An outbreak of bovine tuberculosis in a fallow deer herd (Dama dama)
Benedetta Amatoa*, Sebastian Alessandro Mignaccaa, Maria Lodovica Pacciarinic Gaetano Guarneria, Salvatore Antocia, Salvatore Cucinottaa, Roberto Puleioa, Elena Biasibettib, Michele Fiasconaroa, Maria Teresa Capucchiob, Di Marco Lo Presti Vincenzoa
aIstituto Zooprofilattico Sperimentale della Sicilia, via S. Andrea 96, Barcellona Pozzo di Gotto, ME, 98051, Italy
bDipartimento di Scienze Veterinarie - Università degli Studi di Torino, Largo Braccini 2 Grugliasco, TO 10095, Italy
cNational Reference Centre for Bovine Tuberculosis, Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, via Bianchi 7/9, Brescia, 25124, Italy
Abstract
Wild ruminants have an important role in the epidemiology of bovine tuberculosis. Aim of this study was to describe an outbreak of bovine tuberculosis occurring in a fallow deer herd in Sicily. In 2012 a sicilian herd of 47 animals was referred for cachexia. Pathological investigations performed on two dead animals allowed the observation of disseminated granulomas particularly involving skin and subcutaneous tissues. Tissue samples were submitted to histological, bacteriological culture and biomolecular assay. PCR analysis identified the presence of mycobacteria. Genotyping performed by spoligotyping and MIRU-VNTR profiles confirmed the presence of
Mycobacteriumbovis spoligotype SB0120 in both subjects. In 2014 28 deer of the same group were submitted to
skin test. Four animals were considered positive reactors and 4 inconclusive reactors. The 8 positive/inconclusive animals were submitted to euthanasia showing granulomatous disseminated lesions in 6 cases. Three animals (2 positives and 1 negative to skin test) showed, also, cutaneous lesions. Mycobacterium bovis spoligotype SB0120 was identified from all subjects in which were observed tuberculous-like lesions included two deer resulted negatives to the skin test. The high number of animals involved and the detection of diffuse skin lesions as potential route of Mycobacterium bovis infection, suggest further studies to develop a control and monitoring plan of this zoonosis in wildlife species considering the epidemiological role carried out by the wildlife in the maintenance of bovine tuberculosis infection and the potential risk for humans.
Introduction
Bovine tuberculosis (bTB) is a chronic worldwide disease caused by Mycobacterium bovis and Mycobacterium
caprae, that shows a wide host range affecting domestic and wild mammals and humans (Boniotti et al., 2009;
Gortazar et al., 2003; Prodinger et al., 2002).
Although the infection has been controlled in most countries, the complete eradication is complicated by the persistence of the Mycobacterium bovis infection in wild animals. The role of reservoir or spillover host done by some wild species is closely related to the ecosystem in which it lives and to the related ecological factors (animal, environment and population factors). Therefore, in different ecosystems the same species can assume different epidemiological roles (Gortazar et al., 2011;Nugent et al., 2002). Particularly some wild animals have already been recognized as reservoirs such as badger in the United Kingdom, wild boar in the Iberian Peninsula, brush tail possum in the New Zealand, and white tailed deer and fallow deer in Spain (Aranaz et al., 2004; Gortazar et al., 2011). In Italy Mycobacterium bovis in wildlife is mainly reported in wild boar, wild ungulates and feral pigs (Balseiro et al., 2009; Chiari et al., 2014; Di Marco et al., 2012; Dondo et al., 2006; Serraino et al., 1999; Thoen et al., 2014).
Sicily records a Mycobacterium bovis prevalence between 7.2% in Nebrodi Black pigs (Di Marco et al., 2006) and 4.19% in cattle. Public lands and natural parks in this region are the natural habitat of many wild species such as wild boar, feral pigs, fallow deer, hares, wild rabbits, hedgehogs and foxes. The regional livestock system, mainly in the province of Messina, is characterized by herds reared in a feral and semi-feral condition with ample opportunities of sharing mycobacteria between domestic animals and wildlife.
