Serologic Prevalence of Dirofilaria immitis, Ehrlichia canis, and Borrelia burgdorferi Infections in Brazil*

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1 Serologic Prevalence of Dirofilaria immitis, Ehrlichia canis, and Borrelia burgdorferi Infections in Brazil* Norma Labarthe, DVM, DSc a Marcelo de Campos Pereira, DVM, PhD b Oclydes Barbarini, DVM c William McKee, DVM d Carlos Alberto Coimbra, BM, MSc e Johnny Hoskins, DVM, PhD f a Universidade Federal Fluminense Vital Brazil Filho 64 Niterói, RJ, Brazil b Universidade de São Paulo Instituto de Ciências Biomédicas Departamento de Parasitologia São Paulo, SP, Brazil c Laboratórios Pfizer Ltda. Divisão de Saúde Animal Alexandre Dumas, 1860 Chácara Santo Antonio São Paulo, SP, Brazil d IDEXX Laboratories One Idexx Drive Westbrook, Maine e Museum de Astronomia e Ciências Afins MCT Gen. Bruce 258 Rio de Janeiro, RJ, Brazil f DocuTech Services, Inc. PO Box Baton Rouge, LA ABSTRACT Dogs infected with Dirofilaria immitis, Ehrlichia canis, or Borrelia burgdorferi may show nonspecific clinical signs or may be asymptomatic. In Brazil, E. canis and D. immitis infections are frequently diagnosed based on the presence of classical signs; however, serologic tests are seldom performed to confirm the presence of infection. To estimate the seroprevalence of these three canine diseases in Brazil, 2,553 dogs presented at veterinary practices for various tests, routine treatments, or *This study was supported by funds from IDEXX Laboratories, Westbrook ME and Laboratórios Pfizer Ltda. Divisão de Saúde Animal, São Paulo, Brazil. examinations were evaluated by an in-office commercial ELISA test kit (SNAP 3Dx, IDEXX Laboratories). Each dog was examined by the veterinarian, and a whole-blood sample was collected and immediately tested for the simultaneous detection of B. burgdorferi and E. canis antibodies and D. immitis antigen. D. immitis infection was detected in 51 dogs (2.0%) and E. canis antibodies were present in 505 dogs (19.8%). Only one dog tested positive for B. burgdorferi antibodies. INTRODUCTION Dirofilariasis, ehrlichiosis, and Lyme borreliosis are arthropod-borne diseases that can 67

2 Veterinary Therapeutics Vol. 4, No. 1, Spring 2003 cause clinical signs in a variety of animal species, including feral and domestic dogs. 1,2 Dogs infected with any of these parasites may exhibit glaring classic signs of the disease or nonspecific clinical signs that could be indicative of a number of conditions, or may be asymptomatic. The most common early sign of dirofilariasis is a nonproductive cough, which worsens with exercise; however, infections with Dirofilaria immitis in the United States are often detected by routine serology testing of asymptomatic animals. In Brazil, however, it is common for the diagnosis of D. immitis infection to be made by the observation of typical clinical signs in conjunction with a known risk factor for the region; it is rare for infection to be confirmed by serology testing of asymptomatic dogs. A number of parasitologic or serologic (detection of circulating microfilariae or D. immitis antigen in the blood) and necropsy surveys conducted in areas of Brazil have reported from 1.3% to 46% of the feral and pet dog populations positive for D. immitis In addition, a survey conducted in Itacoatiara in Rio de Janeiro identified six mosquito species infected with D. immitis. 8 Canine ehrlichiosis is frequently a chronic and insidious disease, and clinical signs will vary according to the phase of the infection. 11 In the acute and chronic phases, various hematologic and biochemical abnormalities are often noted, and clinically, the dog may exhibit fever, neurologic signs, weight loss, spontaneous bleeding, pallor, or intermittent limb edema. These signs are also indicative of multiple myeloma or chronic lymphocytic leukemia, however, and serologic identification of the organism in the affected dog is often necessary to confirm the diagnosis. Clinical signs are generally absent in the subclinical phase of ehrlichiosis. In one survey 12 conducted in a rural area of Rio de Janeiro, Ehrlichia canis was identified in 4.8% of 250 dogs evaluated by examination of a blood smear. In another Brazilian (Paraná) study, of 69 dogs infested with ticks (Rhipicephalus sanguineus or Amblyomma cajennense or both) tested positive for E. canis using polymerase chain reaction methods. In the same study, 12 of 61 dogs with anemia (20%) and four of 19 with thrombocytopenia (21%) were confirmed positive for E. canis. Three clinical cases of human Lyme disease were identified in 1996 in Mato Grosso do Sul, Brazil. 