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The proportion of CPV-2-positive unvaccinated dogs varies between 32.63 and 84.98%, whereas CPV-2 positive vaccinated dogs (vaccinated at least once) varies between 15.02 and 48.42% (Figure 7). Both vaccinated and unvaccinated dogs can be affected by CPV-2 (60). The positive rates of CPV in unvaccinated dogs were significantly higher than those in vaccinated animals (41–43, 47, 50, 55, 56, 58, 60). The above studies show that vaccination is vitally important for the prevention and control of CPV-2. In China, insufficient attention is often paid to dogs, and therefore, individuals have not yet realized the importance of immunizing their pets. This in turn leads to a large proportion of unimmunized sick dogs. In conclusion, the key to preventing canine parvovirus is to establish a timely and functional immune response.
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.
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.
In the present study, we verified the frequency of five canine viral enteropathogens in a dog population with low vaccination coverage where CPV-2 was the most frequently detected virus (Table 2). The data presented herein agree with previous studies where CPV-2 is reported as the most common cause of severe diarrhea in puppies.7, 15, 16, 22 However, the frequency of CPV-2 found in our study (54.3%) was higher when compared with the ranges reported in other studies (16–48.7%).7, 15, 16, 22 The CPV-2 prevalence varies between studies, depending on the inclusion criteria for participation. However, since vaccination against canine pathogens other than rabies is not mandatory in Brazil, the vaccine coverage for these viruses must be drastically lower, which contributes to the high rate of detection observed in our study.
CCoV is generally the second most common viral agent detected in diarrheic dogs,7, 15, 16, 22 but in the present study, CDV had a higher frequency of detection (Table 2). However, the frequency of detection observed for CCoV was still higher than that reported other studies.7, 15, 16, 22 Some CCoV strains can cause severe diarrhea and intestinal damage indistinguishable from those caused by CPV-2.6
CDV is endemic to Brazil8, 23 but not to the regions sampled in previous studies.7, 16, 22 This could explain the high rates of CDV infection detected in the present study. Moreover, CDV causes a multisystemic disease with immunodepression that can favor infection by other pathogens including other diarrhea-associated viruses.24
CRV and CAdV were also identified in 8.2 and 4.9% of the dogs tested, respectively. Both viral agents are linked to diarrhea and vomiting13 and should not be excluded in the diagnosis of canine diarrhea. Moreover, CRV is a zoonotic pathogen, and its frequency in dogs needs to be determined to better assess the risk of infection in humans.
The samples analyzed in the present study were obtained by convenience from veterinary clinics which could bias information regarding sanity. Nevertheless, CPV-2 remained as the most frequent viral agent in the different dog groups (Table 1). The only exception was in dogs apparently healthy where CCoV and CDV were more frequent than CPV-2 but with no statistical significance.
More than one-third of the dogs tested for CPV-2, CCoV, CDV, CRV and CAdV were positive for co-infections (Table 2). No significant difference in co-infection rates were observed between the groups regarding clinical signs and vaccination status (Table 3). The occurrence of co-infection can increase the pathogenicity of the disease since these viral agents can act as immunosuppressants.12, 24, 25 There is a lack of studies searching for multiple viral pathogens in dogs, which could uncover the real etiology and interactions in the clinics. Moreover, a single search for the more common pathogens can reveal the real epidemiology of less common viral enteropathogens. Canine diarrhea can have other etiologies such as bacteria and parasites that are frequently reported.15, 16 Despite CPV-2 being the most frequently detected diarrhea-associated viral pathogen in the present study, a high degree of co-infections was observed. These co-infection rates reinforce that the search for more common individual pathogens could uncover the real epidemiology of less common viral enteropathogens.
In the present study, stool samples from dogs were evaluated for the presence of viral pathogens, and CPV-2 was found to be the most common. In contrast with previous studies, CDV was the second most common viral agent detected, probably since it is endemic to Brazil and not to the regions sampled in other studies. Moreover, the high frequency of co-infected dogs that was observed can increase the pathogenicity of the disease. The data presented herein can improve the clinical knowledge in regions with low vaccine coverage and highlight the need to improve the methods of controlling infectious diseases in domestic dogs.
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.
No conflicts of interest have been declared.
Despite the frequent occurrence of Canine Adenovirus worldwide, in Turkey, the only notified cases of Canine Adenovirus (CAV) infection are those reported by Okuyan and Gür and Acar. The antibody prevalence of the infection is reported to vary between 30 and 82% worldwide. Gür and Acar reported that out of 94 dogs belonging to the Kangal, Turkish Greyhound, and Akbaş breeds, which were sampled in Konya and Eskişehir provinces, 82 (82.7%) were positive for CAV antibodies.
Although, in most cases, CAV-1 and CAV-2 infections are not difficult to discriminate clinically from each other, they have the same morphological features under the electron microscope and the same cytopathogenic effects on cell cultures. There have been reports that CAV-2 can also infect the intestinal tract, one of the major target organs for CAV-1 [2, 19]. Diagnosis of CAV infections is usually based on serological tests, virus isolation, and negative staining.
In this study, the blood samples which are inoculated into cells were examined by direct immunofluorescence test for virus isolation. Viruses were not isolated from blood samples by direct immunofluorescence test. The adenovirus replicates in the lymph tissue and then spreads into the bloodstream. The replication reaches peak levels in 3–6 days after infection. Viral load decreases rapidly with respect to antibody production and longer CAV-2 cannot be isolated after 9 days [9, 20]. In the present study, the reason for unavailability of virus isolation may be due to the time of sampling.
The CAV-2 is a highly contagious viral agent that is incriminated in canine respiratory tract disease, particularly in young dogs kept in a crowded environment, such as pet stores, boarding kennels, and veterinary hospitals. This classical syndrome is commonly referred to as kennel cough. Infection with CAV-2 is generally transient and seldom fatal, unless it is complicated with a secondary bacterial bronchopneumonia [21, 22].
