Molecular diagnosis of Ehrlichia canis and Babesia vogeli, and serological diagnosis of Neospora caninum and Toxoplasma gondii infection in dogs from the municipality of Cândido Sales-BA and microregion

[Diagnóstico molecular de infecção por Ehrlichia canis e Babesia vogeli e diagnóstico sorológico de infecção por Neospora caninum e Toxoplasma gondii em cães provenientes do município de Cândido Sales-BA e microrregião]

ABSTRACT

The occurrence of Toxoplasma gondii, Neospora caninum, Ehrlichia canis and Babesia vogeli in dogs from the municipality of Cândido Sales, Bahia, was investigated. Total and peripheral blood samples were obtained from 131 dogs. Blood smears were performed to check for morulae. Toxoplasma gondii and N. caninum infections were determined by an Indirect Immunofluorescence Reaction. Nested-PCR and PCR were used for the diagnoses of E. canis and B. vogeli infections, respectively. Additionally, the risk factors associated with infection by these agents were analyzed. The frequency of infection was 70.2% for N. caninum, 67.9% for T. gondii and 37.4% for E. canis. None of the dogs tested positive for B. vogeli. Morulae of Ehrlichia spp. were identified in two animals and the piroplasm in one animal. Age (> 3 years) was considered a risk factor for infections by E. canis and N. caninum and the rural habitat for infections by N. caninum. Co-infections were frequent, mainly with N. caninum and T. gondii (45.03% of dogs). Ehrlichia canis infection was significantly associated with N. caninum and T. gondii infection. These findings indicated a high occurrence of T. gondii, N. caninum, and E. canis in the studied region.

Keywords:
babesiosis; epidemiology; ehrlichiosis; neosporosis; toxoplasmosis

RESUMO

Avaliou-se a ocorrência de infecções por Toxoplasma gondii, Neospora caninum, Ehrlichia canis e Babesia vogeli em cães do município de Cândido Sales - BA. Para isso, foram obtidas amostras de sangue total e periférico de 131 cães. Esfregaços sanguíneos foram confeccionados para verificação de mórulas. Infecções por T. gondii e N. caninum foram determinadas pela reação de imunofluorescência indireta. O diagnóstico das infecções por E. canis e B. vogeli foi feito por meio de Nested-PCR e PCR, respectivamente. Adicionalmente, foram analisados os fatores de risco associados à infecção por esses agentes. Verificou-se frequência de infecção de 70,2% para N. caninum, 67,9% para T. gondii e de 37,4% para E. canis. Nenhum cão foi positivo para B. vogeli. Mórulas de Ehrlichia spp. foram identificadas em dois animais, e piroplasma em um. A idade (acima de 3 anos) foi fator de risco para infecções por E. canis e N. caninum, e hábitat rural para N. caninum. Coinfecções foram frequentes, principalmente por N. caninum e T. gondii (45,03%). Infecção por E. canis foi associada significativamente às infecções por N. caninum e T. gondii. Os achados indicam elevada ocorrência de T. gondii, N. caninum, e E. canis na região.

Palavras-chave:
babesiose; epidemiologia; erliquiose; neosporose; toxoplasmose

INTRODUCTION

The protozoan parasites Toxoplasma gondii and Neospora caninum belong to the phylum Apicomplexa and are obligate intracellular parasites of major importance in veterinary medicine. They have a wide geographical distribution and are found in several parts of the world (Tenter et al., 2000). Dogs, humans, and other species are considered intermediate hosts of T. gondii. In these animals, the infection can either show no symptoms or have a major clinical presentation, particularly in immunocompromised individuals. The evaluation of the seroprevalence of this agent in canine species is of great importance because the diagnosis in these animals can reveal environmental and human contamination (Ratzlaff et al., 2018). In addition, dogs can act as definitive hosts of N. caninum which, in areas with cattle, can lead to severe economic losses because infection by this agent is mainly related to episodes of pregnancy loss in cows (Dubey et al., 2007).

