Stray Dogs as Reservoirs of the Zoonotic Agents Leptospira interrogans,Trypanosoma cruzi,and Aspergillus spp. in an Urban Area of Chiapas in Southern Mexico

Abstract

This investigation determined the presence and prevalence of the zoonotic agents Leptospira interrogans, Trypanosoma cruzi, and Aspergillus spp. in the stray dog population (a total of 224 stray dogs) in an urban area of Southern Mexico. Blood serum samples were taken from all dogs, and root hair samples were taken from dogs with skin lesions and partial alopecia. IgG antibodies for L. interrogans from 10 serovars were detected using the microscopic agglutination test. Immunofluorescence antibody test and Western blot assay were used for serologic diagnosis of T. cruzi. The Sabouraud medium was used to isolate Aspergillus spp. Prevalence of L. interrogans was 4.9%, which was determined by identifying only serovars Pyrogenes, which accounted for 3.6%, and Tarassovi, which constituted 1.3%, with titers from 1:100 to 1:800. Additionally, T. cruzi antibodies were detected in 4.5% of the dogs. Skin lesions were found in 43% of the dogs (98/224), and 35 cultures were positive for Aspergillus spp. (35.7%, p < 0.05, 95% confidence interval 2.45–3.67), identified as A. niger (82.8%), A. flavus (14.3%), and A. terreus (2.9%). This study demonstrates the presence of certain zoonotic agents (bacteria, protozoa, and fungi) in stray dogs living within the studied area. Dogs play an important role in the transmission of diseases that are potentially harmful to humans. Although the prevalence of canine leptospirosis and trypanosomiasis is not high in Southern Mexico compared with other tropical regions of Mexico, the presence of these zoonotic agents in the stray dog population demonstrates that the stray dog population in this region is a significant reservoir and potential source of infection in humans. Special care should be taken when handling stray dogs that exhibit skin lesions with partial alopecia, since a pathological Aspergillus sp. fungus may be present.

Introduction

S tray dogs (Canis familiaris) are widely scattered throughout the urban and rural areas of Chiapas, Southern Mexico (Ortega-Pacheco et al. 2007). However, no precise numbers of urban or rural dog populations have been counted in the State of Chiapas. These dogs survive in the streets as their natural habitat and, thus, are in close contact with humans sharing the same environment. For this reason, stray dogs are potential sources of infectious diseases that can be transmitted to people.

Apart from rabies, at least 65 other zoonoses, including ancylostomiasis, echinococcosis, leptospirosis, and salmonellosis, may be transmitted to humans by direct contact or by contact with pet secretions and excretions (Faulkner 1975). Some of these diseases are widespread and may cause symptoms that are severe or even fatal (Chomel and Arzt 2001). Leptospirosis is a serious zoonotic disease caused by a gram-negative spirochete of the species Leptospira interrogans sensu lato, of which around 8 serovars, from a total of approximately 240, may be pathogenic in dogs. The most commonly associated pathogenic Leptospira serovars in dogs are Icterohaemorrhagiae and Canicola, which are capable of producing clinical signs of the disease. However, dogs can serve as accidental hosts of other serovars as well (Sessions and Greene 2004). In Mexico, leptospirosis is widely distributed throughout dog and human populations. In Mexico city, 68% of dog owners show reactive titers for Leptospira spp., involving serovars Canicola, Pomona, and Icterohaemorrhagiae; the same serovars have also been found in rural regions of Chiapas (Flisser et al. 2002).

American trypanosomiasis (also known as Chagas disease) is an infectious parasitosis that is widely distributed in Latin America. American trypanosomiasis may be responsible for severe cardiopathies and is produced by the hemoflagellate protozoa Trypanosoma cruzi, which is mainly transmitted by vector insects belonging to the Reduviidae family. Insects obtain nutrition from a variety of mammalian species, including dogs, which are susceptible to becoming infected with the protozoa (Gürtler et al. 1998). American trypanosomiasis has been reported in dogs from urban and rural areas in some states in Mexico, with a prevalence between 9.8% and 21% (Estrada-Franco et al. 2006, Jimenez-Coello et al. 2008a); infected dogs also play an important role in the intra-domiciliary epidemiology of Chagas disease in humans (Sosa-Jurado et al. 2004, Estrada-Franco et al. 2006). However, other infected mammals, such as rodents, marsupials (Pinto et al. 2006), and cats (Gürtler et al. 2007), may also play a significant part in the intra-domiciliary epidemiology of the disease. Chiapas is considered a T. cruzi–endemic state, with human seroprevalence between 1.27% in the coastal and central rural regions and 32.1% in rural communities in the tropical forest and central mountain regions (Velasco-Castrejon et al. 1992, Mazariego-Arana et al. 2001).

