A serological survey of canine respiratory coronavirus in New Zealand

ABSTRACT

Aims: To determine the seroprevalence of canine respiratory coronavirus (CRCoV) in New Zealand dogs, and to explore associations with age, sex, breed, month, and geographical region of sampling and reported presence of clinical signs suggestive of respiratory disease.

Methods: A total of 1,015 canine serum samples were randomly selected from submissions to a diagnostic laboratory between March and December 2014, and were analysed for CRCoV antibodies using a competitive ELISA. Logistic regression analysis was used to determine associations between seroprevalence of CRCoV and breed category, age, sex, sampling month, region, and reported health status of dogs.

Results: Overall, 538/1,015 (53.0%) samples were seropositive for CRCoV, with 492/921 (53.4%) positive dogs in the North Island and 46/94 (49%) in the South Island. Age of dog, sampling month, region, and presence of abnormal respiratory signs were included in the initial logistic regression model. Seroprevalence was higher in dogs aged ≥3 compared with ≤2 years (p?<?0.01). The lowest seroprevalence was observed in July (30/105; 28.5%) and August (32/100; 32%), and the highest in June (74/100; 74%). Seroprevalence in dogs from Auckland was higher than in dogs from the Hawkes Bay, Manawatu, Marlborough, and Waikato regions (p?<?0.05). Abnormal respiratory signs (coughing, nasal discharge, or sneezing) were reported for 28/1,015 (2.8%) dogs sampled. Seroprevalence for CRCoV tended to be higher among dogs with respiratory signs (67.9 (95% CI?=?47.6–83.4)%) than dogs with no reported respiratory signs (52.6 (95% CI?=?49.5–55.7)%).

Conclusions: Serological evidence of infection with CRCoV was present in more than half of the dogs tested from throughout New Zealand. Differences in CRCoV seroprevalence between regions and lack of seasonal pattern indicate that factors other than external temperatures may be important in the epidemiology of CRCoV in New Zealand.

Clinical relevance: Our data suggest that CRCoV should be included in investigations of cases of infectious canine tracheobronchitis, particularly if these occur among dogs vaccinated with current vaccines, which do not include CRCoV antigens.     

Serological prevalence of canine respiratory coronavirus

Canine respiratory coronavirus (CRCoV) has recently been detected in dogs; it is a group 2 coronavirus showing similarity to bovine coronavirus (BCoV) but is distinct from canine enteric coronavirus (CECoV). CRCoV may play an important role in canine infectious respiratory disease (CIRD) either by predisposing to further and potentially more serious viral and bacterial infections or possibly as a primary pathogen. The prevalence of serum antibodies to CRCoV, in a population of dogs in the south east of England, has been shown previously to be 30.1% on the first day of entry to a rehoming kennel [Erles, K., Toomey, C., Brooks, H.W., Brownlie, J., 2003. Detection of a group 2 coronavirus in dogs with canine infectious respiratory disease. Virology 310, 216-223]. The purpose of this study was to establish the prevalence of CRCoV in the general canine population within as well as outside the UK. An ELISA, used to test for the presence of antibodies to CRCoV in canine serum samples, identified seropositive dogs in UK, USA, Canada, Republic of Ireland and Greece. The development of an ELISA based on CRCoV antigen and immunofluorescence assay are described here. 54.7% (547/1000) of North American and 36.0% (297/824) of United Kingdom dogs were seropositive for CRCoV. The age and geographical distribution of seropositive dogs was also assessed. The cross-reactivity demonstrated between CRCoV antibodies from different countries and a UK viral isolate suggests immunological similarity. The overall prevalence of this virus in both North America and the UK suggests that CRCoV has international significance and that further epidemiological studies are required.     

Serological prevalence of canine respiratory coronavirus in southern Italy and epidemiological relationship with canine enteric coronavirus.

Canine respiratory coronavirus (CRCoV) has been detected in dogs suffering from respiratory disease and is thought to be involved in canine infectious respiratory disease (CIRD) complex. Canine enteric coronavirus (CECoV) is a widespread pathogen of dogs, responsible for mild to severe diarrhea in pups. The purpose of this study was to establish the seroprevalence of CRCoV in Italy and its relationship to CECoV type II seroprevalence. The age and year of sample collection from seropositive dogs was also assessed. Of adult domestic dogs, 23.3% had antibodies to CRCoV, compared with 86.1% with antibodies to CECoV. Amongst a population of kenneled pups, 4.0% had antibodies to CRCoV, and 97.0% had antibodies to CECoV.     