As adenoviruses do not have a lipid envelope, they are very resistant to environmental conditions and maintain their viability for an extended period in the external environment. Animals that recover from adenoviral infection continue to shed the virus for an extended period of time, and it has been even reported that vaccinated animals also shed the virus. Therefore, the prevalence of CAV infection has been reported to be rather high in dog shelters that lack reliable vaccination records.
CAV-1, which causes infectious canine hepatitis (ICH), is eliminated from the body of infected animals by saliva, urine, and faeces and is transmitted to susceptible animals by direct contact with contaminated material. In young animals, CAV-1 causes serious clinical symptoms, including anorexia, ataxia, and paralysis, which may result in death; and compared with older animals, the clinical course of the infection is more severe in the young. In the present study, antibody prevalence was found to be higher in animals aged 2 years and above. In this study, out of the 60 animals below 2 years of age, 23 (38%) and out of the 128 animals aged 2 years and above, 80 (62.5%) were confirmed to be positive for CAV antibodies. In particular, puppies below the age of 1, even if equipped with passive immunity through maternal antibodies, are not able to be protected against CAV infection when exposed, and mortalities may occur. Clinical ICH infection is more severe in young canids, as compared with adults.
The prevalence of the disease being higher in older animals that survive could be attributed to the high mortality in the young. In this study, few animals below 1 year of age were able to be sampled. Of the very few that were sampled, only 1 (a 2-month-old puppy) was determined to be positive for CAV antibodies. It was considered that this puppy had been protected by maternal immunity in early life. The remaining animals either could not have been exposed to the virus during life or could have suffered from low antibody levels in early life. No information was able to be obtained on whether the other animals survived.
In a study conducted in jackals in California, Cypher et al. determined that the prevalence of CAV antibodies was higher in adult animals. Higher CAV antibody prevalence among older age classes may be a function of greater mortality among pups resulting in a lower proportion of seropositive survivors in younger age classes [25, 26].
In Turkey, dogs both in rural areas and in dog shelters are not able to be regularly vaccinated against CAV-1 and CAV-2. In the present study, dogs that were admitted to the Internal Medicine Clinic of Selcuk University, Faculty of Veterinary Medicine, with complaints including fever (above 40°C), coughing, nasal changes, mucopurulent conjunctivitis, listlessness, inappetence, weight loss, pain and sensitivity of the abdominal region, vomiting, and diarrhea were sampled. Of the 111 dogs, which were admitted to the clinic, 37 were owned animals tended to by their owners. Twelve owned animals which were declared to have been vaccinated according to anamnesis were found to be negative for CAV antibodies. No reliable information was able to be accessed for the vaccination status of the remaining animals. Similarly, no reliable information was available for the vaccination status of the 77 dogs, which were sampled at the dog shelters in Isparta and Burdur provinces. The blood parameters of the animals that were admitted to the Internal Medicine Clinic of Selcuk University, Faculty of Veterinary Medicine, revealed the presence of lymphocytosis in some of the animals (5.84–12.61 m/mm3, reference range for dogs 0.6–5.1 m/mm3) and leucopenia (2.2–4.6 m/mm3, reference range for dogs 6.0–17.0 m/mm3) and anemia (0.99–5.24 m/mm3, reference range for dogs 5.5–8.5 m/mm3) in some others. Based on these findings, viral infection was suspected and the animals were tested for CAV. Two of the dogs exhibited photophobia and corneal opacity. In these 2 animals, although the presence of CAV viral antigen was not detected, the presence of CAV antibodies was confirmed. It is considered that these 2 unvaccinated animals were exposed to the disease and managed to survive the infection. For, in general, 7–10 days after being exposed to CAV, the acute signs of infection are replaced by corneal edema, which presents with a blue and rather opalescent appearance of the cornea. This appearance, which is generally observed in the convalescence period, disappears spontaneously. In some cases of mild infection, no clinical symptom is observed other than corneal edema. Although, rarely, this clinical picture may also develop as a vaccination complication, this option was discarded as the 2 animals that presented with corneal edema were unvaccinated. This clinical picture, specifically referred to as “Hepatitis Blue Eye” is known to be caused by CAV-1. However, as early detection was not possible, the CAV antibodies were not able to be typed in the animals that presented with leukopenia and lymphopenia.
According to the vaccination schedule applied in Turkey, new-born puppies are vaccinated as from 2 months of age, 3 times at 21-day intervals with live multivalent vaccines (CAV-2, Distemper, Parvovirus, Parainfluenza, and Leptospira). Literature reports are available, which suggest that canine adenovirus antibodies produced against the two different CAV types provide cross-protection, owing to the antigenic similarity of CAV-1 and CAV-2.
In the present study, apart from the correlation between age and antibody prevalence, the correlation between sex and antibody prevalence was also investigated. Accordingly, it was determined that antibody prevalence was higher in females (41%) in comparison with males (36%). Due to the number of female animals sampled in this study being greater than the number of sampled males, it is considered that the difference observed in antibody prevalence for sex is not significant (P > 0.05). On the other hand, in previously conducted studies, the correlation of sex with antibody prevalence was neither not investigated nor found to be statistically insignificant. Therefore, a comparative assessment was not able to be made in this study.
