Pérez de Val et al. BMC Veterinary Research (2022) 18:352 https://doi.org/10.1186/s12917-022-03454-7 CASE REPORT Open Access Generalized tuberculosis due to Mycobacterium caprae in a red fox phylogenetically related to livestock breakdowns Bernat Pérez de Val1,2* , Claudia Perea3, Josep Estruch4, Carlos Solano‑Manrique5, Carles Riera6, Albert Sanz6, Enric Vidal1,2 and Roser Velarde4 Abstract Background: Tuberculosis (TB) due to Mycobacterium caprae is endemic in goat herds and free-ranging wild boars in Spain, causing infections in other livestock or wild animals to a lesser extent. TB infection in foxes is infrequently reported and they are usually considered spillover hosts of TB. Case presentation: A blind, depressed and severely emaciated red fox (Vulpes vulpes) was admitted to a rehabilita‑ tion center. After clinical examination it was humanely sacrificed. At necropsy, generalized TB lesions were observed that were subsequently confirmed by histopathology along with a co-infection with canine distemper virus. M. caprae was isolated from mycobacterial culture and spoligotype SB0415 was identified. Whole genome sequencing (WGS) of the isolated M. caprae was carried out and single nucleotide polymorphisms (SNP) were compared with other sequences of M. caprae isolated from livestock and wildlife of the same area throughout the last decade. Conclusions: This is the first reported case of TB due to M. caprae in a fox in the Iberian Peninsula. WGS and SNP analysis, together with spatial-temporal investigations, associated this case with recent M. caprae outbreaks in cattle and goat herds of the area. The results indicated transmission of M. caprae between livestock and the fox, suggesting that this species may occasionally play a role in the epidemiology of animal TB. Keywords: Tuberculosis, Mycobacterium caprae, Fox, Livestock, Whole genome sequencing Background human TB [5], mainly through inhalation of mycobacte- Mycobacterium caprae is the main cause of goat tubercu- ria, but ingestion from contaminated dairy products can- losis (TB) in Spain [1] and has also been involved in TB not be ruled out. outbreaks in other livestock and wildlife species in con- The circulation of Mycobacterium tuberculosis com- tact with infected goats, such as cattle [2], sheep [3], and plex (MTBC), mainly M. bovis and M. caprae, in non- wild boars [4]. Moreover, M. caprae may be a source of bovine domestic and wild hosts is hampering the success of bovine TB eradication campaigns in Spain, which has been practically stagnant for the last two decades [6]. The *Correspondence: bernat.perez@irta.cat red fox (Vulpes vulpes) has been usually considered a 2 IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal spillover host of TB and it is rarely included in the active (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, surveillance of the disease, therefore only sporadic cases Catalonia, Spain Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pérez de Val et al. BMC Veterinary Research (2022) 18:352 Page 2 of 6 of TB due to M. bovis have been detected to date in Spain swabs in Löwenstein-Jensen with pyruvate and Coletsos [7, 8]. solid media (BD Diagnostics,) and incubated at 37 °C. Nowadays, the use of whole genome sequencing Culture growth was confirmed as MTBC by multi- (WGS) for epidemiological investigation of animal TB plex PCR [9]. DNA from MTBC growth was molecu- outbreaks and to study putative transmission events is larly characterized by DVR-spoligotyping, at VISAVET becoming more widespread. A case of red fox (Vulpes Health Surveillance Centre (Madrid, Spain), and WGS vulpes) with disseminated TB in Catalonia (Spain), posed followed by Single Nucleotide Polymorphisms (SNPs), at the first detection of M. caprae in this species in the Ibe- the National Veterinary Services Laboratories of the US rian Peninsula, and the investigation of its origin by using Department of Agriculture (Ames, IA, USA). epidemiological and WGS analysis aimed this study. At necropsy, the fox was severely emaciated. In the eyes, diffuse corneal edema, hypopyon, and the presence Case presentation of a few filiform worms in the conjunctival sac, consistent In February 2021, an