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

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

The obtained data are initially presented in a descriptive way and a 95% confidence interval was calculated. Analysis was performed using IBM SPSS.

The results are presented as the mean ± standard deviation (SD). Statistical significance between different vaccination groups was calculated by Student's t-test and Kaplan–Meier analysis using the statistical package for the social sciences (International Business Machine Corporation, Armonk, NY, USA) statistical software. P-values less than 0.05 were considered significant.

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

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

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

Sample processing and virus isolation were performed as previously described (54). RNA was directly extracted from nasal swabs for screening. All of the M-gene-positive samples were incubated on a monolayer of Madin-Darby canine kidney (MDCK) cells for virus isolation. The HA and NA subtypes were determined by direct sequencing of the PCR products. Whole genomes were amplified by PCR and sequenced by segment-specific primers.

Study population: The 51 dogs with respiratory clinical illness included

in this study were 29 males and 22 females. Most of the dogs were puppies (37.3%) or senile

(23.5%). Most presented with a nasal discharge (80.4%), coughing (47.1%), loss of appetite

(56.9%) and bronchopneumonia (41.2%). Only 29.4% (15/51) of dogs were vaccinated.

Optimized and analytical performances of simplex and multiplex PCR assays:

Optimization of each simplex PCR was undertaken using positive controls and clinical samples

with different cycling conditions. Different annealing temperatures were evaluated, with the

optimum Ta for all virus detections being 58°C, at which temperature no primer dimers or

non-specific amplicons were detected (data not shown). In silico and

in vitro analytical specificity tests revealed that each primer was able

to amplify the specific target DNA without any cross amplification among the CIRDC viruses,

CPV, CCoV and B. bronchiseptica. In addition, the sequenced amplicons

showed 100% sequence identity with their respective corresponding sequence in the GenBank

database.

Analytical sensitivity, specificity and reproducibility: The sensitivity

of the multiplex PCR was tested by detection of the various viruses in serial dilutions and

compared with that using the simplex PCR for each particular virus. The multiplex PCR

products of the tested viruses were observed at the same template dilutions as with the

simplex PCR, suggesting a similar sensitivity for the simplex and multiplex PCRs (Figs. 1 and 2). The highest detection threshold was found for CDV and CRCoV, then CaHV-1 and CIV,

and finally by CPIV and CAdV-2.

The specificity of the tested PCRs was evaluated by using other pathogens as mentioned

above. No specific amplicons were detected in all reactions. For evaluation of the

reproducibility, both intra- and inter-assay variations revealed similar results among the

assays (data not shown).

Evaluation of the multiplex PCR using clinical specimens: The multiplex

PCRs were tested on the 51 NS and 51 OS samples (Fig.

3) and compared with the simplex PCR assays for each respective virus (Table 2). The CAdV-2 and CRCoV detection had 100% sensitivity and specificity for both

the NS and OS sampling sites. False negative results were observed in CaHV-1, CIV, CPIV and

CDV detection when performing multiplex PCRs, which resulted in a lower sensitivity of

87.5–97.7%. The PPV (100%) of all multiplex PCRs was consistent with the specificity (100%),

while the NPV (89.5–99.0%) of those reactions was contrary with their sensitivity. Neither

the multiplex RT-PCR nor the multiplex PCR showed false positive results when compared with

its simplex counterpart.

The comparison between the multiplex PCRs and the rapid three-antigen test kit (CAdV-2, CIV

and CDV) was performed on the same samples (Table

3). With the clinical samples tested in this study, the rapid test kit yielded

100% sensitivity and a relatively high specificity for CAdV-2 and CDV. However, for CIV,

there were high numbers of PCR-positive samples detected by multiplex PCR (83/102), whereas

the test kits showed negative results.

Detection of CIRDC viruses in clinical samples by multiplex PCR: In single

infection CIV was the predominant virus detected and accounted for 23.5% (12/51) and 19.6%

(10/51) positive NS and OS samples, respectively. The next most common virus was CPIV,

detected at 3.9% (2/51) and 5.8% (3/51) of NS and OS samples, respectively, with 2% (1/51)

being positive for CRCoV infection in both NS and OS samples. Even though the CDV, CAdV-2

and CaHV-1 were not detected as a single infection, they were detected in multiple

infections in these tested samples (Table

4).

