In February 2021, an adult female free-ranging red fox was found blind and depressed in southern Catalonia (Spain). The animal was admitted to Vallcalent Wildlife Rehabilitation Centre (Lleida, Catalonia). The fox was severely emaciated (2.94 kg), with abundant ectoparasites (ticks and fleas) and filiform worms consistent with Thelazia callipaeda in both conjunctival sacs. After clinical examination, it was humanely euthanized. Afterwards, according to the Catalan Wildlife Health Surveillance Plan, it was submitted to the Veterinary Faculty of the Autonomous University of Barcelona (Bellaterra, Spain) to carry out a complete necropsy. Tissue samples from brain, eyes, retropharyngeal, thoracic, and mesenteric lymph nodes, heart, pericardium, trachea, lungs, liver, spleen, kidneys, urinary bladder, adrenal glands, and bone marrow were fixed in 4% neutral buffered formalin and processed and embedded in paraffin for routine microscopic examination. Intestinal samples were not collected since they were too autolytic for their precise evaluation. Sections of 3–4 μm thick were stained with Mayer’s haematoxylin and eosin. Ziehl-Neelsen staining was also carried out to detect acid-fast bacilli. Immunohistochemistry was used to evaluate the presence of canine distemper virus (CDV) antigen in selected sections from the brain and lung. A monoclonal antibody against the nucleoprotein of CDV was used (from VMRD, Ref.: CDV-NP, Ascites fluid, 1 mg/mL, IgG2b, kappa light chain, dilution used 1:5000).
Fresh samples of kidney and mediastinal lymph node with TB compatible lesions were processed for bacteriology. Tissues were mechanically homogenized in 10 ml of sterile water using an automatic homogenizer (Masticator, IUL, Barcelona, Spain), decontaminated with 10 ml of 5% w/v oxalic acid for 30 min. in orbital shaking, neutralized with 5 ml of sodium hydroxide 1 M and centrifuged at 2471×g for 30 min. Supernatants were discarded, pellets were resuspended with 1 ml of sterile phosphate buffered saline, and an aliquot of 0.5 ml was inoculated in BBL MGIT supplemented tubes and incubated in the BACTEC MGIT 320 system (BD Diagnostics, Sparks, MD, USA). The remaining 0.5 ml was cultured using swabs in Löwenstein-Jensen with pyruvate and Coletsos solid media (BD Diagnostics,) and incubated at 37 °C.
Culture growth was confirmed as MTBC by multiplex PCR [9]. DNA from MTBC growth was molecularly characterized by DVR-spoligotyping, at VISAVET Health Surveillance Centre (Madrid, Spain), and WGS followed by Single Nucleotide Polymorphisms (SNPs), at the National Veterinary Services Laboratories of the US Department of Agriculture (Ames, IA, USA).
At necropsy, the fox was severely emaciated. In the eyes, diffuse corneal edema, hypopyon, and the presence of a few filiform worms in the conjunctival sac, consistent with Thelazia callipaeda, were noted (Fig.1a). Internally, retropharyngeal, prescapular, and mesenteric lymph nodes were moderately enlarged, and multifocal yellow areas of caseous necrosis were seen in the cranial mediastinal lymph node (Fig.1b). There was a diffuse severe thickening of the pericardium and abundant caseopurulent exudate filled the pericardial cavity (Fig.1e). In the heart, white mild nodular thickening of the left atrio-ventricular and aortic valves was detected. From these affected valves, the inflammation extended locally into the myocardium. In the lungs, an increased consistency and brown-gray discoloration of the caudo-dorsal areas, consistent with verminous pneumonia, was recognized. Several discrete white granulomas, 2 to 4 mm in diameter, were noted in the left parietal pleural surface. The liver was pale orange, had slightly rounded margins, and subtle multifocal to coalescent inflammatory infiltrates. A diffuse, mild splenomegaly was also present. Severe multifocal to coalescent foci of caseous-purulent necrosis were found in both kidneys (Fig. 1c). A moderate number of cestodes was seen in the small intestine.
