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Prevalence of selected zoonotic and vector-borne agents in dogs and cats in Costa Rica
Veterinary Parasitology 183 (2011) 178–183
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Prevalence of selected zoonotic and vector-borne agents in dogs and cats in Costa Rica Andrea V. Scorza ∗ , Colleen Duncan, Laura Miles, Michael R. Lappin Veterinary Teaching Hospital, Colorado State University, 300 West Drake Road, Fort Collins, CO 80523, USA
a r t i c l e
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Article history: Received 26 July 2010 Received in revised form 30 March 2011 Accepted 30 June 2011 Keywords: Cats Dogs Zoonoses Vector-borne Costa Rica
a b s t r a c t To estimate the prevalence of enteric parasites and selected vector-borne agents of dogs and cats in San Isidro de El General, Costa Rica, fecal and serum samples were collected from animals voluntarily undergoing sterilization. Each fecal sample was examined for parasites by microscopic examination after fecal flotation and for Giardia and Cryptosporidium using an immunofluorescence assay (IFA). Giardia and Cryptosporidium IFA positive samples were genotyped after PCR amplification of specific DNA if possible. The seroprevalence rates for the vector-borne agents (Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum) were estimated based on results from a commercially available ELISA. Enteric parasites were detected in samples from 75% of the dogs; Ancylostoma caninum, Trichuris vulpis, Giardia, and Toxocara canis were detected. Of the cats, 67.5% harbored Giardia spp., Cryptosporidium spp., Ancylostoma tubaeforme, or Toxocara cati. Both Cryptosporidium spp. isolates that could be sequenced were Cryptosporidium parvum (one dog isolate and one cat isolate). Of the Giardia spp. isolates that were successfully sequenced, the 2 cat isolates were assemblage A and the 2 dog isolates were assemblage D. D. immitis antigen and E. canis antibodies were identified in 2.3% and 3.5% of the serum samples, respectively. The prevalence of enteric zoonotic parasites in San Isidro de El General in Costa Rica is high in companion animals and this information should be used to mitigate public health risks. © 2011 Published by Elsevier B.V.
1. Introduction There are more than 150 zoonoses, some of which are harbored by companion animals (Robertson and Thompson, 2002; Traub et al., 2002). Several parasites that are present in the feces of dogs and cats can cause serious public health concerns. For example, Toxocara canis, Toxocara cati, and Baylisascaris procynosis can induce visceral or ocular larva migrans. Ancylostoma caninum, Ancylostoma braziliense, Ancylostoma tubaeforme, Uncinaria
∗ Corresponding author. Tel.: +1 970 297 4247; fax: +1 970 297 1275. E-mail addresses: [email protected], [email protected] (A.V. Scorza). 0304-4017/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.vetpar.2011.06.025
stenocephala and Strongyloides stercoralis can induce cutaneous or visceral larva migrans. The common enteric protozoans Cryptosporidium spp. and Giardia spp. can also be zoonotic. Previous studies reported enteric zoonotic agents in humans, animals and the environment in Costa Rica. Studies performed on samples from humans during 1960–1977 found that over 95% of those studied harbored intestinal parasites; Trichuris (73%), Ascaris (42%), Entamoeba histolytica (41%), hookworms (33%) and Giardia (22%) were common in one historical study (Botero, 1981). Dog or cat feces could have been the source of some of these infections. Toxocara spp. and A. caninum eggs have been found in dog fecal and environmental samples (sand and grass) in all geographical regions of Costa Rica that have been
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studied (Vargas and Contreras, 1998; Paquet-Durand et al., 2007). Additionally, previous studies have shown Toxocara spp. antibody prevalence rates are high in humans with suspected larva migrans (Paquet-Durand et al., 2007). Dogs and cats can harbor both host-specific and zoonotic genotypes of Cryptosporidium and Giardia (Monis and Thompson, 2003). Cryptosporidium oocysts were reported in water, contaminated fruits and vegetables, and feces of humans and calves in Costa Rica (Luna et al., 2002; Calvo et al., 2004; Gutierrez et al., 1997; Pérez et al., 1998). However, to our knowledge, no studies have been published reporting Cryptosporidium oocysts in feces of dogs and cats in Costa Rica. While Giardia cysts were detected in the feces of children and dogs in Costa Rica, none of the studies genotyped the Giardia isolates and the potential for dogs and cats to harbor zoonotic assemblages is unknown in this country (Aguilar et al., 1988). Vector-borne infections, including Dirofilaria immitis, have been detected in humans and dogs in Costa Rica (Beaver et al., 1986; Sancho, 1989; Rodriguez, 2002, 2003). Additionally, dogs with clinical signs of ehrlichiosis are common in Costa Rica with the first case being reported in 1995 (Menses, 1995). Ehrlichia canis is thought to be the most common Ehrlichia spp. in the region and this tick borne agent is also potentially zoonotic (Menses, 1995; Sambri et al., 2004; Labruna et al., 2007). The presence of other rickettsial diseases, particularly Brazilian spotted fever (BSF; Rocky Mountain spotted fever) has also been reported in Costa Rica (Fuentes, 1986). However, to our knowledge, Borrelia burgdorferi and Anaplasma phagocytophilum seroprevalence rates in dogs of Costa Rica are unknown. The objectives of this study were to determine the prevalence of enteric parasites in dogs and cats and the seroprevalence rates of the vector-borne agents D. immitis, B. burgdorferi, E. canis and A. phagocytophilum in the dogs of San Isidro de El General, San Jose, Costa Rica.
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education. This program was approved by the Costa Rica agricultural branch and the Costa Rica Veterinary College. The dogs and cats were owned by local people and were voluntarily brought to the temporary clinic for sterilization. Samples were collected from all dogs and cats regardless of their health status. Animals that came in to the clinic had unknown deworming schedules. Blood and fecal samples were collected when the animals were under general anesthesia for sterilization or recovering after surgery. Approximately 2 g of feces were collected from the rectum using a gloved finger and stored in individual, labeled containers. Blood (5–10 ml) was collected and placed into a clot tube and a tube containing EDTA. The clotted blood samples were centrifuged and then the serum was pipetted into 1.5 ml tubes. The samples were stored at 4 ◦ C until transported to the laboratory where processing occurred between 2 and 4 weeks after collection. After recovering from surgery, all the animals were administered doramectin using the dose schedule suggested by the company. (Doramec L.A, Agrovet Market Animal Health, Lima, Peru).