For dual infections, the most frequently detected viruses were CIV co-infected with CRCoV

at 13.7% and 21.6% in NS and OS, respectively, followed by CIV with CPIV at 9.8% and 7.8% in

NS and OS samples, respectively. For triple infections, CIV and CRCoV were frequently found

together co-infected with other viruses, and especially with CDV and CPIV. However, one dog

was negative for all tested viruses in both the NS and OS samples.

Generally, dual infections were predominant in CIRDC suffering dogs (42.2%), followed by

single (28.4%) and triple (22.6%) infections. With regards to the sampling site, the

frequency of positive results was not statistically different between the OS and NS sampling

sites (P>0.05).

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.

Cynda Crawford

Maddie's Shelter Medicine Program, University of Florida, Gainesville, FL, USA

Diagnosis of FIV infection is based on detection of circulating FIV antibodies. Introduction of FIV vaccines created a diagnostic dilemma because vaccine antibodies were indistinguishable from those induced by infection using existing serological assays. New point‐of‐care tests for FIV antibody are now available to practitioners. The purpose of this study was to determine the accuracy of FIV diagnostic tests in FIV‐vaccinated cats and whether any of the tests could serve as a DIVA test to differentiate infected from vaccinated animals.

Plasma samples were collected from 104 uninfected SPF cats vaccinated 3 times with a killed dual‐subtype FIV vaccine (Fel‐O‐Vax, Boehringer Ingelheim) according to manufacturer instructions. Age at vaccination ranged from 2.5 to 13 months. The interval between vaccination and sample collection ranged from 1.75 to 14 months. The FIV‐free infection status of all cats was confirmed by virus culture. Three FIV‐infected cats were similarly vaccinated and tested. The plasma samples were tested in 4 commercial point‐of‐care assays: SNAP® Feline Combo FeLV Ag/FIV Ab Test, WITNESS® FeLV‐FIV Test, Anigen® Rapid FIV Ab/FeLV Ag Test Kit, and VetScan® Feline FeLV/FIV Rapid Test. All testing was performed by personnel blinded to sample status. Each test result was independently interpreted by 2 personnel.

The SNAP® and VetScan® tests detected FIV antibodies in 102/104 and 88/104 uninfected vaccinated cats, respectively. This would lead to misclassification of most of the vaccinated cats as infected. The WITNESS® and Anigen® tests had very high specificity, indicating that nearly all vaccinated cats were correctly identified as uninfected. All 4 tests correctly identified the 3 FIV‐infected cats that were vaccinated against FIV.

Accurate detection of FIV‐infected cats is essential to providing appropriate care and segregation from uninfected cats. Since euthanasia is a tool employed in the control of FIV, especially in animal shelters where the previous vaccination history of cats is usually unknown, misidentification of vaccinated cats as being infected may have unwarranted fatal consequences. Based on this study, the WITNESS® FeLV‐FIV Test and the Anigen® FIV Ab/FeLV Ag Test are the most accurate for differentiating FIV‐infected from FIV‐vaccinated cats and meet the criteria for DIVA tests.

First discovered in 1953 by Rowe et al., Ads are non-enveloped, double-stranded DNA viruses with 57 unique serotypes, some of which are specific for attacking the respiratory track, conjunctiva, or gastrointestinal track (40). Key features of Ad infections include various symptoms of disease, including rhinorrhea, nasal congestion, cough, sneezing, pharyngitis, keratoconjunctivitis, pneumonia, meningitis, gastroenteritis, cystitis, and encephalitis. Illnesses may be asymptomatic, mild, or severe; however, immunocompromised patients and infants are at increased risk of severe morbidity and death.

Rhinoviruses are small, single-stranded RNA viruses in the picornavirus family that are responsible for more than half of all upper respiratory tract infections. In addition to exacerbating asthma and chronic obstructive pulmonary disease, rhinoviruses have also been associated with acute respiratory hospitalizations among children (30). In a large prospective study of US pneumonias, rhinoviruses have been identified as the second most prevalent etiology of pneumonia in children after respiratory syncytial virus and the first most common etiology among adults (31). There are more than 150 unique types of rhinoviruses. Among the three genotypes (A, B, and C) types A and C are most often associated with increased morbidity and bacterial secondary infection. In animals, rhinovirus type C has been associated with morbidity in chimpanzees (32). With an array of unique serotypes no vaccines or approved antiviral therapies have been commercially produced; however, experiments have suggested that vaccines and antiviral therapy may be possible (33, 34).

