兽研背景：猫博卡病毒（FBoV）被认为是影响猫的新发病毒病原体之一。方法：本观察性研究通过对2020—2025年意大利洛迪兽医教学医院收治的224只猫进行便利抽样，采用靶向NP1基因的PCR方法调查FBoV的发生情况。阳性样本进行了系统发育分析。结果：在17/224（7.6%）只猫中观察到FBoV阳性。鉴定出FBoV基因型1（4%）、2（0.9%）和3（2.7%）。在采集粪便样本的动物中，FBoV阳性猫为17/53（32.1%），而采集血液（n=69）、呼吸道（n=54）、尿液（n=46）或其他（n=2）样本的猫均为FBoV阴性。在采集了不止一份粪便样本的部分猫中，病毒携带状态持续长达两周。幼龄、流浪/收容所状态和住院与FBoV阳性显著相关（分别为p=0.0008、p=0.0139和p=0.0023）。在分析了粪便样本的猫中，猫泛白细胞减少症病毒（FPV）阳性与FBoV阳性相关（p<0.001），而腹泻的存在与FBoV发生无关。结论：采样策略无法估算猫群体中FBoV的流行率。尽管如此，意大利猫群中遗传多样性FBoV毒株的循环、FBoV阳性与住院的关联以及提示FBoV可能不是胃肠道疾病原发性病原体的结果，凸显了全面监测研究对加深理解FBoV进化特征、流行病学和致病作用的重要性。
1. 引言
博卡细小病毒（BoVs），曾称为博卡病毒，是细小病毒科中新出现的一组病毒，于2005年首次在患有急性下呼吸道感染和胃肠炎的儿童中鉴定，随后在多种动物物种中检测到，表明其宿主范围广泛[1,2,3,4]。BoVs的传播通常通过粪口途径发生，导致幼龄动物和人类出现呼吸道和胃肠道症状，而成人感染常为亚临床[5,6,7]。人博卡病毒（HBoV）也偶尔在感染儿童的血浆和尿液中检测到，提示存在其他可能的HBoV相关疾病[8,9]。
猫博卡病毒（FBoV）于2012年首次在香港有或无临床症状的流浪猫的粪便、尿液、肾脏、鼻腔和血液样本中鉴定[10]，现被认为是影响猫的新发病毒病原体之一[4,11,12]。迄今为止，除其他在中国猫中检测到FBoVs的研究外[2,3,13,14]，FBoVs已在多个国家报道，包括美国[15]、加拿大[11]、日本[16]、泰国[17,18]、越南[19]、比利时[20]、葡萄牙[21]、土耳其[22]和意大利南部[23]。
全世界猫群体中已描述了三种FBoV基因型（FBoV-1、FBoV-2和FBoV-3）[16,19]。FBoV-1、FBoV-2和FBoV-3已分别重新分类为食肉动物博卡细小病毒3、食肉动物博卡细小病毒4和食肉动物博卡细小病毒5[24]。
FBoV的致病作用尚不清楚，因为FBoV已在健康猫和患有临床疾病的猫中鉴定[10]。关于潜在致病性，与其他博卡病毒类似，FBoV已在表现为腹泻（包括出血性肠炎）的猫中鉴定[13,16,17,23]。在猫中已观察到与FBoV感染相关的神经系统感染[20]，最近FBoV也被认为是潜在的嗜神经病毒[3,14,18]。
FBoV在猫中的流行病学、进化、多样性和致病性仍在研究中。与其他动物博卡病毒感染类似，仍需进一步研究以更好地理解FBoV在猫呼吸道和泌尿道疾病中的潜在作用。
由于意大利关于FBoV的研究数量有限，本观察性研究主要基于PCR检测，目的为：（i）使用PCR方法和测序分析调查意大利猫中FBoV病毒DNA的存在，以表征循环FBoV毒株；（ii）通过调查猫粪便、尿液、呼吸道和血液样本中的FBoV病毒DNA，探讨FBoV的潜在致病作用；（iii）评估FBoV阳性、临床状态和患者变量之间的关联。
2. 材料与方法
2.1. 动物与采样
本观察性研究通过对2020年至2025年间意大利洛迪兽医教学医院因常规诊断程序收治的224只猫进行便利抽样，调查FBoV的发生情况。从猫采集血液、粪便、尿液、呼吸道标本或其他类型样本。
所有样本均在临床病理学检查背景下或作为转诊兽医要求的健康检查的一部分采集，并事先获得主人知情同意。诊断程序完成后，样本和/或提取的DNA随后储存于−80°C以备进一步分子分析。
所有样本均为诊断目的或作为健康检查的一部分采集，并获得主人知情同意，因此根据米兰大学机构动物护理和使用委员会指南，无需正式批准。
每只动物均记录了采样时间、性别、年龄和所有权类型的数据。年龄分为幼年（<1岁）和成年（≥1岁）[14]。还记录了根据已发表方案[25]基于qPCR检测粪便样本中猫泛白细胞减少症病毒（FPV）的常规诊断分析数据。仅对粪便样本，还考虑了腹泻的发生和FPV阳性情况。
2.2. 通过PCR检测FBoV
使用商业DNA提取试剂盒（NucleoSpin Blood® kit，Macherey-Nagel，Düren，Germany）按照制造商说明从全血、呼吸道和尿液样本中提取DNA。使用商业DNA提取试剂盒（NucleoSpin® Tissue Kit，Macherey-Nagel，Düren，Germany）按照制造商说明从粪便样本中提取DNA。提取的DNA储存于−20 °C直至进一步分子分析。通过针对脊椎动物12S rRNA基因座的PCR对所有样本进行DNA分析前质量控制[26]。
如前所述，进行了能够通过靶向NP1基因的一部分同时检测FBoV-1、FBoV-2和FBoV-3的PCR。该PCR分别扩增FBoV-1、FBoV-2和FBoV-3的464、503和491 bp片段[27]。该PCR assay在本实验室未进行敏感性和特异性的分析验证。PCR在25 μL的最终反应体积中进行，包含1x Phire Green Hot Start II PCR（Thermo Fisher Scientific，Segrate –Milan，Italy）、1 μM正向引物FBoV1-2-3 NP1 F（5′- AGA ACC RCC RAT CAC ART CCACT-3′）、1 μM反向引物FBoV1-2-3 NP1 R（5′- TGG CRA CCG CYA GCA TTT CA-3′）和5 μL模板DNA。扩增程序为：初始变性步骤98 °C 30 s，随后40个循环的变性98 °C 5 s、退火51 °C 5 s和延伸72 °C 10 s。最终延伸步骤为72 °C 1 min。PCR产物通过1.5%琼脂糖凝胶电泳分离并在紫外光下可视化。PCR反应中还包含阳性对照（本实验室先前获得的FBoV田间样本，经测序确认为FBoV-3）和空白对照（无DNA酶水样本）。
当从同一动物采集并分析多个样本时，如果至少一个样本为阳性，则该猫被视为FBoV阳性。
显示预期大小条带的样本进一步进行测序以表征FBoV基因型。
2.3. FBoV基因型与系统发育
PCR阳性结果的扩增产物使用DNA扩增中相同的内部正向和内部反向引物进行纯化和测序。Sanger测序由商业测序机构（Microsynth Seqlab，Germany）完成。使用BioEdit软件版本7.0 https://thalljiscience.github.io/（访问于2026年3月3日）组装序列数据。使用BLAST https://blast.ncbi.nlm.nih.gov/Blast.cgi（访问于2026年7月10日）将序列与GenBank中可用的序列进行比较。使用BioEdit软件v.7.0中的Clustal X将所有共识序列与从GenBank检索的FBoV基因型进行比对，以鉴定菌株的FBoV基因型。
