Journal of Advanced Veterinary Research

Volume 10, Issue 2, 2020, Pages: 61-65


(ISSN: 2090-6277/2090-6269/ © 2011-2020 JAVR)


Serological Survey of Ehrlichia canis, Babesia canis and Leishmania infantum in a Brazilian Canine Population


Tainá Luana Vieira Lopes Zuchi*, Luciana Corassa, Gustavo Bonetto, Cláudia Luana Lopatini, Júlia Balena Spricigo, Soraya Regina Sacco Surian, Diogenes Dezen, Joice Lara Maia Faria


Federal Institute of Santa Catarina, Concórdia campus, Highway SC 283, km 17, Concórdia, Santa Catarina 89703-720, Brazil.


Received: 23 January 2020; Accepted: 18 March 2020

(*Corresponding author:




This study evaluated occurrences of babesiosis, ehrlichiosis and leishmaniasis, and the presence of coinfection due to these diseases among dogs in the municipality of Concordia, in the west of the state of Santa Catarina (SC), Brazil. Blood was collected from 424 dogs that were attended at the Clinical Practice Center of the Federal Institute of Santa Catarina, Concordia, and also in clinics in the city and its rural zone. The presence of antibodies against Leishmania infantum was investigated using the Indirect Immunofluorescence reaction and against Babesia canis and Ehrlichia canis using the Enzyme-Linked Immunosorbent Assay. The chi-square test at the significance level of 5% was used to compare the positive and negative animals in the sampled population. Out of the 424 samples evaluated, 170 (40.09%) were positive for ehrlichiosis, 178 (41.98%) were positive for babesiosis, and 59 (13.91%) were positive for leishmaniasis. There were statistical differences regarding ehrlichiosis in relation to the variables of area, street access and breed. Moreover, among the positive samples, there were coinfection reactions. From these results, it can be suggested that these three diseases are present in the municipality of Concordia, SC.


Keywords: Babesiosis, Dog, Ehrlichiosis, Leishmaniasis.




Canine ehrlichiosis, babesiosis and leishmaniasis are diseases transmitted through vectors that have a high incidence in many countries around the world (Greene, 2012). The diseases unleashed by these hemoparasites can trigger similar clinical-laboratory alterations in their hosts, and this may lead to conflicting diagnoses (Miró et al., 2013; Maggi et al., 2014).

The agent that causes canine ehrlichiosis is Ehrlichia canis (Dumler et al., 2001), which is mainly transmitted by the tick Rhipicephalus sanguineus (Lewis et al., 1977). In its subclinical phase, antibody titers remain in serum for periods greater than six months, thus indicating that the duration of infection is prolonged and that antigen stimulation occurs (Waner et al., 1997; Harrus et al., 1998). Thus, dogs are left incapable of setting up an effective immune response and remain chronically infected (Harrus et al., 1998). For these cases, Enzyme-Linked Immunosorbent Assay (ELISA) is one of the serological methods that mostly used for diagnosis of the disease (Waner et al., 2001).

Canine babesiosis is caused by the protozoa Babesia canis and Babesia gibsoni, and it is transmitted by the tick R. sanguineus (Ayoob et al., 2010). In babesiosis, some animals may remain in the subclinical phase (Macintire et al., 2002), which makes the ELISA test useful in these cases as well. Moreover, this test also makes it possible to diagnose babesiosis in situations of low parasitemia in both the chronic and the subacute phase (Olicheski, 2003).

The vectors involved in the transmission of leishmaniasis that are found in Brazil are Lutzomyia longipalpis and Lutzomyia cruzi (Santos et al., 1998; Lainson and Shaw, 1987). Domestic dogs are the main reservoir for Leishmania infantum, and they play an essential role in the transmission of this disease (Castro-Junior et al., 2014), both because of their proximity to humans and because many dogs are positive but asymptomatic. Domestic dogs act as carriers of the parasite and form a source of contamination for the vectors that transmit the disease (Marzochi et al., 2009). Serological tests like Indirect Immunofluorescence (IIF) are frequently used to detect this disease in epidemiological surveys (Alves and Bevilacqua, 2004).

