Epidemiological Study of the Occurrence of Typhus Group Rickettsia Natural Infection in Domiciliated Dogs from a Rural Community in South-Eastern Mexico

Abstract

Simple Summary

Rickettsioses are relevant emergent and reemergent zoonoses in the Americas, including Mexico. Murine typhus caused by typhus group (TG) Rickettsia is prevalent in humans and their companion animals, such as dogs. This study found that 23.9% of the dogs (34/142) were infected by TG Rickettsia in Maxcanú, Yucatan (southeastern Mexico). Statistical analyses showed that reduced outdoor activities, age, sex and previous antiparasitic treatment are associated factors with less risk of TG Rickettsia infection in the sampled dogs. Monitoring and controlling these factors could help to restrict the enzootic transmission risk and prevent the potential zoonotic transmission.

Abstract

The aim is to describe the Typhus group (TG) Rickettsia infection in dogs and to identify factors associated with this infection. We collected blood samples and gathered exposure and clinical data of 142 dogs from a rural community of Yucatan. The Rickettsia group was determined by semi-nested PCR. Generalized linear models with binomial error distribution were used to model the associated factors from the dog sample for risk ratio (RR) estimation. Thirty-four dogs (23.9%) showed molecular evidence of TG Rickettsia DNA. The multivariate model showed that mixed-breed dogs (RR = 0.06) and dogs that had received antiparasitic treatment (RR = 0.049) had a lower risk of getting infected, taking as reference the purebred group and the non-treated dogs, respectively. Looking at variable interactions, adult dogs without outdoor activities had a lower infection risk than puppies (RR = 0.26). Among dogs with antiparasitic treatment, females had a higher infection risk than male dogs (RR = 26.2). The results showed enzootic TG Rickettsia circulation in dogs of a rural community. The factors outdoor activities, age and previous antiparasitic treatment, as well as the clinical variables signs of hemorrhages and epistaxis, were associated with a less chance of natural infection in the studied dogs. Prevention and control of the enzootic transmission risk of TG Rickettsia should help to reduce the potential zoonotic transmission of this pathogen.

1. Introduction

The typhus group (TG) Rickettsia is integrated by Rickettsia prowazekii and Rickettsia typhi, which are relevant species for public health because they are the causative agents of epidemic typhus in humans and a neglected zoonosis known as murine typhus, endemic typhus or flea-borne typhus, respectively [1].

After an extensive analysis of TG Rickettsia species in Mexico, Sánchez-Montes et al. [1] showed that the most common Rickettsia species of this group occurring in dogs is R. typhi, whereas R. prowazekii occurs only in the human population. In this regard, murine typhus disease is widely distributed in countries of the Mediterranean region, Africa, Southeast Asia and North America, but is more important in coastal or urban areas with high densities of synanthropic rats (Rattus rattus and R. norvegicus) [2,3].

The clinical manifestations in infected people include fever, headache, myalgia and rashes, among others. This leads to the misidentification or habitual confusion of this disease with other febrile diseases such as the deadly Rocky Mountain spotted fever, Dengue fever, Zika fever, West Nile fever, leptospirosis, meningitis, syphilis and other less common diseases [3,4,5]. However, R. typhi infection in dogs is subclinical [6], and many studies provide no description of clinical signs [7,8,9,10]; except for some cases such as a recent description by Juhasz et al. [11] for a clinically ill dog with intermittent fever, lethargy, inappetence and lymphadenopathy, with molecular and serological tests showing infection by R. typhi. Nonetheless, dogs are asymptomatic carriers, according to several clinical and epidemiological studies [8,12,13].

The classical enzootic transmission cycle of murine typhus causal agent (R. typhi) occurs naturally between the synanthropic rats and the Oriental rat flea, Xenopsylla cheopis, in whose intestines it multiplies and is then excreted in faeces while it feeds, infecting the new susceptible host by contamination of the mucous membranes or auto-inoculation by skin abrasion [2,5,7]. However, in some regions of North America, an alternative cycle has been described, in which humans accidentally participate in the spreading of the enzootic cycle to dogs, cats, and peridomestic opossums through the common cat flea, Ctenocephalides felis [14,15,16,17] since this arthropod has a generalist feeding habit [5], which facilitates the transmission among various susceptible hosts [3].

Recently, studies in a rural locality in the State of Yucatan, Mexico, performed by Torres-Castro et al. [18,19,20], found that 90% of the people exposed to Rickettsia sp. infection (antibodies) were dog owners, and 70% had more than one dog in their household. There is also molecular evidence of two members of the spotted fever group (SFG) Rickettsia (Rickettsia rickettsii and Rickettsia parkeri) and a single member of the TG (R. typhi) circulation, raising questions about the risk of the dog population regarding Rickettsia infection.

Dogs are epidemiologically significant as sentinels of R. typhi circulation in both endemic and non-endemic areas, making them incidental hosts of the bacteria and temporary hosts of the transmitting fleas. Nevertheless, there are few global studies assessing the general epidemiological factors and clinical aspects of naturally infected dogs with murine typhus [8,9,10], mainly in rural settlements.

Since people from Maxcanú showed exposure to TG Rickettsia [18], and there is evidence of R. typhi infecting domiciliated dogs in the studied region [9,10,21], this research addresses the following questions: what is the occurrence of the TG Rickettsia natural infection in domiciliated dogs? Are there factors that help to predict the relative risk (RR) of TG Rickettsia natural infection in the canine population from the studied locality?

2. Materials and Methods

2.1. Study Site

This study was carried out in Maxcanú, a rural community located in the state of Yucatan, Mexico (20°24′–20°50′ N, 89°54′–90°21′ W), which has a maximum elevation of 100 m above sea level. The climate is tropical sub-humid with rain in summer; the average annual temperature ranges from 26 to 28 °C, with an average annual rainfall from 700 to 1100 mm. The predominant vegetation surrounding the community is tropical deciduous forest and its secondary stages. Land use is primarily for extensive and traditional agriculture, and some areas are used for livestock [22].

2.2. Collection of Clinical and Exposure Variables of Dogs

The research protocol and the informed consent letter were evaluated and approved by the Research Ethics Committee (CEI) of the Centro de Investigaciones Regionales “Dr. Hideyo Noguchi” (CIR) of the Universidad Autónoma de Yucatán (UADY) (Protocol number CIRB-07-2018), Merida, Mexico.

The study design was a cross-sectional epidemiological survey. From February to April 2019, several public outreach events were held to verbally inform the public about rickettsial diseases and to recruit participants for this study. The people who voluntarily accepted the invitation were visited by our work team and answered an epidemiological questionnaire with the exposure variables for each examined dog. Subsequently, a group of veterinary practitioners inspected each dog (general physical examination) for the data collection on clinical variables. The physical examination was performed with all necessary measures to protect the physical well-being of the dogs and the workgroup’s safety.