We used the sequence-derived phenotype markers provided by the Influenza Research Database (IRD) (61) to compare known phenotype-associated amino acid changes in the CIVs collected for this study and other host species and lineages (available at https://www.fludb.org). We also used IRD’s HA subtype numbering tool (62).

The titers of M2e-specific antibodies were detected by ELISA as described previously.24 Briefly, a 96-well micro-titer plate (Sigma, St. Louis, SO, USA) was coated with either H5N1–, H7N9–, or HK/156-M2e (GL Biochem Ltd, Shanghai, China) at a concentration of 1 µg/well and incubated overnight at 4 °C. After the coated plate was blocked with 3% bovine serum albumin in PBS for 2 h at room temperature (RT), serially diluted sera were added to the plate and incubated at RT for 2 h. Horseradish peroxide-conjugated goat anti-mouse immunoglobulin G antibody (Dako, Glostrup, Denmark) was added to the plate and incubated at RT for 1 h. Substrate 3,3′,5,5′-tetramethylbenzidine (Life Technologies, Carlsbad, CA, USA) was added to the plate and incubated at RT for 0.5 h. The reaction was stopped by adding 1 M H2SO4, and the results were measured at absorbance of 450 nm using an ELISA reader (Beckman Coulter, Brea, CA, USA).

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

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

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

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

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

Rhonda LaFleur1, Tamara Davis1, Patrick Tuma1, Huchappa Jayappa1, Mike Francis2, Ian Tarpey2

1Merck Animal Health, Elkhorn, NE, USA, 2MSD Animal Health, Milton Keynes, UK

A study was conducted in dogs to evaluate the efficacy of an inactivated Canine Influenza Virus (CIV) H3N2 vaccine following experimental challenge with a virulent heterologous strain of CIV H3N2, isolated from the recent CIV H3N2 outbreak in the United States. Eleven dogs, 7–8 weeks of age, were vaccinated with 2 doses of an inactivated CIV H3N2 vaccine, 3 weeks apart, and 19 dogs were vaccinated with a placebo. Two weeks after the second vaccination, all dogs were challenged intranasally with virulent CIV H3N2 and then monitored daily for 10 days for clinical signs including fever, nasal discharge, sneezing, coughing, depression, and dyspnea. Nasal swabs were collected to evaluate viral shedding, and serum samples were collected at various time points to determine antibody titers. At necropsy, lungs were scored for consolidation. Following the booster vaccination, the placebo‐vaccinated control dogs remained seronegative (<10) to CIV H3N2, while 10 of the 11 vaccinated dogs developed an antibody titer to CIV H3N2 (GMT = <80; Range = <10 ‐ 320). Antibody titers in dogs from both treatment groups increased following challenge, but the increase was greater in the vaccinated dogs

(GMT = >1452; Range = 40 ‐ >10,240). Following challenge, 8 (42%) of the 19 placebo‐vaccinated control dogs were euthanized prior to the 10‐day post‐challenge observation period due to severe clinical signs, including difficulty breathing, depression, fever, and severe coughing with retching; whereas, none (0%) of the vaccinates had to be euthanized (P = 0.014). Clinical signs were evaluated based on a weighted scoring system. The mean clinical score for the placebo‐vaccinated control group was 24.9, compared to only 8.7 for the vaccinated group (P = 0.036). The placebo‐vaccinated control group shed CIV H3N2 virus for a mean of 1.9 days, compared to 1.4 days for the vaccinated group (P = 0.507). The median lung consolidation score for the placebo‐vaccinated control group was 7.4, compared to 0.0 for the vaccinated group (P = 0.026). Results of this study demonstrate that this inactivated CIV H3N2 vaccine significantly protects dogs against severe clinical disease and lung consolidation associated with a virulent CIV H3N2 infection.

