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Putative risk factors for infections with Toggenburg Orbivirus in goat herds in Southern Switzerland (Canton of Ticino)
Veterinary Microbiology 160 (2012) 29–34
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Putative risk factors for infections with Toggenburg Orbivirus in goat herds in Southern Switzerland (Canton of Ticino) Antonella Reber a, Lothar Kreienbrock b, Simona Casati c, Vale´rie Chaignat d, Heinzpeter Schwermer e,* a
Veterinary Public Health Institute, Schwarzenburgstrasse 155, 3003 Liebefeld, Switzerland Institute for Biometry, Epidemiology and Information Processing, WHO-Collaborating Centre for Research and Training in Veterinary Public Health, University of Veterinary Medicine, Bu¨nteweg 2, D-30559 Hannover, Federal Republic of Germany c Cantonal Institute of Microbiology, Via Mirasole 22A, 6500 Bellinzona, Switzerland d Institute of Virology and Immunoprophylaxis, Sensemattstrasse 293, 3147 Mittelha¨usern, Switzerland e Swiss Federal Veterinary Office, Schwarzenburgstrasse 155, 3003 Liebefeld, Switzerland b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 January 2011 Received in revised form 9 May 2012 Accepted 18 May 2012
Toggenburg Orbivirus (TOV), only detected in goats, has been described as a member of the Bluetongue virus (BTV) serogroup. The transmission pathway of the virus seems different from other Bluetongue viruses (BTVs). The objective of this study was to explore risk factors, especially the influence of alpine pasture and the presence of other livestock species, for the presence of TOV infected goats on farms. Between February 2008 and September 2009, blood samples were collected and analyzed for TOV and hereupon a total of 60 goat farm owners (37 TOV-positive and 23 TOV-negative holdings) were interviewed. Additionally, goatlings were tested for TOV by rRT-PCR prior and after alpine pasture in 2009. These goatlings were positive for TOV only after the alpine pasture. The final logistic regression model included: ‘‘exposure to goats from other farms’’ (OR = 10.12, p = 0.007), ‘‘exposure of the goats to red deer’’ (OR = 4.79, p = 0.04) and ‘‘exposure to sheep from other farms’’ (OR = 0.05, p = 0.002). These variables do not implicitly include direct contact, and the findings are only vaguely indicative for a contact-driven transmission. Furthermore, it is likely that they are only associated with, and thus indicative for, an unknown risk factor associated with alpine pasture not measured in the study. The results of this screening study do not indicate iatrogenic transmission pathways as a main transmission mode and stimulate the formulation of hypotheses on the origin, the transmission pathway and other host species for TOV. Especially, the involvement of an insect vector in transmission on alpine pasture and the relevance of vertical transmission are to be clarified. ß 2012 Elsevier B.V. All rights reserved.
Keywords: Switzerland Bluetongue virus Goat Questionnaire Logistic regression Transmission Toggenburg Orbivirus
1. Introduction The Bluetongue virus (BTV) serogroup holds some of the most important pathogens of the genus Orbivirus. Recently, a new member of the Bluetongue virus (BTV) serogroup named Toggenburg Orbivirus (TOV) has been
* Corresponding author. Tel.: +41 31 323 30 53; fax: +41 31 323 95 43. E-mail address: [email protected] (H. Schwermer). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.05.027
described in goats from Switzerland (Hofmann et al., 2008a; Chaignat et al., 2009). The index cases were detected on two distinct goat holdings north of the Alps, in the regions of Solothurn and Toggenburg (Hofmann et al., 2008b; Chaignat et al., 2009). Adult goats, in the field and after experimental TOV infection, only occasionally show clinical symptoms or gross pathological alterations. However, it could be demonstrated that the virus is transmissible to and replicates in goats. In contrast to adult goats, several stillborn and weak born kids were reported on the index
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farms. Several facts indicate a vertical transmission on these farms (Chaignat et al., 2009). Although it was possible to infect sheep experimentally, no evidence of an infection process outside the goat population could be found in the field despite extensive testing in cattle, sheep or other domestic ruminants (Chaignat et al., 2010). These field observations revealed no indication of clinical disease, but a seroprevalence rate of 4% in sheep on farms with high seroprevalence of TOV in goats (Chaignat et al., 2010). Following the discovery of the new virus, a serological prevalence study for TOV in the Swiss goat population was conducted to gain insight into the spatial distribution of the virus. Although TOV seems to be of minor importance as a pathogen in the field, even in the absence of clinical disease TOV interferes with the diagnostics for BTV in goats and thus the distribution of TOV is of general interest. The national study showed that cantons north of the Alps were free, cantons in the Alps had very