The present research was approved by the Ethical Committee of the Universidad Autónoma de Ciudad Juárez. The handling of animals was carried out based on the regulation of laboratory animal handling and animal welfare (NORMA Oficial Mexicana NOM-062-ZOO-1999) [42] and in accordance with the International Guiding Principles for Biomedical Research Involving Animals.
An analysis of the variation in infection by E. canis in four tissues was carried out, in two groups of dogs: positive and negative by PCR in blood.
Animals
Fifty-nine dogs obtained from the municipal Anti-Rabies Centre of Juárez were used in this study. Based on the Centre’s internal regulations, animals that were not adopted eight weeks after their arrival were euthanised. Euthanasia was performed by an overdose of sodium pentobarbital according to national and international animal welfare regulations.
In order to increase the possibility that dogs will present the subclinical phase of the disease, the inclusion criteria were that the dogs should have ticks but be clinically healthy; therefore, dogs without ticks or with signs of any disease were excluded.
Sample collection
Whole blood samples were collected in tubes containing EDTA (Vacutainer BD®, Mexico City, Mexico) by cephalic venepuncture with prior administration of sodium pentobarbital. The other tissue samples were acquired by biopsies immediately after euthanasia, following the steps of surgical asepsis in order to prevent cross-contamination. In addition, with the same purpose, a change of instruments was made for the biopsy of each tissue, and particular attention was taken to avoid blood or other fluid from the dog coming into contact with the tissue samples.
Bone marrow aspirates were obtained with bone marrow aspiration needles (Argon Medical Devices®, Dallas, TX, USA) from the greater tubercle of the humerus, as described by Raskin and Messickin in 2013 [43]. Hepatic and splenic biopsies were obtained by celiotomy and with the ligature fracture technique [44]. Finally, prescapular lymph node were biopsied with a biopsy punch (Premier®, Plymouth Meeting, PA, USA) as previously described [45]. Tissues samples were marked and frozen at –20 °C for future extraction of DNA and PCR analysis.
Biopsies obtained from spleen, liver and lymph node had an average weight of 200 mg (range 150–210 mg). The amount of whole blood obtained was 1.5 mL and the bone marrow biopsy obtained 0.6 mL on average (range 0.4–0.7 mL)
DNA extraction
In the blood samples, the extraction of genomic DNA from the cellular package of the dogs’ samples was performed with the UltraClean Blood DNA Isolation Kit (MoBio Lab®, Carlsbad, CA, USA), according to the manufacturer’s instructions.
The other tissues were handled in sterile fashion prior to the extraction of DNA. For the extraction of DNA from the biopsies, the protocol was modified with the previous addition of lysis reagents [46]. The tissues were then macerated with the use of a low-velocity drill (Jorvet Lab®, Loveland, CO, USA) and a dental burn (JOTA Technical®, Rüthi
Switzerland). Once each tissue was macerated, DNA extraction was performed in the same way as for the blood.
PCR amplification and analysis
Detection of E. canis DNA was achieved with the use of the nested PCR molecular test. Initially, to amplify the Ehrlichia genus 16SrRNA gene, 2 pmol of primer ECC (5´-AGA-ACGAACG-CTGGCGGCCAAGC-3´) and ECB (5´-CGTATTACC GCGGCTGCT-3´) were used [28]. In the second PCR, to amplify the E. canis 16SrRNA gene, 2 pmol of primer HE-3(5´TATAGGTACCGTCATTATCTTCCCTAT-3’) combined with the reverse primer ECA (5´-CAATTATTTATAGCCTCTGGCTATAGGAA-3´) were used [28,47].
Initially, PCR was performed in a thermocycler (BIO-RAD® C-1000 Touch, Hercules, CA, USA) starting at 94 °C for 1 min followed by 35 cycles of 94 °C for 1 min (denaturation), 60 °C for 1 min (hybridisation) and 72 °C for 3 min (extension). This was followed by 94 °C for 5 min and then 40 cycles of 94 °C for 1 min (denaturation), 60 °C for 1 min (hybridisation), and 72 °C for 1 min (extension), as described previously [28,47,48].
Statistical analyses
A multivariate logistic regression model was used for the response variable ‘infection’ which was binary (variable dummy) with y = 1 if positive, and y = 0 if negative), depending on two explanatory variables: blood positivity (two levels) and positivity in four separate tissues (four levels). Therefore, the model was: infection = blood + tissue + error.
The model analysed separately infection in both groups of dogs. In each group, the model compared infection among four tissues using statistical tests ‘z’ between pairs of tissues, using a multiple-comparison Scheffe test.
Comparison of the proportions of positive and negative results in blood, lymph node, liver and spleen samples were performed using Chi square and Fisher’s exact tests with the FREQ procedure of SAS (9.0). Significance was considered with a P value of <0.05.