2.3. Fecal assays 2.3.1. Fecal flotation Sheather’s sugar centrifugation was performed using 1 g of feces followed by microscopic examination for parasites.
2.3.2. Giardia and Cryptosporidium IFA Approximately 0.25 g of feces were mixed with 1 ml of PBS, a thin fecal smear was made, and each sample was stained with monoclonal antibodies for detection of Cryptosporidium parvum and Giardia and examined microscopically for the parasites following the manufacturer’s guidelines (Merifluor Crypto/Giardia kit, Meridian Diagnostic Corporation, Cincinnati, OH).
2. Materials and methods 2.1. Study area Fecal and serum samples were collected from dogs and cats that resided in San Isidro de El General, which is the capital city of Perez Zeledon County in San Jose province and the second largest city in Costa Rica. Pérez Zeledón covers an area of 1905.51 km2 and it has a population of 130,982 inhabitants (Wikipedia). The ecological map of Costa Rica comprises 12 life zones following the World Life Zone System by Holdridge (Paquet-Durand et al., 2007). These 12 regions can be grouped into three main areas according to precipitation and humidity, which are the main factors that influence parasite survival. The province of San Jose is considered to be tropical moist forest (PaquetDurand et al., 2007). 2.2. Samples/data collection Samples were collected in January 2009 by veterinary students and veterinarians involved in an outreach program that combined community service and veterinary
2.3.3. Cryptosporidium spp. PCR assays and genotyping PCR assays for the 18S rRNA, and the HSP-70 genes were performed on IFA positive samples following published protocols (Morgan et al., 2001; Scorza et al., 2003). Positive amplicons were further evaluated by genetic sequencing using a commercially available service (Macromolecular Resources, Colorado State University). The DNA sequences were analyzed both forward and reverse direction using an ABI3100 Genetic Analyzer (Applied Biosystems, Foster City, California). The sequence data were compared with those in the Genebank database by BLAST analysis (http://blast.ncbi.nlm.nih.gov/) to determine the Cryptosporidium species.
2.3.4. Giardia spp. PCR assays and genotyping PCR assays for the -giardin and GDH genes were performed were performed on IFA positive samples following published protocols (Lalle et al., 2005; Read et al., 2004). The Giardia assemblages were determined as described for Cryptosporidium.
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Table 1 Prevalence of enteric parasites in 60 dogs and 9 cats of San Isidro de El General, Costa Rica. Parasite
Dogs # pos
CIa
Cats # pos
CI
Overall Ancylostoma spp. Cryptosporidium spp.b Giardia spp.b Toxocara spp. Trichuris vulpis
45 (75%) 45 (75%) 1 (1.7%) 5 (8.6%) 3 (5%) 16 (26.7%)
62.1–85.3 62.1–85.3
6 (66.7%) 1 (1.1%) 1 (1.4%) 4 (57.1%) 1 (1.1%) 0
29.9–92.5
a b
95% confidence intervals. Giardia and Cryptosporidium were detected only by IFA and so the prevalence rates are based on evaluation of 58 dog samples and 7 cat samples.
2.4. Serological assays All canine sera were assayed in a commercially available ELISA (SNAP® 4Dx test; IDEXX Laboratories, Westbrook, ME) for simultaneous qualitative detection of. D. immitis antigen, and antibodies against B. burgdorferi, E. canis, and A. phagocytophilum. 2.5. Statistical analysis The overall prevalence rate for enteric parasitic infections in dogs and cats was defined as the percentage of fecal samples that tested positive for any parasite by any of the diagnostic tests. Specific enteric parasite prevalence rates were also calculated. For dogs, age was categorized as young (less that 1-year old) or adult (more than 1-year old) and enteric parasite prevalence rates compared between young and adult dogs using the X2 test with significance defined as p ≤ 0.05. 95%. There were only two adult cats and so statistical comparisons were not made. Confidence intervals were calculated when possible (Rickard et al., 1999). Seroprevalence rates were reported descriptively. 3. Results Fecal flotation was performed on samples collected from 60 dogs and 9 cats; adequate feces for IFA testing were available for 58 dogs and 7 cats. The overall prevalence rates for enteric parasites in dogs and cats were 75% and 66.7%, respectively (Table 1). In dogs, Ancylostoma caninum was most prevalent, followed by Trichuris vulpis, Giardia spp., and T. canis. Adult dogs were more likely than puppies to harbor T. vulpis (Table 2). In cats, Giardia spp., A. tubaeforme, Cryptosporidium spp. and T. cati were detected (Table 1). The nematodes were only detected in kittens. Sixteen of the 60 dogs (26.7%) had dual infections, 12 of those infections were caused by A. caninum and T. vulpis and triple infections were detected in three dogs. Cryptosporidium spp. was detected by IFA and by the 18srRNA PCR assay in one dog and one cat; these isolates were C. parvum. The HSP-70 and PCR failed to amplify Cryptosporidium DNA from the IFA positive samples. Giardia was detected in four cats by IFA and two of them were amplified by both PCR assays. In dogs, Giardia was detected in five samples by IFA and two of them were amplified by both PCR assays. The sequence analysis of the two genes identified the cat isolates as assemblage A (zoonotic genotype) and the dog isolates as assemblage D (dog specific genotype).
