兽研引言
孢子丝菌病是一种在巴西具有重要且日益增长的公共卫生意义的地下真菌病，由孢子丝菌属真菌引起。过去二十年间，巴西已成为人兽共患孢子丝菌病的中心，这主要归因于巴西孢子丝菌的出现，该菌种以其高毒力和人兽共患潜力而著称[1]。该病原体通过家猫建立了强大的传播循环，家猫既是感染宿主又是传播媒介，尤其是在城市地区，人与猫的密切接触促进了其传播[2]。
在里约热内卢，孢子丝菌病已达到流行程度，其驱动因素是在猫群数量庞大的密集人口区域中人猫频繁接触[3]。圣保罗是另一个主要热点地区，也经历了类似模式，研究表明由于与感染猫的接近，对动物和人类健康产生了显著影响[4]。在巴西东南部沿海州圣埃斯皮里图，孢子丝菌病病例不断增加，这与不受监管的城市扩张和猫群快速增长有关[5]。
南里奥格兰德州（RS）是巴西孢子丝菌病的主要中心之一，与里约热内卢并列，该病在那里具有显著的人兽共患影响[6, 7]。该州位于温带，拥有独特的生态系统，如潘帕生物群落[8]，孢子丝菌病发病率高，尤其是在家猫中[6]。近年来孢子丝菌病显著增加，尤其是在南里奥格兰德州南部和大都市地区[9]。在南里奥格兰德州，该病影响人类和动物，猫是人兽共患的主要来源[10, 11]。
由于缺乏强制报告，真实患病率仍不清楚，尽管该病被认为是地方性流行病，在七个卫生区的40个城市有病例报告[9]。里奥格兰德 feline病例发病率从2010年每月0.75例新发病例显著上升至2014年每月3.33例，凸显了快速的城市传播[12]。巴西南部的孢子丝菌病，尤其是南里奥格兰德州，是一个发病率不断增加、地理分布广泛的新发公共卫生问题。尽管专业参考服务改善了诊断和治疗[7]，但积极的公共卫生政策和更高的认识对于遏制这种人兽共患病的传播至关重要[1, 9]。
然而，针对南里奥格兰德州的专项研究仍然稀缺，主要局限于临床病例报告和一般流行病学数据，缺乏对该地区流行的孢子丝菌属物种多样性的全面分子研究[13]。这一区域特异性数据的空白阻碍了对巴西南部地方性孢子丝菌变异株的识别，并限制了针对性公共卫生干预措施的制定。此外，南里奥格兰德州与乌拉圭和阿根廷的地理邻近——这些国家也报告了孢子丝菌病病例[14–17]——凸显了进行严格分子监测和综合公共卫生策略的必要性，以降低跨境传播风险并加强区域对孢子丝菌病的卫生应对。
本研究填补了南里奥格兰德州南部孢子丝菌属分子流行病学的关键空白，该地区对于巴西孢子丝菌病的监测和控制具有战略重要性。该地区位于与乌拉圭和阿根廷的边境，在跨境传播动态中发挥关键作用，使其成为新发和再发人兽共患病监测的焦点。通过对物种分布、传播动态和临床表现的综合分析，本研究将区域流行病学和临床特征置于国家和国际背景中。本文呈现的研究结果为制定针对性控制策略和加强当前流行场景中的人兽共患风险管理提供了基础。
方法
本研究在巴西南里奥格兰德州南部进行，该地区为温带气候（Köppen Cfa），与乌拉圭、阿根廷、圣卡塔琳娜州和大西洋接壤。该地区包括沿海平原、低丘和帕托斯湖盆地，冬季温和，夏季炎热，降雨分布均匀。潘帕生物群落占主导地位，支持广泛的养牛业和农业，维持着佩洛塔斯、里奥格兰德和巴热等人口稀疏的城市。该地区靠近乌拉圭和阿根廷，通过塔伊姆沼泽等湿地促进了跨境互动和生物多样性交流[8]。
临床和流行病学数据回顾性取自佩洛塔斯联邦大学兽医真菌学实验室（MICVET/UFPel）的被动病例送检记录，该实验室是动物孢子丝菌病的区域诊断参考中心。记录对应2019年至2023年间确诊的病例，包括宿主物种（犬或猫）、年龄、性别、来源市镇、病变类型、受累解剖部位以及呼吸道症状的有无。病变根据两个主要标准进行分类：（i）解剖分布，定义为局限性（单一部位）或播散性（多个区域），以及（ii）形态学模式，分为溃疡型、结节型、混合型或其他表现（如脓肿和脓疱）。每只动物被分配一个主要病变类别，以避免重复并确保分析的一致性。
真菌分离与培养条件
从2019年至2023年间巴西佩洛塔斯联邦大学（UFPel）真菌学实验室临床真菌学保藏库记录的猫和犬病例中，共选取了100株先前在属水平鉴定的孢子丝菌分离株。初步鉴定基于孢子丝菌属的大体形态学和显微形态学特征。为在分子分析前确认活力，将冷冻保存的分离株解冻并传代接种于添加氯霉素（Sigma-Aldrich，圣路易斯，密苏里州，美国）的沙氏葡萄糖琼脂（SDA；Becton, Dickinson and Company，富兰克林湖，新泽西州，美国）上，于25 °C孵育7–14天，以促使适合后续DNA提取的年轻、有活力菌落发育。
分子鉴定与表征
使用Brazol试剂（LCG Biotecnologia，巴西）按照制造商说明从马铃薯葡萄糖琼脂上生长的菌落中提取基因组DNA。通过使用种特异性引物PCR扩增钙调蛋白（CAL）基因进行物种鉴定，扩增产物为469 bp片段[18]。每个20 µL反应体系包含2× PCR MasterMix（LCG Biotecnologia，巴西）、引物（3–10 pmol）、模板DNA和无核酸酶水（NFW）。应用标准PCR循环条件，扩增产物在紫外光下于1.5%琼脂糖凝胶上观察，并使用100 bp分子量标记（Ladder 100 bp 500 µl，Ludwig Biotechnology，阿雷格里港，南里奥格兰德州，巴西）。PCR产物从凝胶中切出，使用凝胶纯化试剂盒（Ludwig Biotechnology，阿雷格里港，南里奥格兰德州，巴西）按照供应商说明进行纯化。使用BigDye Terminator Cycle Sequencing Kit v3.1（Thermo Fisher Scientific，美国）在ABI 3500遗传分析仪（Applied Biosystems，美国）上进行测序。
