Introduction

Brucellosis is a contagious disease which affects many species of mammals, including man. It has been reported in many domestic and wild animal species from different continents (Thorne 1982). In Europe, the main concern is due to Brucella suis biovar 2 infection in wild boar (Sus scrofa) and European brown hare (Lepus europaeus) that are the natural host of B. suis (Hoflechner-Poltl et al. 2000). In France both the range and the prevalence of B. suis biovar 2 infection in wild boar had increased with a rate of 350% from 1987 to 1998 (Hars and Garin-Bastuji 2001).

On the contrary, brucellosis has been reported sporadically in wild ruminants in Europe, and reports usually refer to alpine chamois (Rupicapra rupicapra) from Switzerland, France, and Italy (Bouvier et al. 1958; Garin-Bastuji et al. 1990; Ferroglio et al. 2003). Notwithstanding, Alpine ibex (Capra ibex) is considered to be susceptible to brucellosis. The infection in this species, due to Brucella melitensis 2, has been only recently reported in a 7-year-old male ibex in the Orco Valley of the Gran Paradiso National Park (GPNP) in Western Italian Alps (Ferroglio et al. 1998). Further studies demonstrated the presence and persistence of the infection in ibex of the GPNP territory. In 2002 a serological survey, by complement fixation test (CFT) and Rose Bengala test (RBT), was carried out in two areas of the GPNP and 7 out of 140 (5.0%) ibex tested positive in at least one test (Bassano and Ferroglio 2002).

Concerns arise from breeders about the persistence of infection in GPNP ibex and the risk of a spill back from wildlife to livestock and, to a less extend, of the possibility of B. melitensis 2 transmission to human beings.

Considering that sheep and goat flocks, which in summer range in the GPNP, share pastures with more than 3,500 ibex and 9,000 chamois living in the park, we deemed it interesting to demonstrate, under controlled conditions, if Brucella infection can be easily transmitted from ibex to sheep and goat.

Materials and methods

In an area of the GPNP, a big enclosure of above 5,000 m2 surrounded by a wire netting 300 m long and 2.7 m tall was built. High-ridged rocks, forest trees, shrubs, and grazing ground have been included in the enclosure to ensure natural habitat for animal welfare.

During normally planned capture activities carried out for research purpose, a 7-year-old male alpine ibex , serologically positive to B. melitensis (>1:80 dilution to CFT) in a previous capture and testing, was recaptured.

This ibex showed a visible precarpal igroma at the right leg and testicular monolateral (right) enlargement. The animal serum was retested by CFT and RBT and infection was confirmed (CFT>1:80). It was put in the enclosure with five goats and two sheep rams (day 0) which come from flocks that were at least negative for 5 years and resulted negative twice in 1 month at the CFT for Brucella spp.

The animals were allowed to graze and browse in the wooded and pasture area of the enclosure. Fresh water in drinking troughs, hay, and fodder in mangers were available for all animals in the center of the enclosure.

For the first 3 weeks, domestic animals were blood sampled once a week for serological screening. After that time, blood was collected every 2 weeks. Blood for serology was collected from the jugular vein (Vacutainer system, Belliver Industrial Estate, Plymouth, UK).

Due to poor conditions, loss of weight, ataxia, loss of appetite, the ibex , after deep anaesthesia induced, was euthanized (intravenous Tanax® injection; 5 ml containing embutramide 200 mg/ml, mebezone iodure 50 mg/ml, and tetracaina chloridrate 5 mg/ml; Hoechst Roussel Vet Gmbh, Munich, Germany) at day 40 and immediately necropsied. Precarpal bursa (igroma), epididymis, testicles, spleen, liver, bone marrow, kidneys, lymph nodes, and heart tissues samples were collected at necropsy for bacterial culture.

Domestic ruminants stayed into the enclosure potentially contaminated for further 38 days. At day 78, they were all blood sampled, ethically euthanized (same way of ibex), and immediately necropsied and sampled for udder, uterus, prescapular and iliac lymph nodes, liver, spleen, and hearth.

Sera collected during the trial were tested by RBT and CFT according to Alton et al. (1988a).