14 In 1997, Yoshinari 15 and colleagues from the University of São Paulo reported that Lyme disease had been confirmed in 25 human patients since A recent evaluation 16 of 237 dogs in São Paulo revealed 23 (9.7%) were positive for antibodies to Borrelia burgdorferi by ELISA, and 20 of these were confirmed by Western blot (WB). Canine Lyme disease is often diagnosed using laboratory tests to confirm the infection when clinical signs and epidemiologic risks suggest a high probability of infection. 17 Problems encountered with some of the serologic tests include positive findings for clinically normal dogs, persistence of antibodies, and the use of vaccines that induce antibodies detectable by several assay methods. To compensate for some of these issues, some laboratories first conduct an immunofluorescent antibody assay or whole-cell ELISA tests to detect antibodies directed against B. burgdorferi and then use a WB test to differentiate between infection and vaccination. However, this two-step technique requires extra time and is more expensive, and the WB assay can be subject to various interpretations by individual laboratories and readers. An ELISA test (SNAP 3Dx, IDEXX Laboratories) has been developed for simultaneous diagnosis of E. canis and B. burgdorferi antibodies and D. immitis antigen. This ELISA uses a synthetic peptide (C 6 ) derived from invariable region (IR 6 ) as a diagnostic antigen and is highly specific for B. burgdorferi. 17 The test also provides for early diagnosis of E. canis (specificity 68

3 98.2%) 18 and D. immitis (specificity 100% 19 and sensitivity 67% to 100%, depending on the number of adult female worms present) In the study described here, this commercially available in-office ELISA test kit was used for simultaneous detection of B. burgdorferi and E. canis antibodies and D. immitis antigen to estimate the prevalence of these parasites in dogs in areas of Brazil and to evaluate specific criteria for correlations with infection rates. MATERIALS AND METHODS Study Dogs A total of 2,553 dogs presented to 138 vet- erinary practices in 12 Brazilian states (Table 1) were examined between July and October All dogs were owned by clients of the veterinary practice and were brought to the clinic for routine vaccinations, examinations, or other procedures. Although some dogs included in the survey exhibited signs characteristic of one or more of the organisms of interest, no attempt was made to select dogs on this basis. After obtaining owner s consent for the dog to participate in the survey, the veterinarian examined each dog and recorded identification and other information that could be considered predictive of infections (name, residence, sex, TABLE 1. Dirofilaria immitis and Ehrlichia canis Infection Detected in Dogs Residing in Areas of Brazil Using In-Office C 6 ELISA Test D. immitis Antigen E. canis Antibodies No Positive/ No Positive/ Region/State Total Tested (%) Total Tested (%) Northeast Ceará 1/11 (9.1) 5/11 (45.5) Pernambuco 0/105 (0.0) 52/105 (49.5) Alagoas 0/11 (0.0) 6/11 (54.5) Bahia 5/117 (4.3) 42/117 (35.9) Combined 6/244 (2.5) 105/244 (43.0) Southeast Minas Gerais 0/446 (0.0) 93/446 (20.9) Rio de Janeiro 16/421* (3.8) 125/422 (29.6) São Paulo 18/671 (2.7) 104/671 (15.5) Combined 34/1,518* (2.2) 322/1,539 (20.9) South Paraná 3/44 (6.8) 2/43 (4.7) Santa Catarina 7/142 (4.9) 1/142 (0.7) Rio Grande do Sul 1/356 (0.3) 6/356 (1.7) Combined 11/542 (2.0) 9/541 (1.7) Central Mato Grosso do Sul 0/126 (0.0) 45/126 (35.7) Distrito Federal 0/101 (0.0) 24/101 (23.8) Combined 0/227 (0.0) 69/227 (30.4) Total 51/2,551* (2.0) 505/2,551* (19.8) *No results were available for two dogs. No results were available for one dog. 69