与从GenBank检索的FBoV参考序列比对的序列使用BioEdit软件v.7.0中的Clustal X进行系统发育分析。在存在来自同一动物的相同序列时，仅选择一条代表性序列/猫进行系统发育分析。使用MEGA v.12的最大似然（ML）方法及1000次bootstrap重复用于系统发育分析[28,29,30]。基于ML方法的系统发育分析所使用的General Time Reversible模型（含Gamma分布和不变位点）根据最低AIC值选择。
对于序列比较，使用MEGA v.12计算成对进化距离的核苷酸相似性百分比。
本研究产生的序列已提交至GenBank，登录号为PZ787303-PZ787319。
2.4. 血液学和生化检测
为了调查可能与FBoV阳性相关的血液学或生化变化的存在，检查了VTH的数据库以评估PCR阳性的猫是否也进行了血液学或生化分析。
当临床表现需要时，在采集用于PCR的样本的同一天采集血液。更具体地说，将1 mL血液置于EDTA管中，剩余量置于无抗凝剂的管中。试管立即送至VTH的临床病理学实验室。EDTA管用于使用激光分析仪Sysmex XN1000V（Sysmex Corporation，Kobe，Japan）进行全血细胞计数（CBC）。无抗凝剂的管离心（10 min，4500 rpm），血清用于使用分光光度计BT3500（Futurlab Srl，Limena PD，Italy）进行常规临床化学检测。
2.5. 数据分析
对于猫的FBoV检测，使用WinEpi（http://www.winepi.net/uk/index.htm，访问于2025年10月1日）计算了224只动物的样本量，考虑2020-2025年VTH收治的约6000只猫进行常规诊断分析的种群规模、根据低阳性率猫样本中的平均阳性率计算的最低流行率1.3%[10]以及95%置信水平。
采用Pearson卡方检验或Fisher精确检验评估FBoV阳性动物比例与性别、年龄类别及住院状态之间的差异。在具有可分析粪便样本的猫亚组中，亦采用Fisher精确检验评估FBoV阳性动物比例与细小病毒阳性及腹泻存在之间的差异。当同一只猫存在多个粪便样本时，若至少一个样本为FPV阳性，则该猫被视为FPV阳性。基于Pearson卡方检验或Fisher精确检验的统计比较以及优势比（OR）及95%置信区间（CI）的计算使用Epitools进行[31]，以p < 0.05为显著性标准。每次分析的统计单位以动物为代表。
对于有腹泻和无腹泻猫的样本中FBoV和FPV单一阳性或共同阳性的检测，亦以纯描述性结果报告样本水平数据，这些数据未进行统计分析。
3. 结果
3.1. 受检动物与样本
在总共224只接受FBoV检测的猫中，中位年龄为5岁2个月，范围为1个月16天至18岁。基于分析猫数量的动物特征数据，包括采集粪便样本的猫中腹泻的存在及FPV阳性情况，见表1。
从这224只猫中共采集了263份样本。样本包括80份粪便、76份血液、54份呼吸道、51份尿液和2份其他类型样本，分别采集自53、69、54、46和2只猫。每只动物采集的样本数量不一，201只（89.7%）猫采集了一份样本，而23只（10.3%）猫采集了多份粪便、血液或尿液样本。关于53只具有可分析粪便样本的猫亚组，39只（73.6%）猫采集了一份粪便样本，14只（26.4%）猫采集了多份样本，范围为2至6份（表2）。
3.2. FBoV的检出及基因型
在本观察性研究中，通过PCR检测并经测序确认，在17/224只猫（7.6%；95% C.I：4.1–11.1%）中检测到FBoV，对应19/263份（7.2%）阳性样本。FBoV阳性猫的结果见表1。
在53只具有可分析粪便样本的猫中，仅考虑采集单一粪便样本的39只猫时，结果显示10只（25.6%）猫为FBoV阳性。对于其余14只接受多次粪便采样的猫，每份样本的FBoV PCR结果见表2。表2结果显示，在7只（50%）FBoV阳性猫中，5只（71.4%）猫仅在一份样本中检测到FBoV，2只（28.6%）猫在两份样本中检测到FBoV。这两份阳性样本之间的采集间隔在一只猫（36号猫）中为一周，在另一只猫（78号猫）中为两周。所有7只具有多份可分析粪便样本的FBoV阳性猫在整个样本采集期间均住院。
所有FBoV-PCR阳性样本均成功获得了用于FBoV基因型鉴定的测序结果。对本研究序列NP1片段的BLAST分析显示，与GenBank中 deposited 的FBoV序列核苷酸同一性最高，范围为96.4%至99.4%。本研究中的10条序列与近期在意大利报道的FBoV序列[23]观察到最高核苷酸相似性。此外，本研究中的4条序列与来自美国的序列（GenBank登录号KM017745）显示最高核苷酸同源性，2条与来自中国的FBoV序列（GenBank登录号OR544348）显示最高同源性，1条与来自泰国的FBoV序列（GenBank登录号PP592986）显示最高同源性。
本研究序列之间的核苷酸同一性范围为55.2%至100%。从同一只猫（36号猫和78号猫）采集的两份阳性样本中获得了相同的FBoV序列。在不同猫的序列之间，在三条（214、218、219号猫）、两条（60、62号猫）和两条序列（63、66号猫）中观察到100%核苷酸同一性。
根据测序结果和BLAST分析，分别有9只（4%）、2只（0.9%）和6只（2.7%）猫检测到FBoV-1、FBoV-2和FBoV-3。在17只阳性猫中，超过半数（9只，52.9%）携带FBoV-1，其次为6只（35.3%）携带FBoV-3和2只（11.8%）携带FBoV-2。
基于本研究中检测到的FBoV序列（每只猫一条序列）的ML分析系统发育结果见图1。系统发育分析证实，本研究中从猫获得的序列与GenBank中检索到的FBoV-1、FBoV-2或FBoV-3序列聚类在一起。
系统发育分析显示，本研究中部分序列存在显著的亚分支，其bootstrap值>90%，这些序列与核苷酸同一性为100%的序列相对应。这些序列分别来自三个FBoV-3序列（猫编号214、218和219）、两个FBoV-1序列（猫编号60和62）以及两个FBoV-2序列（猫编号63和66），这些猫要么来自同一家庭（猫编号218和219），要么在同一时间段住院。具有相同FBoV-1和FBoV-2序列的猫的样本属于同一提取/PCR批次，该批次还包括其他结果为阴性或FBoV阳性但基因型和序列不同的样本。具有相同FBoV-3序列的猫的样本也属于同一提取/PCR批次，该批次还包括其他阴性样本。
在53只采集了粪便样本的猫中，根据腹泻状态统计的FBoV或FPV单一阳性以及FBoV基因型与FPV共同阳性的结果，在部分多次采样的猫中存在不一致。在这些动物中，同一只猫的不同样本显示，一个样本为FBoV单一阳性而其他样本为FPV单一阳性，或者一个样本为FBoV或FPV单一阳性而其他样本同时检测到FBoV和FPV（补充表S1）。
