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Serological and molecular diagnosis of Ehrlichia canis and associated risk factors in dogs domiciled in western Cuba
Veterinary Parasitology: Regional Studies and Reports 14 (2018) 170–175
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Original article
Serological and molecular diagnosis of Ehrlichia canis and associated risk factors in dogs domiciled in western Cuba
T
Maylin G. Navarretea, Matheus D. Cordeirob, Claudia B. Silvab, Carlos Luiz Massardb, Eugenio R. Lópeza, Julio César A. Rodrígueza, Carla C.D.U. Ribeirob, Osvaldo F. Rodríguezc, ⁎ Adivaldo H. Fonsecab, a
Department of Animal Prevention, Veterinary Medicine College, Agrarian University of Habana, Mayabeque, Cuba Department of Epidemiology and Public Health, Veterinary Institute, Federal Rural University of Rio de Janeiro, Br 465, km 7, Seropedica, Rio de Janeiro 23897-000, Brazil c National Center for Animal and Plant Health, Mayabeque, Cuba b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ehrlichia canis Domestic dogs Diagnosis Risk factors
Ehrlichia canis is a rickettsia transmitted by the tick, Rhipicephalus sanguineus, and is the causative agent of canine monocytic ehrlichiosis (CME). In Cuba, the first diagnosis of CME was made in 2001, but few studies have since investigated this disease locally. The objective of this study was to estimate the prevalence of E. canis in dogs domiciled in four municipalities within the western region of Cuba and determine the associated risk factors. Blood was drawn from 378 selected dogs living in four municipalities in two provinces of western Cuba. From the total number of samples, 206 plasma samples were selected to perform an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against E. canis. Using the original 378 samples of extracted DNA, a nested polymerase chain reaction (nPCR) was performed to amplify a specific fragment of the 16S rRNA gene of E. canis. Analysis of the 206 plasma samples revealed a total of 162 animals that were seropositive for E. canis (78.64%) with a density index between 109.5 and 970.7. In contrast, 179 samples were positive based on the nPCR assay (47.35%). As well, there was a high concordance (kappa = 0.7), calculated through the Kappa index, between the animals found to be positive based on nPCR and those determined based on ELISA. The analysis of risk factors showed that residing in the municipality of Boyeros in addition to having a history of infestation by ticks increases the probability of having a positive result based on nPCR.
1. Introduction Ehrlichia canis is an obligate intracellular bacterium that infects monocytes and macrophages. It is the etiological agent of canine monocytic ehrlichiosis (CME), a cosmopolitan and infectious disease that severely affects dogs (Parola et al., 2013). E. canis is transmitted by the tick, Rhipicephalus sanguineus (Nicholson et al., 2010), mainly in the tropical and subtropical regions (Parola et al., 2013). R. sanguineus is the most common species of tick to affect dogs from several Caribbean islands, including Aruba, the British West Indies, Grenada, Haiti, Puerto Rico, Saint Kitts and Nevis, Trinidad, and the Turks and Caicos Islands, and is responsible for the transmission of agents that have been reported in this region (Navarrete et al., 2017). According to George et al. (2002) and Gondard et al. (2017), the Caribbean region is a cosmopolitan area, at the crossroads of
intercontinental exchanges between the Americas, Europe, and Africa, which thus poses risks of the introduction and dispersal of ticks and tick-transmitted pathogens, primarily through legal or illegal animal trade and bird migration. Barros-Battesti et al. (2009) reported that the tick, R. sanguineus, is geographically distributed towards the western zone of Cuba. In Cuba, the first diagnosis of CME was made by Pérez et al. (2002) based on clinical and anatomopathological findings. Six years later, León Goñi and Gómez Rosales (2008) visualized rickettsia in blood smears, detected E. canis seropositive animals and established factors associated with infection, such as age, sex, race, time of year, and the presence of ticks in dogs with clinical symptoms. Nevertheless, this background does not clearly demonstrate the scenario involving this disease in Cuba owing to the low sensitivity and specificity of the diagnostic methods used and intentionality in the selection of the sample.
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Corresponding author. E-mail addresses: [email protected] (M.G. Navarrete), [email protected] (C.L. Massard), [email protected] (E.R. López), [email protected] (J.C.A. Rodríguez), [email protected] (A.H. Fonseca). https://doi.org/10.1016/j.vprsr.2018.10.005 Received 1 July 2018; Received in revised form 24 October 2018; Accepted 25 October 2018 Available online 29 October 2018 2405-9390/ © 2018 Elsevier B.V. All rights reserved.
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Fig. 1. Smaller map: Island of Cuba. Larger map: Location of the municipalities in which blood was collected from dogs in Habana (Cotorro, Boyeros, Habana del Este) and Mayabeque (San José de las Lajas) provinces. Scale bar = 20 Km.
molecular diagnostic study of tick-borne pathogens in dogs from Saint Kitts Island in the Caribbean (Loftis et al., 2013). The number of animals was determined by a simple random sampling formula with a 95% confidence interval and maximum error of 5% (Sampaio, 2007). In this way, a sample size of 378 dogs was determined to be sufficient. A sample of 378 selected dogs was established regardless of sex, breed, age, or presence of clinical symptoms related to canine ehrlichiosis. One-hundred-and-four dogs were from Habana del Este, 102 were from Boyeros, 90 from San José de las Lajas, and 82 from Cotorro. The distribution of the total number of animals to be sampled in relation to municipality was proportional to the estimated population of dogs, as according to Pino-Rodríguez et al. (2017).
Considering the limited knowledge regarding the diagnosis of E. canis infection in dogs from Cuba and the scarcity of epidemiological studies of this agent in the western region of the country, the aim of this study was to estimate the prevalence of E. canis in dogs domiciled in four municipalities of the western region of Cuba and determine the associated risk factors. 2. Material and methods 2.1. Location and animals under study A cross-sectional study was conducted between October and November 2013 in four municipalities located in the western region of Cuba: Habana del Este, Boyeros, Cotorro (belonging to the province of Havana), and San José de las Lajas (belonging to the Mayabeque province; Fig. 1).
2.3. Epidemiological data An epidemiological questionnaire was provided to dog owners who voluntarily agreed to participate in the study. The questions were organized according to aspects inherent to the animal (municipality of origin, age, breed definition, including mixed breed or defined breed, and gender), features related to the tick (infestation, history of infestation six months before the study, history of transmitted diseases,
2.2. Sample size calculation Based on the lack of data with respect to the prevalence of E. canis in Cuba, an expected prevalence of 40% was considered based on a 171
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indicate no agreement. Values of Kappa interpretation follow the categorization of Landis and Koch (1977) and consider < 0 (no agreement); 0–0.19 (poor agreement); 0.20–0.39 (fair agreement); 0.40–0.59 (moderate agreement); 0.60–0.79 (substantial agreement); 0.80–1.00 (almost perfect agreement). We used a generalized linear s model with binomial distribution and logarithmic link function (GLM log-binomial) to identify the risk factors associated with E. canis infection in dogs. First, univariate analysis was performed with the epidemiological data obtained in the questionnaire as independent variables, with the dependent variable being the result of the molecular diagnosis (positive/negative). The variables that showed a value of p < 0.25 based on the Wald value of the χ2 test were introduced in a multinomial model. Within the multinomial model, only those significant variables were retained (p < 0.05). The statistical analyses were carried out with the statistical program, BioEstat 5.0 R software (R Development Core Team, 2018) (Team, 2013).
and use of tick treatment) and elements related to the breeding and handling of the dog (contact with other dogs, number of dogs per house, place where it resides, the environment in which it spends the most time, and ways in which the owner maintains it). 2.4. Blood sample collection A total of 4 mL of venous blood was drawn by puncture from the cephalic vein employing hypodermic needles of caliber 25 × 0.8 mm (21G) and tubes for blood extraction by vacuum system (BD Vacutainer®), with 7.2 mg of K2 EDTA added to each sample as an anticoagulant. Of the 378 animals, 206 were selected to have plasma obtained from their extracted blood, and was stored at −20 °C until later use. 2.5. Enzyme-linked immunosorbent assay (ELISA)
3. Results
A total of 206 plasma samples were utilized as in the remains occurred hemolysis compromising the fidelity of the optical reading. The detection of antibodies against the crude antigen of a Brazilian strain of E. canis isolated in cell culture was carried out with an in-house enzyme-linked immunosorbent assay (ELISA) according to the methodology described by Voller et al. (1976), albeit with modifications. The sonicated and purified antigen was diluted in carbonate/bicarbonate buffer (pH = 9.6) at 10 mg/mL; the serum was diluted in phosphate buffered saline (PBS Tween 20 0.05%; pH 7.4) at 1:400 and the conjugate was diluted at 1:5000 (anti-dog IgG, whole molecule, alkaline phosphatase, Sigma®). Twelve negative samples from previously tested animals and one positive control of a naturally infected animal were used from Seropédica city, Brazil. The cut-off point for the test was calculated according to Frey et al. (1998) employing the t-Student distribution with a confidence level of 99.99%, and the optical density index that was used was based on the formula: DO x 100/cut point.