系统发育分析
使用Phred碱基识别和Phrap组装工具对电泳图进行双向分析以获得一致序列，随后使用ClustalW方法在MEGA12软件（分子进化遗传学分析第12版）[19]中进行比对。使用BLAST工具（//blast.ncbi.nlm.nih.gov/Blast.cgi）在美国国家生物技术信息中心（NCBI）数据库中进行序列相似性搜索。获得的CAL基因序列以登录号PQ512086–PQ512099存入NCBI GenBank。使用MEGA12[21]中实施的Tamura 3参数（T92）模型[20]的最大似然（ML）法推断孢子丝菌分离株之间的系统发育关系。位点间速率异质性使用离散伽马分布建模，以白色孢子丝菌（Sporothrix albicans）和红褐蛇口壳菌（Ophiostoma rufum）作为外群。分支支持度使用1000次自举重复[22]评估，≥ 60%的值被认为显著。最终比对包含27条序列和994 nt。从NCBI GenBank数据库检索来自巴西、阿根廷和巴拉圭的人类和动物分离株参考序列，并纳入系统发育分析以进行比较。基于部分CAL序列（一种用于区分申克孢子丝菌复合群内物种的验证标记[18]）的系统发育树显示，巴西孢子丝菌（S. brasiliensis）形成一个支持良好的单系分支，与申克孢子丝菌（S. schenckii）和白色孢子丝菌（S. albicans）明显不同。
描述性与统计分析
使用描述性统计（包括频率和百分比）总结分类变量，如物种分布、性别、病变形态、解剖部位和呼吸道症状。统计分析使用IBM SPSS Statistics第25版（IBM Corp.，阿蒙克，纽约州，美国）进行。分类变量之间的关联使用卡方检验或当预期单元格计数< 5时使用Fisher精确检验进行评估。病例数量的时间趋势使用卡方检验评估。猫与犬之间的比较分析侧重于病变形态和解剖分布，统计显著性设定为p < 0.05。
伦理批准
伦理审批
本研究仅依赖于从常规兽医诊断送检至兽医真菌学实验室（MICVET/UFPel）的存档孢子丝菌分离株。未直接涉及活体动物或人类受试者，也未为研究目的进行任何实验操作。因此，无需机构动物护理与使用委员会（IACUC）或机构审查委员会（IRB）的批准。所有程序均符合巴西临床兽医实践法规及佩洛塔斯联邦大学（UFPel）的机构生物安全标准。
结果
真菌分离与病例构成
共从2019年至2023年间巴西南里奥格兰德州南部确诊的猫和犬孢子丝菌病病例中回收了100株孢子丝菌分离株。所有分离株均来自UFPel兽医真菌学实验室的临床菌种保藏。其中，75株（75%）来自猫，24株（24%）来自犬，1株未进行菌种鉴定。从冷冻保存中复苏后，菌落在25 °C沙氏葡萄糖琼脂上培养7–14天后表现出典型的孢子丝菌大体形态，确认其活力可用于下游分子和系统发育分析。
临床与流行病学发现
时间与空间分布
2019年至2023年间，兽医真菌学实验室（MICVET/UFPel）共诊断99例孢子丝菌病病例，包括75例猫和24例犬。整个研究期间确诊病例数逐步增加（图1）。猫的病例数从2019年的5例增至2023年的42例，增长了八倍。犬的病例也呈稳定上升趋势，从2019年的3例增至2023年的10例。两种宿主的时间趋势均具有统计学显著性（趋势χ2检验，p < 0.001），表明该地区诊断的孢子丝菌病病例数持续增长。这一模式提示当地地方性流行范围正在扩大，2021年后实验室确诊病例显著增加。可获得78只动物的饲养条件信息，包括全部24只犬。其中73只（93%）为半散养，有定期户外活动或自由漫游行为。这一特征反映了地方性流行城市地区常见的典型管理条件，可能促进了与感染动物的持续接触及孢子丝菌属的本地传播网络。
[图：2019年至（年份截断）期间在巴西南部诊断的猫（n = 75）和犬（n = 24）孢子丝菌病病例的时间趋势]
因此，空间分布反映的是送检病例的地理来源，而非主动监测设计。大多数病例来自佩洛塔斯及周边市镇，其他南部沿海地区有零星送检。空间上，病例高度集中在佩洛塔斯（80%），其次为卡庞杜莱昂（13%），里奥格兰德、莫罗雷东杜、圣维多利亚杜帕尔马和舒伊有零星报告（1–3%）。与所有其他市镇合计的病例分布相比，佩洛塔斯城市范围内的猫病例空间聚集具有统计学显著性（χ2(1) = 29.03，p < 0.0001）（图2）。
[图：从兽医真菌学实验室培养物保藏中心分析的100株孢子丝菌属分离株的地理来源]
宿主、性别与病变特征
