In this work, of the 59 clinically healthy dogs analyzed, 47.45% had a positive result with blood PCR to Ehrlichia canis. In addition, PCR recognized a higher prevalence of E. canis in different tissues of naturally infected dogs, both in dogs with positive and negative results by blood PCR. With these results it was demonstrated that some dogs suspected of presenting subclinical ehrlichiosis, presented E. canis DNA in various tissues, even though they had negative PCR results in blood.
At the present time, diagnosis by PCR is more useful than serology for the differentiation of concurrent infections and co-infections with diverse Ehrlichia spp. and is used for treatment monitoring [47]. However, in naturally-occurring CME, the diagnostic sensitivity and optimal tissue for PCR testing in the untreated dog or in the post-treatment setting has not yet been clarified [47]. Results obtained at this point demonstrate that, in dogs with naturally-occurring CME infection, it is feasible to detect E. canis in different tissues, even if they have had negative blood tests. Additionally, in the acute phase of infection, E. canis is easily detected in blood, while in the subclinical and chronic phases, there is the possibility of false negatives. Therefore, some tissues are more appropriate for sampling, such as the bone marrow and the spleen [8,9,13,49], argument that has been corroborated in the present investigation. This research does not suggest performing PCR of tissues for routine diagnosis of CME in dogs, because perform biopsies in dogs with no clinical signs is unpractical. However, sampling tissues may be relevant to understand the distribution of CME in do
Comparative information on the spread and presence of E. canis by PCR analysis in multiple organs is limited, especially in dogs with the natural form of the disease, although some research has been done in experimentally inoculated dogs. For example, it is proven that PCR is effective in detecting E. canis from diverse tissues of dogs with experimental disease [12]. In the same way, it has been described that the spleen is a tissue that can be useful to demonstrate the presence of E. canis DNA by PCR [8,9]. In addition, the possibility of dogs in the subclinical phase being negative to the PCR in blood and positive to the PCR of splenic aspirates has also been established [8]. Splenic aspirates were previously performed to detect E. canis DNA by PCR. Previous research has shown that dogs that were blood positive were also to splecnic aspirates, compared to those that were negative in blood. [50]. These results differ from those obtained in the present investigation, where a prevalence of 42.37% (n = 25) was obtained. Furthermore, of the 19 PCR blood negative dogs, 9 (47.36%) were positive by PCR in the splenic biopsies.
It has been revealed that in the acute phase of disease the splenic aspirates were not superior to blood samples for detection of ehrlichial DNA by PCR. However, splenic aspirates are superior to blood in the evaluation of response to therapy in experimentally treated dogs, because E. canis DNA could be detected in the spleen after its elimination from the blood [8].
The results of the present study also differ from previous reports, where the number of dogs positive and negative for E. canis PCR is similar in blood samples and splenic aspirates. The results revealed that the E. canis DNA was isolated in 29 (72.5%) spleen samples and in 30 (75%) whole blood samples; and ehrlichia DNA was not isolated in 11 (27.5%) spleen samples and in 10 (25%) whole blood samples [10].
The difference between the others studies and the present investigation is the spleen tissue analysed. In our study, DNA was obtained through splenic biopsy, whereas in others DNA was obtained from blood through splenic aspirates. In another investigation, it was found that out of 78 dogs with splenic disease, only one was positive for E. canis by PCR in a splenic biopsy [51]. The present study creates the expectation of performing researches that establish the most adequate technique to obtain E. canis DNA from the spleen in dogs. Comparing splenic aspirates with biopsy, including those taken with minimally invasive techniques, such as ultrasound-guided or laparoscopic.
Furthermore, another important difference in our study is that the tissue with the highest number of positive samples was the bone marrow, in contrast to a previous report that obtained more positives in the aspirates of the spleen [8]. Nevertheless, other studies have demonstrated that other tissues besides the spleen are better to detect E. canis by PCR. For example, some authors describe results similar to those obtained in the present study, where it was shown that E. canis DNA was most often amplified from bone marrow [52,53]. But in these cases there was experimental disease, and PCR was performed by aspirates. On the other hand, in one study performed on biopsies of dog cadavers, contrary to the results of the present study, none of the bone marrow biopsies were positive for E. canis by PCR [10].
An important limitation of the present study is the absence of blood analysis, especially blood counts. This could have established in a more accurate way the dogs presenting with the subclinical phase of monocytic ehrlichiosis [47]. However, it can be assumed that positive dogs are in this phase, since they are clinically healthy.
E. canis is widespread throughout the different body systems of infected dogs. In addition, the molecular detection of E. canis DNA has shown that it can be present in different target organs [13,54,55]. In the subclinical and chronic phases, E. canis could be “hiding” in splenic macrophages [8]. In this case the spleen may be the principal reservoir of E. canis, probably because it has an abundance of macrophages. Moreover, some studies propose that it is the last organ to contain the microorganism before its elimination [8,56]. Therefore, when containing a large number of bacteria, the spleen is considered by some authors as the organ of choice for molecular detection in different phases of the disease [4,8,49,57]. Although, in our study E. canis DNA was detected in the spleen, the results differ slightly with this statement, since it was the third most affected organ, surpassed by the bone marrow and liver. However, the results are similar with other studies, which suggested that the spleen was inferior when compared to other tissues [10,12,51,52].