3.1. Animals The average age of the dogs investigated was 4.5 ± 2.1 years (minimum age two months, maximum 16 years). Overall, 33.06% of the dogs sampled were 2 to 5 years of age, 29.10% were 5 to 10 years of age, 23.01% were between 6 months and 2 years of age, 31% were > 10 years of age, and just 4.49% were < 6 months of age. This population was represented by 210 females (55.5%) and 168 males (44.4%); of them, 183 (48.41%) were dogs with a defined breed, while 195 (51.58%) were without defined breed. Dog breeds were distributed as follows: Tibetan (9.52%); German Shepherd (6.61%); Dachshund (5.29%); Chihuahua (3.7%); Rottweiler (2.64%); Cocker Spaniel (2.38%); Siberian Husky, Pointer, Dalmatian, and Boxer (2.11% each); Labrador, Pitbull, and Belgian Shepherd (1.32% each), Mexican Nude, Chow Chow, and Stanford (1.05% each); Pekinese (0.79%); Doberman (0.52%); and Maltese, Poodle, Shar Pei, Beagle, and Bulldog (0.26% each). More than half of the dogs (58.99%) had been in contact with other dogs, 38.09% were tick-infested, and 44.44% of the owners reported that they had applied tick treatment to their dogs.
2.6. Nested PCR The extraction of the genomic DNA was carried out from 300 μL of whole blood, using the Wizard Genomic DNA Purification Kit (Promega®, Madison, USA) according to the manufacturer's recommendations. The quality and concentration of the DNA were determined with a Nanodrop ND-2000® (Thermo Fisher Scientific, Wilmington, USA) and all samples were diluted to a concentration of 100 ng/μL. For the first PCR reaction (PCR1), ECC primers (5’-AGAACGAACG CTGGCGGCAAGC-3′) and ECB primers (5’-CGTATTACCGCGGCTGCTG GCA-3′) were used, which amplify a 479-bp fragment of the 16S rRNA gene. To identify the species of Ehrlichia, a second PCR reaction (PCR2) was conducted with ECAN primers (5’-CAATTATTTATAGCCTCTGGCT ATAGGA-3′) and HE3 primers (5’-TATAGGTACCGTCATTATCTTCCC TAT-3′), which amplify a 389-bp region of the 16S rRNA of E. canis according to previous methodology described by Murphy et al. (1998). The nPCR products were applied to 2% agarose gels (Sigma-Aldrich, St. Louis, USA) in 0.5× TBE buffer containing ethidium bromide (0.4 μg/mL), and the results were visualized in a UV 302 NM ultraviolet light transilluminator (KASVI - Curitiba, Brazil). In all cases, a 100-bp molecular weight marker (Promega, Madison, USA) was employed (data not shown).
3.2. ELISA Anti-E. canis antibodies were detected in 162/206 dogs belonging to the four municipalities (78.6%; 95% CI: 72.55–83.69). At the “cut-off” point, three plates each underwent an optical reading in the different ELISA conditions (0.119, 0.116 and 0.143), and they were transformed into 100 by this DO × 100/cut-off formula to compare all samples of the various plates with respect to density index. The distribution of the optical densities around the “cut-off” (DO × 100 / “cut-off”) obtained from the ELISA test for E. canis in sera from positive dogs resulted in density indices ranging from 109.5 to 970.7. 3.3. Nested PCR Of the total 378 samples analyzed, 179 were positive for E. canis based on the PCR assay (47.35%; 95% CI: 42.37–52.39). 3.4. Concordance between the two techniques Of the dogs examined, 109 of the seropositive animals (67.28%) were positive for E. canis based on nPCR, whereas 53% were positive for E. canis according to both ELISA and nPCR; 37 (18%) were negative for both tests. When the results of the molecular and serological tests were compared, a high concordance was observed (kappa = 0.7) according to the criteria of Kramer and Feinstein (1981) and Landis and Koch (1977). Of the two diagnostic techniques, a higher proportion (p = 0.0001) of
2.7. Statistical analysis The frequencies of E. canis-positive animals determined by ELISA and nPCR were compared via McNemar's, χ2 test for 95% confidence. The concordance between the frequencies of positive animals in the nPCR and ELISA was evaluated by the Kappa index (Kramer and Feinstein, 1981). This measure of agreement has a maximum value of 1, where this value 1 represents total agreement and values close to 0 172
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Table 1 Univariate analysis (GLM log-binomial) of the potential risk factors for infection by Ehrlichia canis in dogs, diagnosed by nested PCR, from four municipalities located in the western region of Cuba. Variables analyzed
Aspects related to the inherent characteristics of the dogs Municipality
Age
Breed definition Gender Aspects related to the tick Infestation by R. sanguineus Background of infestation History of diseases and control of ticks History of diseases transmitted by ticks Acaricide treatment Aspects related to the breeding and handling of the dog Contact with other dogs Number of dogs per house
Local access dog
The environment in which the dog spends most of the time
Habits
E. canis (nPCR) Positives/n (%)
PR
Cotorro Habana del Este San José de las Lajas Boyeros > 10 years ≥ 5 years to 10 years ≥ 2 years to < 5 years ≥ 6 months to < 2 years < 6 months Mixed breed Defined breed Male Female
28/82 (34.1) 42/104 (40.4) 42/90 (46.7) 67/102 (65.7) 19/39 (48.72) 53/110 (48.18) 61/125 (48.80) 49/83 (52.7) 5/17 (29.41) 96/197 (48.7) 83/181 (45.9) 86/174 (49.4) 93/204 (45.6)
⁎ 1.18 1.37 1.92 ⁎ 1.02 0.92 1.08 0.43 ⁎ 0.94 ⁎ 0.92
No Yes No Yes
102/231 (44.2) 77/147 (52.4) 37/103 (35.9) 142/275 (51.6)
⁎ 1.19 ⁎ 1.44
No Yes No Yes
130/281 (46.3) 49/97 (50.5) 105/233 (45.1) 74/145 (51.0)
⁎ 1.09 ⁎ 1.13
No Yes 1 dog 2 to 3 dogs > 3 dogs Urban environment Urban environment/At the house Confined in the house Land Grass or garden In cement constructions Loose Always confined Confined part of time
75/160 (46.9) 104/218 (47.7) 72/145 (49.7) 75/162 (46.3) 32/71 (45.1) 13/24 (54.2) 86/174 (49.4) 80/180 (44.4) 35/72 (48.6) 104/226 (46.0) 40/80 (50.0) 84/177 (47.5) 31/59 (52.5) 64/142 (45.1)
⁎ 1.02 ⁎ 0.93 0.91 ⁎ 0.91 0.82 ⁎ 0.95 1.03 ⁎ 1.11 0.95
95% CI
P-value
0.81–1.73 0.94–1.98 1.38–2.62
0.3876 0.1007 0.0001
0.7–1.48 0.63–1.34 0.74–1.57 0.17–1.09
0.9282 0.6611 0.6824 0.0765
0.76–1.16
0.5767
0.75–1.14
0.4556
0.96–1.47
0.1137
1.08–1.91
0.0117
0.86–1.38
0.4611
0.91–1.4
0.2531
0.82–1.26
0.8731
0.74–1.18 0.67–1.23
0.556 0.5331
0.61–1.36 0.55–1.23
0.6515 0.3356
0.72–1.25 0.74–1.42
0.6973 0.8643
0.83–1.48 0.75–1.21
0.4882 0.6718
nPCR: nested PCR; n: total number of samples; PR: Prevalence Ratio; CI: Confidence Interval. ⁎ Category reference.
seropositive and seronegative animals were detected by ELISA than nPCR.
Table 2 Risk factors for infection by Ehrlichia canis in dogs from four municipalities located in the western region of Cuba, based on nested PCR. Results of the multivariate analysis (GLM log-binomial).
3.5. Associated risk factors
Variables
Categories
PR
95% CI
p-value
The most common risk factor associated with E. canis infection was place of residence, as the population of dogs that resided in the Boyeros municipality exhibited a risk ratio of E. canis, 1.84 times higher (RR = 1.84; 95% CI: 1.32–2.56; p = 0.0003) than that found in the population of dogs within the Cotorro municipality. The prevalence of E. canis in the municipalities of San José de las Lajas and Habana del Este did not show significant differences from that of Cotorro municipality. A history of tick infestation was another significant risk factor (p = 0.007). Dogs that lived in the Boyeros municipality that had a history of tick infestation were more likely to contract infection via E. canis (risk factors), with a risk ratio 1.38 times higher (RR = 1.38; 95% CI: 1.04–1.81; p = 0.023) than that of animals with no history of ticks (Tables 1 and 2). There was no significant association between E. canis infection and age, breed, or sex.
Municipality
Cotorro Habana del Este San José de las Lajas Boyeros No Yes
⁎⁎ 1.13 1.34
0.78–1.66 0.92–1.94
0.521 0.123
Background of tick infestation
1.84 1.32–2.56 0.0003⁎⁎⁎ Reference category 1.38 1.04–1.81 0.023⁎
PR: Prevalence Ratio; CI: confidence interval. ⁎ p < 0,05. ⁎⁎ Category Reference. ⁎⁎⁎ p < 0,001.