全部99只动物的性别均已确定。雄性猫的感染率（52/75，69.3%）显著高于雌性猫（23/75，30.7%）（χ2 = 12.13，p < 0.001）。犬的感染未显示性别偏倚（雄性16/24，66.7% vs. 雌性8/24，33.3%，p = 0.102）。可获得71只猫（94.7%）和24只犬的解剖部位数据。猫以播散性病变为主（49.3%），其次为口吻部（19.7%）、面部（11.3%）、四肢（9.9%）和耳/颈部（7.0%）；其他局部部位占2.8%。犬的播散性和面部病变同样常见（各占25.0%），而口吻部（16.7%）和四肢（12.5%）受累较少。猫的播散性病变发生率显著高于犬（χ2 = 4.6，p = 0.032）。病变形态在宿主间也存在差异。猫以溃疡性病变为主（53.0%），其次为结节性（22.7%）、混合性（15.2%）和非典型性（9.1%）病变。犬以结节性病变最常见（41.7%），其次为溃疡性（33.3%）、混合性（16.7%）和非典型性（8.3%），宿主间差异显著（χ2 = 5.12，p = 0.024）。呼吸道症状见于9.3%的猫和12.5%的犬，无种间差异（p = 0.589）。在有鼻部病变的猫中，50%（7/14）出现呼吸道受累。然而，该关联无统计学显著性（Fisher精确检验，p = 0.754）。
孢子丝菌属菌种的分子鉴定
钙调蛋白（CAL）基因的PCR扩增在所有分离株中均产生预期的469 bp扩增产物。阴性对照未见扩增，证实操作过程无菌。分子分型鉴定巴西孢子丝菌为优势菌种（97/100；97%），其中包括来自猫的73株（97.3%）和犬的23株（95.8%）分离株。严格意义申克孢子丝菌在3株分离株中检出（3%）。宿主物种与菌种鉴定之间未观察到统计学关联（Fisher精确检验，p=0.611）。这些发现证实巴西孢子丝菌是巴西南部动物孢子丝菌病的主要病原体。
系统发育分析
选取14条代表性CAL序列（约800 bp），在Tamura 3参数（T92）模型下进行最大似然（ML）法重建[18]。所得拓扑结构 delineated 一个稳健的巴西孢子丝菌单系分支（自举支持率，BS ≥ 84%），与申克孢子丝菌和白色孢子丝菌清晰分离。在巴西孢子丝菌内部，中等自举值（BS = 60–84%）揭示有限的种内结构。一个由南里奥格兰德州分离株（以猫源为主，PQ512086–PQ512097）组成的紧凑南部亚分支表现出极小的核苷酸分歧（BS = 75–84）。来自圣保罗、伯南布哥、米纳斯吉拉斯、巴拉那和联邦区的序列形成一个凝聚的东南部簇（BS = 64–81）。来自巴拉圭（OQ266365）和阿根廷（MK850448）的两条序列嵌套于巴西孢子丝菌分支内，靠近巴西南部分离株，与南锥体地区的跨境传播一致。人源与猫源序列交织分布，表明不存在宿主隔离，支持该地区由家猫介导的持续人兽共患传播循环。总体而言，基于CAL的系统发育分析证实巴西孢子丝菌是一个遗传上凝聚但具有区域结构的谱系（图3）。
[图：基于巴西南部孢子丝菌分离株部分钙调蛋白（CAL）基因序列推断的最大似然系统发育]
讨论
巴西孢子丝菌在巴西南部的优势地位，强化了该菌种作为该国孢子丝菌病主要人兽共患病原体持续领土扩张的趋势[23]。申克孢子丝菌的残余检出表明生态置换尚未完成，祖先孢子丝菌谱系可能持续存在于潘帕生物群系内受环境保护的微生境中[1, 24]。这种共存反映的是生态韧性而非竞争排斥，与巴西孢子丝菌通过高效的猫-猫传播占据城市和城郊生态位的适应能力一致[5]。相比之下，申克孢子丝菌可能以低水平持续存在于环境和替代宿主中。
行为和管理变量似乎是维持这些动态的核心因素。雄性猫（67%）和犬（75%）的感染优势与先前将领地行为和攻击性互动与接种风险联系起来的证据一致[2, 6]。半散养动物比例极高（93%），其中大多数可无限制外出，进一步支持了持续暴露于感染动物和短距离传播网络的模型[2]。这些行为模式是城市地方性孢子丝菌病的典型特征，其中自由漫游的雄性动物充当孢子丝菌传播的局部放大器。这一解释与巴西南部近期的建模数据一致，佩洛塔斯猫孢子丝菌病的基本再生数（R₀）估计为1.45[25]，证实该地区在缺乏协调控制策略的情况下传播仍可自我维持。将行为生态学和饲养管理纳入控制策略仍然至关重要[23]，其中圈养、绝育和社区教育代表与分子和临床监测互补的可行干预措施。
纳入24株犬源分离株扩展了巴西南部孢子丝菌病的分子流行病学框架。与先前来自巴西西南部和南部的研究一致[26, 27]，巴西孢子丝菌是唯一检出的菌种，证实了统一的家养传播网络而非宿主特异性循环的持续存在。猫源与犬源分离株之间缺乏系统发育隔离，表明存在共享的流行池，其中犬作为偶然但具有流行病学信息价值的宿主。这些发现强化了将犬孢子丝菌病纳入流行区One Health分子监测系统的重要性。
解剖学模式也支持这种攻击行为模型。病变集中于面部、口吻部和四肢，这些是打斗中创伤性接种的常见部位，证实巴西南部的传播仍由行为介导且生态稳定[2, 6, 26, 28]。在空间上，病例集中于佩洛塔斯与巴西东南部的观察结果平行，那里密集的猫种群和无管制流动维持着孢子丝菌属的地方性循环[2, 5, 23]。总之，这些发现勾勒出一个在温带条件下持续存在的地方性疫源地，而温带条件此前被认为对国家疫情而言处于边缘地位。
