INTRODUCTION

Zoonoses involve several diseases that represent a significant worldwide problem affecting human and animal health (García et al., 2013). Such diseases include Ehrlichiosis, Anaplasmosis and Rickettsiosis which are caused by a gramnegative bacteria characterized by intracellular obligated growth (genus Rickettsia, family Rickettsiaceae; genus Ehrlichia and Anaplasma, family Anaplasmataceae). These are mainly transmitted by ectoparasites, including Riphicephalus sanguineus, the brown dog’s thick, which affect ground vertebrates (^{Parola et al., 2009}; ^{Alvarez, 2017}). Several studies have demonstrated these ectoparasites act as vectors from zoonotic agents including Erlichia canis, Anaplasma platys and Rickettsia rickettsii (^{Gaunt et al., 2010}).

E. canis causes a zoonotic disease in dogs, cats and rodents; then, the human is an accidental victim after being stung by the Rhipicephalus stick hosted in these animals. After the infection, the microorganism is incubated during 1-2 weeks and enters into the blood and lymphatic vessels; then, it travel to the spleen, liver and lymph nodes to be multiplied by binary fusion for spreading to other body organs (^{Castro et al., 2004}).

E. canis is considered as a bacterium of cosmopolitan distribution. In Mexico, it was first described in 1996 and classified as endemic because it has been reported in the whole country, but mainly in northwest states such as Sonora and Sinaloa. In Sonora, E. canis is considered as a health emergency and a growing public health issue. Since 2002, more than 600 cases have been reported, the vast majority in municipalities in the north of the state (^{Álvarez, 2017}; ^{Sosa et al., 2013}).

Clinical signs of the acute phase of the disease are hematologic alterations, leukopenia, thrombocytopenia and mild to moderate anemia; the chronic phase is characterized by thrombocytopenia, epistaxis, nephropathy, dyspnea, hepatomegaly, splenomegaly or lymphadenopathy, inflammatory or hemorrhagic meningitis, among others (^{Ismail et al., 2010}).

A. platys is distributed worldwide and it is also transmitted by the ticks R. sanguineus. It was first described in 1978 in dogs from United States (^{Sánchez & Tesouro, 2001}). This bacteria belong to the Anaplasma genus (^{Ábrego et al., 2009}). It causes canine infectious cyclic thrombocytopenia. The disease may present with fever, anorexia, petechiae, uveitis, generalized lymphadenopathy, leukopenia, moderate anemia and especially thrombocytopenia, occurring in episodes of 3-4 days at intervals of 7-21 days, eventually leading to chronic thrombocytopenia with slow recovery (^{Cicuttin et al. 2014}).

Currently, A. platys has been detected with low incidence in the states of Coahuila, Durango and Sonora (^{Almazán et al., 2016}; ^{Murrieta et al., 2017}). As zoonotic disease, ^{Arraga et al. (2014)} reported two women in Venezuela who were exposed to R. sanguineus. Intraplatelet inclusion bodies suggestive of A. platys were subsequently observed in smears and A. platys DNA was amplified and sequenced from whole blood, although treatment with doxycycline did not alleviate their symptoms. These cases provide additional support for A. platys as a tick-borne zoonotic pathogen, most likely of low pathogenicity; however, the cause of A. platys disease in humans has not been confirmed.

R. rickettsii is the main agent of spotted fever in the Americas; cases have been reported in the U.S., Canada, Mexico, Costa Rica, Panama, Colombia, Brazil and Argentina (^{López et al., 2007}; ^{Herrero et al., 2010}; ^{Lebruna et al., 2011}), with a high mortality rate due to late detection of the pathogen (^{Oteo et al., 2014}). It is considered that Mexico has the ideal conditions for the transmission cycle of the disease, commonly associated with living conditions (poverty) since most of the cases presented have been detected in marginalized and rural areas of Mexico (^{Peniche et al., 2015}). Recent studies demonstrated the presence of Rickettsia spp. in ticks from the state of Sonora by PCR technique (^{Foley et al., 2019}).