4. Discussion In this study, a high percentage (78.6%) of dogs were found to possess anti-E. canis antibodies. This suggests that the dog populations belonging to the two studied provinces in the western region of Cuba 173
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(Nakaghi et al., 2008)]. This difference between the number of seropositive animals and percentage confirmed by nPCR may also be based on the stage of infection of the disease. During the acute stage, Ehrlichia infection is detectable by PCR in blood, whereas in the subclinical and chronic stages, the most appropriate samples are from the spleen and bone marrow (Harrus et al., 2004) (Mylonakis et al., 2004). As the results of this investigation were based on venous blood samples from a randomly selected population without clinical symptoms, it is possible that several dogs were in the subclinical or chronic phase, when it is not always possible to detect the agent in the blood, which would have resulted in false negatives when using PCR. During the ELISA test, cross-reactions may take place between Ehrlichia species, such as between E. ewingii and E. chaffeensis. This technique can also generate cross-reactions with other agents of the Anaplasmataceae family. Similar results to those obtained in this investigation were reported in Cameroon and the United States by Wen et al. (1997) and Ndip et al. (2005), respectively, who confirmed an agreement between PCR infection and indirect fluorescence antibody (IFAT) detection techniques in 41% of dogs diagnosed positively. Iqbal et al. (1994) compared the sensitivity of IFAT and PCR methods and concluded that a combination of both optimized the diagnosis. Similarly, Guedes et al. (2015) diagnosed canine ehrlichiosis using blood smears and both serological and molecular techniques, concluding that these are complementary methods owing to the differences in sensitivity and specificity exhibited by the methods in relation to the clinical phase of the infection. Dogs resident in the Boyeros municipality featured a higher probability of infection. This could be because of the breeding conditions in this area, where approximately 50% of the animals are loose or semiconfined, favoring contact with other dogs and exposure to ticks, just as reported by de Azevedo et al. (2011) and Cardoso et al. (2012). Huerto-Medina and Dámaso-Mata (2015) and Guedes et al. (2015) established that contact with other dogs is a risk factor associated with E. canis infection. In addition, dogs that have a history of tick infestation are more likely to be infected with E. canis, making tick infestation another risk factor. In previous studies conducted by (Dagnone et al., 2003) and (Ueno et al., 2009), however, possessing a history of tick infestation was not identified as a risk factor. Similar results in relation to age, breed, and sex were reported by (Procajło et al., 2011) and (Dahmani et al., 2015), neither of which determined a statistical association between positive PCR results and age or sex.
are highly exposed to infection. Although studies carried out in Cuba are scarce, the results of this investigation are similar to those of León Goñi and Gómez Rosales (2008), who also found high values of seroprevalence (81.65%). The animals evaluated by those authors were of defined breeds, had a history of infestation by ticks, and exhibited hemorrhagic and febrile symptoms. Those high values of seroprevalence may be based on the clinical condition of the animals that were investigated. Furthermore, 55.04% of the seropositive dogs were German Shepherds, the breed considered most susceptible to E. canis, with the disease being more severe and having a worse prognosis because the humoral immune response is depressed. Wen et al. (1997) and Ndip et al. (2005) reported that, among animals treated in clinics and veterinary hospitals in Cameroon and the United States, prevalence fluctuated between 32 and 76%, whereas prevalence was < 50% in certain regions of Brazil. Vieira et al. (2011) and Melo et al. (2011) put forth a seroprevalence value of 74.3% in urban areas of the Pantanal region, Mato Grosso, Brazil. In the north of Trinidad, Asgarali et al. (2012) also found values lower than 44.6% in stray dogs and Martínez-Vega et al. (2016) observed a seroprevalence value of 64% in Mexico. In this way, it can be observed that prevalence rates are variable in different regions of the American continent. However, in Cuba, the prevalence found indicated that E. canis is present on the island and that its distribution should attract the attention of the veterinary medical surveillance services given its high proportion and mainly because this is the first epidemiological study of canine ehrlichiosis in the country. This variability in seroprevalence likely occurs because the epidemiology of canine ehrlichiosis is related to vector distribution and associated with tropical and subtropical zones as well as being affected by animal behavior, breed, and living environment (Costa Jr et al., 2007). The results of the nPCR detected E. canis DNA in 47.35% of samples. The prevalence values obtained here are higher than those reported by Wen et al. (1997), who determined that 44% of dogs in the United States were positive for E. canis. The results presented herein are also higher than those from studies in other Caribbean regions, such as the 24.7% prevalence found in Granada (Yabsley et al., 2008), 14% prevalence found in Trinidad (Georges et al., 2008), 38.9% prevalence in Ribeirão Preto (Santos et al., 2009), and 27% prevalence in the Saint Kitts Islands (Kelly et al., 2013). In contrast, the current results concerning E. canis infection were lower than those obtained by (de Paiva Diniz et al., 2007) in dogs from the state of São Paulo (76.8%) and lower than those of (Ramos et al., 2009), who noted prevalence values of 57% using nPCR with dogs clinically suspected of the disease. These variations in the percentages of positivity to rickettsia may be related to the condition of the canine population, exposure to the vector, R. sanguineus, and the diagnostic methods along with their sensitivity (Solano-Gallego et al., 2006). Of the four municipalities investigated, Boyeros had the highest prevalence value (65.7%), followed by San José de las Lajas (46.7%), Havana del Este (40.4%), and Cotorro (34.1%). This finding is influenced by the handling and conditions of dogs. Despite the fact that 100% of those sampled in Boyeros had owners, 43.28% of them roamed the streets all day, affecting control and making contact with other dogs infested with the vector more likely in addition to having access to environments favorable for the development and maintenance of the disease (Dantas-Torres, 2008, 2010; Silveira et al., 2009). Employing nPCR, 109 (67.28%) dogs were confirmed to be positive for E. canis, whereas 162 (78.6%) were diagnosed seropositive by ELISA. The absence of E. canis DNA in 53 samples that were positive by serology can be explained by the occurrence of non-specific reactions that cause false positives through serological testing (Al-Adhami et al., 2011). Additionally, serological techniques only discern specific antibodies, and these can remain systemically for a long time after treatment and cure, or elimination of the agent by the immune system [although results of PCR would be negative (Rikihisa et al., 1994);
5. Conclusions In the studied area, a high prevalence of E. canis was detected in the canine population. In addition, residing in the Boyeros municipality and featuring a history of tick infestation was associated with E. canis infection. Furthermore, the results of ELISA testing in the population studied exhibited high concordance with the results of the nPCR to detect infected animals. Conflict of interest The authors declare there are no conflicts of interest. Acknowledgements We would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes/MES-Cuba – Proc. 188/2013) and Fundação de Apoio à Pesquisa do estado do Rio de Janeiro (FAPERJ – Proc. E26/ 201.144/2014) for the financial support. References Al-Adhami, B., Scandrett, W.B., Lobanov, V.A., Gajadhar, A.A., 2011. Serological cross-