结构性局限不可避免地限制了这些发现的普适性。由于采样依赖于被动实验室送检，来自周边市镇的分离株可能代表性不足，导致全州发病率可能被低估[7, 9]。此外，猫孢子丝菌病常通过细胞学检查诊断，该方法快速且高度敏感，通常可支持在不需真菌培养的情况下启动治疗。在这些情况下，可能不进行培养和分子鉴定，尤其是在难以获得专业实验室服务的地区。因此，仅基于培养确诊分离株的研究可能低估该地区孢子丝菌病的真实负担和地理分布[29]。在COVID-19大流行期间（2019–2021年），诊断可及性进一步降低[29]，而Rio Grande另一诊断机构的邻近可能导致了病例地理分布的不均衡。尽管如此，这些局限性凸显了区域监测能力的关键缺口，并强调需要建立连接兽医和人类实验室的协调网络，以加强流行病学监测并促进早期发现。
系统发育重建证实，S. brasiliensis是一个遗传上紧密凝聚、具有区域结构的谱系。来自Rio Grande do Sul的分离株紧密聚类，提示基因流动受限和局部传播链占主导，这些特征是由家养宿主维持的扩张性人兽共患谱系的典型特征。该模式与先前在流行区观察到的低单倍型多样性一致，支持S. brasiliensis流行病由强烈的克隆传播而非持续的环境重组驱动的观点[1, 16]。
巴西南部、阿根廷和巴拉圭分离株之间密切的系统发育亲缘关系强化了南锥体传播走廊的假说，在该走廊中生态连续性和伴侣动物流动性促进了跨境病原体流动。这种遗传接近性而非随机扩散，可能反映了由人类相关流动和区域贸易介导的历史或当代播散事件。分离株之间缺乏明显分化也支持近期地理扩张的情景，这与S. brasiliensis流行病推断出的较短进化时间尺度一致[14, 23]。
尽管未纳入人类分离株，但动物源S. brasiliensis序列与南美洲人类参考株的接近性表明存在共享的流行病学关系。这种一致性佐证了先前关于在流行环境中动物与人类之间循环着遗传同质种群的证据[2, 16, 23]，强调了兽医分子监测作为One Health框架中人兽共患风险早期指标的价值。尽管依赖于CAL测序和被动送检，本研究为巴西南部提供了一致的分子-流行病学信号。
S. brasiliensis的优势地位、其与邻国南美洲分离株的系统发育聚类以及半家养猫的行为特征，共同描绘了该大陆最南部地区稳定的人兽共患传播循环。在这些边界内，CAL位点仍是物种鉴定和谱系凝聚性检测的稳健且可重复的靶标，验证了其作为兽医和公共卫生实验室分子监测一线工具的应用价值。持续的多中心测序工作，结合标准化临床元数据、强制报告以及常规真菌培养和物种鉴定，对于界定S. brasiliensis传播的地理界限、减少报告偏倚并监测其在南锥体的生态持续性至关重要。
作者贡献
所有作者对本研究做出了实质性贡献。A.R.G.起草、编辑并批判性审阅了手稿。G.L.S.起草并排版了手稿，并与K.R.M.和G.B.F.M.共同实施了实验程序。B. C.、J. T. F.、I. S. M.、L. C. G. M.、L. P. R.和L. S. S.整理了数据。S. J.、R. C. C.和R. O. F.提供了批判性审阅和反馈，以确保手稿的科学严谨性。
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本研究出版的文章处理费（APC）由Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil（CAPES）资助（ROR标识符：00x0ma614）。本研究得到了Fundação de Amparo à Pesquisa e Inovação do Espírito Santo（FAPES）和国家科学技术发展委员会（CNPq/MCTI）的支持，资助编号408595/2023-0，征集编号10/2023。
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Introduction
Sporotrichosis, a subcutaneous mycosis of significant and growing public health concern in Brazil, is caused by fungi of the genus Sporothrix. Over the past two decades, Brazil has become the epicenter of zoonotic sporotrichosis, largely because of the emergence of Sporothrix brasiliensis, a species recognized for its high virulence and zoonotic potential [1]. This pathogen has established a robust transmission cycle through domestic cats, which act as both reservoirs and vectors of infection, particularly in urban areas, where close human-cat interactions facilitate its spread [2].
