The study area was chosen with care. Fumacê, Anjo da Guarda and Mauro Fecury lie in the Itaqui-Bacanga region of São Luís, a zone of residential districts interwoven with port and industrial activity, limited sanitation infrastructure and restricted sewage coverage. The municipality has a tropical humid climate with marked seasonal rainfall and a long history of visceral leishmaniasis endemicity. According to the Brazilian Notifiable Diseases Information System, 205 confirmed human cases of visceral leishmaniasis were reported among São Luís residents between 2020 and 2025, underscoring that transmission remains active. Previous entomological work in the metropolitan region has documented Lutzomyia longipalpis and Lutzomyia whitmani among the most abundant sand fly species, together with Lutzomyia flaviscutellata, the classical vector associated with L. amazonensis transmission. This combination of competent vectors, environmental degradation and dense human-animal contact makes the region an ideal natural laboratory for asking whether more than one Leishmania species circulates among domestic animals.

Between July and October 2020, the team recruited households door-to-door with the help of local Community Health Agents, sampling 110 dogs in each of the three neighborhoods for a total of 330 animals, alongside 21 cats. The canine sample size was calculated with Epi Info using a conservative expected prevalence of 50 percent, a 95 percent confidence level and 5 percent precision, based on an estimated local dog population of roughly 2,035 animals. Feline sampling was exploratory, constrained by the lower availability of cats and by owner concerns about restraint and blood collection. Peripheral blood was drawn by cephalic or jugular venipuncture; serum was frozen at minus 20 degrees Celsius for serology, while whole blood aliquots were preserved in ethanol in the field for molecular work. Owners also completed structured questionnaires covering animal age, sex, clinical signs, street access, peridomiciliary conditions, the presence of wild animals around the household and the use of preventive measures such as insecticide-impregnated collars.

Serological testing used the indirect immunofluorescence antibody test, or IFAT, with in vitro-cultured promastigotes of both L. infantum and L. amazonensis as antigens and a standardized cutoff dilution of 1:80 for both, in line with Brazilian Ministry of Health guidance. The results were striking. Among dogs, 125 of 330 animals, or 37.9 percent, were seroreactive to the L. amazonensis antigen, while 76, or 23.0 percent, reacted to L. infantum. Breaking the overlap down, 75 dogs reacted only to L. amazonensis, 26 only to L. infantum, and 50 to both antigens. Antibody titers ranged from 1:80 up to 1:5,120. In cats, the corresponding figures were 28.6 percent seroreactive to L. amazonensis and 14.3 percent to L. infantum, with titers between 1:80 and 1:160. Clinical signs compatible with canine leishmaniasis, including cutaneous ulcers, diffuse alopecia, onychogryphosis, cachexia, hepatosplenomegaly and lymphadenomegaly, were recorded in 16 of the 76 dogs seroreactive to L. infantum antigen, and one seroreactive cat showed cutaneous lesions.

Molecular detection told a complementary but more sobering story. Using primers targeting the internal transcribed spacer 1 region, or ITS1, of the parasite genome, the researchers amplified Leishmania DNA in 58 of 330 dogs, or 17.6 percent, and in 2 of 21 cats, or 9.5 percent. To identify species, all PCR-positive products were digested with the restriction enzyme HaeIII and compared against reference profiles from L. infantum and L. amazonensis controls obtained from the Brazilian Trypanosomatid Collection. Only nine of the 58 canine samples yielded restriction profiles clear enough for reliable species assignment. Of these, eight showed profiles characteristic of L. infantum and one displayed a mixed pattern suggesting the presence of genomic fragments from both L. infantum and L. amazonensis. Both PCR-positive cats were characterized as L. infantum, and notably, both were seronegative by IFAT for either antigen. The remaining 49 PCR-positive canine samples produced absent, weak or poorly defined restriction profiles and could not be assigned to any species.

The integration of serological and molecular data revealed substantial discordance. Among the 58 PCR-positive dogs, only 14 were also seroreactive to L. infantum antigen and 20 to L. amazonensis antigen, including eight reacting to both, while 32, or 55.2 percent, were seronegative on both tests. Such PCR-positive but seronegative animals may reflect differences in infection stage, immune response or sample type, a pattern reported previously in endemic regions. Conversely, seroreactivity alone cannot be taken as proof of active or species-specific infection, particularly where multiple Leishmania species coexist and antibodies may cross-react between related species. The authors emphasize that the higher seroreactivity to L. amazonensis antigen, although noteworthy in an area traditionally recognized as endemic for visceral leishmaniasis, must be interpreted cautiously precisely because of this potential cross-reactivity and the limited success of species-level molecular characterization.

