Outbreak of canine visceral leishmaniasis in Barra Mansa , State of Rio de Janeiro Cintia

Introduction: In Brazil, visceral leishmaniasis (VL) has spread to various regions. This study reports canine cases of VL in Barra Mansa, where human VL cases were recently reported. Methods: Using the human index case, a canine survey was performed by dual-path platform immunochromatography and enzyme-linked immunosorbent assay. Seropositive animals were euthanized. Cultures were collected to detect Leishmania parasites. Results: Serological tests detected 141 canine VL cases, and Leishmania chagasi were isolated from 82.2% animals. Conclusions: Leishmania chagasi is in circulation in Barra Mansa. This study broadens information on the parasite’s distribution in the State of Rio de Janeiro.

Visceral leishmaniasis (VL) is of serious public health importance, and affects approximately 1-2 million people annually around the world 1 .Autochthonous transmission has been recorded in nearly all Brazilian states.The domestic dog is an important reservoir of infection, therefore constituting a primary target for disease control 2 .
Human cases are rare in the State of Rio de Janeiro, although canine cases have often been recorded over the years in municipalities such as Angra dos Reis, Mangaratiba 3 , Maricá 4 , and Volta Redonda 5 , and in different urban areas of the capital 3,6 .Canine VL cases historically precede spatial and temporal human cases 7 ; therefore, the geographical expansion of canine cases in the State of Rio de Janeiro has put epidemiological surveillance authorities on alert 8 .
Barra Mansa is a municipality in the Southern region of the state where nine human cases of VL were recently reported 9 .Confi rmation of these cases triggered a series of actions in Barra Mansa, in particular, the initiation of a canine survey directed at tracking the occurrence and extent of VL among domestic dogs.In this paper, our focus was to report the initial results of the canine survey in Barra Mansa during the years 2011-2013 and to identify Leishmania species in seroreactive animals.During the study period, 3,103 dogs were evaluated.Of these, 141 (4.5%) dogs were seropositive and hence euthanized.Promastigote forms were isolated from different sites in 116 (82.2%) euthanized animals (Table 1).Twenty-fi ve euthanized dogs were culturenegative.Among these, 15 dogs were asymptomatic, nine dogs were oligosymptomatic, and one dog was symptomatic.
At least one sample of the isolated organisms from each animal was processed by multilocus enzyme electrophoresis.Leishmania chagasi was confi rmed in 100% (n = 116) of dogs.
The expansion of VL has been reported in various regions of Brazil, and the results of this study corroborate this.In 1977, in the State of Rio de Janeiro, the fi rst human case of VL occurred in the municipality of Rio de Janeiro.From this case, the protocol of the canine survey was offi cially established in 1980 in this state 6 .At that time and in subsequent years, this survey was performed only in certain areas of the City of Rio de Janeiro.However, it has recently been extended to other regions of the state such as Barra Mansa because of the discovery of human and/or canine cases in these regions.The displacement of infected animals and the high adaptability of the vector have been the main reasons for the dispersal of VL in Brazil, and the State of Rio de Janeiro is no exception to this rule.Barra Mansa is bordered by the municipalities of Resende and Volta Redonda where VL cases have also recently been described 5 .
In this respect, a point that should be emphasized is the need for epidemiological surveillance in regions with favorable conditions for the establishment of VL.In Barra Mansa, the presence of L. chagasi has been confi rmed, which places the region on alert.
Canine seropositivity may vary, depending on the epidemiological characteristics of the region.In Belo Horizonte, is approximately 7% which is an area of intense transmission 11 .In Barra Mansa, we reported a prevalence of 4.5%.It is interesting that by 2009 canine leishmaniasis seropositivity had already been reported at levels that reached 10% in Barra Mansa 12 .At that time, L. chagasi was not detected in the dogs studied; however, one dog was infected with Leishmania braziliensis.This suggests that canine visceral leishmaniasis (CVL) was perhaps already spreading silently in Barra Mansa, and led to an explosion in the number of cases such as those reported in this paper.
The limitation of serological testing is a major argument against euthanasia of seropositive dogs 11 .In our study, L. chagasi was confirmed by culture in 82% of the 141 euthanized seropositive dogs.This speaks in favor of the routine -DPP followed by enzyme-linked immunosorbent assay that is currently employed for the diagnosis of CVL in Brazil 8 .In 25 seropositive dogs, parasitological confi rmation was not possible.This may be explained by the clinical condition of the animal at the time of euthanasia because 15 (60%) dogs were classifi ed as asymptomatic animals.Studies have shown that the chance of parasite isolation is less in asymptomatic dogs than in symptomatic animals 13 .
Another point that should be emphasized is the need for laboratory confi rmation, primarily in areas with no record of VL.Human cases have been reported in Barra Mansa; however, this is the fi rst time that L. chagasi has been shown in VL cases in this region.
The culture is the standard reference tool for the diagnosis of leishmaniasis; however, this approach is not used in epidemiologic studies 14,15 .
The isolation of L. chagasi from the assessed dogs reinforces the involvement of this animal in the transmission cycle of VL in Barra Mansa.Despite reports of L. braziliensis in this region, this study did not detect it in any of the evaluated dogs.In the present study we evaluated a variety of clinical specimen, and the highest rates of positivity were in lymph node fragments, whereas interestingly the lowest rates were in lymph node aspirates.We believe this result may be because of variations in the volume of material collected by puncture.In recent years, Barra Mansa is a Brazilian municipality in the southern State of Rio de Janeiro.It is located at latitude 22°32′39″ S, longitude 44°10′17″ W, altitude 381m, and has an area of 547,226km².Bio Manguinhos/FIOCRUZ/Rio de Janeiro, Brazil] at the Central Laboratory Noel Nutels [Laboratório Central (LACEN), Rio de Janeiro, Brazil], as recommended by the Ministry of Health 8 .All animals confi rmed as seropositive by enzymelinked immunosorbent assay (ELISA) were collected and sent for euthanasia at the Laboratório de Pesquisa Clínica em Dermatozoonoses/FIOCRUZ, Rio de Janeiro, Brazil.Euthanasia followed the technical norms defi ned by Resolution no 714/02 from the Federal Council of Veterinary Medicine and by the screening-culling procedure adopted by the Brazilian Ministry of Health in its Program for Visceral Leishmaniasis Control 2 .
Before euthanasia, the animals were sedated with ketamine (10mg/kg) mixed with acepromazine (0.2mg/kg) and clinically classifi ed as asymptomatic (i.e., dogs without clinical signs Mello CX et al. -Canine visceral leishmaniasis in Barra Mansa

TABLE 1 -Results of parasitological cultures using different clinical specimens from euthanized seropositive dogs.
3L upon physical examination), oligosymptomatic (i.e., dogs with 1-3 clinical signs), and symptomatic (i.e., dogs that had >3 clinical signs of VL such as emaciation, skin lesions, lethargy, lymph node enlargement, or signs of hepatomegaly or splenomegaly).Euthanasia was performed with an overdose of sodium thiopental 5%.Tissue samples (from liver, spleen, and intact skin fragments) and bone marrow and lymph node aspirates were collected and processed by culture for the isolation of parasites using the protocol described by Madeira et al.3.Tissue samples wer e fi rst placed in sterile saline solution with antibiotics and an antifungal, and later transferred to a CI95%: 95% confi dence interval.ofLeishmania braziliensis (MHOM/BR/75/M2903) and Leishmania chagasi (MHOM/BR/74/PP75) were the reference samples in all experiments.