In Rio de Janeiro, sporotrichosis has reached epidemic proportions, driven by frequent contact between humans and cats in densely populated areas with large feline populations [3]. São Paulo, another major hotspot, has experienced similar patterns, with studies indicating significant impacts on animal and human health due to proximity to infected cats [4]. In Espírito Santo, a coastal state in southeastern Brazil, increasing sporotrichosis cases have been linked to unregulated urban expansion and the rapid growth of feline populations [5].
Rio Grande do Sul (RS) is the primary epicenter of sporotrichosis in Brazil, alongside Rio de Janeiro, where the disease has a significant zoonotic impact [6, 7]. Situated in a temperate zone with unique ecosystems, such as the Pampa biome [8], the state has a high incidence of sporotrichosis, particularly among domestic cats [6]. Sporotrichosis has shown a marked increase in recent years, particularly in the southern and metropolitan regions of RS [9]. In RS, the disease affects both humans and animals, with cats being the primary source of zoonosis [10, 11].
Owing to a lack of mandatory reporting, the true prevalence remains unclear, although the disease is considered endemic, with cases reported in 40 cities across seven health districts [9]. The incidence of feline cases in Rio Grande increased significantly from 0.75 new cases per month in 2010 to 3.33 cases per month in 2014, highlighting the rapid urban spread [12]. Sporotrichosis in southern Brazil, particularly in RS, is an emerging public health issue with an increasing incidence and wide geographic distribution. Although specialized reference services have improved diagnosis and treatment [7], aggressive public health policies and greater awareness are essential to curb the spread of this zoonotic disease [1, 9].
However, studies specific to Rio Grande do Sul remain scarce, mainly limited to clinical case reports and general epidemiological data, and lack comprehensive molecular investigations of the diversity of Sporothrix species circulating in the region [13]. This gap in region-specific data hinders the identification of Sporothrix variants endemic to Southern Brazil and constrains the development of targeted public health interventions. Additionally, the geographical proximity of Rio Grande do Sul to Uruguay and Argentina, which also reported sporotrichosis cases [14–17], underscores the need for rigorous molecular surveillance and integrated public health strategies to mitigate cross-border transmission risks and strengthen regional health responses to sporotrichosis.
This study addresses critical gaps in the molecular epidemiology of Sporothrix spp. in southern Rio Grande do Sul, a region of strategic importance for monitoring and controlling sporotrichosis in Brazil. Situated at the border with Uruguay and Argentina, this area plays a pivotal role in cross-border transmission dynamics, positioning it as a focal point for surveillance of emerging and re-emerging zoonoses. By conducting a comprehensive analysis of species distribution, transmission dynamics, and clinical manifestations, this study situates regional epidemiological and clinical profiles within both national and international contexts. The findings presented herein provide a foundation for developing targeted control strategies and enhancing zoonotic risk management in ongoing epidemic scenarios.
Methodology
The study was conducted in southern Rio Grande do Sul, Brazil, a temperate region (Köppen Cfa) bordered by Uruguay, Argentina, Santa Catarina, and the Atlantic Ocean. The area encompasses coastal plains, low hills, and the Lagoa dos Patos Basin, with mild winters, hot summers, and evenly distributed rainfall. The Pampa biome predominates, supporting extensive cattle ranching and agriculture that sustains sparsely populated cities such as Pelotas, Rio Grande, and Bagé. The region’s proximity to Uruguay and Argentina facilitates cross-border interactions and biodiversity exchanges through wetlands, such as the Banhado do Taim [8].
Clinical and epidemiological data were retrospectively retrieved from passive case submissions to the Veterinary Mycology Laboratory of the Federal University of Pelotas (MICVET/UFPel), a regional diagnostic reference center for animal sporotrichosis. The records corresponded to confirmed cases diagnosed between 2019 and 2023 and included host species (dog or cat), age, sex, municipality of origin, lesion type, affected anatomical site, and the presence or absence of respiratory signs. Lesions were classified according to two main criteria: (i) anatomical distribution, defined as localized (single site) or disseminated (multiple regions), and (ii) morphological pattern, categorized as ulcerative, nodular, mixed, or other presentations (e.g., abscesses and pustules). Each animal was assigned a predominant lesion category to avoid duplication and ensure consistency in the analysis.
Fungal Isolation and Culture Conditions
A total of 100 Sporothrix isolates previously identified at the genus level were selected from feline and canine cases recorded between 2019 and 2023 in the Clinical Mycological Collection at the Mycology Laboratory of the Federal University of Pelotas (UFPel), Pelotas, Brazil. Primary identification was based on the macromorphological and micromorphological characteristics of Sporothrix spp. To confirm viability before molecular analysis, cryopreserved isolates were thawed and subcultured on Sabouraud Dextrose Agar (SDA; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) supplemented with chloramphenicol (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 25 °C for 7–14 days to allow the development of young, viable colonies suitable for subsequent DNA extraction.