因此，关于FBoV基因型和FPV单一阳性或共同阳性检测的描述性结果按单个粪便样本报告（表3）。结果显示，在腹泻猫的FBoV阳性样本中，FBoV-1与FPV共同阳性占阳性样本的最高比例。
3.3. 与FBoV阳性相关的变量
基于Pearson卡方检验或Fisher精确检验评估不同变量间FBoV阳性猫比例差异的统计分析结果见表1。在分析粪便样本的猫中，年轻年龄、流浪/收容所状态、住院以及FPV阳性与FBoV阳性显著相关。未检测到FBoV阳性与其他所考虑变量之间的相关性。
未发现与临床病理变化有明显关联；17只阳性猫中有12只有可用的血液学和生化数据。12只猫中有3只的临床病理检测几乎可以忽略：其中两只轻度贫血，一只尿素和红细胞压积升高，可能与脱水一致。其余9/12只猫表现为高血糖，其中5例与全白细胞减少症相关，3例与淋巴细胞减少和伴有毒性改变的中性粒细胞性白细胞增多相关。在大多数病例中，这些变化与临床症状、FPV或FIV或FeLV阳性相关（表4）。
4. 讨论
猫博卡病毒（FBoV）被分为三种不同的基因型，目前被认为是影响猫的新发病毒性病原之一，尽管其致病潜力仍不明确。本研究调查了意大利北部的FBoV，证明在洛迪兽医教学医院检查的猫中检测到了FBoV，表明该病毒并非如近期报道的那样仅在意大利南部流行[23]。
在便利抽样研究的猫群体中，PCR分析观察到的总阳性率为7.6%，对应7.2%的阳性样本，该结果基于粪便、呼吸道、尿液和血液样本的分析，可能受到本研究采样方法的影响。事实上，动物是因诊断目的而被招募的，便利抽样无法推断意大利猫群体中FBoV的流行率。这种采样类型可能导致高估偏倚。然而，本研究中检测到的总阳性率与中国364只猫不同类型样本中观察到的2.5%阳性率一致[10]。
根据具体样本类型，本研究中粪便样本中FBoV的检测是预期之中的，且此前已有报道。本研究中分析粪便样本的猫中观察到的32%阳性率，与中国猫粪便样本中25.9%的流行率[2]以及意大利急性胃肠炎猫直肠拭子中22.2%的流行率[23]的研究一致。然而，也有报道低于本研究的流行率，中国、越南和日本猫直肠拭子/粪便样本的阳性率范围为2.4%至9.9%[10,16,19]。除便利抽样可能相关的高估外，不同研究间阳性率的差异可能由多种因素导致，包括猫群体、地理区域以及用于检测FBoV的诊断方法的差异。事实上，本研究使用的PCR检测方法未在当前实验室进行灵敏度和特异性的分析验证，因此不能排除假阳性和假阴性结果，这可能影响了本研究中FBoV频率的估计。
在分析的猫血液、呼吸道和尿液样本中，FBoV检测结果为阴性，这表明在所采用的检测方法下，相对有限的受检样本中未检测到FBoV。因此，不能排除这些样本类型中存在低水平或间歇性检出的可能性，需要开展涉及大量样本的进一步研究来证实这些结果。事实上，虽然已有报道称猫的血液、呼吸道和尿液样本中FBoV阳性率极低[10]，但也有一些研究观察到较高的阳性率[14,22,32]。
我们的结果显示，在来自意大利的猫中检测到了全部三种FBoV基因型，其中FBoV-1检出频率最高，这证实了近期在意大利猫群中报道的以及此前在其他国家报道的全部三种FBoV基因型的流行情况。FBoV-1也是本研究中检出频率最高的FBoV基因型，与此前的发现一致[23,33]。我们对连续编号的猫样本中检测到的相同FBoV序列进行了验证。基于实验室检测流程和所获得的结果，排除了潜在的批次相关假象，但同一家庭中在同一天连续处理的两只猫除外。
本研究中大多数动物从不同猫中检测到不同毒株，这支持了此前报道的宿主间遗传多样性的假说[10]。然而，使用单一NP1靶标可能影响了对遗传上存在差异的毒株的检测，本研究中鉴定出的序列可能无法全面代表意大利流行的FBoV的遗传多样性。
年轻年龄和流浪/收容所状态作为与FBoV阳性相关的变量，此前已被报道为FBoV感染的危险因素[2,4,32,33]，尽管其他研究未发现年龄或饲养状态与FBoV感染相关[16,19]。年轻猫的FBoV阳性可能归因于其免疫系统尚未成熟，而流浪猫/收容所猫与有主猫相比，FBoV阳性风险可能更高，原因是居住条件拥挤且持续暴露于其他感染猫，提示高应激环境可能是促进FBoV传播的重要因素[2]。然而，由于本研究未测量应激水平，需要进一步研究来证实这一方面。
关于FBoV的致病性，应当指出，这是一项主要基于PCR检测的观察性研究，而非旨在确立因果关系的研究。尽管如此，仅在粪便样本中发现FBoV可能提示其在胃肠道疾病中具有潜在的致病作用。不同国家的流行病学调查提示，FBoV可能导致猫腹泻，患病率为5.5%–25.9%[2,10,15,16,23]，并且可能与出血性肠炎的暴发相关[17]。然而，我们的研究未能证实此前在中国猫中报道的FBoV感染与腹泻之间的总体关联[2,27]。另一方面，我们的结果与此前的研究一致，即从有和无胃肠道症状的猫采集的粪便样本中FBoV阳性率相似[19]，提示FBoV可能不是引起猫腹泻的原发病原体。此外，血液学或生化数据（如有）为非特异性的（例如，高血糖不伴随其他血液学或生化改变，可能由应激引起），或与炎症性肠道疾病相关，尤其是在细小病毒阳性和泛白细胞减少症的猫中。这些发现提示，可能的FBoV阳性除原发肠道疾病（如有）引起的变化外，不诱导特定的血液学或生化改变。然而，血液学和生化结果应谨慎解读，因为17只阳性猫中仅有12只有完整的临床病理学数据，且缺乏特异性异常可能不能排除FBoV的临床相关影响。此外，这些数据是根据临床指征获取的，而非在标准化的前瞻性方案下获得。因此，需要进一步研究来系统评估FBoV感染是否与特定的血液学或生化改变相关。
流行病学调查表明，FPV与FBoV的混合感染较为常见[23,34]。在腹泻猫中，FPV与FBoV混合感染的检出频率较高，而健康猫中则以单一感染更为常见[27]。本研究中在腹泻猫样本中观察到的FBoV和FPV单一阳性或共同阳性结果，需要进一步研究以证实我们的发现。事实上，本研究结果纯属描述性，来自极少数个体粪便样本，且部分观察基于同一只猫的多个样本，不同样本间有时出现不一致的结果。因此，本粪便样本分析中的观察结果不能视为独立，也无法得出推断性结论。此外，腹泻猫样本数量有限，无法判断FBoV-1是否比其他FBoV基因型具有更强的致病性。其他研究发现，FBoV-1和FBoV-2与幼猫腹泻的关联显著高于FBoV-3[33]。FBoV不同型别的致病性差异需要进一步研究，以确定这些差异是否可能仅仅是由于FBoV-1检出频率较高所致，因为我们的研究也显示FBoV-1是唯一以单一阳性检出的FBoV基因型。确实，腹泻猫中FBoV-1检出率较高可能反映的是基因型频率的差异，而非毒力或致病性的差异。
