兽研引言
内脏利什曼病（VL）是一种严重的人兽共患疾病，在巴西具有重大公共卫生意义，其特征为慢性进展，若不治疗可能导致致命后果。历史上，VL与农村和森林环境相关，但自二十世纪下半叶以来，由于环境改变、无序城市扩张、人口迁移以及媒介对人为改造区域的适应，VL经历了剧烈的城市化进程1,2。
犬在内脏利什曼病的传播循环中发挥核心作用，是利什曼原虫（L. infantum）的主要城市储存宿主。犬类感染常先于人类病例出现，使犬成为流行病学监测的重要哨兵指示动物。因此，即使没有确诊人类病例，有犬类感染证据的地区也应被视为监测和控制行动的优先区域，尤其是在被归类为新发或静默传播区的地区3,4。
在巴西，VL传播呈现明显的区域异质性，受影响媒介分布和丰度的生态与环境因素影响。在亚马逊地区，森林砍伐、城市扩张和栖息地破碎化等过程加剧了媒介、储存宿主和人类群体之间的相互作用，促成了新的传播场景的出现5-9。
利什曼原虫的传播动态涉及媒介、储存宿主、宿主和环境条件之间的复杂相互作用，尤其是在经历快速生态转型的地区10。这些过程有利于在城市增长和环境扰动影响的地区建立和维持传播循环11,12。
尽管朗多尼亚州历史上被认为无本土内脏利什曼病传播，但近期研究已证明利什曼原虫（L. infantum）在吉帕拉纳和卡科阿尔等市的犬中流行，此外在城市和城郊环境中存在 competent 媒介1,13,14。
此外，波多韦柳报告的犬内脏利什曼病病例涉及无流行区旅行史的动物，提示可能存在本地传播14。综合来看，这些发现表明该地区的流行病学脆弱性增加，并可能出现新的传播疫点。
尽管证据日益增多，但整合朗多尼亚内陆小城市犬类感染、媒介出现和当地环境条件数据的研究仍然匮乏。这一空白限制了对可能被定性为静默传播区中内脏利什曼病传播早期建立的理解13,15。
因此，本研究旨在采用整合的血清学、分子学、血液学和昆虫学方法，评估朗多尼亚州蒙特内格罗市犬内脏利什曼病的流行病学状况。
伦理
所有涉及动物的程序均按照巴西国家动物实验控制委员会（CONCEA）制定的现行动物研究伦理标准执行。本研究经圣保罗大学生物医学科学研究所动物使用伦理委员会（CEUA-ICB/USP）批准，方案编号1290291123（ID 053138），修正案于2025年5月13日批准。
材料与方法
研究区域
本研究在巴西朗多尼亚州蒙特内格罗市（南纬10°15'37.8"，西经63°17'6.3"）开展。根据巴西地理与统计研究所（IBGE）的估计，该市估计人口为12,168人，其中约5,520人居住在城市地区16。本研究共纳入450只家养犬和流浪犬，在社区1、2、3、4和贝拉维斯塔区进行采样，这些区域是该市城市区的代表性区域。数据收集时间为2024年2月至2025年5月（图1）。
[图：2024-2025年采集犬血液样本的蒙特内格罗邻近地区。]
犬类采样与血清学
共从朗多尼亚州蒙特内格罗市不同城市区的家养犬和流浪犬采集450份血液样本。为进行空间流行病学分析，根据现场采样时采用的当地地域划分，将该市城市区按区域分层。样本采集通过颈静脉和/或头静脉穿刺进行，每只动物采集约5 mL血液，分别置于含EDTA的试管和普通试管中。
根据巴西卫生部的技术指南，估计犬与人的比例约为1：6.6；因此，蒙特内格罗的城市犬群估计约为1,840只。因此，本研究评估的样本约占估计城市犬群的24.4%，根据巴西卫生部的方案，这在流行病学意义上代表了相当大的覆盖率17。
采集于普通管中的样本以5，000 rpm离心10分钟以分离血清，血清分装为三份，置于无DNA的PVC管中于−20 °C保存。第一份用于按照制造商推荐进行TR-DPP®快速血清学检测（Bio-Manguinhos/FIOCRUZ）。
对于采集于含抗凝剂管中的血液样本，使用IDEXX®分析仪进行自动化全血细胞计数。在玻璃载玻片上制备血涂片，吉姆萨染色，并在光学显微镜（Zeiss®）下以1，000倍放大（油镜）检查以检测血液寄生虫。根据既定诊断标准，基于形态学特征（包括红细胞内型和白细胞包涵体）鉴定巴贝斯虫属、犬埃立克体及其他血液寄生虫。在入户访视期间，动物主人或照护者填写流行病学问卷，内容包括家庭特征、与林地的接近程度、家族或犬内脏利什曼病病史、年龄、性别、疫苗接种状态以及与疾病相符的临床症状的存在情况。在样本采集时，已获得动物主人或监护人的知情同意。
分子分析
对TR-DPP®免疫层析检测反应性的犬只血液样本随后通过酶联免疫吸附试验（ELISA）（EIE-LVC®，Bio-Manguinhos/FIOCRUZ）进行分析，按照制造商说明书进行。该试验用作确认性血清学检测，用于检测抗利什曼原虫属IgG抗体。
使用酶标仪测定光密度，值高于试剂盒所确立临界值的样本被视为阳性。