Serum samples were collected from 84 dogs. Of these, two of 84 serum samples (2.3%) tested positive for D. immitis antigen and three of 84 serum samples (3.5%) were positive for E. canis antibody. 4. Discussion Results of this study prove that the prevalence of gastrointestinal parasites in companion animals of San Isidro de El General in Costa Rica is very high. It is unknown whether any of the dogs or cats had been administered antihelmintics, which may have lessened the prevalence rates for the nematodes but this was considered unlikely. Worldwide, there is significant variation in the prevalence of intestinal parasites with ranges between 26% and 85% (Papazahariadou et al., 2007; Eguia-Aguilar et al., 2005). Causes for this variation may be related to both the environmental conditions and regional animal management practices. As many of the identified parasites can have significant health implications it is important to have an understanding of regional parasite burdens such that public health effects can be minimized. The present study supports the findings of previous researchers stating that Ancylostoma spp. are the most common parasite of carnivores (55% prevalence rate in dogs and cats) in Costa Rica (Paquet-Durand et al., 2007). In another study of dogs in Costa Rica, prevalence of A. caninum was 93% (Vargas and Contreras, 1998). In addition, hookworm eggs were found in sand and in grass samples from all geographical regions of the country (Paquet-Durand et al., 2007). A. tubaeforme was detected in the feces of only one kitten but neither of the 2 adults. T. vulpis eggs were detected in 26% of the dog samples, which is similar to previous studies that detected the parasite in 15% and 19% of the samples collected (Vargas and Contreras, 1998; Paquet-Durand et al., 2007). As for A. caninum, the prevalence of Trichuris spp. in dogs varies regionally with rates varying from 3.7% in Brazil to 49.5% in Gabon (Katagiri and Oliveira-Sequeira, 2008; Davoust et al., 2008; Vasquez Rojas and Zumbado, 1980). In the study described here, T. vulpis was more likely to be found in feces from adult dogs than puppies (p-value = 0.024). This has been documented in other studies as well and may reflect an increase chance of exposure over time (Sauerland et al., 2001; Katagiri and Oliveira-Sequeira, 2008; Mohamed et al., 2009). Toxocara spp. eggs were detected in 5% of the dogs and 18.1% of cats. While T. canis was found in puppies and dogs, only kittens were positive for T. cati. In 1998, T. canis eggs
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Table 2 Prevalence of gastrointestinal parasites in puppies and adult dogs of San Isidro de El General, Costa Rica by fecal flotation. Parasites
Puppies (n = 21)
CI
Adults (n = 39)
CI
p-value
Overall Giardia Cryptosporidium Ancylostoma caninum Trichuris vulpis Toxocara canis
5 (71.4%) 1 (4.7%) 1 (4.7%) 15 (71.4%) 2 (9.5%) 4.8 (22.9%)
47.8–88.7
30 (76.9%) 4 (10.3%) 0 30 (76.9%) 16 (35.9%) 5.1 (13.1%)
60.7–88.9
0.65
60.7–88.9 21.2–52.9
0.64 0.024 0.95
47.8–88.7 1.2–30.4
CI = 95% confidence intervals.
were present in 8% of the dog fecal samples in 4 districts of San Jose, Costa Rica (Vargas and Contreras, 1998) and subsequently, it was identified in 7% fecal samples from dogs and cats from four different regions in Costa Rica (Paquet-Durand et al., 2007). Both adult dogs and puppies were shedding T. canis eggs emphasizing that deworming should be considered for all ages as recommended by some organizations like the Companion Animal Parasite Council (www.capcvet.org). Soil-transmitted helminthiasis affects more than 2 billion people worldwide (World Health Organization, 2006). The clinical signs of toxocariasis in humans can range from asymptomatic infection to two severe clinical syndromes that are visceral larva migrans and ocular larva migrans. Although toxocariasis is the most prevalent human helminthiasis in some industrialized countries, public awareness of this syndrome is poor (RubinstenskyElefant et al., 2010). Human toxocariasis have a tendency to be more prevalent in tropical climates and in rural areas (Rubinstensky-Elefant et al., 2010). Hookworms (A. caninum, A. braziliense, Ancylostoma ceylanicum, A. tubaeforme and U. stenocephala) can also cause soil transmitted zoonoses. The migration of hookworm larva can produce cutaneous larva migrans and less frequently eosinophilic pneumonitis, localized myositis, folliculitis, erythema multiforme or ophthalmological manifestations (Bowman et al., 2010). Information regarding the transmission, clinical manifestations of disease and prevention of these common zoonotic diseases should be available to the general public (Lee et al., 2010). Additionally owners should be given confidence that pet ownership is safe as long as their pets are healthy (Lee et al., 2010). A major goal of the One Health Program stress the importance of generating closer ties between the public health professionals, veterinarians, and the general public to help disseminate this information (Paul et al., 2010). In previous studies in Costa Rica, Cryptosporidium oocysts were identified on 4.3% of the children with diarrhea, 7.4% of HIV positive patients, and 11.76% of calves with diarrhea (Mata et al., 1984; Gutierrez et al., 1997; Pérez et al., 1998). In addition, Cryptosporidium oocysts were found in untreated water samples and in nonchlorinated water samples from a water plant in San Jose, Costa Rica and oocysts were also detected in lettuce, parsley, cilantro, and blackberries acquired in local markets of the Central Valley of Costa Rica (Luna et al., 2002; Calvo et al., 2004). Based on the results of this study, Cryptosporidium spp. infections may also be detected in dogs and cats which were expected as this parasite has been documented in feces from dogs and cats from many other countries