4 Veterinary Therapeutics Vol. 4, No. 1, Spring 2003 life style [outside, inside, outside/inside], age, breed, clinical signs, traveling experience, previous treatments for E. canis infection, and heartworm chemoprophylaxis history) on a form specifically designed for this purpose. A wholeblood sample was collected from each dog and immediately tested at the clinic with the in-office C 6 ELISA diagnostic kit for D. immitis antigen and E. canis and B. burgdorferi antibodies. Results were recorded on the report forms. The C 6 ELISA Test Kit The in-office C 6 ELISA test can be conducted with canine serum, plasma, or whole blood. The C 6 synthetic peptide was conjugated to bovine serum albumin (BSA) and to horseradish peroxidase (HRP), using standard methods. The HRP C 6 peptide conjugate was contained in a conjugate diluent containing HRP-labeled antiheartworm antibody, HRP-labeled E. canis peptide conjugate, nonspecific proteins, and detergent. The B. burgdorferi or E. canis antibody or D. immitis antigen (if present) in the sample bind to the synthetic peptide HRP conjugate and to the synthetic peptide BSA conjugate. Two drops of blood, serum, or plasma were dispensed into a sample tube using the pipette provided with the kit. Five drops of conjugate were added to the sample, and this mixture was dispensed into a sample well in the test device. The deposited blood sample and conjugate mixture flowed through the matrix of the test device, which contained substrate reagents. The C 6 ELISA test was considered positive for B. burgdorferi, E. canis, or D. immitis if color developed in the designated reaction area of the matrix. A positive control area in the device was used to verify that the sample had been properly prepared and that the reagents were adequately reactive. Statistical Analysis Test results were tabulated using Proc FREQ (SAS Version 8.1). Probabilities for the overall comparison of categories within each factor (including Not Recorded as a valid level) were computed using Fisher's exact test. Differences were declared significant when P <.05. Percentages of animals positive for D. immitis and E. canis were calculated and tabulated by state and region, but were not tested for significance according to these criteria. Other factors evaluated for their influence on the frequency of infected dogs included age group, sex, lifestyle, travel history, preventative/medication, and clinical signs. RESULTS Several breeds were represented in the study, but the majority were mongrel, poodle, Labrador retriever, Doberman pinscher, German shepherd, cocker spaniel, rottweiler, dachshund, and boxer. Ages ranged from 3 months to 18 years; however, age was not recorded for 95 of the dogs. The distribution of sexes was similar, with 1,244 males and 1,297 females. Sex was not recorded for 10 dogs. Dirofilaria immitis Infection Test results for two dogs (both from Rio de Janeiro) were not available. Findings for D. immitis and E. canis are summarized in Tables 1 and 2. For all states and regions combined, 2% of the 2,551 dogs tested were positive for D. immitis antigen (Table 1). Infected dogs were identified in seven of the 10 states surveyed in the northeast, southeast, and south regions, and none of the 227 dogs from the two states represented in the central region tested positive for D. immitis (Table 1). There was some variability in infection rates among states, but some of this could be attributed to differences in sample size. Overall, the combined percentage of infected dogs was relatively similar among the three regions (2.0% to 2.5%). The proportion of heartworm-infected dogs detected by the test tended to increase signifi- 70