To evaluate the reproducibility of the assay, the detection mPCRs for both CRV and CEV were performed as three independent mPCR assays by using three different PCR instruments at different times. Three premixed plasmids for CRV (Fig 5A) and CEV (Fig 5B), with different dilutions, could be amplified under different conditions and showed similar results among the assays.

The clinical signs most frequently associated with enteric CCoVs are not easily differentiated from those associated with other enteric pathogens such as CPV2 or canine rotavirus and canine adenovirus. Consequently, CCoV diagnosis requires laboratory confirmation. The diagnostic techniques employed for the detection of CCoVs in fecal samples include electron microscopy (EM), virus isolation (VI) in cell cultures, and biomolecular analysis. EM examination of negatively stained fecal suspensions and immune electron microscopy are rapid procedures for detecting coronavirus and appear to be valuable diagnostic tools. However, coronavirus-like particles in intestinal contents often resemble coronaviruses, and EM examination required specialized laboratories and technicians. VI is the most commonly used technique for diagnosis of CCoVs infection, but is more complex, more time-consuming and less sensitive than other methods. CCoV type 2 grows on several cell lines of canine and feline origin, and the identification of an isolate requires neutralization of the cytopathic effects and/or immunofluorescence test with a reference serum or monoclonal antibodies. Failure to isolate CCoV type 1 in cell cultures reduces the changes of identifying many forms of enteritis caused by this virus, so the frequency of CCoV type 1 disease is probably underestimated. Such difficulties and limitations prevent an authentic evaluation of the immunological characteristics of this new genotype and hinder the acquisition of key information on its pathogenetic role in dogs.

In the past decade, several PCR-based methods have been developed for detecting CCoV RNA in the feces of dogs, allowing the detection limits of virus isolation to be overcome. PCR has been identified as the gold standard because of the improvement in both sensitivity and specificity when compared to conventional methods [20, 23, 61, 62]. Therefore, none of the developed PCRs were designed to be quantitative. Moreover, conventional PCR contains a certain risk of carryover contamination due to post-PCR manipulations and to a second amplification step in nested PCR systems, especially when a high sample throughput is required. Conversely, real-time TaqMan RT-PCR enables a sensitive and specific quantitation of viral RNA [63–66]. Decaro et al. developed a real-time fluorogenic RT-PCR, a simple, rapid, and reproducible method for the detection and quantitation of CCoV RNA in the feces of infected dogs, based on the TaqMan technology. In comparison to conventional RT-PCR, the fluorogenic assay is a closed system in which the tube is never opened postamplification, ruling out the possibility of cross-contamination. The main advantage of the fluorogenic dye system consists of quantifying CCoV RNA amounts in fecal samples with a high degree of reproducibility and precision compared to quantitative gel-based PCR assays [68, 69].

Recently, two genotype-specific fluorogenic RT-PCR assays were developed for the detection, discrimination, and quantitation of CCoV type 1 and CCoV type 2 RNAs in the feces of dogs with diarrhea. The assay showed high specificity, sensitivity, and reproducibility, allowing a precise quantitation of CCoVs RNA over a linear range of about eight orders of magnitude, from 101 to 108 copies of standard RNA. The genotype-specific fluorogenic assay can be useful to detect and measure viral loads in fecal samples collected from dogs naturally or experimentally infected with type 1, type 2, or both genotypes.

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

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

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

BALB/c mice were used to determine the protective efficacy of mAb D7. Intranasal inoculations with 107 TCID50 of virus strains JS/10, GD/12 and SD/05 were given to experimental groups I (n = 45), II (n = 45) and III (n = 45), respectively; the control group received PBS (n = 15). Each experimental group was divided into three subgroups (n = 15 for each subgroup), which were the virus-infected, mAb D7 and irrelevant mAb IgG subgroups, respectively. Mice injected with PBS or irrelevant mAb IgG were considered as blank and negative controls, respectively. For the mAb D7 and irrelevant mAb IgG subgroups, mice were pretreated intraperitoneally with mAb D7 (36 μg/mL) or irrelevant mAb IgG (32 μg/mL) against IgM from Chinese breams developed in our laboratory, at a dose of 20 mg per kg of body weight in 100 μL of PBS before the viral challenge. After 24 h, mice were challenged with three different H3N2 strains. Mice were observed daily to monitor body weight and clinical symptoms for up to 14 days.

