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Spring 2014 Distribution of Ticks (Ixodida) of Canis lupus familiaris L. (Carnivora: Canidae) from Seven Towns in Chiriquí Province, Panamá Michelle Ferrell SIT Study Abroad
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Recommended Citation Ferrell, Michelle, "Distribution of Ticks (Ixodida) of Canis lupus familiaris L. (Carnivora: Canidae) from Seven Towns in Chiriquí Province, Panamá" (2014). Independent Study Project (ISP) Collection. 1874. https://digitalcollections.sit.edu/isp_collection/1874
This Unpublished Paper is brought to you for free and open access by the SIT Study Abroad at SIT Digital Collections. It has been accepted for inclusion in Independent Study Project (ISP) Collection by an authorized administrator of SIT Digital Collections. For more information, please contact [email protected]. SIT PANAMA SPRING 2014, UNIVERSITY OF RICHMOND, EL LABORATORIO DE ENTOMOLOGIA DEL INSTITUTO CONMEMORATIVO GORGAS DE ESTUDIOS EN SALUD
Distribution of Ticks (Ixodida) of Canis lupus familiaris L. (Carnivora: Canidae) from Seven Towns in Chiriquí Province, Panamá
La distribución de garrapatas (Ixodida) del Canis lupus familiaris L. (Carnivora: Canidae) en siete pueblos de la Provincia de Chiriquí, Panamá
Michelle Ferrell
Ferrell i
Table of Contents
Abstract ...... ii Resumen Ejecutivo...... iii Acknowledgements ...... vi Introduction ...... 1 History of Spotted Fever Group Rickettsiae and Anaplasmataceae in Panama ...... 5 Literature Review ...... 8 Importance of studying R. sanguineus as the Primary Vector ...... 8
Amblyomma ovale as the Second Most Prominent Vector ...... 10
Methods and Materials ...... 10
Study Sites ...... 10
Removal of Ticks ...... 13
Future Molecular Assays ...... 14
Results ...... 15
Discussion...... 16
Concluding Remarks and Future Recommendations ...... 19
References ...... 21
Appendix A. Figures and Tables ...... 31
Ferrell ii
Abstract
In Panama, there are cases of ticks that can act as vectors or reservoirs for rickettsioses.
This study hopes to verify the effects of urbanization in correlation with elevation on the distribution of populations of different species of ticks on domesticated dogs in the Chiriquí
Province of Panama. Seven towns, three in the lowlands (La Concepcion, David and Dolega) and four in the highlands (Boquete, Cerro Punta, Potrerillos Arriba, and Volcan) were chosen according to an urbanizational and elevational gradient. A total of 280 dogs (n=40 for each town) were examined for all life stages of ticks (Acari: Ixodida) with the permission of the owners.
Following removal, ticks were stored in glass tubes with 95% ethanol. 37.14% of all the dogs were parasitized with ticks (16.88 % in highlands, 64.16% in lowlands). A total of 1,823 ticks were collected, belonging to three species: Rhicelphilus sanguineus s.l., R. microplus,
Amblyomma ovale, and additionally, one unidentified species of Amblyomma. Rhipicephalus sanguineus was the most widespread and prevalent species, except in Potrerillos Arriba where just one specimen was collected, possibly for lack of an ecologically environment suitable for R. sanguineus . The environmental conditions in Panama can affect the portions of different species of ticks parasitizing dogs which can increase the risk of transmission for tick-borne diseases in some areas.
Ferrell iii
Resumen Ejecutivo
Las enfermedades transmitidas por vectores son difíciles de prevenir porque hay muchas variables que afectan a la transmisión del patógeno a una persona (Mills et al. 2010). Una comprensión completa de las complicadas interacciones entre el vector, el hospedero, el medio ambiente, y el patógeno es necesaria para la creación de un método de prevención exitoso
(Institute of Medicine 2011). La domesticación de animales ha creado interacciones ecológicas nuevas entre los ectoparásitos de mascotas y humanos (Bermúdez and Miranda 2011). En el caso de las enfermedades transmitas por garrapatas, la circulación de los patógenos es afectada por la ecología entre distintas especies domésticas y silvestres que interactúan con las especies de garrapatas, además de conductas humanas que puedan propiciar el parasitismo (Mills et al.
2010, Gage et al. 2008). De esta forma, las variables en el ambiente alrededor de poblados puede propiciar el establecimiento de ciertas especies de garrapatas, provocando diferentes niveles de riesgos según las especies involucradas (Dantas-Torres 2010, Bermudez and Miranda 2010,
Bermudez and Miranda 2012).
Las garrapatas de la familia Ixodidae transmiten una amplia variedad de patógenos, destacándose en América Latina las rickettsioses, tanto por el número de casos por año, como por el reconocimiento de nuevos agentes (Guimaraes et al 2001, Bermudez et al. 2009,
Bermudez et al. 2011). Estas enfermedades son provocadas por bacterias del género Rickettsia , la cual mantiene descrita hasta el momento 27 especies, 16 capaces de causar casos febriles de distinta magnitud (Azad and Beard 1998, Parola et al. 2005, Bermudez 2009, Santamaria et al.2014). Los síntomas son compatibles con otras enfermedades febriles, ya que inicia de forma súbita con cefaleas, fiebres altas, los cuales son comunes con otras dolencias (Parola et al.
2005). En Panamá las rickettsiosis son conocidas desde mediados del siglo pasado cuando se Ferrell iv reportó un brote de tifus murino ( R. typhi transmitida por pulgas) en Ciudad de Panamá (Calero
1948). Posteriormente se reconocieron los primeros casos de fiebre manchada provocada por R. rickettsii (Rodaniche and Rodaniche 1950, Calero et al. 1952, Rodaniche 1953). A diferencia de las infecciones causadas por R. typhi , donde no se registraron decesos, un 50% de los pacientes murió por R. rickettsii . Recientemente se ha reportado la re-emergencia de infecciones por R. rickettsii, registrándose 11 casos confirmados con 10 decesos (Estripeaut et al. 2007; Tribaldos et al. 2011), además de reportarse otras especies como R. felis , y R. amblyommii (Bermudez
2012b).