These pathogens could be present previously in the tick, causing simultaneous co- infections in the host (i.e. more than one agent can be transmitted), which will be causing the disease at the same time. This can result in the manifestation of clinical signs, in a more severe and non-specific way, being a disadvantage for the clinical diagnosis by human and veterinary physicians attending these cases (^{Alleman & Wamsley, 2008}; ^{Mutz et al., 2009}).

Considering the zoonotic importance of these diseases, and that no previous studies have been reported in the municipality of Cajeme, Sonora, the presence of vectors of E. canis,

A. platys and R. rickettsii in domestic dogs in the municipality is suggestive of the presence of these zoonotic diseases. Therefore, the objective was the molecular identification of the species E. canis, A. platys and R. rickettsii to determine their co-infection in domestic dogs in the municipality of Cajeme, Sonora.

MATERIAL AND METHODS

RESULTS

It was observed by electrophoresis that all the extracted DNA showed a good integrity of the molecules and yielded an average quantification by spectrophotometry of 248.32 ng/µl in the leukoplatelet layer samples. A purity value of 1.85 measured through the ratio 260- 280 indicate the DNA as “pure”, using samples from whole blood with EDTA.

For the PCR technique, a concentration of 0.5 ng/µl of each gblocks was used as positive controls, obtaining the corresponding base pairs of each agent, achieving good efficiency and speed for molecular standardization.

The number of positive cases and frequency of Ehrlichis spp., Ehrlichia canis, Anaplasma platys and Rickettsia spp. obtained from molecular analysis of canine blood samples (n=170) are described in Table 2. Of the 92 samples that tested positive for Ehrlichia spp. 12 co-infections (Table 3) of Ehrlichia canis with Anaplasma platys (Genus EA) were found (13%).

In addition, sequencing of 1 sample of each positive for the different species of microorganisms analyzed was performed, obtaining pure chromatograms analyzed with the SanpGene Viewer program. The analysis of the sequences in the BLASTn program (National Center for Biotechnology Information) detected a similarity between 99 and 100% with the previously reported sequences.

DISCUSSION

Microorganisms of the order Rickettsiales have zoonotic conditions called Rickettsiosis, Ehrlichiosis and Anaplasmosis that are due to several pathogens of veterinary importance that have gained ground not only in our region but also worldwide (^{Rodríguez et al. 2016}), this is mainly due to its vector Rhipicephalus sanguineus, being the most commonly reported tick species and with the greatest geographic distribution (^{Sosa et al., 2016}; ^{Cabezas-Cruz et al., 2019}). These pathogens are increasing due to the activity of human beings that has generated radical changes in the environment, favoring vector-borne diseases (^{Suthers, 2004}) and increasing their prevalence in late spring and summer. Due to the fact that these ectoparasites increase their activity in high ambient temperatures during these seasons of the year, inoculating the mentioned hemoparasites more quickly (^{Parola et al., 2009}), necessitating the standardization of precise techniques for the detection of these zoonotic microorganisms of the Rickettsiales order in Tropicales and Subtropicales regions.

Currently using gBlocks gene fragments as positive controls used in this study have become popular as standards and synthetic positives for the detection of bacterial microorganisms. Currently similar data were reported in Barcelona where this type of fragments were used for detection of E. coli, E. faecalis and Legionella pneumiphilia (^{Cardenas, 2018}); also, in Australia such fragments were used as standards in the multiplex PCR detection of Taenia spp. (^{Ng-Nguyen et al., 2017}) Therefore, the use of gBlocks for PCR standardization is recommended as it increased the speed of the bioassay in detection due to the lack of availability of biological isolates.