Further, diseases transmitted by ticks are of great importance because the same species can transmit several pathogens, favoring the presence of co-infections in animals. Among various hemoparasites, babesiosis and ehrlichiosis are transmitted by ticks. In Brazil, canine babesiosis has great prominence, and despite reports showing sporadic occurrence of other species, like B. gibsoni reports in the south of the country, B. vogeli is mainly responsible for the disease in dogs (Dantas-Torres et al., 2021). Similarly, E. canis is of great epidemiological importance, and is transmitted mainly through Rhipicephalus sanguineus, which is known as the “brown tick,” and in Brazil, uses dogs predominantly as its hosts (Dantas-Torres et al., 2004; Vieira et al., 2021). The occurrence of these diseases in dogs in Brazil is highlighted by the tropical climate, which favors the vector's life cycle (Soares et al., 2017).

Moreover, the close proximity between dogs and humans may also favor the transmission of common agents between both species, such as E. canis and other hemoparasites (Cunha et al., 2009).

Several coinfections in dogs have been described, which can lead to clinical problems and are common in areas where several etiological agents occur concomitantly. Epidemiological studies related to these pathogens are of great importance because of the growing connection between canine species and humans, which necessitates the establishment of better care and strategic actions for the prevention of zoonoses and other important diseases for animal health (Ratzlaff et al., 2018).

To date, there have been few studies in Bahia on the frequency of infection by T. gondii, N. caninum, E. canis, and B. vogeli in dogs, while no studies have been conducted specific to the south-central mesoregion of the state. The objective of this study was to evaluate the occurrence of infection by these agents in the region of Cândido Sales, Bahia, Brazil.

MATERIALS AND METHODS

This study included 131 domesticated domiciled and semi-domiciled dogs from urban and rural areas of the south-central region of Bahia, including the municipality of Cândido Sales and a rural district of Vitória da Conquista (Cercadinho), located near the city of Cândido Sales. At the time of the study, the city of Cândido Sales, Bahia, had no medical veterinary assistance. The methodology was approved by the Ethics Committee on Animal Use of the Universidade Estadual de Santa Cruz - UESC (CEUA/UESC) (protocol 005/21). The inclusion of the animals in the study and the sampling of biological material occurred only after authorization by the tutors and obtaining written informed consent.

After the animals were physically restrained, approximately 5 ml of venous blood was collected by cephalic puncture, which was dispensed into two tubes, one with the anticoagulant ethylenediaminetetraacetic acid (EDTA) and the other with a clot activator for later molecular and serological tests, respectively.

Blood smears were prepared from the whole and peripheral blood samples (ear tips). The slides were stained with fast panoptic dye and analyzed under a binocular optical microscope (1600x Olen K55ba Kasvi) to study and identify hematozoa or other inclusions. Additionally, a questionnaire was completed to investigate the history and management of the animals and verify the risk factors associated with infections.

The blood samples stored in the tubes with clot activator were centrifuged to obtain the serum, which was stored in 2 ml microtubes at a temperature of -20°C until the IFA serological technique was performed to detect the IgG antibodies anti-N. caninum and anti-T. gondii.

The IFA technique for the detection of anti-N. caninum and anti-T. gondii antibodies were used as previously described by Gondim et al. (2001), and Valadas et al. (2010), respectively. To visualize the reactions, anti-Dog IgG-F7884 conjugate (Sigma Aldrich ®) with a 1:128 dilution, labeled with fluorescein isothiocyanate, was used. The slides were examined under an epifluorescence microscope (BX51 ®; Olympus). Reactions in which total peripheral fluorescence was observed in > 50% of tachyzoites were considered positive. Positive and negative controls were obtained from samples from previous studies in the region. Benchmarks were set at 1:50 for N. caninum and 1:16 for T. gondii.

The blood samples packed in tubes with an anticoagulant agent were subjected to DNA extraction using the phenol-chloroform-isoamyl alcohol method (Ghaheri et al., 2016). The final DNA concentration in each sample was determined by spectrophotometry using a Nanodrop spectrophotometer (260 nm optical density). E. canis infection was diagnosed using nested-PCR technique, as previously described by Murphy et al. (1998). The following primers were used for the first reaction: ECC (5’-AGAACGAACGCTGGCGGCAAGC-3’) and ECB (5’-CGTATTACCGCGGCTGCTGGCA-3’), which amplify a fragment of the 16S gene of the genus Ehrlichia. For the second reaction, the primers ECAN (5’-CAATTATTTATAGCCTCTGGCTATAGGA-3’) and HE3 (5-‘TATAGGTACCGTCATTATCTTCCCTAT-3’) were used to obtain a final product of 396 bp of E. canis DNA. The sensibility and specificity of this technique was established by Murphy et al. (1998). Samples positive for E. canis were used as the positive control, while ultrapure water was used as the negative control.

