It is estimated that vector-borne diseases account for approximately 17% of infectious diseases worldwide, being especially prevalent in impoverished tropical regions with poor sanitation1. One such disease is visceral leishmaniasis, caused by the parasite Leishmania infantum (syn. L. chagasi). In urban environments, domestic dogs are the main reservoir of this zoonosis2. The parasite is transmitted by phlebotomine insects belonging to the genus Lutzomyia (New World) and Phlebotomus (Old World)2. Phlebotomines related to visceral leishmaniasis are mainly Lutzomyia longipalpis and L. cruzi2. Lutzomyia longipalpis has a wide geographical distribution and seems to be well adapted to the urban environments of the southeast region of Brazil, living in and around human domiciles, henhouses, pigsties, and kennels2.
Another vector-borne disease with an impact on public health is Brazilian spotted fever (BSF), a highly fatal disease caused by the bacterium Rickettsia rickettsii3. The main vectors of R. rickettsii in Brazil are ticks of the Amblyomma cajennense species complex (A. cajennense sensu lato), followed by those of the species A. aureolatum. Rhipicephalus sanguineus is a suspected vector and may play a role in transmission in certain situations3. Dogs, which live in close proximity to humans and many possible vectors, are considered reservoirs for canine visceral leishmaniasis (CVL) and good sentinels of BSF4.
Barra Mansa (22°32′39″ S, 44°10′17″ W) is a Brazilian municipality in the south of the state of Rio de Janeiro, with a mild climate (annual mean temperature 20.7 °C), rainy summers, and Atlantic Forest vegetation. Human cases of visceral leishmaniasis, with high mortality rates, have been reported in this municipality5. In addition, Barra Mansa is within the Vale do Paraíba region, an area where human cases of BSF have been reported since 20076, making the region endemic for both diseases. This has raised awareness of the need for surveillance in dogs, which should be screened for CVL and BSF.
Given the zoonotic potential of CVL and BSF together with the participation of dogs in their epidemiological cycles, the aim of this study was to verify the presence of anti-Rickettsia spp. antibodies in dogs with a previous diagnosis of CVL that lived in a region endemic for both diseases, independent of clinical symptoms. The development of this study was approved by the Ethics Committee on Animal Research of the Universidade Federal Fluminense (CEUA 438/2013 and 525/2014).
This study included 26 dogs, unvaccinated against CVL, that tested positive for Leishmania spp. on a dual-path platform immunochromatographic test (DPP rapid test; Bio Manguinhos/Fundação Oswaldo Cruz, Rio de Janeiro, Brazil) and on an enzyme-linked immunosorbent assay for CVL (EIE-LVC; Bio Manguinhos/Fundação Oswaldo Cruz). These dogs were sent to the Fundação Oswaldo Cruz by the municipal government of Barra Mansa for euthanasia; prior to the euthanasia, the animals were submitted to physical examination and collection of blood samples.
Classification according to the presence of typical CVL clinical signs was performed as previously suggested2. The dogs were classified as asymptomatic (absence of clinical signs); oligosymptomatic (discrete clinical manifestations and adenopathy); or symptomatic (presenting all or some of the more common signs of the disease).
Blood samples were centrifuged and sera were diluted to 1:64 in phosphate buffered saline to perform an indirect immunofluorescence assay (IFA), which was conducted as previously described7. Slides used for IFA were prepared with R. rickettsii str. Taiaçu, and R. parkeri str. AT-24 infected vero cells8,9. Serum samples showing uniform and coccoid-, bacillary-, or coccobacillary-shaped fluorescent spots at 1:64 titer were considered to be positive7. The frequency of dogs with a positive serological reaction was calculated; variables were stored in an electronic worksheet (EXCEL 2007, Microsoft®, São Paulo, São Paulo, Brazil) and analyzed using Fisher’s exact test (BioEstat, version 5.0, Instituto Mamirauá, Belém, Pará, Brazil).
