virus using the IDEXX canine diarrhea profile real-time PCR tests.

Dog throat, rectal and penile swabs were collected, placed in 2–5 ml of veal infusion broth transport media (Difco Laboratories Inc., Detroit, MI) and frozen at < -60 °C until processed for virus isolation and identification. Blood specimens were collected from each dog and dogs in each cohort at the time and 14–28 days later.

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].

Each plasmid was used as a template to evaluate the sensitivity of the mPCRs. For CRV, the minimum detection limits for pMD-CAV-2, pMD-CDV, pMD-CIV and pMD-CPIV were 1×103 (Fig 3A), 1×103 (Fig 3B), 1×104 (Fig 3C) and 1×104 (Fig 3D) viral DNA copies, respectively. For CEV, the minimum detection limits for pMD-CAV-2, pMD-CanineCV, pMD-CCoV and pMD-CPV were 1×104 (Fig 4A), 1×104 (Fig 4B), 1×103 (Fig 4C) and 1×103 (Fig 4D) viral DNA copies, respectively. The sensitivity test results revealed that the minimum simultaneous detection limit for mPCR of CRV was 1 × 104 viral copies (Fig 3E), and the limit for CEV (Fig 4E) was also 1×104 viral copies.

Canine infectious respiratory disease (CIRD) may be associated with single virus infections or with a multifactorial etiology and are assigned to infectious agents that replicate sequentially or in synergy.1 The main viral agents involved in CIRD include Canine distemper virus (CDV), Canine parainfluenza virus (CPIV), Canine adenovirus type 2 (CAdV-2) and Canid herpesvirus 1 (CaHV-1).2

In Brazil, CDV infection is endemic in dog populations, is associated with respiratory and/or multisystemic disease, and causes thousands of deaths each year.3, 4 Due to its impact on animal health, CDV is one of the most important infectious diseases in dogs.2, 5 Similarly to CDV, CAdV-2 has a worldwide distribution and is a major agent of canine infectious tracheobronchitis (CIT) or “kennel cough”, a disease characterized by restricted infection of the respiratory system.6 CPIV has a wide distribution in canine populations with an estimated seroprevalence ranging from 30 to 70%.7 CPIV infection is related to high population density; the virus is highly transmissible and presents with rapid dissemination between animals.2 CaHV-1 has a worldwide distribution and is associated with respiratory and reproductive disease.8 Like other Alphaherpesviruses, CaHV-1 establishes latent infections in nerve ganglia and can periodically reactivate the infection.9 An estimated 30–100% of domestic dogs have antibodies to CaHV-1.10

The transmission of respiratory viruses occurs through direct or indirect contact between animals, primarily through contaminated nasal secretions and aerosols.1 CIRD may affect dogs of both genders and ages; puppies under 90 days old are more susceptible, as well as immunosuppressed dogs, animals without a history of vaccination; vaccination failures or maternal immunity may also contribute.11 The disease presents a seasonal pattern with a higher incidence in cold months.12

The diagnosis of CIRD is largely based on the epidemiology, clinical signs and response to therapy. However, an etiologic diagnosis requires the identification of the agent or its products (proteins or nucleic acids).4 Vaccination is largely used to prevent or control respiratory infections in dogs and helps minimize clinical disease; however, current vaccines are not always effective.11

In Brazil, despite the wide distribution of these infections and informal reports by veterinarians, very few reports describe viral respiratory disease in dogs.13, 14, 15, 16, 17, 18 Additionally, there is little information regarding these infections in local environments with high densities and constant animal movement such as dog shelters. The identification of the more common respiratory viruses in dogs in various epidemiological conditions is essential for developing efficient control and prevention measures.

Thus, the objective of this study was to investigate respiratory viral infections in dogs in shelters. For this, three shelters located in Rio Grande do Sul state, Brazil, presenting diverse sanitary and nutrition conditions were included in an attempt to associate the occurrence of viral infections with the conditions observed. The viruses were detected in nasal secretions via polymerase chain reaction (PCR) and focused on the main agents involved in this condition (CDV, CPIV, CAdV-2 and CaHV-1).

Canine adenovirus indirect ELISA Kit (EVL/European Veterinary Laboratory-Netherlands, catalogue no. D1003-AB01) was used for detecting CAV antibodies in dogs. The test was performed as per the manufacturer's instructions. The plates were then read on an automatic plate reader at 450 nm.

To evaluate the specificity of the mPCRs, we performed specificity assays on CRV and CEV with CRV- and CEV-specific primers, respectively. Similar procedures were used to detect possible cross-reaction of CRV and CEV primers with RNA/DNA extracted from MDCK cells or from other pathogens (RABV, E. coli and Salmonella enterica). The nucleic acid extraction products of the MDCK cells, E. coli and Salmonella enterica were used directly as PCR templates. In contrast, the viral RNA extraction products of RABV required RT prior to use as templates. Both the individual plasmid and premixed plasmids were tested separately in this assay. The empty pMD-18T vector was used as a negative control.

Canine enteritis can be caused by a number of viral, bacterial or parasitic agents. The most common viral entero-pathogens are canine parvovirus (CPV) and coronavirus (CCoV),, although other agents, such as canine adenovirus (CAdV) type 1, canine distemper virus (CDV), rotaviruses, reoviruses, and caliciviruses, have been associated with enteric disease in dogs. In recent years, novel viruses have been discovered from dogs with enteritis, namely noroviruses, sapoviruses, astroviruses, and kobuviruses,.

More recently, a dog circovirus (DogCV) was detected in dogs with vasculitis and/or hemorrhagic diarrhoea in the US (13). Circoviruses (family Circoviridae, genus Circovirus) are non-enveloped, spherical viruses with a small monomeric single-strand circular DNA genome of about 2 kb in length. According to the most recent release of the Universal Virus Database of the International Committee on Taxonomy of Viruses, the genus Circovirus consists of eleven recognized species, including Porcine circovirus 1 (PCV-1), Porcine circovirus 2 (PCV-2), Canary circovirus (CaCV), Beak and feather disease virus (BFDV), and other viruses of domestic and wild birds (http://ictvdb.bio-mirror.cn/Ictv/fs_circo.htm). Porcine and avian circovirus infections are characterized by clinical courses that may vary from asymptomatic infections to lethal disease.

Two independent studies have shown that, similar to other animal circoviruses, DogCV possesses an ambisense genomic organization with 2 major inversely arranged ORFs encoding for the replicase and capsid proteins, respectively,. The canine virus, firstly detected in serum samples, was later recognized as causative agent of necrotizing vasculitis and granulomatous lymphadenitis.

The aim of this paper is to report the detection and molecular characterisation of DogCV in dogs with acute gastroenteritis in Italy. The full-length genome of the Italian prototype strain was determined and analyzed in comparison with American strains and other circoviruses.

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.

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

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

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