兽研摘要
具有医学和兽医意义的媒介传播病原体（VBPs）大多在城市犬群中进行过研究，而农村环境中接触体外寄生虫预防措施有限的动物则受到较少关注。本研究评估了巴西农村景观中犬只对VBPs的暴露情况。2024年8月至10月，我们从巴西东北部伯南布哥州加兰洪斯市农村地区的家养犬中采集了155份血液样本。样本通过免疫荧光抗体试验（IFAT）检测抗克氏锥虫抗体，并通过快速免疫层析试验结合ELISA检测利什曼原虫（L. infantum）抗体。此外，每份样本均通过定性ELISA检测D. immitis抗原以及抗嗜吞噬细胞无形体/扁平无形体、犬埃立克体/尤因埃立克体和新疏螺旋体广义种的抗体。共有56.1%（87/155）的样本对至少一种病原体呈反应性。具体而言，5.1%（8/155）检测到抗克氏锥虫抗体，其中四份样本（2.5%，4/155）同时检测到抗利什曼原虫（L. infantum）抗体。观察到抗埃立克体属抗体占50.9%（79/155），无形体属占8.3%（13/155），伯氏疏螺旋体占0.6%（1/155）。检测到D. immitis抗原的占1.2%（2/155）。未观察到变量（性别、流动性、过夜停留和体外寄生虫存在）与病原体血清阳性率之间的统计学关联。总之，农村地区的犬只暴露于VBPs，需要使用体外寄生虫杀虫预防化合物以避免体外寄生虫侵染并降低病原体暴露风险。
关键词：美洲锥虫病，内脏利什曼病，犬，抗体，同一健康
引言
影响犬只的媒介传播病原体（VBPs）包括多种致病因子，主要由细菌（如埃立克体属、无形体属、立克次体属、疏螺旋体属）、原虫（如肝簇虫属、巴贝斯虫属、利什曼原虫属、克氏锥虫）和线虫（如恶丝虫属、棘唇线虫属）代表（Ramos et al. 2010, 2022; Macedo et al. 2022）。这些病原体呈全球分布，在全年媒介暴露常见的热带地区患病率更高（Rocha et al. 2024）。
这些病原体主要在常规接受预防产品的城市犬群中进行过研究，而涉及获得此类措施有限的农村犬只的研究仍然稀少。在热带地区的农村环境中，体外寄生虫预防有限，加上全年存在媒介，以及家犬与森林环境中常见的野生动物的接触，增加了暴露于自然生态系统中流行的已知和较少已知VBPs的风险。
将这一关注扩展到巴西，利什曼原虫属和克氏锥虫是影响人类和家犬的最重要病原体之一，尤其是在农村地区（Pavan et al. 2025）。两种寄生虫可能在同一地区同域分布，对这些感染的流行病学理解构成额外挑战。
除了这两种人畜共患寄生虫（利什曼原虫属和克氏锥虫）外，其他VBPs也值得考虑。蜱传细菌（埃立克体属和无形体属）和蚊传寄生虫（D. immitis）在巴西已有充分记录（Macedo et al. 2022; Ramos et al. 2022）。由血红扇头蜱广义种传播的埃立克体属和无形体属引起一种以多种临床症状为特征的疾病，包括发热、出血、血红蛋白尿、瘀点、黄疸、死亡以及血液学异常如贫血和血小板减少。相比之下，D. immitis引起心丝虫病，是全球犬只获得性心肌病的主要原因（Esteves-Guimarães et al. 2024）。在本研究中，我们评估了巴西东北部农村景观中犬只对主要VBPs的暴露情况。
材料与方法
研究区域与伦理方面
本研究中评估的动物来自加兰洪斯市（南纬08°53′25″，西经36°29′34″）的一个农村社区，位于巴西东北部伯南布哥州阿格雷斯特地区。该地区海拔896米，属于卡廷加生物群落，具有高地湿地的典型特征，并残留有大西洋森林。年平均气温约为21.7°C，年降水量为660至890毫米。降雨具有季节性，主要集中在4月至7月之间，而相对湿度全年保持较高水平。
采样与实验室程序
根据Canatto等人（2012）描述的先前建立的1只犬对应4.4人的比例，最小样本量估计为150只动物。研究区域的人口估计为2，100名居民（IBGE，2025），而犬类种群估计为477只动物。所需样本量使用Epi Info™（版本7.2）中的StatCalc工具计算，基于预期患病率和95%置信水平（Thrusfield 2007）。为确保完整的地理覆盖，在研究区域内随机选择采样点，每户采样一只动物。犬只在主人提供书面知情同意后纳入研究。
2024年8月至10月，从155只家养犬的头静脉采集血液（n=5 mL），分别置于含乙二胺四乙酸（EDTA）和不含抗凝剂的试管中。不含抗凝剂的试管经离心（2,000 g，10分钟）并分离血清后，样品于-20 °C保存至血清学分析。记录性别、活动性、过夜留宿情况及体外寄生虫存在情况。
克氏锥虫抗体检测采用Camargo（1966）所述方案的免疫荧光抗体试验（IFAT）进行。当抗原整个外周表面在 cutoff ≥ 1:40 时呈现荧光，样品判定为阳性。读数在落射荧光显微镜（OLYMPUS BX60-FLA，Westborough，MA，美国）下进行。每张玻片均加入阳性对照（流行区犬的反应性血清）和阴性对照（非流行区犬的非反应性血清）。该试验使用巴西里约热内卢Oswaldo Cruz基金会锥虫生物学实验室提供的克氏锥虫全抗原。
为检测抗利什曼原虫（L. infantum）抗体，血清样品首先按照制造商说明书使用快速免疫层析试验（TR-DPP®，Bio-Manguinhos-Fiocruz，里约热内卢，RJ，巴西）进行筛查。随后，反应性样品按照制造商推荐使用间接酶联免疫吸附试验（ELISA/S7®，Biogene，Recife，PE，巴西）进行检测。所有样品均设复孔检测，包括空白、阴性和阳性对照。当样品同时经两种试验判定为反应性时，判定为阳性。
样品还按照制造商说明书使用定性ELISA（SNAP® 4Dx® Plus，IDEXX Laboratory，Westbrook，ME，美国）进行单独检测。该试验可检测犬恶丝虫抗原以及针对嗜吞噬细胞无形体/扁平无形体、犬埃立克体/尤因埃立克体和伯氏疏螺旋体广义种的抗体。