Microscopically, diffuse granulomatous infiltrates and/or granulomas compatible with TB lesions were seen in most tissues. Diffuse granulomatous infiltrates were present in the pericardium, epicardium, heart valves, interstitial renal medulla, and choroid layer in the eyes. Discrete miliary (up to 200 μm), round to oval granulomas, characterized by a central core of macrophages surrounded by a thin rim of lymphocytes and plasma cells were numerous in the cortical area of the kidneys, liver, lymph nodes and spleen. A few small granulomas were also found in the brain, bone marrow and lung. None of the granulomas were encapsulated. The number of multinucleated giant cells was low and most of them had only two to three nuclei (small). Areas of caseous necrosis containing few degenerate neutrophils were present in both granulomatous infiltrates and granulomas. Small foci of mineralization were only seen in the valves. The pericardial sac and epicardium were markedly thickened with fibroblasts proliferation and fibrosis, suggesting that this was the oldest lesion.
In the eyes, a severe granulomatous choroiditis and vitritis were established as the cause of the blindness (Fig.1d). The exudate in the vitreous chamber caused the detachment and degeneration of the retina in both eyes. In the right eye, the granulomatous infiltrate and exudate extended to the anterior uvea and chamber explaining the hypopyon. A mild keratitis characterized by mild superficial edema and neutrophilic infiltrate with incipient neovascularization at the limbus was also present. No inclusion bodies were seen in the corneal or conjunctival epithelium.
Numerous acid-fast and rod-shaped bacilli were seen within the macrophages and necrotic areas in all tissues, including foamy macrophages in the lumen of the bronchi and renal pelvis consistent with an active shedding of mycobacteria (Fig.1f).
Additionally, lesions consistent with CDV co-infection were also present in the brain and the lungs. Mild perivascular lymphocytic cuffing was noted in the section from the hippocampus in the brain. In the lung, the presence of patchy necrosis, and attenuation of bronchiolar epithelium along with lymphocytes around bronchioles and vessels, alveolar edema, and the occasional alveolar epithelial syncytial cell were consistent with multifocal viral subacute bronchiolo-interstitial pneumonia. Canine distemper virus antigen was detected in bronchiolar epithelial cells, alveolar macrophages in the lung and in the occasional neuron, glial, and ependymal cell in the brain.
Verminous pneumonia was also confirmed. Arterial damage within the lesions suggested Angiostrongylus vasorum as the possible etiology, although no adult parasites were present in the sections. The main final diagnosis was a generalized TB infection with a co-infection with canine distemper virus.
Culture results revealed growth of M. tuberculosis complex (MTBC) in both tissues, and the spoligopattern M. caprae SB0415 (www.mbovis.org) was identified. Then, the whole genome sequences were analyzed together with other 9 recent M. caprae SB0415 isolates (2018-2021) from cattle (1), goat (1) and wild boar (7) of the same area and other 17 available whole genome sequences of previous M. caprae isolates in Catalonia (2008-2020) [4].
WGS analysis showed a close phylogenetic relationship between the fox isolate and another one (8 SNP pairwise distance) isolated 1 year before from a cattle herd located at the same municipality where the ill fox was found (Fig. 2). In addition, the strains isolated from two goat herds in 2015 and 2019, located at 56 and 18 Km from the cattle-fox outbreak, respectively, also showed a close phylogenetical relationship with the fox strain (9 SNP pairwise distance, Fig. 2). These four isolates share a common ancestor within 6.5 SNP (range 5-8).
Additionally, in the context of the Catalan wildlife health surveillance plan, 3 out of the 9 (33%) wild boar sera sampled in the outbreak municipality in 2020 were seropositive to TB by ELISA [10], while all wild boar sera obtained before the cattle breakdown (18, between 2015 and 2019) and after the detection of the diseased fox (3 in 2021) were seronegative. Even though no wild boar tissues were sampled to confirm the infection, these results suggest that mycobacteria could be circulating among wild boars after the cattle outbreak.