The study was the first work using PCR to detect the species Ehrlichia canis, Anaplasma platys and Rickettsia rickettsi in blood samples from dogs in the municipality of Cajeme and in Sonora, allowing to know the expanded geographical distribution of the three pathogens in the state. The percentage of 54% to at least one pathogenic agent in our study agrees with investigations performed using molecular tools in some South and Central American countries, where Brazil reported 69% (^{Tanikawa et al., 2013}), Nicaragua 80% (^{Wei et al., 2014}), Panama 70.6% (^{Santamaria et al., 2014}), Costa rica 45% (Rojas et al., 2015) and El Salvador 60% (^{Miranda et al., 2018}). Our prevalence percentage differed when was compared with high percentages of other countries, probably because they worked with dogs without owners and being more in contact with the vector of the diseases as it is associated with living conditions in marginalized areas (^{Peniche et al., 2015}). Prevalence to hemoparasites have been reported in some other states of Mexico, where Sinaloa reports 74.3% (^{Sosa-Gutiérrez et al., 2013}), Coahuila and Durango 41% (^{Almazán et al., 2016}), Yucatán 69% (^{Díaz et al., 2016}), Chihuahua 40% (^{Escárcega et al., 2018}) and Sonora 33% (^{Murrieta et al., 2017}). As early mentioned, Cajeme yielded 57% of infection, positioning Sonora at this time as one of the main states with the highest incidence in tropical and subtropical regions where parasites and the vector are present.

Regarding the percentage of presence of each hemoparasite, our study evidenced the higher prevalence of E. canis reported in Mexico (28%). Other studies reported lower results. In the municipality of San Luis Rio Colorado, 8% of infection for E. canis was found in blood samples from 235 dogs (^{Murrieta et al., 2017}); also, in the State of Coahuila and Durango 10% was found in a population of 100 healthy dogs infested with ticks (^{Almazán et al., 2016}). In Yucatan, 36% was determined in a population of 50 dogs (10 domestic dogs and 40 in an animal control center), where all positive dogs were from samples collected from the animal shelter, representing a prevalence of 45% for this sampling site (^{Path et al., 2015}). There are also findings in other countries such as Buenos Aires, Argentina, where the results were similarly lower in a population of 223 dogs, obtaining 6.7% of positives to E. Canis (^{Cicuttin, 2016}), Uruguay is one of the few countries which reported the presence of other hemoparasites, but not the presence of E. Canis in a population of 191 dogs (^{Carvalho, 2017}). In contrast to Brazil, the percentage in our study was lower because the worked with 472 dogs in the Brazilian northeast detecting 34.5% of positive dogs (^{Silva et al., 2010}), Colombia represents the largest area with high prevalence reviewed on E. canis, mainly the municipalities of Palmira (92.8%) and Cartago (90%) (^{Rojas et al., 2013}). However, it is worth mentioning that this work was performed on stray dogs unlike our study.

The presence of Anaplasma platys (11%) in our study, turned out to be higher than those reported in Buenos Aires, Argentina of 223 dogs, being positive for A. platys 7.2% (^{Cicuttin, 2016}). In Uruguay, out of 191 dogs, 4.2% were positive (^{Carvalho, 2017}). Also in San Luis Rio Colorado, Sonora in 235 canine samples were positive for 18% (^{Murrieta et al., 2017}). Other publications show similar values to Costa Rica with 10% (^{Wei et al., 2014}), Nicaragua 13% (^{Rojas et al., 2014}), Cuba 16% (^{Silva et al., 2016}) and El Salvador with 17% (^{Murrieta, 2017}), but proved to be lower with respect to Panama with 21.3%. In Brazil, in a population of 100 dogs, a higher prevalence was obtained for Anaplasma platys with 21% and 9% for Ehrlichia, being one of the few studies that differs in our results, since, in our work and most of the research conducted in Central and South America, they report a lower incidence of A. platys than E. canis.

The molecular diagnosis in our research of hemoparasites related to Ehrlichia and Anaplasma has been easy to identify, unlike the bacterium Rickettsia rickettsii with the PCR technique from blood samples in canines. Our prevalence percentage to this agent was lower (0.8%) compared to E. canis and A. platys, this may be because it is typically mentioned that low numbers of rickettsiae circulate in the blood in the absence of advanced disease or fulminant infection (^{CDC, 2017}; ^{Tinoco et al., 2018}).