Canine respiratory coronavirus: an emerging pathogen in the canine infectious respiratory disease complex.

Infectious respiratory disease in dogs is a constant challenge because of the involvement of several pathogens and environmental factors. Canine respiratory coronavirus (CRCoV) is a new coronavirus of dogs, which is widespread in North America, Japan, and several European countries. CRCoV has been associated with respiratory disease, particularly in kenneled dog populations. The virus is genetically and antigenically distinct from enteric canine coronavirus; therefore, specific tests are required for diagnosis.     

Seroepidemiology of respiratory (group 2) canine coronavirus, canine parainfluenza virus, and Bordetella bronchiseptica infections in urban dogs in a humane shelter and in rural dogs in small communities

This prospective study evaluated seroepidemiologic features of canine respiratory coronavirus (CRCoV), canine parainfluenza virus (CPIV), and Bordetella bronchiseptica infections in dogs in an urban humane shelter and in rural/small community dog populations in western Canada. Seroprevalence of CRCoV and CPIV was low compared with other countries; seroprevalence of B. bronchiseptica was moderate to high in most populations examined. Rural dogs were 0.421 times (P ≤ 0.0001) less likely to be positive for CRCoV than dogs admitted to the shelter. There were no statistical differences in prevalence of antibodies to B. bronchiseptica and CPIV between urban and rural populations. Dogs from Fort Resolution, NWT were significantly (P < 0.05) less likely to have moderate or high antibody titers to the 3 agents than dogs in the shelter. Seroconversion to CRCoV was common in dogs in the shelter, but was not associated (P = 0.18) with respiratory disease. Antibodies to CRCoV, CPIV, or B. bronchiseptica on arrival were not significantly (P > 0.05) associated with disease-sparing after entry into the shelter.     

Serological and molecular evidence that canine respiratory coronavirus is circulating in Italy

Canine respiratory coronavirus (CRCoV) is a group II coronavirus that was firstly identified in lung samples of dogs with canine infectious respiratory disease (CIRD) in UK in 2003. We report for the first time the identification of CRCoV in Italy, together with serological evidence that the virus has been circulating in the Italian dog population as from 2005. Serological investigations on 216 dog sera, carried out by an ELISA test using the strictly related bovine coronavirus (BCoV) as antigen, revealed an overall CRCoV seroprevalence of 32.06% in the last 2 years. RT-PCR targeting the S-gene of CRCoV was carried out on 109 lung samples collected from carcasses of dogs submitted for diagnostic investigations. Positive results were obtained from the lungs of a dog of the Apulia region that was co-infected with canine parvovirus type 2. Sequence analysis of the S-gene fragment amplified by RT-PCR (595bp) showed similarity to group II coronaviruses, with the highest nucleotide identity (98%) to the only CRCoV strain currently available in the GenBank database (strain T101). The results of the present study show that CRCoV is present also in continental Europe, although further studies are required to determine the real pathogenic potential of the virus.     

Serological evidence of H3N8 canine influenza-like virus circulation in USA dogs prior to 2004

H3N8 canine influenza virus (H3N8 CIV) was first reported as a novel canine respiratory pathogen in racing greyhounds and shelter dogs in the U.S.A. in 2004. Phylogenetic analyses determined that this host-adapted pathogen originated from interspecies transmission of an equine influenza virus (EIV), but it is unknown when the transmission occurred prior to discovery in 2004. The objective of this study was to determine if racing greyhound and shelter dog sera collected from 1984 to 2004 had serological evidence of exposure to H3N8 CIV or EIV. Archived sera from 702 racing greyhounds and 1568 shelter dogs were tested for H3 antibodies to the original 2004 CIV isolate, as well as EIV isolates from 1991 to 1999. None of the racing greyhounds from 1984 and 1985 had detectable H3 antibodies. One of the shelter dogs, which entered a north Florida shelter in 2004, was seropositive. For racing greyhounds sampled from 1999 to 2004, 133/520 (26%) dogs had antibodies to both CIV and EIV H3 proteins. The annual seroprevalence was 27% in 1999, 28% in 2000, 10% in 2001, 1% in 2002, 41% in 2003, and 28% in 2004. The odds of H3 seropositivity were greater among dogs that raced > or =6 months, raced on > or =2 tracks, and raced in 1998, 2002, and 2003. Many of the seropositive dogs raced at tracks that were involved in 'kennel cough' epidemics in 1998-1999 and 2002-2003. Based on serological evidence, a H3N8 canine influenza-like virus was circulating in racing greyhounds in the U.S.A. as early as 1999.     