In this light, the present study had the aim of evaluating occurrences of E. canis, B. canis and L. infantum, in dogs in the municipality of Concordia, in the west of the state of Santa Catarina, and also the presence of coinfection among the hemoparasites.


Materials and methods


Experimental animals


Serum from 424 dogs (Canis familiaris) was used in this study. All of these samples were used in evaluating occurrences of ehrlichiosis, leishmaniasis and babesiosis. The dogs selected could either be presenting clinical alterations compatible with hemoparasites, or could be asymptomatic. All of them were attended either at the Clinical Practice Center of the Federal Institute of Santa Catarina, Concordia or at private clinics in the city or on farms in this municipality's rural zone. From each dog, 5 mL of total blood was collected. The serum was then separated and was used to detect anti-E. canis, anti-B. canis and anti-L. infantum antibodies. From the animals that were sampled, information on sex, access to the streets, place of residence, age and breed was compiled. The samples collected were stored at -20 °C until the time of processing. This study was approved by the Ethics Committee for the use of animals, at the Federal Institute of Santa Catarina, under protocol number 31/2014.


ELISA for the detection of anti-E. canis and anti-Babesia canis vogeli antibodies


To detect anti-E. canis and anti-Babesia canis vogeli antibodies, the ELISA test was used, through kits marketed by the Imunodot laboratory (Imunotest, Brazil). A sample dilution of 1:200 μL was used, and the reaction was conducted following the manufacturer's instructions. In the end, the plates were read in an ELISA reader with a filter of 405 nm (Mindray MR-96A). To interpret the results, a cutoff index was calculated (average optical density of the negative control serum, multiplied by 2.5). The samples that presented intense yellowish coloration and an optical density greater than or equal to the cutoff index were considered to be positive for E. canis and B. canis.


Indirect immunofluorescence (IIF) test for the detection of anti-L. infantum antibodies


To perform the IIF test, kits marketed by Imunodot (Imunotest, Brazil) for L. infantum were acquired. The tests were run using a sample dilution of 1:40 μL. The readings were performed under an optical microscope using inverted light/fluorescence (BEL Photonics- FLUO 3). The samples were considered positive if greenish fluorescence from the parasites was present across the entire surface, and negative if there was no fluorescence.


Statistical analysis


The chi-square test at the significance level of 5% was used to compare the variables (sex, street access, place of residence, age and breed of the animals) between the positive and negative animals of the sampled population, using Minitab 19 statistical software.




Out of the 424 samples evaluated, 170 (40.09%) were positive for ehrlichiosis, 178 (41.98%) for babesiosis and 59 (13.91%) for leishmaniasis, as displayed in Tables 1, 2 and 3, respectively. Only for ehrlichiosis were there statistically significant differences (P ≤ 0.05) in relation to the place of residence, street access and breed (P = 0.0001, 0.0010 and 0.0081 respectively). Moreover, among the 424 animals, 10 (2.36%) presented co-infection among the three diseases: 69 (16.27%) between babesiosis and ehrlichiosis, 19 (4.48%) between ehrlichiosis and leishmaniasis and 17 (4.00%) between babesiosis and leishmaniasis.


Table 1. Seropositivity for anti-E. canis antibodies, according to ELISA, among dogs in the municipality of Concordia, Santa Catarina.




Positive (n/%)

Negative (n/%)



100/170 (58.82)

169/254 (66.53)


70/170 (41.18)

85/254 (33.46)


Urban areaa

113/170 (66.47)

223/254 (87.80)

Rural areab

57/170 (33.53)

31/254 (12.20)

Street access

With street accessa

86/170 (50.59)

88/254 (34.64)

Without street accessb

84/170 (49.41)

166/254 (65.35)



96/170 (56.47)

125/254 (49.21)


10/170 (5.88)

38/254 (14.96)

German Shepherdb

8/170 (4.70)

2/254 (0.79)


6/170 (3.53)

21/254 (8.27)

Other breedsa

50/170 (29.41)

68/254 (26.77)


Less than 1 yeara

8/170 (4.70)

15/254 (5.90)

Between 1 and 5 yearsa

67/170 (39.41)

78/254 (30.70)

Between 5 and 10 yearsa

49/170 (28.82)

74/254 (29.13)

Over 10 yearsa

35/170 (20.59)

74/254 (29.13)

Not informeda

11/170 (6.47)

13/254 (5.12)


a,b Different lowercase letters indicate the presence of a statistically significant difference, with a significance level of 5% (P < 0.05).