5 cantly (P <.01) with age (Table 2). No dog 2 years of age or younger was infected, whereas 1.5% of dogs 2.1 to 5.0 years of age, 3.2% of dogs 5.1 to 8 years of age, and 3.5% of dogs more than 8 years of age were infected. The prevalence of D. immitis was similar for males (1.8%), females (2.3%), and dogs for which sex was not recorded (Table 2). The dog s lifestyle appeared to play a significant (P <.01) role in the prevalence of D. immitis infection. Among dogs that lived outside, 3.1% were infected with D. immitis, compared with 1.9% of dogs that had access to inside and outside the owner s house and 0.1% for dogs kept inside with very restricted access to the outdoors (Table 2). Dogs that did not travel were more likely to be infected (P <.01) than were those that traveled with the owner or those for which travel data were not provided. Two dogs routinely given a heartworm preventative were positive for D. immitis infection. The name of the product was not recorded by the participating veterinarian. Both of these infected dogs were kept outside and lived in a coastal resort area in São Paulo. Clinical signs commonly associated with D. immitis infection were present in 21 of the 51 infected dogs (41.2%) as well as 774 of the 2,382 dogs (32.5%) not infected with D. immitis. Clinical signs were absent in 30 (58.8%) of the infected dogs. Ehrlichia canis Infection E. canis antibodies were detected in 19.8% of the dogs tested (Table 1). Infected dogs were from all four regions and in all 12 states. The highest prevalence was observed in the northeast region (43.0%) and the lowest infection rate (1.7%) was in the south. All age groups had positive dogs, and the prevalence was relatively similar among all ages (Table 2). Most E. canis antibody positive dogs (86.9%) had never been treated for ehrlichiosis before this study; however, 37 dogs (7.3%) had previously been treated for ehrlichiosis. Antibodies to E. canis were detected in 22.0% of the males and 17.4% of the female dogs surveyed. Dogs that lived outside had a slightly higher prevalence of E. canis (21.7%) than dogs that lived inside (17.4%) or those that lived inside/outside (18.3%), but the difference was not significant. Traveling experience did not increase the frequency of E. canis antibodies (18.2% for dogs that traveled versus 22.4% for those that did not). Clinical signs believed by the veterinary practitioner to represent those commonly seen with ehrlichiosis were present in 44.2% of the dogs positive for E. canis antibodies as well as in 12.2% of the dogs with no evidence of E. canis infection. Conversely, 49.5% of the E. canis infected dogs exhibited no clinical signs of infection. Eight dogs positive for D. immitis antigen were also positive for E. canis antibodies. Although these dogs were only 0.3% of the total number of dogs examined, they represented 16.3% of the heartworm-infected population. All eight dogs lived in the southeastern region of the country, either in Rio de Janeiro (n = 6) or São Paulo (n = 2). None of these positive dogs had previously received treatment for ehrlichiosis, nor had they received heartworm chemoprophylaxis. All eight lived outside. Borrelia burgdorferi Infection Only one dog tested positive for B. burgdorferi antibodies. This dog was a mixed breed, negative for E. canis and D. immitis, with no history of heartworm preventive medication or treatment for E. canis. The dog resided in a suburb of São Paulo. DISCUSSION D. immitis infection was detected in dogs residing in seven of the 12 Brazilian states surveyed in this study. For every state surveyed, the frequency of infection was much lower in the 71

6 Veterinary Therapeutics Vol. 4, No. 1, Spring 2003 TABLE 2. Results of In-Office C 6 ELISA Testing for Dogs Surveyed for Dirofilaria immitis and Ehrlichia canis Infections in Brazil Grouped by Various Categories D. immitis Antigen E. canis Antibodies No. Positive/ No. Positive/ Category Category Total (%) Category Total (%) Age (yr) < 2 0/367 (0.0) 73/367 (19.8) /1,042* (1.5) 207/1,043 (19.7) /565 (3.2) 122/563 (21.5) >8.0 17/481 (3.5) 86/481 (17.9) Not recorded 0/96* (0.0) 17/97 (17.9) P <.01 Sex Male 28/1,243 (2.3) 274/1,244 (22.0) Female 23/1,298 (1.8) 226/1,297 (17.4) Not recorded 0/10 (0.0) 5/10 (50.0) NS Lifestyle Inside 1/682 (0.1) 119/682 (17.4) Outside 39/1,248 (3.1) 270/1,247 (21.7) Both 9/471 (1.9) 86/470 (18.3) Unknown 2/150 (1.3) 30/152 (19.7) P <.01 Travel Yes 18/1146 (1.6) 208/1146 (18.2) No 33/1164 (2.8) 260/1162 (22.4) Not recorded 0/241 (0.0) 37/243 (15.2) P <.01 Clinical signs Yes 21/796 (2.6) 223/794 (28.1) No 30/1638 (1.8) 250/1638 (15.3) Not recorded 0/116 (0.0) 32/118 (27.1) NS Received preventive medication (D. immitis) or previous treatment (E. canis) Yes 2/65 (3.0) 37/85 (43.5) No 46/2,378 (1.9) 439/2,377 (18.5) Not recorded 3/108* (2.8) 29/89 (32.6) NS Total 51/2,551* (2.0) 505/2,551 (19.8) NS = not significant. *Findings for two dogs (one 5 years of age and one for which age was not recorded) were excluded from all categories for D. immitis. Test results for two dogs not available. 72