few cases, but in the Canton of Ticino (TI), south of the Alps, an exceptionally high prevalence of around 60% on the farm level and 49% on the animal level was observed (Chaignat et al., 2010). In TI, the positive farms were distributed over the whole canton. However, cases were more abundant in areas with summer alpine pasture (Chaignat et al., 2010). Evidence for the long presence of TOV in goats in TI was found after testing serum stored in the national serum bank, collected in 1998: twenty percent of the tested samples were positive in BT ELISA (Chaignat et al., 2010). Given the negative results of the surveillance for BT in this region (Cagienard et al., 2006), and given TOV is serologically indistinguishable from other BTV, the most likely reason for these results are TOV infections. Additionally, it is known that BT disease is either rare or not recognized in many regions with enzootic BTV infection (Maclachlan et al., 2009). Thus, it is most likely that TOV stayed undiscovered for a long period of time. Given the recent detection and identification, but albeit high regional and within-herd prevalence only in goats even more than ten years ago, questions about the origin and transmission pathway of the virus raised. Before the beginning of our study, different hypotheses on the epidemiology and transmission pathway of TOV had already been discussed. Because of the high prevalence in that region, we focused and carried out our study in TI. The purpose of this cross-sectional screening study was to identify putative risk factors for the presence of TOV infected goats on the farm, to unravel particular epidemiological features of this virus and to appraise the hypothesized transmission pathways. The results should direct further investigations on the epidemiology of this virus and should support future studies rather than being finally conclusive. 2. Materials and methods 2.1. Definition of the outcome variable (case definition) The unit of analysis was the goat farm. A goat farm was declared to be a case farm, if at least one goat was tested seropositive. Otherwise the farm was declared and
identified as negative. All samples were taken by official veterinarians and selected randomly. If there were up to five goats on the farm, samples were taken from all animals. On farms with more than five and less than 15 goats, at least five samples were taken. On farms with at least 15 goats, 8–75 goats were tested serologically. The higher number of tested animals refers to farms with high seroprevalence, where we tried to identify freshly infected animals for virus isolation. With an assumed sensitivity of 95% of the ELISA and a design prevalence on the farm of 40%, these figures correspond to probabilities between 95% and 99% to detect at least one positive goat in herds of 15, respectively, more than 15, goats (Freecalc 2.0, Australian Centre for International Agricultural Research, Canberra, Australia). 2.2. Serological analysis All blood samples were tested at the Institute of Virology and Immunoprophylaxis (IVI, Mittelha¨usern, Switzerland) or at the Cantonal Institute of Microbiology (Bellinzona, Switzerland). The testing procedure was conducted as described by Chaignat et al. (2010). Briefly, the diagnostic kit in use was Bluetongue virus Antibody Test Kit (VMRD, Pullman, WA, USA) (BT ELISA VMRD) and positive sera were confirmed with the ID.Screen Blue Tongue Competition ELISA Kit (ID.VET, Montpellier, France) and the double recognition ELISA INGEZIM BTV DR1 (INGENASA, Madrid, Spain) (BT ELISA INGEZIM). If weak positive results were obtained using BT ELISA VMRD, Blue Tongue Competitive ELISA Kit (B.D.S.L., Irvine, UK) (BT ELISA BDSL) was used as confirmation tests. The tests were used according to the manufacturer’s instructions. No TOV specific serology assay is available but as TI did not have any BTV infection up to the study period and did not vaccinate goats against BTV, seropositivity is a decisive hint for TOV infection. Additionally, in 81 seropositive samples from seven farms with high seroprevalence, TOV genome could be detected by a TOV rRT-PCR and confirmed by sequencing. Viral RNA was detected using RT-qPCR-panBTV and a first version of RT-qPCR-TOV (Hofmann et al., 2010). 2.3. Study population Farms that had a testing result for TOV in 2008 and 2009 were included into the sampling frame. The study population comprised farms sampled in the 2008 annual Caprine Artheritis Encephalitis (CAE) survey, a crosssectional survey (sample of 2008). For that survey, the herds were chosen randomly from the database of the AGIS (Swiss agricultural information system; Federal Office for Agriculture). In 2008, serum samples were collected between February and March, prior to the vaccination of cattle and sheep against BTV-8. Five samples per herd were tested with BT ELISA. In the 2009 sampling, farms with seropositive goats were sampled again. Goats and, if present, sheep and cattle were sampled. On these farms, five to ten samples from yet untested goats and additional five to ten samples from unvaccinated sheep and cattle were collected.