Table 2. Seropositivity for anti-B. canis antibodies, according to ELISA, among dogs in the municipality of Concordia, Santa Catarina.




Positive (n/%)

Negative (n/%)



117/178 (65.73)

151/246 (61.38)


61/178 (34.27)

95/246 (38.61)


Urban area

146/178 (82.02)

190/246 (77.23)

Rural area

32/178 (17.98)

56/246 (22.76)

Street access

With street access

71/178 (39.89)

104/246 (42.28)

Without street access

107/178 (60.11)

142/246 (57.72)



91/178 (51.12)

130/246 (52.84)


17/178 (9.55)

31/246 (12.60)


8/178 (4.49)

20/246 (8.13)

Other breeds

62/178 (34.83)

65/246 (26.42)


Less than 1 year

5/178 (2.81)

19/246 (7.72)

Between 1 and 5 years

64/178 (35.95)

81/246 (32.93)

Between 5 and 10 years

56/178 (31.46)

73/246 (29.67)

Over 10 years

39/178 (21.91)

63/246 (25.61)

Not informed

14/178 (7.86)

10/246 (4.06)



Table 3. Seropositivity for anti-L. infantum antibodies, according to the Indirect Immunofluorescence (IIF) reaction, among dogs in the municipality of Concordia, Santa Catarina.



Positive (n/%)

Negative (n/%)



37/59 (62.71)

232/365 (63.56)


22/59 (37.29)

133/365 (36.44)


Urban area

48/59 (81.35)

288/365 (78.90)

Rural area

11/59 (18.65)

77/365 (21.10)

Street access

With street access

19/59 (32.20)

156/365 (42.74)

Without street access

40/59 (67.80)

209/365 (57.26)



36/59 (61.02)

185/365 (50.68)


5/59 (8.47)

43/365 (11.78)


4/59 (6.77)

25/365 (6.85)

Other breeds

14/59 (23.73)

112/365 (30.68)


Less than 1 year


22/365 (6.03)

Between 1 and 5 years

23/59 (38.98)

122/365 (33.42)

Between 5 and 10 years

17/59 (28.81)

114/365 (31.23)

Over 10 years

17/59 (28.81)

85/365 (23.29)

Not informed

2/59 (3.39)

22/365 (6.03)






Studies conducted in Brazil have shown that the seroprevalence of ehrlichiosis in dogs is similar among the regions, for example: 31.2% in northern Brazil, 42.5% in the central region, 35.6% in the northeast and 44.7% in the southeast (Aguiar et al., 2007; Costa et al., 2007; Saito et al., 2008; Silva et al., 2010; Souza et al., 2010). However, in one study conducted in the southern region by Saito et al. (2008), among the 389 dogs surveyed, only 19 (4.8%) were positive for E. canis. Another study developed by Labarthe et al. (2003), also showed a low prevalence of E. canis in the southern region, comprising 4.7% in Parana, 1.7% in Rio Grande do Sul and 0.7% in Santa Catarina. The present study is divergent from theirs, in that we observed seroprevalence of 40.09% among the animals. Thus, the prevalence found in the municipality of Concordia is similar to that of other regions of Brazil. This may suggest that the disease is occurring in an endemic form.

Regarding the seropositivity for babesiosis that was found in this study, it was observed that this finding corroborates the results from studies conducted in other regions, such as 42.2% in Sao Paulo, 51.4% in Minas Gerais and 35.7% on Paraná. In all of these studies, the IIF technique was used (Dell’ Porto et al., 1993; Milken et al., 2004; Trapp et al., 2006). Based on these results, it can be suggested that this disease is endemic in this municipality.