数据分析
血清反应性和阳性动物的患病率采用Wilson法计算，考虑95%置信区间（https://epitools.ausvet.com.au/ciproportion）。为比较各组（性别、活动性、过夜留宿情况和体外寄生虫存在情况）之间的阳性频率，使用带Yates校正的卡方检验（χ²）；采用的显著性水平为5%。所有分析均使用BioEstat软件5.0版进行。
结果
共有56.1%（n=87；95% CI=48.2–63.7）的样品对至少一种病原体呈反应性。具体而言，5.1%（n=8；95% CI=2.6–9.8）呈现抗克氏锥虫抗体，其中2.5%（n=4；95% CI=1.0–6.4）的样品同时检出抗利什曼原虫（L. infantum）抗体。在7份利什曼原虫（L. infantum）抗体筛查阳性的样品中，4份经ELISA确认。仅这4份双阳性样品用于计算血清阳性率。
抗埃立克体属、无形体属和伯氏疏螺旋体抗体分别在50.9%（n=79；95% CI=43.1–58.2）、8.3%（n=13；95% CI=4.9–13.8）和0.6%（n=1；95% CI=0.1–3.5）的样品中检出。犬恶丝虫抗原在1.2%（n=2；95% CI=0.3–4.5）的受检动物中检出。
共有10.9%（n=17；95% CI=6.9–16.8）呈现两种或三种病原体的混合感染（表1）。所调查病原体按性别、活动性、体外寄生虫存在情况和过夜留宿情况分的血清阳性率见表2。总体而言，所评估变量与阳性之间未观察到统计学显著关联（p > 0.05）。由于数量较少，伯氏疏螺旋体（n=1）和犬恶丝虫（n=2）阳性样品被排除在表2之外。由于蜱是体格检查中发现的唯一体外寄生虫，本文其余部分将直接以蜱指代。
Table 1. Single and simultaneous positivity by canine vector-borne pathogens in animals residing in a rural landscape
| Pathogen | Prevalence % ( n ) | 95% CI |
|---|---|---|
| Leishmania infantum | 1.94 (3) | 0.66–5.54 |
| Ehrlichia canis | 40.00 (63) | 32.62–7.86 |
| Anaplasma spp. | 0.65 (1) | 0.11–3.56 |
| Trypanosoma cruzi | 1.29 (2) | 0.35–4.58 |
| *Dirofilaria immitis | 1.29 (2) | 0.35–4.58 |
| Leishmania infantum + Ehrlichia canis | 0.65 (1) | 0.11–3.56 |
| Ehrlichia canis + Anaplasma spp. | 7.10 (11) | 4.01–12.26 |
| Ehrlichia canis + Trypanosoma cruzi | 2.58 (4) | 1.01–6.45 |
| Anaplasma spp. + Borrelia burgdorferi + Trypanosoma cruzi | 0.65 (1) | 0.11–3.56 |
Table 2. Absolute and relative frequency of seroreactive dogs for Leishmania infantum , Ehrlichia canis , Anaplasma spp. and Trypanosoma cruzi , according to sex, mobility, presence of ticks and overnight stay
| Variables | Leishmania infantum n / N (%; 95% CI) | χ2; p value | Ehrlichia canis n / N (%; 95% CI) | χ2; p value | Anaplasma spp. n / N (%; 95% CI) | χ2; p value | Trypanosoma cruzi n / N (%; 95% CI) | χ2 p value |
|---|---|---|---|---|---|---|---|---|
| Sex | ||||||||
| Male | 3/86 (3.4%; 1.1–9.7) | 0.0818; p = 0.77 | 39/86 (45.3%; 35.2–55.8) | 1.9617; p = 0.16 | 5/86 (5.8%; 2.5–12.9) | 0.9974; p = 0.32 | 4/86 (4.6%; 1.8–11.3) | 0.002; p = 0.96 |
| Female | 1/69 (1.4%; 0.2–7.7) | 40/69 (57.9%; 46.2–68.8) | 8/69 (11.5%; 5.9–21.2) | 4/69 (5.8%; 2.2–13.9) | ||||
| Mobility | ||||||||
| Semi-restricted | 4/147 (2.7%; 1.0–6.8) | NP | 74/147 (50.6%; 42.6–58 − 6) | 0.0942; p = 0.75 | 13/147 (8.9%; 5.2–14.6) | NP | 8/147 (5.4%; 2.8–10.4) | NP |
| Unrestricted | 0/8 (0) | 5/8 (62.5%; 30.5–86.3) | 0/8 (0) | 0/8 (0) | ||||
| Ticks | ||||||||