Of the 188 animals sampled in the present study, the majority were unvaccinated dogs housed at shelters. In view of the animals that had been admitted to the internal medicine clinic of Selcuk University being vaccinated dogs, the antibody presence confirmed in these animals was considered as an indicator of the vaccination schedule having been properly applied in these animals. On the other hand, the antibody prevalence detected in the unvaccinated animals sampled at the dog shelters in Isparta and Burdur provinces was considered as an indicator of the presence of CAV infection in these dog shelters. This study clearly demonstrates the high prevalence of CAV infection in dogs, which live in groups and are not vaccinated on a regular basis. It is considered that regular vaccination would provide protection against the disease for a certain time period in dogs, and in particular in puppies, which live in groups under unfavorable conditions. Furthermore, as the transmission of the CAV occurs by environmental contamination (contact with infected faeces, urine, etc.), both the maintenance of hygiene conditions and the prevention of contact among dogs are of great importance in the control of the disease.
Although most FHV-1 strains produce a relatively uniform disease seen primarily in the respiratory tract, pancreatitis and generalized disease may be seen occasionally in debilitated animals or in neonatal kittens. The FHV-1 described in this study, was isolated from a captive tiger that exhibited respiratory signs that were suspected to be due to rhinotracheitis. By PCR/RT-PCR, the only virus detected in the trachea homogenates was FHV-1, which was confirmed afterwards by virus isolation, the TEM examination of cell cultures showing CPE, and a challenge experiment in cats. Furthermore, the full genome of the virus isolated in cell culture is being sequenced, and the obtained sequences are 90%–100% homologous with that of PCR products either for TK gene or for gB gene. Challenged cats exhibited uniform clinical symptoms and shed FHV-1 virus. Other agents causing respiratory disease were excluded as possible pathogens.
The tiger was born in Shenzhen Wildlife Zoo, and had chances to contact stray cats and other felids during its lifetime. On its infection, the authors supposed two possible sources. One was that the tiger got the virus in captivity perhaps due to stray cats in the zoo, because FHV-1 was shed in ocular, nasal, and oral secretions, and transmission was primarily by direct contact with an infected cat. The other was an endogenous infection originating from a latent viral infection. As with other alphaherpesviruses, latency is common, and periodic viral reactivation is sometimes associated with the reoccurrence of clinical signs. To validate the first supposition, a retrospective investigation was conducted after the virus isolation and identification. Although negative PCR results were found for the swab samples collected from stray cats and captive leopards and tigers, the contagious source could not be easily obviated. It is very much regrettable that neither prior sera nor post mortem sera were stored enabling the seroconversion analysis, making our second supposition to be theoretic.
FHV-1 is relatively fragile in the environment and is highly susceptible to the effects of common disinfectants. Even though negative PCR results of the retrospective survey indicated that widespread infection was unlikely to occur in the zoo, serological data of felid animals would be obtained in the next research to assess the epidemic risk for tigers better and more precisely.
Currently available evidence indicates that the host range of FHV-1 includes several members of the Felid such as cheetahs, lions, and wild and domestic cats, but to our knowledge, FHV-1 infection has not been reported in tigers previously. This report describes the first occurrence of FHV-1 in a South China tiger in China and extends the species range, confirming that this virus poses a risk to this species.
All twelve dogs that were tested for CPV at the initial presentation were PCR-negative (Table 5). Vector-borne infections were detected in 4 dogs (31 %, Table 5): infection with Babesia spp. was detected in Dogs 3 and 8; infection with L. infantum was diagnosed in Dog 4 and infection with Dirofilaria immitis was found in Dog 13. Dog 13, which tested positive in D. immitis antigen and Knott tests, had received a certificate from a laboratory in Budapest, Hungary, that stated a negative result in the Knott test in August 2013. None of the rescue dogs tested positive for Ehrlichia canis (Table 5).
The present study investigated the presence of respiratory viruses in dogs of three shelters in Rio Grande do Sul state, Brazil, through virus detection in nasal secretions via PCR. Considering the previous serological studies on canine respiratory viruses in Brazil,14, 15 the primary difference of the present study was the direct demonstration and identification of the viruses involved in CIRD.
Our results showed the occurrence of the main canine respiratory viruses in these shelters with varying frequencies and combinations of single and mixed infections. In shelter #1, 78% of the 74 samples were positive for at least one virus; CPIV was the most frequent agent (71% of the samples). CPIV was detected in single (30%) or in mixed infections and was associated with CAdV-2 (23%), CDV (4%), or both (14%). CDV and CAdV-2 were found in a high percentage of animals, especially in coinfections (Table 2). In shelters #2 and #3, unlike shelter #1, a small percentage of samples were positive for the virus and only in single infections. In shelter #2, CPIV was detected in 9% of the samples and CaHV-1 was detected in 6%. In shelter #3, 9% of the samples were positive for CAdV-2 and 1% for CDV (Table 2). The varying sanitary and nutritional conditions and the dog crowding/density of the respective shelters may explain the important differences in the rates of positive animals.
Shelter #1 had precarious nutritional and sanitary conditions, poor infrastructure and poor food quality (Fig. 1A). In shelter #2, the animals had a wide outdoors area in which to play and exercise; however, the dogs of varying ages had direct contact (Fig. 1B). Fig. 1C shows shelter #3 with individual dog houses and cages with a low population density and good sanitary conditions (approximately six dogs/cage). Factors associated with animal overcrowding, such as excessive noise, poor air quality and diet, in addition to bad kennel cleaning, may cause stress, which may in turn promote the establishment and dissemination of viral infections.23, 24, 25 Thus, the poor sanitary and nutritional conditions of shelter #1 may have favored the high rate of respiratory viruses.