No Serologic Evidence for Zoonotic Canine Respiratory Coronavirus Infections among Immunocompetent Adults

Zoonotic diseases continue to emerge and threaten both human and animal health. Overcrowded shelters and breeding kennels create the perfect environment for amplified infectious disease transmission among dogs and present a critical opportunity for zoonotic pathogens to emerge and infect people who work in close contact with dogs. Coronaviruses’ widespread prevalence, extensive host range, various disease manifestations and increased frequency of recombination events all underline their potential for interspecies transmission (Methods Mol. Biol. 2008, 454, 43). The objectives of this study were to determine whether people with occupational contact with dogs were more likely to have antibodies against canine respiratory coronavirus (CRCoV) compared to persons with no dog exposure. A seroepidemiological cohort study was completed, for which 302 canine‐exposed and 99 non‐canine‐exposed study subjects enrolled in the study by providing a serum sample and completing a self‐administered questionnaire. A competitive enzyme‐linked immunosorbent assay (ELISA) was developed to detect human antibodies against CRCoV while controlling for cross‐reacting antibodies against the human coronavirus OC43. All study subjects were negative for antibodies against CRCoV by this competitive ELISA. This study supports the premise that humans are not at risk for CRCoV infections; however, infrequent cross‐species transmission of CRCoV cannot be ruled out.     

Serum antibody responses to vaccinal antigens in lean and obese geriatric dogs

The immune responses in control dogs [1 to 4 years of age, body condition score (BCS): 4 to 5 out of 9] were compared to those of aging dogs (based on breed and body size) either categorized as lean (BCS: 4 to 5 out of 9) or obese (BCS: 8 to 9 out of 9). Of interest were the serum titers to the following common agents found in vaccines, canine parainfluenza virus (CPIV), canine parvovirus (CPV), canine distemper virus (CDV), canine respiratory coronavirus (CRCoV), and Bordetella bronchiseptica. There were no statistical differences in the antibodies to CPIV, B. bronchispetica, and CRCoV, among the age/weight categories, nor among the age/weight categories and the time, in days, between the date of sample collection and the date of the last recorded vaccination for CPIV, B. bronchiseptica, CPV, and CDV. For CPV, the control dogs had significantly (P < 0.002) higher serum neutralization (SN) titers than the lean geriatric dogs and the obese geriatric dogs. For CDV SN titers, the only statistically significant (P = 0.01) difference was that the control dogs had higher SN titers than the lean geriatric dogs.     

Seroprevalence of canine influenza virus (H3N8) in Iditarod racing sled dogs

We conducted a cross-sectional convenience sampling study of dogs racing in the 2010 Iditarod to determine the seroprevalence of canine influenza virus (CIV) in the sled dog population. Questionnaires were completed detailing medical and CIV vaccination history, kennel size and location, travel history, and social interactions for each team. A total of 399 dogs were tested for CIV antibodies by hemagglutination inhibition assay. None of these, including 39 samples from dogs reported as CIV vaccinated, were seropositive for CIV antibodies. All of the vaccinated dogs were also negative on virus microneutralization assay. Risk factors for CIV seropositivity could not be determined due to a lack of positive samples. This is the first published study investigating the prevalence of CIV in sled dogs and additional studies are warranted to assess CIV infection among racing sled dogs and to evaluate the ecology of CIV and the vaccine efficacy in this population of dogs.     

