Introduction

Canine monocytic ehrlichiosis (CME) is a worldwide distributed infectious disease of domestic dogs caused by Ehrlichia canis that infects blood mononuclear cells. The infection is transmitted to susceptible dogs by the brown dog tick Rhipicephalus sanguineus. This one-host tick preferentially feeds on dogs in all three stages of its life cycle and can live indoors in domiciled environments in which dogs are housed (Neer & Harrus 2004). The ticks acquire E. canis as either larvae or nymphs, by feeding on rickettsemic dogs. Once infected, they transmit infection to susceptible dogs throughout their life. Transtadial transmission of E. canis occurs in the tick (Bremer et al. 1995) but transovarial spread does not occur. In the dog, after a short incubation period, E. canis infection may progress into three consecutive clinical stages: acute, subclinical and chronic. In the acute phase, clinical signs may vary from mild to severe and include non-specific signs such as anorexia, depression, lethargy, mild weight loss, fever, splenomegaly and lymphadenomegaly (Castro et al. 2004). Hematological abnormalities in this phase may include thrombocytopenia, mild anemia and mild leucopenia. Appropriate treatment results in complete recovery however, with improper treatment or lack thereof, the disease may progress to the subclinical phase, which may last for years (Shipov et al. 2008). In the latter phase dogs appear healthy, however mild thrombocytopenia may persist. Dogs in this phase may remain carriers for life, may spontaneously recover or progress to the chronic severe form of the disease. Common clinical signs in the chronic phase include anemia severe thrombocytopenia and leucopenia due to impaired bone marrow production. In natural occurring infections, accurate staging of the disease could be difficult. Mild infection may progress without any clinical signs. High percentage of dogs in an enzootic area may develop subclinical ehrlichiosis that may last several years (Neer et al. 2002). Being a domiciled tick, and having the preference of all stages feeding on dogs, R. sanguineus is important in the maintenance of E. canis infection in kennels environment. Control in endemic areas can be accomplished by maintaining strict tick control programs for dogs and premises, by using the indirect test to identify exposed and infected dogs and by treating all infected dogs with therapeutic regimen of tetracycline or doxicycline. If these guidelines are followed, the cycle of E. canis infection in the tick should be broken, because transovarial transmission of E. canis does not occur in the Rhipicephalus sanguineus (Neer & Harrus 2006) The occurrence of two cases of acute clinical form of E. canis infection in a kennel in which about thirty adult dogs shared the same environment led us to investigate the prevalence of infection among the dogs and to introduce a control program of ehrlichial infection in the premise, based on the previous statements (Neer et al. 2002).

Material and Methods

Dogs and control program. The control program was based on the identification of infected animals by PCR and indirect IFA test, treatment of infected animals with doxycycline and tick control in the environment and on the dogs. Twenty eight dogs, male (n = 17) and female (n = 11), three to eight years old, were screened for E. canis infection. Almost all of the dogs were Rottweillers. None of the dogs showed clinical signs at the moment when blood was taken for hematological, serological and PCR tests. All dogs infected by Ehrlichia canis (positive results for E. canis 16sRNA) were submitted to antimicrobial treatment regimen with doxycycline (5mg/kg/bid for thirty days) and imidocarb dipropionate (5mg/kg, subcutaneously, twice, fifteen days apart) and re-tested after the treatment was completed. Complete CBC, IFA test and PCR were repeated three, six, nine and twelve months later.

Tick control. For tick control on the dog, fipronil was used monthly, during six months and for kennel environment, lambdacialotrina 0.025% was applied as recommended by the manufacturer four times at two week intervals.

Ehrlichia canis nucleic acid detection. Polymerase chain reaction (PCR) on peripheral blood was done as described by Wen et al. (1997). ECA/HE3 primers were used to amplify DNA products of approximately 400 pb of E. canis 16sRNA gene.

Detection of anti-Ehrlichia canis antibodies. Indirect immunofluorescence antibody (IFA) test for detection of anti-E. canis antibodies was done (Hasegawa 2005). Ehrlichia canis infected DH80 cells were used as antigens. The cut-off for IFA test was 80.

