ABSTRACT

The aim of the study was to determine the frequency and associated factors of Ehrlichia canis infection in dogs. Blood samples from 150 dogs infested with ticks in 10 veterinary clinics in the city of Huanuco in Peru were collected. The dogs were randomly selected without regard to breed, age or sex. Ehrlichia canis antibodies were detected by chromatographic immunoassay.51.3% of dogs were infected with Ehrlichia canis. In the multivariate analysis, factors associated with the presence of Ehrlichia canis were: poor health of the dog (p = 0.049), a higher average of tick infestation (p = 0.018), and adult dogs (p = 0.038). The frequency of Ehrlichia canis in dogs of this city is high. Control of the brown dog tick (Rhipicephalus sanguineus) vector of Ehrlichia canis is recommended.

Key words: Ehrlichia canis; Rhipicephalus sanguineus; Prevalence; Immunoenzyme techniques (source: MeSH NLM).

INTRODUCTION

Canine ehrlichiosis is a disease of domestic and wild canines and occurs worldwide. It is caused by Ehrlichia canis and is also called “tracker dog disease,” “tropical canine pancytopenia,” “canine hemorrhagic fever,” and “canine typhus” ⁽¹⁾. E. canis is a gram-negative pleomorphic microorganism of the Rickettsiaceae family and infects circulating monocytes within their cytoplasm in aggregates known as morula. E. canis is transmitted by the brown dog tick, Rhipicephalus sanguineus, which occurs in the transstadial form but not in the transovarial form ⁽²⁾. The infection in the animal disseminates through the blood or the lymph system within infected mononuclear cells, thus reaching other organ systems ⁽³⁾. The majority of cases occurs in endemic areas in spring and summer months, when ticks are most active. Because the transmission of ehrlichiosis is mechanical and nonbiological, transfusions with infected blood can result in high rates of infection ⁽²⁾.

The brown dog tick, R. sanguineus, is widespread in Peru ⁽⁴⁾. Some studies of E. canis have shown increased susceptibility of the German shepherd dogs to this disease, as compared to other breeds ⁽⁵⁾. Although E. canis was identified for the first time in Algeria in 1935, it received broad attention in 1987 when E. chaffeensis, a closely related microorganism (shares 98.2% homology with ribosomal DNA [16S rDNA] of E. canis), was identified as the cause of human monocytic ehrlichiosis ⁽²⁾. In recent years, a new species of monocytic Ehrlichia was isolated and characterized in Venezuela; this organism may be a subspecies of E. canis; consequently, human ehrlichiosis is considered a disease of zoonotic importance by the Pan-American Health Organization (PAHO). In 2006, by PCR analysis of the rRNA 16S gene, researchers found that 30% of seropositive patients have symptoms consistent with ehrlichiosis; this was the first report of E. canis infection in humans ⁽⁶⁾

Ehrlichiosis has been reported as an emerging zoonotic disease, and multiple cases have been described worldwide. In Peru, ehrlichiosis has been detected in dogs (canine monocytic ehrlichiosis) since 1982 ⁽⁷⁾, and the number of reported cases has increased since then.

In 2001, 16.5% seroprevalence was discovered in metropolitan Lima in the districts of Chorrillos, La Molina, and San Juan de Miraflores ⁽⁸⁾. In 2005, a dog with canine granulocytic ehrlichiosis (CGE) was identified in the district of La Molina. In addition, seroprevalence of up to 76% was reported in 2006 for Sullana, Piura ⁽⁹⁾.

This disease is highly likely to develop in our environment; therefore, serological detection of antibodies to E. canis in dogs should be useful in the fields of veterinary medicine and public health. The objective of this study was to determine the frequency of and factors associated with canine ehrlichiosis in the province of Huanuco, Peru.

THE STUDY

This was a descriptive cross-sectional study. The city of Huanuco is located in north-central Peru. According to the National Institute of Statistics and Informatics, in 2012, this city had an estimated population of 301,396. It is located 1800 m above the sea level, in the valley formed by the Huallaga River. With the average temperature of 24ºC, the lowest temperature is observed in winter, during the months of July and August (21ºC during the day and 17ºC at night). The highest temperatures are in spring, during November and December (30ºC during the day): endemic conditions for tick infestation. In the rural area, people have a habit of keeping multiple dogs per household, and the majority of these owners do not treat their dogs for ticks. In addition, there are many stray dogs, making it easier for the R. sanguineus tick to reproduce. Therefore, the presence of appropriate vectors for known ticks as well as host reservoirs largely determine where E. canis occurs (it is primarily transmitted by R. sanguineus) ⁽¹¹⁾. The estimated canine population is 57,850 (according to The Office of Environmental Health of the Huanuco Health Network; 2012).