PCR分子分析在朗多尼亚州Oswaldo Cruz基金会（Fiocruz）昆虫学实验室进行。从保存于70%乙醇中的血凝块样本中提取基因组DNA，采用基于蛋白酶K消化和SDS介导裂解的方案，随后进行有机萃取和乙醇沉淀。简言之，将70-100 mg血凝块与蛋白酶K（20 mg/mL）和20% SDS在65 °C孵育1小时。通过氯仿萃取进行DNA纯化，随后用冷乙醇沉淀并随后洗涤。DNA沉淀重悬于含RNase的TE缓冲液中，于−30 °C保存直至使用。
鉴于样本数量庞大，DNA提取物被组织成最多含五个样本的混合池，共计103个混合池。采用该策略以优化大规模筛查；然而，它可能降低分析灵敏度，尤其是在寄生虫载量低的样本中。
利什曼原虫DNA的检测通过聚合酶链反应（PCR）进行，靶向Hsp70基因，使用引物HSP70-240F（5’-GGACGAGATCGAGCGCATGGT-3z’）和HSP70-240R（5’-TCCTTCGACGCCTCCTGGTTG-3’），扩增约240 bp的片段，如Graça等18所述。PCR反应在25 μL的终体积中进行，含12.5 μL GoTaq Green Master Mix（Promega）、各1.0 μL引物、5.5 μL超纯水和5 μL模板DNA。
扩增在热循环仪中按以下条件进行：94 °C初始变性5分钟；随后35个循环的94 °C变性30秒、61 °C退火30秒和72 °C延伸45秒；最后72 °C终延伸10分钟。
以利什曼原虫（L. infantum）基因组DNA作为阳性对照，超纯水作为阴性对照。为评估潜在的PCR抑制，在一部分样本中使用内部扩增对照，降低了假阴性结果的可能性。
PCR产物通过溴化乙锭染色的琼脂糖凝胶电泳分析，并在紫外光下观察。
昆虫学研究
昆虫学采集在两种主要类型的环境中进行：户周环境，定义为住宅的紧邻周围，包括后院、家畜棚舍和繁殖区；以及林区，对应于与住户相邻的森林片段。该分类允许比较白蛉在家庭环境和附近森林环境中的发生情况。
使用CDC光诱捕器捕获白蛉。诱捕器连续运行72小时，在每次月度采样活动中连续运行三天，最好在新月期间，置于距地面1.5米高度，表征整个研究期间标准化的采样努力。
采集后，昆虫被送往位于Monte Negro市的ICB-5/USP朗多尼亚单位，在那里在Zeiss®体视显微镜下以40倍放大进行初步筛查和初步鉴定，旨在将白蛉与其他节肢动物分开。随后，标本单独置于含无水乙醇的瓶中，并冷藏保存以防止酒精蒸发。
随后，昆虫学材料被送往位于亚马孙州马瑙斯的ILMD/FIOCRUZ亚马孙分校传播性疾病生态学实验室，在那里进行了物种分类鉴定和性别判定。捕获的白蛉分类鉴定基于外部形态特征和生殖器结构。标本事先经透明处理并 mounted 于显微镜载玻片上，以便观察雄虫末端、雌虫受精囊、食窦、咽和翅脉等特征，遵循医学和兽医昆虫学广泛使用的标准。物种判定使用专业分类检索表，包括Galati及其合作者描述的形态学方法以及先前建立的新热带白蛉亚科图解检索表，这些检索表涵盖了雄虫和雌虫的主要鉴别特征19。
血清阳性犬主人的随访
在拥有血清阳性犬（TR-DPP®）的家庭中，居民接受了检查，并按照巴西卫生部制定的方案受邀进行血清学筛查17。所有个体均未出现血清学阳性或与人间内脏利什曼病相符的体征或症状。
结果
在检查的血液样本中，70份表现为血小板减少，7份嗜酸性粒细胞增多，55份贫血。此外，检测到3例梨形虫属感染、1例无浆体属感染、26例犬埃里希体感染和5例肝簇虫属感染（表1）。
表1. 巴西朗多尼亚州蒙特内格罗市犬的血液学改变，2024-2025年。
在朗多尼亚州蒙特内格罗市城区共评估了450只犬。在本地命名为第2、3和4区的街区中发现了血清反应阳性犬，并对血清学结果进行了分层分析。
在检查的犬中，45只（10.0%）在TR-DPP®免疫层析试验中检测为反应性。这些样本随后通过ELISA分析，其中34只（75.6%）抗利什曼原虫属抗体阳性。第2区呈现最多的TR-DPP®反应性动物（n=25），其中22只经ELISA确认。第3区有11只TR-DPP®反应性犬，均经ELISA确认，而第4区有10只TR-DPP®反应性犬，其中1只经ELISA确认（表2）。
表2. 巴西朗多尼亚州蒙特内格罗市按城区划分的血清反应阳性犬分布。
在所有分析的样本中均未观察到预期Hsp70基因片段的扩增。
蒙特内格罗市记录的白蛉区系由六个物种组成：Psychodopygus davisi、Nyssomyia antunesi、Migonemyia migonei、Evandromyia piperiformis、Brumptomyia pintoi和Evandromyia saulensis，包括5只雄性和11只雌性（表3）。Nyssomyia antunesi为最常见物种，占捕获白蛉的50.0%。
表3. 2024年2月至2025年4月在巴西朗多尼亚州蒙特内格罗市捕获的白蛉物种。
在各采样环境中，大多数白蛉采集于住区周围区域，占总数81.3%（13/16），而森林区域占18.7%（3/16）。Nyssomyia antunesi主要采集于住区周围环境（75.0%；6/8），其余标本鉴定于森林区域（25.0%；2/8）。