around the world (Ballweber et al., 2009; McReynolds et al., 1999; Arai et al., 1990; Svobodovà et al., 1994). Genotyping of Cryptosporidium and Giardia is traditionally done by PCR, followed by sequence analysis of the amplicons. Most of the genotyping done on isolates from dogs have been performed using one target gene and sometimes one isolate can be identified as a different genotype depending on the gene of choice. The use of multilocus genotyping of Giardia and Cryptosporidium increases the ability to identify potential zoonotic agents. The 2 isolates that were able to be sequenced in this study were C. parvum. However, the 18S rRNA PCR used is accurate for confirming the IFA positive results but is not optimal for determining the infective genotype because it amplifies a short and highly conserved region of the Cryptosporidium spp. 18S rRNA gene. Unfortunately, PCR for the HSP-70 gene was negative and so whether the Cryptosporidium spp. isolates were truly C. parvum rather than the host adapted genotypes C. felis or C. canis could not be definitively confirmed. The fecal samples were processed between 2 and 4 weeks after being collected and so it is possible that Giardia cysts were deformed and not recognized or did not float properly which could explain the discordant results between fecal flotation and the IFA. However, the IFA was reported more sensitive than fecal centrifugationfloatation for the detection of Giardia in dog samples, which may also explain the results (Geurden et al., 2008). Cats and dogs can harbor both the zoonotic (assemblage A) and the host-specific genotype (assemblage C and D for dogs and F for cats). The dogs in the study were infected with the dog-specific genotype and the cats were infected with the zoonotic genotype. It is estimated that the transmission of the dog-specific genotype can be predominant when there is intense contact between large numbers of dogs living together. Under these conditions the dog adapted genotypes can out-compete the other genotypes (Monis and Thompson, 2003). On the contrary, household dogs were infected with the zoonotic genotype: assemblage A. However, other researchers found that household dogs were infected with dog-specific assemblages as well (Itagaki et al., 2005; Rimhanen-Finne et al., 2007). Two of the 4 IFA Giardia positive cats in the study were positive by PCR and harbored the zoonotic genotype: assemblage A suggesting acquisition of the infection from contact with humans or their excrement. Assemblage A is commonly found in cats, 41 of 106 Giardia isolates from cats around the world were assemblage A (Biancardi et al., 2004; Vasilopulos et al., 2007; Souza et al., 2007; Fayer et al., 2006; Itagaki et al., 2005; Read et al., 2002; Ravagnan et al., 2009; Scorza et al., 2009; Lebbad et al., 2010). There are no reports of infected
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cats and humans living in the same house with the A genotype. However, assemblage A was found in a human and in a dog sharing the same house in India and also in a child and his own dog in Brazil (Traub et al., 2002; Volotão et al., 2007). Giardia is one of the most common water-borne parasites worldwide and it was previously detected in Costa Rica. For example, in a previous study, Giardia was detected in 15% of the dogs in central Costa Rica (Aguilar et al., 1988). Giardia lamblia was identified among the pathogens associated with travelers’ diarrhea in Latin America (Black, 1986). G. lamblia was detected in the feces of pre-school children 15–17% (Vasquez Rojas and Zumbado, 1980). Of the sera, 2% tested positive for D. immitis antigen. These results were not confirmed by microfilaria testing and so potentially could reflect false positive reactions. Antigen positive dogs can also be tested by the presence of microfilaria in blood to validate the serologic results. The deworming status of the animals that attended the clinic was unknown and so the results of this study could have been affected by the administration of heartworm preventatives. This prevalence rate is similar to an earlier study finding of a 2.3% D. immitis infection rate of dogs in the Costa Rican central plateau (Sancho, 1989). The presence of heartworm is not surprising as the climate of Costa Rica, and particularly San Isidro, is capable of supporting mosquito populations. Past research has shown that populations of adult mosquitoes and their larvae can be high in urban areas of Costa Rica regardless of public education campaigns or mosquito control (Troyo, 2008). Case reports from Costa Rica of human infections with D. immitis highlight the continued zoonotic potential of Dirofilaria species. Dirofilaria worms have been removed from an index finger artery, the heart, pulmonary tissues, peritoneum, and periorbital tissues of human patients (Black, 1986; Rodriguez, 2002, 2003). E. canis antibody was detected in 3.5% of the serum samples. A literature search did not reveal published prevalence studies of E. canis in Costa Rica since the first reported case in 1995 (Menses, 1995). Nevertheless, Rhipicephalus sanguineus and other tick species capable of acting as vectors for E. canis are widespread in Costa Rica and support the current results. The presence of other rickettsial diseases, particularly BSF (Rocky Mountain spotted fever) have also been reported in Costa Rica (Fuentes, 1986). Rickettsia rickettsii, the causative agent of BSF, and E. canis has the potential to share the same vectors and can produce similar clinical signs. Thus, E. canis infection may have been previously under diagnosed because it was misdiagnosed as BSF. These results might be influenced by the tick control policy, which was extensively studied in Costa Rica (Ramirez, 1981; Wilson, 1996; Alvarez et al., 1999). The seropositive dogs were asymptomatic when the samples were collected. The failure to detect antibodies against A. phagocytophilum or B. burgdorferi may reflect the absence of an appropriate tick vector in the area or the relatively small sample size. Awareness of the prevalence, routes of transmission and preventive measurements to control parasite is important to the welfare of cats and dogs in all countries, including Costa Rica. The mechanism of infection of the helminthes is through fecal contamination of the soil and poor environmental sanitation; whereas, the mechanism
of infection of intestinal protozoa is by the fecal oral route by direct or indirect contact with contaminated food or water. Monitoring parasite burden in domestic pets should be a continuous task due to the aspects of zoonotic infections and the potential impact on public and human health. A preventive medicine plan providing information about parasites and the importance of regular deworming for pets should help their owners and eventually the community in the understanding these infections. The prevalence of zoonoses transmitted from dogs and cats to humans is too complicated to estimate as it depends on factors including route of transmission of the agent, number of infected animals, behavioral characteristics of the owners and knowledge on the measures of prevention. In communities where animals live in close proximity to people, and there is lack of knowledge of the transmission of zoonotic agents, the risk for transmission of zoonotic diseases is high.
Acknowledgments The authors would like to thank Dr. Pedro Boscan, Dr. Adrian Solano, Mariam Corrales, Dr. Alejandro Acevedo, Rosaura Barrantes, Dr. Roberto Carranza, Dr. Lora Ballweber, Juliette Hart and Suzie Friedman. IDEXX Laboratories, Portland ME, donated the SNAP 4DX kits used in the study. Other funding was provided by the Center for Companion Animal Studies at Colorado State University (http://csuvets.colostate.edu/companion/index.htm).