The selection of the sample was not probabilistic (via casual or accidental sampling); the dogs were included consecutively as they were brought to the clinics. We used a confidence level of 95%, an estimate of 6%, and prevalence of 16.5% ⁽⁸⁾. The statistical sample consisted of 150 dogs.

Ten veterinary clinics in the province of Huanuco participated on a voluntary basis. The study included 15 dogs from each clinic. Only tick-infested dogs were included, and the owner was queried to establish the factors associated with this infection.

The study variables were the following:

Lifestyle: defined as the way of life, describing generically the style, form, or manner in which living is understood (street and home).

Degree of tick infestation: the brown dog ticks (R. sanguineus) were counted at all stages (larvae, nymphs, adults, or blood-filled females) because they can act as a vector for E. canis at any stage. The criterion used for the count was cranial-to-caudal occurrence; no validated methodology for the calculation of tick infestation was found in the literature. The results were categorized in ordinal form (from 0 to 10, 11 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, and from 101 to 110).

Health condition of the dog: categorized as poor, good, or excellent. The condition was deemed good when the dog had a normal physical appearance, the diet was adequate, it had no clinical signs of disease, and the owner stated that the dog was in good physical form. The condition was deemed excellent when the dog was in good condition, and the owner followed its vaccination and antiparasitic treatment schedule properly, in addition to a proper physical appearance of the dog. The condition was deemed poor when the dog was not healthy, according to the presence of one or more clinical signs such as diarrhea, vomiting, cachexia, ecchymosis, jaundice, ataxia, dyspnea, or weight loss.

Age group: defined according to the period that the dog has lived since birth.

Type of diet: defined as the intake of any nutrient by the dog (homemade, mixed, or balanced).

Central tendency and dispersion measures for quantitative variables as well as percentages for categorical variables were used for data analysis. The bivariate analysis was carried out using the Pearson chi squared test, which assesses the existence of a relation between ehrlichiosis and relevant factors. A multivariate analysis with nonconditional logistic regression was performed with the variables that were significant in the bivariate analysis. The effect of collinearity between the evaluated variables was taken into account. We used statistical software Stata v. 11.

A rapid test for detection of anti-E. canis antibodies was used to diagnose E. canis infection; the test consists of an immunochromatographic assay on a solid phase for detection of anti-E. canis antibodies in serum, plasma, or blood of dogs. The duration of reading the results was 20 minutes. This test is made by the Bionote® Company, South Korea ⁽¹⁰⁾.

RESULTS

The frequency of E. canis infection in dogs infested with ticks in the province of Huanuco was 51.3% (95% CI: 43.0–59.6).

There was a statistically significant difference between the presence of E. canis and the following variables: the health condition of a dog (for poor condition, p = 0.016); lifestyle of the dog (for strays, p = 0.011); average number of ticks (i.e., infestation degree; p = 0.009); age (p = 0.006), and type of diet (p = 0.004; Table 1). No statistically significant correlation was detected between the prevalence of E. canis infection and the following variables: sex (p = 0.35), breed (p = 0.296), social status of the owner (p = 55.521), and educational level of the owner (p = 0.378).

In the multivariate analysis, independent associations were found between the prevalence of E. canis infection and these variables: a poor health condition of the dog (p = 0.049), average number of ticks (p = 0.018), age (p = 0.038), and the type of balanced diet (p = 0.015; Table 2).

DISCUSSION

The high prevalence of E. canis infection (51.3%) among tick-infested dogs in the Huanuco province could be due to its climatic characteristics and its sanitation conditions because the incubation process of R. sanguineus eggs depends mainly on environmental temperature and humidity. The optimal conditions for oviposition and progression through the various life cycle stages are the temperature range from 20°C to 30°C ^(11,12) and the environmental humidity range from 20% to 93% ⁽¹³⁾. The average temperature in Huanuco is 24°C, similar to the annual average temperature in Belo Horizonte in Brazil, 23°C, where 7318 ticks were collected from infested dogs, and 100% of the ticks were identified as R. sanguineus ⁽¹⁴⁾. These similar climatic conditions, in addition to the rural sanitation conditions of the dwellings, can account for greater tick proliferation.

