INTRODUCTION

Equine granulocytic anaplasmosis (EGA) and equine piroplasmosis (EP) are sidesases tramsitted by ticks, and affects equines in several places worldwide (1,2). EGA is caused by Anaplasma phagocytophilum, a bacteria of the Anaplasmataceae family, comprised by 4 genuses: Ehrlichia, Anaplasma, Wolbachia and Neorickettsia.all of them are obligate intracellular bacteria that replicate in small vacuoles derived from the host cell membrane. Each species may replicate inside vertebrate hosts, with the exception of Wolbachia, which has not been reported infecting mammals. Vectors for each species have been well studied, generally ticks or trematodes; however, the study of Wolbachia has not been clear due to the wide variety of invertebrate hosts where it has been found (3).

Within the Anaplasmataceae, family, Ehrlichia y Anaplasma have been reported as the main pathogens in various types of wild and domestic mammals, including humans (4,5). Depending on the species, such genuses affect different blood cell types, to wit: neutrophils, monocytes, erythrocytes, macrophages, platelets, and endothelial cells of host mammals. Ehrlichiosis and anaplasmosis are recognized as emergent and re-emergent diseases with veterinary and human medical relevance (3).

From the geographic standpoint within the Anaplasma genus, A. phagocytophilum is the most widely spread, and it has been reported in Asia, Europe, and America (4). A. phagocytophilum has been long recognized as a pathogen in domestic ruminants in Europe and equines in the USA, but it has recently been detected in several species of mammals, including humans. Molecular studies suggest that some strains of these bacteria, pathogenic for humans and domestic animals, circulate in nature through different hosts (2,6).

Equines infected with A. phagocytophilum show clinical signs such as fever, depression, anorexia, petechial hemorrhages and jaundice. It is believed that the incubation period of these bacteria is close to 14 days, with clinical manifestations starting at day 7 (5). EGA is a disease of veterinary relevance, and, with the appearance of human anaplasmosis caused by the same bacteria, there is an increasing interest in its effects on public health. This pathogen has been reported affecting sheep and bovine cattle in countries such as Scotland, Ireland, Austria, Switzerland, Spain, and France (3). Evidence of agents causing equine ehrlichiosis y anaplasmosis in America has been reported (7).

On the other hand, EP is caused by hemoparasites Theileria equi and Babesia caballi of the Apicomplexa phylum, which includes a large number of obligated intracellular eukaryote microorganisms in vertebrates and invertebrates. The common characteristic between these microorganisms is an apical complex which contains organelles deemed important in the invasion or establishment of the microorganism in the host cell. The phylum is divided into 4 main groups: Coccidia (subclass), Gregarinasida (subclass), Haemosporida (order), and piroplasmida (order). Piroplasmida order has two main genuses, Babesia and Theileria, which are responsible for major animal diseases and economic losses (8).

Piroplasmosis is a disease with high economic, medical, and veterinary relevance and it is the second most common parasitic disease found in the blood of mammals, after trypanosomes (9). Clinical symptoms in animals infected with this disease include fever, anemia, dyspnea, jaundice, splenomegaly and hepatomegaly, edema, intravascular hemolysis, hemoglobinuria, mucosa hemorrhage, and finally, death. Piroplasma affect animal health causing loss of appetite, and reducing their work capabilities (10). After suffering an acute infection, animals then enter into a chronic stage with no clinical signs, becoming reservoirs for the microorganisms (11).

EP is distributed worldwide and it is endemic in most tropical, subtropical, and mild climate areas (12); however, a few countries are considered EP-free, such as Canada, Japan, Auatraliam England, United States, and Ireland. One of the impacts of piroplasmosis is the impediment for international mobilization of seroreactive equine animals, since countries free of piroplasmosis assume the presence of its vectors (13).

Very Little information is available in Colombia regarding the Anaplasmataceae family, although one clinical case of EGA was diagnosed in Florencia (Caquetá) through a clinical test and peripheral blood smear (14). On the other hand, EP has been reported in some countries such as Costa Rica, Venezuela, and Brazil (15,16,17); in Colombia, studies have been performed in the departments of Antioquia, Córdoba, and Santander (18,19).

Regarding the diagnosis of these agents, it is noteworthy that clinic-based findings are non-specific, and peripheral blood smears have low sensitivity (20). On the other hand, serological diagnosis depends on the production of antibodies during the acute phase of the disease, and shows a cross reaction with other closely related microorganisms (21). Also, the above techniques do not allow, in most cases, to discriminate the species of these tick-transmitted agents. The purpose of this study was to detect agents of the Anaplasmataceae family and piroplasms in quines in a slaughterhouse in the municipality of Rionegro (Antioquia).