In this shelter, CDV, CPIV and CAdV-2 were detected in single or mixed infections, corresponding to 78% of the positive dogs. CPIV was detected in 71% of the samples, of which 30% were single infections and 41% were associated with CAdV-2 and/or CDV. CPIV is considered the primary virus involved in respiratory disease in dogs,2, 7, 26, 27, 28, 29 and has been most frequently reported in conditions of high animal density.2 CPIV infection produces pathology in the tracheal epithelium15 and favors secondary respiratory infections by other pathogens such as CAdV-2.6
In shelter #1, CDV was detected only associated with CAdV-2 and/or CPIV, corresponding to 21% of the positive samples. CDV replication occurs in epithelial cells and macrophages of the upper respiratory system, pharynx and tonsils, followed by lymph node infection and systemic dissemination that can evolve to multisystem disease and immunosuppression.30 For this reason, bacterial secondary infections are often detected in dogs with distemper in addition to coinfection by other viruses, such as CAdV-2 and CaHV-1.5, 31, 32
CAdV-2 detection in 45% of the samples from shelter #1 may have been influenced by the high CPIV and CDV infections in the kennel because CPIV promotes primary lesions in the tracheal epithelium15 and CDV induces immunosuppression.30 Additionally, adenoviruses are highly resistant in environmental conditions and remain viable in the environment for an extended duration, thereby favoring dissemination among animals.6 Notably, a high prevalence of CAdV in dog populations has been reported in shelters without a history of vaccination.33, 34
An investigation of respiratory viruses in dogs in Germany analyzed 58 samples of shelter animals with and without respiratory signs and detected 22.4% (13/58) to be positive for CPIV and one positive for CAdV-2 and CPIV.29 A similar study performed in Germany examined 68 nasal swabs of domestic dogs28; in this study, 7.4% (5/68) of the samples were positive for CPIV, 2.9% (2/68) for CAdV-2 and 1.5% (1/68) for CDV. Despite varying frequencies, these studies reported CPIV to be the most frequent respiratory virus in dogs, followed by CAdV-2 and CDV.
There are few reports of direct diagnosis of respiratory viruses in dogs; however, some serological studies have been performed in Brazil.14, 15 In southern Brazil, a serological investigation of 817 domestic dogs without a vaccination history showed that 43% of the animals were seropositive to CAdV and 27.3% to CDV.14 A similar study was conducted in a population of 173 dogs in shelters, also from the RS state, in which antibodies to CPIV and CDV were detected in 51.4% and 4.1–9.3% of the samples, respectively.15 These studies showed that respiratory viruses circulate among domestic and shelter dogs in southern Brazil in varying combinations and prevalences that likely reflect environmental and epidemiological differences between regions and dog populations.
In shelters #2 and #3, the respiratory viruses were detected only in single infections with 14% of infections caused by CPIV or CaHV-1 (shelter #2) and 9% by CDV or CAdV-2 (shelter #3). CaHV-1 was detected in samples collected only from the dogs of shelter #2, corresponding to 6% of the collected samples. Although CaHV-1 may cause respiratory disease, the infection has also been associated with other clinical outcomes, including reproductive disease.35, 36 Due to the ability of CaHV-1 to remain latent in the host, its diagnosis in dog populations should preferentially be performed via serological testing.37, 38, 39, 40 In this sense, we detected positive serology for CaHV-1 in 7 out of 8 dogs in shelter #1 (not shown). A two-year longitudinal investigation in a shelter in the United States involving 211 necropsied dogs showed CaHV-1 involvement in 12.8% and 9.6% of trachea and lung samples, respectively, reinforcing the involvement of CaHV-1 in respiratory disease in dogs.2
The identity of the sequenced matrix of the shelter samples with sequences deposited in GenBank revealed 96 to 99% identity (KU341102, KU341103, KU341104 and KU341105) with the N gene (AJ009656.1, JF965338.1, KC590511.1, AY386315, JF965338.1, KF856711.1, KP738610.1 and JN381190.1) of CDV, 98 to 99% identity (KU341106, KU341107, KU341108, KU341109) with the N gene (EF543648.1, EF546391.1 and AY581307.1) of CPIV, 97–100% identity (KU315333, KU315334, KU315335, KU315336 and KU315337) with the E3 gene (KF676978.1, JX416842.1, JX416842.1, U77082.1 and GQ915311.1) of CAdV-2, and 99–100% identity (KU315338 and KU315339) with the gB gene (KJ946357.1, KJ676506.1, JX908000.1, HQ846625.1, AF361073.1 and AY582737.1) of CaHV-1.
Thus, the results obtained in this research showed that respiratory virus infections (CPIV, CDV, CAdV-2 and CaHV-1) are common in dogs housed in public shelters. The frequency and dissemination of these viral infections appear to be related to a high population density and poor sanitary and nutritional conditions. These results also indicate the need for control/prevention measures, such as vaccination and good environmental conditions, to minimize these infections in shelter dogs. CPIV infection appears to play an especially predominant role in winter respiratory infections in dog shelters and warrants further preventive measures.
Infectious causes of respiratory disease are common in dogs; canine distemper virus, adenovirus 2, parainfluenza, influenza, herpesvirus, pneumovirus, respiratory coronavirus, Bordetella bronchiseptica, various Mycoplasma spp., and Streptococcus equi var. zooepidemicus are documented causes.1 Molecular diagnostic assays to detect viral and bacterial pathogens are available for these agents. In the United States, modified live vaccines (MLVx) for intranasal (IN) administration are currently available for adenovirus 2, B. bronchiseptica, and parainfluenza. These vaccines do not induce sterilizing immunity, and vaccinated dogs can still develop clinical signs of disease if exposed to virulent strains of the organisms.2 It is currently unknown if IN administration of MLVx against these agents results in positive molecular diagnostic assay results in dogs without previous vaccination. If transient positive molecular diagnostic assay results are common after vaccination, the positive predictive value of the diagnostic assays to predict disease caused by these agents in dogs would be decreased.