北京地区犬呼吸道冠状病毒感染状况的监测与分析

本研究旨在分析犬呼吸道冠状病毒(canine respiratory coronavirus, CRCoV)的感染情况和在北京地区的流行病学状况。采用RT-PCR方法,对自2015年12月—2017年3月于中国农业大学动物医院采集的487份犬咽鼻拭子进行了CRCoV检测,同时,对部分样本进行了犬副流感病毒(canine parainfluenza virus, CPIV)、犬腺病毒II型(canine adenovirus type 2,CAV-2)、犬瘟热病毒(canine distemper virus, CDV)等病毒的混合感染情况调查。结果表明：CRCoV的阳性率为21.36%(104/487);CRCoV与CPIV、CAV-2、CDV的混合感染率分别为3.43%(11/321),0%(0/156)和3.85%(6/156)。在所有呼吸道症状记录完整的455例样本中,无呼吸道症状的犬CRCoV携带率为27.19%(31/114);呈轻度呼吸道症状(鼻液、咳嗽)的犬CRCoV感染率为19.73%(59/299);呈中至重度呼吸道症状(肺炎)的犬CRCoV感染率为14.28%(6/...     

成都地区宠物犬感染犬瘟热病毒和犬呼吸道冠状病毒的分子流行病学调查

为了解成都地区宠物犬犬瘟热病毒（canine distemper virus,CDV）和犬呼吸道冠状病毒（canine respiratory coronavirus,CRCoV）的感染情况,本试验应用RT-PCR对采自成都地区8家动物医院共计420份出现呼吸道症状的宠物犬鼻腔棉拭子样本进行分子检测。结果发现,从420份样本中,检出213份CDV阳性,检出率为50.71%;检出247份CRCoV阳性,检出率为58.81%;CDV和CRCoV混合感染的检出率为41.19%。表明成都地区宠物犬感染CDV和CRCoV较为严重,且二者混合感染率较高。宠物犬CDV和CRCoV的检出率与年龄、性别、品种、季节和免疫状况等因素的关系存在不同程度的差异。其中,1~3月龄幼犬检出率最高,分别为74.40%和79.20%;纯种犬的检出率较其他犬种高,分别为59.37%和62.22%;春季CDV的检出率较高,为56.19%,而冬季CRCoV的检出率较高,为67.59%;未免疫犬的检出率较高,分别为64.42%和63.46%。该研究丰富了成都地区宠物犬CDV和CRCoV的流行病学资料,为该地区宠物犬CDV和CRCoV的诊断及防控提供了基本数据。     

Factors associated with development of Canine Infectious Respiratory Disease Complex (CIRDC) in dogs in 5 Canadian small animal clinics

This study investigated the association between presence of respiratory pathogens and development of Canine Infectious Respiratory Disease Complex (CIRDC) in dogs in 5 Canadian small animal clinics. In total, 86 dogs were tested using a commercial PCR respiratory panel; 64 dogs were considered as cases and 22 were control dogs matched by veterinary clinic. No control animals (0/22) were positive for canine parainfluenza virus (CPIV), whereas 27/64 (42%) CIRDC cases were positive. Furthermore, 81% of case dogs tested positive for Mycoplasma cynos, compared with 73% of control dogs. Canine respiratory corona virus (CRCoV) was detected in no control dogs compared with 9.4% of clinical dogs. No animals were positive for any influenza virus type A present in the diagnostic panel. Presence of CPIV was associated (P < 0.01) with the occurrence of CIRDC after adjustment for demographic factors and presence of CRCoV (P = 0.09).     

Seroprevalence and risk factors for canine H3N8 influenza virus exposure in household dogs in Colorado

Background: Since 2004, canine influenza virus (CIV) has spread throughout the United States. While studies suggest that CIV is commonly detected in shelter dogs, little is known about its prevalence in household dogs. Objectives: To evaluate the seroprevalence of CIV in pet dogs presented for care in a veterinary hospital in Colorado and to investigate risk factors that might predispose these dogs to CIV infection. Animals: One hundred and forty dogs presenting to the Community Practice service, 110 dogs seen at other clinical services at Colorado State University's Veterinary Teaching Hospital in 2009, and samples from 75 dogs seen before 2004. Methods: In this prospective study, samples were tested with hemagglutination inhibition assays, using 3 CIV isolates. To identify risk factors for CIV infection, 140 owners completed questionnaires at time of sampling. Results: CIV seroprevalence was 2.9% (4/140) for dogs seen by the Community Practice service and 4.5% (5/110) for dogs seen by other hospital services (P= .48). All sera collected before 2004 tested negative for CIV. No differences were seen in antibody titers to the 3 CIV isolates tested. Data from the questionnaires indicated an association between CIV seropositivity and canine daycare visits (P < .001). Conclusion and Clinical Importance: CIV seropositivity in household dogs in Colorado is low, although it has increased since 2004. Antibody titers to the 3 CIV isolates were comparable, suggesting that measurable antigenic drift has not yet occurred. Finally, dogs boarded in kennels or attending daycare might be at an increased risk of CIV infection.     