Infection by B. vogeli was diagnosed using conventional PCR, in which the primers CAN 626R (5'-GAA CTC GAA AAA GCC AAA CGA-3') and CAN 172F (5'-GTT TAT TAG TTT GAA ACC CGC-3'), specific to B. vogeli, were used to amplify a fragment of the 18s ribosomal RNA gene (450 bp) (Inokuma et al., 2004). Positive samples for B. vogeli were used as the positive control, whereas ultrapure water was used as the negative control.

The amplified products in the nested-PCR reactions for E. canis and in the PCR reactions for B. vogeli were subjected to 2% agarose gel electrophoresis containing SYBR® Safe DNA Gel Stain (Invitrogen®). The TAE buffer was used for running, and electrophoresis was performed at 75V/200mA for 40 min. A 1 Kb Plus DNA Ladder marker (Invitrogen®) was used to determine the size of the amplified products. The bands were verified using an ultraviolet transilluminator, followed by evaluation using a Locus Biotechnology L-Pix Transilluminator.

Statistical analysis of the risk factors associated with infections was performed with bivariate and multivariate analyses using Epi Info 3.5.2. For bivariate analysis, the chi-square test was used to associate each independent variable with the dependent variables (positive or negative animals). Variables with a p-value ≤ 0.3 were selected for multivariate analysis using unconditional logistic regression. Statistical significance was set at p < 0.05.

RESULTS

In the evaluation of blood smears, structures compatible with morulae of Ehrlichia spp. were identified in the neutrophils of the whole blood of two animals. Piroplasma was identified in red blood cells in an ear tip blood smear of a dog. Through serological diagnosis, a frequency of 70.2% (92/131) and 67.9% (89/131) was obtained for N. caninum and T. gondii, respectively. Nested-PCR detected a positivity rate of 37.4% (49/131) in dogs tested for E. canis. None of the evaluated animals tested positive for B. vogeli. Of the two dogs that presented morulae of Ehrlichia spp. in the evaluation of blood smears, only one tested positive for E. canis in the molecular diagnosis.

Considering the results obtained by combined serological and molecular techniques, 94.65% (124/131) of the dogs were infected with at least one of the studied agents. Infections by a single etiologic agent occurred in 31.29% (l41/131) of dogs, 19.84% (26/131) by T. gondii, 9.92% (13/131) by N. caninum and 1.52% (2/131) by E. canis. Regarding co-infections in dogs, 45.03% (59/131) presented N. caninum and T. gondii, 32.82% (43/131) presented N. caninum and E. canis, and 20.61% (27/131) presented T. gondii and E. canis co-infections. Co-infections with three of the agents (E. canis, T. gondii, and N. caninum) were identified in 17.55% (23/131) of the animals.

The presence of E. canis infection in this study was significant associated with T. gondii infection (p = 0.025; odds ratio [OR] (0.3959; 95% CI 0.18-0.84), as well as for N. caninum (p = 0.0014; OR 4.8265; 95% CI 1.84-12.62). Although more frequent, co-infections with N. caninum and T. gondii showed no statistically significant association (p = 0.218; OR 0.5364; 95% CI 0.22-1.26).

Considering the two dogs that tested positive for the genus Ehrlichia spp. in the parasitological diagnosis, both were seropositive for N. caninum, and one was seropositive for T. gondii. The dog that presented with Piroplasma in red blood cells tested positive for E. canis and N. caninum.

Statistical evaluation of risk factors associated with E. canis infection in dogs showed that only age (> 3 years old) was significant in the bivariate and multivariate analyses (p = 0.011; OR 0.3176; 95% CI 0.13-0.74 and p= 0.0079; OR 0.3176; 95% CI 0.1363-0.7403, respectively) (Table 1a and 1b, Supplementary Material).