The purpose of this study was to determine the impact of administration of a single IN dose of a commercially available MLVx adenovirus 2, B. bronchiseptica, and parainfluenza containing vaccine,1 included as part of a facility standard initial vaccination series with a parenteral administration of MLVx containing adenovirus 2, canine distemper virus, and parvovirus, on the results of a commercially available polymerase chain reaction (PCR) panel that amplifies the RNA or DNA of the agents.2
The study was completed with Institutional Animal Care and Use approval. Beagle puppies housed at a commercial breeding facility were used.3 The puppies were housed in a closed facility without contact with other dogs and staff members followed facility barrier precautions over the course of the study. A sterile cotton swab was gently rubbed at the entrance to the external nares, and a second swab gently rubbed against the mucosa of the oropharynx in nonsedated puppies. The swabs were stored separately at 4°C in sterile plastic tubes and stored until shipped by overnight express on cold packs for performance of the molecular assays.2
A total of 12 puppies were screened twice as described, 1 week apart, and all were negative for nucleic acids of the target organisms. Eight puppies were randomly selected for the study and housed in a separate room at the breeding facility for the duration of the study. The puppies were approximately 9 weeks of age when samples were collected on Day 0 before the SQ administration of a MLVx containing adenovirus 2, canine distemper virus, and parvovirus4 and the IN administration of a MLVx1 containing adenovirus 2, B. bronchiseptica, and parainfluenza following manufacturer's instructions (approximately ½ mL per nares). Nasal and pharyngeal swabs were then collected on days 1, 2, 3, 4, 5, 6, 7, 10, 14, 17, 21, 24, and 28 for molecular analysis.2
Sneezing or coughing which have been associated with IN MLVx administration was not noted over the course of the study. Adverse effects associated with the collection of the nasal and oropharyngeal swabs were not noted. At the time the study was performed, the PCR panel utilized also included primers for canine distemper virus RNA; and none of the samples collected over the course of the study were positive. In contrast, nucleic acids of adenovirus 2, B. bronchiseptica, and parainfluenza were amplified from both sampling sites, from all 8 puppies, on multiple days after vaccine administration (Table 1). Because adenovirus 2 was administered in both vaccine types, source of that virus cannot be determined. Increasing numbers of positive samples after vaccination suggest local replication of the vaccinal strains. Decreasing numbers of positive samples over time suggest immune responses inhibiting organism replication. However, quantitative PCR assays normalized to total DNA/RNA on the swab would be needed to confirm or deny these hypotheses. The PCR laboratory adheres to standard operating procedures including use of positive and negative controls thus erroneous results are unlikely.
Agents considered most common for kennel cough syndrome include canine distemper virus, adenovirus 2, parainfluenza, and B. bronchiseptica. However, emerging pathogens include influenza, herpesvirus, respiratory coronavirus, pantropic coronavirus, pneumovirus, and others.1 All of these agents, as well as S. equi var. zooepidemicus and Mycoplasma spp., have been identified as causes of canine infectious respiratory disease. Determination of the agent is important for targeting treatment, particularly for dogs who fail to respond to standard treatment recommendations.2 In animal shelter environments, agent identification is critical for outbreak control and individual case management.3
Bacterial and viral shedding postvaccine administration complicates diagnostic testing and treatment. This is especially problematic in shelter environments as dogs are routinely vaccinated on intake. Viral shedding after vaccination has been detected in cats,4 people,5 cattle,6 pigs,7 and dogs.8 A vaccine strain of B. bronchiseptica was detected via nasal culture up to 4 weeks after IN vaccination of 2–week‐old puppies.9
Commercially available respiratory PCR panels are a relatively cost and time effective diagnostic method for identifying multiple respiratory pathogens. However, amplification of nucleic acids may inherently lead to inaccurate clinical diagnosis because small amounts can be amplified from some animals even though the agent may not be present in sufficient quantity to cause disease. In this study, nucleic acids of all 3 organisms contained in the IN vaccine were amplified from both sites on multiple days via PCR, although no clinical signs of respiratory disease were observed. Thus, interpretation of PCR panel results for diagnoses should include consideration of recent vaccination status and clinical signs of disease. Use of quantitative PCR and wild‐type sequence differences may be able to differentiate between vaccine and pathogenic agent shedding and may be used diagnostically in the future.
Real‐time reverse transcriptase PCR has been used to amplify canine distemper virus RNA in blood, urine, and conjunctival swabs after administration of SQ MLVx.10 In this study, the PCR panel did not amplify distemper virus RNA from nasal or pharyngeal swabs. Further studies are needed to determine whether the negative result is because this strain of vaccine virus does not reach the nasal or pharyngeal tissues or was present at levels below the detectable limit of the assay used.
Canine 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. 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, 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.
Over the past few years, efforts have been made towards a better understanding of the health status of animal populations, particularly regarding viral infections. Due to their high mutation rate and replication strategies, viruses are responsible for recently recognized emerging diseases, posing a danger not only to domestic and wild animals, but also to humans.
The high density of domestic and stray animals in urban areas enables viral dissemination and maintenance in these populations. Consequently, these animals can act as reservoirs of diseases, with the possibility of transmission to wildlife populations through occasional contact.
Canine parvovirus (CPV) was first identified in the late 1970s and was responsible for severe hemorrhagic gastroenteritis and myocarditis in dogs. Parvoviruses are extremely stable in the environment and indirect transmission assumes a critical role in spreading and maintenance of the virus in animal populations, especially in wild carnivores, in which contact rates between animals are lower. Shortly after its initial detection, CPV-2 was replaced by two antigenic variants, CPV-2a and CPV-2b and more recently a third variant was described CPV-2c.
Canine distemper virus (CDV) is the etiological agent of canine distemper, a highly contagious disease, responsible for high mortality rates in dogs worldwide. Sequence analysis of CDV strains originated in different geographical areas from several animal species, showed that the hemagglutinin gene has undergone a genetic drift according to the geographic location. Phylogenetic analysis based on this gene revealed the existence of at least nine strains in different geographical areas, namely America-1, America-2, Asia-1, Asia-2, Europe-1/South America 1, European wildlife, Arctic-like, South America 2 and Southern Africa.