观察到的FBoV阳性与住院之间的关联并不令人意外。部分住院猫有多个样本，首次采样阴性而第2次或第3次采样阳性，这值得进一步前瞻性研究，以明确FBoV阳性是否可能发生在猫入院之后。在同一天住院的部分猫中检出相同的FBoV序列，进一步需要额外研究以探讨住院期间猫与猫之间潜在传播的可能性。事实上，本观察性设计未在入院前检测FBoV，无法确定所有猫的阳性是否发生在入院之后。同样无法排除以下可能性：猫因自身临床状况而住院，而这些状况本身增加了FBoV检出的可能性，或者因为这些猫更可能接受粪便采样。此外，重要的潜在混杂因素，包括年龄、饲养状态、临床疾病、采样类型和FPV感染，可能与住院相关。因此，本研究中观察到的FBoV阳性与住院之间的关联应谨慎解读，需要进一步的前瞻性研究来探讨院内传播的可能性。兽医医院和诊所中已报道了不同的院内感染，也称为医疗保健相关感染（HAIs）[35]。迄今为止，关注重点集中在细菌感染，尤其是抗菌药物耐药菌引起的感染，但院内感染也可由非细菌性病原体引起，包括病毒、真菌和寄生虫[36]。在兽医环境中，FPV已成为院内传播风险最高的病毒之一，而病毒尤其令人关注，因为细小病毒具有环境稳定性高、易于传播以及往往在医院群体中快速扩散的特点[37]。考虑到FBoV与FPV一样属于细小病毒科，且最近已有FBoV环境污染的报道[38]，FBoV在兽医医院中的传播风险需要进一步研究。
FBoV在猫之间的传播可能通过粪口途径[13,39]。虽然本研究未重点关注FBoV粪便排毒的持续时间，但我们在一小部分采集了多个粪便样本的猫中获得的初步结果表明，FBoV阳性通常持续数天，而少数猫可能出现延长的粪便排毒，持续至少两周。类似地，最近有报道称临床康复后FBoV病毒排毒时间延长，其中一只幼猫的FBoV DNA检出时间长达65天[38]。这与细小病毒科其他病毒报道的排毒持续时间一致，例如猫的FPV，其排毒通常持续1–2天或5–7天，罕见情况下可长达6周，早期研究中曾有一只猫的报道[40]。然而，仍需进一步研究以确认猫FBoV粪便排毒的持续时间以及环境污染的存在（和持续时间），从而进一步了解传播风险。
本研究存在一些局限性。首先，这是一项观察性研究，并非旨在确立FBoV与患病猫之间的因果关系。鉴于FBoV阳性猫的数量有限，未进行基于多变量模型的统计分析。因此，所识别的变量与FBoV阳性之间的关联无法被确认为独立危险因素。此外，由于未对其他猫腹泻、呼吸道或CKD相关病原体进行调查，疾病病因未能确定。本研究未调查同一动物不同样本中FBoV的存在情况，以确认先前报道的广泛组织分布[10]。在本研究中，仅使用NP1基因的一个片段进行FBoV基因型鉴定，可能影响了对毒株间遗传差异的检测。因此，本研究中检测到的FBoV毒株的遗传多样性可能被低估。基于全基因组测序的进一步研究对于全面表征意大利流行的FBoV毒株的遗传特征至关重要。
5. 结论
这项观察性研究的结果显示，在来自意大利的猫中检测到了FBoV-1、FBoV-2和FBoV-3，需谨慎解读，因为便利采样限制了所观察到的FBoV阳性率向更广泛意大利猫群体推广的能力。本研究未确立FBoV在胃肠道疾病中的因果作用。然而，FBoV的检测凸显了进一步研究来自意大利的FBoV阳性猫的遗传特征、流行病学、排毒动态和致病性的必要性。
查看英文原文▾
Background: Feline bocavirus (FBoV) is considered one of the emerging viral agents affecting cats. Methods: this observational study investigated FBoV occurrence by convenience sampling of 224 cats admitted to the Veterinary Teaching Hospital of Lodi, Italy, in 2020–2025 using PCR targeting the NP1 gene. Positive samples were phylogenetically analyzed. Results: FBoV positivity was observed in 17/224 (7.6%) cats. FBoV genotypes 1 (4%), 2 (0.9%) and 3 (2.7%) were identified. Out of the animals with collection of fecal samples, FBoV-positive cats were 17/53 (32.1%), whereas cats with blood (n. 69), respiratory (n. 54), urine (n. 46), or other (n. 2) samples were FBoV-negative. In a subset of cats with more than one fecal sample collected, viral carriage was detected for up to two weeks. Young age, stray/shelter condition and hospitalization were significantly associated with FBoV positivity (p = 0.0008, p = 0.0139 and p = 0.0023, respectively). In cats with analyzed fecal samples, Feline panleukopenia virus (FPV) positivity was associated with FBoV positivity (p < 0.001), whereas presence of diarrhea was not associated with FBoV occurrence. Conclusions: the sampling strategy did not allow us to estimate FBoV prevalence in the cat population. Nevertheless, the circulation of genetically diverse FBoV strains among cats in Italy, the association of FBoV positivity with hospitalization and results suggesting that FBoV may not be a primary pathogen for gastrointestinal disease underscore the importance of comprehensive surveillance studies to enhance the understanding of the evolutionary characteristics, epidemiology and pathogenic role of FBoV.