adult female free-ranging red fox with Thelazia callipaeda, were noted (Fig.1a). Internally, was found blind and depressed in southern Catalonia retropharyngeal, prescapular, and mesenteric lymph (Spain). The animal was admitted to Vallcalent Wildlife nodes were moderately enlarged, and multifocal yellow Rehabilitation Centre (Lleida, Catalonia). The fox was areas of caseous necrosis were seen in the cranial medi- severely emaciated (2.94 kg), with abundant ectopara- astinal lymph node (Fig.1b). There was a diffuse severe sites (ticks and fleas) and filiform worms consistent with thickening of the pericardium and abundant caseopuru- Thelazia callipaeda in both conjunctival sacs. After lent exudate filled the pericardial cavity (Fig.1e). In the clinical examination, it was humanely euthanized. After- heart, white mild nodular thickening of the left atrio- wards, according to the Catalan Wildlife Health Surveil- ventricular and aortic valves was detected. From these lance Plan, it was submitted to the Veterinary Faculty affected valves, the inflammation extended locally into of the Autonomous University of Barcelona (Bellaterra, the myocardium. In the lungs, an increased consistency Spain) to carry out a complete necropsy. Tissue samples and brown-gray discoloration of the caudo-dorsal areas, from brain, eyes, retropharyngeal, thoracic, and mesen- consistent with verminous pneumonia, was recognized. teric lymph nodes, heart, pericardium, trachea, lungs, Several discrete white granulomas, 2 to 4 mm in diam- liver, spleen, kidneys, urinary bladder, adrenal glands, eter, were noted in the left parietal pleural surface. The and bone marrow were fixed in 4% neutral buffered for- liver was pale orange, had slightly rounded margins, and malin and processed and embedded in paraffin for rou- subtle multifocal to coalescent inflammatory infiltrates. tine microscopic examination. Intestinal samples were A diffuse, mild splenomegaly was also present. Severe not collected since they were too autolytic for their pre- multifocal to coalescent foci of caseous-purulent necrosis cise evaluation. Sections of 3–4 μm thick were stained were found in both kidneys (Fig. 1c). A moderate number with Mayer’s haematoxylin and eosin. Ziehl-Neelsen of cestodes was seen in the small intestine. staining was also carried out to detect acid-fast bacilli. Microscopically, diffuse granulomatous infiltrates and/ Immunohistochemistry was used to evaluate the pres- or granulomas compatible with TB lesions were seen ence of canine distemper virus (CDV) antigen in selected in most tissues. Diffuse granulomatous infiltrates were sections from the brain and lung. A monoclonal anti- present in the pericardium, epicardium, heart valves, body against the nucleoprotein of CDV was used (from interstitial renal medulla, and choroid layer in the eyes. VMRD, Ref.: CDV-NP, Ascites fluid, 1 mg/mL, IgG2b, Discrete miliary (up to 200 μm), round to oval granulo- kappa light chain, dilution used 1:5000). mas, characterized by a central core of macrophages sur- Fresh samples of kidney and mediastinal lymph node rounded by a thin rim of lymphocytes and plasma cells with TB compatible lesions were processed for bacteriol- were numerous in the cortical area of the kidneys, liver, ogy. Tissues were mechanically homogenized in 10 ml of lymph nodes and spleen. A few small granulomas were sterile water using an automatic homogenizer (Mastica- also found in the brain, bone marrow and lung. None of tor, IUL, Barcelona, Spain), decontaminated with 10 ml of the granulomas were encapsulated. The number of multi- 5% w/v oxalic acid for 30 min. in orbital shaking, neutral- nucleated giant cells was low and most of them had only ized with 5 ml of sodium hydroxide 1 M and centrifuged two to three nuclei (small). Areas of caseous necrosis at 2471×g for 30 min. Supernatants were discarded, containing few degenerate neutrophils were present in pellets were resuspended with 1 ml of sterile phosphate both granulomatous infiltrates and granulomas. Small buffered saline, and an aliquot of 0.5 ml was inoculated foci of mineralization were only seen in the valves. The in BBL MGIT supplemented tubes and incubated in the pericardial