| Yes | 1/35 (2.8%; 0.5–14.5) | 0.2387; p = 0.23 | 17/35 (48.5%; 32.9–64.4) | 0.0169; p = 0.89 | 3/35 (8.5%; 2.9–22.3) | 0.0911; p = 0.76 | 1/35 (2.8%; 0.5–14.5) | 0.0708; p = 0.79 |
| Not | 3/120 (2.5%; 0.8–7.0) | 62/120 (51.6%; 42.8–60.4) | 10/120 (8.3%; 4.5–14.6) | 7/120 (5.8%; 2.8–11.5) | ||||
| Overnight stay | ||||||||
| Peridomicile | 4/139 (2.8%; 1.1–7.1) | NP | 71/139 (51.0%; 42.8–59.2) | 0.0332; p = 0.85 | 11/139 (7.9%; 4.4–13.6) | 0.0227; p = 0.88 | 7/139 (5.0%; 2.4–10.0) | 0.1511; p = 0.69 |
| Intradomicile | 0/16 (0) | 8/16 (50.0%; 28.0–72.0) | 2/16 (12.5%; 35.0–36.0) | 1/16 (6.2%; 1.1–28.3) |
讨论
本研究结果表明，生活在农村地区的犬只暴露于多种蜱传病原体，其中一些是One Health关注的重要病原体。克氏锥虫、利什曼原虫（L. infantum）、无浆体属、犬埃立克体、伯氏疏螺旋体抗体及犬恶丝虫抗原的检测结果凸显了威胁该动物种群的病原体多样性。总体血清阳性率高达56.1%（87/155），表明通过使用外寄生虫杀虫剂化合物进行有效节肢动物控制存在不足。这些发现与近期关于农村-城郊过渡区蜱传病原体高流行率的报道一致，这些地区卫生基础设施有限且气候适宜，全年维持媒介种群（Silva et al. 2023）。
克氏锥虫血清阳性率（5.1%）与研究区域邻近州如阿拉戈斯州（3.8%）和帕拉伊巴州（4.0%）的检出率相符（Oliveira Neto et al. 2026），但与塞阿拉州（22%）和皮奥伊州（71%）差异显著。尽管本研究中未评估媒介，这些差异很可能归因于当地气候影响锥蝽种群，进而影响犬只对克氏锥虫的暴露（Oliveira Neto et al. 2026）。虽然未观察到流行病学变量与阳性结果之间的统计学显著关联，但仅在半限制活动犬中检出且主要见于庭院周围生活的动物，这强化了锥蝽对庭院周围区域的定殖适应性（Silva et al. 2024），凸显了犬作为家庭储存宿主的作用。
利什曼原虫（L. infantum）血清阳性率（2.5%）与此前检测到的2.4%动物呈抗体阳性的流行率一致（Lins et al. 2018），但远低于近二十年前一项研究的16%（Santos et al. 2010）。尽管这些差异可能源于传播动力学变化或控制及诊断方法的改进等多种因素，但早期研究相比近期研究使用不同血清学技术可能是其高阳性率的原因。在研究区域，已报道一例犬内脏利什曼病本土病例（Ubirajara Filho et al. 2021），且监测卫生机构已通报人内脏和皮肤利什曼病病例（Ubirajara Filho et al. 2023）。雄性频率高于雌性，且在庭院周围过夜的半限制活动动物中频率更高，提示其与媒介接触更多。有趣的是，研究区域开展的昆虫学调查仅发现一种白蛉（即Lutzomyia evandroi）（Ubirajara Filho et al. 2020），提示该物种可能作为媒介发挥作用。该假说有待进一步验证。
四只动物中同时检出克氏锥虫和利什曼原虫（L. infantum）抗体。在流行区，暴露于这两种病原体的动物之间的交叉反应已有充分文献记载（Zanette et al. 2014）。由于所用血清学检测方法的局限性，无法确定这四只动物是真正的双重感染还是交叉反应所致。需要直接检测病原体的诊断方法（如分子检测或显微镜分析）来确认真实感染状态。
犬埃立克体（E. canis）血清阳性率高达50.9%，无形体属（Anaplasma spp.）为8.3%，这表明该犬群高度暴露于血红扇头蜱（R. sanguineus s.l.）（Araújo et al. 2026）。两种病原体抗体的共同检出凸显了暴露于单一媒介所带来的临床和流行病学复杂性（Perera et al. 2025）。所采用的诊断试验并不能确认活动性感染，而是提示抗体的存在，对于某些病原体如犬埃立克体，抗体在感染后可能持续数月（Evaristo et al. 2024）。仅在单只动物中检出抗伯氏疏螺旋体（B. burgdorferi）抗体（0.6%），这与同一研究区域此前的检测结果相符（Macedo et al. 2022）。
在两只动物中诊断出犬恶丝虫（D. immitis）抗原（1.2%），证实了该线虫在此高海拔地区的流行，与此前报道一致（Macedo et al. 2022）。对伯南布哥州七个沿海城市的研究显示犬感染率为3.6%，证实了该线虫在该地区不同区域的持续存在（Rocha et al. 2024）。研究区域位于高海拔地区（海拔896米），其独特的微气候，包括平均气温略低于20°C的季节性湿润期（5月至8月），减少了蚊科昆虫种群，这解释了当地血清学流行率较低的原因。
缺乏动物的临床数据，且未对采样时发现的蜱进行采集、鉴定和评估，是本研究的 important 局限性。此外，除犬恶丝虫外，几乎所有病原体所用的诊断技术均无法确定活动性感染，仅能反映病原体暴露情况。尽管存在局限性，本研究中观察到的媒介传播病原体（VBPs）总体血清流行率较高，可能对犬具有重要的临床意义。这些发现对“同一健康”至关重要，因为犬是人兽共患病原体的储存宿主。需要采取战略性媒介控制措施，强调“同一健康”框架，包括公共卫生教育、昆虫学监测以及全年使用体外寄生虫杀虫剂化合物。总之，该地区的犬暴露于媒介传播病原体，鉴于其与人类密切共居，它们可能作为重要的流行病学哨兵，其感染模式反映了某些病原体如利什曼原虫和锥虫的人兽共患风险。
