VETERINARY THERAPEUTICS SPRING 2003 (VOL 4, NO 1)

Serologic Prevalence of Dirofilaria immitis, Ehrlichia

canis, and Borrelia burgdorferi Infections in Brazil
by Norma Labarthe, DVM, DSc, Marcelo Pereira, DVM, PhD, Oclydes Barbarini, DVM, William McKee, DVM,
Carlos Coimbra, BM, MSc, Johnny Hoskins, DVM, PhD

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 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. canisantibodies and D. immitis antigen. D. immitis infection was detected in
51 dogs (2.0%) and E. canisantibodies were present in 505 dogs (19.8%). Only one dog tested
positive for B. burgdorferiantibodies.

Introduction
Dirofilariasis, ehrlichiosis, and Lyme borreliosis are arthropod-borne diseases that can 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. immitisantigen 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.3-10 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
survey12conducted 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 (Paran) study,13 14
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, Yoshinari15 and colleagues from the University of So Paulo reported that
Lyme disease had been confirmed in 25 human patients since 1989. A recent evaluation 16 of
237 dogs in So 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 ofE. canis and B. burgdorferi antibodies and D. immitis antigen. This ELISA uses a
synthetic peptide (C6) derived from invariable region (IR6) as a diagnostic antigen and is highly
specific for B. burgdorferi.17 The test also provides for early diagnosis of E. canis (specificity
98.2%)18 and D. immitis (specificity 100%19 and sensitivity 67% to 100%, depending on the
number of adult female worms present).18-21 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. immitisantigen 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 veterinary practices in 12 Brazilian states (Table 1) were
examined between July and October 2001. 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, 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 whole-blood
sample was collected from each dog and immediately tested at the clinic with the in-office
C6 ELISA diagnostic kit for D. immitisantigen and E. canis and B. burgdorferi antibodies.
Results were recorded on the report forms.
The C6 ELISA Test Kit
The in-office C6 ELISA test can be conducted with canine serum, plasma, or whole blood.
The C6synthetic peptide was conjugated to bovine serum albumin (BSA) and to horseradish
peroxidase (HRP), using standard methods. The HRP-C6 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 peptideHRP 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
C6ELISA 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 andE. canis are summarized in Table 1 and Table 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 significantly
(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.
immitisinfection. 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. immitisinfection. 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 So 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 forE. 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 So 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 So 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
present study than that reported for dogs evaluated from 1986 through 2000. 3-10 The overall
prevalence (2%) found in this study was less than previously reported (7.9%) for 2,160 dogs
from So Paulo, Rio de Janeiro, Santa Catarina, Rio Grande do Sul, Mato Grosso, and Minas
Gerais between 1986 and 1988.3 In a 1990 study in Rio de Janeiro,4 124 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 So
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 199222 and antigen in-house kits in 1997. 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 So 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.23-28 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 ofR. 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 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 So 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.
canisantibodies as well as being highly specific (99.6%) and sensitive (94.4%) for B.
burgdorferi.17,18Vaccination of dogs with currently available vaccines against B. burgdorferi did
not induce a cross-reactive antibody response in the C6 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.
This study was supported by funds from IDEXX Laboratories, Westbrook ME and Laboratrios
Pfizer Ltda.Diviso de Sade Animal, So Paulo, Brazil.

Phillips T: Tick-borne zoonoses. Four potential problems for pets and people. Pet Vet 3(2):1828, 1991.
2. Abraham D: Biology of Dirofilaria immitis, in Boreham PFL, Atwell RB (eds): Dirofilariasis.
Boca Raton, CRC Press, 1988, pp 29-46.
3. Guerrero J, Vezzoni A, Ducos de Lahitte J, et al: Distribution of Dirofilaria immitis in selected
areas of Europe and South America. Proc Heartworm Symp '89:13-18, 1989.
4. Guerrero J, Ducos de Lahitte J, et al: Update on the distribution of Dirofilaria immitis in dogs
from Southern Europe and Latin America. Proc Heartworm Symp '92:31-37, 1992.
5. Alves LC, Silva LVA, Faustino MAG, et al: Survey of canine heartworm in the city of Recife,
Pernambuco, Brazil. Mem Inst Oswaldo Cruz 94 (5):587-590, 1999.
6. Labarthe NV, Arajo AM, Bordin EL, et al: Update on the distribution of Dirofilaria immitis in
dogs in Brazil. Proc XVII WSAVA:287-289, 1992.