讨论
本研究结果应在巴西内脏利什曼病的流行病学背景下解读，其特征为显著的空间异质性以及储存宿主、媒介和环境因素之间复杂的相互作用。自20世纪70年代以来，内脏利什曼病的城市化已被广泛记录，并与无计划的城市扩张、移民流动加剧、环境变化和自然生态屏障减少相关，促进了以前被认为无病地区传播疫源地的出现20。
鉴定出45只TR-DPP®试验反应性犬，相当于评估群体的10%，提示既往暴露于利什曼原虫属抗原。然而，也应考虑与巴西犬中常见的其他病原体的血清学交叉反应，尤其是血液寄生虫如巴贝斯虫属和犬埃里希体，因为这些病原体可能诱导非特异性免疫反应21,22。在本研究中，贫血和血小板减少等血液学改变，连同血涂片中巴贝斯虫属的鉴定，强化了这种可能性22。
犬感染已被认为是寄生虫循环的重要哨兵标志，通常先于人间病例的发生，尤其是在新发或初期传播地区3,4。在巴西不同地区进行的研究报告，在疾病扩散早期、家庭传播循环巩固和人间病例发生之前，犬血清阳性率范围为5%至15%23。
TR-DPP®与ELISA检测之间观察到的高度一致性，在反应性犬中有34份样本经ELISA确认，强化了既往暴露于利什曼原虫属抗原的证据。既往研究报告，在犬内脏利什曼病监测中联合使用TR-DPP®和ELISA的敏感性和特异性均高于90% 24。然而，所有分析样本均未通过PCR进行分子检测，表明仅血清阳性不应被解释为活动性感染的确定性证据 21。
在新发传播地区开展的研究中也报告了类似发现，当以外周血作为生物样本时，血清阳性犬常呈现PCR阴性结果 23。这一发现可能与亚临床或慢性感染中的低寄生虫血症有关，也与寄生虫在组织中的不均匀分布有关。在犬内脏利什曼病中，脾、淋巴结和骨髓等组织通常对分子检测表现出更高的敏感性 18。
此外，本研究采用的方法学策略涉及最多包含五个个体的混合样本，可能降低了PCR的分析敏感性，尤其是在寄生虫负荷低的样本中。既往研究表明，由于目标DNA的稀释，混合样本可能降低PCR检测的分析敏感性，尤其是当病原体载量低时，增加了亚临床感染中出现假阴性结果的可能性 25。
选择Hsp70基因作为分子靶标，是因为其适用于利什曼原虫种的检测和鉴定。然而，与kDNA微环等高拷贝靶标相比，该标记的分析敏感性较低，尤其是在分析外周血样本时。因此，未出现扩增并不能完全排除寄生虫在该市的循环，而只是提示在所采用的分析条件下未检测到感染 18, 25。
在区域背景下，尽管朗多尼亚州历史上被认为是无犬内脏利什曼病本地传播的地区，但近期证据表明该病在该州呈地理扩张趋势。Porto Velho、Ji-Parana和Cacoal等市报告的本地犬病例，加上在城市和城郊地区发现 competent vectors，表明流行病学脆弱性过程正在持续 1, 13, 14。
本研究中鉴定的白蛉区系组成进一步支持了这一情况。在巴西不同地区开展的研究表明，即使没有高密度的Lutzomyia longipalpis，Migonemyia migonei和Nyssomyia antunesi等物种也可能在人类改造环境中作为潜在或次要媒介 26, 27。近期在Porto Velho开展的研究在雌性Ny. antunesi中检测到L. infantum DNA，提示其可能参与当地传播循环的维持 17。
尽管本研究中捕获的白蛉标本数量有限，可能是由于环境和操作条件所致，但这些物种的存在表明昆虫学背景与潜在传播风险相符 10, 14, 26。
从One Health角度看，犬血清反应性、具有流行病学意义的白蛉存在以及环境改变相结合，可能促成有利于内脏利什曼病出现的条件。综合性研究表明，家庭传播循环的建立可能是逐渐发生的，并且可能先经历犬群中沉默或亚临床循环的时期 11。因此，即使没有分子确认，鉴定出与潜在媒介存在相关的血清反应性犬也可能构成需要持续监测的流行病学警示信号 3, 13, 17, 23。
因此，这些发现强化了在Monte Negro市开展内脏利什曼病综合监测的重要性，包括对犬进行血清学和分子监测、持续昆虫学监测以及针对当地居民的健康教育活动，正如巴西卫生部所建议的那样 17。建议未来开展更大规模昆虫学采样工作并使用对分子检测更敏感的组织样本的研究，以更好地描述当地传播循环的潜在建立情况。
结论
本研究结果表明，在所评估期间，朗多尼亚州Monte Negro市没有证据显示利什曼原虫（L. infantum）存在 active transmission。然而，在家周围环境和森林片段中检测到血清阳性犬以及作为潜在媒介的白蛉存在，尽管丰度较低，仍表明存在潜在的流行病学风险，提示西亚马逊地区可能出现内脏利什曼病本地疫源地。
在此背景下，建议加强持续监测策略，包括对犬进行血清学和分子学监测、系统性昆虫学监测以及针对当地居民的健康教育活动。此外，当地卫生当局与研究机构之间的协调对于防止疾病传播的建立和保护脆弱的亚马逊人群至关重要。
查看英文原文▾
INTRODUCTION