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Short communication
Prevalence of selected zoonotic and vector-borne agents in dogs and cats in Costa Rica Andrea V. Scorza ∗ , Colleen Duncan, Laura Miles, Michael R. Lappin Veterinary Teaching Hospital, Colorado State University, 300 West Drake Road, Fort Collins, CO 80523, USA
a r t i c l e
i n f o
Article history: Received 26 July 2010 Received in revised form 30 March 2011 Accepted 30 June 2011 Keywords: Cats Dogs Zoonoses Vector-borne Costa Rica
a b s t r a c t To estimate the prevalence of enteric parasites and selected vector-borne agents of dogs and cats in San Isidro de El General, Costa Rica, fecal and serum samples were collected from animals voluntarily undergoing sterilization. Each fecal sample was examined for parasites by microscopic examination after fecal flotation and for Giardia and Cryptosporidium using an immunofluorescence assay (IFA). Giardia and Cryptosporidium IFA positive samples were genotyped after PCR amplification of specific DNA if possible. The seroprevalence rates for the vector-borne agents (Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum) were estimated based on results from a commercially available ELISA. Enteric parasites were detected in samples from 75% of the dogs; Ancylostoma caninum, Trichuris vulpis, Giardia, and Toxocara canis were detected. Of the cats, 67.5% harbored Giardia spp., Cryptosporidium spp., Ancylostoma tubaeforme, or Toxocara cati. Both Cryptosporidium spp. isolates that could be sequenced were Cryptosporidium parvum (one dog isolate and one cat isolate). Of the Giardia spp. isolates that were successfully sequenced, the 2 cat isolates were assemblage A and the 2 dog isolates were assemblage D. D. immitis antigen and E. canis antibodies were identified in 2.3% and 3.5% of the serum samples, respectively. The prevalence of enteric zoonotic parasites in San Isidro de El General in Costa Rica is high in companion animals and this information should be used to mitigate public health risks. © 2011 Published by Elsevier B.V.
1. Introduction There are more than 150 zoonoses, some of which are harbored by companion animals (Robertson and Thompson, 2002; Traub et al., 2002). Several parasites that are present in the feces of dogs and cats can cause serious public health concerns. For example, Toxocara canis, Toxocara cati, and Baylisascaris procynosis can induce visceral or ocular larva migrans. Ancylostoma caninum, Ancylostoma braziliense, Ancylostoma tubaeforme, Uncinaria
∗ Corresponding author. Tel.: +1 970 297 4247; fax: +1 970 297 1275. E-mail addresses: [email protected], [email protected] (A.V. Scorza). 0304-4017/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.vetpar.2011.06.025
stenocephala and Strongyloides stercoralis can induce cutaneous or visceral larva migrans. The common enteric protozoans Cryptosporidium spp. and Giardia spp. can also be zoonotic. Previous studies reported enteric zoonotic agents in humans, animals and the environment in Costa Rica. Studies performed on samples from humans during 1960–1977 found that over 95% of those studied harbored intestinal parasites; Trichuris (73%), Ascaris (42%), Entamoeba histolytica (41%), hookworms (33%) and Giardia (22%) were common in one historical study (Botero, 1981). Dog or cat feces could have been the source of some of these infections. Toxocara spp. and A. caninum eggs have been found in dog fecal and environmental samples (sand and grass) in all geographical regions of Costa Rica that have been
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studied (Vargas and Contreras, 1998; Paquet-Durand et al., 2007). Additionally, previous studies have shown Toxocara spp. antibody prevalence rates are high in humans with suspected larva migrans (Paquet-Durand et al., 2007). Dogs and cats can harbor both host-specific and zoonotic genotypes of Cryptosporidium and Giardia (Monis and Thompson, 2003). Cryptosporidium oocysts were reported in water, contaminated fruits and vegetables, and feces of humans and calves in Costa Rica (Luna et al., 2002; Calvo et al., 2004; Gutierrez et al., 1997; Pérez et al., 1998). However, to our knowledge, no studies have been published reporting Cryptosporidium oocysts in feces of dogs and cats in Costa Rica. While Giardia cysts were detected in the feces of children and dogs in Costa Rica, none of the studies genotyped the Giardia isolates and the potential for dogs and cats to harbor zoonotic assemblages is unknown in this country (Aguilar et al., 1988). Vector-borne infections, including Dirofilaria immitis, have been detected in humans and dogs in Costa Rica (Beaver et al., 1986; Sancho, 1989; Rodriguez, 2002, 2003). Additionally, dogs with clinical signs of ehrlichiosis are common in Costa Rica with the first case being reported in 1995 (Menses, 1995). Ehrlichia canis is thought to be the most common Ehrlichia spp. in the region and this tick borne agent is also potentially zoonotic (Menses, 1995; Sambri et al., 2004; Labruna et al., 2007). The presence of other rickettsial diseases, particularly Brazilian spotted fever (BSF; Rocky Mountain spotted fever) has also been reported in Costa Rica (Fuentes, 1986). However, to our knowledge, Borrelia burgdorferi and Anaplasma phagocytophilum seroprevalence rates in dogs of Costa Rica are unknown. The objectives of this study were to determine the prevalence of enteric parasites in dogs and cats and the seroprevalence rates of the vector-borne agents D. immitis, B. burgdorferi, E. canis and A. phagocytophilum in the dogs of San Isidro de El General, San Jose, Costa Rica.
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education. This program was approved by the Costa Rica agricultural branch and the Costa Rica Veterinary College. The dogs and cats were owned by local people and were voluntarily brought to the temporary clinic for sterilization. Samples were collected from all dogs and cats regardless of their health status. Animals that came in to the clinic had unknown deworming schedules. Blood and fecal samples were collected when the animals were under general anesthesia for sterilization or recovering after surgery. Approximately 2 g of feces were collected from the rectum using a gloved finger and stored in individual, labeled containers. Blood (5–10 ml) was collected and placed into a clot tube and a tube containing EDTA. The clotted blood samples were centrifuged and then the serum was pipetted into 1.5 ml tubes. The samples were stored at 4 ◦ C until transported to the laboratory where processing occurred between 2 and 4 weeks after collection. After recovering from surgery, all the animals were administered doramectin using the dose schedule suggested by the company. (Doramec L.A, Agrovet Market Animal Health, Lima, Peru).