sac and epicardium were markedly thickened BACTEC MGIT 320 system (BD Diagnostics, Sparks, with fibroblasts proliferation and fibrosis, suggesting that MD, USA). The remaining 0.5 ml was cultured using this was the oldest lesion. Pérez de Val et al. BMC Veterinary Research (2022) 18:352 Page 3 of 6 Fig. 1 Red fox with generalized tuberculosis and canine distemper virus coinfection. Gross and histopathological findings. a Right eye, mild corneal edema and hypopyon, and few filiform worms consistent with Thelazia callipaeda in the conjunctival sac. b Cranial mediastinal lymph node with multifocal yellow areas of caseous necrosis. c Multiple foci of caseous-purulent necrosis in both kidneys. d Right eye, dense perivascular to diffuse infiltration of the choroid with histiocytes and lymphocytes, and a dense subretinal pyogranulomatous exudate (Hematoxylin and eosin stain, original magnification × 200, bar = 100 μm), Inset, pyogranulomatous exudate with multiple purple-stained acid-alcohol resistant bacteria (Ziehl-Neelsen stain, original magnification × 200, bar = 100 μm). e Diffuse thickening of the pericardium and abundant caseous-purulent exudate filling the pericardial cavity. f High quantity of acid-alcohol resistant bacteria within macrophages and necrotic areas in the kidneys (Ziehl-Neelsen stain, original magnification × 400, bar = 100 μm). All images taken by the authors In the eyes, a severe granulomatous choroiditis and distemper virus antigen was detected in bronchiolar epi- vitritis were established as the cause of the blindness thelial cells, alveolar macrophages in the lung and in the (Fig.1d). The exudate in the vitreous chamber caused the occasional neuron, glial, and ependymal cell in the brain. detachment and degeneration of the retina in both eyes. Verminous pneumonia was also confirmed. Arterial In the right eye, the granulomatous infiltrate and exu- damage within the lesions suggested Angiostrongylus date extended to the anterior uvea and chamber explain- vasorum as the possible etiology, although no adult par- ing the hypopyon. A mild keratitis characterized by mild asites were present in the sections. The main final diag- superficial edema and neutrophilic infiltrate with incipi- nosis was a generalized TB infection with a co-infection ent neovascularization at the limbus was also present. No with canine distemper virus. inclusion bodies were seen in the corneal or conjunctival Culture results revealed growth of M. tuberculosis epithelium. complex (MTBC) in both tissues, and the spoligopat- Numerous acid-fast and rod-shaped bacilli were seen tern M. caprae SB0415 (www.​mbovis.​org) was identi- within the macrophages and necrotic areas in all tissues, fied. Then, the whole genome sequences were analyzed including foamy macrophages in the lumen of the bron- together with other 9 recent M. caprae SB0415 isolates chi and renal pelvis consistent with an active shedding of (2018-2021) from cattle (1), goat (1) and wild boar (7) mycobacteria (Fig.1f ). of the same area and other 17 available whole genome Additionally, lesions consistent with CDV co-infec- sequences of previous M. caprae isolates in Catalonia tion were also present in the brain and the lungs. Mild (2008-2020) [4]. perivascular lymphocytic cuffing was noted in the section WGS analysis showed a close phylogenetic relation- from the hippocampus in the brain. In the lung, the pres- ship between the fox isolate and another one (8 SNP pair- ence of patchy necrosis, and attenuation of bronchiolar wise distance) isolated 1 year before from a cattle herd epithelium along with lymphocytes around bronchioles located at the same municipality where the ill fox was and vessels, alveolar edema, and the occasional alveolar found (Fig. 2). In addition, the strains isolated from two epithelial syncytial cell were consistent with multifocal goat herds in 2015 and 2019, located at 56 and 18 Km viral subacute bronchiolo-interstitial pneumonia. Canine from the cattle-fox outbreak, respectively, also showed Pérez de Val et al. BMC Veterinary Research (2022) 18:352 Page 4 of 6 Fig. 2 Phylogenetic and spatiotemporal relationships of M. caprae strains. a Phylogenetic tree of M. caprae