Members of deer family are particularly sensible towards bovine tuberculosis (Hardstaff et al., 2014; Palmer et al., 2012). The prevalence varies from 50% in farmed deer to 20% in wild herds (Martin Hernando et al., 2010). In Italy the presence of Mycobacterium bovis infection in the same species is sporadically documented (Balseiro et al., 2009; Chiari et al., 2014). Gross lesions were most frequently observed in the medial retropharyngeal lymph nodes and in lungs (Balseiro et al., 2009; Hardstaff et al., 2014; Vicente et al., 2006; Zanella et al., 2008). Lesions also occur in the oropharyngeal tonsils, the mediastinal, bronchial and mesenteric lymph nodes (Mackintosh et al., 2004).
Cutaneous tuberculosis (CTB) is one of the less common extra-pulmonary form of BT. Resurgence of CTB in human subjects and in other species (cats, dogs and livestock) has been well documented in recent years (Gupta et al., 2013; Hardstaff et al., 2014; Lloret et al., 2013; Solsona Peirò et al., 2014). The clinical appearance of CTB is quite varied (Palmer et al., 2009). In addition to a systemic dissemination, and pulmonary involvement, CTB is characterized by the presence of nodules in the skin, draining tracts, ulceration and local lymphadenopathy (Puri,
2011; Santos et al., 2014). However there are two general categories of CTB. The first includes cutaneous lesions characterized by an active proliferation of the tubercle bacilli into the skin. The second category comprises the tuberculids, a group of clinical lesions representative of phenomena of hypersensitivity reactions to a haematogenous small numbers of tubercle bacilli or bacilli antigens in cutaneous vessels (erythema induratum, papulo-necrotic tuberculid and lichen scrofulosorum) (Garcìa Rodrìguez et al., 2008). The CTB can be acquired by exogenous or endogenous mechanisms: exogenous infection results from direct inoculation of bacilli into the skin of susceptible individuals, endogenous infection is secondary to a preexisting primary focus and may results from contiguous, hematogenous or lymphatic dissemination (Santos et al., 2014). In author's opinion the CTB in fallow deer was not reported.
The aim of this study is to describe an outbreak of bovine tuberculosis characterized by both skin and generalised multi-organ lesions due to Mycobacterium bovis in a fallow deer herd reared in semi-captivity in a natural park of Sicily.
Material and Methods Animals
In July 2012 a group of 47 fallow deer aging from 2 months to 10 years old, reared in semi-captivity into a natural park of the Peloritani mountains (Messina, Sicily), during a routine veterinary control, presented severe cachexia and premature death. No other remarkable clinical signs were reported except for a severe tick infestation.
Gross Pathology
Two carcasses were sent to the Istituto Zooprofilattico of Barcellona P.G. (Messina) for diagnostic investigations. The carcasses were submitted to a complete necropsy, with detailed macroscopic inspection of lymph nodes and thoracic and abdominal organs. Granulomatous or abscess–like lesions were collected and submitted to histological and bacteriological investigations.
Histopathology
Tissues for histological examination were fixed in 10% neutral buffered formalin, routinely processed and embedded in paraffin wax to obtain 4-μm sections which were stained with haematoxylin and eosin and
Ziehl-Neelsen stain. Granulomas were classified on the basis of the cell composition, the caseous necrosis and extensive mineralization (García-Jiménez et al., 2012).
Bacteriological and molecular analysis
Smears from fresh tissues were prepared, stained with Ziehl-Neelsen and assessed for the presence of mycobacteria.
For microbiological investigations, according to the official international protocol (Manual of diagnostic tests and vaccines OIE, 2009) 1-4 gr. of each sample were homogenized, decontaminated, centrifuged, resuspended in PBS and cultured at 37 ̊C in the liquid medium Middlebrook 7H9 broth (BBL MGIT, Becton, Dickinson), Bactec MGIT 960 system (Becton, Dickinson and Company, Sparks, MD). After 12-14 days of incubation the positive cultures were submitted to molecular analysis for the identification of the isolates.