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In total, test results for TOV from 95 farms were available, with 57 farms having at least one seropositive goat. With a statistical power analysis with two independent proportions using the statistical software PASS 2008 (http:// www.ncss.com/pass.html) we determined that a sample size of 60 farms has to be provided (sample size group 1 = 30, sample size group 2 = 30) to detect an odds ratio of 4 with a power of approximately 80% (target alpha at 5%). As we were interested in factors having a robust effect on the outcome, we decided to conduct the study albeit the low number of data sets. The 60 farms were selected randomly. Additionally, as preliminary results indicated that infection might took place on alpine pasture, 36 goatlings originating from three TOV-affected holdings (holding A [in-herd seroprevalence 42%], B [23%] and C [38%]) were tested for TOV by rRT-PCR prior and after alpine pasture in 2009. These animals were born during the vector-free period in January and February 2009 and were not vaccinated against BTV-8. 2.4. Questionnaire and data collection For the collection of concomitant data, one out of three interviewers filled out a questionnaire in Italian during a telephone interview with the owner. The gathered information was: stable location, minimum and maximum altitude of the farm pasture; the number and breed of goats, sheep, cattle, other ruminants and equines on the farm; introduction of animals in the previous three years, with date, species and origin; frequency of exposure to animals from other farms with species, period and motive; frequency of exposure to wild animals with species, period and kind of exposure; detailed information on vaccination history; the name of the veterinarian. Additionally, we also asked if there had been sanitary problems or special observations in the last three years. The interview took approximately ten minutes. Many farmers had problems to
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answer the question about vaccination and often did not recollect whether it was a vaccination or another injection, or the name of the vaccine. 2.5. Statistical analysis For general data description mean, median, and coefficient of variation were calculated for the continuous variables and frequency tables were set up for the categorical variables. Calculation of odds ratio for the categorical variables and further (risk factor) analyses were only performed, if each category comprised at least five units. As the number of observations was small and the distributions of continuous variables were clumps at low numbers with an extreme skewed distribution, the continuous variables were categorized with their median to avoid a loss of power within the analyses. Because of the uncertainty on the data for vaccination, we simplified this factor and made a binary categorization into general presence or absence of vaccination on the farm. A basic set of variables was suitable for further analysis, i.e. ‘‘size of the goat farm’’ (farm_size) with a median of 20 and categorized in two sizes, ‘‘number of sheep on the goat farm’’ (sheep_size) with a median of 30 and three categories, ‘‘alpine pasture’’, ‘‘vaccinated’’, ‘‘purchase of goats in the last 3 years’’, ‘‘exposure of the goats to red deer’’, ‘‘exposure to sheep from other farms’’ and ‘‘exposure to goats from other farms’’ (Table 1). Model selection for the risk factor analyses was conducted by an automated forward selection approach using an entry p-value of 25% and a staying p-value of 15%. These analyses were improved by systematically mixing backward and forward selection to check for model sensitivity, if no different final model was found. All statistical analyses were conducted with SAS 9.2 (http://www.sas.com) with the use of PROC LOGISTIC for performing ordinary logistic regression analyses.