El objetivo de esta investigación fue evaluar la composición de especies de garrapatas que parasitan perros en poblados con diferencias ambientales en la provincia de Chiriquí. De esta manera se escogieron siete poblados con diferentes grados de perturbación antrópica y elevación, siendo considerados tres localidades en áreas de tierras bajas (La Concepción, David, Dolega) y cuatro en tierras altas (Boquete, Cerro Punta, Potrerillos Arriba, y Volcan). Un total de 280 perros (n=40 por cada pueblo) fueron revisado por todas las etapas de la vida de las garrapatas
(Ixodida) con el permiso de los dueños. Las garrapatas se preservaron en etanol al 95%.
Los resultados muestran que 37.14% de los perros (16.88% en tierras altas y 64.16% en tierras bajas) fueron parasitados con garrapatas. En total, 1,823 garrapatas fueron recolectadas, pertenecido a tres especies diferentes: Rhicelphilus sanguineus s.l., R. microplus, Amblyomma ovale, y adicionalmente, una especie inidentificada de Amblyomma . La garrapata con mayor distribución fue R. sanguineus, excepto en Potrerillos Arriba donde solamente una espécimen fue colectado, posiblemente por la falta de un medio ambiente apropiado para R. sanguineus en la presencia del pueblo rural. Ferrell v
En Panamá, la mayoría de la población vive en sitios urbanos dentro de las tierras
bajas, los mismos lugares que tienen la más diversidad y abundancia de garrapatas (Bermudez
and Miranda 2011). Este estudio apoya estos datos, excepto la diversidad alta en las tierra bajas
porque no había muchos co-parasitismos en ninguno sitio. R. sanguineus es como una plaga de las ciudades a través de su comportamiento endophilico y el suceso de la ovoposición en las temperaturas altas (20-35 centígrados) (Dantas-Torro et al. 2006, Demma et al. 2005). Un estudio pasado sugiere que haya un límite alta de R. sanguineus a 1000 metros; sin embargo, esta especie fue encontrado en Alto Boquete, Boquete (altitud: 1100-1500 m), un lugar que no tuvo
R. sanguineus durante el tiempo del estudio pasado. Es posible que una aumenta de la población humana creyera un ambiente mejor para el establecimiento de las garrapatas aquí. Potrerillos
Arriba (950-1100 metros de altitud) no tuvo una población de R. sanguineus , sino Amblyomma ovale , un vector de Rickettsia en Panamá. El ambiente rural del área es más preferido por A. ovale que R. sanguineus porque no hay el cemento para dar protección a R. sanguineus. El número de ratones y perros, hospederos preferidos de las ninfas y adultas de A ovale , es ideal para la sobrevivencia de A. ovale en este área.
Debido al tiempo corto de este estudio, no puedo generalizar en las poblaciones de las garrapatas en estas áreas o en la Provincia de Chiriquí. Por eso, más investigación es necesaria para tener una cobertura mejor de las especies de garrapatas en Chiriquí. Los medios ambientales de los pueblos afectan a las especies de garrapatas que parasitan a los perros, dando la oportunidad para una tasa de transmisión más alta en algunas áreas. En la ausencia de estudios epidemiólogos, una encuesta de las garrapatas en todos lugares es importante para saber el nivel de riesgo de las enfermedades en lugares diferentes. En general, más educación sobre las Ferrell vi enfermedades transmitidas por las garrapatas es necesaria porque la gente común no sabe que las garrapatas dan enfermedades a los seres humanos (Bermúdez et al. 2013).
Acknowledgements
I would like to thank el Laboratorio de Entomología del Instituto Conmemorativo Gorgas de Estudios en Salud for allowing me to use their collection supplies and their lab in identifying my ticks. The members of this lab did a lot to help me identify the destroyed ticks and in getting comfortable in telling the difference between the genera and genders. Pictures were provided by
Gleydis Garcia and Roberto Miranda. A big thanks to Senor Javier of Potrerillos Arriba, Miguel
Santamaria of la Concepcion, the Cabellero Family of Cerro Punta, Senora Maria de Boquete, and Hiasbeth Castillo de David is needed for helping me to collect ticks and to find houses with dogs in their respective home towns. I want to thank the owners of all the dogs for allowing me to search their dogs for ticks. I want to thank SIT Panama for financing my project and for giving me the chance to have this experience. I want to thank Ruben Gonzalez, my SIT director, for all the advice and help he gave me throughout my project. Finally, a sincere thank you to Sergio
Bermudez, my advisor, for teaching me everything I know on neotropical ticks, for helping me to complete all aspects of this project, and most of all for being so selfless in his time whenever I had questions or needed help on something.
Ferrell 7
Introduction
While no natural relationship between people and ticks exists, ticks are one of the main
vectors for zoonotic diseases (Fairchild et al. 1966). In total, there are around 900 spp. in 22 genera and three main families: Ixodidae (called “hard ticks”), Argasidae (“soft ticks”), and the
Nutalliellidae, comprised of one species from South Africa and Tanzania (Evans 1992, Keirans,
1992; Keirans and Robbins 1999). Soft ticks can experience up to seven different life stages while hard ticks go through only three molts (Oliver 1989, Sonenshine 1991, Institute of
Medicine 2011). The duration of a life cycle changes depending on the climate and availability of hosts (Institute of Medicine 2011). In cold climates, ticks can go for years at a time without feeding due to the lack of hosts and the shortened months of tick activity (Sonenshine 1991). For each life stage, a tick has to have a blood meal. For Ixodid ticks, larvae are hatched from eggs.
Larvae will generally search for a smaller host for their first blood meal because they are closer to the ground and easier to climb on to. Following the blood meal, the tick will drop to the ground and molt into a nymph. The nymph will find another host and feed again. It then falls off and molts into an adult. After the last meal, the adult drops down to the ground and the females lay eggs that will restart the process again (Figure 1, Institute of Medicine 2011).