Visceral leishmaniasis (VL) is a severe zoonotic disease of major public health importance in Brazil, characterized by chronic progression and potentially fatal outcomes when left untreated. Historically associated with rural and sylvatic environments, VL has undergone an intense urbanization process since the second half of the twentieth century, driven by environmental modifications, unplanned urban expansion, population migration, and the adaptation of vectors to anthropized areas 1 , 2 .
Dogs play a central role in the transmission cycle of visceral leishmaniasis, acting as the main urban reservoirs of Leishmania infantum. Canine infection frequently precedes human cases, making dogs important sentinel indicators for epidemiological surveillance. Consequently, areas with evidence of canine infection, even in the absence of confirmed human cases, should be considered priorities for surveillance and control actions, especially in regions classified as emerging or silent transmission areas 3 , 4 .
In Brazil, VL transmission exhibits marked regional heterogeneity influenced by ecological and environmental factors affecting vector distribution and abundance. In the Amazon region, processes such as deforestation, urban expansion, and habitat fragmentation have intensified interactions among vectors, reservoirs, and human populations, contributing to the emergence of new transmission scenarios 5 - 9 .
The transmission dynamics of Leishmania involve complex interactions among vectors, reservoirs, hosts, and environmental conditions, particularly in regions undergoing rapid ecological transformation 10 . These processes favor the establishment and maintenance of transmission cycles in areas affected by urban growth and environmental disturbance 11 , 12 .
Although Rondonia State has historically been considered free of autochthonous visceral leishmaniasis transmission, recent studies have demonstrated the circulation of Leishmania infantum in dogs from municipalities such as Ji-Parana and Cacoal, in addition to the presence of competent vectors in urban and peri-urban environments 1 , 13 , 14 .