1. Introduction
Bocaparvoviruses (BoVs), formerly known as bocaviruses, are an emerging group of viruses in the family Parvoviridae that were first identified in 2005 in children affected by acute lower respiratory tract infections and gastroenteritis and were later detected in a variety of animal species, indicating a broad host range [1,2,3,4]. Transmission of BoVs commonly occurs via the fecal–oral route, leading to respiratory and gastrointestinal signs in young animals and humans, whereas infections in adults are frequently subclinical [5,6,7]. Human bocavirus (HBoV) has also been occasionally detected in serum and urine of infected children, suggesting the presence of other possible HBoV-associated disease [8,9].
Feline bocavirus (FBoV) was first identified in 2012 in fecal, urine, kidney, nasal and blood samples from stray cats with or without clinical signs in Hong Kong [10] and is now considered one of the emerging viral agents affecting cats [4,11,12]. To date, besides other studies detecting FBoVs in cats from China [2,3,13,14], FBoVs have been reported in several countries, including the USA [15], Canada [11], Japan [16], Thailand [17,18], Vietnam [19], Belgium [20], Portugal [21], Turkey [22] and Southern Italy [23].
Three genotypes of FBoV (FBoV-1, FBoV-2, and FBoV-3) have been described worldwide in feline populations [16,19]. FBoV-1, FBoV-2 and FBoV-3 have been reclassified as carnivore bocaparvovirus-3, carnivore bocaparvovirus-4 and carnivore bocaparvovirus-5, respectively [24].
The pathogenic role of FBoV is unclear as FBoV has been identified in both healthy cats and cats with clinical disease [10]. Regarding the potential pathogenicity, similarly to other bocaviruses, FBoV has been identified in cats presenting with diarrhea, including hemorrhagic enteritis [13,16,17,23]. Nervous system infection associated with FBoV infection has been observed in cats [20] and recently FBoV has also been considered a potentially neurotropic virus [3,14,18].
The epidemiology, evolution, diversity and pathogenicity of FBoV in cats are still under investigation. Mirroring bocavirus infections in other animals, further studies are still required to better understand the potential role of FBoV also in respiratory and urinary tract disorders in cats.
Due to the limited number of studies on FBoV in Italy, the aims of this observational study based primarily on PCR detection were (i) to investigate the presence of FBoV viral DNA in cats from Italy using PCR methods and sequencing analysis to characterize circulating FBoV strains, (ii) to investigate the potential pathogenic role of FBoV by investigating FBoV viral DNA in fecal, urine, respiratory and blood samples from cats and (iii) to assess the association between FBoV positivity, clinical status and patient variables.
2. Materials and Methods
2.1. Animals and Sampling
This observational study investigated FBoV occurrence by convenience sampling of 224 cats admitted to the Veterinary Teaching Hospital of Lodi, Italy for routine diagnostic procedures between 2020 and 2025. Blood, feces, urine, respiratory specimens or other types of samples were collected from cats.
All samples were collected in the context of clinicopathological investigations or as part of health checks requested by the referring veterinarians following informed consent of the owners. Following diagnostic procedures, samples and/or extracted DNA were subsequently stored at −80 °C for further molecular analysis.
All samples were collected for diagnostic purposes or as part of health checks and with informed consent of the owners and therefore according to the guidelines of the Institutional Animal Care and Use Committee of the University of Milan, a formal approval was not required.
Data regarding time of sampling, sex, age, and type of ownership were recorded for each animal. Age was categorized as juvenile (<1 year) and adult (≥1 year) [14]. Data from routine diagnostic analysis based on qPCR to detect Feline panleukopenia virus (FPV) in fecal samples, according to a published protocol [25], were also recorded. For fecal samples only, the occurrence of diarrhea and FPV positivity was also considered.
2.2. FBoV Detection by PCR
DNA was extracted from whole blood, respiratory and urine samples using a commercial DNA extraction kit (NucleoSpin Blood® kit, Macherey-Nagel, Düren, Germany), following manufacturer’s instructions. DNA was extracted from fecal samples using a commercial DNA extraction kit (NucleoSpin® Tissue Kit, Macherey-Nagel, Düren, Germany), according to the manufacturer’s instructions. The extracted DNA was stored at −20 °C until further molecular analysis. DNA pre-analytical quality control was performed on all samples by a PCR targeting vertebrate 12S rRNA locus [26].
A PCR capable of simultaneously detecting FBoV-1, FBoV-2, and FBoV-3 by targeting a proportion of the NP1 gene was performed as previously described. The PCR amplifies 464, 503 and 491 bp fragments for FBoV-1, FBoV-2 and FBoV-3, respectively [27]. The PCR assay was not analytically validated for sensitivity and specificity in the present laboratory. PCR was performed in a final reaction volume of 25 μL containing 1x Phire Green Hot Start II PCR (Thermo Fisher Scientific, Segrate –Milan, Italy), 1 μM of forward primer FBoV1-2-3 NP1 F (5′- AGA ACC RCC RAT CAC ART CCACT-3′), 1 μM reverse primer FBoV1-2-3 NP1 R (5′- TGG CRA CCG CYA GCA TTT CA-3′), and 5 μL of template DNA. Amplification was performed with an initial denaturation step at 98 °C for 30 s, followed by 40 cycles of denaturation at 98 °C for 5 s, annealing at 51 °C for 5 s, and extension at 72 °C for 10 s. A final extension step was performed at 72 °C for 1 min. PCR products were separated by electrophoresis on 1.5% agarose gel and visualized under UV light. A positive control (a FBoV field sample previously obtained in our laboratory and confirmed by sequencing as FBoV-3) and a blank control (DNAse-free water sample) were also included in the PCR reaction.