Table 1a
Factors associated with E. canis infection in positive dogs

Variable		N	Positive dogs	Frequency %	OR	95%CI	P-value
Gender	Male Female	67 64	27 22	40.30% 34.38%	1.2886	0.63 -2.62	0.6032210227
Age	<3 years old >3 years old	43 88	9 40	20.93% 45.45%	3.15	1.35-7.38	0.0113507327
Breed	No definite breed With definite breed	107 24	42 7	39.25% 29.17%	1.5692	0.59 -4,10	0.4905287771
Habitat	Urban area Rural area	72 59	26 23	36.11% 38.98%	0.8847	0.43- 1.80	0.8756183165
Outdoor access	Yes No	91 40	34 15	37.36% 37.50%	0.9942	0.46 -2.14	1.0000000000
Presence of ticks	Yes No	76 55	33 16	43.42% 29.09%	1.8706	0.89- 3.91	0.1362326291
Ectoparasite control	Yes No	29 102	8 41	27.59% 40.20%	0.5668	0.22- 1.40	0.3073090445
Contact with other animals	Yes No	109 22	42 7	38.53% 31.82%	1.3433	0.50 - 3.5	0.5527448634

Table 1b
Association between dogs positive for E. canis and the age variable

Variable	Odds ratio	Confidence interval 95%	P valor (<0.05)
Age	3.15	1.35-7.33	0.0079

• P= 0,0065 Likelihood: 0,0052

Regarding the evaluation of risk factors related to N. caninum infection, there was a statistically significant difference in the bivariate analysis related to age above 3 years (p = 0.020; OR 0.3715; 95% CI 0.17-0.81) and the animal's habitat, given that animals from the rural area had a higher risk of infection than animals from the urban area (p = 0.019; OR 0.3601; 95% CI 0.16-0.80). The values were confirmed by multivariate analysis (p = 0.0042; OR 0.2922; 95% CI 0.12-0.67 for age, and p = 0.0046; OR 0.2861; 95% CI 0.12-0.68 for habitat) (Tables 2a and 2b, Supplementary Material).

No risk factors associated with T. gondii infection were identified in dogs in the study region (Table 3, Supplementary Material).

Table 2a
Factors associated with N. caninum infection in positive dogs

Variable		N	Dogs	Frequency	OR	95%CI	P-valor
Gender	Male Female	67 64	46 46	68.66% 71.88%	0.8571	0.40- 1.81	0.8324554528
Age	<3 years old >3 years old	43 88	24 68	55.81% 77.27%	2.69	1.23- 5.88	0.0204057711
Breed	No definite breed With definite breed	107 24	77 15	71.96% 62.50%	1.5400	0.60- 3.89	0.5033144929
Habitat	Urban area Rural area	72 59	44 48	61.11% 81.36%	2.78	1.24- 6.23	0.0198472528
Outdoor access	Yes No	91 40	68 24	74.73% 60.00%	1.9710	0.89- 4.34	0.1361952816
Contact with other animals	Yes No	109 22	77 15	70.64% 68.18%	1.1229	0.41- 3.01	1.0000000000
Contact with dogs	Yes No	98 33	71 21	72.45% 63.64%	1.5026	0.65- 3.46	0.4608060352
Contact with bovine	Yes No	22 108	18 74	81.82% 68.52%	2.0676	0.65- 6.57	0.3207241959
Hunting habit/raw meat intake	Yes No	38 93	31 61	81.58% 65.59%	2.3232	0.92- 5.85	0.1083827233

Table 2b
Association between dogs positive for N. caninum and the variables age and habitat

Variable	Odds ratio	Confidence interval 95%	P valor (<0.05)
Age	3.42	1.47-7.95	0.0042
Habitat	3.50	1.47-8.30	0.0046