Depending on the host life cycle of a tick, ticks can live on preferential hosts, in distinct environments, and spend varying amounts of time off host. Hard ticks can be endophilic (adapted to living indoors) or exophilic (adapted to outside environments), monotrophic (have the same host species for all life stages) or polytrophic (have more than one host species) or one-, two-, or three-host (number of life stages in which a new host is required) (Figure 1, Oliver 1989). A variety of combinations of these traits can be attributed to different species of ticks. However, these typical behaviors can change when a tick is in unideal conditions. For example, Ferrell 8
Rhipicephalus sanguineus s.l. is an endophilic, monotrophic (parasitizes domestic dogs), three host species (Dantas-Torres et al. 2006, Demma et al. 2005), but it can adapt to live outdoors and to occasionally feed on other host species when ticks populations are high or hosts are scarce
(Figure 1, Dantas-Torres et al. 2006, 2010a, 2010b). Adaptations like these can enable ticks to transmit diseases in ways that can’t be understood without knowing the natural history or behavior of the ticks (Dantes-Torres 2010).
Ticks are known to carry a variety of different pathogens (including different species of bacteria, helminthes, protozoa, and viruses) which can be transmitted to humans or domestic animals during a blood meal (Guimaraes et al 2001). In general terms, during each feeding stage, the tick has the chance of becoming infected from a pathogen present in the blood of a reservoir host. A pathogen can also get passed from an infected adult female to her ovules via transovarial transmission, as is the case for Rocky Mountain spotted fever (Institute of Medicine 2011).
Following infection, a tick can transmit a pathogen into another reservoir animal during its next blood meal. Evolutionarily, ticks have developed saliva with anti-coagulates to maintain blood flow during feeding (Kaufman and Nuttall 1996) . The pathogens have their own substances to affect the inflammation and immunity responses of the host species as a way to aid in successful transmission. Sometimes, the pathogen can even be helped by the saliva’s attributes and this is what causes serious or fatal illness (Kaufman and Nattall 1996). After being infected, this host animal can go on to infect more ticks, creating a continuous cycle of infection (Institute of
Medicine 2011).
Currently, tick-borne pathogens are the most important in veterinary science and the second most important in human health. To understand the epidemiology of these diseases, it is important to know the relationship between pathogens and human and animal hosts (Bermudez Ferrell 9
et al. 2009, Guimaraes et al. 2001, Labruna and Machado 2006). In fact, knowledge of natural
history is a key element in the prevention and control of these kinds of diseases (Gage et al.
2008, Mills and Childs 1998) .
The domestication of animals has led to the creation of new ecological interactions
between the ectoparasites of pets and humans (Bermudez and Miranda 2011). In the case of tick-
borne diseases, circulation of diseases would be affected by the interactions between the
Rickettsia infected ticks with domestic and wild vertebrates, according to the environment and
human behaviors that allow contact between ticks and people (Mills et al. 2010, Gage et al.
2008). Different species of ticks will parasitize pets according to the type of town and
environment that surrounds them (Pereira et al. 2000, Szabo et al. 2003). For example, dogs in
urban environments are usually parasitized by Rhicelphilus sanguineus, but in areas close to
cattle, species like Amblyomma cajennense can also be found (Dantas-Torres et al. 2006, 2010a,
2010b, Bermudez and Miranda 2011). On the other hand, it has also been suggested that even some breeds of dogs are more prone to getting bitten than others (Tinoco-Garcia et al. 2009,
Dantas-Torres et al. 2009). Therefore, differences in the population density and abundance of ticks and host species, can lead to variances in the transmission of the diseases to humans and pets and the infection prevalence and abundance of the ticks (Bermudez et al. 2012a).
Among the tick-borne diseases, the rickettsioses are most relevant in Latin America. Of the 27 species, around 16 in Rickettsia are capable of causing high fevers in Latin America. The
Rickettsiales are a group of different obligate intracellular gram-negative bacteria that have close
associations with arthropod vectors that infect reservoir host species; other genera include
Orientia, Ehrlichia, Neorickettsia, Neoehrlichia, and Anaplasma (Raolt and Parola 2007).
Among the zoonotic diseases transmitted by ticks in the Americas, rickettsioses including Ferrell 10
Rickettsia, Ehrlichia, and Anaplasma are becoming increasingly important to study as a multitude of them can and have infected a variety of animals and humans with sicknesses ranging from small to deadly infections in areas where they were previously not reported (Azad and Beard 1998, Parola et al. 2005, Bermudez 2009, Santamaria et al.2014).
Tick-borne rickettsial diseases ( Rickettsia and Anaplasmataceae) have similar symptoms, but different cycles of transmission and ways of infecting individuals (Figure 2, Dumler et al.
2001, Chapman et al. 2006). Members of Rickettsia are gram-negative coccobacilli that can only multiply in eukaryotic cells due to their leaky membranes which require the intracellular osmolarity (Fournier and Raoult 2007). According to Fournier and Raolt (2009), there are three main groups separated according to the clinical symptom of the disease: the typhus group is associated with lice and fleas; the spotted fever group is found mostly in ticks; and the scrub typhus group is carried by mites and other arthropods (Figure 3). The tick-borne spotted fever group (SFG) has been recorded on every continent except Antarctica (Fournier and Raolt 2009).
The Anaplasmataceae ( Ehrlichia, Anaplasma, Neorickettsia, and Wolbachia) have morphologies ranging from short rods to coccobacilli. Generally this group of pathogens attacks the mononuclear and polymorphonuclear leukocytes in a person’s blood (Mott et al. 1999,
Webster et al. 1998). These species tend to give “nonspecific, febrile, influenza-like illnesses” to people, but symptoms can range from asymptomatic to mortal disease (Fritz et al. 1997, Peterson et al. 1989, Standaert et al. 1995). Typical clinical symptoms of canine ehrlichiosis are fever, anorexia, lymphadenopathy, anemia, weight loss, edema, and coagulopathies (Dantas-Torres
2008).
In many countries, there is no real understanding for the burden these diseases have because the countries lack on-going epidemiological surveillance programs. When there is a no Ferrell 11
specific diagnosis, the presence of many rickettsial agents found around an area can help to give
ambiguous epidemiological information for an area without the surveillance (Parola et al. 2005).
For example, if high infection prevalence is found in hosts or vectors around an area, then a
person has a greater chance of coming into contact with the infection, increasing the risk for
transmission. Therefore, a complete understanding of the complex relationships between the
tick, the hosts, the environment, and the pathogen is necessary for creating prevention measures
against tick-borne diseases (Institute of Medicine 2011).