Canine coronavirus (CCoV) causes a mild to moderate enteritis in dogs and its infection is characterized by high morbidity and low mortality. CCoV is transmitted by faecal-oral route and spreads rapidly through a group of susceptible animals. Stressful environments with large concentrations of animals and poor hygienic conditions, often seen in kennels, favour the development of this disease. Although a higher mortality rate is observed in animals with multiple infections with other pathogens such as CPV-2, canine adenovirus type 1 and CDV, CCoV represents per si a major infectious agent responsible for several epidemics.
Virological surveys are conducted throughout the world, allowing the detection and analysis of a large variety of viruses in different animal populations. In Cape Verde archipelago to our knowledge, no similar study had been conducted so far. In order to detect the presence of canine viruses on Maio island, samples collected from stray dogs from Vila do Maio were tested for canine parvovirus (CPV), canine distemper virus (CDV) and canine coronavirus (CCoV), to estimate the viral prevalence in this population and investigate the role of these animals in the maintenance and potential spread of common viral pathogens.
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).
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.
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–, 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
. 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–. 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.
Kapoor et al. reported for the first time the detection of CanineCV in the serum of healthy dogs in the USA. Over the last five years, CanineCV has been detected in Italy, Germany, China, Thailand, Taiwan and Brazil [3–6, 8, 12, 13]. In the present study, we report an outbreak of a fatal disease associated with the presence of CanineCV in Argentina.
Although virus isolation was not attempted since it was proved unsuccessful in previous reports, the full-length genome of strain UBA-Baires was determined, revealing close similarity to European viruses. We found only one unique mutation in the rep gene, but surprisingly, four unique aa substitutions in the Cap protein, after two independent experiments using different sequencing facilities. Very recently, nine major variable regions have been proposed to exist within the cap gene of CanineCV. Only one of the four mutations present in the cap gene of strain UBA-Baires is encompassed at these proposed locations (residue 95), suggesting that, until sequences from more extent geographical areas are analyzed, these variable regions need to be taken cautiously. Point mutations in the cap gene have been described for PCV2 and are a potential source of virus evolution, especially due to selective pressure caused by vaccination. In the case of CanineCV, where vaccination has not been used, the mechanisms behind these aa substitutions are unclear; virus variability under natural infection has been shown to be very complex for PCV-2. In addition, the relevance of these changes, if any, is difficult to be evaluated at this moment when it is still under investigation the role of CanineCV as a sole cause of disease.
In the case of PCV2, coinfection with other pathogens has been associated with different clinical outcomes. Special attention was given to coinfection with porcine parvovirus and the syndrome could be reproduced experimentally [26, 27]. Similarly, CanineCV has been found in most cases accompanying other viral pathogens. In hemorrhagic enteritis, it is often associated to CPV, but also canine distemper virus and canine coronavirus with coinfection rates of 100% [4, 6], 70–80% [8, 9, 11] and 50%. Only one report so far has found CanineCV as a primary agent. Case-control studies carried out in different population of dogs suggest a high association between diarrhea and CanineCV, but CanineCV is still found in feces and serum from healthy dogs [8–11]. Nevertheless, one study showed that mortality rate was higher when CanineCV was found together with CPV. Interestingly, in dogs with respiratory symptoms from Thailand, CanineCV was detected together with respiratory pathogens (canine influenza virus, canine parainfluenza virus, and canine respiratory coronavirus). In our case, all organs tested except for stomach, were also positive for CPV. Unfortunately, no histopathology was performed on these cases in order to confirm the presence of some of the lesions observed in previously described cases involving CanineCV, in particular vasculitis. Surprisingly, in our case, sick animals had been vaccinated against CPV. This phenomenon was also described in a breeding colony in the USA where two severe outbreaks of hemorrhagic gastroenteritis occurred among animals vaccinated against CPV. Again, the role of CanineCV in the context of vaccine efficacy against co-infecting viruses is still unclear and needs to be carefully analyzed. ISH studies carried out with clinical samples show that CanineCV infects mainly lymphoid tissues and cells [2, 5] suggesting that Infection with circovirus could cause immunosuppression and allow infection with CPV even in the case of vaccinated animals. Nevertheless, further experimental infections are necessary to elucidate the pathogenic involvement of CanineCV in disease pathogenesis.
To our knowledge, this is the first report of a complete genome sequence of CanineCV associated with disease from Latin America. A very recent paper published by Weber and col. characterizing the dog’s virome found CanineCV in the sera from seven healthy dogs from Brazil. Phylogenetic analysis using a partial sequence from Rep revealed that the Brazilian virus seemed to be related to strains from China and the USA, among others. The tree topology is somehow different form the one we present in this study based on the full-length genome, in which the Argentinian strain grouped closer to European viruses. Hopefully, additional sequences from South America will be soon deposited in GenBank allowing a more thorough analysis of the epidemiology and evolution of this emerging virus and will aid in the comprehension of its role in disease.
Records were only available for the specimens sampled in 2011. Of the 125 dogs, all them of undetermined or mixed breeds, 65 were females (52%) and 57 males (46%). For 3 dogs (2%) no data was registered regarding gender.
Diaharreic feaces were described for 4 animals (3%).Only two dogs had been vaccinated, both with Tetradog® vaccine and no information regarding vaccination of the rest of the animals was available (NA).
The percentage of positivity for CPV-DNA was very similar in the 2010 and 2011 sampling; 23/53 (43.3%) and 41/93 (44.1%, respectively). From the 88 sera sampling collected during 2011, 63 (71.6%) tested positive for CPV antibodies, with 10 animals included in the first ELISA Unit (EU) class (100–1000 EU), 29 in the second EU class (1000–10000 EU) and 24 in the third EU class (>10000 EU) (Tables 1 and 2).