Data on physical exams, epidemiological questionnaire, and blood samples from dogs were collected during a single visit to each owner’s household. All studied dogs were manipulated after authorization and consent signatures by the owners, who were always present during the physical exam and blood collection.

The exposure variables collected in the epidemiological questionnaire were as follows: sex (male or female); age (puppy: under 1 year old, or adult: over 1 year old); body size (small: less than 5 kg, or large: more than 5 kg); hair length (short: less than 5 cm from the body to top, or long: more than 5 cm from the body to top); hair color (dark: more than 50% body, or light: more than 50% body); breed (purebred or mixed breed); ectoparasite infestation (no infestation, only ticks, only fleas or fleas and ticks); level of infestation (no infestation: zero ectoparasites, mild: 1 to 10 ectoparasites, or severe: more than 10 ectoparasites); anatomical site of the infestation (no infestation, one infested body site or several body sites); outdoor activities (if the dog accompanies or not the owners to carry out recreational activities, agricultural activities or hunting, regardless of the time or frequency); specific resting place (inside or outside the dwelling, regardless of the time or frequency); and social cohabitation with other dogs in the dwelling (more than one dog in the dwelling or not).

The clinical variables collected in the physical examination included vaccination (yes or no, without quantifying the number or type of vaccine, according to the information provided by the owners); antiparasitic treatments against ectoparasites and endoparasites, applied anytime in the 3 months before the day of the physical examination (yes or no, regardless of the doses or commercial brands, according to the information provided by the owners); fever (no (≤39 °C) or yes (>39 °C) by the use of a thermometer via the rectum); hemorrhages and epistaxis (no or yes if any sign in the skin or the mucosa, including oral, conjunctival, perianal and vulval in females); body condition (skinny; below 20% of the ideal body condition, ideal and overweight, regarding the breed [23]); skin lesions (no or yes if present in any body site, regardless of severity and distribution); bruising, petechiae and ecchymoses (no or yes, including oral mucosa, conjunctiva, perianal and vulvar mucosa in females); and other clinical signs such as nervous and locomotor disorders (yes or no) compatible with Rickettsia infection as previously described [24,25,26,27].

2.3. Blood Sampling in Studied Dogs and Genomic DNA Extraction

During the visits to the dwellings, after the physical examination, a sample of whole blood was collected directly from the saphenous vein (maximum 7 mL) with the help of a syringe and then placed in a sterile centrifuge tube (BD Vacutainer^TM, Franklin Lakes, NJ, USA) with EDTA. Before bleeding, the area was cleaned with hydrogen peroxide and iodine to ensure that the blood was not exogenously contaminated with ectoparasite faeces during venipuncture.

The samples were preserved in hermetically sealed plastic portable refrigerators with refrigerants at approximately 4 °C during the fieldwork and subsequently transferred to the laboratory, where they were centrifuged at 1500 g at room temperature for 10 min. The white cell layer was collected and stored in a sterile 1.5-mL vial (Eppendorf^®, Hamburg, Germany) at −80 °C.

Genomic DNA was extracted from the white cell layer, using the commercial Kit QIAamp DNA Mini Kit^® (QIAGEN, Hilden, Germany) protocol “DNA Purification from Liquids and Fluids”, following the manufacturer’s specifications. The DNA was quantified with the help of a spectrophotometer NanoDrop 2000^® (Thermo Scientific^®, Waltham, MA, USA).

2.4. Molecular Identification of Rickettsia DNA

Rickettsia DNA molecular identification was performed by a multiple semi-nested polymerase chain reaction (snPCR), which allowed differentiation between the SFG and TG Rickettsia [28] by amplifying fragments of the sca5 gene [29]. The primers, reagent concentrations and thermocycler conditions were the same as those described by Choi et al. [28]. The snPCR enables determining the infective Rickettsia group: bands of 420 base pairs (bp) for species belonging to the SFG and 237 bp for species belonging to the TG [10,28]. In all PCRs, the DNA of Rickettsia conorii was used as a positive control. The negative control was the reaction mixture (primers plus reagents) without DNA [10,18,19,20].

Electrophoresis was performed in 10% polyacrylamide gels (Sigma-Aldrich Products^®, Taufkirchen, Germany) deposited in vertical cameras (Bio-Rad Laboratories^®, Hercules, CA, USA) and stained with 1.1 molar silver nitrate. For the final visualization and recording of the results, a UV light transilluminator was used (Hoefer^®, Holliston, MA, USA).

2.5. Statistical Analysis

All exposure variables collected in the epidemiological questionnaire, the clinical variables obtained in the physical examination and the results of the laboratory tests (snPCR) were captured in a digital database using the Excel^® software (Microsoft^®, Bellevue, WA, USA). All categorical variables were analyzed with descriptive statistics. The Rickettsia TG infection frequency in the studied dog population was determined according to the results of the snPCR, and 95% confidence intervals were estimated with the Clopper-Pearson procedure [30,31].

A generalized linear model (GLM) with binomial error distribution and log link function, using the backward stepwise selection procedure, was used to select the predictor exposure variables for TG Rickettsia infection in dogs, accounting for those variables with a p value < 0.15 [32]. Subsequently, we explored the interactions between the most informative variables stratifying the analysis. Different models were constructed using Akaike’s criterion for model selection. The statistical significance for the modelled variables was set with a p < 0.05 value to estimate risk ratios (RR) and 95% confidence intervals. Statistical analyses were executed in R version 3.6.0 [33].

3. Results

A total of 142 dogs living in 58 households from Maxcanú were included in the sample. The frequencies of all categorical variables accounted from the survey are presented in Table 1. As can be seen, 76 (53.5%) dogs were males and 66 (46.5%) were females. According to age, 51 (35.9%) were puppies and 91 (64.1%) were adults; the average age for the studied dog population was 3.1 years, with a range from 2 months to 12 years. However, some owners were unable to refer the exact age of their dog. Regarding the body size, 45 (31.7%) dogs were small and 97 (68.3%) were large.

Most of the studied dogs had long (woolly) hair (92, 64.8%), and dark coloration was the most frequent (79, 55.6%). The dominant breed was the mixed breed (137, 96.5%). Eighteen individuals (12.7%) had no ectoparasites, and 56 (39.4%) had an infestation with fleas and ticks. The severe infestation level was the most frequent (82, 57.7%), followed by mild (42, 29.6%). In total, 106 (74.6%) dogs had an infestation in several body sites. Most of the studied dogs did not join their owners for outdoor activities (98, 69%) and did not have a specific place to sleep or rest inside the house (128, 90.1%). Finally, 119 (83.8%) dogs lived in the same house or in social convivence with at least one dog. None of the examined dogs were sterilized.