• P=0.007 Likelihood: 0.0005

Table 3
Factors associated with T. gondii infection in positive dogs

Variable		N	Positive dogs	Frequency%	OR	95%CI	P-valor
Gender	Male Female	67 64	47 42	70.15% 65.63%	1.2310	0.59- 2.56	0.7133505849
Age	<3 years old >3 years old	43 88	31 58	72.09% 65.91%	1.3362	0.60-2.97	0.6080975703
Breed	No definite breed With definite breed	107 24	75 14	70.09% 58.33%	1.6741	0.67- 4.16	0.3822955696
Habitat	Urban area Rural area	72 59	49 40	68.06% 67.80%	1.0120	0.48- 2.11	1.0000000000
Outdoor access	Yes No	91 40	62 27	68.13% 67.50%	1.0294	0.46- 2,27	1.0000000000
Contact with other animals	Yes No	109 22	76 13	69.72% 59.09%	1.5944	0.62- 4.09	0.4687974907
Contact with cats	Yes No	38 93	24 65	63.16% 69.89%	0.7385	0.33- 1.63	0.5869817177
Hunting habit/raw meat intake	Yes No	38 93	24 65	63.16% 69.89%	0.,7385	0.33- 1.63	0.5869817177

DISCUSSION

The low frequency of Ehrlichia spp. in parasitological diagnosis may be due to the low parasitemia presented by the animals, possibly due to the stage of infection, in addition to the low sensitivity of the technique. On the other hand, in some animals, morulae suggestive of Ehrlichia spp. can be observed in non-mononuclear leukocytes, and the animal may still test negative for E. canis using molecular techniques, as observed in one of the animals in this study. In this case, the findings can be interpreted as another species of Ehrlichia spp. or can also be characterized as other types of intracytoplasmic inclusions, such as azurophilic granules, platelets, and material from phagocytosis (Mylonakis et al., 2003).

The frequency of E. canis infection found in this study is similar to that described by Ramos et al. (2010) in Recife, Pernambuco, who reported positivity in 38.04% (78/205) of dogs evaluated using the PCR technique, and by Santos et al. (2009) in Ribeirão Preto, São Paulo, who found a prevalence of 38.9% (86/221) using the nested-PCR technique. In the state of Bahia, the present study demonstrated the highest frequency of E. canis infection through molecular diagnosis, compared to the findings described by Cordeiro et al. (2020), who reported a prevalence of 16.54% in Itabuna; Guedes et al. (2015), who observed a prevalence of 25.6% in Ituberá; and Carvalho et al. (2008), who reported a prevalence of 10.7% and 4.3% in Ilhéus and Itabuna, respectively.

Previous studies conducted in Brazil reported a low prevalence of infection by B. vogeli in dogs (below 10%), using the molecular diagnosis technique (conventional PCR) (Ramos et al., 2010; Bahiense et al., 2020). Despite the absence of positivity for B. vogeli by PCR in this study, the observation of Piroplasma in the blood smear of an animal that tested negative for B. vogeli in the molecular diagnosis indicates the occurrence of other species or subspecies of Babesia spp. in the studied region.

The frequency of N. caninum infection found in the animals in this study was higher than that reported in other studies carried out in the last 20 years in Brazil, including the study conducted by Benetti et al. (2009), which reported an infection prevalence of 67.56% (25/37) in dogs in the rural area of the southwest region of Mato Grosso using the serological technique. This result is highly important for the Cândido Sales region, especially for rural properties, as it warns about the possibility of infection in cattle, which can cause episodes of pregnancy loss in this species, resulting in economic losses.

The frequency of T. gondii infection found in the present study was similar to the results obtained by Valadas et al. (2010) in Pará, Amazonas, and by Barbosa et al. (2003) in Salvador, Bahia, who reported a prevalence of 69.8% (90/120) and 63.55% (143/225), respectively. The high frequency of anti-Toxoplasma gondii antibodies found in dogs in the region of Cândido Sales warns of the possible risk to humans, since dogs and humans can share the same infection sources.

The high rate of infections by these agents in dogs in the studied region may also be related to the lack of veterinary assistance and consequent lack of knowledge on the part of owners about the prophylaxis of these infections. Aspects related to sanitary management and animal life habits, as well as contact with other species, such as cattle and cats, feeding habits, ectoparasite control, lack of environmental hygiene, basic sanitation, and water treatment, can also favor the transmission and persistence of agents in the environment.