Three mice from each subgroup were euthanized humanely according to a pre-designated schedule. At 2, 4, 6, 10 and 14 days post-infection (dpi), blood samples and tissues including heart, spleen, lung, brain and intestine, as well as feces were collected. Virus shedding was detected by screening fecal samples. Detection of viral RNA was used to determine tissue distribution and virus shedding. Tissues and feces were homogenized in lysates at a ratio of 1:1 (g/mL), respectively, centrifuged at 10 000 × g for 30 min, and the supernatants were collected for the extraction of viral RNA using the Virus Nucleic Acid Extraction Kit II (Geneaid, Taiwan). All tissues collected above, including blood, were used for virus titration; the lung, brain and heart were also used for histological and immunohistochemical analysis at 6 dpi.

The CIRDC is an important disease that impacts on dogs, especially puppies or

immunosuppressed dogs, and is frequently associated with viral infections. It has gained

attention recently, because many viruses have been discovered and co-infections with

multiple pathogens are often fatal. Thus, the development of diagnostic tools for

CIRDC-associated virus detection is necessary to enhance the diagnosis coverage. In this

study, multiplex RT-PCR and multiplex PCR for the detection of CIRDC-associated RNA and DNA

viruses, respectively, were developed and compared with conventional methods. Both developed

multiplex PCRs could detect several viruses associated with CIRDC efficiently. The two

multiplex PCRs gave similar results equivalent to that obtained from the conventional

simplex PCRs that could only detect one pathogen per reaction and so required six separate

reactions per sample. Nested amplification was performed for CRCoV detection in order to

increase the sensitivity of detection (Poovorawan, personal communication). Although

multiplex PCR has been developed previously to detect several pathogens of CIRDC, such as

CIV, CDV and CRCoV, its application remained

limited because of the narrow range of viruses covered, with other CIRDC-associated viruses

being neither detected nor ruled out. Thus, our study might provide a novel platform for

whole CIRDC-virus detection.

The overall sensitivity of the multiplex RT-PCR and multiplex PCR was more than 90% and

87%, respectively, compared to their simplex counterparts. However, the detection of CRCoV

was modified as a hemi-nested RT-PCR to increase its sensitivity. The false negative

reactions when performing multiplex PCRs in this study might be resulted from the selection

of the single optimized Ta for several primer pairs and the low amount of particular target

genes. These suggested for the decreased

sensitivity of the developed multiplex PCRs. Moreover, there was 100% specificity in both

modalities for clinical sample detection. Thus, these platforms could likely be used

effectively in practice. Recently, some multiplex PCR assays were developed in order to

detect the CIRDC pathogens; however, the test

might be immature, because only CIV, CDV and CRCoV could be detected but not for others.

Thus, our study expanded the coverage of CIRDC virus detection. In an evaluation of the

commercially available three-antigen rapid test kit (CAdV-2, CIV and CDV), we found only CIV

detection showed an unexpected sensitivity and specificity. A previous study reported that

the developed multiplex RT-PCR for H3N2 CIV, CDV and CRCoV detection had an almost 100%

sensitivity and specificity compared with the conventional RT-PCR and rapid antigen test kit

[10]. In contrast, our study showed that the

CIV-positive samples by multiplex RT-PCR were negative when tested with the rapid antigen

test kit. This is consistent with reports that many rapid test kits might have a low

sensitivity to detect the influenza virus, but could still be suitable for rapid in-house

clinical applications [11, 15]. This reflects that the type of kit, viral copy number, duration of

storage, route of sample collection, and type or virus strain may all influence the test

results. Interestingly, in this study, about 70%

(71/102) of samples from the clinical respiratory illness dogs were found to have multiple

infections. This finding supports that symptomatically, the CIRDC is a complex disease,

which is mostly caused by co-infection with more than one pathogen. Recently, Jeoung

et al. (2013) used both NS and whole blood samples for CIRDC virus

detection, but found that only CDV (and not CIV and CRCoV) could be detected from the whole

blood samples. Correspondingly, respiratory

swabs have been reported to be appropriate samples for the detection of respiratory

pathogens [9, 16]. Thus, NS and OS served as appropriate sample sources in our study due to

their ease of and non-invasive sampling nature and that they lie on the viral shedding

routes. This study also suggested that the virus should be screened for in NS and OS, with

detection levels at each site depending on the type of virus. The CAdV-2 and CaHV-1 mostly

replicate in the lower respiratory tracts and shed via respiratory discharge, consisting

with our finding that they were mostly detected in the OS, even though NS could often detect

these viruses as well. However, the CAdV-2 primer pair used in this study was able to

amplify CAdV-1 DNA virus which also shows airborne transmission and replicates in tonsil