Aspergillosis is one of the most common fungal infections in cats and dogs and is produced by Aspergillus spp., a hyphomycete that is widely spread throughout the environment. Several species are considered to be opportunistic pathogens, causing localized and general infection in animals and in humans (Solyst and Summer-Smith 1971). In dogs, the most commonly reported mycosis involves Aspergillus, which infects the nasal cavities and paranasal sinuses (Barrett et al. 1977). Additionally, generalized aspergillosis has also been reported (Watt et al. 1995, Bruchim et al. 2006) and is especially associated with immunosuppressed animals (Mullaney et al. 1983). The most frequently reported Aspergillus spp. in dogs include the following: A. terreus, A. fumigatus, A. niger, A. deflectus, and A. flavus. Although the pathogenicity of these organisms is low in most cases, neonates and immunocompromised persons can be at risk of developing severe clinical symptoms. In Mexico, a few cases of Aspergillus spp. in humans have been reported (Hernandez-Hernandez et al. 2003), but no information is available regarding the presence of Aspergillus spp. in free-roaming stray dogs.

The objective of this study was to determine the presence and prevalence of the zoonotic agents Leptospira spp., T. cruzi, and Aspergillus spp. in the stray dog population of an urban area of Chiapas, Tuxtla Gutierrez, in Southern Mexico.

Material and Methods

Study area

This study was performed in Tuxtla Gutierrez, the capital city of the State of Chiapas, Mexico (93°07′ and 16°45′N latitude; 93°07′ and 94°15′W longitude). The climate in the area is sub-humid, with a well-defined rainy season during the summer.

Animals

A convenience sampling of 224 stray dogs was chosen from the population at the dog pound of the municipality of Tuxtla Gutierrez, Chiapas, at the end of dry season, which runs through January, February, March, and April, and at the beginning of the rainy season, consisting of May and June. The dogs at the pound had been relinquished by owners or had been found free roaming in the streets and causing a nuisance. Dogs were randomly selected and examined immediately after euthanasia. Sex was determined by examination, and age was estimated based on dentition and tartar deposition on the teeth.

Sample collection

Blood samples (5 mL) were taken by either the cephalic or saphenous vein, collected in sterile tubes, and centrifuged at 400 g for 15 min; the serum was then separated and stored at −80°C until analysis. Root hair samples were taken from dogs with skin lesions and partial alopecia.

Microscopic agglutination test

The microscopic agglutination test (MAT) was considered the reference test for leptospirosis in the sampled population and was performed using live antigens, as has been described (Turner 1968). The antigens were from the following serovars: Canicola, Hardjo, Pyrogenes, Panama, Pomona, Tarassovi, Icterohaemorrhagiae, Grippotyphosa, Wolffi, and Bratislava. The MAT was performed as previously described (Brandao et al. 1998).

Immunofluorescence antibody test

For the serological diagnosis of Chagas disease, the immunofluorescence antibody test (IFAT) was performed as previously described (Jimenez-Coello et al. 2008a). Cultured parasites of T. cruzi strain Y were used as antigens. Parasites were harvested from an axenic culture and poured onto a multiwell glass slide (Glass 8-well multi-test slide; ICN Biomedicals, Aurora, OH). The slides were blocked with 50% horse serum in a phosphate buffer (PBS-HS50) and washed three times with PBS before adding serum samples at 1:32 dilution in PBS-HS50. They were then incubated for 45 min at room temperature. After being extensively washed with PBS, the slides were incubated with a sheep anti-dog IgG conjugated with fluorescein isothiocyanate (Sigma & Co., St. Louis, MO) that was diluted 1:80 in PBS-HS50 and 0.01% Evan's blue. Slides were covered with Vectashield^® mounting medium and were examined using a Nikon TE300 microscope fitted with an epifluorescence attachment. Minimal diagnosis titers for T. cruzi were considered as a dilution of ≥1:32 (Montenegro et al. 2002).

Western blot

For confirmation of serologic diagnosis of Chagas disease, the Western blot (WB) method was used as indicated (WHO 2002) and performed according to a methodology that has been previously described (Reiche et al. 1998, Jimenez-Coello et al. 2008a). The WB detected antigens for epimastigotes of T. cruzi (Y strain), which were cultivated and spread in a liver infusion tryptose medium with 10% fetal bovine serum. The parasites were solubilized in Laemmli Buffer 1× in the presence of 5% (v/v) 2-mercaptoethanol and heated at 95°C for 10 min.