isolates in Catalonia. A rooted phylogenetic tree, based on maximum likelihood model (GTR-CAT, RAxML), shows the SNP distance of the 27 M. caprae strains isolated in Catalonia between 2008 and 2021. Infected animal species (cattle, goats, wild boars or fox) are shown with silhouettes next to each branch. The strain isolated from the fox (blue silhouette) formed a clade together with the strain isolated from a cattle herd (blue silhouette, < 1Km) and two goat herds (blue silhouettes, 18 and 56 Km, respectively). The dates of isolation are shown next to the silhouettes. b Location of the M. caprae clade isolates. The inset shows a region of southwestern Catalonia. Species are shown by silhouettes. Dates of isolation and distance between the fox and livestock herds are indicated in the map a close phylogenetical relationship with the fox strain (9 between both species. In addition, at least two goat herds SNP pairwise distance, Fig. 2). These four isolates share a might be related with the origin of this outbreak accord- common ancestor within 6.5 SNP (range 5-8). ing to SNP pairwise distance, estimated in < 12 SNP Additionally, in the context of the Catalan wildlife distance between individuals in other studies [4, 15]. health surveillance plan, 3 out of the 9 (33%) wild boar Unfortunately, only fragmentated data on goat TB cases sera sampled in the outbreak municipality in 2020 were is available due to the absence of a mandatory caprine seropositive to TB by ELISA [10], while all wild boar sera TB eradication program, thus limiting a more accurate obtained before the cattle breakdown (18, between 2015 understanding of the outbreak. and 2019) and after the detection of the diseased fox (3 In Southern Catalonia, there is a predominance of in 2021) were seronegative. Even though no wild boar tis- livestock herds with extensive management, sometimes sues were sampled to confirm the infection, these results with limited biosecurity, thus facilitating the interac- suggest that mycobacteria could be circulating among tion between wildlife and livestock. Maintenance of TB wild boars after the cattle outbreak. within multi-host communities has been identified in some European regions [16]. Indeed, in the epidemiologi- Discussion and conclusions cal context of the outbreak region, cattle, goats and wild The involvement of the red fox in the epidemiology of boars may constitute a multi-host maintenance com- animal TB remains largely unknown. However, cases munity of both M. bovis and M. caprae, with particular of TB in foxes due to M. bovis have been increasingly emphasis on goat herds which are not yet subjected to reported in European endemic areas of bovine TB [7, 8, an official eradication program [10, 17]. Even though an 11–14]. There is limited evidence for cattle-to-fox trans- infected cattle herd could be the most likely source of missions and vice versa [13]. Here, we demonstrated infection for the fox, the participation of wild boars as close spatiotemporal and phylogenetic relationships intermediate hosts cannot be ruled out since seropositive that suggest direct or indirect transmission of M. caprae animals were detected in the same municipality between Pérez de Val et al. BMC Veterinary Research (2022) 18:352 Page 5 of 6 the detection of the infection in the cattle herd and the Abbreviations TB: Tuberculosis; MTBC: Mycobacterium tuberculosis complex; WGS: Whole fox. Genome Sequencing; SNP: Single Nucleotide Polymorphisms. M. caprae infection in wildlife has been recently reported in Catalonia [4] as well as in Central and East- Acknowledgments We are grateful to Zoraida Cervera, Maite Martín, Abel Muñoz, Mònica Pérez, ern European regions [18, 19]. This study further sup- Rosa López and Anna Barceló for their outstanding technical assistance. ports the evidence that foxes are susceptible to M. caprae infection that may evolve to disseminated TB. A case of Authors’ contributions RV, JE, CS-M and BPdV contributed to clinical, pathological and bacteriologi‑ generalized TB due to M. caprae was recently reported in cal analysis and data collection. BPdV, CP, AS, CR, EV and RV, analyzed and Austria [20], where M. caprae is the main causative agent