2.3. Fecal assays 2.3.1. Fecal flotation Sheather’s sugar centrifugation was performed using 1 g of feces followed by microscopic examination for parasites.
2.3.2. Giardia and Cryptosporidium IFA Approximately 0.25 g of feces were mixed with 1 ml of PBS, a thin fecal smear was made, and each sample was stained with monoclonal antibodies for detection of Cryptosporidium parvum and Giardia and examined microscopically for the parasites following the manufacturer’s guidelines (Merifluor Crypto/Giardia kit, Meridian Diagnostic Corporation, Cincinnati, OH).
2. Materials and methods 2.1. Study area Fecal and serum samples were collected from dogs and cats that resided in San Isidro de El General, which is the capital city of Perez Zeledon County in San Jose province and the second largest city in Costa Rica. Pérez Zeledón covers an area of 1905.51 km2 and it has a population of 130,982 inhabitants (Wikipedia). The ecological map of Costa Rica comprises 12 life zones following the World Life Zone System by Holdridge (Paquet-Durand et al., 2007). These 12 regions can be grouped into three main areas according to precipitation and humidity, which are the main factors that influence parasite survival. The province of San Jose is considered to be tropical moist forest (PaquetDurand et al., 2007). 2.2. Samples/data collection Samples were collected in January 2009 by veterinary students and veterinarians involved in an outreach program that combined community service and veterinary
2.3.3. Cryptosporidium spp. PCR assays and genotyping PCR assays for the 18S rRNA, and the HSP-70 genes were performed on IFA positive samples following published protocols (Morgan et al., 2001; Scorza et al., 2003). Positive amplicons were further evaluated by genetic sequencing using a commercially available service (Macromolecular Resources, Colorado State University). The DNA sequences were analyzed both forward and reverse direction using an ABI3100 Genetic Analyzer (Applied Biosystems, Foster City, California). The sequence data were compared with those in the Genebank database by BLAST analysis (http://blast.ncbi.nlm.nih.gov/) to determine the Cryptosporidium species.
2.3.4. Giardia spp. PCR assays and genotyping PCR assays for the -giardin and GDH genes were performed were performed on IFA positive samples following published protocols (Lalle et al., 2005; Read et al., 2004). The Giardia assemblages were determined as described for Cryptosporidium.
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Table 1 Prevalence of enteric parasites in 60 dogs and 9 cats of San Isidro de El General, Costa Rica. Parasite
Dogs # pos
CIa
Cats # pos
CI
Overall Ancylostoma spp. Cryptosporidium spp.b Giardia spp.b Toxocara spp. Trichuris vulpis
45 (75%) 45 (75%) 1 (1.7%) 5 (8.6%) 3 (5%) 16 (26.7%)
62.1–85.3 62.1–85.3
6 (66.7%) 1 (1.1%) 1 (1.4%) 4 (57.1%) 1 (1.1%) 0
29.9–92.5
a b
95% confidence intervals. Giardia and Cryptosporidium were detected only by IFA and so the prevalence rates are based on evaluation of 58 dog samples and 7 cat samples.
2.4. Serological assays All canine sera were assayed in a commercially available ELISA (SNAP® 4Dx test; IDEXX Laboratories, Westbrook, ME) for simultaneous qualitative detection of. D. immitis antigen, and antibodies against B. burgdorferi, E. canis, and A. phagocytophilum. 2.5. Statistical analysis The overall prevalence rate for enteric parasitic infections in dogs and cats was defined as the percentage of fecal samples that tested positive for any parasite by any of the diagnostic tests. Specific enteric parasite prevalence rates were also calculated. For dogs, age was categorized as young (less that 1-year old) or adult (more than 1-year old) and enteric parasite prevalence rates compared between young and adult dogs using the X2 test with significance defined as p ≤ 0.05. 95%. There were only two adult cats and so statistical comparisons were not made. Confidence intervals were calculated when possible (Rickard et al., 1999). Seroprevalence rates were reported descriptively. 3. Results Fecal flotation was performed on samples collected from 60 dogs and 9 cats; adequate feces for IFA testing were available for 58 dogs and 7 cats. The overall prevalence rates for enteric parasites in dogs and cats were 75% and 66.7%, respectively (Table 1). In dogs, Ancylostoma caninum was most prevalent, followed by Trichuris vulpis, Giardia spp., and T. canis. Adult dogs were more likely than puppies to harbor T. vulpis (Table 2). In cats, Giardia spp., A. tubaeforme, Cryptosporidium spp. and T. cati were detected (Table 1). The nematodes were only detected in kittens. Sixteen of the 60 dogs (26.7%) had dual infections, 12 of those infections were caused by A. caninum and T. vulpis and triple infections were detected in three dogs. Cryptosporidium spp. was detected by IFA and by the 18srRNA PCR assay in one dog and one cat; these isolates were C. parvum. The HSP-70 and PCR failed to amplify Cryptosporidium DNA from the IFA positive samples. Giardia was detected in four cats by IFA and two of them were amplified by both PCR assays. In dogs, Giardia was detected in five samples by IFA and two of them were amplified by both PCR assays. The sequence analysis of the two genes identified the cat isolates as assemblage A (zoonotic genotype) and the dog isolates as assemblage D (dog specific genotype).