7 present study than that reported for dogs evaluated from 1986 through The overall prevalence (2%) found in this study was less than previously reported (7.9%) for 2,160 dogs from São Paulo, Rio de Janeiro, Santa Catarina, Rio Grande do Sul, Mato Grosso, and Minas Gerais between 1986 and In a 1990 study in Rio de Janeiro, dogs (21.3%) were positive for heartworm (either circulating microfilariae of D. immitis or occult infection), compared with 3.8% infection rate in the present study. The prevalence of D. immitis in São Paulo decreased from 8.8% 3 in a survey conducted between 1986 and 1988 to 2.7% in the present study. In Santa Catarina, a prevalence of 12% was reported in 1992, 6 compared with the current infection rate of 4.9%. In that same study, 6 the prevalence in Rio Grande do Sul was 1.1%, compared with 0.3% in the present study. No infected dogs were identified in Pernambuco or Alagoas in the present study, whereas infection rates were previously reported to be 2.3% 5 and 12.5%, 10 respectively, in earlier studies. This decrease in prevalence of D. immitis infection may reflect an increased awareness of veterinarians and dog owners about heartworm disease because of the more extensive research devoted to control and diagnosis of this parasite and the publication of beneficial information on annual antigen testing and monthly prevention. The availability of the more-convenient heartworm preventatives and better compliance in their administration provide opportunities for more efficient control of D. immitis. 8 Despite a substantial decrease in the prevalence of heartworm in this study compared with findings of surveys conducted between 1986 and 1999, it is evident that most dog owners in these areas of Brazil are not administering heartworm preventatives to their dogs. Preventive medication was first introduced in Brazil in and antigen in-house kits in The value of these measures may not be evident at first glance of the data in this study, with 3.0% of the dogs receiving a heartworm preventative found to be positive for D. immitis antigens versus 1.9% infection rate for dogs not receiving a heartworm preventative. However, closer examination of the data reveals that the sample sizes are highly skewed because the majority of the dogs in the study (>93%) were not receiving any prophylactic medication. There were actually only two dogs on chemoprophylaxis that tested positive for heartworm. These dogs lived outside at a coastal resort in the state of São Paulo, where the prevalence has been previously reported to be as high as 45%. 23 It is quite possible these two dogs became infected because of lack of compliance by the owner in the administration of the heartworm preventative Interviews in the past with dog owners conducted outside of this study have indicated that preventatives are often given only during months of the year with the highest expected risk. However, no attempt was made to interview the owners of these two infected dogs in this study, and no conclusions can be drawn regarding the efficacy (or lack thereof) of any heartworm preventative in these cases. Clinical signs commonly associated with D. immitis infection were present in several infected dogs and in a similar proportion of dogs that were not infected with D. immitis. Conversely, clinical signs were absent in the majority of infected dogs. Serologic screening for D. immitis should be part of the annual profile for dogs in Brazil regardless of the presence or absence of clinical signs of infection. A relatively high serologic prevalence (19.8%) of E. canis antibodies was found in all 12 Brazilian states surveyed. The lowest prevalence was found in the southern region states where the climate is more temperate than for the other regions included. Data are not available for the distribution of R. sanguineus ticks and other tick species that may transmit E. canis in areas of Brazil, but it can be speculated that there would be fewer ticks in an area with a temperate climate 73