The occurrence of co-infection with T. gondii and E. canis observed in the dogs in this study was similar to that found by Deiró et al. (2018). These researchers used IFA and ELISA serological techniques to diagnose infection by T. gondii and E. canis, respectively, in 353 dogs from Bahia and found co-infection by these agents in 20.1% (71/353) of the evaluated animals. The relationship between co-infection by E. canis and T. gondii or N. caninum may be due to the opportunistic characteristics of the parasites (Ratzlaff et al., 2018).

The co-infection rate of N. caninum and T. gondii observed in this study was much higher when compared to the rates obtained by Mineo et al. (2001), who described only 3.1% (5/163) of reactive samples for both parasites in dogs with neuromuscular signs in Uberlândia, Minas Gerais, and Acosta et al. (2016), who described only one animal with co-infection by N. caninum and T. gondii in Espírito Santo. N. caninum infection, however, did not present a statistically significant association with the simultaneous occurrence of T. gondii, as was also previously described by Bresciani et al. (2007) in a serological study conducted in the city of Araçatuba, São Paulo. Although there was no significant correlation, the high occurrence of this co-infection may contribute to the clinical manifestation of the disease (Girardi et al., 2014). The high rate of concomitant infection with N. caninum and T. gondii observed in this study suggests that both etiological agents should be considered in differential clinical diagnoses, especially in dogs with neuromuscular, respiratory, and/or gastrointestinal disorders (Mineo et al., 2001).

In this study, age (> 3 years) was considered a risk factor for infection by E. canis and N. caninum, respectively, corroborating with the studies carried out by Deiró et al. (2018) and Souza et al. (2002) who described that adult dogs may have a higher prevalence of infection due to longer exposure to pathogens.

In addition, rural habitat was considered a risk factor for N. caninum infection, and this result corroborates those obtained by Cunha Filho et al. (2008) in a survey carried out in Pelotas, Rio Grande do Sul. The greater risk of infection in dogs from rural environments can be explained by the animals' easier access to sources of infection, with a greater possibility of ingestion of carcasses, aborted bovine fetuses, and placental remains (Souza et al., 2002; Cunha Filho et al., 2008).

In this study, no variable was identified as a risk factor for T. gondii infection. However, according to Fábrega et al. (2020) the risk of infection increases according to the animals' lifetime, due to greater exposure to pathogens.

Furthermore, it should be considered that risk factors related to infections by different pathogens in dogs can be influenced by inherent characteristics of each region. Socioeconomic and environmental issues, for example, may favor different risks for infections (Fábrega et al., 2020).

CONCLUSIONS

The results of this study confirm the presence of high rates of infection and co-infections with N. caninum, T. gondii, and E. canis in the region of Cândido Sales, Bahia, with no occurrence of B. vogeli. Age (> 3 years old) was considered a risk factor for infection by E. canis and N. caninum. In addition, the (rural) habitat was considered a risk factor for N. caninum infection. These findings reaffirm that specific diagnostic techniques should be used in veterinary practice to reduce the high rate of infections and/or co-infections caused by immunosuppressive or opportunistic agents in animals. The risk factors described should be observed and prophylactic measures should be taken to avoid the persistence and transmission of these agents in the study region. Further studies with a larger number of animals are needed to elucidate the risk factors, especially for T. gondii infections, and to confirm the absence or rare occurrence of B. vogeli in dogs in the Cândido Sales-BA region.