Regarding the clinical-oriented variables, in 84 (59.2%) studied dogs, the owners indicated that at least one vaccine was applied (mainly against the Rabies virus), and 41 (28.9%) had received antiparasitic treatment at least 3 months before blood sampling (ivermectin in different commercial presentations and doses, according to owners); 87 (61.3%) dogs had a skinny body condition. The most frequent clinical signs observed in the physical examination were skin lesions (42, 29.6%), followed by bruising, petechiae or ecchymosis (27, 19.0%), fever (25, 17.6%), other clinical signs related to Rickettsia infection (24, 17%), signs of hemorrhages and epistaxis (22, 15.5%) and pruritus (19, 13.4%).

The snPCR showed that 34 (23.9%) (95% CI = 17.2–31.8) of the studied dogs were positive for Rickettsia species belonging to the TG.

Of the 34 infected dogs, 18 (52.9%) were female and 16 (47.1%) were male. Regarding the ages, 15 (44.1%) were puppies and 19 (55.9%) were adults. The large-body size was most frequent in the infected dogs (23, 67.6%). Twenty-two dogs (64.7%) had long (woolly) hair, and 20 dogs (58.8%) had dark hair. The majority were mixed breeds (28, 90.3%), whereas the remaining three individuals were two Chihuahuas (5.8%) and a Golden Retriever (2.9%).

Tick and flea co-infestations were most frequent in the infected dogs (14, 41.2%), followed by only flea or only tick infestation (8, 23.5%). The severe infestation level was the most frequent (20, 58.8%). Similarly, most of the infected dogs had ectoparasites in several anatomical areas (27, 79.4%). Twenty-seven (79.4%) infected dogs did not join their owners in outdoor activities, 31 (91.2%) did not have a resting place inside the household, and 30 (88.2%) cohabitated with at least one other dog in the household.

Regarding clinical variables, 17 (50%) infected dogs had no vaccination, and 25 dogs (73.5%) had not received any antiparasitic treatments. Seven (20.6%) had a fever and three (8.8%) had signs of bleeding (epistaxis, hemorrhage, among others). In the infected dogs, the most frequent body condition was skinny (19, 55.9%), followed by ideal (15, 44.1%). Skin lesions, pruritus and petechiae or ecchymosis occurred in eight (23.5%), six (17.6%) and six (17.6%) dogs, respectively. Finally, three (8.8%) infected dogs showed other clinical but uncommon signs compatible with Rickettsia infection.

The GLM analysis showed that the most informative associated factors, sex, age, breed and outdoor activities, together with clinical variables, antiparasitic treatment at least 3 months before the sampling, vaccination, as well as hemorrhages and epistaxis, were statistically significant (p < 0.15) and were included to find a less complex multivariate model (Table 1).

After fitting different stratified models with interactions between variables, the final model included the following variable combinations: outdoor activities by age, breed, vaccination and antiparasitic treatment according to the dog’s sex (male or female). The multivariate model showed that mixed-breed dogs (RR = 0.06) and dogs that had received antiparasitic treatment 3 months before sampling (RR = 0.049) had a lower risk of Rickettsia TG infection, taking as reference the group of purebred dogs and the non-treated dogs, respectively. Looking at the interaction of the variables in the final model, we estimated that adult dogs without outdoor activities were at lower risk of Rickettsia TG infection than puppies (RR = 0.26), and among dogs with antiparasitic treatment, females had a higher risk of Rickettsia TG infection than male dogs (RR = 26.2) (Table 2).

4. Discussion

Dogs are some of the most frequent hosts of the TG Rickettsia. Sánchez-Montes et al. [1], in a recent bibliographic review on the Rickettsia occurrence in Mexico, showed their contribution to the transmission cycles of different Rickettsia species as incidental or temporary hosts of arthropod vectors, such as ticks and fleas [9,21,34,35].

Dogs suffer from intermittent infestations with C. felis fleas [36], making them susceptible hosts to TG Rickettsia infection such as R. typhi [8], acquiring epidemiological relevance for the occurrence of murine typhus in human populations, particularly for people who usually live with them [7]. However, reports with PCR tests of the occurrence of R. typhi natural infection in these companion animals are scarce [8,9,10].

The frequency of infection with TG Rickettsia found in the studied dogs from Maxcanú was higher than that reported by Martínez-Ortiz et al. [9] in domiciliated dogs from Bolmay, Yucatan (5.5% vs. 21.8%). Additionally, only one Rickettsia group (TG) was found circulating in the dog population from Maxcanú. The variation in the infection frequency might be due to changes in flea vector population densities and host susceptibility [7,37] or the neglected living of dogs in rural localities of Yucatan [38,39]. When fleas have generalist feeding habits (such as C. felis), the contact with mammals such as domiciliated dogs facilitates the enzootic spread of the bacteria [8].

The TG Rickettsia transmission cycle mainly depends on the ecology and behavior of its vector populations [3,5]. In this context, one of the most common fleas that infest domiciliated dogs from the State of Yucatan is C. felis [40], which, in some endemic regions, is the primary vector and reservoir of R. typhi [15,41] as well as the most abundant ectoparasite inside households and their surroundings [42], increasing the chances of Rickettsia TG transmission in the domiciliated dogs [21].

Most of the studied dogs from Maxcanú did not have veterinary services such as antiparasitic treatments. This circumstance increases the chances of finding infestation with ectoparasites (including fleas) in these animals [43]. However, the findings of this study showed the significance of antiparasitic treatments in these companion animals as treated dogs displayed a lower risk of having a Rickettsia TG infection.

When we adjusted the multivariate model for sex, we found that treated female dogs had an increased risk of infection with TG Rickettsia than treated male dogs. This finding can be partially explained by the less pronounced roaming habits of the female dogs compared to male dogs, as documented by Muinde et al. [44], because of several factors such as mating, pregnancy and the care of the young. Regarding that, extensive roaming behavior is a factor that modifies the risk for rickettsial infection [45,46,47]. In this context, female dogs could be spending more time in the peridomicile area, where exposure to flea vectors is frequent, as evidenced by the infestation levels of other hosts of C. felis and murine typhus, such as synanthropic opossums and small rodents in several localities of Yucatan [48,49,50,51]. However, the dynamics among flea-dog interactions in the peridomiciles of households should be addressed in future studies to obtain further information about the peridomicile cycles of the TG Rickettsia.

The multivariate model showed that mixed-breed dogs had a lower risk of occurrence of TG Rickettsia infection, taking as reference the group of purebred dogs; nonetheless, this result should be taken with caution because of the total number (n = 5) of purebred dogs in the sample was low. Our findings show the need for a deeper understanding of the physiopathology of TG Rickettsia infection in controlled experimental studies with companion animals to achieve a better understanding of the possible signs of infection by TG Rickettsia.

Outdoor activities such as hunting, going with the owners to agricultural areas or roaming into the surrounding patches of sylvatic vegetation, are suggested to have an effect on infection chances in the studied dogs. Likewise, age was an informative variable for the occurrence of TG Rickettsia infection in the studied dogs, but only when adjusted by outdoor activities. Concerning the multivariate results, adult dogs with outdoor activities have a lower risk of TG Rickettsia infection. In the state of Yucatan, dogs are exposed at an early age (puppies) to infestations with fleas and ticks [38], which might help to explain the increase in the infection risk in this group. Age probably plays a conditioning role in the susceptibility and immunological memory response in animals against various infections [52] and seems to influence their levels of infestation with ectoparasites [53,54].