This study hopes to verify the effects of urbanization in conjunction with elevation on the distribution of populations of different species of ticks on domesticated dogs in the Chiriquí
Province of Panama. It is hypothesized that the ticks will follow the same patterns that were found in Bermudez and Miranda (2011) and Bermudez et al (2011). By comparing the compositions of ticks on the dogs of each town, this study hopes to learn more about the potential vectors that are present at varying levels of urbanization. The focus of this investigation will be on R. sanguineus due to its widespread population across the urban and peridomestic
environments of Panama (discussed more below).
History of Spotted Fever Group Rickettsiae and Anaplasmataceae in Panama
The tick-borne rickettsial diseases have been recorded in Panama since the early1950’s
when five cases of spotted fever caused by Rickettsia rickettsii were reported from Ollas Arriba,
including three mortal cases (Rodaniche and Rodaniche 1950, Calero et al. 1952, Rodaniche
1953). Amblyomma cajennense (Fabricius) was responsible for the transmission of the disease in
these cases (Rodaniche 1953). On the other hand, sero-surveillance studies using complement
fixation with the R. rickettsii antigen during 1951-52 established a relatively high seroprevalence
of 5.4-15.2% in nine provinces of Panama (Calero and Silva-Goytia 1956). Ferrell 12
After the 1950’s, there were no new cases of rickettsioses in Panama for over 51 years until the early 2000’s (Estripeaut et al. 2007; Tribaldos et al. 2011). Since then, 11 cases of spotted fever have been described in Panama with a high mortality associate. The case fatality rate recorded for the past six cases is 100 percent (Tribaldos et al. 2011). Ticks and their associated diseases still are a danger to Panamanian citizens as seen by recently reported fatal cases of Rock Mountain Spotted Fever in Panama City and surrounding areas (Estripeaut et al.
2007; Tribaldos et al. 2011). The need to study tick-borne rickettsial diseases has increased in
Panama with the recent reporting of other strains of the SFG and Anaplasmataceae (Ehrlichia and Anaplasma) in numerous tick species throughout Panama (Bermudez et al. 2009, 2011).A high positivity for Rickettsia was reported in sites with reported ectoparasites infected with
Rickettsia species (Bermudez et al. 2013). This prevalence is much higher than the low numbers of Rocky Mountain spotted fever cases recorded in Panama pointing to the possibility that other strains in the Spotted Fever group Rickettsiae are responsible (Eremeeva et al. 2009).
However, little epidemiological data exists showing the extent of the human rickettsial cases or of the species of ticks involved in human infection with the rickettsial diseases
(Bermudez et al. 2013). In Panama, Rickettsia rickettsii, Rickettsia felis, and R. amblyommii have been found in ecological reports (Bermudez et al. 2012b). A recent study done in areas known to have infected ticks found that rickettsial diseases circulate in Panama between people in close contact with domestic and zoo animals and wildlife most likely due to the presence of high numbers of infected ticks (Bermudez et al. 2012a). Another study found that the ticks most frequently parasitizing people are Rhipicephalus sanguineus s.l., A. cajennense s.l., and A. tapirellum in urban and rural areas and natural forested areas, respectively (Bermudez et al.
2012a). However, this particular study is limited because it can only shows these interactions in Ferrell 13
one region of Panama. More studies are needed showing the effect of different environments or
varying levels on urbanization on the populations of ticks in other parts of the country.
A study by Bermudez et al. (2009) recently found Ehrlichia and Anaplasma in different tick species of Panama. It gave evidence in Panama for the presence of the pathogen of human monocytic ehrlichiosis, E. chaffensis in A. cajennense ticks ; this has also been found in ticks from cows and horses (Eremeeva et al. 2009) . Cases of ehrlichiosis may have occurred in Darien
Province among patients who were experiencing a fever with an unknown cause (Bermudez et al. 2009).
Currently, the epidemiology of ehrlichiosis and anaplasmosis in canines is not well understood in Panama (Santamaria et al., 2014). In the past, canine cases with Ehrlichia and
Anaplasma were suspected based on the cases received in private practices and dogs getting
better after doxycycline treatment (Santamaria et al., 2014). Panama was considered to be
endemic for canine ehrlichiosis based on “clinical signs, hematology, positive tick samples, and
limited serology” (Santamaria et al. 2014). A recent study (Santamaria et al. 2014) found
Ehrlichia and Anaplasma infections in dogs exhibiting the typical symptoms of Ehrlichia in
Panama City (70.6% of symptomatic dogs were positive). It was found that E. canis (64.2% of
positives) and A. platys infections (21.4%) were the most prevalent. In comparison to other Latin
American countries that used a similar methodology to this study (Romero et al. 2011, Vargas-
Hernandez et al. 2012), the prevalence for E. canis was higher while the prevalence for A. platys
was similar. The high prevalence for these two infections could be due to the high populations of
R. sanguineus, a known vector for these pathogens (Santamaria et al. 2014, Dantas-Torres 2008,
Bermudez and Miranda 2011).
Ferrell 14
Literature Review
Importance of Studying R. sanguineus as the Primary Vector
Commonly known as the brown dog tick, Rhipicephalus sanguineus serves as a vector for
many different types of pathogens (Dantes-Torres 2007). This species also is the most widely
dispersed tick in the world, (Walker et al. 2000, Guglielmone et al. 2003, Estrada-Pena et al.
2004); nevertheless, the current information supports that R sanguineus has at least three species
inside the taxon (Pegram et al. 1987, Burlini et al. 2010). In America, for example, R. sanguineus
was introduced during the Colonial Era (Walker et al. 2000, Guglielmone et al., 2003), and now
it is known to have two genetic species separated in clades from Africa (in North and South
America) and Europe (in southern part of South America) (Oliveira et al. 2005, Szabo et al.,
2005, Moraes-Filho et al. 2011).