Molecular Identification and Characterization
Genomic DNA was extracted from colonies grown on Potato Dextrose Agar using Brazol reagent (LCG Biotecnologia, Brazil) according to the manufacturer’s instructions. Species identification was performed by PCR amplification of the calmodulin (CAL) gene using species-specific primers that generated a 469 bp fragment [18]. Each 20 µL reaction contained 2× PCR MasterMix (LCG Biotecnologia, Brazil), primers (3–10 pmol), template DNA, and nuclease-free water (NFW). Standard PCR cycling conditions were applied, and the amplicons were visualized on 1.5% agarose gels under UV illumination, and a 100 bp molecular weight marker (Ladder 100 bp 500 µl, Ludwig Biotechnology, Porto Alegre, Rio Grande do Sul, Brazil) was used. The PCR products were excised from the gel and purified using the Gel Purification Kit (Ludwig Biotechnology, Porto Alegre, Rio Grande do Sul, Brazil), following the supplier’s instructions. Sequencing was performed using the BigDye Terminator Cycle Sequencing Kit v3.1 (Thermo Fisher Scientific, USA) on an ABI 3500 Genetic Analyzer (Applied Biosystems, USA).
Phylogenetic Analysis
The electropherograms were bidirectionally analyzed using the Phred base calling and Phrap assembly tool to obtain consensus sequences, which were subsequently aligned with the MEGA12 software (Molecular Evolutionary Genetics Analysis version 12) [19] using the ClustalW method. Sequence similarity searches were performed in the National Center for Biotechnology Information (NCBI) database using the BLAST tool (//blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences obtained for the CAL gene were deposited in the NCBI GenBank under accession numbers PQ512086–PQ512099.
Phylogenetic relationships among Sporothrix isolates were inferred using the Maximum Likelihood (ML) method with the Tamura 3-parameter (T92) model [20], implemented in MEGA12 [21]. Rate heterogeneity among sites was modeled using a discrete gamma distribution, and Sporothrix albicans and Ophiostoma rufum were used as outgroups. Branch support was evaluated using 1000 bootstrap replicates [22], with values ≥ 60% considered significant. The final alignment included 27 sequences and 994 nt. Reference sequences from human and animal isolates from Brazil, Argentina, and Paraguay were retrieved from the NCBI GenBank database and included in the phylogenetic analysis for comparative purposes. The phylogenetic tree based on partial CAL sequences, a validated marker for discriminating species within the Sporothrix schenckii complex [18], showed that S. brasiliensis formed a well-supported monophyletic clade, clearly distinct from S. schenckii and S. albicans.
Descriptive and Statistical Analysis
Descriptive statistics, including frequencies and percentages, were used to summarize categorical variables such as species distribution, sex, lesion morphology, anatomical site, and respiratory signs. Statistical analyses were performed using IBM SPSS Statistics version 25 (IBM Corp., Armonk, NY, USA). Associations between categorical variables were evaluated using the chi-square or Fisher’s exact test when the expected cell counts were < 5. Temporal trends in case numbers were assessed using the chi-square test. Comparative analyses between cats and dogs focused on lesion morphology and anatomical distribution, with statistical significance set at p < 0.05.
Ethical Approval
This study relied exclusively on archived Sporothrix isolates obtained from routine veterinary diagnostic submissions to the Veterinary Mycology Laboratory (MICVET/UFPel). No live animals or human subjects were directly involved, and no experimental procedures were performed for research. Therefore, approval by an Institutional Animal Care and Use Committee (IACUC) or an Institutional Review Board (IRB) was not required. All procedures complied with Brazilian regulations for clinical veterinary practice and the institutional biosafety standards of the Federal University of Pelotas (UFPel).
Results
Fungal Isolation and Case Composition
A total of 100 Sporothrix isolates were recovered from confirmed cases of feline and canine sporotrichosis diagnosed between 2019 and 2023 in southern Rio Grande do Sul, Brazil. All isolates originated from the clinical collection of the UFPel Veterinary Mycology Laboratory. Of these, 75 (75%) were from felines, 24 (24%) from canines, and one lacked species identification. Following revival from cryostorage, the colonies exhibited typical Sporothrix macromorphology after 7–14 days of incubation at 25 °C on Sabouraud Dextrose Agar, confirming viability for downstream molecular and phylogenetic analyses.
Clinical and Epidemiological Findings
Temporal and Spatial Distribution
Between 2019 and 2023, 99 sporotrichosis cases were diagnosed at the Veterinary Mycology Laboratory (MICVET/UFPel), including 75 feline and 24 canine cases. The number of confirmed cases progressively increased throughout the study period (Fig. 1). In felines, the number of cases increased from five in 2019 to 42 in 2023, representing an eight-fold increase. Canine cases also showed a steady upward trend, increasing from three in 2019 to ten in 2023. The temporal trend was statistically significant for both hosts (χ2 test for trend, p < 0.001), indicating sustained growth in the number of diagnosed sporotrichosis cases in the region. This pattern suggests an expanding local endemic scenario, with a marked increase in laboratory-confirmed cases after 2021. Information on housing conditions was available for 78 animals, including all 24 dogs. Among these, 73 (93%) were semi-domiciled, with regular outdoor access or free-roaming behavior. This profile reflects the typical management conditions observed in endemic urban areas and likely contributes to sustained exposure to infected animals and local transmission networks of Sporothrix spp.
[图：Temporal trends of feline (n = 75) and canine (n = 24) sporotrichosis cases diagnosed in southern Brazil between 2019 an]
Therefore, the spatial distribution reflects the geographic origin of the submitted cases rather than an active surveillance design. Most cases originated from Pelotas and the surrounding municipalities, with sporadic submissions from other localities across the southern coastal region. Spatially, cases were highly concentrated in Pelotas (80%), followed by Capão do Leão (13%), with sporadic reports in Rio Grande, Morro Redondo, Santa Vitória do Palmar, and Chuí (1–3%). The spatial clustering of feline cases within the urban perimeter of Pelotas was statistically significant compared to the distribution of cases in all other municipalities combined (χ2(1) = 29.03, p < 0.0001) (Fig. 2).