The clinical signs caused by the natural infection with TG Rickettsia are not fully described [8,11] and, as well as other rickettsial infections such as R. rickettsii, R. felis and R. conorii, are usually subclinical, hardly recognizable and can cause non-specific and highly variable clinical signs such as fever, diarrhea, anemia, weight loss and skin rashes. However, these infections are generally self-limiting or disappear with the correct antimicrobial treatment [11,24,25,26,27,34]. It is also important to consider that previous infections with other non-pathogenic Rickettsia species could favor an immunological memory against future infections, including pathogenic Rickettsia species [1,12].

Some of those observed clinical signs agree with the findings in some infected dogs from Maxcanú; however, hemorrhages and epistaxis showed a lesser chance of being related with infection by TG Rickettsia because those are clinical signs compatible with those caused by Ehrlichia canis [8,24,27,55]. This microorganism is widely distributed in dogs from urban and rural areas of Yucatan [56,57,58,59]. Likewise, some studied dogs from Maxcanú showed flea and tick infestations, enabling natural infection with E. canis since natural coinfection with etiological agents transmitted by ticks and fleas has been documented in domiciliated dogs [27,53].

The present study showed interesting findings on TG Rickettsia-infected dogs. However, the study’s design had some limitations. The first was the lack of a priori sample size calculation. The sample included only dogs whose owners voluntarily agreed to participate in our study. Second, therefore, the sample did not have specific selection criteria for tick or flea-borne diseases, making some of the recorded clinical signs broad for other infectious diseases. Addressing these constraints, we estimated a posteriori sample size to dimension the effect of the sample power with 138 dogs from an infinite population with 0.21 of observed prevalence for TG Rickettsia infection (empirical) and 0.05 precision, leaving 0.85 confidence for the study’s result. In this regard, the classical epidemiological approach [60] recommends at least 0.9 confidence for acceptable observational field epidemiological studies, and our sample size was slightly smaller than suggested (142 dogs). On the second constraint, we could not specifically address detailed selection criteria for sampled dogs since the dog population from the study site was very heterogeneous, as documented previously by several regional studies [9,38,43]. Nonetheless, future studies must address the described constraints to increase power and refine model estimations.

The detection of TG Rickettsia DNA in dogs from Maxcanú suggests that they could be sentinels for the detection of risk areas or future potential outbreaks of murine typhus in humans [12,13] due to its proximity to human populations [45,61,62]. The epidemiological monitoring of pathogens transmitted by ectoparasites (ticks and fleas) in domiciliated dogs could be outstanding to prevent and control their transmission to human populations [45,46,63].

5. Conclusions

The Rickettsia TG species circulated actively in the studied dogs from a rural community of Yucatan State, representing a remarkable veterinary health problem and a potential zoonotic risk to their owners and the community, given the high frequency of animals naturally infected and infested with ectoparasites. Infection by TG Rickettsia in the studied dogs was subclinical. The factors of outdoor activities, age and previous antiparasitic treatment, as well as the clinical variables signs of hemorrhages and epistaxis, were associated with less chance of natural infection in the studied dogs.

Author Contributions

Conceptualization, M.T.-C., R.I.R.-V. and E.R.-N.; methodology, M.T.-C., R.I.R.-V. and E.R.-N.; software, E.R.-N.; formal analysis, M.T.-C. and E.R.-N.; investigation, M.T.-C. and H.N.-P.; resources, R.I.R.-V., P.C.-S., S.S.-M., M.B.-G., C.L.-C., K.D.-R. and F.I.P.; data curation, M.T.-C. and E.R.-N.; writing—original draft preparation, M.T.-C. and E.R.-N.; writing—review and editing, M.T.-C., E.R.-N., R.I.R.-V., P.C.-S. and S.S.-M.; visualization, R.I.R.-V., P.C.-S., S.S.-M., M.B.-G., C.L.-C., K.D.-R. and F.I.P.; supervision, R.I.R.-V., P.C.-S., S.S.-M., M.B.-G., C.L.-C., K.D.-R. and F.I.P.; project administration, M.T.-C. and R.I.R.-V. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The animal study protocol was approved by the Ethics Committee of Centro de Investigaciones Regionales “Dr. Hideyo Noguchi” (CIR) of the Universidad Autónoma de Yucatán (UADY) (CIRB-07-2018 approved 06-19-2018).

Informed Consent Statement

Not applicable.

Data Availability Statement

Informed consent was obtained from all subjects involved in the study.

Acknowledgments

To W. Arcila Fuentes, R. Puc, J. Mena, W. Carrillo and G. Ortiz for their help in the fieldwork. To B. Balam for his help in the laboratory. To the municipal authorities and families from Maxcanú for participating in the study.

Conflicts of Interest

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Tables

Table 1

Frequency of typhus group (TG) Rickettsia detected by semi-nested polymerase chain reaction (Sca5 gene) relative to clinical and exposure variables of dogs from Maxcanú, Yucatan, Mexico, and their resulting p-values from the generalized linear model (GLM) with backward stepwise selection of variables fitted for the association analysis.

Variables	Numberof Dogs (%)	TG-InfectedDogs (%)	GLMp-Value
Exposure variables
Sex
Male	76 (53.5)	16 (47.1)
Female	66 (46.5)	18 (52.9)	0.1366
Age
Puppy	51 (35.9)	15 (44.1)
Adult	91 (64.1)	19 (55.9)	0.0439
Body size
Small	45 (31.7)	11 (32.4)
Large	97 (68.3)	23 (67.6)
Hair length
Short	50 (35.2)	12 (35.3)
Long	92 (64.8)	22 (64.7)
Hair color
Dark	79 (55.6)	20 (58.8)
Light	63 (44.4)	14 (41.2)
Breed
Purebred	5 (3.5)	3 (8.8)
Mixed breed	137 (96.5)	31 (91.2)	0.0885
Ectoparasite infestation
No infestation	18 (12.7)	4 (11.8)
Only ticks	25 (17.6)	8 (23.5)
Only fleas	43 (30.3)	8 (23.5)
Ticks and fleas	56 (39.4)	14 (41.2)
Level of infestation
No infestation	18 (12.7)	4 (11.8)
Mild	42 (29.6)	10 (29.4)
Severe	82 (57.7)	20 (58.8)
Anatomical site of infestation
No infestation	18 (12.7)	4 (11.8)
One site	18 (12.7)	3 (8.8)
Several sites	106 (74.6)	27 (79.4)
Outdoor activities
No	98 (69)	27 (79.4)
Yes	44 (31)	7 (20.6)	0.0954
Specific resting place
Outside the household	128 (90.1)	31 (91.2)
Inside the household	14 (9.9)	3 (8.8)
Social coexistence with other dogs
No	23 (16.2)	4 (11.8)
Yes	119 (83.8)	30 (88.2)
Clinical variables
Vaccination
No	84 (59.2)	17 (50)
Yes	59 (41.5)	17 (50)	0.0330
Antiparasitic treatments (< 3 months)
No	101 (71.1)	25 (73.5)
Yes	41 (28.9)	9 (26.5)	0.0847
Fever
No	117 (82.4)	27 (79.4)
Yes	25 (17.6)	7 (20.6)
Hemorrhages and epistaxis
No	120 (84.5)	31 (91.2)
Yes	22 (15.5)	3 (8.8)	0.0708
Body condition
Skinny	87 (61.3)	19 (55.9)
Ideal	55 (38.7)	15 (44.1)
Skin lesions
No	100 (70.4)	26 (76.5)
Yes	42 (29.6)	8 (23.5)
Pruritus
No	123 (86.6)	28 (82.4)
Yes	19 (13.4)	6 (17.6)
Bruising, petechiae and ecchymoses
No	115 (81)	28 (82.4)
Yes	27 (19)	6 (17.6)
Other clinical signs 1
No	118 (83)	31 (91.2)
Yes	24 (17)	3 (8.8)