[3]. Therefore, the positive PCR reaction for canine

adenovirus could not discriminate between CAdV-1 and CAdV-2 in this study. Additionally,

CaHV-1 can be latent in various nerve ganglions, resulting in negative results from nucleic

acid-based CaHV-1 detection in respiratory discharges in non-symptomatic dogs.

In this study, 3 out of 15 vaccinated dogs receiving, at least once, combined vaccine

against CPIV, CDV and CAdV-2 showed PCR positive results for CIRDC virus detection (2 CDV

positive dogs and 1 CPIV positive dog). Even though live attenuated vaccines can give false

positive results with molecular testing, it is essential to discriminate between wild-type

infection and recent vaccination for the prevention of false positivity in the future.

This study documented CaHV-1 and CRCoV circulation in Thailand for the first time. In 2012,

CIV H3N2 was discovered in Thailand from dogs with flu-like symptoms. Here, CIV and CRCoV were the most frequently detected viruses in

CIRDC-infected dogs, suggesting that the viruses might spread rapidly. These viruses were

not only found in single infections, but they were also found as co-infections together or

with other viruses.

This study also exhibited a higher level of infections compared with a previous report

[15], although this might be caused by the

different timing of sample collection, population size and locations. However, it has

previously been reported that infection with CRCoV and CPIV might facilitate or initiate the

disease and, subsequently, enhance the entry of other pathogens, so the prevalence of infected dogs is then increased. Moreover, we

found that the dogs that were infected with CIV, CPIV, CDV and CRCoV showed a greater

severity of clinical symptoms, such as marked bronchopneumonia and sudden death (data not

shown). This finding is consistent with other investigations suggesting that co-infections

might augment the severity of clinical symptoms [7,

16]. Thus, advanced genetic-based detection

methods, such as multiplex PCR assays, are considered as an alternative diagnostic platform

for a panel of suspected CIRDC causing viruses with a high sensitivity and specificity.

Because of the cost benefit and practical usage, the developed multiplex PCR assays are

suitable for a screening test for disease diagnosis, quarantine and prevention measures,

especially in developing countries.

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.

Nine canine diarrheal samples from an unexplained outbreak of diarrhea were analyzed by viral metagenomics using three pools of three diarrhea samples each. Based on the BLASTx results, one of the three pools showed the presence of viral sequences most closely related to different chapparvoviruses reported from different vertebrates (0.05% of all reads). Other eukaryotic viral sequences observed were from Gyrovirus 4 (0.0003% of all reads), which has been reported in both chicken meat and human stool, indicating that it likely represents a dietary contaminant, and Torque teno canis virus (0.002% of reads), a common commensal canine blood virus.

Using de novo assembly and PCR paired with Sanger sequencing, a near complete genome of 4,123 bases containing the two main open reading frames of chapparvoviruses was generated (Figure 1, panel A). The available genome consisted of a 516 bases partial 5’UTR followed by an ORF encoding a 663 aa non-structural protein (NS) possessing the ATP binding Walker loop motif GPSNTGKS followed by a second ORF encoding a 505 aa viral capsid (VP) finishing with a 108 bases partial 3’UTR (Figure 1, panel A). When NS1 and VP1 proteins were compared to all available parvovirus sequences, the closest relative was from a Cameroonian fruit bat chapparvovirus (MG693107.1) with an amino acid identity of 61 and 63% respectively (Table S1). A 210 amino acid ORF that is missing a start codon and is overlapping the NS1 ORF was also detected showing 57% identity to its homologue protein in mouse kidney parvovirus (AXX39021) (Figure 1, panel A). This NP ORF is widely conserved among chapparvoviruses. The 5’ UTR DNA sequence was 68% identical to that of the bat parvovirus sequence (MG693107.1)). The virus was named cachavirus (canine chapparvovirus) strain 1A (CachaV-1A).