For data analysis, the animals were grouped as dogs with absence of CVL clinical signs (asymptomatic dogs), and dogs presenting clinical manifestations (symptomatic and oligosymptomatic). Asymptomatic dogs accounted for 19.2% of the present study, while symptomatic and oligosymptomatic dogs together accounted for 80.8%. Among these dogs, the most common clinical abnormality was onychogryphosis (53.8%), followed by poor physical condition and exfoliative dermatitis (50.0%); dehydration and skin lesions (42.3%); hypertrophy of the lymph nodes (38.5%); pallor of mucous membranes (30.8%); and hepatosplenomegaly (23.1%).
Of the 26 CVL-positive dogs, two (7.7%) presented a serological reaction to R. rickettsii, at a titer of 1:64. Although no immunoglobulin G (IgG) antibodies against R. parkeri were detected, it has been already demonstrated that there is an association between the seroreactivity for these two antigens by IFA and it is therefore not possible to identify the species involved4.
No ticks were found on the animals at the time of sampling, although tick ectoparasitism had been consistently reported by the dog owners. Of the two animals in which anti-R. rickettsii IgG antibodies were detected, one was asymptomatic at the time of sampling, without obvious signs of rickettsiosis or CVL, and the other was oligosymptomatic, presenting nonspecific clinical signs (pallor of mucous membranes and poor physical condition). There was no significant difference (p = 1.000) between dogs seroreactive for R. rickettsii and the presence of clinical signs for CVL (asymptomatic and symptomatic/oligosymptomatic).
The most common symptoms in the present study were also reported by other authors10,11, which emphasizes that skin lesions, onychogryphosis, and lymphadenopathy, for example, are promptly recognized in cases of CVL and are typical signs of disease in endemic regions. The presence of hepatosplenomegaly in 23.1% of the dogs is in agreement with studies that showed spleen injury in dogs with CVL11. Ultimately, we cannot rule out the fact that the clinical signs observed in the present study may also be related to other diseases of dogs, which unfortunately were not investigated.
Given that CVL is a severe, debilitating disease for dogs, we expected that dogs testing positive for Leishmaniaspp. would also present a serological response to other diseases. However, in our study, the proportion of dogs with anti-spotted fever group (SFG) rickettsiae antibodies was low. Similarly, Souza and Almeida12 found a rate of co-infection between CVL and canine monocytic ehrlichiosis of only 2.5%.
Even though this was a limited study, we suggest that living in an area endemic for BSF is the most important determinant of seropositivity for SFG rickettsiae. One recent study showed that R. rickettsii antibody titers tend to be very high among dogs living in an area where there has been a recent outbreak of human BSF13, which was not the case in the present study.
To our knowledge, the present study is the first to detect a serological reaction to R. rickettsii and Leishmaniaspp. simultaneously among dogs in Barra Mansa. Interestingly, in addition to the expansion and urbanization of Leishmania spp.2, epidemiological data also suggest urbanization of SFG-Rickettsia spp.; this has been reported previously in a study that did not observe any significant difference between the frequency of anti-Rickettsia spp. antibodies in regions with different degrees of environmental preservation4. It is noteworthy that for both CVL and rickettsiosis, asymptomatic dogs may be important links in the epidemiological chain, as they are highly competent in establishing the infection of the vectors14.
CVL and BSF are both vector-borne diseases; although these vectors are distinct on a first analysis, it has been proposed that R. sanguineus might be involved in the epidemiology of CVL15, which is supported by the persistence of L. infantum kinetoplast DNA in unfed nymphs and adults of R. sanguineus and in the association between infestation by this tick and the anti-Leishmania antibodies in dogs. In the present study, we suggest that CVL and BSF are probably diseases with peculiar characteristics. It is likely that there is no epidemiological overlap between the two, and the diagnosis of one has no influence on the risk of the other.
We believe that the municipality of Barra Mansa has low circulation of antibodies to R. rickettsii. Even so, we reinforce the concept that dogs serve as sentinels for the presence of SFG rickettsiae and we recommend that serological studies in dogs should continue in order to monitor this scenario.