¹ Infrequent reported clinical signs in dogs infected with Rickettsia.

Table 2

Results of the multivariate stratified analysis to identify the association between exposure and clinical variables with the occurrence of the typhus group (TG) Rickettsia infection in domiciliated dogs from Maxcanú, Yucatan, Mexico.

Variables	Coefficients	Multivariatep (<0.05)	RR (95% CI)
Outdoor activities 1
Yes	−18.38	0.99	1.04 × 10−8 (4.9 × 10−250–364.5)
Breed 2
Mixed breed	−2.9	0.03	0.06 (0.002–0.67)
Antiparasitic treatment 1
Yes	−3.01	0.03	0.049 (0.001–0.43)
Hemorrhages and epistaxis 1Yes	−1.45	0.11	0.23 (0.03–1.12)
Vaccination 1Yes	2.39	0.06	10.9 (1.23–251.8)
Interactions 3
Adult without outdoor activitiesAdult with vaccination	−1.35−1.78	0.040.19	0.26 (0.007–0.89)0.16 (0.0006–1.9)
Female with antiparasitic treatment	3.27	0.02	26.2 (2.6–911)

¹ The negative category was used as a reference variable. ² Purebred used as a reference variable. ³ Puppy without outdoor activities, puppy without vaccination and male with antiparasitic treatment were used as a reference variable. Coefficients = multivariate binomial regression coefficients; RR (95% CI) = risk ratio with 95% confidence interval.

References

1. Sánchez-Montes, S.; Colunga-Salas, P.; Lozano-Sardaneta, Y.N.; Zazueta-Islas, H.M.; Ballados-González, G.G.; Salceda-Sánchez, B.; Huerta-Jiménez, H.; Torres-Castro, M.; Panti-May, J.A.; Peniche-Lara, G. et al. The genus Rickettsia in Mexico: Current knowledge and perspectives. Ticks Tick-Borne Dis.; 2021; 12, 101633. [DOI: https://dx.doi.org/10.1016/j.ttbdis.2020.101633] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33388556]

2. Azad, A.F. Epidemiology of murine typhus. Annu. Rev. Entomol.; 1990; 35, pp. 553-570. [DOI: https://dx.doi.org/10.1146/annurev.en.35.010190.003005] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/2105686]

3. Civen, R.; Ngo, V. Murine typhus: An unrecognized suburban vectorborne disease. Clin. Infect. Dis.; 2008; 46, pp. 913-918. [DOI: https://dx.doi.org/10.1086/527443] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/18260783]

4. Dumler, J.S.; Taylor, J.P.; Walker, D.H. Clinical and laboratory features of murine typhus in south Texas, 1980 through 1987. JAMA J. Am. Med. Assoc.; 1991; 266, pp. 1365-1370. [DOI: https://dx.doi.org/10.1001/jama.1991.03470100057033]

5. Bitam, I.; Dittmar, K.; Parola, P.; Whiting, M.F.; Raoult, D. Fleas and flea-borne diseases. Int. J. Infect. Dis.; 2010; 14, pp. e667-e676. [DOI: https://dx.doi.org/10.1016/j.ijid.2009.11.011]

6. Foley, J.E. Murine Typhus in Animals. 2020; Available online: https://www.msdvetmanual.com/generalized-conditions/rickettsial-diseases/murine-typhus-in-animals (accessed on 13 September 2022).

7. Lledó, L.; Gegundez, M.I.; Serrano, J.L.; Saz, J.v.; Beltrán, M. A Sero-epidemiological study of Rickettsia typhi infection in dogs from Soria province, central Spain. Ann. Trop. Med. Parasitol.; 2003; 97, pp. 861-864. [DOI: https://dx.doi.org/10.1179/000349803225002679]

8. Nogueras, M.M.; Pons, I.; Pla, J.; Ortuño, A.; Miret, J.; Sanfeliu, I.; Segura, F. The role of dogs in the eco-epidemiology of Rickettsia typhi, etiological agent of murine typhus. Vet. Microbiol.; 2013; 163, pp. 97-102. [DOI: https://dx.doi.org/10.1016/j.vetmic.2012.11.043]

9. Martínez-Ortiz, D.; Torres-Castro, M.; Koyoc-Cardeña, E.; López, K.; Panti-May, A.; Rodríguez-Vivas, I.; Puc, A.; Dzul, K.; Zavala-Castro, J.; Medina-Barreiro, A. et al. Molecular evidence of Rickettsia typhi infection in dogs from a rural community in Yucatán, México. Biomedica; 2016; 36, pp. 45-50. [DOI: https://dx.doi.org/10.7705/biomedica.v36i2.2913]

10. Dzul-Rosado, K.; Lugo-Caballero, C.; Tello-Martin, R.; López-Avila, K.; Zavala-Castro, J. Direct evidence of Rickettsia typhi infection in Rhipicephalus sanguineus ticks and their canine hosts. Open Vet. J.; 2017; 7, pp. 165-169. [DOI: https://dx.doi.org/10.4314/ovj.v7i2.14]

11. Juhasz, N.B.; Wilson, J.M.; Haney, K.N.; Clark, M.H.; Davenport, A.C.; Breitschwerdt, E.B.; Qurollo, B.A. Rickettsia typhi infection in a clinically ill dog from Houston, Texas. Vet. Parasitol. Reg. Stud. Rep.; 2022; 35, 100781. [DOI: https://dx.doi.org/10.1016/j.vprsr.2022.100781]