Antibodies against CPV were detected in 20% of the animals aged less than 6 months (2/10), in 57.1% in dogs aged between 6 months and 1 year (8/14), in 87.5% in dogs with 1 to 2 years (14/16), in 85.3% in dogs with 2 to 5 years (29/34), in 75% in dogs with 5 to 7 years (6/8) and in 1/1 dog older than 7 years (Figure 1). The proportion of seropositive animals was significantly higher in older animals (p < 0.05). No differences were found between the seroprevalence and gender (p > 0.05). From the 56 samples that were tested for virology and serology, 46.6% (26/56) were positive for CPV-DNA and 64.3% (36/56) were seropositive. Out of the 26 dogs that were excreting the virus at the time of collection, 7 were seronegative for CPV specific IgG.
Regarding the 2010 samples tested for CDV-RNA (n = 53), 6 animals were positive (11.3%), of which 2 were also co-infected with CPV. All samples from 2011 were found CDV-RNA negative. As for serology, 45 of the 88 animals sampled during 2011 were seropositive for CDV (51.1%). Two groups were identified according to the antibody (Ab) titer: 1) low Ab titer (IIF values 1/20-1/40: n = 43 (96%)); and 2) medium Ab titer (IIF values 1/80-1/160: n = 2 (4%)).
Antibodies against CDV were detected in 30% of animals aged less than 6 months (3/10), in 50% of dogs aged between 6 months and 1 year (7/14), in 56.3% of dogs with 1 to 2 years (9/16), in 53% of dogs with 2 to 5 years (18/34), in 75% in dogs with 5 to 7 years (6/8) and in 1/1 dog older than 7 years (2011 samples) (Figure 2). Although there was a linear increase of seropositive animals with age, this association was not statistically significant (p > 0.05). The presence of antibodies was independent of gender (p > 0.05).
Only two samples, collected in each year of the survey, tested positive for CCoV-RNA, (2010 (1/53, 1.9%) and 2011(1/93,1.1%)).
Pet dogs play an important role in humans’ daily lives. Recently, the emergence of new pathogens and the continuous circulation of common etiological agents in dog populations have complicated canine diseases. Among these diseases, canine infectious respiratory diseases (CIRD) and viral enteritis pose notable threats to dog health.
CIRD are complex and include canine adenovirus type 2 (CAV-2), canine distemper virus (CDV), canine influenza virus (CIV), canine parainfluenza virus (CPIV), canine herpesvirus (CHV), canine reovirus, Bordetella bronchiseptica and other pathogenic agents [2–4]. Among these, CAV-2, CDV or CPIV have frequently been detected in dogs with CIRD, according to previous studies [5, 6]. Avian-origin H3N2 CIV has been detected in domestic dogs in South Korea and China since 2007 [7, 8]. H3N2 CIV is now circulating in dog populations in China, South Korea, Thailand, and even the United States [9–11]. Distinguishing these pathogens can be challenging, because dogs often show similar clinical signs of infection with these viruses, such as low-grade fever, nasal discharge and cough. These respiratory symptoms are flu-like and difficult to diagnose.
Canine viral enteritis is common in dogs with acute vomiting and diarrhea. Canine parvovirus (CPV) is one of the major viruses leading to acute gastroenteritis in dogs; CPV infection is characterized by fever, severe diarrhea and vomiting, with high morbidity. Puppies tend to be intolerant of CPV infection and have higher mortality than adult dogs because of myocarditis and dehydration [14, 15]. Canine coronavirus (CCoV) is characterized by high morbidity and low mortality. Dogs infected with CCoV alone are likely to have mild diarrhea, whereas the disease may be fatal when coinfection by CCoV and CPV, CDV or canine adenovirus type 1 (CAV-1) occurs [16, 17]. CAV-2 is associated with mild respiratory infection and episodic enteritis [18, 19]. Canine circovirus (CanineCV), a newly discovered mammalian circovirus, was first reported by Kapoor et al. in 2012. CanineCV has been detected in dogs with severe hemorrhagic diarrhea, and it is more common in puppies than in adults [21, 22]. Coinfection of CanineCV with other intestinal pathogens (CPV or CCoV) is closely related to the occurrence of intestinal diseases [23, 24]. Dogs with intestinal diseases are often infected with one or more viruses, and their clinical symptoms are similar [17, 25, 26], making clinical differential diagnosis difficult. To date, no multiplex PCR (mPCR) method has been developed to detect CanineCV and other enteropathogens.
An effective diagnostic tool is important for the prevention, control and treatment of CIRD and viral-enteritis-related viral diseases. Although many methods exist to detect CIRD and canine viral enteritis, most can detect only 2 or 3 pathogens, and the current lack of systematic and comprehensive detection methods makes diagnosis impractical and time consuming [4, 27, 28]. Because mPCR can simultaneously detect multiple pathogens in a timely and inexpensive manner, this technique has become increasingly popular. Therefore, in this study, two new mPCR methods were developed for the detection of canine respiratory viruses (CRV, including CAV-2, CDV, CIV and CPIV) and canine enteric viruses (CEV, including CAV-2, CanineCV, CCoV and CPV), and we indicated that the mPCR methods established here are simple and effective tools for detecting the viruses of interest.
Equine Rhinopneumonitis is caused by equine herpes virus Type 1 (EHV-1). It occurs in horses of all ages but is more common in horses less than three years old. Sporadic outbreaks come from inhalation of the virus particles. Following a respiratory infection, the virus can cause abortions. Death of the foetus occurs two weeks to four months after exposure to the virus, or during the last three months of pregnancy. Abortion storms have a sudden onset with no additional clinical signs. The virus also cause respiratory problems in foals, and infections near birth can produce weak foals that die within 24 hours.