Univariable analysis identified distinct risk profiles for the two antigens. Seroreactivity to L. infantum was significantly associated with age greater than one year, with an odds ratio of 2.27, male sex, with an odds ratio of 1.81, and the presence of clinical signs compatible with canine leishmaniasis, with an odds ratio of 2.87. Seroreactivity to L. amazonensis antigen was associated with age greater than one year, male sex, access to the street and the reported presence of wild animals around the household. These patterns are biologically plausible: adult animals accumulate vector exposure over time, roaming behavior increases opportunities for contact with infected sand flies, and peridomestic environments hosting animals such as house mice and opossums may help maintain vector populations. The associations, however, are exploratory, since no multivariable adjustment was performed and potential confounding among factors cannot be excluded.

The vector biology adds another layer of complexity. Lutzomyia longipalpis, the principal vector of L. infantum in the Americas, is considered a permissive sand fly, capable of supporting the development of several Leishmania species, whereas selective vectors exhibit more restricted parasite compatibility. Although L. amazonensis is classically associated with Lutzomyia flaviscutellata, its DNA has been detected in naturally infected Lu. longipalpis in northeastern Brazil, hinting that vector-parasite associations may be looser than a strict one-vector, one-parasite correspondence. The authors caution, however, that permissiveness under experimental conditions or the detection of parasite DNA in field-caught sand flies does not establish a role in natural transmission, and because no entomological survey accompanied this study, the animal findings cannot be attributed to any particular vector species. Environmental conditions in the Itaqui-Bacanga region, including poor sanitation and the reported presence of synanthropic animals around houses, are nonetheless consistent with conditions favoring vector maintenance.

The feline results, though limited by the small sample of 21 cats, confirm that infection occurs in cats in the area. Molecular detection of L. infantum in two seronegative cats aligns with previous reports that feline leishmaniasis is generally subclinical or associated with dermatological signs, and reports of L. amazonensis in cats remain rare in Brazil. Whether cats contribute meaningfully to transmission dynamics remains unresolved, and the authors are explicit that their sample size precludes inference about the epidemiological role of cats. The study’s other limitations include the exclusive use of peripheral blood, which can carry low parasite loads and hamper molecular detection, the reduced success rate of PCR-RFLP species identification, and the absence of tissue sampling and entomological investigation, all of which may have constrained the detection of parasite diversity.

Taken together, the findings do not overturn the centrality of L. infantum in the visceral leishmaniasis cycle of São Luís, where the predominance of L. infantum among characterized samples fits the endemic context and the documented history of urban expansion of the disease, including human cases in the Anjo da Guarda neighborhood during earlier phases of urbanization. But the elevated seroreactivity to L. amazonensis antigen, combined with a single mixed PCR-RFLP profile in one dog, raises the possibility that Leishmania occurrence in domestic animals in this endemic area may not be restricted to a single species, even if that mixed profile alone does not confirm coinfection. The study’s broader message is methodological as much as epidemiological: combining serology with species-level molecular characterization is essential in settings where multiple Leishmania species may coexist, because relying on either approach alone risks misreading the true diversity of parasites circulating among the animals that share our cities.

Subject of Research: Serological and molecular detection of Leishmania species in domestic dogs and cats in a visceral leishmaniasis-endemic urban area of northeastern Brazil

Article Title: Looking Beyond Leishmania infantum: Serological and Molecular Investigation of Leishmania spp. in Domestic Dogs and Cats from a Visceral Leishmaniasis-Endemic Area in Northeastern Brazil

Article References: Rocha, A. V. V. O., Costa, T. F., de Oliveira, R. M., Jordão, F. T., Speranca, M. A., Laurenti, M. D., Cabral, A. D., Costa, F. B., de Maria Seabra Nogueira, R., & da Costa, A. P. (2026). Looking Beyond Leishmania infantum: Serological and Molecular Investigation of Leishmania spp. in Domestic Dogs and Cats from a Visceral Leishmaniasis-Endemic Area in Northeastern Brazil. Acta Parasitologica, 71(5), Article 226. https://doi.org/10.1007/s11686-026-01408-9

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01408-9

Keywords: Leishmania infantum, Leishmania amazonensis, visceral leishmaniasis, canine leishmaniasis, feline leishmaniasis, IFAT, ITS1 PCR-RFLP, sand flies, Lutzomyia longipalpis, São Luís, Brazil, zoonosis