12. Nanayakkara, D.M.; Rajapakse, R.P.V.J.; Wickramasinghe, S.; Kularatne, S.A.M. Serological evidence for exposure of dogs to Rickettsia conorii, Rickettsia typhi, and Orientia tsutsugamushi in Sri Lanka. Vector-Borne Zoonotic Dis.; 2013; 13, pp. 545-549. [DOI: https://dx.doi.org/10.1089/vbz.2012.1049]

13. Nogueras, M.M.; Pons, I.; Ortuño, A.; Segura, F. Seroprevalence of Rickettsia typhi and Rickettsia felis in dogs from North-Eastern Spain. Clin. Microbiol. Infect.; 2009; 15, pp. 237-238. [DOI: https://dx.doi.org/10.1111/j.1469-0691.2008.02156.x] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19392898]

14. Adjemian, J.; Parks, S.; McElroy, K.; Campbell, J.; Eremeeva, M.E.; Nicholson, W.L.; McQuiston, J.; Taylor, J. Murine typhus in Austin, Texas, USA, 2008. Emerg. Infect. Dis.; 2010; 16, pp. 412-417. [DOI: https://dx.doi.org/10.3201/eid1603.091028] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20202415]

15. Noden, B.H.; Davidson, S.; Smith, J.L.; Williams, F. First detection of Rickettsia typhi and Rickettsia felis in fleas collected from client-owned companion animals in the Southern Great Plains. J. Med. Entomol.; 2017; 54, pp. 1093-1097. [DOI: https://dx.doi.org/10.1093/jme/tjx069]

16. Azad, A.F.; Radulovic, S.; Higgins, J.A.; Noden, B.H.; Troyer, J.M. Flea-borne rickettsioses: Ecologic considerations. Emerg. Infect. Dis.; 1997; 3, pp. 319-327. [DOI: https://dx.doi.org/10.3201/eid0303.970308]

17. Maina, A.N.; Fogarty, C.; Krueger, L.; Macaluso, K.R.; Odhiambo, A.; Nguyen, K.; Farris, C.M.; Luce-Fedrow, A.; Bennett, S.; Jiang, J. et al. Rickettsial infections among Ctenocephalides felis and host animals during a flea-borne rickettsioses outbreak in Orange county, California. PLoS ONE; 2016; 11, e0160604. [DOI: https://dx.doi.org/10.1371/journal.pone.0160604]

18. Torres-Castro, M.; Reyes-Novelo, E.; Noh-Pech, H.; Tello-Martín, R.; Lugo-Caballero, C.; Dzul-Rosado, K.; Puerto-Manzano, F.; Rodríguez-Vivas, R.I. Personal and household factors involved in recent Rickettsia exposure in a rural population from Yucatán, Mexico. Zoonoses Public Health; 2020; 67, pp. 506-515. [DOI: https://dx.doi.org/10.1111/zph.12714]

19. Torres-Castro, M.; Reyes-Novelo, E.; Noh-Pech, H.; Sánchez-Montes, S.; Colunga-Salas, P.; Lugo-Caballero, C.; Rodríguez-Vivas, R.I. Rickettsia rickettsii y Rickettsia typhi en habitantes de una comunidad rural del sureste de México. Rev. Peru. Med. Exp. Salud Publica; 2022; 39, pp. 124-125. [DOI: https://dx.doi.org/10.17843/rpmesp.2022.391.10519]

20. Torres-Castro, M.; Sánchez-Montes, S.; Colunga-Salas, P.; Noh-Pech, H.; Reyes-Novelo, E.; Rodríguez-Vivas, R.I. Molecular confirmation of Rickettsia parkeri in humans from Southern Mexico. Zoonoses Public Health; 2022; 69, pp. 382-386. [DOI: https://dx.doi.org/10.1111/zph.12927] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35142079]

21. Dzul-Rosado, K.R.; Reyes-Novelo, E.; Lugo-Caballero, C.; Cuxim-Koyoc, A.D.; Collí-Padrón, F.; Tello-Martín, R.; López-Ávila, K.B.; Palma-Chan, A.; Peniche-Lara, G.; Ruiz-Piña, H.A. Urban ecology of hosts and vectors of Rickettsia in a rickettsiosis-endemic city of the Yucatan Peninsula, Mexico. Acta Trop.; 2021; 216, 105832. [DOI: https://dx.doi.org/10.1016/j.actatropica.2021.105832]

22. INAFED. Enciclopedia de los Municipios y Delegaciones de México, 2019. Maxcanú. Available online: http://www.snim.rami.gob.mx/enciclopedia_v2/PruebaPropuesta.php?id=2379&tipo=m&e=31&m=48 (accessed on 13 September 2022).

23. Cline, M.G.; Burns, K.M.; Coe, J.B.; Downing, R.; Durzi, T.; Murphy, M.; Parker, V. 2021 AAHA Nutrition and weight management guidelines for dogs and cats. J. Am. Anim. Hosp. Assoc.; 2021; 57, pp. 153-178. [DOI: https://dx.doi.org/10.5326/JAAHA-MS-7232] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34228790]

24. Solano-Gallego, L.; Caprì, A.; Pennisi, M.G.; Caldin, M.; Furlanello, T.; Trotta, M. Acute febrile illness is associated with Rickettsia spp infection in dogs. Parasites Vectors; 2015; 8, 216. [DOI: https://dx.doi.org/10.1186/s13071-015-0824-3] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25886403]

25. Solano-Gallego, L.; Kidd, L.; Trotta, M.; di Marco, M.; Caldin, M.; Furlanello, T.; Breitschwerdt, E. Febrile illness associated with Rickettsia conorii infection in dogs from Sicily. Emerg. Infect. Dis.; 2006; 12, pp. 1985-1988. [DOI: https://dx.doi.org/10.3201/eid1212.060326] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/17326960]

26. Labruna, M.B.; Kamakura, O.; Moraes-Filho, J.; Horta, M.C.; Pacheco, R.C. Rocky mountain spotted fever in dogs, Brazil. Emerg. Infect. Dis.; 2009; 15, pp. 458-460. [DOI: https://dx.doi.org/10.3201/eid1503.081227] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19239764]

27. López Del, P.J.; Abarca, V.K.; Azócar, A.T. Evidencia clínica y serológica de rickettsiosis canina en Chile. Rev. Chil. Infectol.; 2007; 24, pp. 189-193. [DOI: https://dx.doi.org/10.4067/S0716-10182007000300002]

28. Choi, Y.J.; Jang, W.J.; Kim, J.H.; Ryu, J.S.; Lee, S.H.; Park, K.H.; Paik, H.S.; Koh, Y.S.; Choi, M.S.; Kim, I.S. Spotted fever group and typhus group rickettsioses in humans, South Korea. Emerg. Infect. Dis.; 2005; 11, pp. 237-244. [DOI: https://dx.doi.org/10.3201/eid1102.040603]