8 Veterinary Therapeutics Vol. 4, No. 1, Spring 2003 than in areas with a humid tropical climate. Lifestyle, age group, or traveling experience did not seem to influence the prevalence of E. canis antibodies in this population of dogs. The most effective means of preventing ehrlichiosis is by controlling tick populations. 11 Therefore, veterinarians should educate dog owners regarding effective tick control programs. In addition, periodic surveillance studies on the prevalence of ticks and E. canis should be continued in these areas. Several dogs (n = 37) with E. canis antibodies had been previously treated for ehrlichiosis. Most E. canis antibody positive dogs (52.9%) were free of clinical signs, and several dogs (29.1%) displaying clinical signs believed to be indicative of E. canis infection were negative for this organism. In the clinical situation, this inconsistency makes clinical diagnosis difficult. Because prevention of E. canis is currently limited to tick prophylaxis, which cannot be accomplished in an efficient manner, annually laboratory screening should be considered for all dogs as good preventive medicine. The eight dogs infected with both D. immitis and E. canis lived outside in a heartworm-enzootic area. 6,23 Dogs living in high-risk situations require closer surveillance and should be considered prime candidates for chemoprophylaxis. Antibodies to B. burgdorferi were detected in only one dog in this study. However, other researchers have reported a higher incidence of B. burgdorferi antibodies in a smaller population of dogs in São Paulo. 14 Further investigation is needed to confirm the presence of Lyme borreliosis in the Brazilian dog population. There are no vaccines against B. burgdorferi currently available in Brazil. The in-office ELISA test kit was sensitive for D. immitis antigen, even with fewer than four female worms present; however, maximum sensitivity was achieved with burdens that included three or more female worms. 20,21 The test kit also is reported to be highly specific (98.2%) for E. canis antibodies as well as being highly specific (99.6%) and sensitive (94.4%) for B. burgdorferi. 17,18 Vaccination of dogs with currently available vaccines against B. burgdorferi did not induce a cross-reactive antibody response in the C 6 assays. 17,29 CONCLUSIONS Despite a downward trend in the prevalence of heartworm-infected dogs in several regions of Brazil over the past few years, it seems prudent to recommend that dogs in these regions should be maintained on monthly heartworm preventive medication, and testing for D. immitis antigen should be conducted annually. The high prevalence of ehrlichiosis in Brazil requires preventive measures (tick prophylaxis on a regular basis) and annual serologic testing for antibodies in dogs, even in the absence of clinical signs. There was little evidence of B. burgdorferi in the dogs in this study; however, results of previous investigations indicate that dogs in Brazil should continue to be tested for this organism. The in-office C 6 ELISA diagnostic kit for the simultaneous detection of B. burgdorferi and E. canis antibodies and D. immitis antigen was satisfactory for conducting these multiple screenings in a large number of dogs. The majority of criteria evaluated in this population sample (i.e., age, lifestyle, travel history) were correlated with the presence of absence of D. immitis, but did not appear to be predictive for the presence of E. canis. Clinical signs were unreliable indicators of D. immitis and E. canis infection in these dogs. Routine serology testing is recommended as the most reliable diagnostic method for identifying these organisms in dogs residing in high-risk areas of Brazil. ACKNOWLEDGMENTS The authors would like to thank the clinical veterinarians and dog owners in Brazil that participated in the conduct of this survey. 74

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