Table 1 Univariate statistics of factors associated with the infection of goats with Toggenburg Orbivirus (TOV) in the canton of Ticino. OR, odds ratio; CI, confidence interval. Factor description
Category
Number of sheep on the goat farm
0 1–30 >30
TOV-negative n (%)
TOV-positive n (%)
OR
p-Value
95% CI
9 (27.27) 5 (38.46) 9 (64.29)
24 (72.73) 8 (61.54) 5 (35.71)
1 0.6 0.21
0.46 0.02
0.16–2.33 0.06–0.79
Vaccinated (any vaccination)
No Yes
20 (44.44) 3 (20)
25 (55.56) 12(80)
1 3.20
0.10
0.79–12.91
Alpine pasture
<500 m >500 m
7 (58.33) 16 33.33)
5 (41.67) 32 (66.67)
1 2.80
0.12
0.77–10.22
Size of the goat farm
1–20 >20
18 (54.55) 5 (18.52)
15 (45.45) 22 (81.48)
1 5.28
0.006
1.61–17.33
Exposure of the goats to red deer
No Yes
10 (62.5) 11 (26.83)
6 (37.5) 30 (73.17)
1 4.68
0.009
1.47–14.92
Purchase of goats in the past 3 years
No Yes
16 (50) 6 (26.09
16 (50) 17 (73.91)
1 2.41
0.13
0.78–7.48
Exposure to goats from other farms
No Yes
16 (59.26) 7 (21.21)
11 (40.74) 26 (78.79)
1 5.40
0.004
1.74–16.79
Exposure to sheep from other farms
No Yes
13 (28.89 10 (66.67)
32 (71.11 5 (33.33)
1 0.20
0.01
0.06–0.71
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3. Results From the 60 goat farms tested, 37 farms were positive by the serological blood test and 23 were negative. Nera Verzasca (28 farms), the Chamois-coloured (15 farms) and Saanen (11 farms) were the mostly often kept breeds. In addition, most breeders also had crossbreeds (36 farms). Rare breeds were Tibetan goat, Peacock goat, Stieffelgeiss goat, Boer-goat, Mountain goat und Grisons. We could observe small (20; 20 = the median of number of goats on the farm) and also big goat farms (>20) with goats only. On most of the mixed farms, the number of goats on the farm and the number of sheep on the goat farm were inversely proportional, e.g. in large flocks of goats, the number of sheep on the farm was small, and in small goat flocks the number of sheep was large. None of our 60 goat farms had small ruminants imported in the last three years. Many farmers reported exposure of their goats to wild animals. The species observed were the Western European red deer (Cervus elaphus elaphus) (41 farms), roe deer (Capreolus capreolus) (34 farms), chamois (Rupicapra rupicapra) (29 farms), wild boar (Sus scrofa) (16 farms) and fox (Vulpes vulpes) (10 farms). The type and closeness of the exposure varied from ‘‘occasionally seen on the pasture’’ to ‘‘regularly staying together with the goats’’. Only 16 farms reported to have had their goats vaccinated. The used vaccines were OVILIS1 HEPTAVAC ad us. vet. against enterotoxemia (12 farms), ECTHYBEL1 ad us. vet. against Ecthyma contagiosum (2 farms) and FOOTVAX1 ad us. vet. against foot rot (2 farms). Fifty-one goat farms had a veterinarian. Generally, only one or two farmers mentioned to have the same veterinarian, and the three veterinarians mentioned by more farmers served both on positive and negative farms. Thus, we could not find any connection between positive farms and a specific veterinarian. The distribution and respective numbers of the eight variables suitable for statistical analysis are given in Table 1. Sixty-five percent of the positive farms, but only 39% of the negative farms, had no sheep. The preventive effect of
‘‘sheep size’’ increased with the categories of higher number of sheep on the farm. Additionally, exposure to sheep from other farms also showed a preventive effect. Most farms reported to have sent their goats on alpine pasture; 69% for negative farms and 80% for positive farms. Half of the negative farms reported exposure to red deer, but 93% of positive farms. The proportion of small farms among the negative farms (78%) doubled that of positive farms (40%). Purchase of goats or exposure to goats from other farms increased the risk of a farm to be positive. The final model included the following factors: ‘‘exposure to goats from other farms’’ with an odds ratio (OR) of 10.12 (p = 0.0073), ‘‘exposure of the goats to red deer’’ with an OR of 4.793 (p = 0.0446) and ‘‘exposure to sheep from other farms’’ with an OR of 0.047 (p = 0.0022) (Table 2). Within the selection process odds ratios from the multi-factorial model show huge differences to nonadjusted values, indicating the confounding