Studying this species is imperative to veterinary and public health for understanding tick-
borne diseases. R. sanguineus parasitizes dogs in high numbers, acts as the primary vector for
Ehrlichia canis, the pathogen for canine monocytic ehrlichiosis, Babesia canis vogeli, the pathogen for canine babesiosis, and members of the rickettsial spotted fever group, like R.
rickettsii in the Americas (Otranto et al. 2009).
In Panama, R. sanguineus s.l., a lineage from South Africa, appears to be the only species
to exist of the R. sanguineus taxon (Moraes-Filho et al. 2011). The DNA of Rickettsia
amblyommii has been found in R. sanguineus (Eremeeva et al. 2009, Bermudez et al. 2009,
Bermudez et al. 2011) , and it is currently thought that this could cause a mild fever or rash around the bite site (Moraes-Filho et al. 2011). However, no recorded cases of this currently exist in Panama. Ferrell 15
The range for the feeding time of R. sanguineus (2 days to several weeks) changes according to the life stage of the tick and to the species of the host (Koch 1982, Troughton and
Levin 2007). Typically, the longer a tick spends on a host, the higher the chance of passing on the disease. Risk of disease transmission increases with the presence of more male R. sanguineus ticks on a host. Male ticks spend more time on the hosts and can take multiple blood meals. This enables males to be able to move from feeding on one pet dog to another. If the first dog is already infected, the tick that bit the first dog can now pass the disease onto the second dog or to a human owner (Little et al. 2007). The presence of males also seems to increase the biting rate of immature stages of the ticks (Rechav and Nuttall 2000).If these nymphs happen to be on an infected dog, the increased biting rates will lead to more infected ticks.
Due to its ability to live off host following feeding, R. sanguineus s.l. is able to colonize new areas and hosts relatively easily (Dantas-Torro et al. 2006, Demma et al. 2005). Cities with wide urban zones in hot climates are the best suited environments for this species (Bermudez and
Miranda 2011). This tick species spends a lot of time in close proximity with people in urban and suburban environments because it is endophilic, or best suited for living indoors (Dantas-Torro et al. 2006, Demma et al. 2005). However, R. sanguineus can still survive outside of houses if safe havens, like walls or rocks on the ground, are available to them (Dantas-Torro et al. 2006,
Demma et al. 2005, Demma et al. 2006, Uspensky and Ioffe-Uspensky 2002, Okoli et al. 2006).
Usually dogs are the primary host for all three life stages of this species, but R. sanguineus also parasitizes humans when tick and dog populations are high (Dantas-Torro et al. 2006, Demma et al. 2005, Uspensky and Ioffe-Uspensky 2002, Okoli et al. 2006). There is a higher risk for human parasitism when the viable dog population is diminished in an area with high levels of ticks (Sergio Bermudez 2014, pers. comm.). This demonstrates that R. sanguineus is able to Ferrell 16
change its typical host-seeking behavior to questing when hosts are scarce (Parola et al. 2008).
Therefore, R. sanguineus is able to adapt new behaviors to survive, making it even more important to study its ethology under specific circumstances.
Amblyomma ovale as the Second Most Prominent Vector
As one of the most widespread ticks in the Americas, Amblyomma ovale is found in a variety of different habitats ranging from northern Mexico to Northern Argentina (Guglielmone et al. 2003, 2004). Unlike R. sanguineus, this species can parasitize a variety of different hosts depending on the life stage. In this sense, the immatures prefer smaller vertebrates, like mice, while the adults feed especially on Carnivora (Guglielmone et al. 2003, 2004). In Panama,
Murgas et al. (2013) reported 21 different hosts, with canines as the most common host in rural areas, disturbed rainforests, and the outskirts of urban localities ranging from the lowlands up to
800 meters. It is suggested that this species is second only to R. sanguineus as being the most
widespread and prominent tick species of Panama (Bermudez and Miranda 2011, Mugas et al.
2013).
Only a few epidemiological studies exist for this species in all of Latin America, with the
majority of them coming from Brazil (Spolidorio et al. 2010, Sabatini et al. 2010, Silva et
al.2011). In Panama, this species was implicated in a case of human paralysis (Baeza 1979) and
has recently been shown to be infected with Rickettsia species in Panama (Bermudez et al.
2009). In the absence of epidemiological data, surveillance of this species is extremely important for inferring the level of risk for these diseases in Panama.
Materials and Methods
Study Sites: Ferrell 17
Study sites were chosen according to a gradient based on urbanization and elevation in the Chiriquí Province of Panama. The climate switches between tropical and humid depending on the time of year in these areas with an average temperature is 18-22 C (Panama. Empresa de
Transmision Electrica).
The city of David is the third largest city in Panama hosting a population of 163,019 persons (Panama. Contraloría General de la República) in a surface area of 869.1 square kilometers (Visual Adventures). It is located 30 meters above sea level on the coastal plain of the western part of the Chiriquí province placing it into the 26.8-27 degrees Celsius temperature range (Figure 4, Panama. Empresa de Transmision Electrica). As the capital of this province, the city of David is divided into ten smaller areas: David, Bijagual, Chiriquí, Cochea, Guaca, Las
Lomas, Pedregal, San Carlos, San Pablo Nuevo and San Pablo Viejo (Visual Adventures). The streets are filled with vendors of common household items and touristy trinkets. Restruants, clothing stores, and large super markets make up the majority of the city center. At a typical house, about 50% or more is covered in concrete with very few trees or plants.
La Concepcion is roughly 20 minutes by car from the border with Costa Rica. It has a population of 22,286 people (Panama. Contraloría General de la República). The town sits around 200-250 meters above sea level with a temperature range of 26-26.3 degrees Celsius
(Figure 4, Panama. Empresa de Transmision Electrica), and has a relatively well developed town center with a number of stores, churches, and grocerias (See it 360). The town is most well- known for its handmade saddles and is an important intermediary for agricultural shipping (Trip
Wolf).
Dolega is a town located 230-250 meters above sea level with a temperature range of
25.1-25.9 (Figure 4, Panama. Empresa de Transmision Electrica) and a population of 4,445 Ferrell 18 people (Panama. Contraloría General de la República). This town acts as a cross roads for travelers heading towards Boquete and Potrerillos. It is made up of lots of small restaurants and tiendas close to the principal highway. As one moves away from the main road, a neighborhood of suburban type housing with lots of trees and a small creek takes over. The majority of the houses has complete lawns and has about 25% of the areas covered in concrete (pers. obser.