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Melo, A.L.T., Martins, T.F., Horta, M.C., Moraes-Filho, J., Pacheco, R.C., Labruna, M.B., Aguiar, D.M., 2011. Seroprevalence and risk factors to Ehrlichia spp. and Rickettsia spp. in dogs from the Pantanal Region of Mato Grosso State, Brazil. Ticks Tick Borne Dis. 2, 213–218. Murphy, G.L., Ewing, S., Whitworth, L.C., Fox, J.C., Kocan, A.A., 1998. A molecular and serologic survey of Ehrlichia canis, E. chaffeensis, and E. ewingii in dogs and ticks from Oklahoma. Vet. Parasitol. 79, 325–339. Mylonakis, M.E., Koutinas, A.F., Baneth, G., Polizopoulou, Z., Fytianou, A., 2004. Mixed Ehrlichia canis, Hepatozoon canis, and presumptive Anaplasma phagocytophilum infection in a dog. Vet. Clin. Pathol. 33, 249–251. Nakaghi, A.C.H., Machado, R.Z., Costa, M.T., André, M.R., Baldani, C.D., 2008. Canine ehrlichiosis: clinical, hematological, serological and molecular aspects. Cienc. Rural 38, 766–770. Navarrete, M.G., Cordeiro, M.D., Batista, Y., Alonso, J.C., Márquez, M., Roque, E., Fonseca, A., 2017. Serological detection of Toxoplasma gondii in domestic dogs in the western region of Cuba. Vet. Parasitol. Reg. Stud. Reports. 9, 9–12. Ndip, L., Ndip, R.N., Esemu, S., Dickmu, V., Fokam, E., Walker, D., McBride, J., 2005. Ehrlichial infection in Cameroonian canines by Ehrlichia canis and Ehrlichia ewingii. Vet. Microbiol. 111, 59–66. Nicholson, W.L., Allen, K.E., McQuiston, J.H., Breitschwerdt, E.B., Little, S.E., 2010. The increasing recognition of rickettsial pathogens in dogs and people. Trends Parasitol. 26, 205–212. Parola, P., Paddock, C.D., Socolovschi, C., Labruna, M.B., Mediannikov, O., Kernif, T., Abdad, M.Y., Stenos, J., Bitam, I., Fournier, P.E., Raoult, D., 2013. Update on TickBorne Rickettsioses around the World: a Geographic Approach. Clin. Microbiol. Rev. 26, 657–702. Pérez, B., Valdés, R., Vitorte, S., 2002. Epidemiología de las enfermedades trasmitidas por garrapatas en. Cuba. Rev. Cubana Cienc. Vet. 20, 78–87. Pino-Rodríguez, D., Márquez-Álvarez, M., Rojas-Hoyos, N.A., 2017. Aspectos demográficos de la población de perros con dueños del municipio Boyeros. Cuba. Rev. Sal. An. 39, 00. Procajło, A., Skupień, E., Bladowski, M., Lew, S., 2011. Monocytic ehrlichiosis in dogs. Polish J. Vet. Sc. 14, 515–520. Ramos, C.A., Ramos, R.A., Araújo, F.R., Guedes Jr., D., Souza, I.I., Ono, T.M., Vieira, A.S., Pimentel, D.S., Rosas, E.O., Faustino, M.A., 2009. Comparação de nested-PCR com o diagnóstico direto na detecção de Ehrlichia canis e Anaplasma platys em cães. Rev. Bras. Parasitol. Vet. 18, 58–62. Rikihisa, Y., Ewing, S., Fox, J., 1994. Western immunoblot analysis of Ehrlichia chaffeensis, E. canis, or E. ewingii infections in dogs and humans. J. Cl. Microbiol. 32, 2107–2112. Sampaio, I.B.M., 2007. Estatística aplicada á experimentaçao animal (Fundaçao de Estudo e Pesquisa em Medicina Veterinaria). pp. 265. Santos, F., Coppede, J.S., Pereira, A.L., Oliveira, L.P., Roberto, P.G., Benedetti, R.B., Zucoloto, L.B., Lucas, F., Sobreira, L., Marins, M., 2009. Molecular evaluation of the incidence of Ehrlichia canis, Anaplasma platys and Babesia spp. in dogs from Ribeirão Preto, Brazil. Vet. J. 179, 145–148. Silveira, J.A.G., Passos, L.M.F., Ribeiro, M.F.B., 2009. Population dynamics of Rhipicephalus sanguineus (Latrielle, 1806) in Belo Horizonte, Minas Gerais state, Brazil. V. Parasit. 161, 270–275. Solano-Gallego, L., Llull, J., Osso, M., Hegarty, B., Breitschwerdt, E., 2006. A serological study of exposure to arthropod-borne pathogens in dogs from northeastern Spain. Vet. Res. 37, 231–244. Team, 2013. R.C. 2014. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria ISBN 3-900051-07-0. http:// www.r-project.org/. Ueno, T.E.H., Aguiar, D.M., Pacheco, R.d.C., Richtzenhain, L.J., Ribeiro, M.G., Paes, A.C., Megid, J., Labruna, M.B., 2009. Ehrlichia canis em cães atendidos em hospital veterinário de Botucatu, Estado de São Paulo, Brasil. Rev. Bras. Parasitol. Vet. 18, 57–61. Vieira, R.F.d.C., Biondo, A.W., Guimarães, A.M.S., Santos, A.P.d., Santos, R.P.d., Dutra, L.H., Diniz, P.P.V.P., Morais, H.A.d., Messick, J.B., Labruna, M.B., 2011. Ehrlichiosis in Brazil. Rev. Bras. Parasitol. Vet. 20, 01–12. Voller, A., Bidwell, D., Bartlett, A., 1976. Enzyme immunoassays in diagnostic medicine: Theory and practice. Bull. Word Health Organ. 53, 55–65. Wen, B., Rikihisa, Y., Mott, J.M., Greene, R., Kim, H.-Y., Zhi, N., Couto, G.C., Unver, A., Bartsch, R., 1997. Comparison of nested PCR with immunofluorescent-antibody assay for detection of Ehrlichia canis infection in dogs treated with doxycycline. J. Clin. Microbiol. 35, 1852–1855. Yabsley, M.J., McKibben, J., Macpherson, C.N., Cattan, P.F., Cherry, N.A., Hegarty, B.C., Breitschwerdt, E.B., O'Connor, T., Chandrashekar, R., Paterson, T., Perea, M.L., Ball, G., Friesen, S., Goedde, J., Henderson, B., Sylvester, W., 2008. Prevalence of Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, Bartonella vinsonii berkhoffii, and Rickettsia spp. in dogs from Grenada. Vet. Parasitol. 151, 279–285.
reactivity between Anaplasma marginale and an Ehrlichia species in naturally and experimentally infected cattle. J. Vet. Diagn. Investig. 23, 1181–1188. Asgarali, Z., Pargass, I., Adam, J., Mutani, A., Ezeokoli, C., 2012. Haematological parameters in stray dogs seropositive and seronegative to Ehrlichia canis in North Trinidad. Ticks Tick-Borne Dis. 3, 207–211. de Azevedo, S.S., Aguiar, D.M., de Aquino, S.F., Orlandelli, R.C., da Fonseca Fernandes, A.R., Uchôa, I.C.P., 2011. Soroprevalência e fatores de risco associados à soropositividade para Ehrlichia canis em cães do semiárido da Paraíba. Brazil. J. Vet. Res. An. Sc. 48, 14–18. Barros-Battesti, D.M., Hernández, M.R., Famadas, K.M., Onofrio, V.C., Beati, L., Guglielmone, A.A., 2009. The ixodid ticks (Acari: Ixodidae) of Cuba. Sys. Appl. Acarol. 14, 101–128. Cardoso, L., Mendão, C., Madeira De Carvalho, L., 2012. Prevalence of Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi sensu lato, Anaplasma spp. and Leishmania infantum in apparently healthy and CVBD-suspect dogs in Portugal - a national serological study. Parasit. Vectors 5, 62. Costa Jr., L.M., Rembeck, K., Ribeiro, M.F.B., Beelitz, P., Pfister, K., Passos, L.M.F., 2007. Sero-prevalence and risk indicators for canine ehrlichiosis in three rural areas of Brazil. Vet. J. 174, 673–676. Dagnone, A.S., De Morais, H.S.A., Vidotto, M.C., Jojima, F.S., Vidotto, O., 2003. Ehrlichiosis in anemic, thrombocytopenic, or tick-infested dogs from a hospital population in South Brazil. Vet. Parasitol. 117, 285–290. Dahmani, M., Loudahi, A., Mediannikov, O., Fenollar, F., Raoult, D., Davoust, B., 2015. Molecular detection of Anaplasma platys and Ehrlichia canis in dogs from Kabylie, Algeria. Ticks Tick-borne Dis. 6, 198–203. Dantas-Torres, F., 2008. Canine vector-borne diseases in Brazil. Parasit. Vectors 1, 25. https://doi.org/10.1186/1756-3305-1-25. Dantas-Torres, F., 2010. Biology and ecology of the brown dog tick, Rhipicephalus sanguineus. Parasit. Vectors 3, 26. https://doi.org/10.1186/1756-3305-3-26. de Paiva Diniz, P.P.V., Schwartz, D.S., De Morais, H.S.A., Breitschwerdt, E.B., 2007. Surveillance for zoonotic vector-borne infections using sick dogs from southeastern Brazil. Vector-Borne Zoon. Dis. 7, 689–698. Frey, A., Di Canzio, J., Zurakowski, D., 1998. A statistically defined endpoint titer determination method for immunoassays. J. Immunol. Methods 221, 35–41. George, J.E., Davey, R.B., Pound, J.M., 2002. Introduced ticks and tick-borne diseases: The threat and approaches to eradication. Vet. Cli. North Am. 18, 401–416. Georges, K., Ezeokoli, C.D., Newaj-Fyzul, A., Campbell, M., Mootoo, N., Mutani, A., Sparagano, O.A., 2008. The application of PCR and reverse line blot hybridization to detect arthropod-borne Hemopathogens of dogs and cats in Trinidad. Ann. N. Y. Acad. Sci. 1149, 196–199. Gondard, M., Cabezas-Cruz, A., Charles, R.A., Vayssier-Taussat, M., Albina, E., Moutailler, S., 2017. Ticks and Tick-Borne Pathogens of the Caribbean: Current understanding and future directions for more comprehensive surveillance. Front. Cell. Infect. Microbiol. 7 (490). https://doi.org/10.3389/fcimb.2017.00490. Guedes, P.E.B., Oliveira, T.N.d.A., Carvalho, F.S., Carlos, R.S.A., Albuquerque, G.R., Munhoz, A.D., Wenceslau, A.A., Silva, F.L., 2015. Canine ehrlichiosis: Prevalence and epidemiology in Northeast Brazil. Braz. J. Vet. Parasitol. 24, 115–121. Harrus, S., Kenny, M., Miara, L., Aizenberg, I., Waner, T., Shaw, S., 2004. Comparison of simultaneous splenic sample PCR with blood sample PCR for diagnosis and treatment of experimental Ehrlichia canis infection. Antimicrob. Agents Chemother. 48, 4488–4490. Huerto-Medina, E., Dámaso-Mata, B., 2015. Factores asociados a la infección por Ehrlichia canis en perros infestados con garrapatas en la ciudad de Huánuco, Perú. Rev. Peruana Med. Exper. Sal. Públ. 32, 756–760. Iqbal, Z., Chaichanasiriwithaya, W., Rikihisa, Y., 1994. Comparison of PCR with other tests for early diagnosis of canine ehrlichiosis. J. Clin. Microbiol. 32, 1658–1662. Kelly, P., Xu, C., Lucas, H., Loftis, A., Abete, J., Zeoli, F., Stevens, A., Jaegersen, K., Ackerson, K., Gessner, A., Kaltenboeck, B., Wang, C., 2013. Ehrlichiosis, Babesiosis, Anaplasmosis and Hepatozoonosis in dogs from St. Kitts, West Indies. PLoS One 8 (1). https://doi.org/10.1371/journal.pone.0053450. Kramer, M.S., Feinstein, A.R., 1981. Clinical biostatistics: LIV. The biostatistics of concordance. Clin. Pharmacol. Therap. 29, 111–123. Landis, J.R., Koch, G.G., 1977. The measurement of observer agreement for categorical data. Biometrics 33, 159–174. León Goñi, A.C., Gómez Rosales, D., 2008. Ehrlichiosis canina. REDVET. Rev. Elect. Vet. 9. Loftis, A.D., Kelly, P.J., Freeman, M.D., Fitzharris, S., Beeler-Marfisi, J., Wang, C., 2013. Tick- borne pathogens and disease in dogs on St. Kitts, West Indies. Vet. Parasitol. 196, 44–49. Martínez-Vega, P.P., Bolio-Gonzalez, M.E., Rodríguez-Vivas, R.I., Gutierrez-Blanco, E., Pérez-Osorio, C., Villegas-Perez, S.L., Sauri-Arceo, C.H., 2016. Associated Factors to Seroprevalence of Ehrlichia spp. in dogs of Quintana Roo, Mexico. J. Trop. Med. 2016, 1–6.