MATERIALS AND METHODS

Type of study and samples. A descriptive cross-sectional study was carried out on 135 equines during June, July, and August, 2015, at the “La Rinconada” slaughterhouse in the municipality of Rionegro (Antioquia), which processes animals from several municipalities of the department of Antioquia and other departments of the Caribbean region. Animals were selected by convenience. Information on species, age, sex, and department and municipality of origin was collected through the mobilization documents (22). Animals were punctured in the jugular vein to draw 4 ml of whole blood with EDTA as an anticoagulant. The Ethics Committee for Animal Experimentation (CEEA, for its acronym in Spanish) of Universidad de Antioquia, Minute No. 89 of 2014 approved the procedures performed on the animals.

Molecular tests. DNA was extracted from 100 µL of whole blood with EDTA. Extraction was performed using the DNeasy Blood and Tissue (QIAGEN, Valencia, CA) kit, following manufacturer recommendations. Concentration in DNA extracts was determined by udrop (Thermo Scientific Multiskan GO, μDrop TM Plate catalog N12391).From the DNA, a 259bp fragment of the β-actin constitutive gene was amplified for internal control. To search for the hemoparasites, we used the 16S rARN gene of the Anaplasmataceae family, and gene rARN 18S of piroplasms.

Amplification of the 360bp of the rARN 16S gene of Anaplasmataceae was made with Ehr-16SD/Ehr-16SR primers, and the amplification of a 450bp of the rARN 18S gene of piroplasms was made with PIRO A1/PIRO B primers. The sequences of all the primers used are shown in Table 1.

For all three PCR tests, each reagent mix contained Buffer 10X [1X], deosynucleotide trifosphate (dNTPs) [0,4mM]), primers [0,4 pM], Magnesium Chloride (MgCl2) [1,6 mM], bovine seric albumin (BSA) [8 x 10 -6 mg/μL], DNAse-free water, and 200 ng of the extracted DNA. DNA of Ehrlichia canis and DNA of Babesia bigemina, donated by Dr. Santiago Nava of Instituto Nacional de Tecnología Agropecuaria (INTA) de Rafaela (Argentina) was used as positive control; DNAse-free water was used as negative control.

PCR amplification was made in a Veriti (Applied Biosystems, Austin, TX, USA) thermocycler, and PCR product visualization was made by electrophoresis in a 2% agarose gel stained with EZ vision® (Amresco, USA) and using a 100bp molecular weight marker (Invitrogen, USA). Gels were ran for 45 minutes at 100 volts.

Genetic analysis. The PCRT products that showed the highest band intensity and quality in the gels were sent for sequencing. Sequences were edited and assembled in the GeneStudio 2.2.0.0 program. The sequences obtained in this work were compared to the GenBank references through the Basic Local Alignment Research Tool (BLASTn) of the National Center for Biotechnology Information (NCBI). The study and reference sequences of hemoparasites obtained from GenBank were aligned with the Clustal W algorithm, and then we created a Neighbor Joining sequence dendrogram with patristic distances and 1000 bootstrap replicates in MEGA 7 (23).

Statistical analysis. The data obtained was subject to a univariate and bivariate descriptive analysis (equine species, sex, age, department, municipality of origin) with respect to its positivity to hemoparasites. Data analysis was made in the InfoStat statistics program.

RESULTS

General results. Out of the 135 equines selected for the study, 78% (n=105) were horses (Equus caballus, Ec), 16% (n=22) were donkeys (Equus asinus, Ea), and 6% (n=8) were mules (E. caballus x E. asinus). The predominating sex was male with 54% (n=73), while females accounted for 46% (n=62). We found animals of various age groups, adults (ages 5 to 18) were the most frequent, 80% (n=108), followed by young animals (<5 years) accounting for 15% (n=20), and elderly animals (>18 years) with 5% (n=7) (Table 1).

As for the origin of the animals, equines came from the departments of Córdoba accounted for 63% (n=90), Antioquia 24% (n=32), and Sucre 10% (n=13). In Antioquia, we received samples from the municipalities of Anzá, Caucasia, Mutatá, Planeta Rica, and Urrao. Equines from Córdoba came from the municipalities of Montería, Planeta Rica and Sahagún, and those from Sucre were brought from Tolú and Tolúviejo (Figure 1 and Table 1).