The multiplex PCR assay described by Kulski et al. (1995), with some modifications, was used for the detection and identification of the members of the genus Mycobacterium as well as for the differentiation between members of the
Mycobacterium Tuberculosis Complex (MTC). Colonies identified as MTC were then PCR typed as described by
Huard and colleagues (2003) in order to detect Mycobacterium bovis genome. All Mycobacteriumbovis isolates were then further typed by both spoligotyping (Kamerbeek et al. 1997) and VNTR analysis . For MIRU-VNTR typing, 11 genomic loci were selected and individually amplified: ETRA, ETRB, ETRC, ETRD, ETRE VNTR2163a, VNTR2163b, VNTR4052, VNTR1895, VNTR3155, and MIRU26. PCRs were performed following the method of Boniotti and colleagues (2009).
Skin test
In January 2014 the skin test was performed in all the remaining subjects (n. 28: 16 females and 12 males, aging from 8 months to 6 years old) using the standard protocol officially applied in cattle. In order to reduce the stress due to the capture a combination of tiletamine–zolazepam and xylazine through telenarcosis was applied using the Janovsky et al. protocol (2000). Firstly the skin of the neck has been shaved and his thickness measured using a caliper. Then 0.1 ml (5,000 tuberculin units (TU)) bovine purified protein derivated (PPD) was intradermally injected at the side of the neck. The correct inoculation was confirmed by the presence of a lentil sized swelling at the injection site. After 72 h the skin fold thickness at the injection site was measured.Results are expressed as the difference in skin thicknesses between the pre- and post-skin test readings considering positive reactions increased thickness ≥ of 4 mm, inconclusive reactions increased thickness between ≥2- ≤4 mm, and negative reactions increased thickness ≤2mm. Clinical signs like oedema, pain, eschar at the injection site as well as hypertrophy of the satellite lymph nodes were also considered.
All positive and inconclusive subjects were submitted to euthanasia performed using an intravenous injection of embutramide, mebezonium iodide and tetracaine chloride at a dose of 0.3ml/kg (TANAX® INTERVET ITALIA Srl, Segrate (MI) ). Gross, histological and microbiological investigations were performed as reported above. Particularly this examination included tonsils, lymph nodes of the head (parotid, retropharyngeal and submandibular lymph nodes), lymph nodes of the thorax (tracheobronchial and mediastinal lymph nodes), and hepatic and mesenteric lymph nodes. Two negative deer dead during the survey activity were submitted to the same diagnostic protocol.
Results
Organs affected by macroscopical lesions were reported in Table 1.
At gross examination both carcasses submitted to diagnostic investigations in 2012 year, revealed subcutaneous granulomas covered by alopecic, brown necrotic-ulcerated areas, widespread throughout the body (Fig. 1). Lesions were similar to abscesses, varied in size (up 1 to 5 cm) and contained creamy whitish-yellowish caseous material (Fig. 2).
Multifocal or diffuse granulomatous lesions affected particularly the lymph nodes, the spleen and the mesentery in one deer. Granulomatous lymphadenitis was characterized by voluminous necrotic centers composed by white-yellowish, fluid exudate, rarely calcified, surrounded by a thin-walled fibrous capsule. Voluminous coalescing granulomas involved the parietal surface of the liver, the lung parenchyma (multiple lobar pneumonia) (Fig .3) and the wall of the large intestine (Fig .4).
Histologically granulomatous lesions consisting of large necrotic areas surrounded by a variable number of neutrophils, epithelioid cells, rare giant cells, macrophages, lymphocytes and peripheral fibroplasia were detected (Fig. 5) . Minimal calcifications in the center were observed . Cutaneous lesions showed more voluminous necrotic core with abundant peripheral neutrophils surrounded by mononuclear cells and macrophages in different stages of development . Granulomas were classified according to García-Jiménez et al. (2012) as a stage III with minimal necrosis.