Furthermore, reports of canine visceral leishmaniasis in Porto Velho involving animals without a history of travel to endemic areas suggest the possibility of local transmission 14 . Together, these findings indicate increasing epidemiological vulnerability and the potential emergence of new transmission foci in the region.
Despite this growing evidence, studies integrating data on canine infection, vector occurrence, and local environmental conditions in small municipalities of inland Rondonia remain scarce. This gap limits the understanding of the early establishment of visceral leishmaniasis transmission in areas that may be characterized as silent transmission zones 13 , 15 .
Therefore, this study aims to evaluate the epidemiological scenario of canine visceral leishmaniasis in Monte Negro municipality, Rondonia, using integrated serological, molecular, hematological, and entomological approaches.
Ethics
All procedures involving animals were conducted in accordance with current ethical standards for the use of animals in research, as established by the Brazilian National Council for the Control of Animal Experimentation (CONCEA). The study was approved by the Ethics Committee on the Use of Animals of the Institute of Biomedical Sciences of the University of Sao Paulo (CEUA-ICB/USP), under protocol Nº 1290291123 (ID 053138), with an amendment approved on May 13, 2025.
MATERIALS AND METHODS
Study area
The study was conducted in Monte Negro municipality, located in Rondonia State, Brazil (10°15’37.8”S, 63°17’6.3”W). The municipality has an estimated population of 12,168 inhabitants, of whom approximately 5,520 live in the urban area, according to estimates from the Brazilian Institute of Geography and Statistics (IBGE) 16 . A total of 450 dogs, domiciled and stray, were included in the study and were sampled across neighborhood sectors 1, 2, 3, 4, and Bela Vista, which are representative areas of the urban zone of the municipality. Data were collected from February 2024 to May 2025 (Figure 1).
[图：Neighborhoods of Monte Negro where dog blood samples were collected, 2024-2025.]
Canine sampling and serology
A total of 450 blood samples were collected from domiciled and stray dogs in different urban sectors of Monte Negro municipality, Rondonia State. For spatial epidemiological analysis, the urban area of the municipality was stratified into sectors according to the local territorial designation adopted during field sampling. Sample collection was performed by jugular and/or cephalic vein puncture, yielding approximately 5 mL of blood per animal, which was placed into tubes containing EDTA and plain tubes.
Based on the technical guidelines of the Brazilian Ministry of Health, an approximate ratio of one dog per 6.6 inhabitants is estimated; therefore, the urban canine population of Monte Negro is estimated at approximately 1,840 dogs. Thus, the sample evaluated in the present study corresponded to approximately 24.4% of the estimated urban canine population, representing substantial coverage for epidemiological purposes, according to the protocol of the Brazilian Ministry of Health 17 .
Samples collected in plain tubes were centrifuged at 5,000 rpm for 10 min to separate the serum, which was aliquoted into three fractions and stored at −20 °C in DNA-free PVC tubes. The first aliquot was used to perform the TR-DPP® rapid serological test (Bio-Manguinhos/FIOCRUZ), according to the manufacturer’s recommendations.
For blood samples collected in tubes containing anticoagulant, automated complete blood counts were performed using an IDEXX® analyzer. Blood smears were prepared on glass slides, stained with Giemsa, and examined under optical microscopy (Zeiss®) at 1,000× magnification (under oil immersion) for the detection of hemoparasites. Identification of Babesia spp., Ehrlichia canis, and other hemoparasites was performed based on morphological characteristics, including intraerythrocytic forms and leukocyte inclusions, according to established diagnostic criteria. During household visits, animal owners or caretakers completed an epidemiological questionnaire containing information on household characteristics, proximity to wooded areas, family or canine history of visceral leishmaniasis, age, sex, vaccination status, and the presence of clinical signs compatible with the disease. At the time of sample collection, informed consent was obtained from the animal owner or guardian.
Molecular analyses
Blood samples from dogs reactive in the TR-DPP® immunochromatographic test were subsequently analyzed by enzyme-linked immunosorbent assay (ELISA) (EIE-LVC®, Bio-Manguinhos/FIOCRUZ), performed according to the manufacturer’s instructions. This assay was used as a confirmatory serological test for the detection of anti-Leishmania spp. IgG antibodies.
Optical density was measured using a microplate reader, and samples with values above the cutoff established by the kit were considered positive.