[图：Geographic origins of the 100 Sporothrix spp. isolates analyzed from the culture collection of the Veterinary Mycology L]
Host, Sex, and Lesion Characteristics
The sex of all 99 animals was determined. Male cats were significantly more affected (52/75, 69.3%) than female cats (23/75, 30.7%) (χ2 = 12.13, p < 0.001). In dogs, infection showed no sex bias (16/24, 66.7% males vs. 8/24, 33.3% females, p = 0.102). Anatomical data were available for 71 cats (94.7%) and 24 dogs. In cats, disseminated lesions predominated (49.3%), followed by those on the muzzle (19.7%), face (11.3%), limbs (9.9%), and ears/neck (7.0%); other localized sites accounted for 2.8% of the cases. In dogs, disseminated and facial lesions were equally common (25.0% each), whereas muzzle (16.7%) and limb (12.5%) involvement was less frequent. Dissemination was significantly more frequent in cats (χ2 = 4.6p, p= 0.032) than in dogs. Lesion morphology also differed among hosts. In cats, ulcerative lesions predominated (53.0%), followed by nodular (22.7%), mixed (15.2%), and atypical (9.1%) lesions. In dogs, nodular lesions were the most frequent (41.7%), followed by ulcerative (33.3%), mixed (16.7%), and atypical (8.3%) forms, with significant differences between hosts (χ2 = 5.12, p = 0.024). Respiratory signs were observed in 9.3% of cats and 12.5% of dogs, with no interspecies difference (p = 0.589). Among cats with nasal lesions, 50% (7/14) presented with respiratory involvement. However, this association was not statistically significant (Fisher’s exact test, p = 0.754).
Molecular Identification of Sporothrix Species
PCR amplification of the calmodulin (CAL) gene produced the expected 469 bp amplicons in all isolates. The negative controls showed no amplification, confirming procedural sterility. Molecular typing identified Sporothrix brasiliensis as the predominant species (97/100; 97%), including 73 feline (97.3%) and 23 canine (95.8%) isolates from the total. S. schenckii sensu stricto was detected in three isolates (3%). No statistical association was observed between host species and identity (Fisher’s exact test, p=0.611). These findings corroborate S. brasiliensis as the dominant etiological agent of sporotrichosis in animals in southern Brazil.
Phylogenetic Analysis
Fourteen representative CAL sequences (~ 800 bp) were included in the Maximum Likelihood (ML) reconstruction under the Tamura 3-parameter (T92) model [18]. The resulting topology delineated a robust S. brasiliensis monophyletic clade (bootstrap support, BS ≥ 84%) clearly separated from S. schenckii and S. albicans. Within S. brasiliensis, moderate bootstrap values (BS = 60–84%) revealed limited intraspecific structure. A compact southern subclade comprising isolates from Rio Grande do Sul (predominantly feline, PQ512086–PQ512097) exhibited minimal nucleotide divergence (BS = 75–84). Sequences from São Paulo, Pernambuco, Minas Gerais, Paraná, and the Federal District formed a cohesive southeastern cluster (BS = 64–81). Two sequences from Paraguay (OQ266365) and Argentina (MK850448) nested within the S. brasiliensis clade proximal to southern Brazilian isolates, consistent with the cross-border dissemination in the Southern Cone. Intermixed human and feline sequences indicated the absence of host segregation, supporting sustained zoonotic transmission cycles mediated by domestic cats in this area. Overall, the CAL-based phylogeny confirmed S. brasiliensis as a genetically cohesive but regionally structured lineage (Fig. 3).
[图：Maximum-likelihood phylogeny inferred from partial calmodulin (CAL) gene sequences of Sporothrix isolates from southern ]
Discussion
The predominance of Sporothrix brasiliensis in southern Brazil reinforces the ongoing territorial expansion of this species as the dominant zoonotic agent of sporotrichosis in the country [23]. The residual detection of S. schenckii suggests that ecological displacement is not yet complete and that ancestral Sporothrix lineages may persist in environmentally protected microhabitats within the Pampa biome [1, 24]. Such coexistence reflects ecological resilience rather than competitive exclusion, consistent with the adaptive capacity of S. brasiliensis to occupy urban and peri-urban niches through efficient cat-to-cat transmission [5]. In contrast, S. schenckii likely persists at low levels in environmental and alternative hosts.
Behavioral and management variables appear to be central to maintaining these dynamics. The predominance of infection in male cats (67%) and dogs (75%) mirrors previous evidence linking territorial behavior and aggressive interactions to the risk of inoculation [2, 6]. The very high proportion of semi-domiciled animals (93%), most of which had unrestricted outdoor access, further supports a model of sustained exposure to infected animals and short-range transmission networks [2].These behavioral patterns typify the urban endemic form of sporotrichosis, in which free-roaming males act as local amplifiers of the propagation of Sporothrix. This interpretation is consistent with recent modeling data from southern Brazil, where the basic reproduction number (R₀) for feline sporotrichosis was estimated to be 1.45 in Pelotas [25], confirming that transmission remains self-sustaining in the absence of coordinated control strategies in the region. Integrating behavioral ecology and housing management into control strategies remains essential [23], with confinement, neutering, and community education representing feasible interventions complementary to molecular and clinical surveillance.