When multiple samples were collected and analyzed from the same animal, cats were considered FBoV-positive if at least one sample was positive.
Samples showing a band of the expected size were further subjected to sequencing for characterization of FBoV genotype.
2.3. FBoV Genotype and Phylogeny
Amplicons from PCR-positive results were purified and sequenced using the same internal forward and internal reverse primers used in the DNA amplification. Sanger sequencing was performed by a commercial sequencing facility (Microsynth Seqlab, Germany). BioEdit software version 7.0 https://thalljiscience.github.io/ (accessed on 3 March 2026) was used to assemble the sequence data. BLAST https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 10 July 2026) was used to compare the sequences with those available in GenBank. Clustal X in BioEdit software v.7.0 was used to align all the consensus sequences against the FBoV genotypes retrieved from GenBank to identify the FBoV genotype of the strain.
Sequences aligned with FBoV reference sequences retrieved from GenBank were subjected to phylogenetic analysis using Clustal X in BioEdit software v.7.0. In the presence of identical sequences from the same animal, only one representative sequence/cat was selected and phylogenetically analyzed. Maximum likelihood (ML) methods with 1000 bootstrap replicates using MEGA v.12 were used for phylogeny [28,29,30]. The General Time Reversible model with Gamma distribution and invariant sites used for phylogeny based on the ML method was selected according to the lowest AIC value.
For sequence comparison, the percentage of nucleotide similarity of pairwise evolutionary distances was calculated using MEGA v.12.
The sequences generated in this study were submitted to GenBank with accession numbers PZ787303- PZ787319.
2.4. Hematological and Biochemical Tests
In order to investigate the presence of hematological or biochemical changes possibly associated with FBoV positivity, the database of the VTH was examined to assess whether cats that were PCR positive also underwent hematological or biochemical analyses.
When requested by the clinical presentation, blood was collected on the same day as the collection of samples submitted for PCR. More specifically, 1 mL of blood was placed in EDTA tubes, and the remaining amount was placed in tubes without anticoagulant. Tubes were immediately submitted to the clinical pathology laboratory of the VTH. EDTA tubes were used to perform a complete blood cell count (CBC) with the laser-based analyzer Sysmex XN1000V (Sysmex Corporation, Kobe, Japan). Tubes without anticoagulant were centrifuged (10 min, 4500 rpm) and serum was used to perform a routine clinical chemistry panel using the spectrophotometer BT3500 (Futurlab Srl, Limena PD, Italy).
2.5. Data Analysis
For FBoV detection in cats, a sample size of 224 animals was calculated using WinEpi (http://www.winepi.net/uk/index.htm, accessed on 1 October 2025), considering a population size of approximately 6000 cats admitted to the VTH in 2020–2025 for routine diagnostic analysis, a minimum prevalence of 1.3% calculated as the mean positivity in cat’s samples with low positivity [10], and 95% confidence level.
Pearson’s chi-square test or Fisher’s exact test was used to evaluate the differences between proportions of FBoV-positive animals and sex, age categories and hospitalization. In the subgroup of cats with analyzed fecal samples, Fisher’s exact test was also used to evaluate the differences between proportions of FBoV-positive animals and parvovirus positivity and presence of diarrhea. In the presence of more than one fecal sample from the same cat, a cat was considered FPV-positive if at least one sample was FPV-positive. Statistical comparisons based on Pearson’s chi-square test or Fisher’s exact test and calculation of odds ratio (OR) with 95% confidence intervals (CI) were carried out using Epitools [31], taking p < 0.05 as significant. The statistical unit used for each analysis was represented by the animal.
Sample-level data were also reported for detection of FBoV and FPV single positivity or co-positivity in samples from cats with and without diarrhea as purely descriptive results, and these data were not statistically analyzed.
3. Results
3.1. Animals and Samples Tested
Out of the 224 total cats tested for FBoV, the median age was five years and two months, ranging from 1 month and 16 days to 18 years of age. Data of the animals’ characteristics based on number of cats analyzed, including presence of diarrhea and FPV positivity in cats from which fecal samples were collected are reported in Table 1.
A total of 263 samples were collected from these 224 cats. The samples comprised 80 fecal, 76 blood, 54 respiratory, 51 urine, and two other types of samples, collected from 53, 69, 54, 46 and two cats, respectively. The number of samples collected from each animal varied, and one sample was collected from 201 (89.7%) cats, whereas more than one fecal, blood or urine sample was collected from 23 (10.3%) cats. Regarding the subgroup of 53 cats with analyzed fecal samples, one fecal sample was collected from 39 (73.6%) cats, and more than one sample was collected from 14 (26.4%) cats, ranging from two to six samples (Table 2).
3.2. FBoV Occurrence and Genotype
In this observational study, FBoV was detected by PCR and confirmed by sequencing in 17/224 cats (7.6%; 95% C.I: 4.1–11.1%), corresponding to 19/263 (7.2%) positive samples. Results of FBoV-positive cats are reported in Table 1.
Among the 53 cats with analyzed fecal samples, when considering the 39 cats with only one fecal sample collected, results showed FBoV positivity in 10 (25.6%) cats. For the remaining 14 cats that underwent multiple fecal sampling, the results of FBoV PCR in each sample are reported in Table 2. Results in Table 2 show that out of the seven (50%) FBoV-positive cats, FBoV was detected in one sample only in five (71.4%) cats, and in two samples in two (28.6%) cats. The interval between collection of these two positive samples was one week in one cat (cat n. 36) and two weeks in the other cat (cat n. 78). All the seven FBoV-positive cats with multiple analyzed fecal samples were hospitalized for the whole period of sample collection.
Sequencing aimed at FBoV genotype identification was successfully obtained for all the FBoV-PCR positive samples. BLAST analysis of the NP1 fragments of the sequences from this study showed highest nucleotide identity ranging from 96.4% to 99.4% with FBoV sequences deposited in GenBank. For 10 sequences from this study, the highest nucleotide similarity was observed with FBoV sequences recently reported in Italy [23]. Moreover, four sequences from this study showed the highest nucleotide homology with a sequence from the USA (GenBank accession KM017745), two with a FBoV sequence from China (GenBank accession n. OR544348) and one with a FBoV sequence from Thailand (GenBank accession n. PP592986).
Nucleotide identity among the sequences from this study ranged from 55.2% to 100%. Identical FBoV sequences obtained from two positive samples collected from the same cats (cat n. 36 and cat n. 78) were observed. Among sequences from different cats, 100% nucleotide identity was observed in three (cats 214, 218, 219), two (cats 60, 62) and in two sequences (cats 63, 66).