interpreted the data. BPdV, EV and RV wrote the original draft. All authors read of TB in cattle and red deer [21]. To our knowledge, we and approved the final manuscript. report here the first case of TB due to M. caprae in a fox Funding in the Iberian Peninsula. This work was supported by the Department of Climate Action, Food, and Rural Blindness caused by ocular TB is a rare presentation of Agenda (DACC) of the Government of Catalonia and the Grant EFA357/INNOTUB (Program Interreg POCTEFA 2004-2020). IRTA is supported by Centres de Recerca de the disease even in domestic animals. Ocular infection Catalunya (CERCA) Programme / Generalitat de Catalunya (www.​cerca.​cat). is usually the result of the spread of the bacteria from the primary site to the eye via the hematogenous route, Availability of data and materials The datasets used and/or analysed during the current study are available from frequently reaching the eye through the choroid [22] the corresponding author on reasonable request. as in this case. Diffuse tuberculous pericarditis is also an uncommon presentation. This lesion is more often Declarations caused by the spread of the bacteria from the medias- tinal lymph nodes directly into the pericardium than Ethics approval and consent to participate The authors confirm that the ethical policies of the journal, as noted on the from tuberculous pneumonia [23]. The pericarditis with journal’s author guidelines page, have been adhered to. No ethical approval fibrous thickening was the oldest lesions seen in this ani- was required for this study as all sampling and handling procedures were mal. The co-infection with canine distemper virus, an conducted by authorized personnel according to Spanish legislation (Royal Decree 2611/1996 and amendments) and European Union laws for protection immunosuppressive virus that can probably influence the of animals used for scientific purposes (2010/63/EU). TB outcome in foxes [20], could have facilitated the sud- den dissemination of the infection. Viral inclusions were Consent for publication Not applicable. not readily seen which was consistent with a later stage of the disease. Co-infections, such as the conspicuous Competing interests parasitism observed in this animal, may have had also an Dr. Bernat Pérez de Val is a member of the editorial board for the BMC Veteri‑ nary Research journal. influence on the generalized presentation of TB in this case [24]. Author details While in some cases MTBC infection does not induce 1 Unitat mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona TB visible lesions in foxes [13], in this report, in con- (UAB), Bellaterra, Catalonia, Spain. 2 IRTA, Programa de Sanitat Animal, Centre sistency with others, it evolves to generalization [8, 16]. de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma Interestingly, even in lower pathogenic forms, myco- de Barcelona (UAB), Bellaterra, Catalonia, Spain. 3 National Veterinary Services Laboratories, U.S. Department of Agriculture, Ames, IA, USA. 4 Wildlife Ecology bacterial shedding has been detected in feces, urine and & Health group (WE&H) and Servei d’Ecopatologia de Fauna Salvatge (SEFaS), oropharyngeal mucus [12, 13], and this excretion can be Departament de Medicina i Cirurgia Animals, Universitat Autònoma de Barce‑ increased in generalized forms. Indeed, lesions in the lona, Bellaterra, Catalonia, Spain. 5 Centre de Fauna de Vallcalent, Generalitat de Catalunya, Lleida, Catalonia, Spain. 6 Departament d’Acció Climàtica, Aliment‑ kidneys of this case had remarkable amounts of mycob- ació i Agenda Rural de la Generalitat de Catalunya, Barcelona, Catalonia, Spain. cateria (Fig. 1), suggestive of extensive bacterial shedding through urine. Received: 11 April 2022 Accepted: 14 September 2022 Therefore, our results further evidence that foxes may act as TB spillovers in endemic areas but also might be an indirect source of infection for livestock and other wild- life species under suitable ecological conditions. Also, the References 1. Aranaz A, Liébana E, Gómez-Mampaso E, Galán JC, Cousins D, Ortega A, risk for human health should not be ignored, especially et al. 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