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Original article
Serological and molecular diagnosis of Ehrlichia canis and associated risk factors in dogs domiciled in western Cuba
T
Maylin G. Navarretea, Matheus D. Cordeirob, Claudia B. Silvab, Carlos Luiz Massardb, Eugenio R. Lópeza, Julio César A. Rodrígueza, Carla C.D.U. Ribeirob, Osvaldo F. Rodríguezc, ⁎ Adivaldo H. Fonsecab, a
Department of Animal Prevention, Veterinary Medicine College, Agrarian University of Habana, Mayabeque, Cuba Department of Epidemiology and Public Health, Veterinary Institute, Federal Rural University of Rio de Janeiro, Br 465, km 7, Seropedica, Rio de Janeiro 23897-000, Brazil c National Center for Animal and Plant Health, Mayabeque, Cuba b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ehrlichia canis Domestic dogs Diagnosis Risk factors
Ehrlichia canis is a rickettsia transmitted by the tick, Rhipicephalus sanguineus, and is the causative agent of canine monocytic ehrlichiosis (CME). In Cuba, the first diagnosis of CME was made in 2001, but few studies have since investigated this disease locally. The objective of this study was to estimate the prevalence of E. canis in dogs domiciled in four municipalities within the western region of Cuba and determine the associated risk factors. Blood was drawn from 378 selected dogs living in four municipalities in two provinces of western Cuba. From the total number of samples, 206 plasma samples were selected to perform an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against E. canis. Using the original 378 samples of extracted DNA, a nested polymerase chain reaction (nPCR) was performed to amplify a specific fragment of the 16S rRNA gene of E. canis. Analysis of the 206 plasma samples revealed a total of 162 animals that were seropositive for E. canis (78.64%) with a density index between 109.5 and 970.7. In contrast, 179 samples were positive based on the nPCR assay (47.35%). As well, there was a high concordance (kappa = 0.7), calculated through the Kappa index, between the animals found to be positive based on nPCR and those determined based on ELISA. The analysis of risk factors showed that residing in the municipality of Boyeros in addition to having a history of infestation by ticks increases the probability of having a positive result based on nPCR.
1. Introduction Ehrlichia canis is an obligate intracellular bacterium that infects monocytes and macrophages. It is the etiological agent of canine monocytic ehrlichiosis (CME), a cosmopolitan and infectious disease that severely affects dogs (Parola et al., 2013). E. canis is transmitted by the tick, Rhipicephalus sanguineus (Nicholson et al., 2010), mainly in the tropical and subtropical regions (Parola et al., 2013). R. sanguineus is the most common species of tick to affect dogs from several Caribbean islands, including Aruba, the British West Indies, Grenada, Haiti, Puerto Rico, Saint Kitts and Nevis, Trinidad, and the Turks and Caicos Islands, and is responsible for the transmission of agents that have been reported in this region (Navarrete et al., 2017). According to George et al. (2002) and Gondard et al. (2017), the Caribbean region is a cosmopolitan area, at the crossroads of
intercontinental exchanges between the Americas, Europe, and Africa, which thus poses risks of the introduction and dispersal of ticks and tick-transmitted pathogens, primarily through legal or illegal animal trade and bird migration. Barros-Battesti et al. (2009) reported that the tick, R. sanguineus, is geographically distributed towards the western zone of Cuba. In Cuba, the first diagnosis of CME was made by Pérez et al. (2002) based on clinical and anatomopathological findings. Six years later, León Goñi and Gómez Rosales (2008) visualized rickettsia in blood smears, detected E. canis seropositive animals and established factors associated with infection, such as age, sex, race, time of year, and the presence of ticks in dogs with clinical symptoms. Nevertheless, this background does not clearly demonstrate the scenario involving this disease in Cuba owing to the low sensitivity and specificity of the diagnostic methods used and intentionality in the selection of the sample.
⁎
Corresponding author. E-mail addresses: [email protected] (M.G. Navarrete), [email protected] (C.L. Massard), [email protected] (E.R. López), [email protected] (J.C.A. Rodríguez), [email protected] (A.H. Fonseca). https://doi.org/10.1016/j.vprsr.2018.10.005 Received 1 July 2018; Received in revised form 24 October 2018; Accepted 25 October 2018 Available online 29 October 2018 2405-9390/ © 2018 Elsevier B.V. All rights reserved.
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Fig. 1. Smaller map: Island of Cuba. Larger map: Location of the municipalities in which blood was collected from dogs in Habana (Cotorro, Boyeros, Habana del Este) and Mayabeque (San José de las Lajas) provinces. Scale bar = 20 Km.
molecular diagnostic study of tick-borne pathogens in dogs from Saint Kitts Island in the Caribbean (Loftis et al., 2013). The number of animals was determined by a simple random sampling formula with a 95% confidence interval and maximum error of 5% (Sampaio, 2007). In this way, a sample size of 378 dogs was determined to be sufficient. A sample of 378 selected dogs was established regardless of sex, breed, age, or presence of clinical symptoms related to canine ehrlichiosis. One-hundred-and-four dogs were from Habana del Este, 102 were from Boyeros, 90 from San José de las Lajas, and 82 from Cotorro. The distribution of the total number of animals to be sampled in relation to municipality was proportional to the estimated population of dogs, as according to Pino-Rodríguez et al. (2017).
Considering the limited knowledge regarding the diagnosis of E. canis infection in dogs from Cuba and the scarcity of epidemiological studies of this agent in the western region of the country, the aim of this study was to estimate the prevalence of E. canis in dogs domiciled in four municipalities of the western region of Cuba and determine the associated risk factors. 2. Material and methods 2.1. Location and animals under study A cross-sectional study was conducted between October and November 2013 in four municipalities located in the western region of Cuba: Habana del Este, Boyeros, Cotorro (belonging to the province of Havana), and San José de las Lajas (belonging to the Mayabeque province; Fig. 1).
2.3. Epidemiological data An epidemiological questionnaire was provided to dog owners who voluntarily agreed to participate in the study. The questions were organized according to aspects inherent to the animal (municipality of origin, age, breed definition, including mixed breed or defined breed, and gender), features related to the tick (infestation, history of infestation six months before the study, history of transmitted diseases,
2.2. Sample size calculation Based on the lack of data with respect to the prevalence of E. canis in Cuba, an expected prevalence of 40% was considered based on a 171
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indicate no agreement. Values of Kappa interpretation follow the categorization of Landis and Koch (1977) and consider < 0 (no agreement); 0–0.19 (poor agreement); 0.20–0.39 (fair agreement); 0.40–0.59 (moderate agreement); 0.60–0.79 (substantial agreement); 0.80–1.00 (almost perfect agreement). We used a generalized linear s model with binomial distribution and logarithmic link function (GLM log-binomial) to identify the risk factors associated with E. canis infection in dogs. First, univariate analysis was performed with the epidemiological data obtained in the questionnaire as independent variables, with the dependent variable being the result of the molecular diagnosis (positive/negative). The variables that showed a value of p < 0.25 based on the Wald value of the χ2 test were introduced in a multinomial model. Within the multinomial model, only those significant variables were retained (p < 0.05). The statistical analyses were carried out with the statistical program, BioEstat 5.0 R software (R Development Core Team, 2018) (Team, 2013).
and use of tick treatment) and elements related to the breeding and handling of the dog (contact with other dogs, number of dogs per house, place where it resides, the environment in which it spends the most time, and ways in which the owner maintains it). 2.4. Blood sample collection A total of 4 mL of venous blood was drawn by puncture from the cephalic vein employing hypodermic needles of caliber 25 × 0.8 mm (21G) and tubes for blood extraction by vacuum system (BD Vacutainer®), with 7.2 mg of K2 EDTA added to each sample as an anticoagulant. Of the 378 animals, 206 were selected to have plasma obtained from their extracted blood, and was stored at −20 °C until later use. 2.5. Enzyme-linked immunosorbent assay (ELISA)
3. Results
A total of 206 plasma samples were utilized as in the remains occurred hemolysis compromising the fidelity of the optical reading. The detection of antibodies against the crude antigen of a Brazilian strain of E. canis isolated in cell culture was carried out with an in-house enzyme-linked immunosorbent assay (ELISA) according to the methodology described by Voller et al. (1976), albeit with modifications. The sonicated and purified antigen was diluted in carbonate/bicarbonate buffer (pH = 9.6) at 10 mg/mL; the serum was diluted in phosphate buffered saline (PBS Tween 20 0.05%; pH 7.4) at 1:400 and the conjugate was diluted at 1:5000 (anti-dog IgG, whole molecule, alkaline phosphatase, Sigma®). Twelve negative samples from previously tested animals and one positive control of a naturally infected animal were used from Seropédica city, Brazil. The cut-off point for the test was calculated according to Frey et al. (1998) employing the t-Student distribution with a confidence level of 99.99%, and the optical density index that was used was based on the formula: DO x 100/cut point.