Detection of bovine herpesvirus type 4 antibodies and bovine lymphotropic herpesvirus in New Zealand dairy cows

AIM: To detect the presence of bovine herpesvirus (BoHV) type 4 in New Zealand dairy cows with clinical metritis.

METHODS: Serum samples taken from 92 dairy cows with clinical metritis, each from a different farm, were tested for the presence of antibodies against BoHV-4 using a commercially available, indirect ELISA. Peripheral blood mononuclear cells (PBMC) were collected from 10 BoHV-4 seropositive cows, and PBMC were examined by a pan-herpesvirus nested PCR to detect herpesvirus. PCR products were sequenced directly and a proportion of the PCR products were cloned and sequenced to identify the virus present.

RESULTS: Antibodies to BoHV-4 were detected in 23/92 (25%) serum samples. The pan-herpesvirus PCR was positive in 8/10 PBMC samples. Cloning and sequencing identified that all of the eight PCR-positive PBMC contained bovine lymphotropic herpesvirus (BLHV); no BoHV-4 DNA was detected.

CONCLUSIONS: This study reports the finding of the presence of apparent antibodies to BoHV-4, and BLHV DNA in New Zealand dairy cows affected by metritis.

CLINICAL RELEVANCE: Bovine herpesvirus type 4 and BLHV are reported to have the potential to cause reproduction failure in cows. This is the first report of apparent BoHV-4 antibodies, and BLHV in New Zealand. The importance and epidemiology of these viruses in cattle in New Zealand requires further investigation.     

No evidence of horizontal infection in horses kept in close contact with dogs experimentally infected with canine influenza A virus (H3N8)

Background

Since equine influenza A virus (H3N8) was transmitted to dogs in the United States in 2004, the causative virus, which is called canine influenza A virus (CIV), has become widespread in dogs. To date, it has remained unclear whether or not CIV-infected dogs could transmit CIV to horses. To address this, we tested whether or not close contact between horses and dogs experimentally infected with CIV would result in its interspecies transmission.

Methods

Three pairs of animals consisting of a dog inoculated with CIV (10^8.3 egg infectious dose₅₀/dog) and a healthy horse were kept together in individual stalls for 15 consecutive days. During the study, all the dogs and horses were clinically observed. Virus titres in nasal swab extracts and serological responses were also evaluated. In addition, all the animals were subjected to a gross pathological examination after euthanasia.

Results

All three dogs inoculated with CIV exhibited clinical signs including, pyrexia, cough, nasal discharge, virus shedding and seroconversion. Gross pathology revealed lung consolidations in all the dogs, and Streptococcus equi subsp. zooepidemicus was isolated from the lesions. Meanwhile, none of the paired horses showed any clinical signs, virus shedding or seroconversion. Moreover, gross pathology revealed no lesions in the respiratory tracts including the lungs of the horses.

Conclusions

These findings may indicate that a single dog infected with CIV is not sufficient to constitute a source of CIV infection in horses.     

Stray dogs as sentinels for canine parvovirus

An attempt to determine the prevalence of canine parvovirus in the stray dog population of Franklin County, Ohio (U.S.A.) was made by sampling dogs during the first 6 months of 1981. Serum and fecal samples, which were collected from 209 strays at time of euthanasia, were tested by serum hemagglutination inhibition (HI) and fecal hemagglutination (HA) techniques to determine canine parvovirus experience (seropositive) or fecal virus shedding, respectively. Sera collected from 93 strays for an unrelated study conducted in 1979 were used as the comparison group. All of the 1979 sera were HI negative (< 1:80) whereas, 139 of 201 (69.2%) sera suitable for testing from the 1981 group of strays were HI positive (? 1:80). The fecal HA results from the 1981 group revealed 26 of the 209 (12.4%) dogs were shedding parvovirus at time of euthanasia (HA titers ? 1:64). Of these 26 of the 209 (12.4%) dogs were shedding parvovirus at time of euthanasia were found to be seropositive. These results indicate that the stray population of Franklin County, Ohio, was not exposed in 1979, but by 1981 had experienced and for the most part, had recovered from canine parvovirus as indicated by a 69.2% seropositive dog population with 12.4% active virus shedders. The stray dog population, if sampled regularly, could thus serve as a sentinel for canine parvovirus activity in a community.     