According to sequencing results and BLAST analysis, FBoV-1, FBoV-2 and FBoV-3 were detected in nine (4%), two (0.9%) and six (2.7%) cats, respectively. Of the 17 positive cats, more than half (nine, 52.9%) harbored FBoV-1, followed by FBoV-3 in six (35.3%) and FBoV-2 in two (11.8%) cats.
Results of phylogeny based on ML analysis of FBoV sequences detected in this work (one sequence/cat) are reported in Figure 1. Phylogeny confirmed that the sequences obtained from cats in this study clustered with either FBoV-1, FBoV-2 or FBoV-3 sequences retrieved from GenBank.
Phylogeny showed the presence of significant subclades with bootstrap values > 90% among some of the sequences from this study corresponding to sequences with 100% nucleotide identity. These sequences were obtained from three FBoV-3 sequences (cats n. 214, n. 218 and n. 219), two FBoV-1 sequences (cats n. 60 and 62) and two FBoV-2 sequences (cats n. 63 and n. 66) detected in cats that were either from the same household (cats n. 218 and n. 219) or hospitalized during the same days. Samples from cats with identical FBoV-1 and FBoV-2 sequences were part of the same extraction/PCR batch including other samples with either negative results or FBoV positivity with different FBoV genotypes and sequences. Samples from cats with identical FBoV-3 sequences were also part of the same extraction/PCR batch including other negative samples.
The results of single positivity of FBoV or FPV and co-positivity of FBoV genotypes and FPV according to diarrhea status in 53 cats with fecal samples collected were discordant in some of the cats with multiple samplings. In these animals, different samples from the same cat showed single positive results for FBoV in one sample and FPV in other samples, or single positive results for FBoV or FPV in one sample and simultaneous detection of FBoV and FPV in other samples (Supplementary Table S1).
Therefore, the descriptive results regarding the detection of single positivity or co-positivity for FBoV genotypes and FPV are reported according to individual fecal samples (Table 3). Results show that, among FBoV-positive samples from cats with diarrhea, FBoV-1 and FPV co-positivity accounted for the highest percentage of positive samples.
3.3. Variables Associated with FBoV Positivity
Results of statistical analysis based on Pearson’s chi-square test or Fisher’s exact tests to evaluate differences in the proportions of FBoV-positive cats according to the variables are reported in Table 1. Young age, stray/shelter status, hospitalization and FPV positivity in cats with fecal samples analyzed were significantly associated with FBoV positivity. No correlation was detected between FBoV positivity and the other variables considered.
No evident association with clinicopathological changes was found; hematological and biochemical data were available for 12 of the 17 positive cats. Clinicopathological testing was almost negligible for 3/12 cats: two of these were slightly anemic and one had increased urea and hematocrit potentially consistent with dehydration. The remaining 9/12 cats presented with hyperglycemia, which was associated with panleukopenia in 5 cases, and with lymphopenia and neutrophilic leukocytosis with toxic changes in 3 cases. In most cases these changes were associated with clinical signs, FPV or FIV or FeLV positivity (Table 4).
4. Discussion
Feline bocavirus (FBoV) is classified into three distinct genotypes and is currently considered one of the emerging viral agents affecting cats, although its pathogenic potential remains unclear. This study investigated FBoV in Northern Italy, demonstrating that FBoV was detected among cats examined at the Veterinary Teaching Hospital of Lodi, showing that the virus is not only circulating in Southern Italy, as recently reported [23].
The 7.6% overall positivity observed by PCR analysis in the study population of cats by convenience sampling, corresponding to 7.2% of positive samples, was based on analysis of fecal, respiratory, urine and blood samples and may have been influenced by the sampling method used in this study. Indeed, animals were recruited for diagnostic purposes, and convenience sampling does not allow us to infer FBoV prevalence in the Italian cat population. A possible overestimation bias due to this type of sampling may have occurred. However, the overall positivity detected in our study is in line with the 2.5% positivity observed in different types of samples in China collected from 364 cats [10].
Depending on the specific sample type, the detection of FBoV in fecal samples in our study was expected and has been reported previously. The 32% positivity observed in the cats with fecal samples analyzed of this work agrees with studies showing prevalence of 25.9% in cats’ fecal samples from China [2] and 22.2% in rectal swabs collected from Italian cats with acute gastroenteritis [23]. However, lower prevalence than that detected in our study has also been reported, with positivity ranging from 2.4% to 9.9% in rectal swabs/fecal samples from cats in China, Vietnam and Japan [10,16,19]. In addition to the potential overestimation related to convenience sampling, variations in positivity among different studies may be due to several factors, including differences in cat populations, geographical areas, and diagnostic approaches used to detect FBoV. Indeed, the PCR assay used in this study was not analytically validated for sensitivity and specificity in the present laboratory and therefore false positive and false negative results cannot be ruled out, which may have affected the estimated FBoV frequency in this study.
The FBoV-negative results in cats with blood, respiratory and urine samples analyzed indicate that FBoV was not detected in the relatively limited number of samples examined using the methodology employed. Therefore, low-level or intermittent detection in these sample types cannot be excluded and further studies involving a large number of samples are required to confirm these results. Indeed, while a very low prevalence has been reported in blood, respiratory and urine samples from cats [10], a few studies have observed higher positivity [14,22,32].
Our results showing the detection of all three FBoV genotypes in cats from Italy, with FBoV-1 being the most frequently detected, confirm the circulation of all three FBoV genotypes in cat populations recently reported in Italy and previously reported in other countries. FBoV-1 was also the most frequently detected FBoV genotype in our study, in agreement with previous findings [23,33]. The detection of identical FBoV sequences in consecutively numbered cat samples was verified. Based on the laboratory processing and the results obtained, potential batch-related artifacts were excluded, except for two cats from the same household that were processed consecutively on the same day.
The detection of different strains from different cats in the majority of animals from this study supports the hypothesis of inter-host genetic diversity previously reported [10]. However, the use of a single NP1 target may have affected the detection of genetically divergent strains and the sequences identified in this study may not comprehensively represent the genetic diversity of FBoV circulating in Italy.
Young age and stray/shelter status as variables associated with FBoV positivity have previously been reported as risk factors for FBoV infection [2,4,32,33], although other studies did not identify age or ownership status as associated with FBoV infection [16,19]. FBoV positivity in young cats may be explained by their immature immune system whereas stray/shelter cats may be at higher risk of FBoV positivity compared to owned cats because of the crowded living conditions and constant exposure to other infected cats, suggesting that a high-stress environment may be an important factor contributing to the circulation of FBoV [2]. However, as stress was not measured in this study, additional investigations are needed to confirm this aspect.