致谢
G.A.C. 获得FACEPE的研究奖学金（Bolsa de Produtividade；批准号BPP-0024-5.05/24），R.A.N.R. 获得CNPq的奖学金（Bolsa de Produtividade；批准号318558/2025-4）。
作者贡献
C.R.C.U.F（方法学、调查、写作——审阅与编辑），G.M.S.（写作——初稿、调查、写作——审阅与编辑），M.L.O.L.（方法学、调查、写作——审阅与编辑），L.O.M.（方法学、调查、写作——审阅与编辑），L.C.A.（写作——审阅与编辑），G.A.C.（写作——审阅与编辑），R.A.N.R.（方法学、调查、写作——初稿、写作——审阅与编辑）。
资助
本研究成果发表的文章处理费（APC）由巴西高等教育人员改进协调委员会（CAPES）资助（ROR标识符：00x0ma614）。
数据可用性
本研究期间未生成或分析任何数据集。
声明
伦理声明
伯南布哥联邦农村大学动物实验伦理委员会（ECAE）批准了本研究方案（批准号：975916112）。
利益冲突
作者声明无利益冲突。
脚注
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参考文献
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关联数据
数据可用性声明
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Abstract
Vector-borne pathogens (VBPs) of medical and veterinary concern have mostly been studied in urban dog populations, while animals in rural settings, with limited access to ectoparasite prevention, have received less attention. In this study, we assessed canine exposure to VBPs in a rural Brazilian landscape. From August to October 2024, we collected 155 blood samples from dogs domiciled in a rural area of the municipality of Garanhuns, Pernambuco, Northeastern Brazil. Samples were assessed using an immunofluorescence antibody test (IFAT) to detect antibodies against Trypanosoma cruzi and using a rapid immunochromatographic test combined with an ELISA to detect Leishmania infantum antibodies. In addition, each sample was tested using a qualitative ELISA to detect D. immitis antigens and antibodies against Anaplasma phagocytophilum/Anaplasma platys, Ehrlichia canis/Ehrlichia ewingii, and Borrelia burgdorferi sensu lato. A total of 56.1% (87/155) of samples were reactive to at least one pathogen. Specifically, in 5.1% (8/155) were detected anti-T. cruzi antibodies, and in four of these samples (2.5%, 4/155) were co-detected antibodies anti-L. infantum. Antibodies against Ehrlichia spp. were observed in 50.9% (79/155), Anaplasma spp. in 8.3% (13/155), and B. burgdorferi in 0.6% (1/155). Antigens for D. immitis were detected in 1.2% (2/155). No statistical associations were observed on variables (sex, mobility, overnight stay and presence of ectoparasites) and pathogen seropositivity. In conclusion, dogs from rural areas are exposed to VBPs, which requires the use of ectoparasiticide preventive compounds to avoid ectoparasite infestation and reduce the risk of pathogen exposure.