3.1. Animals The average age of the dogs investigated was 4.5 ± 2.1 years (minimum age two months, maximum 16 years). Overall, 33.06% of the dogs sampled were 2 to 5 years of age, 29.10% were 5 to 10 years of age, 23.01% were between 6 months and 2 years of age, 31% were > 10 years of age, and just 4.49% were < 6 months of age. This population was represented by 210 females (55.5%) and 168 males (44.4%); of them, 183 (48.41%) were dogs with a defined breed, while 195 (51.58%) were without defined breed. Dog breeds were distributed as follows: Tibetan (9.52%); German Shepherd (6.61%); Dachshund (5.29%); Chihuahua (3.7%); Rottweiler (2.64%); Cocker Spaniel (2.38%); Siberian Husky, Pointer, Dalmatian, and Boxer (2.11% each); Labrador, Pitbull, and Belgian Shepherd (1.32% each), Mexican Nude, Chow Chow, and Stanford (1.05% each); Pekinese (0.79%); Doberman (0.52%); and Maltese, Poodle, Shar Pei, Beagle, and Bulldog (0.26% each). More than half of the dogs (58.99%) had been in contact with other dogs, 38.09% were tick-infested, and 44.44% of the owners reported that they had applied tick treatment to their dogs.
2.6. Nested PCR The extraction of the genomic DNA was carried out from 300 μL of whole blood, using the Wizard Genomic DNA Purification Kit (Promega®, Madison, USA) according to the manufacturer's recommendations. The quality and concentration of the DNA were determined with a Nanodrop ND-2000® (Thermo Fisher Scientific, Wilmington, USA) and all samples were diluted to a concentration of 100 ng/μL. For the first PCR reaction (PCR1), ECC primers (5’-AGAACGAACG CTGGCGGCAAGC-3′) and ECB primers (5’-CGTATTACCGCGGCTGCTG GCA-3′) were used, which amplify a 479-bp fragment of the 16S rRNA gene. To identify the species of Ehrlichia, a second PCR reaction (PCR2) was conducted with ECAN primers (5’-CAATTATTTATAGCCTCTGGCT ATAGGA-3′) and HE3 primers (5’-TATAGGTACCGTCATTATCTTCCC TAT-3′), which amplify a 389-bp region of the 16S rRNA of E. canis according to previous methodology described by Murphy et al. (1998). The nPCR products were applied to 2% agarose gels (Sigma-Aldrich, St. Louis, USA) in 0.5× TBE buffer containing ethidium bromide (0.4 μg/mL), and the results were visualized in a UV 302 NM ultraviolet light transilluminator (KASVI - Curitiba, Brazil). In all cases, a 100-bp molecular weight marker (Promega, Madison, USA) was employed (data not shown).
3.2. ELISA Anti-E. canis antibodies were detected in 162/206 dogs belonging to the four municipalities (78.6%; 95% CI: 72.55–83.69). At the “cut-off” point, three plates each underwent an optical reading in the different ELISA conditions (0.119, 0.116 and 0.143), and they were transformed into 100 by this DO × 100/cut-off formula to compare all samples of the various plates with respect to density index. The distribution of the optical densities around the “cut-off” (DO × 100 / “cut-off”) obtained from the ELISA test for E. canis in sera from positive dogs resulted in density indices ranging from 109.5 to 970.7. 3.3. Nested PCR Of the total 378 samples analyzed, 179 were positive for E. canis based on the PCR assay (47.35%; 95% CI: 42.37–52.39). 3.4. Concordance between the two techniques Of the dogs examined, 109 of the seropositive animals (67.28%) were positive for E. canis based on nPCR, whereas 53% were positive for E. canis according to both ELISA and nPCR; 37 (18%) were negative for both tests. When the results of the molecular and serological tests were compared, a high concordance was observed (kappa = 0.7) according to the criteria of Kramer and Feinstein (1981) and Landis and Koch (1977). Of the two diagnostic techniques, a higher proportion (p = 0.0001) of
2.7. Statistical analysis The frequencies of E. canis-positive animals determined by ELISA and nPCR were compared via McNemar's, χ2 test for 95% confidence. The concordance between the frequencies of positive animals in the nPCR and ELISA was evaluated by the Kappa index (Kramer and Feinstein, 1981). This measure of agreement has a maximum value of 1, where this value 1 represents total agreement and values close to 0 172
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Table 1 Univariate analysis (GLM log-binomial) of the potential risk factors for infection by Ehrlichia canis in dogs, diagnosed by nested PCR, from four municipalities located in the western region of Cuba. Variables analyzed
Aspects related to the inherent characteristics of the dogs Municipality
Age
Breed definition Gender Aspects related to the tick Infestation by R. sanguineus Background of infestation History of diseases and control of ticks History of diseases transmitted by ticks Acaricide treatment Aspects related to the breeding and handling of the dog Contact with other dogs Number of dogs per house
Local access dog
The environment in which the dog spends most of the time
Habits
E. canis (nPCR) Positives/n (%)
PR
Cotorro Habana del Este San José de las Lajas Boyeros > 10 years ≥ 5 years to 10 years ≥ 2 years to < 5 years ≥ 6 months to < 2 years < 6 months Mixed breed Defined breed Male Female
28/82 (34.1) 42/104 (40.4) 42/90 (46.7) 67/102 (65.7) 19/39 (48.72) 53/110 (48.18) 61/125 (48.80) 49/83 (52.7) 5/17 (29.41) 96/197 (48.7) 83/181 (45.9) 86/174 (49.4) 93/204 (45.6)
⁎ 1.18 1.37 1.92 ⁎ 1.02 0.92 1.08 0.43 ⁎ 0.94 ⁎ 0.92
No Yes No Yes
102/231 (44.2) 77/147 (52.4) 37/103 (35.9) 142/275 (51.6)
⁎ 1.19 ⁎ 1.44
No Yes No Yes
130/281 (46.3) 49/97 (50.5) 105/233 (45.1) 74/145 (51.0)
⁎ 1.09 ⁎ 1.13
No Yes 1 dog 2 to 3 dogs > 3 dogs Urban environment Urban environment/At the house Confined in the house Land Grass or garden In cement constructions Loose Always confined Confined part of time
75/160 (46.9) 104/218 (47.7) 72/145 (49.7) 75/162 (46.3) 32/71 (45.1) 13/24 (54.2) 86/174 (49.4) 80/180 (44.4) 35/72 (48.6) 104/226 (46.0) 40/80 (50.0) 84/177 (47.5) 31/59 (52.5) 64/142 (45.1)
⁎ 1.02 ⁎ 0.93 0.91 ⁎ 0.91 0.82 ⁎ 0.95 1.03 ⁎ 1.11 0.95
95% CI
P-value
0.81–1.73 0.94–1.98 1.38–2.62
0.3876 0.1007 0.0001
0.7–1.48 0.63–1.34 0.74–1.57 0.17–1.09
0.9282 0.6611 0.6824 0.0765
0.76–1.16
0.5767
0.75–1.14
0.4556
0.96–1.47
0.1137
1.08–1.91
0.0117
0.86–1.38
0.4611
0.91–1.4
0.2531
0.82–1.26
0.8731
0.74–1.18 0.67–1.23
0.556 0.5331
0.61–1.36 0.55–1.23
0.6515 0.3356
0.72–1.25 0.74–1.42
0.6973 0.8643
0.83–1.48 0.75–1.21
0.4882 0.6718
nPCR: nested PCR; n: total number of samples; PR: Prevalence Ratio; CI: Confidence Interval. ⁎ Category reference.
seropositive and seronegative animals were detected by ELISA than nPCR.
Table 2 Risk factors for infection by Ehrlichia canis in dogs from four municipalities located in the western region of Cuba, based on nested PCR. Results of the multivariate analysis (GLM log-binomial).
3.5. Associated risk factors
Variables
Categories
PR
95% CI
p-value
The most common risk factor associated with E. canis infection was place of residence, as the population of dogs that resided in the Boyeros municipality exhibited a risk ratio of E. canis, 1.84 times higher (RR = 1.84; 95% CI: 1.32–2.56; p = 0.0003) than that found in the population of dogs within the Cotorro municipality. The prevalence of E. canis in the municipalities of San José de las Lajas and Habana del Este did not show significant differences from that of Cotorro municipality. A history of tick infestation was another significant risk factor (p = 0.007). Dogs that lived in the Boyeros municipality that had a history of tick infestation were more likely to contract infection via E. canis (risk factors), with a risk ratio 1.38 times higher (RR = 1.38; 95% CI: 1.04–1.81; p = 0.023) than that of animals with no history of ticks (Tables 1 and 2). There was no significant association between E. canis infection and age, breed, or sex.
Municipality
Cotorro Habana del Este San José de las Lajas Boyeros No Yes
⁎⁎ 1.13 1.34
0.78–1.66 0.92–1.94
0.521 0.123
Background of tick infestation
1.84 1.32–2.56 0.0003⁎⁎⁎ Reference category 1.38 1.04–1.81 0.023⁎
PR: Prevalence Ratio; CI: confidence interval. ⁎ p < 0,05. ⁎⁎ Category Reference. ⁎⁎⁎ p < 0,001.