Proteins from the parasites were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane for immunodetection.

The serum samples were incubated in a 1:100 dilution in TBST-milk (150 mM NaCl, 0.05% Tween 20, 10 mM Tris-HCl pH 7.4, and 1% nonfat milk). Next, the membranes were washed three times with PBS for 10 min each. The antibodies that were bound to the membrane were detected by the use of an anti-dog IgG alkaline phosphatase conjugate labeled (Santa Cruz Inc., Santa Cruz, CA) at a 1:5000 dilution. Antibody reactivity was viewed using a Nitro Blue Tetrazolium chloride-5-Bromo-4-chloro-3-indolyl phosphate substrate. A conjugate anti-dog IgG control was used to discard nonspecific bands. Interpretation of the WB results was based on previously established criteria (Jimenez-Coello et al. 2008a).

Aspergillus spp. isolation

Hair root samples were collected from dogs with partial alopecic lesions. Aspergillus spp. isolation was performed on Sabouraud dextrose agar (Difco Laboratory, Detroit, MI), and samples were incubated at 37°C for 10 days. Positive samples were microscopically evaluated to observe the hyphae structure and to detect the presence of spherical accessory conidia. The isolated strains were identified based on the macromorphology of the colonies after growth on Sabouraud dextrose agar and based on the micromorphology of the conidia after growth on potato dextrose agar, as described by Riddel (1950).

Statistical analysis

To determine the prevalence of antibodies for L. interrogans in dogs, the results of the MAT serological test were analyzed using descriptive statistics, and each serovar was identified and recorded. The prevalence of dogs that were seroreactive to T.cruzi antigens was calculated by IFAT and WB from the serum samples. The positive isolation cultures of Aspergillus spp., from hair roots obtained from dogs with partial alopecic lesions, were quantified to estimate the frequency of different species of the Aspergillus genera. The risk of infection for each dog by Leptospira sp., T. cruzi, and Aspergillus sp., according to sex, age, and presence of skin lesion(s), was analyzed using a χ ²-test or Fisher's exact test, as appropriate. Analysis was performed using Epi-Info software (Version 6.0; CDC, Atlanta, GA).

Results

The overall seroprevalence for L. interrogans was 4.9% (11/224), with titers ≥1:100. Only two serovars were found: Pyrogenes at 3.6% (8/224) and Tarassovi at 1.3% (3/224) (Table 1). Positive cases, identified using MAT, demonstrated titers between 1:100 and 1:800. Prevalence according to sex was 6.3% for males and 3.5% for females, with no statistical difference (Table 2).

Table 1.

Prevalence of Leptospira interrogans, Trypanosoma cruzi, and Aspergillus spp. in Stray Dogs in Tuxtla Gutierrez, Chiapas, Mexico

Dogs (n)	Dogs with skin lesions (%)	Microorganism	Serovar	Positive (%)	Overall prevalence
224		Leptospira interrogans	Pyrogenes	8 (3.6%)	4.9%
			Tarassovi	3 (1.3%)
224		Trypanosoma cruzi		10 (4.5%)	4.5%
224	98 (43.7%)	Aspergillus niger		29 (29.5%)	35.7%
		Aspergillus flavus		5 (5.1%)
		Aspergillus terreus		1 (1.02%)

Table 2.

Antibody Titers for L. interrogans of Serovars Pyrogenes and Tarassovi in 224 Stray Dogs from Tuxtla Gutierrez, Chiapas, Mexico

Serovar	Positives/n (%)	Ab titers	Positives	Sex
L. interrogans	8/11 (73%)	1:400	3	F
Pyrogenes				M
				M
		1:200	3	F
				M
				F
		1:100	2	M
				M
L. interrogans	3/11 (27%)			F
Tarassovi		1:800	2	M
		1:100	1	M
		Total	11

Ab, antibody; F, female; M, male.

Dog sera that were evaluated to detect T. cruzi IgG antibodies, using the IFAT and WB techniques, showed a prevalence of 4.5% (10/224) (Table 1). Positive cases showed a frequency of 60% (6/10) in females and 40% (4/10) in males.

Skin lesions with partial alopecia were found in 43% (98/224) of the stray dog population. Of the hair root cultures, 35 of those collected from affected areas were positive for Aspergillus spp. (34.6%, p < 0.05) (Table 3) and were identified as A. niger (82.8%, 29/35), A. flavus (14.3%, 5/35), and A. terreus (2.9%, 1/35). No mixed infections of leptospirosis, Chagas, and aspergillosis were found.

