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INTRODUCTION
Infectious diseases are the leading cause of death among dogs (1), and the simultaneous occurrence of more than one disease is common, especially in immunocompromised or predisposed dogs. Although in clinical practice, diagnosis is usually based on the evolution of clinical signs and laboratory findings, many dog diseases present non-specific signs such as apathy, anorexia and occasionally fever, which progress to respiratory, gastrointestinal and neurological manifestations (2,3).
These clinical signs are usually related to viruses such as canine distemper and infection by agents of the genus Ehrlichia, in addition to the possible presence of other infectious agents such as Borrelia spp.
In general, these may begin with general signs of apathy and anorexia, but when they appear with fever and ocular-nasal discharge, they may be associated with respiratory manifestations of canine distemper (4), ehrlichiosis (5) and borreliosis, which may include neurological manifestations (6).
This study is aimed at detecting canine distemper, Ehrlichia canis and Borrelia burgdorferi sensu lato (s.l.) in stray dogs in the cities of Rio de Janeiro and Seropédica, State of Rio de Janeiro, Brazil
MATERIALS AND METHODS
The study was carried out at the Zoonosis Control Center of Paulo Dacorso Filho (CCZ) located in the Santa Cruz neighborhood, and at the Campus of the Federal Rural University of Rio de Janeiro (UFRRJ), located in Seropédica, both places belong to the State of Rio de Janeiro, Brazil. One hundred and fifty-eight canine blood samples from the two cities were collected and analyzed. Of these, 96 from stray dogs located at CCZ and 62 from free-roaming animals near the Campus of UFRRJ. The animals sampled were of different ages, sexes, undefined breeds and with an unknown vaccination history.
Blood samples were obtained by puncture of the jugular or cephalic vein into vacuum tubes with and without anticoagulant. The blood collected without anticoagulant was placed in a tilted support and left at room temperature until clot formation. Then, serum was obtained by centrifugation at 2,600xg for 5 minutes, and it was stored at -20ºC (-4ºF) until use. During collections, canines were clinically evaluated.
To research hemoparasites, peripheral blood thin smears were obtained from the first drop of blood from the capillary vessels of the atrial region. The smears were air-dried, fixed in methanol and stained with Giemsa. These slides were examined under light microscopy using a 100x objective.
The sera were thawed at room temperature to be analyzed by serology and to detect canine distemper virus antigens. Thirty-eight samples from CCZ were tested for anti-E. canis antibodies and for the qualitative detection of canine distemper virus antigen with the chromatographic immunoassay test “Antigen Rapid CDV Ag Test Kit,” according to manufacturer's recommendation.
For the serological diagnosis of E. canis, the Indirect Fluorescent Antibody Test (IFAT) was performed, using the E. canis São Paulo reference strain (7). For the detection of antibodies of homologous IgG class against B. burgdorferi s.l., an indirect ELISA serological test was performed, according to a previously developed methodology (8).
RESULTS AND DISCUSSION
The young female dog (1/38), a stray animal that presented apathy, anorexia, fever, and ocular-nasal discharge at the time of collection, was the only animal with a positive CDV antigen test. This animal tested positive for both eye-drainage and serum samples. It should be noted that with the exception of this animal, the other dogs were asymptomatic while living in the same environment. When the similarity of the clinical manifestations of the diseases studied and the possibility of co-infections were confirmed, the animal tested positive for E. canis antibodies with a 1:40 titer in IFAT.
With regard to the results of the study by Hartmann et al. (9), which was carried out on breeding dogs of different ages with an unknown vaccination history and using a neutralising antibody test, the authors concluded that most of the dogs did not have specific CDV antibody titers, indicating that there was no contact with canine distemper virus antigen due to natural infection or previous immunisation (10). In addition to this finding, the authors suggested that the absence of CDV neutralizing antibodies in unvaccinated animals may lead to a high risk of exposure to subsequent CDV infection.
In this study, only one animal tested positive among the 38 dogs tested (2.63%) and the rate, although close to that obtained by Headley et al. (11), was contrary to expectations due to the conditions of the population studied. In an epidemiological study by Borba et al. (12), an infection rate of 2.07 % was identified among animals in veterinary clinics in Maringa, State of Paraná, between 1998 and 2001. The highest frequency of infection occurred in winter. Headley et al. (11) have also reported low infection rates (1.98 %). CDV infection has a pre-patent period of about 20 days, followed by fever peaks and a subsequent asymptomatic state (3). The antigen can only be detected in animals with viraemia, which occurs between 3 and 10 days after infection, clinically manifested by fever and apathy, among other signs of systemic involvement, which was not common among the animals in this study despite coexistence with the positive and symptomatic animal.