4. Discussion In this study, a high percentage (78.6%) of dogs were found to possess anti-E. canis antibodies. This suggests that the dog populations belonging to the two studied provinces in the western region of Cuba 173
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(Nakaghi et al., 2008)]. This difference between the number of seropositive animals and percentage confirmed by nPCR may also be based on the stage of infection of the disease. During the acute stage, Ehrlichia infection is detectable by PCR in blood, whereas in the subclinical and chronic stages, the most appropriate samples are from the spleen and bone marrow (Harrus et al., 2004) (Mylonakis et al., 2004). As the results of this investigation were based on venous blood samples from a randomly selected population without clinical symptoms, it is possible that several dogs were in the subclinical or chronic phase, when it is not always possible to detect the agent in the blood, which would have resulted in false negatives when using PCR. During the ELISA test, cross-reactions may take place between Ehrlichia species, such as between E. ewingii and E. chaffeensis. This technique can also generate cross-reactions with other agents of the Anaplasmataceae family. Similar results to those obtained in this investigation were reported in Cameroon and the United States by Wen et al. (1997) and Ndip et al. (2005), respectively, who confirmed an agreement between PCR infection and indirect fluorescence antibody (IFAT) detection techniques in 41% of dogs diagnosed positively. Iqbal et al. (1994) compared the sensitivity of IFAT and PCR methods and concluded that a combination of both optimized the diagnosis. Similarly, Guedes et al. (2015) diagnosed canine ehrlichiosis using blood smears and both serological and molecular techniques, concluding that these are complementary methods owing to the differences in sensitivity and specificity exhibited by the methods in relation to the clinical phase of the infection. Dogs resident in the Boyeros municipality featured a higher probability of infection. This could be because of the breeding conditions in this area, where approximately 50% of the animals are loose or semiconfined, favoring contact with other dogs and exposure to ticks, just as reported by de Azevedo et al. (2011) and Cardoso et al. (2012). Huerto-Medina and Dámaso-Mata (2015) and Guedes et al. (2015) established that contact with other dogs is a risk factor associated with E. canis infection. In addition, dogs that have a history of tick infestation are more likely to be infected with E. canis, making tick infestation another risk factor. In previous studies conducted by (Dagnone et al., 2003) and (Ueno et al., 2009), however, possessing a history of tick infestation was not identified as a risk factor. Similar results in relation to age, breed, and sex were reported by (Procajło et al., 2011) and (Dahmani et al., 2015), neither of which determined a statistical association between positive PCR results and age or sex.
are highly exposed to infection. Although studies carried out in Cuba are scarce, the results of this investigation are similar to those of León Goñi and Gómez Rosales (2008), who also found high values of seroprevalence (81.65%). The animals evaluated by those authors were of defined breeds, had a history of infestation by ticks, and exhibited hemorrhagic and febrile symptoms. Those high values of seroprevalence may be based on the clinical condition of the animals that were investigated. Furthermore, 55.04% of the seropositive dogs were German Shepherds, the breed considered most susceptible to E. canis, with the disease being more severe and having a worse prognosis because the humoral immune response is depressed. Wen et al. (1997) and Ndip et al. (2005) reported that, among animals treated in clinics and veterinary hospitals in Cameroon and the United States, prevalence fluctuated between 32 and 76%, whereas prevalence was < 50% in certain regions of Brazil. Vieira et al. (2011) and Melo et al. (2011) put forth a seroprevalence value of 74.3% in urban areas of the Pantanal region, Mato Grosso, Brazil. In the north of Trinidad, Asgarali et al. (2012) also found values lower than 44.6% in stray dogs and Martínez-Vega et al. (2016) observed a seroprevalence value of 64% in Mexico. In this way, it can be observed that prevalence rates are variable in different regions of the American continent. However, in Cuba, the prevalence found indicated that E. canis is present on the island and that its distribution should attract the attention of the veterinary medical surveillance services given its high proportion and mainly because this is the first epidemiological study of canine ehrlichiosis in the country. This variability in seroprevalence likely occurs because the epidemiology of canine ehrlichiosis is related to vector distribution and associated with tropical and subtropical zones as well as being affected by animal behavior, breed, and living environment (Costa Jr et al., 2007). The results of the nPCR detected E. canis DNA in 47.35% of samples. The prevalence values obtained here are higher than those reported by Wen et al. (1997), who determined that 44% of dogs in the United States were positive for E. canis. The results presented herein are also higher than those from studies in other Caribbean regions, such as the 24.7% prevalence found in Granada (Yabsley et al., 2008), 14% prevalence found in Trinidad (Georges et al., 2008), 38.9% prevalence in Ribeirão Preto (Santos et al., 2009), and 27% prevalence in the Saint Kitts Islands (Kelly et al., 2013). In contrast, the current results concerning E. canis infection were lower than those obtained by (de Paiva Diniz et al., 2007) in dogs from the state of São Paulo (76.8%) and lower than those of (Ramos et al., 2009), who noted prevalence values of 57% using nPCR with dogs clinically suspected of the disease. These variations in the percentages of positivity to rickettsia may be related to the condition of the canine population, exposure to the vector, R. sanguineus, and the diagnostic methods along with their sensitivity (Solano-Gallego et al., 2006). Of the four municipalities investigated, Boyeros had the highest prevalence value (65.7%), followed by San José de las Lajas (46.7%), Havana del Este (40.4%), and Cotorro (34.1%). This finding is influenced by the handling and conditions of dogs. Despite the fact that 100% of those sampled in Boyeros had owners, 43.28% of them roamed the streets all day, affecting control and making contact with other dogs infested with the vector more likely in addition to having access to environments favorable for the development and maintenance of the disease (Dantas-Torres, 2008, 2010; Silveira et al., 2009). Employing nPCR, 109 (67.28%) dogs were confirmed to be positive for E. canis, whereas 162 (78.6%) were diagnosed seropositive by ELISA. The absence of E. canis DNA in 53 samples that were positive by serology can be explained by the occurrence of non-specific reactions that cause false positives through serological testing (Al-Adhami et al., 2011). Additionally, serological techniques only discern specific antibodies, and these can remain systemically for a long time after treatment and cure, or elimination of the agent by the immune system [although results of PCR would be negative (Rikihisa et al., 1994);
5. Conclusions In the studied area, a high prevalence of E. canis was detected in the canine population. In addition, residing in the Boyeros municipality and featuring a history of tick infestation was associated with E. canis infection. Furthermore, the results of ELISA testing in the population studied exhibited high concordance with the results of the nPCR to detect infected animals. Conflict of interest The authors declare there are no conflicts of interest. Acknowledgements We would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes/MES-Cuba – Proc. 188/2013) and Fundação de Apoio à Pesquisa do estado do Rio de Janeiro (FAPERJ – Proc. E26/ 201.144/2014) for the financial support. References Al-Adhami, B., Scandrett, W.B., Lobanov, V.A., Gajadhar, A.A., 2011. Serological cross-