On the basis of macroscopical and histological features the diagnosis of tuberculosis was hypothesized.
Bacteriological culture and biomolecular assay confirmed the presence of Mycobacterium bovis spoligotype SB0120/MIRU-VNTR profile 4,4,5,3,3,10,4,4,4,1,7,5.
In 2014, a total of 4 fallow deer (adult males), failed the skin test (increased thickness ≥ of 4 mm) and were considered reactors to bovine PPD presenting eschar and oedema at the injection site and hypertrophy of the prescapular lymph nodes on the same side of the PPD injection.
4 animals (3 adult males and 1 adult female) showed an inconclusive reaction (increased thickness between ≥2- ≤4 mm).
These 8 deer were euthanatized and, with other 2 subjects negative to skin test dead during the survey activity, were submitted to diagnostic investigations. 8 of them (80%) showed diffuse granulomatous lesions. Only 2 carcasses of inconclusive reactive subjects showed no pathological changes. Interesting finding was the presence of diffuse granulomas of different size, isolated or confluent, involving the cutaneous and subcutaneous tissues in three of these subjects (2 positive and 1 negative to skin test) .
Bacteriological investigations performed on granulomatous lesions of the eight deer with macroscopical lesions confirmed the presence of Mycobacterium bovis spoligotype SB0120/MIRU-VNTR profile 4,4,5,3,3,10,4,4,4,1,7,5. The remaining 18 animals were negatives to the skin test and the following TB controls so they were considered in good health.
Discussion
Wild animals carry and spread various infectious and parasitic diseases.
The spread of these diseases in free and semi-free range animal populations can represent a risk for public health and particularly a potential zoonotic risk in the context of hunting, farming and tourism, an animal health risk for the potential interference with eradication plans in cattle and a potential risk for the possible involvement of susceptible wild species. Bovine tuberculosis prevalence in wildlife and the severity of the lesions are indicatives of the circulation of mycobacteria in a territory. High prevalences and phenomena of generalisation support the presence of the disease in domestic animals and the consequent passage from them to the wildlife, low prevalences and localized lesions could be indicative of the presence of mycobacteria in wildlife with potential risk of transmission of the infection to livestock.
Mycobacterium bovis infections in fallow deer have been reported in different countries and play an important role
in the epidemiology of bovine tuberculosis, together with other deer species. Cervids seem in fact to be highly susceptible to bTB (Jaroso et al., 2010) and its role as a wild reservoir might be very important due to the lesion patterns that can facilitate the spread of infection within the population (Zanella et al., 2008). The histological
characterization of the induced lesions is well reported in the available literature (Johnson et al., 2008; Martín-Hernando et al., 2010; Rhyan and Saari, 1995).
The present study reports pathological, microbiological and molecular investigations performed on a TB infected herd of fallow deer .
Most of the animals showed generalised lesions (67%), presenting as abscess like–granulomas involving both organs and lymph nodes, with a poor calcification and with the tendency to disseminate infected macrophages and tuberculous bacilli throughout the host body. According to literature these lesions are often reported in chronic infection, in farms and environment where bTB control is poor and the infectious pressure is high, but also in animals with high susceptibility (Mackintosh et al., 2004) indicating a reduced genetic resistance (Acevedo-Whitehouse et al., 2005; Naranjo et al., 2008). The presence of these lesions confirm the susceptibility of the deer to
Mycobacterium bovis and it’s reduced ability to contain the infection.
Unusual sites of infection observed in the present study were represented by the heart left auricle affected in one case, aortic lymph nodes involved in another subject and skin lesions observed in five animals. Heart lesions appeared as small, isolated, white yellowish granulomas of 1-2 cm in diameter while skin lesions appeared as skin fistulous tracts burrowing from subcutaneous granulomas characterized by a voluminous necrotic core with abundant peripheral neutrophils surrounded by mononuclear cells.