Michelle Ferrell 2014).
Potrerillos Arriba is a rural town covering 56.2 kilometers at 750-1000 meters above sea level with a temperature range of 22.2-25 (Figure 4, Infoplaza 54: Potrerillos Arriba, Panama.
Empresa de Transmision Electrica). It is made up of a small town center with small tiendas and a school; the surrounding areas are made up of gravel roads leading to houses and fields (pers. obser. Michelle Ferrell 2014). The houses have hardly any concrete surfaces and tend to have chickens, cats, and occasionally, goats. The town has a population of 1,566 people (Panama.
Contraloría General de la República).
Boquete is located within a valley in the highlands of Chiriquí. It covers an area of 514 km with a population of a 6,990 people (Panama. Contraloría General de la República). The altitude of the Boquete is between 1100-1500 m above sea level with an average temperature range of 19.6-22.1 degrees Celsius (Figure 4, Panama. Empresa de Transmision Electrica). The town center is filled with restaurants, small stores, and churches aimed towards tourists. The surrounding mountainsides are covered in coffee and flowers, two of the main exports of the region (Boquete Chiriqui, Panama). Alto Boquete, the collection area, appears to be more modern than the rest of the Boquete region. The majority of Boquete is made up of the large coffee farms, and has houses that are farther apart with lots of trees. However, Alto Boquete Ferrell 19
appears like a suburban neighborhood with concrete in the housing areas covering about 25-50% of the outside area in some areas.
Volcan has a population of 13,604 people (Panama. Contraloría General de la República)
and sits at 1500 m above sea level with an average temperature range of 15.1-19.5 degrees
Celsius (Figure 4, Panama. Empresa de Transmision Electrica). This town is made of many small
stores, churches, and restaurants along the principal thoroughfare and the streets coming from
this main road are covered with suburban style housing (pers. obser. Michelle Ferrell 2014). The
roads transition from gravel to paved depending on the area of the neighborhood. The majority of
the jobs appear to be constructing wooden boxes and repairing cars. The houses didn’t have a lot
of trees or concrete. The ground was generally covered in small plants and earth (pers. obser.
Michelle Ferrell).
Cerro Punta is a small rural town located in the highlands of Chiriquí, and is located just
outside of el Parque International la AMISTAD and el Parque Nacional Volcan-Baru
(cerropunta.chiriqui.org). The town has a population of 8,144 people and sits between 2100-2200
meters above sea level with an average temperature range of 10.1-15 degrees Celsius (Figure 4
Panama. Contraloría General de la República ). The area surrounding the town is made up of farms and forest interlaced with rivers. The farms here produce about 80% of the entire country’s produce, and this has caused a lot of deforestation on the mountainsides surrounding the town
(cerropunta.chiriqui.org). The town itself has hotels, small hotels, and houses close together. The amount of concrete for houses is roughly 25%.
Removal of Ticks:
Two trips were made via bus to the seven study sites between April 14 and 25 in 2014.
For the majority of the towns, I travelled with a local to ensure my safety walking alone in a Ferrell 20 place for which I was unfamiliar. All ticks species (Ixodida) of all life stages were removed using pincers from pet dogs (n=40 for each town) with the permission of the owners. To get the permission of the owners, I told them a brief description of my research and that I’m a student working with el Instituto del GORGAS. I offered to call them following molecular analysis of the ticks, so they could know whether the ticks on their dogs were positive for Rickettsia or
Anaplasmataceae. During the collection process, the dogs were held by the owners to ensure the health of the collector and the comfort of the dog. If the owner ever felt that their dog was too upset, the collection process was stopped immediately. On the dogs, particular attention was paid to the ticks’ preferred attaching sites: the ears, around the neck, the nose, the axila, and between the toes on the dog (Dantas-Torres 2010). Three dog samples were received from a veterinarian’s office in Dolega after the workers agreed to save ticks for me.
Following removal from the dogs, ticks were stored in glass tubes with 95% ethanol. The ticks were identified according to the Fairchild et al (1966) taxonomic key. Specimens were separated according to species and life stage. The type of lone or co-parasitism was determined for each individual dog. For each town, the percentage of parasitized dogs and number of ticks per each species and life stage was found.
Future Molecular Assays:
In the future, these specimens will be shipped to the R. Jory Brinkerhoff lab at the
University of Richmond for further analysis. Each individual tick will be pulverized with a sterile plastic pestle after flash freezing in liquid nitrogen. DNA will be extracted from individual ticks using the Dneasy tissue kit (Guiagen, Hilden, Germany), following the product’s listed instructions for animal tissues. Similar to the procedure conducted in Bermudez and Miranda
(2011), SYBR-Green polymerase chain reaction (PCR) assays targeting a fragment of the 401-bp Ferrell 21
rickettsial citrate synthase gene (gltA ) will be completed using primers DS-78 and CS-323 to detect the DNA of the spotted fever group rickettsiae (SFGR) and Anaplasmataceae ( Anaplasma and Ehrlichia ). Any samples showing visible results in the first PCR will be tested with primers
CS-239 and CS-1069 aiming for the overlapping 821-bp fragment of the gltA gene and primers
Rr190.70 and Rr190.701 targeting the 632-bp region of the rickettsial outer membrane protein A gene (OmpA). Gene sequencing via BLAST will determine the most closely related species for the pathogen. The infection prevalence for each tick species, the number of co-infections, and infection prevalence of the specific town will be determined. These results will be written and presented as part of a senior thesis at the University of Richmond for the 2014-2015 school year.
Results
A total of 280 dogs were examined and 1,823 ticks were collected from 7 towns. 37.14% of the dogs were parasitized by ticks (16.88 % in highlands, 64.16% in lowlands) (Table 1).