Sampled tissue specimens were cultured according to (Corbel and Brinley Morgan 1984; Alton et al. 1988b). Isolated strains had been subcultured, colored, biochemical, and PCR tested according to Corbel and Brinley Morgan (1984), Corbel et al. (1988), and Bricker and Halling (1994).

Furthermore, behavior observations were done to verify the occurring of direct or indirect (spatial and temporal superimpositions) contact between wild and domestics. During the first 3 weeks, once a week, animals’ position and behavior were examined for 5 min every half hour from 7 a.m. to 7 p.m.

Results

None of the domestic animals exhibited obvious clinical signs of brucellosis during the study; they all tested negative at serology, at all sampling time, and at bacteriological culture. At necropsy the ibex showed a precarpal bursitis on right leg with a thick connective capsule containing necrotic and suppurative tissue, enlargement and hardening of a testicle with granulomatous calcified purulent confluenting necrosis foci surrounded by a diffuse fibrosis of the parenchyma.

B. melitensis biovar 3 was isolated from ibex precarpal bursa (igroma), epididymis, testicles, and spleen samples.

Behavior observations allow to verify the occurring of direct and indirect (spatial and temporal superimpositions) contact between the ibex and the domestics, on pasture, on mangers, and in drinking troughs.

Discussion

Davis (1990) said that probably the last reservoir of Brucella on the hearth will be a wild specie. Considering that Brucella is eradicated, or in the verge of eradication, in many countries, the risk of its transmission from wild ibex to livestock represents a potential treat for who is involved in farming or public health.

Our data show that also after 40 days of strict cohabitation and 38 days of permanence on the same enclosure an infected, and culture positive ibex, did not infect sheep and goat. Also, if the risk of Brucella transmission between wildlife and livestock is mainly due to infection of pastures during abortion (Nicoletti 1980), in the area interaction between ibex and livestock, moreover sheep and goat, these are mainly done by male ibex. This is confirmed by a previous survey carried out in the GPNP which evidences that, during the 4 months of the summer trial, out of 1,500 ibex contact observations, only three cases of interaction with domestics were observed. All these three contacts involved young male ibex (Lamorgia 2001). During the summer, females live at higher altitudes than males and do not share pasture with livestock. So, we choose to use a male ibex in our trial because it represents the real possibility of Brucella transmission from ibex to livestock. This is the second isolation of B. melitensis from ibex. The first case comes from the same area, but it was classified as B. melitensis biovar 2 (Ferroglio et al. 1998), while in this case, we found B. melitensis biovar 3. In Italy, B. melitensis biovar 3 is really uncommon, moreover in our study area (Gennero, unpublished data). Conversely, on the French side of Alps, B. melitensis biovar 3 is the strain that has been more frequently isolated from sheep ranging on alpine pastures (Garin-Bastuji et al. 1993). Considering that GPS-collared male and female ibex overstep regularly and seasonally between GPNP and the neighbouring Vanoise National Park (France) (Girard et al. 2004), it is possible that this ibex contracted infection from sheep in the French range.

In the GPNP, 9 out of 199 (4.5%) ibex sampled from 1998 to 2005 tested positive for Brucella at CFT or RBT (Bassano, unpublished data). All positive animals came from the Orco Valley where the prevalence (9/51, 17.6%) was significantly greater than in other part of the GPNP (0/148) (χ 2 = 22.4, p = 0.000001).

Analyzing retrospective data, it emerge that 26 ibex captured from 1977 to 1979 (Lanfranchi, unpublished data) and 84 ibex captured from 1985 to 1994 tested all negative (Peracino et al. 1996). The differences among the prevalence observed in these two survey and recent data are statistically significant, χ 2 = 3.8, p = 0.05 and χ 2 = 13.17, p = 0.0003, respectively, and suggest that the ibex became infected in the second half of 1990s.

Although limitation of our field trial obtained data indicate that, even if it could not be completely refused, the transmission of B. melitensis from ibex to livestock appears at least to be very remote. Further study carried out with more animals during a more prolonged time can allow to better evaluate the effective risk of B. melitensis transmission from ibex to livestock and between ibex.