Keywords: American Trypanosomiasis, Visceral Leishmaniasis, Dogs, Antibodies, One Health
Introduction
Vector-borne pathogens (VBPs) affecting dogs include a diverse range of causative agents, represented primarily by bacteria (e.g., Ehrlichia spp., Anaplasma spp., Rickettsia spp., Borrelia spp.), protozoa (e.g., Hepatozoon spp., Babesia spp., Leishmania spp., Trypanosoma cruzi), and nematodes (e.g., Dirofilaria spp., Acanthocheilonema spp.) (Ramos et al. 2010, 2022; Macedo et al. 2022). These pathogens have a global distribution, with a higher prevalence in tropical regions where year-round vector exposure is common (Rocha et al. 2024).
These pathogens have been studied primarily in urban dog populations that routinely receive preventive products, whereas studies involving rural dogs with limited access to such measures remain scarce. In rural settings of tropical regions, limited ectoparasite prevention, combined with the presence of vectors year-round, and contact of domestic dogs and wild animals commonly found in forested environments, increases the risk of exposure to both well-known and lesser-known VBPs circulating in natural ecosystems.
Extending this focus to Brazil, Leishmania spp. and T. cruzi are among the most significant pathogens affecting both humans and domestic dogs, particularly in rural areas (Pavan et al. 2025). Both parasites may occur in sympatry in the same area, posing an additional challenge to the epidemiological understanding of these infections.
Besides these two zoonotic parasites (Leishmania spp. and T. cruzi), other VBPs warrant consideration. Tick-borne bacteria (Ehrlichia spp. and Anaplasma spp.) and the mosquito-borne parasite (D. immitis) are well documented in Brazil (Macedo et al. 2022; Ramos et al. 2022). Ehrlichia spp. and Anaplasma spp., transmitted by Rhipicephalus sanguineus sensu lato ticks, cause a disease marked by diverse clinical signs, including fever, bleeding, hemoglobinuria, petechiae, jaundice, death, and hematological abnormalities such as anemia and thrombocytopenia. In contrast, D. immitis causes heartworm disease, the leading cause of acquired cardiomyopathy in dogs worldwide (Esteves-Guimarães et al. 2024). In the present study, we assessed the exposure to the main VBPs of dogs in a rural landscape, in the Northeastern region of Brazil.
Materials and methods
Study area and ethical aspects
Animals assessed in this study were from a rural community of the municipality of Garanhuns (08°53′25″ S, 36°29′34″ W), located in the Agreste region of the state of Pernambuco, Northeastern Brazil. The region is at an altitude of 896 m above sea level, it is part of the Caatinga biome and presents typical characteristics of highland wetlands, with remnants of the Atlantic Forest. The mean annual temperature is approximately 21.7 °C, with annual precipitation ranging from 660 to 890 mm. Rainfall is seasonal, concentrated mainly between April and July, while relative humidity remains high throughout the year.
Sampling and laboratory procedures
The minimum sample size was estimated at 150 animals, based on the previously established ratio of 1 dog per 4.4 humans, as described by Canatto et al. (2012). The human population of the study area was estimated at 2,100 inhabitants (IBGE, 2025), while the canine population was estimated at 477 animals. The required sample size was calculated using the StatCalc utility in Epi Info™ (version 7.2), based on the expected prevalence and a 95% confidence level (Thrusfield 2007). To ensure full geographic coverage, sampling points were randomly selected within the study area, with one animal sampled per household. Dogs were enrolled in the study after their owners provided written informed consent.