Confirmation of the occurrence of the chewing louse Bovicola (Lepikentron) breviceps (Insecta: Phthiraptera: Trichodectidae) on alpacas (Lama pacos) in New Zealand

Abstract

AIM: To investigate the cause of classical swine fever (CSF) virus-seropositive animals in a nucleus pig-breeding herd in New Zealand, where porcine circovirus-associated disease had been diagnosed.

CASE HISTORY AND CLINICAL FINDINGS: An exotic disease investigation was undertaken to exclude CSF and porcine reproductive and respiratory syndrome (PRRS) on a nucleus pig-breeding herd comprising approximately 300 breeding sows, 1,000 weaners, and 650 grower pigs. The herd was experiencing poor reproductive performance in sows, and breeding records showed a declining farrowing rate attributable to a single manager. The growing pigs (10–15 weeks old) were experiencing respiratory disease and wasting, and the mortality rate by pen varied between 9 and 20%. Post-mortem changes in affected grower pigs were consistent with circovirus-associated diseases.

DIAGNOSTIC TESTING: Serological screening using an IDEXX-ELISA gave negative results for PRRS virus antibodies, but two grower pigs and one sow tested positive for CSF virus antibodies. These three seropositive animals remained positive to CSF virus, using three commercial ELISA test kits, over 27 weeks. A newly developed virus neutralisation test (VNT), using a New Zealand isolate of border disease (BD) virus, demonstrated that the seropositive pig sera had higher antibody titres to BD virus than to bovine viral diarrhoea (BVD) virus and CSF virus.

PCR performed on tonsil, kidney, ileum and spleen gave negative results for CSF virus, and histopathology on lymph nodes, intestine, lung, kidney, liver and brain showed no evidence of the disease. Virus isolation performed on a number of samples was negative.

CLINICAL RELEVANCE: The seropositive samples for CSF virus found in this investigation were likely to be a cross reaction to a pestivirus other than CSF virus. The finding of a possible endemic pestivirus capable of being transmitted between sheep and pigs on this farm may explain findings from previous serological survey work in New Zealand, and supports experience elsewhere, where BD virus was found to be the predominant ruminant pestivirus infecting pigs. The results show that pestivirus cross reactivity can result in unexpectedly high titres, and that testing with a full set of (local) pestiviruses is necessary to reach the correct conclusion. The investigation has direct relevance where pig herds with a low seroprevalence are encountered during surveillance for CSF.     

Prevalence of antibodies to four major canine viral diseases in dogs in a Liverpool hospital population

To determine the prevalence of antibodies to four major canine viruses, serum samples were obtained from 190 dogs presented to the Small Animal Hospital at the University of Liverpool. Antibodies to canine coronavirus (CCV), canine distemper virus (CDV), canine parvovirus (CPV) and rotavirus (RV) were assayed using serum neutralisation (CCV and CDV), haemagglutina-tion inhibition (CPV) and indirect fluorescent antibody (RV) techniques. Overall 54 per cent of dogs were seropositive to CCV, 84 per cent to CDV, 70 per cent to CPV and 86 per cent to RV, The antibody titres obtained were analysed with respect to a number of different parameters including: age, sex, breed, vaccination status, exercise regime, diet, Liverpool district in which the dog resided and the presence of diarrhoea, The prevalence and titres of antibodies to CCV, CDV and RV appeared to be influenced by age, CDV by vaccination status, and CCV by the presence of diarrhoea; no other influencing parameters were found.     

Antibody titers for canine parvovirus type-2, canine distemper virus, and canine adenovirus type-1 in adult household dogs

Serum antibody titers for canine parvovirus type-2 (CPV-2), canine distemper virus (CDV) and canine adenovirus type-1 (CAV-1) were investigated in 1031 healthy adult household dogs (2 to 18 years old) given an annual inoculation in the previous 11 to 13 months. The number of dogs retaining significant titers of antibodies against CPV-2, CDV, and CAV-1 were 888 (86%), 744 (72%), and 732 (71%), respectively. There were no differences between males and females in antibody titers against the 3 viruses. Antibody titer for CPV-2 was significantly higher in younger dogs than in older dogs, CDV antibody was significantly higher in older dogs than in younger dogs, and CAV titer was not associated with age.