Upon evaluating the relation between the health condition of the dogs and the presence of E. canis, we found a significant association because the poor health condition in 63.9% of cases is related to a positive diagnostic test result. The poor health condition of a dog is a risk factor of E. canis infection. Infection is likely to depend on transmission via tick bites.

Homelessness of a dog is also a risk factor of E. canis infection. This is because when an animal is away from home, being in contact with “street dogs,” which are usually infested with ticks, the risk of infection is greater. This association was not proven by the multivariate analysis.

The factor “greater number of ticks” was found to be linked to E. canis infection. This is because with a greater number of ticks, the risk of transmission is greater. It is difficult to control ticks due to the complexity of their life cycle and the favorable climate in the province of Huanuco; thus, reinfestation by ticks and transmission of ehrlichiosis are possible.

Upon evaluating the relation between the age of the dog, categorized by groups, and the diagnosis of E. canis infection, we found a significant association. To be precise, adult age of a dog is linked to E. canis infection. In a study conducted at the Veterinary Teaching Hospital of Londrina State University, State of Parana, southern Brazil, 87 of 381 dogs (22.8%) tested positive for ehrlichiosis; according to that study, groups at increased risk of being seropositive for ehrlichiosis compared with the general population included dogs over 1 year of age, previously exposed to ticks, and showing neurological signs ⁽¹⁵⁾. Middle-aged dogs have a greater chance of being exposed to the vector than pups do. This situation is due to the habit of the owners to let the animals out on the street when they complete their vaccinations, believing that such dogs are at a lower risk of infectious diseases. In this study, the breeds (including the cross-breeds) showed no significant relation to the presence of E. canis infection; i.e., when it comes to the breed, animals have the same likelihood of infection ⁽¹⁶⁾. This result contradicts studies where breed analysis indicated that the German Shepherd is at a greater risk.

The type of homemade food that the dog receives is related to E. canis infection. This finding can be explained: an animal that consumes balanced food takes in a greater amount of nutrients and thus the immune response triggered by the host is better as compared to an animal that consumes homemade food only. Nevertheless, infection is still likely to depend on transmission via a tick bite.

Finally, upon evaluating the relation between the educational level of the dog’s owner and E. canis infection in the dog, we found no significant association. Therefore, the educational level of dogs’ owners is not related to E. canis infection. This is because in an area endemic for ticks, the ticks are present in any home, regardless of the educational level of the dogs’ owners.

As for the risk to humans, E. canis, or an organism indistinguishable from it because it has been isolated from a human being in Venezuela, seems to carry a zoonotic risk, in line with another study ⁽¹⁷⁾. Later, in 2006, by means of PCR analysis of the 16S rRNA gene, some researchers demonstrated that 30% of seropositive patients have symptoms consistent with ehrlichiosis; that was the first report of E. canis infection in humans ⁽¹⁸⁾. In addition, genetic characterization of the 16S rRNA gene in two strains of E. canis in the region of Botucatu, São Paulo State, Brazil, revealed that a strain that naturally infects dogs is identical to the Venezuelan strain that infects humans ⁽¹⁹⁾. Thus, the E. canis strains of Brazil may infect human beings.

According to a study by De Souza et al., the indirect immunofluorescence test is the gold standard for the diagnosis of human monocytic ehrlichiosis. In this test, there is cross-reactivity between E. canis and E. chaffeensis. There is only one description of E. chaffeensis (from a deer) confirmed by sequencing in Brazil. In contrast, E. canis is common and widespread among dogs. Those authors hypothesized that some, if not all of the human cases attributed to E. chaffeensis in Brazil are actually caused by E. canis ⁽¹⁹⁾.

The main limitation of our study is the risk of low representativeness due to the small sample size, in addition to nonprobabilistic sampling.

In conclusion, the prevalence of E. canis infection among dogs in the province of Huanuco is 51.3%. The variables that are related independently to E. canis infection are the poor health condition of a dog, the average number of ticks (infestation degree), adult age, and homemade diet of the dog.

Acknowledgements: we thank veterinarian doctors of the 10 offices for assistance with the analyses performed.

Author contributions: EHM participated in the conception and design of the study, laboratory work, and collection of data. EHM and BDM performed analysis and interpretation of data, writing of the manuscript, critical review, and approval of its final version. BDM also provided technical, statistical, and administrative advice.

Funding source: self-funded by the main author (EHM).

Conflicts of interest: the authors have no conflicts of interest related to the publication of this article.

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Correspondence: Edward Huerto-Medina

Address: Jr. Jorge Chávez 332, Paucarbamba, Amarilis, Huánuco, Peru

Phone Number: 062-962700265