In the present study, it was observed that 59 (13.91%) of the animals tested showed antibodies for L. infantum. This result allows us to suggest the disease is occurring in Concordia, and this serves as a warning: if a focus of the transmitting agent is present, this municipality will be in a situation of risk regarding the transmission of this disease between dogs and humans.

Most animals that are seropositive for leishmaniasis are asymptomatic (Barata et al., 2013; Penaforte et al., 2013). This emphasizes the importance of dogs as domestic reservoirs for L. infantum (Silva, 2007). The data of the present study reinforce this, since not all the positive animals showed clinical signs consistent with the disease.

Maziero et al. (2014) conducted a study in the west of the state of Santa Catarina on 252 animals and found that, among them, 43 showed seropositivity for L. infantum through the IIF test. These authors also suggested this region is exposed to the agent, which was thus in line with the findings of the present study. Another occurrence that reinforces the suggestion that the western region is exposed to the agent was the leishmaniasis case that was confirmed in the municipality of Chapeco.

Coinfection between babesiosis and ehrlichiosis was found in 69 (16.27%) of the animals, and this rate was higher than what has been reported in the literature. In a study conducted by Krawczak et al. (2015), nine (9.37%) out of 96 animals showed coinfection, using the IIF test. Fonseca et al. (2017) detected a rate of 5.6% in their study, using the same serological test. Krawczak et al. (2015) also observed (via the IIF and ELISA tests) that 4.3% of the dogs examined were positive for ehrlichiosis and leishmaniasis. The findings of the present study were in line with theirs, since coinfection between ehrlichiosis and leishmaniasis occurred in 4.48% (19/424) of our animals.

In Campo Grande, Mato Grosso do Sul, a study conducted by Sousa (2012), demonstrated that some dogs that were serologically positive for leishmaniasis also showed antibodies against babesiosis and ehrlichiosis, among which 39 (65%) were co-infected with ehrlichiosis, 26 (43.33%) with babesiosis and 20 (33.33%) with both of these. In the present study, considering the same perspective, the percentage of the dogs that were serologically positive for leishmaniasis and presented coinfection was lower, such that 32.2% (19/59) were coinfected with ehrlichiosis, 28.81% (17/59) with babesiosis and 16.95% (10/59) with both of these diseases. This was possibly due to the favorable climatic conditions in the Campo Grande region for vector development (Salgado, 2006). Moreover, the situation regarding leishmaniasis in the state of Mato Grosso do Sul is “worrisome”, according to this state’s health department (Sousa, 2012).

The existence of cross-reactions among antibodies is questionable in relation to diagnosing blood parasitoses. Ferreira et al. (2007) reported that cross-reactivity due to E. Canis occurred in animals that were seropositive for leishmaniasis. Mettler et al. (2005) identified reactivity due to B. canis in relation to the same disease. However, studies conducted in endemic areas have found through serological tests that there are no cross-reactions between E. canis, B. canis and L. infantum but, rather, occurrences of coinfection (Oliveira et al., 2000, Krawczak et al., 2015). Because of these discrepancies in the results, additional studies on the samples from Concordia are needed. Such studies would use techniques like molecular biology to identify the DNA of these agents in the samples obtained through the present study. Only in this manner will it become possible to ascertain how disseminated these diseases are and how many of these seropositive samples might have resulted from cross-reactions with other pathogens.




From the present study, it can be suggested that canine visceral leishmaniasis is a disease that is present in the municipality of Concordia. Given that canine cases precede human cases, there is a need to study the presence of Phlebotominae in this city. Regarding babesiosis and ehrlichiosis, it can be suggested that both of these diseases are endemic in this municipality. Lastly, studies using molecular biology techniques are needed in order to evaluate the possibility of cross-reactions with other pathogens and thus ascertain the real seropositivity.




Preliminary results were presented as an Abstract at the X MICTI - Mostra Nacional de Iniciação Científica e Tecnológica Interdisciplinar e III IFCultura, Balneário Camboriú, 8th-9th November 2017 and at the VII Mostra de Iniciação Científica, Concórdia, 20th September 2017.


Conflict of Interests


Authors declared no conflict of interests exists.




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