IBR is a serious contagious herpes virus disease of cattle that can cause a variety of different disease syndromes, the most common of which is respiratory disease (pneumonia) and the genital form of the disease complex in both males and females (Infectious pustuloes vulvovaginitis). IBR is a commonly diagnosed viral cause of abortions in cattle. Abortions most commonly occur from 4 months to term, and may occur weeks after the disease has gone through the herd. A cow can also abort if she develops an infectious condition that does not directly affect the foetus.
Bats have become the subject of an increasing number of field-based epidemiological studies due to their association with zoonotic pathogens such as Ebola virus, Marburg virus, Nipah virus, Hendra virus, SARS coronavirus, and most recently a novel bat coronavirus in the Middle East– all of which cause mortality in humans,. It has been suggested that outbreaks of viruses within bat populations have been related to the waning of immunity in juvenile cohorts. Understanding the duration of maternal antibodies in pteropid bats (and the age of the bat) will help determine whether anti-henipavirus IgG in juveniles is maternally-derived rather than the result of viral exposure. Each of the two experiments presented here provide valuable data related to bat immunology, however, there were limitations to interpreting observed differences or similarities between the results from the two experiments since they each involved different bat species and different methodologies. Experiment 1 used a canarypox vectored canine distemper virus vaccine as a proxy for Nipah virus infection, which although it was the safest option, may not have generated the same results had we been able to follow pups born to P. hypomelanus dams naturally infected with NiV (as with HeV in Experiment 2). Since the completion of Experiment 1, a canarypox vectored Hendra virus vaccine has been developed, and this may serve as a better surrogate for future studies requiring lower biosafety conditions. We expect that the results from Experiment 2, which was based on a natural infection of a pteropid bat with Hendra virus, are more likely to be comparable to immune dynamics in closely related species infected with Nipah virus compared to those from Experiment 1, though both studies provided a controlled opportunity to measure immune system dynamics in key Henipavirus reservoir species.
Previous age-stratified serological studies of henipaviruses in pteropid bats have found that the sero-status of neonates matches their dam,,,. Plowright et al., described the annual occurrence of HeV spillover events in Australia as coinciding with the presence of a susceptible juvenile population of bats and estimated that maternal antibody had waned by approximately 6 months post-partum, coinciding with annual HeV spillover events. Similarly, distinct pulses of Marburg virus transmission in juvenile Rousettus aegyptiacus fruit bats at approximately 6 months post-partum have been reported. A wave of virus infection has also been detected in Myotis myotis bats approximately one month after parturition which the authors speculated may be associated with waning maternal antibody. A study of Nipah virus in captive P. vampyrus found maternal IgG to last approximately 14 months; however the exact age of the pups in that study was uncertain, and two of the four died before titers became negative. We found that the calculated half-life values of maternally derived antibody did not differ significantly between pups from vaccinated bats (Experiment 1) and naturally infected bats (Experiment 2). In experiment 2, four pups whose titers did not fall below the negative threshold during the period of measurement were omitted from calculation. However, if they had an endpoint and were included, the mean duration of immunity would have been lengthened, although it cannot be determined if this would have created a statistically significant difference from Experiment 1.
Bats in both experiments showed a similar duration of maternal antibodies between 7.5 and 8.5 months in Experiments 1 and 2 respectively. Duration of maternal immunity is influenced by multiple factors including the magnitude of the mother’s titer during gestation (which can be affected by vaccination vs. natural infection), the age of the neonate at parturition (premature offspring tend to receive fewer antibodies) as well as antibody decay rate in neonates. The duration of maternal antibodies to measles virus in human infants has been shown to be longer in those born to naturally infected mothers versus mothers who were vaccinated. The timeframes we observed are longer than the suggested six months estimated at the population level for Hendra virus in P. scapulatus
. However, our data from Experiment 2 do show a significant decrease in titer by six months, which represents approximately 3.5 terminal half-life periods for Hendra virus antibodies, or a decay to less than 1/8 of the starting titer, which may result in sufficiently decreased herd immunity at the population level to allow for viral circulation among the juvenile cohort.
A direct correlation was observed between the seropositivity of dams and their pups, with antibodies against CDV being detected in all five pups born to vaccinated dams, and anti-HeV antibodies detected in the 12 pups born to 12 seropositive dams and no HeV antibodies in the one pup born to a seronegative dam. This result is consistent with an earlier Hendra virus study demonstrating a strong association between dam and pup serostatus. It appears from the data that the pups’ titers correlate with their dam’s. Interestingly, we found that Charisma, who had the lowest titer of the dams, produced twins (Chesa and Cayha) that had significantly lower titers than their peers. We hypothesize that there was correlation between the titers of dams and their pups, however, in this case we did not have enough data to test this statistically as the titers of Chesa and Cahya were not independent of each other and would likely skew the correlation coefficient towards significance.
We did not measure the decay rates of antibodies in adult bats, however, we would expect them to be slower than that observed in pups. Faster decay of maternally derived antibodies has been reported in human infants born to vaccinated mothers compared with naturally immune mothers,.
Henipaviruses are an important group of zoonotic viruses carried by Pteropus species, and understanding pteropid immunology is important for modeling the dynamics of viral infections within flying fox populations. Waning immunity to henipaviruses in juvenile cohorts may be critical to the timing of outbreaks within colonies, and therefore related to risk of spillover to humans and other animals. Further study of bat immunology will be helpful both for ecological studies of viral pathogens and also for understanding how bats respond to viral infections.
The study did not involve any animal experiment. Only sample collection from naturally infected dogs was carried out, consisting of a single nasal swab per dog. This was needed for the laboratory analyses and did not involve any suffering of the sampled animals.
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.