Due to this we found in the literature different investigations, mostly directed to the diagnosis of Rickettsia spp. and R. rickettsii in R. sanguineus ticks in dogs from different regions, as is the case of Yucatan, selected 28 dogs where 106 Rhipicephalus sanguineus ticks were collected with a 26% incidence (^{Peniche et al., 2015}) In Matamoros, Coahuila, the endpoint PCR technique was performed for the analysis of 100 ticks (Rhipicephalus sanguineus) giving as positive to Rickettsia spp 4% of the samples analyzed (^{Castillo et al., 2015}). In Mexicali Baja California, Mexico, dog ticks belonging to the morphology of Rhipicephalus sanguineus were analyzed by means of PCR, resulting in positive samples with 100% homology to R. rickettsii (^{Foley et al., 2019}). Currently Sonora leads the entities with the most reports of Rickettsiasis in the country, being Cajeme one of the main cities with more cases of death, despite being a data associated with public health, it is important to know the incidence of R. rickettsii in animals (^{PSS, 2014}), giving greater importance to our work, because not only Rickettsia spp. but R. rickettsii was also detected, with 100% homology to the genus with the gltA gene and the species with the OmpA gene in blood samples from 2 dogs (n = 170), these genes are the most used for the detection of the bacteria that cause spotted fever.

The results obtained from the three agents, increases the alert and the incidence of cases associated with Rickettsiales in the state of Sonora transmitted by the brown tick Riphicephalus sanguineus. It is currently the most important vector of Rickettsiales in Mexico (^{Labruna, 2009}), constituting a public health problem, since R. rickettsii, E. canis and A. platys are considered zoonotic disease by the CDC in 2017 and that over the years have acquired greater territory of infection. As mentioned above, these pathogens can be present in the tick, causing simultaneous co-infections in the host, i.e. more than one single agent can be transmitted, as demonstrated in the present investigation, finding 13% of co-infection to the agents E. canis and A. platys.

The presence of co-infection with these bacteria in our study is not a strange finding, since they are the most common in Latin America. In our study we have similar percentages obtained in Panama, which report a co-infection between E. canis and A. platys of 7.5% (^{Santamaría et al., 2014}), El Salvador with 4.5% (^{Miranda et al., 2018}) and San Luis Rio Colorado, Sonora with 12.2% (^{Murrieta et al., 2017}), mentioning that the latter is very similar to our co-infection results and is from the same state.

It is essential to consider that the remaining 46% of the negative samples in the study to genus and species, may be due to the absence of the pathogens or the presence of other diseases, because in the investigation all blood samples came from presumptive dogs or manifested clinical symptoms, although it may also be due to the presence in undetectable low quantities of the pathological agents.

Finally, it is important to highlight that the differences found between the samples positive to genus and negative to E. canis, A. platys and R. rickettsi species are probably due to the fact that the primers used in the first genus PCR (ECC/ECB) also amplify other species, which could indicate infection by other Ehrlichia species (E. ewingii or others) or other species of the Anaplasmataceae family (A. phagocytophilum or others).

CONCLUSIONS

The percentage of haemoparasites in domestic dogs reported in this study, currently positions Sonora as one of the main states with the highest frequency in tropical and subtropical regions. The three agents were detected by molecular technics in the blood of suspected dogs from the city of Cajeme, Sonora, identifying Ehrlichia canis, Anaplasma platys and Rickettsia rickettsii with frequencies of 28%, 11% and 0.8%, respectively. In addition, these three agents were confirmed by sequencing. Regarding co-infections, only Ehrlichia canis and Anaplasma platys were detected simultaneously with 13% of frequency.

To our knowledge, this is the first molecular identification investigation in our state, confirming the presence of Ehrlichia canis, Anaplasma platys and Rickettsia rickettsii starting from whole blood of canines. However, further studies are suggested to explore the frequency of these infectious agents in other regions from the state on Sonora.