From August to October 2024, blood (n = 5 mL) was drawn from the cephalic veins of 155 domiciled dogs and kept in tubes with (Ethylenediaminetetraacetic acid - EDTA) and without anticoagulant. After centrifugation (2,000 g for 10 min) of tubes with no anticoagulant and separation of serum, samples were stored at -20 °C until serological analysis. Data on sex, mobility, overnight stay, and presence of ectoparasites were recorded.
The T. cruzi antibody detection was performed using the Immunofluorescence Antibody Test (IFAT) according the protocol described by Camargo (1966). Samples were considered positive when the entire peripheral surface of the antigen showed fluorescence at the at a cut-off ≥ 1:40. Readings were performed in an epifluorescent microscope (OLYMPUS BX60-FLA, Westborough, MA, United States). On each slide, positive (reactive serum of a dog from endemic area) and negative control (non-reactive serum of a dog from non-endemic area) were added. For this assay were used total T. cruzi antigens provided by the Laboratory of Biology of Tripanosomatids of the Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
For the detection of antibodies anti-Leishmania infantum, serum samples were initially screened using a rapid immunochromatographic test (TR-DPP®, Bio-Manguinhos-Fiocruz, Rio de Janeiro, RJ, Brazil), according to the manufacturer’s instructions. Afterwards, reactive samples were tested using an indirect enzyme-linked immunosorbent assay (ELISA/S7®, Biogene, Recife, PE, Brazil) following manufacturer’s recommendations. All samples were tested in duplicate, including blank, negative, and positive controls. Samples were considered positive when scored reactive simultaneously for both assays.
Samples were also individually tested using a qualitative ELISA (SNAP® 4Dx® Plus, IDEXX Laboratory, Westbrook, ME, United States), according to the manufacturer’s instructions. The test can detect D. immitis antigens and antibodies against Anaplasma phagocytophilum/Anaplasma platys, Ehrlichia canis/Ehrlichia ewingii, and Borrelia burgdorferi sensu lato.
Data analysis
The prevalence of seroreactive and positive animals was calculated using the Wilson method, considering a 95% confidence interval (https://epitools.ausvet.com.au/ciproportion). To compare the frequency of positivity across groups (sex, mobility, overnight stay and presence of ectoparasites), the Chi-square test (χ²) with Yates’ correction was used; the adopted significance level was 5%. All analyses were performed using BioEstat software version 5.0.
Results
A total of 56.1% (n = 87; 95% CI = 48.2–63.7) of samples were reactive to at least one pathogen. Specifically, 5.1% (n = 8; 95% CI = 2.6–9.8) presented anti-T. cruzi antibodies, and in 2.5% (n = 4; 95% CI = 1.0–6.4) of these samples, antibodies anti-L. infantum were co-detected. Of the seven samples that screened positive for L. infantum antibodies, four were confirmed by ELISA. Only these four dual-positive samples were used to calculate seroprevalence.
Antibodies anti-Ehrlichia spp., Anaplasma spp., and B. burgdorferi were detected in 50.9% (n = 79; 95% CI = 43.1–58.2), 8.3% (n = 13; 95% CI = 4.9–13.8), and 0.6% (n = 1; 95% CI = 0.1–3.5) of the samples, respectively. Antigens for D. immitis were detected in 1.2% (n = 2; 95% CI = 0.3–4.5) of the evaluated animals.
A total of 10.9% (n = 17; 95% CI = 6.9–16.8) presented co-infections by two or three pathogens (Table 1). The seroprevalence of the investigated pathogens according to sex, mobility, presence of ectoparasites, and overnight stay is presented in the Table 2. Overall, no statistically significant associations were observed between the evaluated variables and positivity (p > 0.05). Due to the low number, positive samples for B. burgdorferi (n = 1) and D. immitis (n = 2) were excluded from the Table 2. Because ticks were the only ectoparasites found during the physical examination, they will be referred to directly throughout the remainder of this text.