Although normal cleaning and disinfection procedures are usually effective against the canine distemper virus (5), the presence of an infected animal poses a risk for those living in the same environment, especially since it is a chronic, low immunogenic disease involving different organs and systems.
A prevalence of 22.8% (13/158) of E. canis was found by the observation of blood smears. Thus, frequencies of 4.22 % (3/62) and 10.42 % (10/96) were observed in UFRRJ and CCZ, respectively. In the serological test for E. canis, 92.1 % (35/38) of the animals had antibodies against the antigen of E. canis São Paulo reference strain. Most of the seropositive animals had titers equal to or higher than 1:10,240 (23/38), which may suggest a field exposure to the infectious agent. Therefore, these data were above the perspectives reported to those found in the northeastern region of Brazil, where the frequency was 35.6 % (13). In a comparative study of 30 dogs treated at the UNESP Veterinary Teaching Hospital in Jaboticabal, São Paulo State, 63.3% of the samples were positive by IFAT, 70% by DOT-ELISA and 53.3% by nPCR. The authors highlighted the importance of clinical and haematological tests for the diagnosis of canine ehrlichiosis (14).
In the present study, a large number of asymptomatic positive animals indicate a high prevalence of subclinical infection among the animals tested. According to Harrus et al. (2), clinical ehrlichiosis should be considered for differential diagnosis in dogs from endemic areas with typical clinical signs and hematological and biochemical abnormalities. Traditional diagnostic techniques, including hematology, cytology, serology, and isolation are important diagnostic tools; however, the definitive diagnosis of the infection by E. canis requires molecular techniques.
Of the 158 sera tested, 85 were positive (53.8 %) with IgG homologous class titers against B. burgdorferi s.l. They were closer than those already reported in Brazil, using either the indirect ELISA method used in this study (8,15-17) or the ELISA method used in the Snap 3 Dx® test kit (IDEXX Laboratories) (18). Antibodies to Borrelia spp. were detected in stray dogs at UFRRJ in 48.39 % (30/62) of the animals tested, while at CCZ, the frequency of positives was 57.29 % (55/96).
Using an indirect ELISA test, Jopper et al. (15) found 9.7 % seropositivity in the city of São Paulo, and Carlos et al. (18) observed a positivity of only 1 % of the animals studied by an ELISA test in Ilhéus, state of Bahia. In the rural areas of seven municipalities in the State of Rio de Janeiro, O'Dwyer et al. (16) found 15.85 % of seropositive dogs. Among the dogs from the municipalities of Baixada Fluminense, Rio de Janeiro, Soares et al. (8) and Alveset et al. (19) found a positivity of 20 % and 48.25 %, respectively. Cordeiro et al. (17) found an infection rate of 52.56 % in companion dogs in the metropolitan area of Rio de Janeiro.
In Lyme borreliosis, the dog may act as an epidemiological sentinel, hosting the spirochete, behaving as a reservoir in the domestic environment, and the tick vector to vehicle the pathogen to humans and other animals (19). The origin of the animals in this study was unknown, and most animals were captured in urban areas, which enhanced the possibility of them to act as reservoir of infection to both animals and humans.
The animals sheltered in kennels with dividing collective bays were exposed to a stressful situation because of the cohabitation condition. Despite having a good management, disputes over food and water may occur. In addition, the unknown vaccination history situation predisposed susceptible animals to disease and to develop a clinical, sub-clinical or chronic form of disease. These conditions favored a state of immunosuppression that enabled co-infections.
Moretti et al. (20) reported triple agent co-infection with canine distemper, ehrlichiosis and toxoplasmosis in a dog with a neuropathological clinical picture. The authors analyzed canine distemper as a primary disease and its association with ehrlichiosis based on clinical and epidemiological data, inadequate immunological prophylactic protocol and the role of these diseases in immunosuppression. Canine distemper and ehrlichiosis were diagnosed on the basis of the epidemiological situation of the region and compatible clinical signs, combined with blood count and cytology results. In this study, animals with good to poor nutritional status, but good mood and active, had antibody titers for more than one disease-causing agent.
The presence of an animal positive to canine distemper virus and without clinical cases indicated the presence of the agent and suggested that dogs were naturally immunized or sick at a stage when it was not possible to detect circulating antigens. The high number of seropositive animals, although asymptomatic to ehrlichiosis, showed a high prevalence of infection among the dogs examined. The presence of anti-B. burgdorferi s.l. antibodies also showed the circulation of the spirochete Borrelia spp. in the dogs in the studied region.