Serum samples were collected from 84 dogs. Of these, two of 84 serum samples (2.3%) tested positive for D. immitis antigen and three of 84 serum samples (3.5%) were positive for E. canis antibody. 4. Discussion Results of this study prove that the prevalence of gastrointestinal parasites in companion animals of San Isidro de El General in Costa Rica is very high. It is unknown whether any of the dogs or cats had been administered antihelmintics, which may have lessened the prevalence rates for the nematodes but this was considered unlikely. Worldwide, there is significant variation in the prevalence of intestinal parasites with ranges between 26% and 85% (Papazahariadou et al., 2007; Eguia-Aguilar et al., 2005). Causes for this variation may be related to both the environmental conditions and regional animal management practices. As many of the identified parasites can have significant health implications it is important to have an understanding of regional parasite burdens such that public health effects can be minimized. The present study supports the findings of previous researchers stating that Ancylostoma spp. are the most common parasite of carnivores (55% prevalence rate in dogs and cats) in Costa Rica (Paquet-Durand et al., 2007). In another study of dogs in Costa Rica, prevalence of A. caninum was 93% (Vargas and Contreras, 1998). In addition, hookworm eggs were found in sand and in grass samples from all geographical regions of the country (Paquet-Durand et al., 2007). A. tubaeforme was detected in the feces of only one kitten but neither of the 2 adults. T. vulpis eggs were detected in 26% of the dog samples, which is similar to previous studies that detected the parasite in 15% and 19% of the samples collected (Vargas and Contreras, 1998; Paquet-Durand et al., 2007). As for A. caninum, the prevalence of Trichuris spp. in dogs varies regionally with rates varying from 3.7% in Brazil to 49.5% in Gabon (Katagiri and Oliveira-Sequeira, 2008; Davoust et al., 2008; Vasquez Rojas and Zumbado, 1980). In the study described here, T. vulpis was more likely to be found in feces from adult dogs than puppies (p-value = 0.024). This has been documented in other studies as well and may reflect an increase chance of exposure over time (Sauerland et al., 2001; Katagiri and Oliveira-Sequeira, 2008; Mohamed et al., 2009). Toxocara spp. eggs were detected in 5% of the dogs and 18.1% of cats. While T. canis was found in puppies and dogs, only kittens were positive for T. cati. In 1998, T. canis eggs
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Table 2 Prevalence of gastrointestinal parasites in puppies and adult dogs of San Isidro de El General, Costa Rica by fecal flotation. Parasites
Puppies (n = 21)
CI
Adults (n = 39)
CI
p-value
Overall Giardia Cryptosporidium Ancylostoma caninum Trichuris vulpis Toxocara canis
5 (71.4%) 1 (4.7%) 1 (4.7%) 15 (71.4%) 2 (9.5%) 4.8 (22.9%)
47.8–88.7
30 (76.9%) 4 (10.3%) 0 30 (76.9%) 16 (35.9%) 5.1 (13.1%)
60.7–88.9
0.65
60.7–88.9 21.2–52.9
0.64 0.024 0.95
47.8–88.7 1.2–30.4
CI = 95% confidence intervals.
were present in 8% of the dog fecal samples in 4 districts of San Jose, Costa Rica (Vargas and Contreras, 1998) and subsequently, it was identified in 7% fecal samples from dogs and cats from four different regions in Costa Rica (Paquet-Durand et al., 2007). Both adult dogs and puppies were shedding T. canis eggs emphasizing that deworming should be considered for all ages as recommended by some organizations like the Companion Animal Parasite Council (www.capcvet.org). Soil-transmitted helminthiasis affects more than 2 billion people worldwide (World Health Organization, 2006). The clinical signs of toxocariasis in humans can range from asymptomatic infection to two severe clinical syndromes that are visceral larva migrans and ocular larva migrans. Although toxocariasis is the most prevalent human helminthiasis in some industrialized countries, public awareness of this syndrome is poor (RubinstenskyElefant et al., 2010). Human toxocariasis have a tendency to be more prevalent in tropical climates and in rural areas (Rubinstensky-Elefant et al., 2010). Hookworms (A. caninum, A. braziliense, Ancylostoma ceylanicum, A. tubaeforme and U. stenocephala) can also cause soil transmitted zoonoses. The migration of hookworm larva can produce cutaneous larva migrans and less frequently eosinophilic pneumonitis, localized myositis, folliculitis, erythema multiforme or ophthalmological manifestations (Bowman et al., 2010). Information regarding the transmission, clinical manifestations of disease and prevention of these common zoonotic diseases should be available to the general public (Lee et al., 2010). Additionally owners should be given confidence that pet ownership is safe as long as their pets are healthy (Lee et al., 2010). A major goal of the One Health Program stress the importance of generating closer ties between the public health professionals, veterinarians, and the general public to help disseminate this information (Paul et al., 2010). In previous studies in Costa Rica, Cryptosporidium oocysts were identified on 4.3% of the children with diarrhea, 7.4% of HIV positive patients, and 11.76% of calves with diarrhea (Mata et al., 1984; Gutierrez et al., 1997; Pérez et al., 1998). In addition, Cryptosporidium oocysts were found in untreated water samples and in nonchlorinated water samples from a water plant in San Jose, Costa Rica and oocysts were also detected in lettuce, parsley, cilantro, and blackberries acquired in local markets of the Central Valley of Costa Rica (Luna et al., 2002; Calvo et al., 2004). Based on the results of this study, Cryptosporidium spp. infections may also be detected in dogs and cats which were expected as this parasite has been documented in feces from dogs and cats from many other countries