Table 1. Single and simultaneous positivity by canine vector-borne pathogens in animals residing in a rural landscape
| Pathogen | Prevalence % ( n ) | 95% CI |
|---|---|---|
| Leishmania infantum | 1.94 (3) | 0.66–5.54 |
| Ehrlichia canis | 40.00 (63) | 32.62–7.86 |
| Anaplasma spp. | 0.65 (1) | 0.11–3.56 |
| Trypanosoma cruzi | 1.29 (2) | 0.35–4.58 |
| *Dirofilaria immitis | 1.29 (2) | 0.35–4.58 |
| Leishmania infantum + Ehrlichia canis | 0.65 (1) | 0.11–3.56 |
| Ehrlichia canis + Anaplasma spp. | 7.10 (11) | 4.01–12.26 |
| Ehrlichia canis + Trypanosoma cruzi | 2.58 (4) | 1.01–6.45 |
| Anaplasma spp. + Borrelia burgdorferi + Trypanosoma cruzi | 0.65 (1) | 0.11–3.56 |
Table 2. Absolute and relative frequency of seroreactive dogs for Leishmania infantum , Ehrlichia canis , Anaplasma spp. and Trypanosoma cruzi , according to sex, mobility, presence of ticks and overnight stay
| Variables | Leishmania infantum n / N (%; 95% CI) | χ2; p value | Ehrlichia canis n / N (%; 95% CI) | χ2; p value | Anaplasma spp. n / N (%; 95% CI) | χ2; p value | Trypanosoma cruzi n / N (%; 95% CI) | χ2 p value |
|---|---|---|---|---|---|---|---|---|
| Sex | ||||||||
| Male | 3/86 (3.4%; 1.1–9.7) | 0.0818; p = 0.77 | 39/86 (45.3%; 35.2–55.8) | 1.9617; p = 0.16 | 5/86 (5.8%; 2.5–12.9) | 0.9974; p = 0.32 | 4/86 (4.6%; 1.8–11.3) | 0.002; p = 0.96 |
| Female | 1/69 (1.4%; 0.2–7.7) | 40/69 (57.9%; 46.2–68.8) | 8/69 (11.5%; 5.9–21.2) | 4/69 (5.8%; 2.2–13.9) | ||||
| Mobility | ||||||||
| Semi-restricted | 4/147 (2.7%; 1.0–6.8) | NP | 74/147 (50.6%; 42.6–58 − 6) | 0.0942; p = 0.75 | 13/147 (8.9%; 5.2–14.6) | NP | 8/147 (5.4%; 2.8–10.4) | NP |
| Unrestricted | 0/8 (0) | 5/8 (62.5%; 30.5–86.3) | 0/8 (0) | 0/8 (0) | ||||
| Ticks | ||||||||
| Yes | 1/35 (2.8%; 0.5–14.5) | 0.2387; p = 0.23 | 17/35 (48.5%; 32.9–64.4) | 0.0169; p = 0.89 | 3/35 (8.5%; 2.9–22.3) | 0.0911; p = 0.76 | 1/35 (2.8%; 0.5–14.5) | 0.0708; p = 0.79 |
| Not | 3/120 (2.5%; 0.8–7.0) | 62/120 (51.6%; 42.8–60.4) | 10/120 (8.3%; 4.5–14.6) | 7/120 (5.8%; 2.8–11.5) | ||||
| Overnight stay | ||||||||
| Peridomicile | 4/139 (2.8%; 1.1–7.1) | NP | 71/139 (51.0%; 42.8–59.2) | 0.0332; p = 0.85 | 11/139 (7.9%; 4.4–13.6) | 0.0227; p = 0.88 | 7/139 (5.0%; 2.4–10.0) | 0.1511; p = 0.69 |
| Intradomicile | 0/16 (0) | 8/16 (50.0%; 28.0–72.0) | 2/16 (12.5%; 35.0–36.0) | 1/16 (6.2%; 1.1–28.3) |
Discussion
Findings from the present study indicates that dogs living in rural areas were exposed to a wide plethora of VBPs, some of them critical agents of One Health concern. The detection of antibodies against T. cruzi, L. infantum, Anaplasma spp., E. canis, B. burgdorferi and D. immitis antigens underscores the diversity of pathogens threatening this animal population. The substantial overall seroprevalence of 56.1% (87/155) suggests the lack of an effective arthropod control, through the use ectoparasiticide compounds. These findings align with recent reports of high prevalence rates for VBPs in rural-peri-urban transition zones, where limited sanitary infrastructure and favorable climate sustain vector populations throughout the year (Silva et al. 2023).
Trypanosoma cruzi seroprevalence (5.1%) matches with rates detected in states nearby the study area such as Alagoas (3.8%) and Paraíba (4.0%) (Oliveira Neto et al. 2026), but differs sharply from the states of Ceará (22%) and Piauí (71%). Even if in the present study, no vectors have been assessed, most likely these differences may be attributed to local climate influencing triatomine population and consequently dog exposure to T. cruzi parasites (Oliveira Neto et al. 2026). Although no statistically significant association was observed between epidemiological variables and positivity, exclusive detection in semi-restricted dogs and predominantly in animals living in the peridomicile reinforces triatomines adaptability to colonize peridomicile areas (Silva et al. 2024), underscoring the role of the dog as a domestic reservoir.