Table 3.

Risk Factors Associated with Positivity for Leptospirosis, Chagas Disease and Aspergillos in Stray Dogs in Tuxtla Gutierrez, Chiapas, Mexico

Risk factor	n	Positive	Prevalence (%)	OR	95% CI	p-Value
Leptospirosis
Sex
Female	113	4	3.5
Male	111	7	6.3	0.55	0.11–2.22	0.33 (NS)
Age (years)
1	6	—	—
2	49	3	6
3	69	3	4
4	38	4	10
5	30	1	3
≥6	32	—	—	—	—	0.42 (NS)
Chagas disease
Sex
Female	113	6	5.3
Male	111	4	3.6	1.50	0.36–6.54	0.74^a (NS)
Age (years)
1	6	—	—
2	49	2	4
3	69	5	7
4	38	2	5
5	30	1	3
≥6	32	—	—	—	—	0.67 (NS)
Aspergillosis
Skin lesion
Presence	98	34	34.6
Absence	126	1	0.7	68	9.62–13.66	<0.05
Sex
Female	113	13	11.5
Male	111	22	19.8	0.53	0.23–1.17	0.08 (NS)
Age (years)
1	6	—	—
2	49	12	24.4
3	69	11	15.9
4	38	5	13.1
5	30	4	13.3
≥6	32	3	9.3	—	—	0.38 (NS)

Fisher test.

NS, not significant.

Discussion

Due to their living environments and poor health conditions, stray dogs may be a significant source of zoonotic diseases, since these animals share the same environment as humans. Thus, stray dogs may serve as effective natural sentinels to assess the risk of human diseases (Castañera et al. 1998, Goossens et al. 2001, Duncan et al. 2004, Millan et al. 2008).

The pattern and distribution of leptospirosis in canine populations can vary greatly, according to observations in different countries. For instance, the serovars Canicola, Icterohaemorrhagiae (Jimenez-Coello et al. 2008b), Pomona (Flisser et al. 2002), and Grippotyphosa (Vado-Solis et al. 2002) have been found in dog populations in Mexico, while the serovar Copenhageni is most commonly associated with clinical signs of dogs in New Zealand (Hilbink et al. 1992). In Mexico City, the serovars Canicola, Pomona, and Icterohaemorrhagiae have been found in dogs and dog owners (Flisser et al. 2002). Human cases of leptospirosis are frequent in endemic tropical and subtropical regions of Mexico (Varela et al. 1972). In rural communities of Chiapas, the prevalence of human leptospirosis can reach up to 37.7% (Leal-Castellanos et al. 2003), involving serovars Canicola, Pomona, and Icterohaemorrhagiae (Flisser et al. 2002). Reactive serovars found in this study are not host specific in dogs; thus, exposure may be accidental and most likely due to contact with rodent reservoirs of these serovars, as has been found in the Pretoria area of South Africa (Myburgh et al. 1993). Since dogs may host leptospira serovars other than Canicola, they may function as reliable sentinels for epidemiological monitoring of leptospirosis (Millan et al. 2008). Although dogs may act as a reservoir of the serovars Pyrogenes and Tarassovi, which can be detected in humans (Brod et al. 2005), these are not normally involved in pathological processes. Due to the low prevalence of serovars, leptospirosis does not appear to be a significant canine disease or constitute a risk of human infection in the studied area. However, even in developed countries, such as Germany, dogs have been implicated in the transmission of leptospirosis to humans (Jansen et al. 2005). High humidity and temperature allow for a longer time of survival for leptospira in the environment studied (Everard and Everard 1993); therefore, an increase in clinical cases of leptospirosis in canines can occur during the rainy season (Adin and Cowgill 2000). Further, a high seroprevalence of canine leptospirosis can be expected after the rainy season (Jimenez-Coello et al. 2008b). The present study was performed before the rainy season, which may be partially responsible for the low seroprevalence demonstrated.