29. Portillo, A.; de Sousa, R.; Santibáñez, S.; Duarte, A.; Edouard, S.; Fonseca, I.P.; Marques, C.; Novakova, M.; Palomar, A.M.; Santos, M. et al. Guidelines for the detection of Rickettsia spp. Vector Borne Zoonotic Dis.; 2017; 17, pp. 23-32. [DOI: https://dx.doi.org/10.1089/vbz.2016.1966]

30. Reiczigel, J.; Marozzi, M.; Fábián, I.; Rózsa, L. Biostatistics for parasitologists—A primer to quantitative parasitology. Trends Parasitol.; 2019; 35, pp. 277-281. [DOI: https://dx.doi.org/10.1016/j.pt.2019.01.003]

31. Rózsa, L.; Reiczigel, J.; Majoros, G. Quantifying parasites in samples of hosts. J. Parasitol.; 2000; 86, pp. 228-232. [DOI: https://dx.doi.org/10.1645/0022-3395(2000)086[0228:QPISOH]2.0.CO;2]

32. Núñez, E.; Steyerberg, E.W.; Núñez, J. Estrategias para la elaboración de modelos estadísticos de regresión. Rev. Esp. Cardiol.; 2011; 64, pp. 501-507. [DOI: https://dx.doi.org/10.1016/j.recesp.2011.01.019]

33. R Development Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2021.

34. Zavala-Castro, J.E.; Zavala-Velázquez, J.E.; del Rosario García, M.; León, J.J.A.; Dzul-Rosado, K.R. A dog naturally infected with Rickettsia akari in Yucatan, México. Vector-Borne Zoonotic Dis.; 2009; 9, pp. 345-347. [DOI: https://dx.doi.org/10.1089/vbz.2008.0189] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19492943]

35. Álvarez-Hernández, G.; Roldán, J.F.G.; Milan, N.S.H.; Lash, R.R.; Behravesh, C.B.; Paddock, C.D. Rocky mountain spotted fever in Mexico: Past, present, and future. Lancet Infect. Dis.; 2017; 17, pp. e189-e196. [DOI: https://dx.doi.org/10.1016/S1473-3099(17)30173-1]

36. Šlapeta, J.; King, J.; McDonell, D.; Malik, R.; Homer, D.; Hannan, P.; Emery, D. The cat flea (Ctenocephalides f. felis) is the dominant flea on domestic dogs and cats in australian veterinary practices. Vet. Parasitol.; 2011; 180, pp. 383-388. [DOI: https://dx.doi.org/10.1016/j.vetpar.2011.03.035] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21515000]

37. Harms, S.; Lior, Y.; Ephros, M.; Grisaru-Soen, G.; Keysary, A.; Strenger, C.; Jongejan, F.; Waner, T.; Baneth, G. Rickettsia conorii in humans and dogs: A seroepidemiologic survey of two rural villages in Israel. Am. J. Trop. Med. Hyg.; 2007; 77, pp. 133-135. [DOI: https://dx.doi.org/10.4269/ajtmh.2007.77.133]

38. Ortega-Pacheco, A.; Rodriguez-Buenfil, J.C.; Bolio-Gonzalez, M.E.; Sauri-Arceo, C.H.; Jiménez-Coello, M.; Forsberg, C.L. A survey of dog populations in urban and rural areas of Yucatan, Mexico. Anthrozoos; 2007; 20, pp. 261-274. [DOI: https://dx.doi.org/10.2752/089279307X224809]

39. Cortez-Aguirre, G.R.; Jiménez-Coello, M.; Gutiérrez-Blanco, E.; Ortega-Pacheco, A. Stray dog population in a city of Southern Mexico and its impact on the contamination of public areas. Veter Med. Int.; 2018; 2018, 2381583. [DOI: https://dx.doi.org/10.1155/2018/2381583]

40. Zavala-Velázquez, J.E.; Zavala-Castro, J.E.; Vado-Solís, I.; Ruiz-Sosa, J.A.; Moron, C.G.; Bouyer, D.H.; Walker, D.H. Identification of Ctenocephalides felis fleas as a host of Rickettsia felis, the agent of a spotted fever rickettsiosis in Yucatan, Mexico. Vector Borne Zoonotic Dis.; 2002; 2, pp. 69-75. [DOI: https://dx.doi.org/10.1089/153036602321131869]

41. Nogueras, M.M.; Pons, I.; Ortuño, A.; Miret, J.; Pla, J.; Castellà, J.; Segura, F. Molecular detection of Rickettsia typhi in cats and fleas. PLoS ONE; 2013; 8, e71386. [DOI: https://dx.doi.org/10.1371/journal.pone.0071386]

42. Vetter, S.M.; Gage, K.L.; Borchert, J.N.; Enscore, R.E.; Wilder, A.P.; Amatre, G.; Atiku, L.A.; Montenieri, J.A.; Eisen, R.J.; van Wyk, K. et al. Early-phase transmission of Yersinia pestis by cat fleas (Ctenocephalides felis) and their potential role as vectors in a plague-endemic region of Uganda. Am. J. Trop. Med. Hyg.; 2008; 78, pp. 949-956. [DOI: https://dx.doi.org/10.4269/ajtmh.2008.78.949]

43. Ojeda-Chi, M.M.; Rodriguez-Vivas, R.I.; Esteve-Gasent, M.D.; Pérez de León, A.A.; Modarelli, J.J.; Villegas-Perez, S.L. Ehrlichia canis in dogs of Mexico: Prevalence, incidence, co-infection and factors associated. Comp. Immunol. Microbiol. Infect. Dis.; 2019; 67, 101351. [DOI: https://dx.doi.org/10.1016/j.cimid.2019.101351]

44. Muinde, P.; Bettridge, J.M.; Sousa, F.M.; Dürr, S.; Dohoo, I.R.; Berezowski, J.; Mutwiri, T.; Odinga, C.O.; Fèvre, E.M.; Falzon, L.C. Who let the dogs out? exploring the spatial ecology of free-roaming domestic dogs in Western Kenya. Ecol. Evol.; 2021; 11, pp. 4218-4231. [DOI: https://dx.doi.org/10.1002/ece3.7317] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33976805]

45. Hii, S.F.; Kopp, S.R.; Thompson, M.F.; O’Leary, C.A.; Rees, R.L.; Traub, R.J. Molecular evidence of Rickettsia felis infection in dogs from northern territory, Australia. Parasites Vectors; 2011; 4, 198. [DOI: https://dx.doi.org/10.1186/1756-3305-4-198] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21989454]

46. Foley, J.; Tinoco-Gracia, L.; Rodriguez-Lomelí, M.; Estrada-Guzmán, J.; Fierro, M.; Mattar-Lopez, E.; Peterson, A.; Pascoe, E.; Gonzalez, Y.; Hori-Oshima, S. et al. Unbiased assessment of abundance of Rhipicephalus sanguineus sensu lato ticks, canine exposure to spotted fever group Rickettsia, and risk factors in Mexicali, México. Am. J. Trop. Med. Hyg.; 2019; 101, pp. 22-32. [DOI: https://dx.doi.org/10.4269/ajtmh.18-0878] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31094313]