Molecular analysis by PCR was conducted at the Oswaldo Cruz Foundation (Fiocruz) Entomology Laboratory in Rondonia. Genomic DNA was extracted from blood clot samples preserved in 70% ethanol using a protocol based on proteinase K digestion and SDS-mediated lysis, followed by organic extraction and ethanol precipitation. Briefly, 70-100 mg of blood clot was incubated with proteinase K (20 mg/mL) and 20% SDS at 65 °C for 1 h. DNA purification was performed by chloroform extraction, followed by precipitation with cold ethanol and subsequent washing steps. The DNA pellet was resuspended in TE buffer containing RNase and stored at −30 °C until use.
Given the large number of samples, DNA extracts were organized into pools containing up to five samples, totaling 103 pools. This strategy was adopted to optimize large-scale screening; however, it may reduce analytical sensitivity, particularly in samples with low parasitic load.
Detection of Leishmania DNA was performed by polymerase chain reaction (PCR), targeting the Hsp70 gene, using the primers HSP70-240F (5’-GGACGAGATCGAGCGCATGGT-3z’) and HSP70-240R (5’-TCCTTCGACGCCTCCTGGTTG-3’), which amplify a fragment of approximately 240 bp, as described by Graça et al. 18 . PCR reactions were carried out in a final volume of 25 μL, containing 12.5 μL of GoTaq Green Master Mix (Promega), 1.0 μL of each primer, 5.5 μL of ultrapure water, and 5 μL of template DNA.
Amplification was performed in a thermocycler under the following conditions: initial denaturation at 94 °C for 5 min; followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 61 °C for 30 s, and extension at 72 °C for 45 s; with a final extension at 72 °C for 10 min.
Genomic DNA of Leishmania infantum was used as a positive control, and ultrapure water as a negative control. To assess potential PCR inhibition, internal amplification controls were used in a subset of samples, reducing the likelihood of false-negative results.
PCR products were analyzed by agarose gel electrophoresis stained with ethidium bromide and visualized under ultraviolet light.
Entomological study
Entomological collections were carried out in two main types of environments: the peridomicile, defined as the immediate surroundings of residences, including backyards, shelters for domestic animals, and breeding areas; and forested areas, corresponding to forest fragments adjacent to households. This categorization allowed comparison of phlebotomine occurrence in domestic and nearby sylvatic environments.
Phlebotomine sand flies were captured using CDC light traps. The traps remained active for 72 consecutive hours and were operated for three consecutive days during each monthly sampling campaign, preferably during new moon periods, at a height of 1.5 m above ground level, characterizing a standardized sampling effort throughout the study.
After collection, the insects were sent to the ICB-5/USP, Rondonia Unit, located in Monte Negro municipality, where initial screening and preliminary identification were performed under a Zeiss® stereomicroscope at 40× magnification, aiming to separate phlebotomines from other arthropods. Subsequently, specimens were individually placed in vials containing absolute ethanol and kept under refrigeration to prevent alcohol evaporation.
The entomological material was then sent to the Laboratory of Ecology of Transmissible Diseases in the Amazon at ILMD/FIOCRUZ Amazonia, in Manaus, Amazonas State, where taxonomic identification of species and sex determination were performed. Taxonomic identification of captured phlebotomine sand flies was based on external morphological characteristics and genital structures. Specimens were previously cleared and mounted on microscope slides to allow observation of characters such as male terminalia, female spermathecae, cibarium, pharynx, and wing venation, following criteria widely used in medical and veterinary entomology. Species determination was carried out using specialized taxonomic keys, including morphological approaches described by Galati and collaborators and previously established illustrated keys for Neotropical Phlebotominae, which encompass the main diagnostic characters of males and females 19 .
Follow-up of owners of seropositive dogs
In households with seropositive dogs (TR-DPP®), residents were examined and invited to undergo serological screening in accordance with protocols established by the Brazilian Ministry of Health 17 . None of the individuals tested positive by serology or presented signs or symptoms compatible with human visceral leishmaniasis.
RESULTS
Among the blood samples examined, 70 presented thrombocytopenia, seven eosinophilia, and 55 anemia. In addition, three cases of infection by Piroplasma spp., one case by Anaplasma spp., 26 cases by Ehrlichia canis, and five cases by Hepatozoon spp. were detected (Table 1).