7. Labarthe N, Serro ML, Melo YF, et al: Heartworm in dogs in the state of Rio de Janeiro,
Brazil.Recent Advances in Heartworm Disease: Symp '98:67-73, 1998.
8. Labarthe N, Serro ML, Melo YF, et al: Potential vectors of Dirofilaria immitis (Leidy, 1856) in
Itacoatiara, oceanic region of Niteri municipality, state of Rio de Janeiro, Brazil. Mem Inst
Oswaldo Cruz 93(4):425-432, 1998.
9. Brito AC, Viana LS, Duarte EM, et al: Dirofilaria immitis infection in dogs from Macei,
Alagoas, Northeast region of Brazil. Arq Bras Med Vet Zootec 52(3):210-211, 2000.
10. Calheiros CML, Duate EM, Tenorio IA, et al: Dirofilaria immitis canina em Macei-AL. Rev
Pat Trop 23:271, 1994.
11. Couto CG: Rickettsial diseases, in Birding SJ, Sherding RG (eds): Saunders Manual of
Small Animal Practice, ed 2. New York, WB Saunders, 2000, pp 126-127.
12. O'Dwyer LH, Massard CL, Pereira de Souza JC: Hepatozoon canis infection associated
with dog ticks of rural areas of Rio de Janeiro State, Brazil. Vet Parasitol 94(3):143-150, 2001.
13. Dagnone AS, Autran de Morais H, Vidotto O, Jojima FS: Ehrlichiosis in anemic,
thrombocytopenic, or tick-infested dogs from a hospital population in south Brazil
[abstract]. Proc 27th WSAVA 2002. Available
at: http://www.vin.com/proceedings/Proceedings.plx?CID=WSAVA2002&PID=2786; accessed
February 1, 2003.
14. Costa IP, Yoshinara NH, Barrow PJ, et al: Lyme disease in Mato Grosso do Sul State,
Brazil: Report of three clinical cases, including the first of Lyme meningitis in Brazil [in
Portuguese]. Rev Hosp Clin Fac Med So Paulo 51(6):253-257, 1996.
15. Yoshinari NJ de Barros PJ, Bonoldi VL, et al: Outline of Lyme disease in Brazil [in
Portuguese].Rev Inst Med Trop So Paulo 52(2):111-117, 1997.
16. Joppert AM, Hagiwara MK, Yoshinari NH: Borrelia burgdorferi antibodies in dogs from Cotia
county, So Paulo, Brazil. Rev Inst Med Trop So Paulo 43(5):251-255, 2001.
17. Levy S, O'Connor TP, Hanscom JL, Shields P: Utility of an in-office C 6 ELISA test kit for
determination of infection status of dogs naturally exposed to Borrelia burgdorferi. Vet
Ther3(3):308-315, 2002.
18. Idexx Laboratories: Testing for tick-borne diseases: Your questions are
answered. Diagnostic Edge 1(2)2-, 2000.
19. Atkins C: Unpublished data. 2000.

20. McCall JW, Supakorndej N, Donaghue AR, Turnbull RK: Study to determine the
performance of commercially available canine heartworm antigen test kits[abstract]. Proc
AAVP 2000.
21. Courtney CH, Zeng Q: Comparison of heartworm antigen test kit performance in dogs
having low heartworm burdens. Vet Parasitol 96(4):317-322, 2001.
22. Labarthe N, Alves LC, Serro ML: Dirofilariose em Pequenos Animais e como Zoonose, in
Almosny NRP, ed. Hemoparasitoses em Pequenos Animais e como Zoonoses. Rio de Janeiro,
LF Livros, 2002, pp 111-135.
23. Duque-Arajo AM, Labarthe N, Luvisrio et al: Filariose canina no Estado de So Paulo
Brasil, in Anais do IV Congresso Ibrico de Parasitologia: 93-94, Santiago de Compostela,
Espanha, 1995.
24. Paul AJ, Todd KS, Wallace DH, et al: Efficacy of a chewable formulation containing
ivermectin and pyrantel pamoate against the development of Dirofilaria immitis in dogs 30 days
post infection.Proc Heartworm Symp '92:197-199, 1992.
25. Plue RE, Jernigan AD, Acre KE, et al: Field efficacy, safety, and acceptability of ivermectin
plus pyrantel in growing and adult dogs. Proc Heartworm Symp '92: 205-208, 1992.
26. Bater AK: Efficacy of oral milbemycin against naturally acquired heartworm infection in
dogs.Proc Heartworm Symposium '89:107-113, 1989.
27. Bradley re: Dose titration and efficacy of milbemycin oxime for prophylaxis
against Dirofilaria immitis infection in dogs. Proc Heartworm Symp '89:115-120, 1989.
28. Clemence RG, Sarasola P, Genchi C, et al: Efficacy of selamectin in the prevention of adult
heartworm (Dirofilaria immitis) infection in dogs in northern Italy. Vet Parasitol 91:251-258;
2000.
29. Liang FT, Jacobson RH, Straubinger RK, et al: Characterization of a Borrelia
burgdorferi VlsE invariable region useful in canine Lyme disease serodiagnosis by enzymelinked immunosorbent assay. J Clin Microbiol 38:4160-4166, 2000.