Results

Initially, blood was collected from twenty six dogs. The remaining two dogs joined the group later. Weight loss, anorexia or reduced physical activity had not been observed by the owners. On physical examination, no apparent abnormalities could be found. Only mild thrombocytopenia was found in some dogs. Neither anemia nor leukopenia was observed. Seven dogs (7/26; 26.9%) were infected by E. canis as showed by positive-PCR results; five of them had anti-Ehrlichia canis antibodies titer ranging from 80 to 80,000, while two dogs did not show any antibody titer, despite the positive-PCR results. Nine dogs were only positive for IFA test (anti-E. canis antibodies titer of 640 to 40,000) and the remaining ten dogs were negative for both tests. After treatment with doxycycline and imidocarb dipropionate, PCR yielded negative results for all infected dogs. Three months later, five additional dogs (5/ 27) had been infected as revealed by PCR; IFA titer ranged from none (1) to 80,000 (1). Three had antibody titer of 640 (1) and 5,120 (2). The previously treated dogs showed declining antibodies titer. Five dogs and the new introduced dog remained negative for both tests. After six months only one dog yielded positive-PCR result, with high antibodies titer (10,240). This dog had been presented increasing antibodies titer since the first screening test (2,560 and 5,120, respectively) and negative result for PCR. Eight dogs remained positive for IFA test. After nine months, two dogs previously infected and treated, whose infection was thought to be cleaned (negative-PCR and negative-IFA test) showed signs of re-infection (positive-PCR and antibody titer). At twelve months, no positive-PCR test was found; nine dogs had antibodies titer (640 to 5,120) and ten had negative-IFA test results; Additional nine dogs had already been moved to another premise. Antimicrobial therapy was apparently successful as all dogs with the exception of the two mentioned dogs became negative for PCR and showed slowly declining titer of antibodies. Tick control of the dogs has been started at the moment of blood collection but technical problems delayed the environment tick control for about forty days. Ticks were found on the dogs two to three weeks after fipronil application, before starting the premise control with lambdacialotrina. The tick control of the kennel was successfully accomplished when both measures were introduced, yet one year later, ticks were scarcely found on the dogs.

Discussion and Conclusions

The occurrence of E. canis infection in dogs in a kennel where Rhipicephalus sanguineus is enzootically found suggests the possibility of ehrlichial infection of other canines sharing the same environment. Actually, about one third of dogs kept as working dogs in the kennel were actively infected, as evidenced by Ehrlichia canis nucleic acid detection in their peripheral blood, in spite of the presence or absence of antibodies. The prevalence of infection was similar to that mentioned by others authors in kennels or shelters (Neer & Harrus 2006). Anti-Ehrlichia canis antibodies were detected in other dogs of the same premise, some of them showing high antibodies titer, at least indicating a recent infection. However, high antibody titers or the absence of antibodies at all were not well correlated to the infectious status of the dog. The infectious status of the dogs could only be determined by PCR analysis, as mentioned elsewhere (Wen et al. 1997). The combination of fluorescent antibody test for E. canis and PCR assay was useful to achieve the diagnosis of active ehrlichial infection, as physical and hematological abnormalities were barely found. Splenomegaly, one of the most prominent features of tick-borne diseases of dogs was not confirmed, because ultrasound image could not be obtained. Many dogs might be exposed and seroconvert, but never show clinical signs (Neer et al. 2002). German shepherd dogs are known to have predisposition to clinical disease in opposition to beagles and mixed breed dogs. Apparently, Rottweilers are also more resistant to develop clinical disease, as could be observed herein. Additionally, some strains of E. canis might cause only mild disease and some animals might harbor the pathogen for months to years without clinical signs. In this case, Ehrlichia canis infection could be maintained for years and only a serological screening might identify the infectious status of the dog (Bartsch & Greene 1996) and minimize the potential for development of the disease within the kennel or shelter. The absence of correlation between antibody titer and active E. canis infection observed in this study strongly suggests the need for PCR analysis for identification and treatment of infected dogs. Otherwise, if is intended to reduce the reservoir of ehrlichial species in the environment, all seropositive dogs should be treated, with high risk of development of drug resistance (Neer et al. 2002). Beside treatment of E. canis infected dogs, the strict tick control program of premises is essential for prevention of susceptible dogs infection and should be continued in endemic areas. Two dogs introduced into the group after successful elimination of ticks from the environment, remained seronegative for E. canis. Notwithstanding, because of the localization of the kennel and the transfer of dogs from one kennel to another owned by the same people, ticks were found in some dogs at the end of experimental period. Despite apparently successful treatment of Ehrlichia canis infection of the dogs (negative-PCR and declining antibodies titer after antimicrobial treatment), two of them presented relapse of infection, with positive PCR and serology. This might be due to reinfection or maintenance of the Ehrlichia canis organism hidden in some part of the host organism, mentioned by several authors (Mylonakis et al. 2004; Gal et al. 2008). This fact emphasizes the importance of sequential molecular and serological evaluation of the dogs to assure host elimination of infection and the strict control of ticks in the environment and on the dogs.

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