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Melo, A.L.T., Martins, T.F., Horta, M.C., Moraes-Filho, J., Pacheco, R.C., Labruna, M.B., Aguiar, D.M., 2011. Seroprevalence and risk factors to Ehrlichia spp. and Rickettsia spp. in dogs from the Pantanal Region of Mato Grosso State, Brazil. Ticks Tick Borne Dis. 2, 213–218. Murphy, G.L., Ewing, S., Whitworth, L.C., Fox, J.C., Kocan, A.A., 1998. A molecular and serologic survey of Ehrlichia canis, E. chaffeensis, and E. ewingii in dogs and ticks from Oklahoma. Vet. Parasitol. 79, 325–339. Mylonakis, M.E., Koutinas, A.F., Baneth, G., Polizopoulou, Z., Fytianou, A., 2004. Mixed Ehrlichia canis, Hepatozoon canis, and presumptive Anaplasma phagocytophilum infection in a dog. Vet. Clin. Pathol. 33, 249–251. Nakaghi, A.C.H., Machado, R.Z., Costa, M.T., André, M.R., Baldani, C.D., 2008. Canine ehrlichiosis: clinical, hematological, serological and molecular aspects. Cienc. Rural 38, 766–770. Navarrete, M.G., Cordeiro, M.D., Batista, Y., Alonso, J.C., Márquez, M., Roque, E., Fonseca, A., 2017. Serological detection of Toxoplasma gondii in domestic dogs in the western region of Cuba. Vet. Parasitol. Reg. Stud. Reports. 9, 9–12. Ndip, L., Ndip, R.N., Esemu, S., Dickmu, V., Fokam, E., Walker, D., McBride, J., 2005. Ehrlichial infection in Cameroonian canines by Ehrlichia canis and Ehrlichia ewingii. Vet. Microbiol. 111, 59–66. Nicholson, W.L., Allen, K.E., McQuiston, J.H., Breitschwerdt, E.B., Little, S.E., 2010. The increasing recognition of rickettsial pathogens in dogs and people. Trends Parasitol. 26, 205–212. Parola, P., Paddock, C.D., Socolovschi, C., Labruna, M.B., Mediannikov, O., Kernif, T., Abdad, M.Y., Stenos, J., Bitam, I., Fournier, P.E., Raoult, D., 2013. Update on TickBorne Rickettsioses around the World: a Geographic Approach. Clin. Microbiol. Rev. 26, 657–702. Pérez, B., Valdés, R., Vitorte, S., 2002. Epidemiología de las enfermedades trasmitidas por garrapatas en. Cuba. Rev. Cubana Cienc. Vet. 20, 78–87. Pino-Rodríguez, D., Márquez-Álvarez, M., Rojas-Hoyos, N.A., 2017. Aspectos demográficos de la población de perros con dueños del municipio Boyeros. Cuba. Rev. Sal. An. 39, 00. Procajło, A., Skupień, E., Bladowski, M., Lew, S., 2011. Monocytic ehrlichiosis in dogs. Polish J. Vet. Sc. 14, 515–520. Ramos, C.A., Ramos, R.A., Araújo, F.R., Guedes Jr., D., Souza, I.I., Ono, T.M., Vieira, A.S., Pimentel, D.S., Rosas, E.O., Faustino, M.A., 2009. Comparação de nested-PCR com o diagnóstico direto na detecção de Ehrlichia canis e Anaplasma platys em cães. Rev. Bras. Parasitol. Vet. 18, 58–62. Rikihisa, Y., Ewing, S., Fox, J., 1994. Western immunoblot analysis of Ehrlichia chaffeensis, E. canis, or E. ewingii infections in dogs and humans. J. Cl. Microbiol. 32, 2107–2112. Sampaio, I.B.M., 2007. Estatística aplicada á experimentaçao animal (Fundaçao de Estudo e Pesquisa em Medicina Veterinaria). pp. 265. Santos, F., Coppede, J.S., Pereira, A.L., Oliveira, L.P., Roberto, P.G., Benedetti, R.B., Zucoloto, L.B., Lucas, F., Sobreira, L., Marins, M., 2009. Molecular evaluation of the incidence of Ehrlichia canis, Anaplasma platys and Babesia spp. in dogs from Ribeirão Preto, Brazil. Vet. J. 179, 145–148. Silveira, J.A.G., Passos, L.M.F., Ribeiro, M.F.B., 2009. Population dynamics of Rhipicephalus sanguineus (Latrielle, 1806) in Belo Horizonte, Minas Gerais state, Brazil. V. Parasit. 161, 270–275. Solano-Gallego, L., Llull, J., Osso, M., Hegarty, B., Breitschwerdt, E., 2006. A serological study of exposure to arthropod-borne pathogens in dogs from northeastern Spain. Vet. Res. 37, 231–244. Team, 2013. R.C. 2014. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria ISBN 3-900051-07-0. http:// www.r-project.org/. Ueno, T.E.H., Aguiar, D.M., Pacheco, R.d.C., Richtzenhain, L.J., Ribeiro, M.G., Paes, A.C., Megid, J., Labruna, M.B., 2009. Ehrlichia canis em cães atendidos em hospital veterinário de Botucatu, Estado de São Paulo, Brasil. Rev. Bras. Parasitol. Vet. 18, 57–61. Vieira, R.F.d.C., Biondo, A.W., Guimarães, A.M.S., Santos, A.P.d., Santos, R.P.d., Dutra, L.H., Diniz, P.P.V.P., Morais, H.A.d., Messick, J.B., Labruna, M.B., 2011. Ehrlichiosis in Brazil. Rev. Bras. Parasitol. Vet. 20, 01–12. Voller, A., Bidwell, D., Bartlett, A., 1976. Enzyme immunoassays in diagnostic medicine: Theory and practice. Bull. Word Health Organ. 53, 55–65. Wen, B., Rikihisa, Y., Mott, J.M., Greene, R., Kim, H.-Y., Zhi, N., Couto, G.C., Unver, A., Bartsch, R., 1997. Comparison of nested PCR with immunofluorescent-antibody assay for detection of Ehrlichia canis infection in dogs treated with doxycycline. J. Clin. Microbiol. 35, 1852–1855. Yabsley, M.J., McKibben, J., Macpherson, C.N., Cattan, P.F., Cherry, N.A., Hegarty, B.C., Breitschwerdt, E.B., O'Connor, T., Chandrashekar, R., Paterson, T., Perea, M.L., Ball, G., Friesen, S., Goedde, J., Henderson, B., Sylvester, W., 2008. Prevalence of Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, Bartonella vinsonii berkhoffii, and Rickettsia spp. in dogs from Grenada. Vet. Parasitol. 151, 279–285.
reactivity between Anaplasma marginale and an Ehrlichia species in naturally and experimentally infected cattle. J. Vet. Diagn. Investig. 23, 1181–1188. Asgarali, Z., Pargass, I., Adam, J., Mutani, A., Ezeokoli, C., 2012. Haematological parameters in stray dogs seropositive and seronegative to Ehrlichia canis in North Trinidad. Ticks Tick-Borne Dis. 3, 207–211. de Azevedo, S.S., Aguiar, D.M., de Aquino, S.F., Orlandelli, R.C., da Fonseca Fernandes, A.R., Uchôa, I.C.P., 2011. Soroprevalência e fatores de risco associados à soropositividade para Ehrlichia canis em cães do semiárido da Paraíba. Brazil. J. Vet. Res. An. Sc. 48, 14–18. Barros-Battesti, D.M., Hernández, M.R., Famadas, K.M., Onofrio, V.C., Beati, L., Guglielmone, A.A., 2009. The ixodid ticks (Acari: Ixodidae) of Cuba. Sys. Appl. Acarol. 14, 101–128. Cardoso, L., Mendão, C., Madeira De Carvalho, L., 2012. Prevalence of Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi sensu lato, Anaplasma spp. and Leishmania infantum in apparently healthy and CVBD-suspect dogs in Portugal - a national serological study. Parasit. Vectors 5, 62. Costa Jr., L.M., Rembeck, K., Ribeiro, M.F.B., Beelitz, P., Pfister, K., Passos, L.M.F., 2007. Sero-prevalence and risk indicators for canine ehrlichiosis in three rural areas of Brazil. Vet. J. 174, 673–676. Dagnone, A.S., De Morais, H.S.A., Vidotto, M.C., Jojima, F.S., Vidotto, O., 2003. Ehrlichiosis in anemic, thrombocytopenic, or tick-infested dogs from a hospital population in South Brazil. Vet. Parasitol. 117, 285–290. Dahmani, M., Loudahi, A., Mediannikov, O., Fenollar, F., Raoult, D., Davoust, B., 2015. Molecular detection of Anaplasma platys and Ehrlichia canis in dogs from Kabylie, Algeria. Ticks Tick-borne Dis. 6, 198–203. Dantas-Torres, F., 2008. Canine vector-borne diseases in Brazil. Parasit. Vectors 1, 25. https://doi.org/10.1186/1756-3305-1-25. Dantas-Torres, F., 2010. Biology and ecology of the brown dog tick, Rhipicephalus sanguineus. Parasit. Vectors 3, 26. https://doi.org/10.1186/1756-3305-3-26. de Paiva Diniz, P.P.V., Schwartz, D.S., De Morais, H.S.A., Breitschwerdt, E.B., 2007. Surveillance for zoonotic vector-borne infections using sick dogs from southeastern Brazil. Vector-Borne Zoon. Dis. 7, 689–698. Frey, A., Di Canzio, J., Zurakowski, D., 1998. A statistically defined endpoint titer determination method for immunoassays. J. Immunol. Methods 221, 35–41. George, J.E., Davey, R.B., Pound, J.M., 2002. Introduced ticks and tick-borne diseases: The threat and approaches to eradication. Vet. Cli. North Am. 18, 401–416. Georges, K., Ezeokoli, C.D., Newaj-Fyzul, A., Campbell, M., Mootoo, N., Mutani, A., Sparagano, O.A., 2008. The application of PCR and reverse line blot hybridization to detect arthropod-borne Hemopathogens of dogs and cats in Trinidad. Ann. N. Y. Acad. Sci. 1149, 196–199. Gondard, M., Cabezas-Cruz, A., Charles, R.A., Vayssier-Taussat, M., Albina, E., Moutailler, S., 2017. Ticks and Tick-Borne Pathogens of the Caribbean: Current understanding and future directions for more comprehensive surveillance. Front. Cell. Infect. Microbiol. 7 (490). https://doi.org/10.3389/fcimb.2017.00490. Guedes, P.E.B., Oliveira, T.N.d.A., Carvalho, F.S., Carlos, R.S.A., Albuquerque, G.R., Munhoz, A.D., Wenceslau, A.A., Silva, F.L., 2015. Canine ehrlichiosis: Prevalence and epidemiology in Northeast Brazil. Braz. J. Vet. Parasitol. 24, 115–121. Harrus, S., Kenny, M., Miara, L., Aizenberg, I., Waner, T., Shaw, S., 2004. Comparison of simultaneous splenic sample PCR with blood sample PCR for diagnosis and treatment of experimental Ehrlichia canis infection. Antimicrob. Agents Chemother. 48, 4488–4490. Huerto-Medina, E., Dámaso-Mata, B., 2015. Factores asociados a la infección por Ehrlichia canis en perros infestados con garrapatas en la ciudad de Huánuco, Perú. Rev. Peruana Med. Exper. Sal. Públ. 32, 756–760. Iqbal, Z., Chaichanasiriwithaya, W., Rikihisa, Y., 1994. Comparison of PCR with other tests for early diagnosis of canine ehrlichiosis. J. Clin. Microbiol. 32, 1658–1662. Kelly, P., Xu, C., Lucas, H., Loftis, A., Abete, J., Zeoli, F., Stevens, A., Jaegersen, K., Ackerson, K., Gessner, A., Kaltenboeck, B., Wang, C., 2013. Ehrlichiosis, Babesiosis, Anaplasmosis and Hepatozoonosis in dogs from St. Kitts, West Indies. PLoS One 8 (1). https://doi.org/10.1371/journal.pone.0053450. Kramer, M.S., Feinstein, A.R., 1981. Clinical biostatistics: LIV. The biostatistics of concordance. Clin. Pharmacol. Therap. 29, 111–123. Landis, J.R., Koch, G.G., 1977. The measurement of observer agreement for categorical data. Biometrics 33, 159–174. León Goñi, A.C., Gómez Rosales, D., 2008. Ehrlichiosis canina. REDVET. Rev. Elect. Vet. 9. Loftis, A.D., Kelly, P.J., Freeman, M.D., Fitzharris, S., Beeler-Marfisi, J., Wang, C., 2013. Tick- borne pathogens and disease in dogs on St. Kitts, West Indies. Vet. Parasitol. 196, 44–49. Martínez-Vega, P.P., Bolio-Gonzalez, M.E., Rodríguez-Vivas, R.I., Gutierrez-Blanco, E., Pérez-Osorio, C., Villegas-Perez, S.L., Sauri-Arceo, C.H., 2016. Associated Factors to Seroprevalence of Ehrlichia spp. in dogs of Quintana Roo, Mexico. J. Trop. Med. 2016, 1–6.
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