Regarding the pathogenicity of FBoV, it should be noted that this was an observational study based primarily on PCR detection rather than a study designed to establish causality. Nevertheless, the finding of FBoV exclusively in fecal samples may suggest a potential pathogenic role in gastrointestinal disease. Epidemiological investigations in different countries have suggested that FBoV may cause diarrhea in cats with a prevalence of 5.5–25.9% [2,10,15,16,23] and may also be associated with outbreaks of hemorrhagic enteritis [17]. However, our study did not confirm the overall association between FBoV infection and diarrhea that was previously reported in cats from China [2,27]. On the other hand, our results are in line with previous studies showing similar FBoV positivity in fecal samples collected from cats with and without gastrointestinal symptoms [19], suggesting that FBoV may not be a primary pathogen to cause diarrhea in cats. Moreover, hematological or biochemical data, when present, were nonspecific (e.g., hyperglycemia not associated with other hematological or biochemical changes, likely induced by stress) or associated with inflammatory intestinal disease, particularly in cats with parvoviral positivity and panleukopenia. These findings suggest that the possible FBoV positivity does not induce specific hematological or biochemical changes other than those induced by the primary intestinal disease, when present. However, hematological and biochemical results should be interpreted cautiously because only 12 of the 17 positive cats had complete clinicopathological data and the absence of specific abnormalities may not exclude clinically relevant effects of FBoV. Furthermore, these data were obtained according to clinical indications rather than under a standardized prospective protocol. Therefore, additional studies are needed to evaluate systematically whether FBoV infection is associated with specific hematological or biochemical alterations.
Epidemiological investigations have shown that coinfections with FPV and FBoV are common [23,34]. FPV and FBoV coinfections have been identified with higher frequency in cats with diarrhea, whereas healthy cats show higher frequency of monoinfections [27]. The detection of FBoV and FPV single positivity or co-positivity observed in our study in samples from cats with diarrhea require further investigations to confirm our findings. Indeed, the results from this study were purely descriptive, were obtained from very small numbers of individual fecal samples and some observations were based on multiple samples from the same cat sometimes showing discordant results in different samples. Therefore, the observations in this fecal-sample analysis cannot be considered independent and no inferential conclusions can be drawn. Moreover, the limited numbers of samples from cats with diarrhea did not allow us to understand if FBoV-1 has greater pathogenicity than the other FBoV genotypes. Other studies have found FBoV-1 and FBoV-2 to be significantly more associated with diarrhea in kittens than FBoV-3 [33]. The different pathogenicity of FBoV types requires further investigation to determine whether these differences may instead be due to the higher frequency of FBoV-1 detection, given that our study also showed that FBoV-1 is the only FBoV genotype detected with single positivity in cats. Indeed, the higher detection of FBoV-1 in cats with diarrhea may reflect differences in genotype frequency rather than differences in virulence or pathogenicity.
The observed association between FBoV positivity and hospitalization was not surprising. The negative results at the first sampling followed by positivity at the 2nd or 3rd sampling in some of the hospitalized cats with multiple samples warrant further prospective investigation to define if FBoV positivity may occur in cats after admission to veterinary hospitals. The detection of the same FBoV sequences in some cats hospitalized on the same days further requires additional study to investigate potential transmission between cats during hospitalization. Indeed, the present observational design did not investigate FBoV prior to admission to determine whether positivity occurred after admission in all cats. It is also not possible to rule out the possibility that cats were hospitalized because of clinical conditions that themselves increased the likelihood of FBoV detection or because they were more likely to undergo fecal sampling. In addition, important potential confounding factors, including age, ownership status, clinical disease, sampling type, and FPV infection, may be associated with hospitalization. Therefore, the association between FBoV positivity and hospitalization observed in this study should be interpreted with caution, and further prospective studies are needed to investigate the possibility of nosocomial transmission. Different nosocomial infections, also referred to as healthcare-associated infections (HAIs), have been reported in veterinary hospitals and clinics [35]. Up to now, attention has focused on bacterial infections, especially those caused by antimicrobial resistant bacteria, but nosocomial infections can also be caused by non-bacterial pathogens, including viruses, fungi, and parasites [36]. In veterinary settings, FPV has emerged as one of the viruses with the highest risk of nosocomial transmission, and viruses are of particular concern because parvoviruses are characterized by high environmental stability, ease of transmission, and often rapid spread within hospital populations [37]. Considering that FBoV, like FPV, belongs to the family Parvoviridae, and that environmental contamination by FBoV has recently been reported [38], the risk of FBoV transmission in veterinary hospitals requires further investigation.
FBoV transmission among cats is likely via the fecal route [13,39]. Although this study did not focus on the duration of FBoV fecal shedding, our preliminary results on a limited number of cats with more than one fecal sample collected suggest that FBoV-positivity generally lasts for a few days, whereas a minority of cats may have prolonged fecal shedding lasting for at least two weeks. Similarly, prolonged FBoV viral shedding after clinical recovery has been recently reported, and in one kitten FBoV DNA has been detected up to 65 days [38]. This aligns with the shedding duration reported for other viruses belonging to the Parvoviridae family, such as FPV in cats, for which shedding typically lasts 1–2 or 5–7 days, and rarely up to 6 weeks as reported in one cat in an early study [40]. However, further studies are needed to confirm the duration of FBoV fecal shedding in cats as well as the presence (and duration) of environmental contamination, to further understand the risk of transmission.
This study has some limitations. First of all, this was an observational study and was not designed to establish causality of FBoV in diseased cats. Given the limited number of FBoV-positive cats, statistical analysis based on a multivariable model was not performed. Therefore, the identified association between variables and FBoV-positivity could not be confirmed as independent risk factors. Moreover, the cause of disease was not determined, as other feline diarrhea-, respiratory- or CKD-associated pathogens were not investigated. Our study did not investigate the presence of FBoV in different samples from the same animal to confirm wide tissue distribution previously reported [10]. In this study, the use of only a fragment of the NP1 gene for FBoV genotype identification may have affected the detection of genetic differences among strains. Therefore, the genetic diversity of the FBoV strains detected in this study may have been underestimated. Further studies based on whole genome sequencing are mandatory to fully characterize the genetic features of FBoV strains circulating in Italy.
5. Conclusions
Results of this observational study showing the detection of FBoV-1, FBoV-2 and FBoV-3 in cats from Italy need to be interpreted with caution as the convenience sampling limits the generalizability of the observed FBoV positivity to the wider Italian cat population. This study did not establish a causal role of FBoV in gastrointestinal disease. Nevertheless, the detection of FBoV highlights the need for further studies on the genetic features, epidemiology, shedding dynamics and pathogenicity in FBoV-positive cats from Italy.