Table 1. Hematological alterations in dogs from Monte Negro municipality, Rondonia State, Brazil, 2024-2025.
| Parameter | Number of cases | Percentage (%) |
|---|---|---|
| Total dogs evaluated | 450 | 100% |
| Thrombocytopenia | 70 | 15.6% |
| Eosinophilia | 7 | 1.6% |
| Anemia | 55 | 12.2% |
| Piroplasma spp. | 3 | 0.7% |
| Anaplasma spp. | 1 | 0.2% |
| Ehrlichia canis | 26 | 5.8% |
| Hepatozoon spp. | 5 | 1.1% |
| Total of hematological findings | 167 | 37.2% |
A total of 450 dogs were evaluated in urban areas of Monte Negro municipality, Rondonia State. Seroreactive dogs were identified in neighborhoods locally designated as Sectors 2, 3, and 4, where stratified analysis of serological results was performed.
Among the examined dogs, 45 (10.0%) tested reactive in the TR-DPP® immunochromatographic test. These samples were subsequently analyzed by ELISA, of which 34 (75.6%) were positive for anti-Leishmania spp. antibodies. Sector 2 presented the highest number of TR-DPP® reactive animals (n = 25), of which 22 were confirmed by ELISA. Sector 3 presented 11 TR-DPP® reactive dogs, all confirmed by ELISA, whereas Sector 4 presented 10 TR-DPP® reactive dogs, with one confirmed by ELISA (Table 2).
Table 2. Distribution of seroreactive dogs according to urban sectors in Monte Negro municipality, Rondonia State, Brazil.
| Sector designation | TR-DPP ® -reactive dogs (n) | ELISA-confirmed reactive dogs (n) |
|---|---|---|
| Sector 2 | 25 | 22 |
| Sector 3 | 10 | 11 |
| Sector 4 | 10 | 1 |
| Total | 45 | 34 |
No amplification of the expected Hsp70 gene fragment was observed in any of the analyzed samples.
The phlebotomine fauna recorded in Monte Negro municipality was composed of six species: Psychodopygus davisi, Nyssomyia antunesi, Migonemyia migonei, Evandromyia piperiformis, Brumptomyia pintoi, and Evandromyia saulensis, comprising five males and 11 females (Table 3). Nyssomyia antunesi was the most frequent species, accounting for 50.0% of the captured phlebotomines.
Table 3. Phlebotomine species captured in Monte Negro municipality, Rondonia State, Brazil, from February 2024 to April 2025.
| Species | Environment | Males | Females | Total | % |
|---|---|---|---|---|---|
| Psychodopygus davisi | Peridomestic | 0 | 1 | 1 | 6.3% |
| Forest | 1 | 0 | 1 | 6.3% | |
| Nyssomyia antunesi | Peridomestic | 2 | 4 | 6 | 37.5% |
| Forest | 0 | 2 | 2 | 12.5% | |
| Migonemyia migonei | Peridomestic | 0 | 1 | 1 | 6.3% |
| Evandromyia piperiformis | Peridomestic | 1 | 1 | 2 | 12.5% |
| Brumptomyia pintoi | Peridomestic | 1 | 1 | 2 | 12.5% |
| Evandromyia saulensis | Peridomestic | 0 | 1 | 1 | 6.3% |
| Total | - | 5 | 11 | 16 | 100% |
Across the sampled environments, most phlebotomines were collected in peridomestic areas, corresponding to 81.3% (13/16) of the total, while forest areas accounted for 18.7% (3/16). Nyssomyia antunesi was predominantly collected in peridomestic environments (75.0%; 6), with the remaining specimens identified in forest areas (25.0%; 2/8).
DISCUSSION
The results of this study should be interpreted in the epidemiological context of VL in Brazil, characterized by marked spatial heterogeneity and complex interactions among reservoirs, vectors, and environmental factors. Since the 1970s, the urbanization of VL has been widely documented and associated with unplanned urban expansion, intensification of migratory flows, environmental changes, and the reduction of natural ecological barriers, favoring the emergence of transmission foci in areas previously considered disease-free 20 .
The identification of 45 dogs reactive in the TR-DPP® test, corresponding to 10% of the evaluated population, suggests previous exposure to Leishmania spp. antigens. However, serological cross-reactions with other pathogens commonly found in dogs in Brazil, especially hemoparasites such as Babesia spp. and Ehrlichia canis, should also be considered, since these agents may induce nonspecific immune responses 21 , 22 . In this study, hematological alterations such as anemia and thrombocytopenia, together with the identification of Babesia spp. in blood smears, reinforce this possibility 22 .
Canine infection has been recognized as an important sentinel marker of parasite circulation, frequently preceding the occurrence of human cases, especially in areas of emerging or incipient transmission 3 , 4 . Studies conducted in different regions of Brazil have reported canine seroprevalence ranging from 5% to 15% during the early stages of disease expansion, prior to the consolidation of the domestic transmission cycle and the occurrence of human cases 23 .