The seropositivity rate for L. infantum (2.5%) is consistent with the prevalence rate previously detected in which 2.4% of animals presented antibodies (Lins et al. 2018), but much lower than 16% of a study conducted nearly two decades prior (Santos et al. 2010). Despite these disparities may result from several factors such as changes in transmission dynamics, or improved control and diagnostic methods, in these studies the use of different serological techniques might be the cause of the high positivity rate of the prior study compared with more recent studies. In the study area, an autochthonous case of canine visceral leishmaniasis has been reported (Ubirajara Filho et al. 2021), and human visceral and cutaneous leishmaniasis have been notified by surveillance health agencies (Ubirajara Filho et al. 2023). The higher frequency in males compared with females, and in semi-restricted animals with access to peridomicile areas overnight, suggest a higher exposure to vectors. Interestingly, entomological surveys carried out in the study area revealed the presence of a single sandfly species (i.e., Lutzomyia evandroi) (Ubirajara Filho et al. 2020), which suggest the potential role of this species as vector. This hypothesis should be further tested.
Antibodies against both T. cruzi and L. infantum were co-detected in four animals. Cross-reactivity between animals exposed to these two pathogens is well-documented in endemic areas (Zanette et al. 2014). Due to the limitations of the serological assays used here, it is impossible to determine whether these four animals experienced a true dual infection or if the results stem from cross-reactivity. Diagnostic methods that directly detect the causative agents (e.g., molecular assays or microscopic analysis) are needed to confirm true infection status.
The substantial seropositivity for E. canis (50.9%) and Anaplasma spp. (8.3%) indicates that this dog population is highly exposed to R. sanguineus s.l. (Araújo et al. 2026). The co-detection of antibodies for both pathogens underscore the clinical and epidemiological complexity posed by exposure to a single vector (Perera et al. 2025). The diagnostic test employed does not confirm active infection, but indicates the presence of antibodies, which for some pathogens as E. canis may persist for months after infection (Evaristo et al. 2024). The unique detection of antibodies anti-B. burgdorferi in a single animal (0.6%) corroborates with a previous detection in the same study area (Macedo et al. 2022).
The diagnose of D. immitis antigen in two animals (1.2%) confirm the circulation of this nematode in this area of high altitude as previously reported (Macedo et al. 2022). Studies across seven coastal cities in the state Pernambuco showed a 3.6% canine infection rate which affirm the nematode persistence in different areas of the region (Rocha et al. 2024). The study area is located in a region of high altitude (896 asl), in which the unique microclimate, including seasonal wet periods with average temperatures slightly below 20 °C (May–August), reduces culicid population, justifying low local serological prevalence.
The absence of clinical data on animals, and no collection, identification and assessment of ticks detected at sampling, are important limitations of this study. Besides that, the diagnostic technique used for almost all pathogens (except for D. immitis), did not allow the determination of active infection, only pathogen exposure. Despite of limitation, the pronounced overall seroprevalence of VBPs observed in this study might has significant clinical consequences for dogs. These findings are critical for One Health, as dogs serve as reservoir of pathogens causing zoonoses. Strategic vector control is needed, emphasizing the One Health framework, with public health education, entomological surveillance and use of ectoparasiticide compounds year-round. In conclusion, dogs of this area are exposed to VBPs, and given their close human cohabitation, they may act as important epidemiological sentinels whose infection patterns mirror for some pathogens as Leishmania and Trypanosoma the zoonotic risk.
Acknowledgements
G.A.C. receives a research fellowship from FACEPE (Bolsa de Produtividade; Grant No. BPP-0024-5.05/24) and R.A.N.R. a fellowship from CNPq (Bolsa de Produtividade; Grant No.318558/2025-4).
Author contributions
C.R.C.U.F (Methodology, Investigation, Writing – review & editing), G.M.S. (Writing – original draft, Investigation, Writing – review & editing), M.L.O.L. (Methodology, Investigation, Writing – review & editing), L.O.M. (Methodology, Investigation, Writing – review & editing), L.C.A. (Writing – review & editing), G.A.C. (Writing – review & editing), R.A.N.R. (Methodology, Investigation, Writing – original draft, Writing – review & editing).
Funding
The Article Processing Charge (APC) for the publication of this research was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) (ROR identifier: 00x0ma614).
Data availability
No datasets were generated or analysed during the current study.
Declarations
Ethical statement
The Ethics Committee for Animal Experimentation (ECAE) of the Universidade Federal Rural de Pernambuco approved the study protocol (approval number: 975916112).
Competing interests
The authors declare no competing interests.
Footnotes
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References
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Associated Data
Data Availability Statement
No datasets were generated or analysed during the current study.