REFERENCES

• ACOSTA, I.C.L.; CENTODUCATTE, L.D.; SOARES, H.S. et al. Occurrence of Neospora caninum and Toxoplasma gondii antibodies in dogs from rural properties surrounding a biological reserve, Espírito Santo, Brasil. Rev. Bras. Parasitol. Vet., v.25, p.536-539, 2016.
• BAHIENSE, C.R.; MATOS, A.C.; ABATE, H.L. et al. Ocorrência de Babesia vogeli, Mycoplasma spp., Ehrlichia canis e Anaplasma spp. em uma população hospitalar de cães do Oeste do Paraná. Semin. Ciênc. Agrár., v.41, p.3133-3144, 2020.
• BARBOSA, M.V.F.; GUIMARÃES, J.E.; ALMEIDA, M.A. O. et al. Freqüência de anticorpos IgG anti-Toxoplasma gondii em soros de cães errantes da cidade de Salvador-Bahia, Brasil. Braz. J. Vet. Res. Anim. Sci., v.40, p.457-465, 2003.
• BENETTI, A. H.; SCHEIN, F.B.; SANTOS, T.R. et al. Pesquisa de anticorpos anti-Neospora caninum em bovinos leiteiros, cães e trabalhadores rurais da região Sudoeste do Estado de Mato Grosso. Rev. Bras. Parasitol. Vet., v.18, p.29-33, 2009.
• BRESCIANI, K.D.S.; COSTA, A.J.; NUNES, C.M. et al. Ocorrência de anticorpos contra Neospora caninum e Toxoplasma gondii e estudo de fatores de risco em cães de Araçatuba - SP. Ars Vet., v.23, p.40-46, 2007.
• CARVALHO, F. S.; WENCESLAU, A. A.; CARLOS, R. S. A.; ALBUQUERQUE, G. R. Epidemiological and molecular study of Ehrlichia canis in dogs in Bahia, Brazil. Genet. Mol. Res., v. 7, p. 657-662, 2008.
• CORDEIRO, J.M.A; GUEDES, P.E.B.; MUNHOZ, A.D.; SILVA, F.L. Molecular diagnosis and risk factors of canine ehrlichiosis in the municipality of Itabuna-Bahia, Brazil. Semin. Ciênc. Agrár., v.41, p.897-906, 2020.
• CUNHA FILHO, N.A.; LUCAS, A.S.; PAPPEN, F.G. et al. Fatores de risco e prevalência de anticorpos anti-Neospora caninum em cães urbanos e rurais do Rio Grande do Sul, Brasil. Rev. Bras. Parasitol. Vet., v.17, p.301-306, 2008.
• CUNHA, N.C.; FONSECA, A.H.; REZENDE, J. et al. First identification of natural infection of Rickettsia rickettsii in the Rhipicephalus sanguineus tick, in the State of Rio de Janeiro. Pesqui. Vet. Bras., v.29, p.105-108, 2009.
• DANTAS-TORRES, F.; ALEXANDRE, J.; MIRANDA, D.E.O. et al. Molecular epidemiology and prevalence of babesial infections in dogs in two hyperendemic foci in Brazil. Parasitol. Res., v.120, n.7, 2021.
• DANTAS-TORRES, F.; FIGUEREDO, L.A.; FAUSTINO, M.A.G. Ectoparasitos de cães provenientes de alguns municípios da região metropolitana do Recife, Pernambuco, Brasil. Rev. Bras. Parasitol. Vet., v.13, p.151-154, 2004.
• DEIRÓ, A.G.J.; MONTARGIL, S.M.A.; CARVALHO, F.S. et al. Antibody occurrence of Anti-Toxoplasma gondii, Leishmania sp. and Ehrlichia canis in dogs in Bahia State. Semin. Ciênc. Agrár., v.39, n.1, 2018.
• DUBEY, J.P.; SCHARES, G.; ORTEGA-MORA, L.M. Epidemiology and control of neosporosis and Neospora caninum. Clin. Microbiol. Rev., v.20, p.323-367, 2007.
• FÁBREGA, L.; RESTREPO, C.M.; TORRES, A. et al. Frequency of Toxoplasma gondii and risk factors associated with the infection in stray dogs and cats of Panama. Microorganisms, v.8, p.927, 2020.
• GHAHERI, M.; KAHRIZI, D.; YARI, K. et al. A comparative evaluation of four DNA extraction protocols for whole blood sample. Cell. Mol. Biol., v.62, p.119-123, 2016.