Molecular results. Out of the total DNA samples extracted, (n=135), 100% were positive for the β-actin gene, demonstrating the amsence of PCR inhibitors in the samples (Figure 2). Also, all of the samples analyzed with 16S rARN gene primers for the Anaplasmataceae family were negative. The general infection frequency for piroplasm infection in the studied animals was 13.3% (18/135). We obtained positive samples in the animals from the departments of Antioquia and Córdoba, and even though the largest number of positive samples came from Córdoba, the department of Antioquia showed a higher percentage of positive samples (Table 2).

We found that horses was the group of animals with the highest percentage of positive samples, with 83.3% (n=15), followed by donkeys with 11.1% (n=2), and finally mules, with 5.5% (n=1). The age group with the highest positive frequency was the adult group with 83.3% (15 animals out of 18 positives), followed by young animals with 16.6% (2animals out of 18 positives), while the elderly group showed no positive sample. The sex with the highest infection rate was females, with 55.5% (n=10) and males showed 45.5% (n=8) (Table 2).

Five samples from horses (Ec) and donkeys (Ea) from Monteria were selected for direct DNA sequencing (Ea035, Ea036, Ec096, Ec121, Ec142). The BLASTn in samples Ea035, Ea036 and Ec096 indicates a full identity with Theileria equi from Brazil (100% with KU240068), whereas the BLASTn in Ec121 and Ec142 shows a high similarity with the same agent from Florida (USA) (97-99% with KU672386). The Neighbor Joining dendrogram confirms circulation of three genotypes (haplotypes of gene18S) of Theileria equi in equines from the northern Colombia with a high branch support (bootstrap 97%) (Figure 3).

DISCUSSION

This work presents the first molecular evidence of at least three genotypes of Theileria equi infecting equines in the northern part of the country. A 13% general piroplamsa infection was detected in these animals, and genetic analysis demonstrate active circulation of Theileria equi in animals from the departments of Antioquia and Córdoba.

There is very few data in Colombia on Ep epidemiology, and the findings in this work demonstrate the active infection of this type of agents in the departments of Antioquia and Córdoba. The zones studies with parasite presence can be considered as having high risk of transmission to healthy animals whose zootechnical use involves activities in EP-free countries, given the mobility restrictions imposed on animals infected with or seropositive to these hemoparasites.

The results of this work are similar to those obtained in other studies in the country regarding piroplasma frequency in equines, but its innovation lies in the implementation of molecular techniques instead of the conventional peripheral blood smears and serological tests (18,19). Thus, while serological tests rely on the search for anti-piroplasma antibodies indicating -depending on the antibody isotype- that the animal experienced a recent or distant infection, this work demonstrates the direct presence of parasite DNA in peripheral blood. Even though there was already knowledge of the circulation of piroplasms in equines in Montería (24), this is the first time in Colombia for molecular identification of these protozoan agents of great significance for the health of equines.

This study was not able to detect agents of the Anaplasmataceae family in equines, despite the broad spectrum of the methodology used. However, these rickettsial agents have been successfully reported in other Latin American countries. For example, O’Nion et al (24) found serological evidence of infection in 51 out of 92 sampled animals (55%) in Nicaragua, and managed to obtain molecular evidence of a potentially new species of Ehrlichia in four of these seroreactive animals (24). In Guatemala, there has been molecular evidence of A. phagocytophilum reported on 13% of 74 analyzed horses (25). Probably, these agents may be circulating in equines in Colombia, but the study design and sample size may be constraints to detect such rickettsial agents in this study.

Colombia has the ideal abiotic conditions for the development of tick-borne diseases, due to its location in a tropical area where temperature, relative humidity and lighting factors are favorable for the survival of disease vectors. For that purpose, in order to clarify the epidemiological scenario of these hemoparasytes in Colombia, further studies are necessary with specific sampling designs and with representation of other regions of the country, also engaging in the analysis of their potential vectors. These searches can be addressed by using techniques that allow a more sensitive and specific diagnosis, such as the one that molecular tests provide.

Acknowledgements

To the CODI of Universidad de Antioquia for financing (project 321-2014). To Colciencias for their support to LAG and LPT through National Doctorates. To Rene Ramírez of Universidad CES and the staff of La Rinconada slaughterhouse for their logistic support

Conflict of interest

The authors hereby declare that they have no conflicts of interest.

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Author notes

leidy.acevedo@udea.edu.co

IR