around the world (Ballweber et al., 2009; McReynolds et al., 1999; Arai et al., 1990; Svobodovà et al., 1994). Genotyping of Cryptosporidium and Giardia is traditionally done by PCR, followed by sequence analysis of the amplicons. Most of the genotyping done on isolates from dogs have been performed using one target gene and sometimes one isolate can be identified as a different genotype depending on the gene of choice. The use of multilocus genotyping of Giardia and Cryptosporidium increases the ability to identify potential zoonotic agents. The 2 isolates that were able to be sequenced in this study were C. parvum. However, the 18S rRNA PCR used is accurate for confirming the IFA positive results but is not optimal for determining the infective genotype because it amplifies a short and highly conserved region of the Cryptosporidium spp. 18S rRNA gene. Unfortunately, PCR for the HSP-70 gene was negative and so whether the Cryptosporidium spp. isolates were truly C. parvum rather than the host adapted genotypes C. felis or C. canis could not be definitively confirmed. The fecal samples were processed between 2 and 4 weeks after being collected and so it is possible that Giardia cysts were deformed and not recognized or did not float properly which could explain the discordant results between fecal flotation and the IFA. However, the IFA was reported more sensitive than fecal centrifugationfloatation for the detection of Giardia in dog samples, which may also explain the results (Geurden et al., 2008). Cats and dogs can harbor both the zoonotic (assemblage A) and the host-specific genotype (assemblage C and D for dogs and F for cats). The dogs in the study were infected with the dog-specific genotype and the cats were infected with the zoonotic genotype. It is estimated that the transmission of the dog-specific genotype can be predominant when there is intense contact between large numbers of dogs living together. Under these conditions the dog adapted genotypes can out-compete the other genotypes (Monis and Thompson, 2003). On the contrary, household dogs were infected with the zoonotic genotype: assemblage A. However, other researchers found that household dogs were infected with dog-specific assemblages as well (Itagaki et al., 2005; Rimhanen-Finne et al., 2007). Two of the 4 IFA Giardia positive cats in the study were positive by PCR and harbored the zoonotic genotype: assemblage A suggesting acquisition of the infection from contact with humans or their excrement. Assemblage A is commonly found in cats, 41 of 106 Giardia isolates from cats around the world were assemblage A (Biancardi et al., 2004; Vasilopulos et al., 2007; Souza et al., 2007; Fayer et al., 2006; Itagaki et al., 2005; Read et al., 2002; Ravagnan et al., 2009; Scorza et al., 2009; Lebbad et al., 2010). There are no reports of infected
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cats and humans living in the same house with the A genotype. However, assemblage A was found in a human and in a dog sharing the same house in India and also in a child and his own dog in Brazil (Traub et al., 2002; Volotão et al., 2007). Giardia is one of the most common water-borne parasites worldwide and it was previously detected in Costa Rica. For example, in a previous study, Giardia was detected in 15% of the dogs in central Costa Rica (Aguilar et al., 1988). Giardia lamblia was identified among the pathogens associated with travelers’ diarrhea in Latin America (Black, 1986). G. lamblia was detected in the feces of pre-school children 15–17% (Vasquez Rojas and Zumbado, 1980). Of the sera, 2% tested positive for D. immitis antigen. These results were not confirmed by microfilaria testing and so potentially could reflect false positive reactions. Antigen positive dogs can also be tested by the presence of microfilaria in blood to validate the serologic results. The deworming status of the animals that attended the clinic was unknown and so the results of this study could have been affected by the administration of heartworm preventatives. This prevalence rate is similar to an earlier study finding of a 2.3% D. immitis infection rate of dogs in the Costa Rican central plateau (Sancho, 1989). The presence of heartworm is not surprising as the climate of Costa Rica, and particularly San Isidro, is capable of supporting mosquito populations. Past research has shown that populations of adult mosquitoes and their larvae can be high in urban areas of Costa Rica regardless of public education campaigns or mosquito control (Troyo, 2008). Case reports from Costa Rica of human infections with D. immitis highlight the continued zoonotic potential of Dirofilaria species. Dirofilaria worms have been removed from an index finger artery, the heart, pulmonary tissues, peritoneum, and periorbital tissues of human patients (Black, 1986; Rodriguez, 2002, 2003). E. canis antibody was detected in 3.5% of the serum samples. A literature search did not reveal published prevalence studies of E. canis in Costa Rica since the first reported case in 1995 (Menses, 1995). Nevertheless, Rhipicephalus sanguineus and other tick species capable of acting as vectors for E. canis are widespread in Costa Rica and support the current results. The presence of other rickettsial diseases, particularly BSF (Rocky Mountain spotted fever) have also been reported in Costa Rica (Fuentes, 1986). Rickettsia rickettsii, the causative agent of BSF, and E. canis has the potential to share the same vectors and can produce similar clinical signs. Thus, E. canis infection may have been previously under diagnosed because it was misdiagnosed as BSF. These results might be influenced by the tick control policy, which was extensively studied in Costa Rica (Ramirez, 1981; Wilson, 1996; Alvarez et al., 1999). The seropositive dogs were asymptomatic when the samples were collected. The failure to detect antibodies against A. phagocytophilum or B. burgdorferi may reflect the absence of an appropriate tick vector in the area or the relatively small sample size. Awareness of the prevalence, routes of transmission and preventive measurements to control parasite is important to the welfare of cats and dogs in all countries, including Costa Rica. The mechanism of infection of the helminthes is through fecal contamination of the soil and poor environmental sanitation; whereas, the mechanism
of infection of intestinal protozoa is by the fecal oral route by direct or indirect contact with contaminated food or water. Monitoring parasite burden in domestic pets should be a continuous task due to the aspects of zoonotic infections and the potential impact on public and human health. A preventive medicine plan providing information about parasites and the importance of regular deworming for pets should help their owners and eventually the community in the understanding these infections. The prevalence of zoonoses transmitted from dogs and cats to humans is too complicated to estimate as it depends on factors including route of transmission of the agent, number of infected animals, behavioral characteristics of the owners and knowledge on the measures of prevention. In communities where animals live in close proximity to people, and there is lack of knowledge of the transmission of zoonotic agents, the risk for transmission of zoonotic diseases is high.
Acknowledgments The authors would like to thank Dr. Pedro Boscan, Dr. Adrian Solano, Mariam Corrales, Dr. Alejandro Acevedo, Rosaura Barrantes, Dr. Roberto Carranza, Dr. Lora Ballweber, Juliette Hart and Suzie Friedman. IDEXX Laboratories, Portland ME, donated the SNAP 4DX kits used in the study. Other funding was provided by the Center for Companion Animal Studies at Colorado State University (http://csuvets.colostate.edu/companion/index.htm).
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