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1 Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna "Bruno Ubertini" (IZSLER), Via Rovelli 53, I-24100 Bergamo, Italy;
2 Wildvet Projects, CH-7605 Stampa, Switzerland;
3 IZSLER, Via Bianchi 9, I-25124 Brescia, Italy;
4 IZSLER, Via Taramelli 7, I-27100 Pavia, Italy;
5 Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università di Milano, Via Celoria 10, I-20133 Milan, Italy
6 Corresponding author (email: alessandra.gaffuri{at}bs.izs.it)
ABSTRACT:
Roe deer (Capreolus capreolus), chamois (Rupicapra rupricapra rupicapra), and domestic sheep in the Orobie Alps, Italy, were serologically tested for antibodies to selected pathogens that may be transmitted across species. Antibodies against Brucella spp. and bovine herpesvirus 1 (roe deer and chamois only) were not detected in any species. In roe deer, antibodies were detected against Toxoplasma gondii (13%) and Neospora caninum (3%). Chamois tested positive for antibodies to T. gondii (5%), N. caninum (21%), bovine respiratory syncytial virus (BRSV) (41%), bovine parainfluenza type-3 virus (17%), pestiviruses (18%), and Mycoplasma conjunctivae (17%). In the sheep, particularly high antibody prevalence rates were found for T. gondii (78%), Chlamydophila spp. (20%), pestiviruses (90%), BRSV (82%), and M. conjunctivae (81%).
Key words: Alpine chamois, domestic sheep, Italian Alps, roe deer, serosurvey.
Interactions between domestic and wild ungulates represent a potential problem in the Alps, due to livestock summer pasturing. Infectious diseases such as brucellosis (Ferroglio et al., 2000) and infectious keratoconjunctivitis (IKC; Belloy et al., 2003) have been found in wild ruminants due to transmission from livestock. In the Alps, sheep are of particular concern due to large herd sizes and the broad movements that often are associated with sheep management. To better understand the potential for pathogen transmission between sheep and wild ungulates in the Alps, a cross-sectional serologic survey of roe deer (Capreolus capreolus), chamois (Rupicapra rupricapra rupicapra), and sheep was done during 1998–2001. The study area (45°40' to 46°10'N, 9°25' to 10°20'E), situated in the Orobie Alps (northern Italy), has three main valleys (Val Brembana, Val Seriana, and Val di Scalve). There are approximately 4,000 roe deer and 5,000 chamois in this area with the highest densities in Val Brembana; an outbreak of pneumonia and IKC recently caused high mortality in the chamois population in Val Brembana. Large wandering sheep flocks as well as smaller groups (approximately 25,000 sheep in total) share habitats with wild ungulates from June to September.
Blood samples were collected by hunters from 207 roe deer and 236 chamois harvested during the 1998–2001 hunting seasons. Refrigerated blood samples were delivered to the laboratory within 2 days; they were centrifuged and sera were stored at –20 C. Sera were collected from 13,565 sheep in 2000 and were stored at –20 C. From these samples, 352 sheep sera from each valley were randomly selected using a computerized random list produced with the "Random" procedure of the PEPI 3.01 software (Abramson and Gahlinger, 1993–2000). For sample size determination (sheep), we used an expected prevalence of 50% for all the tested pathogens; and based on Graat et al. (1997), this sample size provides a precision of 5% at a 95% confidence level.
Wildlife species were tested for antibodies to Brucella spp. by using complement fixation test (CFT) (Ciuchini and Farina, 1991) and the Rose Bengal agglutination test (D.M. 2 Luglio 1992 n.453) depending on the quality and amount of the sera. Ovine sera were tested by CFT; antibody titers 
20 were considered positive.
For Toxoplasma gondii antibody testing, a commercial latex agglutination test (Toxotest-MT®, Eiken Chemical Co., Tokyo, Japan) was used with a cut-off titer of 32 (Walls and Remington, 1983). Antibodies to Neospora caninumin in wildlife species were detected with the indirect fluorescent antibody test performed on antigen slides (Conrad et al., 1993) with species-specific conjugates. Antigen consisted of tachyzoites of the NC-PV1 (N. caninum) Italian isolate (Magnino et al., 1999). Antibody titers were determined as the last dilution showing whole parasite fluorescence. For sheep, a commercial enzyme-linked immunoadsorbent assay (ELISA; Chekit Neospora®, Bommeli Diagnostics, Liebefeld-Bern, Switzerland) was used.
Roe deer and chamois were tested for Chlamydophila spp. antibodies by using a CFT (O.I.E. Manual, 1992); titers 16 were considered positive. Sheep sera were tested using a commercial ELISA (Chekit Chlamydia®, Bommeli Diagnostics).