Chagas disease is endemic to the southern region of Mexico, where the vectors are well established (Guzmán-Marín et al. 1991). Dogs become infected when metacyclic parasites, the infective form, are transferred to the skin during feeding, and they are also common victims of the disease, often developing acute and chronic phases of infection (Andrade et al. 1997). Prevalence of Chagas disease in the present study is low when compared to the 14.4% reported in the State of Yucatan, in southeast Mexico (Jimenez-Coello et al. 2008a). Although the vectors of Chagas' disease, Triatoma dimidiata, Rhodnius prolixus, and Pastrongylus rufotuberculatus, are reported in the State of Chiapas (Cruz and Pickering 2006), it is probable that the vector population is not as well established in the urban area studied as it is in Merida, Yucatan. Results from this study demonstrate the presence of vectors in the city of Tuxtla Gutierrez and the relatively low risk of infection for dogs in that urban environment. Dogs infected with T. cruzi are important reservoirs of the pathogen, particularly within domestic transmission cycles, and are involved in the epidemiology of the disease in humans (Castañera et al. 1998). Since a strong association between the number of infected dogs and infected people living in the same household has been reported (Gürtler et al. 1998), strategies for the control of the vectors should be implemented in areas where they are normally found.

Aspergillosis in dogs commonly manifests itself as a respiratory disease due to conidia inhalation (Solyts and Summer-Smith 1971, Barrett et al. 1977). If not eliminated by the host, Aspergillus spp. can disseminate to other organs (Day et al. 1986, Neer 1988), particularly the lungs (Taubitz et al. 2007), as a result of triggers, such as immunosuppression, starvation, or other stressors (Mullaney et al. 1983, Watt et al. 1995). Seasonal evidence of fungal biota on dogs' fur has been reported (Cabañes et al. 1996); the high prevalence of skin lesions observed in the study dogs may be associated with the increased humidity within the studied area, as well as the type of fungus cultured. Although the most common pathological fungus that produces aspergillosis is associated with A. fumigatus in dogs (Mortellero et al. 1989, Wolf 1992), A. terreus infection in humans has become a growing concern in the past few years (Iwen et al. 1998, Perfect et al. 2001, Hachem et al. 2004, Steinbach et al. 2004). Available data indicate that this fungus may have different epidemiologic features, such as more aggressive clinical behavior and higher mortality rate, from those of other Aspergillus species (Steinbach et al. 2004).

In this study, A. niger was the main isolated species, followed by A. flavus and A. terrus. Neither species is commonly isolated in dogs, but their zoonotic potential is a significant finding. In a study involving 505 isolations from 332 human patients, Caston et al. (2007) described the colonization and infection by A. terreus, A. niger, and A. flavus in 9.1%, 8.1%, and 3.2% of the isolations, respectively. The same three species were found in the present study. On the other hand, Lai et al. (2007) indicated that A. flavus was the second most common species isolated from human patients diagnosed with invasive pulmonary aspergillosis. A. flavus is also associated with ocular disease, neurological deficits, spinal column pain, and urinary system disorders (Kabay et al. 1985, Bruchim et al. 2006), as well as being potentially responsible for cutaneous infections in human neonates (Singh et al. 2004). A. niger has been generally regarded as a nonpathogenic fungus that is widely distributed in nature, and some people are exposed to these conidiospores every day without disease becoming apparent. However, A. niger has been shown to colonize the human body as an opportunistic invader in some cases, which could serve as the origin of invasive aspergillosis. Pulmonary aspergillosis (Kierownik-Kosela et al. 1990, Caston et al. 2007), as well as foot infection and amputation (Louthrenoo et al. 1990) caused by A. niger, have been reported in human patients.

This study demonstrates the presence of bacteria, protozoa, and fungi as potential zoonotic agents from stray dogs in the studied area. Dogs may contract the diseases by contact with wild or other domestic animal reservoirs, by drinking water or by direct contact with the agents, or by being the source of food for the bugs as in the case of Chagas disease. Immunosuppressed people, elderly people, and neonates should avoid contact with stray dogs, particularly with animals that harbor skin lesions, due to the higher probability that these lesions are reservoirs of potentially pathogenic species of Aspergillus. Since the presence of dogs seropositive for Chagas disease is considered a risk factor for humans, vector control where dogs reside is important to reduce the number of reservoir dogs.

The occurrence of the zoonotic agents found in this study illustrates the need for better collaboration between authorities involved in human health, dog owners, and veterinarians for the control of stray dog populations and the prevention of pathological agent transmission.

Footnotes

Acknowledgment

We gratefully acknowledge SIINV-UNACH 2007 (Sistema Institucional de Investigación de la Universidad Autonoma de Chiapas) for financial support: “El cánido de la via pública como factor de riosgo parasitico (infecciones bacterianas y parasitarias) para el ser humano en el Estado de Chiapas” Registration number: 06/VET/SIN/017/07.

Disclosure Statement

No competing financial interests exist.

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