Distance matrices of the NS1 showed that the cachavirus is sufficiently divergent based on ICTV criteria (members of same species showing >85% NS1 identity) to qualify as a member of a tentative new species Carnivore chapparvovirus species 1 in the proposed Chapparvovirus genus (Table S1). A phylogenetic analysis of the NS1 ORF confirms its closest currently known relative is from a Cameroonian fruit bat (Figure 1, panel B).

Using a nested PCR, the other 8 samples were tested for the presence of this virus which was detected in a second diarrheic sample from that outbreak.

A larger set of canine fecal samples were then tested using a real-time PCR assay. Of 2,053 fecal samples tested, a total of 80 were positive (Table 1). Fecal sample submissions from the same time frame as the outbreak (Sept-Oct 2017) were tested in order to determine the prevalence of CachaV-1 during that time. Healthy samples from fecal flotation samples submitted in 2018 for preventive care screening were available. A second set of diarrhea samples that were collected during the same time frame as the healthy samples was also analyzed to check for differences in prevalence across time, as was a set of 83 bloody diarrhea samples.

Three stool samples out of 203 healthy animals tested positive, 32 were positive out of 802 diarrhea submissions from September to October of 2017, and 45 were positive out of 965 diarrhea submissions from September to October of 2018. None of the 83 bloody diarrhea samples tested were positive (Table 1). When the fraction of PCR positive fecal samples was compared between the healthy animals (1.47% positive) and those with diarrhea, a statistically significant difference (p < 0.05) could be detected with the 965 diarrhea cases from 2018 (4.66% positive; p = 0.037), but not with the 803 diarrhea cases collected in 2017 (4.0% positive; p = 0.08). When 2017 and 2018 diarrhea samples were combined (4.35% positive) and compared to the healthy group (1.47% positive), we measured a p-value of 0.05.

Cachavirus viral load as reflected by the Ct value of the real-time PCR were low across all four cohorts, with Ct values ranging from 29 to 39 with an average value of 36 for all positive groups. The five dog samples with the lowest Ct values (highest viral load) were then analyzed by viral metagenomics. All five samples yielded cachavirus reads, but one yielded a near complete genome (cachavirus [1B]). This sample also yielded 0.001% reads that were related to anelloviruses. None of the other four animals showed the presence of other known mammalian viruses. The cachavirus-1B genome showed 98% overall nucleotide identity with the index IDEXX-1A strain. The NS1 and VP encoded protein showed 99 % identity.

Poor surveillance and diagnosis capacity means that (1) data is insufficient to demonstrate disease burden and motivate policy-makers, and (2) impacts of control efforts cannot be evaluated.

Considerable progress has been made in the development of simple and inexpensive techniques for sample preservation and rapid post-mortem diagnosis suitable for laboratories with limited storage and/or diagnostic resources with potential to increase in-country capabilities for surveillance. A new direct rapid immunohistochemical test (dRIT) requires only light microscopes, which are widely available. The test is simple and can be performed by a range of operators if appropriate training is provided. Field evaluation studies in Africa demonstrated that this assay has characteristics equivalent to those of the direct fluorescent antibody (DFA) test, the global standard for rabies diagnosis, including excellent performance on glycerolated field brain material,[73], the preservative of choice under field conditions,[75]. Other simple field-diagnostics that allow rapid screening, including enzyme immunoassays, dot blot enzyme immunoassays and lateral-flow immunodiagnostic test kits,[79] are being evaluated. These tools offer hope of extending diagnostic capacity in resource-limited settings.

Animal-bite injury data from hospitals are an easily accessible source of epidemiological information and have been verified as reliable indicators of animal rabies incidence and human exposures,[14]. Furthermore, increasing availability of communication infrastructure through mobile phone network access in remote areas could enhance surveillance by allowing real-time reporting.

Data were collected and analyzed using MS Excel 2010 and SPSS Statics v20.0 software. Weight loss, viral titers, cytokine levels and histological score were analyzed by analysis of variance (ANOVA), followed by Turkey’s multiple comparison test with P < 0.05 considered to be a significant difference, while P < 0.01 was considered to be statistically extremely significant.