Three species of ticks were observed: Rhicelphilus sanguineus s.l., R. microplus, and
Amblyomma ovale; in addition with one unidentified species of Amblyomma. A total of 810 adult males, 814 adult females and 149 nymphs, and 3 larvae were found of R. sanguineus. For
Amblyomma ovale, 10 adult males and 16 adult females were collected. Eight nymphs of
Amblyomma spp. , and one adult female R. microplus were found (Table 2). No ticks were found in Cerro Punta or Volcan (Table 1). In general terms, the most widespread and prominent tick species was R. sanguineus , except in Potrerillos Arriba where only one specimen was found and
A. ovale was the most abundant (Fig 2, Table 1). Not many co-parasitisms were found in any of the different locations (Figure 4).
Ferrell 22
Discussion
In Panama, around 80% of the population lives in the lowlands, which also are the areas
that have been shown to have the greatest diversity and abundance of ticks on dogs (Bermudez
and Miranda 2011). In this study, it was found that dogs in the lower towns (David, Dolega, and
La Concepcion) were more parasitized than those in the highlands supporting this previous finding. The warmest and most urbanized towns had the highest levels of parasitized dogs.
However, this present study doesn’t support the higher richness of ticks in the lowlands as seen by the small numbers of dogs with co-parasitisms in all of the seven towns (Figure 5, Bermudez and Miranda 2011).
In this study, the populations of R. sanguineus seemed to follow the general patterns of
dispersion associated with it as seen by the highest abundance and prevalence of the tick being in
David (Dantas-Torro et al. 2006, Demma et al. 2005, Demma et al. 2006, Uspensky and Ioffe-
Uspensky 2002, Okoli et al. 2006). As discussed above, these ticks prefer warm centers with
large urban peripheries because of its endophilic behavior and the better success of oviposition in
higher temperature conditions (20-35 degrees Celsius) (Dantas-Torro et al. 2006, Demma et al.
2005). In France, this species has been show to survive best in less condensed areas of houses,
where there aren’t as many large buildings (Gilot et al. 1992). In Japan and France, it has been
suggested that the presence of gardens also helps in the establishment of this species (Gilot et al.
1992, Shimada et al. 2003).
The only town with parasitized dogs to not have R. sanguineus as the prominent tick species was Potrerillos Arriba. This is likely due to the lack of the preferred habitat for R. sanguineus as shown by majority of the town being made up by gravel roads and cattle fields that separate houses distantly from another. Also, the houses have hardly any concrete surfaces Ferrell 23
and tend to have chickens, cats, and occasionally, goats. Many times concrete will make the
temperature of a town hotter due to heat abortion by the cement and more urbanization also
creates weather patterns preferable for R. sanguineus (Dantas-Torres 2010, Bermudez and
Miranda 2011). The lack of this amenity in Potrerillos likely keeps the ticks from being able to
currently establish itself in this area even though R. sanguineus is able to adapt too many
environments in Panama (Dantas-Torro et al. 2006, Demma et al. 2005, Bermudez et al. 2011).
However, it is possible that it may in the future as shown by the one adult female R. sanguineus
that was found. More studies are needed to quantify the population of this species in this area.
A previous study suggested the possibility of an upper limit for R. sanguineus at 1000 m in Panama (Bermudez and Miranda 2011). However, the results of the present study found R. sanguineus in Alto Boquete, Boquete, an area that was uninhabited by R. sanguineus during the time of the Bermudez and Miranda (2011). This suggests that something has changed in this area in the past three years to allow the establishment of this species.
As discussed above, Alto Boquete, the area of collection in Boquete, appears to be a lot more urbanized than the surrounding areas. The majority of the suburb had paved roads, and lots of houses very close together with few trees or large plants, but some of the surrounding areas had only gravel roads and green grass leading up to houses containing chickens and other small domesticated farm animals. The majority of the neighborhood was right in line with the preferred habitat that has been previously observed for R. sanguineus (Dantas-Torro et al. 2006, Demma et al. 2005, Demma et al. 2006, Uspensky and Ioffe-Uspensky 2002, Okoli et al. 2006). It is likely that this area has gone through a period of urbanization or population growth since the last study was conducted here (Bermudez et al. 2011). This demonstrates that the populations for R. Ferrell 24 sanguineus are still being newly established and that regular surveillance for tick populations should be maintained, even in areas that typically don’t have a high prevalence of ticks.
The neighborhood in Volcan appeared to be just as modernized as the one in Boquete; however, no tick specimens, including R. sanguineus, were found. Volcan is slightly higher in elevation than Boquete, putting Volcan in a temperature zone ranging between 15.1-19.5 degrees
Celsius while Boquete is within the temperature zone of 19.6-22.1 degrees Celsius (Figure 4,
Panama. Empresa de Transmision Electrica). Previous findings have suggested that successful oviposition and survival of the immature stages of R. sanguineus occur in temperatures ranging from 20-35 degrees Celsius (Koch and Tuck 1986). It appears that even though Volcan has the correct physical environment for R. sanguineus to survive, it is possible that the climate is just too cold for this species to thrive there. In urban parts of Costa Rica, R. sanguineus has been found in areas at 1200 m, but in the rural areas, this species is not present. Costa Rican areas
(Puricil and Tapanti of the Cartago Province) at altitudes between 1300 and 1400 meters did not have R. sanguineus suggesting that an upper limit may still exist for R. sanguineus (Bermudez and Miranda 2011) . Therefore, more study is needed to verify the populations of ticks in Volcan and Cerro Punta.
In Potrerillos Arriba, every parasitized dog was found to have A. ovale, the most abundant tick species for this area, which supports the findings of Murgas et al. (2013) in which the majority of adult A. ovale were found on canines in rural areas, disturbed rainforests, and the outskirts of urban localities up to 820 meters. It is likely that the high presence of smaller rodents this area helps in facilitating the ability of the immature stages to survive while the large number of dogs enables the establishment of the adults (Guglielmone et al. 2003, 2004). The finding of
A. ovale at this altitude increases the currently known upper limit for this species parasitizing Ferrell 25
dogs in Panama to 950 m. The only other specimen of this species was found in David pointing
to the lack of establishment for this tick in urbanized environments.
Due to the shortness of this study, no broad generalizing statements for tick populations can be made for the test areas or for the province of Chiriquí. Further study is needed to gain a better coverage for the species of ticks in these townships. However, the ability of R. sanguineus
to have established populations in urbanized areas above 1000 m and for A. ovale to be able to
survive at 950 m in rural areas has been supported by these results. The different environments
of the towns affected the species of ticks that parasitized dogs, giving the potential for increased
transmission of diseases in some areas. This is especially important due to the difficulty in
diagnosing these diseases and the lack of epidemiological data in Panama.