47. Abarca, K.; Gárate, D.; López, J.; Acosta-Jamett, G. Flea and ticks species from dogs in urban and rural areas in four districts in Chile. Arch. Med. Vet.; 2016; 48, pp. 109-115. [DOI: https://dx.doi.org/10.4067/S0301-732X2016000200017]

48. Panti-May, J.A.; Hernández-Betancourt, S.F.; Torres-Castro, M.A.; Machaín-Williams, C.; Cigarroa-Toledo, N.; Sodá, L.; López-Manzanero, G.; Meza-Sulú, J.R.; Vidal-Martínez, V.M. Population characteristics of human-commensal rodents present in households from Mérida, Yucatán, México. MANTER J. Parasite Biodiver.; 2016; 5, pp. 1-6. [DOI: https://dx.doi.org/10.13014/K2VD6WCX]

49. Torres-Castro, M.; Cruz-Camargo, B.; Medina-Pinto, R.; Reyes-Hernández, B.; Moguel-Lehmer, C.; Medina, R.; Ortiz-Esquivel, J.; Arcila-Fuentes, W.; López-Ávila, A.; No-Pech, H. et al. Detección molecular de leptospiras patógenas en roedores sinantrópicos y silvestres capturados en Yucatán, México. Biomedica; 2018; 38, pp. 51-58. [DOI: https://dx.doi.org/10.7705/biomedica.v38i3.3938]

50. Boostrom, A.; Beier, M.S.; Macaluso, J.A.; Macaluso, K.R.; Hayes, D.S.; Radulovic, S.; Azad, A.F. Geographic association of Rickettsia felis-infected opossums with human murine typhus, Texas. Emerg. Infect. Dis.; 2002; 8, pp. 549-554. [DOI: https://dx.doi.org/10.3201/eid0806.010350]

51. Canto-Osorio, J.M.; Cuxim-Koyoc, A.; Ruiz-Piña, H.A.; Morales-Malacara, J.B.; Reyes-Novelo, E. Ectoparasites of Didelphis virginiana from Yucatan, Mexico. J. Med. Entomol.; 2020; 57, pp. 1821-1829. [DOI: https://dx.doi.org/10.1093/jme/tjaa106]

52. Solano-Gallego, L.; Trotta, M.; Caldin, M.; Furlanello, T. Molecular survey of Rickettsia spp. in sick dogs in Italy. Zoonoses Public Health; 2008; 55, pp. 521-525. [DOI: https://dx.doi.org/10.1111/j.1863-2378.2008.01149.x]

53. Fortes, F.S.; Silveira, I.; Moraes-Filho, J.; Leite, R.V.; Bonacim, J.E.; Biondo, A.W.; Labruna, M.B.; Molento, M.B. Seroprevalence of Rickettsia bellii and Rickettsia felis in dogs, São José Dos Pinhais, State of Paraná, Brazil. Braz. J. Vet. Parasitol.; 2010; 19, pp. 222-227. [DOI: https://dx.doi.org/10.1590/S1984-29612010000400006]

54. Delgado, S.; Cármenes, P. Canine seroprevalence of Rickettsia conorii infection (mediterranean spotted fever) in Castilla y León (Northwest Spain). Eur. J. Epidemiol.; 1995; 11, pp. 597-600. [DOI: https://dx.doi.org/10.1007/BF01719315] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/8549737]

55. Sosa-Gutierrez, C.G.; Vargas-Sandoval, M.; Torres, J.; Gordillo-Pérez, G. Tick-borne rickettsial pathogens in questing ticks, removed from humans and animals in Mexico. J. Vet. Sci.; 2016; 17, pp. 353-360. [DOI: https://dx.doi.org/10.4142/jvs.2016.17.3.353] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26726019]

56. Rodriguez-Vivas, R.I.; Albornoz, R.E.F.; Bolio, G.M.E. Ehrlichia canis in dogs in Yucatan, Mexico: Seroprevalence, prevalence of infection and associated factors. Vet. Parasitol.; 2005; 127, pp. 75-79. [DOI: https://dx.doi.org/10.1016/j.vetpar.2004.08.022] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15619376]

57. Jiménez-Coello, M.; Pérez-Osorio, C.; Vado-Solís, I.; Rodríguez-Buenfil, J.C.; Ortega-Pacheco, A. Serological survey of Ehrlichia canis in stray dogs from Yucatan, Mexico, using two different diagnostic tests. Vector Borne Zoonotic Dis.; 2009; 9, pp. 209-212. [DOI: https://dx.doi.org/10.1089/vbz.2008.0039]

58. Pat-Nah, H.; Rodriguez-Vivas, R.I.; Bolio-Gonzalez, M.E.; Villegas-Perez, S.L.; Reyes-Novelo, E. Molecular diagnosis of Ehrlichia canis in dogs and ticks Rhipicephalus sanguineus (Acari: Ixodidae) in Yucatan, Mexico. J. Med. Entomol.; 2015; 52, pp. 101-104. [DOI: https://dx.doi.org/10.1093/jme/tju010]

59. Diaz-Medina, O.C.; Bolio-González, M.E.; Rodríguez-Vivas, R.I.; Gutiérrez-Ruiz, E.J.; Pérez-Osorio, C. Molecular survey of Ehrlichia canis in dogs from Mexico: Prevalence of infection and possible factors associated. Ecosist. Rec. Agropecu.; 2016; 3, pp. 251-258. [DOI: https://dx.doi.org/10.19136/era.a3n8.778]

60. Thrusfield, M.; Christley, R. Veterinary Epidemiology; Wiley-Blackwell: Hoboken, NJ, USA, 2018; 896p.

61. Lado, P.; Costa, F.B.; Verdes, J.M.; Labruna, M.B.; Venzal, J.M. Seroepidemiological survey of Rickettsia spp. in dogs from the endemic area of Rickettsia parkeri rickettsiosis in Uruguay. Acta Trop.; 2015; 146, pp. 7-10. [DOI: https://dx.doi.org/10.1016/j.actatropica.2015.02.014]

62. Reyes-Novelo, E.; Ruiz-Piña, H.; Canché-Pool, E.B.; Panti-May, J.A.; Escobedo-Ortegón, F.J. The peridomicile and the zoonoses in Yucatan. Trough the searching for one health. Trop. Subtrop. Agroecosyst.; 2022; 25, 011. [DOI: https://dx.doi.org/10.56369/tsaes.3907]

63. Moonga, L.C.; Hayashida, K.; Nakao, R.; Lisulo, M.; Kaneko, C.; Nakamura, I.; Eshita, Y.; Mweene, A.S.; Namangala, B.; Sugimoto, C. et al. Molecular detection of Rickettsia felis in dogs, rodents and cat fleas in Zambia. Parasites Vectors; 2019; 12, 168. [DOI: https://dx.doi.org/10.1186/s13071-019-3435-6]