The high concordance observed between the TR-DPP® and ELISA tests, with 34 ELISA-confirmed samples among the reactive dogs, reinforces evidence of previous exposure to Leishmania spp. antigens. Previous studies report sensitivity and specificity values above 90% for the combined use of TR-DPP® and ELISA in canine visceral leishmaniasis surveillance 24 . Nevertheless, the absence of molecular detection by PCR in all analyzed samples indicates that seropositivity alone should not be interpreted as definitive evidence of active infection 21 .
Similar findings have been reported in studies conducted in areas of emerging transmission, where seropositive dogs frequently present negative PCR results when peripheral blood is used as the biological sample 23 . This finding may be associated with low parasitemia in subclinical or chronic infections, as well as with the heterogeneous distribution of the parasite in tissues. In canine visceral leishmaniasis, tissues such as spleen, lymph nodes, and bone marrow generally present greater sensitivity for molecular detection 18 .
In addition, the methodological strategy adopted in this study, involving pooled samples containing up to five individuals, may have reduced the analytical sensitivity of PCR, particularly in samples with low parasite burden. Previous studies have demonstrated that pooling samples may reduce the analytical sensitivity of PCR assays due to dilution of target DNA, particularly when pathogen load is low, increasing the likelihood of false-negative results in subclinical infections 25 .
The Hsp70 gene was selected as the molecular target due to its applicability in the detection and characterization of Leishmania species. However, this marker presents lower analytical sensitivity when compared with high-copy targets such as kDNA minicircles, particularly when peripheral blood samples are analyzed. Therefore, the absence of amplification does not completely exclude parasite circulation in the municipality but rather suggests the absence of detectable infection under the analytical conditions employed 18 , 25 .
In the regional context, although Rondonia State has historically been considered an area without autochthonous transmission of canine visceral leishmaniasis, recent evidence indicates geographic expansion of the disease within the state. The reports of autochthonous canine cases in municipalities such as Porto Velho, Ji-Parana, and Cacoal, combined with the identification of competent vectors in urban and peri-urban areas, point to an ongoing process of epidemiological vulnerability 1 , 13 , 14 .
The composition of the phlebotomine fauna identified in this study reinforces this scenario. Studies conducted in different regions of Brazil shows that, even in the absence of high densities of Lutzomyia longipalpis, species such as Migonemyia migonei and Nyssomyia antunesi may act as potential or secondary vectors in anthropized environments 26 , 27 . Recent studies conducted in Porto Velho detected DNA of L. infantum in female Ny. antunesi, suggesting its possible involvement in the maintenance of the local transmission cycle 17 .
Although the number of phlebotomine specimens captured in this study was limited, possibly due to environmental and operational conditions, the presence of these species indicates an entomological context compatible with potential transmission risk 10 , 14 , 26 .
From a One Health perspective, the combination of canine seroreactivity, presence of phlebotomines with epidemiological importance, and environmental modifications may contribute to conditions favorable for the emergence of visceral leishmaniasis. Integrative studies show that the establishment of the domestic transmission cycle may occur gradually and can be preceded by periods of silent or subclinical circulation among canine populations 11 . Therefore, the identification of seroreactive dogs associated with the presence of potential vectors, even in the absence of molecular confirmation, may constitute an epidemiological warning sign requiring continuous surveillance 3 , 13 , 17 , 23 .
Thus, the findings reinforce the importance of integrated surveillance for visceral leishmaniasis in Monte Negro municipality, including serological and molecular monitoring in dogs, continuous entomological surveillance, and health education activities targeting the local population, as recommended by the Brazilian Ministry of Health 17 . Future studies using larger entomological sampling efforts and tissue samples with greater sensitivity for molecular detection are recommended to better characterize the potential establishment of the local transmission cycle.
CONCLUSION
The results of this study indicate the absence of evidence of active transmission of Leishmania infantum in Monte Negro municipality, Rondonia State, during the evaluated period. However, the detection of seropositive dogs and the presence of phlebotomines as potential vectors in peridomestic environments and forest fragments, despite their low abundance, indicate a potential epidemiological risk, suggesting the potential emergence of an autochthonous focus of visceral leishmaniasis in the Western Amazon.
In this context, strengthening continuous surveillance strategies is recommended, including serological and molecular monitoring of dogs, systematic entomological surveillance, and health education activities directed at the local population. In addition, coordination between local health authorities and research institutions is essential to prevent the establishment of disease transmission and protect vulnerable Amazonian populations.