The inclusion of 24 canine isolates expands the molecular–epidemiological framework of sporotrichosis in southern Brazil. Consistent with previous studies from southwest and southern Brazil [26, 27], Sporothrix brasiliensis was the only species detected, confirming the persistence of a unified domestic transmission network rather than host-specific cycles. The absence of phylogenetic segregation between feline and canine isolates indicates a shared epidemic pool in which dogs act as incidental yet epidemiologically informative hosts. These findings reinforce the importance of integrating canine sporotrichosis into One Health molecular surveillance systems in endemic regions.
Anatomical patterns also support this behavioral model of aggression. The lesions concentrated on the face, muzzle, and limbs, which are common sites of traumatic inoculation during fights, confirm that transmission in southern Brazil remains behaviorally mediated and ecologically stable [2, 6, 26, 28]. Spatially, the concentration of cases in Pelotas parallels observations from southeastern Brazil, where dense feline populations and unregulated movement sustain the endemic circulation of Sporothrix spp. [2, 5, 23]. Together, these findings delineate an endemic focus that persists under temperate conditions, which were previously considered marginal to the national epidemic.
Structural constraints inevitably limit the generalizability of these findings. Because sampling relied on passive laboratory submissions, isolates from peripheral municipalities were likely underrepresented, leading to a potential underestimation of the statewide incidence [7, 9]. In addition, feline sporotrichosis is frequently diagnosed by cytological examination, a rapid and highly sensitive method that often supports the initiation of treatment without the need for fungal cultures. In these situations, culture and molecular identification may not be performed, particularly in areas with limited access to specialized laboratories. Consequently, studies based exclusively on culture-confirmed isolates may underestimate the true burden and geographic distribution of sporotrichosis in the region [29]. Diagnostic access was further reduced during the COVID-19 pandemic (2019–2021) [29], and the proximity of another diagnostic facility in Rio Grande may have contributed to the uneven geographic distribution of the cases. Nevertheless, these limitations highlight a critical gap in regional surveillance capacity and underscore the need for a coordinated network linking veterinary and human laboratories to strengthen epidemiological surveillance and facilitate early detection.
Phylogenetic reconstruction confirmed that S. brasiliensis is a genetically cohesive, regionally structured lineage. The compact clustering of the isolates from Rio Grande do Sul suggests restricted gene flow and dominance of local transmission chains, which are features typical of expanding zoonotic lineages maintained by domestic reservoirs. This pattern is consistent with previous observations of low haplotypic diversity in endemic areas, supporting the notion that S. brasiliensis epidemics are driven by intense clonal propagation rather than by ongoing environmental recombination [1, 16].
The close phylogenetic affinity between southern Brazilian, Argentine, and Paraguayan isolates reinforces the hypothesis of a Southern Cone transmission corridor, where ecological continuity and companion animal mobility facilitate cross-border pathogen flow. Rather than stochastic dispersion, this genetic proximity likely reflects historical or contemporary seeding events mediated by human-associated movement and regional trade. The absence of pronounced divergence among isolates also supports the scenario of recent geographic expansion, consistent with the short evolutionary timescale inferred for S. brasiliensis epidemics [14, 23].
Although human isolates were not included, the proximity of animal-derived S. brasiliensis sequences to South American human reference strains indicates shared epidemiological relationships. This coherence corroborates previous evidence of genetically homogeneous populations circulating between animals and humans in endemic settings [2, 16, 23], emphasizing the utility of veterinary molecular surveillance as an early indicator of zoonotic risk in the One Health framework. Despite the reliance on CAL sequencing and passive submissions, the present study provides a consistent molecular–epidemiological signal for southern Brazil.
The predominance of S. brasiliensis, its phylogenetic clustering with neighboring South American isolates, and the behavioral profile of semi-domiciled cats collectively depict a stable zoonotic transmission cycle in the southernmost region of the continent. Within these boundaries, the CAL locus remains a robust and reproducible target for species identification and detection of lineage cohesion, validating its application as a first-line tool for molecular surveillance in veterinary and public health laboratories. Continued multicenter sequencing efforts, coupled with standardized clinical metadata, mandatory notification, and routine fungal culture and species identification, are essential to delineate the geographic limits of S. brasiliensis circulation, reduce reporting bias, and monitor its ecological persistence across the Southern Cone.
Author Contributions
All authors contributed substantially to this study. A.R.G. drafted, edited, and critically reviewed the manuscript. G.L.S. drafted and formatted the manuscript and conducted the experimental procedures with K.R.M. and G.B.F.M.. B. C., J. T. F., I. S. M., L. C. G. M., L. P. R. and L. S. S. curated the data. S. J., R. C. C., and R. O. F. provided critical reviews and feedback to ensure the scientific rigor of the manuscript.
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). This work was supported by the Fundação de Amparo à Pesquisa e Inovação do Espírito Santo (FAPES) and the National Council for Scientific and Technological Development (CNPq/MCTI) under grant No. 408595/2023-0, Call No. 10/2023.
Data Availability
Data supporting the conclusions of this study can be obtained from the corresponding author upon reasonable request.
Declarations
Conflict of interest
The authors declare that they have no affiliations with or involvement in any organization or entity with any financial interest in the subject matter or materials discussed in this manuscript.
Footnotes
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References
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Associated Data
Data Availability Statement
Data supporting the conclusions of this study can be obtained from the corresponding author upon reasonable request.