• GIRARDI, A.F.; LIMA, S.R.; MELO, A.L.T. et al. Ocorrência de anticorpos anti- Toxoplasma gondii e Ehrlichia canis em cães com alterações nervosas atendidos em hospital veterinário universitário. Semin. Ciênc. Agrár., v.35, p.1913-1921, 2014.
• GONDIM, L.F.P.; PINHEIRO, A.M.; SANTOS, P.O.M. et al. Isolation of Neospora caninum from the brain of a naturally infected dog, and production of encysted bradyzoites in gerbils. Vet. Parasitol., v.101, p.1-7, 2001.
• GUEDES, P.E.B.; OLIVEIRA, T.N.A.; CARVALHO, F.S. et al. Canine ehrlichiosis: prevalence and epidemiology in northeast Brazil. Rev. Bras. Parasitol. Vet., v.24, p.115-121, 2015.
• INOKUMA, H.; YOSHIZAKI, Y.; MATSUMOTO, K. et al. Molecular survey of Babesia infection in dogs in Okinawa, Japan. Vet. Parasitol., v.121, p.341-346, 2004.
• MINEO, T.W.P.; SILVA, D.A.O.; COSTA, G.H.N. et al. Detection of IgG antibodies to Neospora caninum and Toxoplasma gondii in dogs examined in a veterinary hospital from Brazil. Vet. Parasitol., v.98, p.239-245, 2001.
• MURPHY, G.L.; EWING, S.A.; WHITWORTH, L.C. et al. A molecular and serologic survey of Ehrlichia canis, E. chaffensis, and E. ewingii in dogs and ticks from Oklahoma. Vet. Parasitol., v.79, p.324-339, 1998.
• MYLONAKIS, M.E.; KOUTINAS, A.F.; BILLINIS, C. et al. Evaluation of citology in the diagnosis of acute canine monocytic ehrlichiosis (Ehrlichia canis): a comparison between five methods. Vet. Microbiol., v.91, p.197-204, 2003.
• RAMOS, R.A.N.; RAMOS, C.A.N.; ARAÚJO, F.R. et al. Molecular survey and genetic characterization of tick-borne pathogens in dogs in metropolitan Recife (North-eastern Brazil). Parasitol. Res., v.107, p.1115-1120, 2010.
• RATZLAFF, F.R.; ENGELMANN, A.M.; LUZ, F.S.; BRÄUNIG, P. et al. Coinfecções por Leishmania infantum, Neospora caninum e Toxoplasma gondii em cães necropsiados da região central do Rio Grande do Sul, Brasil. Arq. Bras. Med. Vet., v.70, p.109-116, 2018.
• SANTOS, F.; COPPEDE, J.S.; PEREIRA, A.L.A. et al. Molecular evaluation of the incidence of Ehrlichia canis, Anaplasma platys and Babesia spp. in dogs from Ribeirão Preto, Brazil. Vet. J., v.179, p.145-148, 2009.
• SOARES, R.; RAMOS, C.A.; PEDROSO, T. et al. Molecular survey of Anaplasma platys and Ehrlichia canis in dogs from Campo Grande, Mato Grosso do Sul, Brazil. An. Acad. Bras. Ciênc., v.89, p.301-306, 2017.
• SOUZA, S.L.P.; GUIMARÃES, J.S.; FERREIRA, F. et al. Prevalence of Neospora caninum antibodies in dogs from dairy cattle farms in Paraná, Brazil. J. Parasitol., v.88, p.408-409, 2002.
• TENTER, A.M.; HECKEROTH, A.R.; WEISS, L.M. Toxoplasma gondii: from animals to humans. Int. J. Parasitol., v.30, p.1217-1258, 2000.
• VALADAS, S.; MINERVINO, A.H.H.; LIMA, V.M.F. et al. Occurrence of antibodies anti-Neospora caninum, anti-Toxoplasma gondii, and anti-Leishmania chagasi in serum of dogs from Pará State, Amazon, Brazil. Parasitol. Res., v.107, p.453-457, 2010.
• VIEIRA, E.M.; ORTEGA, J.R.; ROYO, V.A. et al. Babesiose canina: aspectos hematológicos e comparação de métodos de diagnóstico. Rev. Unimontes Cient., v.23, p.116, 2021.

Publication Dates

• Publication in this collection
  03 May 2024
• Date of issue
  May-Jun 2024

History

• Received
  30 Nov 2022
• Accepted
  14 Aug 2023