Concluding Remarks and Future Recommendations
A myriad of vector-borne diseases can affect dogs and humans; many of them being
transmitted by ticks (Dantas-Torres 2008, Otranto et al. 2009). The interactions between the
ticks, the environment, the hosts, and humans affect the abundance of the tick species and the
level of risk for the transmission of the disease to humans or their pets (Institute of Medicine
2011). Due to this, every individual town or area should have a survey of ticks as a way to
understand the intricacies of these interactions. As a vector of disease, R. sanguineus is extremely important as a carrier of disease to dogs and to humans.
As the main hosts for R. sanguineus, it has been suggested that some breeds of dogs are
more resistant to parasitism that others (Louly et al 2009). Dantas Torres (2010) offers the need
for future studies to learn the importance of specific dog factors that affect the tick burden of
dogs. Because A. ovale specimens have recently been found on wild coyotes in Panama
(Bermudez et al 2013), coyotes could play a role in the maintenance and dispersion of tick-borne Ferrell 26 diseases in Panama (Labruna et al. 2005). More studies of these coyotes in conjunction with domesticated animals could help in determining a more complete picture of the transmission cycles occurring in different areas.
In a survey completed by (Bermudez et al. 2013), it was found that the majority of people (66%) didn’t know that ticks could carry diseases to humans while a 100 percent knew that ticks transmitted disease to to animals. This also was a common observation during the collection process of this study. Scientific data exists for the tick-borne diseases found in ticks, for the general distribution of ticks, and the ticks that attach to humans in Panama. However, the general public has no idea of the role that ticks play in diseases transmission. Therefore, this data should be put into a format that is easily understandable with lots of pictures to explain the current scientific data in Panama for tick populations and disease risk. Also, general education on tick-bone diseases (causes, symptoms, medicines, prevention methods, and affected hosts) those affected by the disease should be readily available and offered to the public. The constant removal of ticks and the regular fumigation of houses should especially be expressed as the best prevention method against tick-borne diseases (Bermudez and Miranda 2011). For example, informational programs on the television would be a good way to reach the common public. For rural areas or the comarcas, it may be best to do in person talks to explain the importance of ticks as hosts for diseases. It was observed in Dolega that the local veterinarian played a key role in giving tick baths and medicine to prevent more parasitisms of dogs. This could be replicated in other areas with the previously mentioned measures.
In conclusion, all of the ideas expressed above should receive more research or action in the future. It would aid in educating the general population and in gaining new understanding for Ferrell 27
the relationship between ticks, their hosts and their surroundings. In the end, it could help in the
development of optimized management strategies for tick vectors and their pathogens in Panama.
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"Rickettsiae and Rickettsial Infections: The Current State of Knowledge." Clinical Infectious Diseases 45.Supplement 1 (2007): S39-44. Print. Walker, Jane B., James E. Keirans, and I. Horak. The Genus Rhipicephalus (Acari, Ixodidae): A Guide to the Brown Ticks of the World . Cambridge: Cambridge UP, 2005. Print. Webster, P., J. W. Ijdo, L. M. Chicoine, and E. Fikrig. "The Agent of Human Granulocytic Ehrlichiosis Resides in an Endosomal Compartment." Journal of Clinical Investigation 101.9 (1998): 1932-941. Print. Ferrell 37 Appendix A. Figures and tables Figure 1. Life and host cycle of R. sanguineus Latreille. Drawing by James Newman and Leah LeFevre, University of Florida. Figure 2 : Evolutionary relationship of the Rickettsiales including the Anaplasmataceae and Rickettsiaceae families (Todar 2012) Ferrell 38 Figure 3. Taxonomy for the genus Rickettsia separated into three groups according to clinical symptoms (Tadar 2012) Figure 4. Map of the annual mean temperature for each of the seven sites in the province of Chiriquí. David is represented by the white dot, and the other towns by white. Adapted from Panama. Empresa de Transmision Electrica . Ferrell 39 Figure 5 . Picture showing the environment in Boquete, Chiriqui, Panama. In the background are coffee farms on the hillsides. The houses in front are shown to be farther apart in comparison with those further in the distance in the left. Photo Credit: Gleydis Garcia, el Laboratorio de Entomología del Instituto Conmemorativo Gorgas de Estudios en Salud. Ferrell 40 Figure 6. Pictures showing the typical environment for the outside of a house in Potrerillos Arriba. Beyond the trees in the bottom picture is a potrero, or field for cows. Photo Credit: Roberto Mirando, el Laboratorio de Entomología del Instituto Conmemorativo Gorgas de Estudios en Salud. Ferrell 41 Figure 5. Percentage of lone and co -parasitisms for the tick-bearing dogs of each town. Ferrell 42 Table 1. The number of parasitized dogs according to each town Town Elevation (m) Town Type Total Dogs Parasitized Dogs % of Parasitized Dogs Boquete 1100-1500 Rural 40 17 42.5 Cerro Punta 2100-2200 Rural 40 0 0 La Concepcion 200-250 Suburban 40 27 67.5 David 30 Urban 40 30 75 Dolega 230-250 Rural 40 20 50 Potrerillos Arriba 950-1100 Rural 40 10 25 Volcan 1500 Rural 40 0 0 Ferrell 1 Table 2. Tick species present on the dogs of the seven towns Town Rhipicephalus sanguineus Rhipicephalus microplus Amblyomma ovale Amblyomma sp. Boquete 427 (176 ♂, 218 ♀, 33 - - - nymph) Cerro Punta - - - - La Concepcion 254 (130 ♂, 79 ♀, 42 1 (♀) - - nymph, 3 larvae) David 943 (441 ♂, 440 ♀, 62 - 1 (♀) 7 (nymph) nymph) Dolega 152 (64 ♂, 76 ♀, 12 nymph) - - - Potrerillos Arriba 1 (♀) - 34 ( 19 ♂, 15 ♀) 1 ( nymph) Volcan - - - -