INTRODUCTION

Ehrlichia canis is an obligate intracellular bacteria, the causative pathogen of Canine monocytic ehrlichiosis (CME), which is a potentially zoonotic vector-borne disease ^1,2. The vector is Rhipicephalus sanguineus ticks. CME distribution is closely related to the distribution of the vector ³. The national seroprevalence of dogs in Mexico is 33% and 74.5% in the northwest of the country ⁴. The clinical symptoms commonly seen with this disease in infected dogs are fever, vomiting, arthralgia, rash, and diarrhea ⁵. Diagnosis is based on visualization of the morula in monocyte and enzymatic-immunoassay ⁴; however, as molecular tools 16SrRNA gene detection by PCR and sequencing have been successfully used for diagnosis ^6,7. In Venezuela, E. canis was isolated from blood of an asymptomatic patient, and another study in infected dogs and ticks from the same region suggest that it is a zoonotic pathogen ^2.8.

Because of the high seroprevalence of infection on dogs from Mexico, is necessary to determine the actual frequency using molecular methods, and knowing the genetic variability of E. canis in dogs and ticks reported from American countries. According to this, the aim of this study was to determine the frequency of Ehrlichia canis in dogs naturally infected, as well as infesting ticks it, comparing the sequences identified in Mexico with those published in GenBank from American countries, and perform phylogenetic analysis looking for the relation between isolates.

MATERIAL AND METHODS

Study areas and samples collection.The study was conducted in Northwestern of Mexico, state of Sinaloa. The study was approved by Comisión Nacional de Investigación (R-2013-069, México). Samples were collected from July 2010 to August 2011. In this study, dogs captured by the pound in three cities of North, Central and South, as well as dogs carried to any veterinary of Sinaloa were included. As well as owners of sick dogs, workers of veterinary and kennels to participate in the study, prior informed consent; 88 owners and kennel workers accepted and we collected their blood samples for finding infection (data not shown). 139 dogs with suggestive clinical manifestations were taken blood samples, and two ticks were collected from each dog.

Ticks were identified morphologically by entomologists, using dichotomous keys. DNA from 139 blood samples dogs of and ticks was extracted with QIAamp(r) DNA Blood kit (Qiagen, CA), and stored at -80 until the processing. For E. canis and Ehrlichia spp. detection, part of 16SrRNA gene was amplified with ECC-ECB and HE3-ECA primers, previously described ^2,8 amplified an expected band 389pb; and sequencing was performed using 15F-842R primers, previously described amplifying the 16SrRNA gene, with an expected band of 800bp ⁹. In each reaction was included a positive and negative (distilled water) control.

Sequencing. Positive samples to Ehrlichia canis were purified by the kit protocol of QIAquick Gel Extration Minikit (Qiagen, CA). Sequences were analyzed with Chromas 233 program, and the search was conducted in GenBank database to determine the species and homology.

Phylogenetic analysis. 15-856 region of the 16SrRNA gene was sequenced and aligned individually with MUSCLE program. The phylogenetic trees were performed with the program MEGA v.5, using the Maximum Likelihood Composite (MLC) method, and Maximum Parsimony method, by assembling data from bootstrap in 1,000 repetitions. The Neighbor-Join algorithm was used to obtain the initial tree, with 10 replicas of the sequences ^10,11.

RESULTS

Of 139 dogs, were obtained 41 positive dogs with clinical manifestations (Table 1). Ehrlichia spp. was found in 41/139 (27.52%) from dogs with clinical suspect, where E. canis was detected in 25/139 (18.0%). The tick R. sanguineus was the most prevalent (92.1%), and were identified Ixodes scapularis, H. leporis-palustris and D. variabilis ticks. Were positive to Ehrlichia spp. 32/139 (23.2%) ticks and 29/139 (20.86) ticks to E. canis (Table 2). The dogs risk to be infected with E. canis when ticks were positive was 8.24 OR (IC 3.2-21.9, 95%).

We sequenced six positive products for E. canis; three dog samples and three tick samples. The 16SrRNA gene amplified from dogs and ticks (GenBank No. KP844657-62) showed a 99.8% homology with Ehrlichia canis strain Jake, E. canis HEV, E. canis VDE and E. canis strain Brazil- COI sequences (Table 3).

Two trees were performed: the first with Maximum likelihood method (Figure 1), in which shows the tree with the highest log likelihood (-1074.2304); was constructed automatically, with the heuristic search by applying Neighbor-Join algorithm with a matrix of pairwise distances estimated, using MCL method; after selecting the topology with a value greater likelihood log. The tree was drawn to scale with branch lengths calculated using the average pathway method and are in the units of the number of changes over the whole sequence.

Figure 1 Phylogenetic analysis of 16SrRNA gene of Ehrlichia canis, constructed with sequences of America countries by Maximum Likelihood method. 

The second tree was constructed by Maximum Parsimony method (Figure 2). Tree number 1 out of 10 most parsimonious trees (length = 91) is showed. The consistency index is 0.967033 (0.936170), the retention index is 0.972973 (0.972973), and the composite index is 0.940897 (0.910868) for all sites and parsimony-informative sites. The Maximum parsimony tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm with search level 0 in which the initial trees were obtained by the random addition of sequences (10 replicates).

Figure 2 Phylogenetic analysis of 16SrRNA gene of Ehrlichia canis, constructed with sequences of America countries by Maximum Parsimony method. 

DISCUSSION

These results confirm the existence of enzootic cycle transmission to E. canis in Sinaloa, Mexico. 92.1% of the ticks identified were R. sanguineus, in which 28 were positive for Ehrlichia spp., and 27 positive to E. canis. Two adult D. variabilis ticks were infected with Ehrlichia spp. This is the first report of this association in Mexico. It was already experimentally demonstrated the competitiveness of this vector to transmit the bacteria ⁴. Antibodies an E. canis can remain elevated during a period of time prolonging, causing false positives in endemic areas.

It is the first evidence of E. canis in H. leporis-palustris ticks in Mexico. It may represent the presence of potential vectors, capable of transmitting E. canis. More information is needed in order to prove if this vector can take on the role of competent vectors; ie, capable of transmitting the pathogen and causing the disease.

The presence of three main groups (I, II and III) for E. canis, shows that this pathogen has few variables in America, similar to that found in other studie ⁶. Although, Mexican isolates show differences between the groups I and II; the sequences found, presented an ancestral relationship with the sequences from Venezuela, Brazil and the United States; hypothesizing that it is possible to find the same variant of E. canis among American countries. It is required more phylogenetic analysis of E. canis between worldwide sequences, and search for other genes with greater variability and observe the behavior.

This study presents the first molecular evidence of E. canis in dogs and ticks from Sinaloa, Mexico. The results show a high risk of infection in tick-infested dogs. In Mexico, recent study in ticks proves a high frequency of infection of E. canis, and could play a hight risk in humans and dogs ¹². The importance of understanding the enzootic cycle of Ehrlichia in ticks, wild and domestic reservoirs, can help implement strategies to prevent infection in dogs ^12,13.

This research provides the assessment for further study of genetic variability in isolated not only from E. canis, but the rest of vector, helping to understand the behavior and potential targets in preventing diseases.