A commercial blocking ELISA (Herd-Check Anti-IBR gE®, IDEXX laboratories, Westbrook, Maine, USA) was used to test for antibodies to bovine herpesvirus 1 (BHV-1). Hemagglutination inhibition, by using SF-4 (ATCC VR281) as a reference virus, was used for bovine parainfluenza 3 virus (BPIV-3) antibody testing; 0.5% guinea pig red blood cells were used in this assay (Contini and Cossu, 1975). For pestivirus antibody testing, a blocking ELISA was performed using anti-p80 monoclonal antibodies as described previously (Brocchi et al., 1993); sera were tested at a 1:4 dilution. A blocking ELISA also was used to test for antibodies to bovine respiratory syncytial virus (BRSV; De Simone et al., 1986).
A commercial ELISA (Chekit M. conjunctivae®, Bommeli Diagnostics) was used to test sheep for antibodies to Mycoplasma conjunctivae (Belloy et al., 2001). For chamois, a monoclonal antibody to sheep and goat IgG that was conjugated to horseradish peroxidase was used (Giacometti et al., 2002).
Because of inadequate serum quantity and poor quality due to hemolysis, individual sera were often not included in all assays. In particular, tests for antibodies against BHV-1 and BPIV-3 virus were not performed for the sheep.
For all antibody prevalence estimates, a 95% confidence interval (CI) was estimated according to a binomial distribution (Henken et al., 1997). Chi-square tests were used to test for differences in seroprevalence between valleys. The Cramer 
coefficient was calculated according to Jekel et al. (1996) to value the effect of the sample size on the estimate of 
2.
Results are shown in Table 1
. Negative results for Brucella spp. antibodies are consistent with observed absence of brucellosis in sheep in this area since 1997. In sheep, antibody prevalence was >70% for T. gondii, pestiviruses, respiratory syncytial virus, and M. conjunctivae. Significant differences in antibody prevalence between sheep sampled in different valleys were detected for T. gondii (2=18.9(df=2), P<0.0001), Chlamydophila spp. (2= 10.2(df=2), P=0.006), and M. conjunctivae (2=21.0(df=2), P<0.0001; Table 2). In all cases, however, contingence coefficients were low (Cramer Toxoplasmosis=0.13, Cramer Chlamydiosis=0.10, and Cramer Keratoconjunctivitis=0.14).
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The prevalence of M. conjunctivae-seropositive chamois observed in this study was higher than prevalence estimates from the eastern Swiss Alps (Giacometti et al., 2002). This is probably due to an IKC outbreak that occurred in the Val Brembana immediately before the collection of our samples.
For chamois, antibody prevalence estimates for pestivirus (2=24.9(df=2), P<0.0001) and BPIV-3 (2=10.5(df=2); P=0.005) differed significantly between the valleys (Table 3). However, we found only moderate values for the contingence coefficients (Cramer Pestivirus=0.54 and Cramer PI3=0.34). Antibodies to pesti-viruses have been detected in chamois in the western Alps (Olde Riekerink, 2005), and pestivirus infections and related disease have been reported from Pyrenean chamois (Hurtado et al., 2004). The presence of antibodies to BRSV in chamois also may be significant, because this virus may have been associated with epidemic pneumonia in chamois in an area bordering west Val Brembana (Citterio et al., 2003).
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Based on the higher prevalence of antibodies in sheep compared with both roe deer and chamois, it is probable that sheep represent a potentially important source of infection for wild ruminants. However, these high prevalence estimates from sheep may be partially influenced by herd reorganization and the routine mixing of small groups of animals. Our results confirm the absence of brucellosis in this area but demonstrate the potential circulation and transmission of several important pathogens between species. To better understand the nature and significance of these interactions, additional clinical data are needed as well as the isolation and molecular characterization of these etiological agents.
The authors are grateful to the Public Veterinary service of the Province of Bergamo, and, in particular, to E. Testa and D. Frosio for collaboration. We also thank T. Ambrosi for contributions as both a veterinarian and a hunter. The hunting districts of the Province of Bergamo and the state gamekeepers also are acknowledged for their cooperation. We thank R. Coates, Centro linguistico dell’Università di Brescia, for linguistic revision. Special thanks are due to C. Garbarino who contributed to the beginning of the study. M.G. was supported by the Fund for Research on Infectious Keratoconjunctivitis, Chur (Switzerland), and P.L. was supported in part by a grant from the University of Milan, Italy (FIRST 2002).
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Received for publication 16 December 2004.
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