Bacteriological investigations confirmed the presence of Mycobacterium bovis spoligotype SB0120 in 10 animals with macroscopical lesions. All the positive subjects were adults, supporting the hypothesis of reduced possibilities of transmission of the disease through milk.
Molecular epidemiological studies performed in the same geographic area allowed to identify the same infection also in wild boar. Wild boar and fallow deer shared the same spoligotype and MIRU-VNTR profile (spoligotype SB0120/VNTR 4,4,5,3,3,10,4,4,4,1,7,5). According to the Bovine Tuberculosis National Reference Centre Database the MIRU-VNTR profile was never reported in cattle of Italy. Supposing the reservoir role of the fallow deer and the spillover role of the wild boar in this ecosystem on the basis of the severity and the generalisation of the lesions, in authors’ opinion the infection was transmitted from fallow deer to wild boar. The main route of infection was, probably, sharing pastures and food.
Fallow deer, extinct in Sicily at the end of the 19th century, in the 80s it was reintroduced by State Forest Service in some areas as result of resettlement policies. Although the role played by deer and wild boar in the epidemiology of bTB is somewhat controversial the possibility to transmit the disease to cattle and humans cannot be excluded.
Considering that the interactions between domestic, wild hosts and environment are always different from country to country, the evaluation of the risk assessment and the creation of control measures should be adapted to every particular situation (Gortazar et al., 2011).
The sensitivity of skin test (80%) was the same observed in Spain (Jaroso et al., 2010). The use of an anesthetic protocol contributed to reduce stress due to capture. Males showed greater increases in skin fold thickness as reported in red deer (Fernandez de Mara et al., 2008; Jaroso et al., 2010). In fact in dimorphic species has been demonstrated that males and females s respond differently to stressful environmental conditions. Males, perhaps for their particular metabolism, are more sensitive to food shortages declining more sharply in the body growth, with increasing population density or with worsening climatic, environmental and health conditions males tend to react before females, resulting in reduction of dimorphism between sexes (Leberg et al., 1993).
The presence of gross lesions in animals negatives to the intra-vitam skin test was probably due to the anergy condition of their immune system.
On the basis of the present results further investigations are required to collect more epidemiological data evaluating the potential transmission routes of the infection and the possibility of survival of the mycobacterium in wildlife, livestockand environment of the Peloritani area establishing the role of wildlife in the maintenance of the disease and the failure of eradication programs in this habitat.
In author’s knowledge this is the first observation of tuberculous skin lesions characterized by abundant necrotic core in fallow deer representing a big concern in the diffusion of infection both in animals and humans.
This report contributes to underline the importance to create more effective and successful eradication programs where wildlife need to be controlled using a multi-integrated approach involving human public health and biodiversity protection/conservation, in a One Health approach.
Acknowledgements
The authors thank Giovanna Cardella, Francesca Mandanici, Giovanna Romeo and Alessandra Sereno for their technical support.
Conflict of interest
Authors disclose any financial and personal relationships with other people or organizations that may compromise or inappropriately influence their work.
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Figure Legend
Fig.1. Skin. Brownish, circular, ulcerated areas spread throughout the body (circle).
Fig. 2. Subcutaneous tissue and musculoskeletal fascia. Multiple granulomatous lesions with abundant whitish necrotic material.
Fig. 3. Lung. Multiple lobar pneumonia.
Fig. 4. Small and large intestine. Granulomas involving parietal surface and mesenteric lymph nodes.
Fig. 5. Intestinal wall. (A) Voluminous granuloma with necrotic center. H&E stain; bar 500µ. (B). Histological features of the granulomatous lesion consisting of large necrotic areas surrounded by a variable number of neutrophils, epithelioid cells, rare giant cells, macrophages, lymphocytes and fibrous capsule. H&E stain; bar 50µ.
Table Legend Table 1.
Skin test results and detailed anatomo-pathological results.