process within the variables selected. But the comparison of the odds ratios on all steps of the selection process and with the odds ratios from the full model of all variables under study showed less variation. In the final model, the two factors ‘‘exposure to goats from other farms’’ and ‘‘exposure to sheep from other farms’’ seemed to be more influential than ‘‘exposure of the goats to red deer’’. All 36 goatlings were rRT-PCR-negative for TOV before they were sent to alpine pasture. In autumn 2009, after the alpine pasture, 8 out of 13 were positive on farm A, 7 out of 13 on farm B (both farms drove their goats on the same alp) and 2 out of 10 on farm C. 4. Discussion This study aimed to clarify some hypotheses of the infection pathway of TOV and direct further investigations. Thus, within the study we sifted the putative risk factors for the presence of goats seropositive for TOV in herds in TI. Major results were to exclude iatrogenic infection and transmission by contaminated vaccines or mechanical
Table 2 Logistic regression models of factors associated with the infection of goats with Toggenburg Orbivirus (TOV) in the canton of Ticino. Single factors, full model and forward model selection (AIC, Akaike information criterion). Factor description
Model selection (AIC) Single risk factors
Full model (68.427)
Step 1 (74.604)
Step 2 (64.707)
Step 3 (62.454)
Step 4 (62.523)
Final model (62.454)
4.793 (0.0446)
5.180 (0.0411) 2.905 (0.1788) 12.046 (0.0057) 0.059 (0.0055)
4.793 (0.0446)
Odds ratio (p-value) Small sheep farms vs. no sheep Large sheep farms vs. no sheep Any vaccination: yes vs. no Alpine pasture: higher vs. lower 500 m Size of the goat farm: larger vs. smaller than 20 animals Exposure of the goats to red deer: yes vs. no Purchase of goats in the past 3 years: yes vs. no Exposure to goats from other farms: yes vs. no Exposure to sheep from other farms: yes vs. no
0.6 (0.46) 0.21 (0.02) 3.20 (0.10) 2.80 (0.12)
1.345 0.544 3.296 0.504
5.28 (0.006)
2.363 (0.2648)
4.68 (0.009)
6.850 (0.0853)
2.41 (0.13)
4.180 (0.1092)
5.40 (0.004)
14.829 (0.0107)
0.20 (0.01)
(0.7543) (0.5780) (0.2412) (0.5673)
0.068 (0.0258)
5.403 (0.0035)
13.344 (0.0016) 0.070 (0.0031)
10.120 0.0073) 0.047 (0.0022)
10.120 (0.0073) 0.047 (0.0022)
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vectors as main mode of infection. Furthermore, the evidence provided was that the transmission of the virus takes place on the alpine pasture. The results are not indicative for a vertical transmission. However, the study design had some weaknesses, mainly due to the limited time and resources. As the study population consisted of 60 farms only, the power to detect a risk factor was limited. Thus, only factors with a high OR could be identified. The many factors with too few observations in at least one category could not be analyzed as they did not fulfill the inclusion criterions for the univariate and multivariate analysis, e.g. veterinarians or breeds. Additionally, the interval between taking blood samples and collecting the questionnaires was two years. However, there was no indication that the situation of the investigated factors changed during that time. All contacted 60 owners answered the questionnaire (response rate 100%). However, it is disappointing that many farmers were not aware of the vaccination history of their goats. In Switzerland, all vaccinations and treatments must be recorded in the stable book. All three interviewers spoke Italian and were aware of the topic and the TOV status of the farms. Thus, interviewer bias cannot be excluded. Another weakness can be seen in the definition of a positive farm, as it is based on a sample from a herd and thus misclassification can occur, as a farm can spuriously be declared as negative. However, as the within herd prevalence in TI was 44%, and our on-farm samples were large enough to detect a 40% prevalence with a probability of at least 95%, a high proportion of farms declared false negative is unlikely. For positive farms, misclassification may have occurred due to a false positive test results or purchase of serologically positive goats. From three farms we observed one seropositive result only. However, two of these farms had only two goats, and only one had 60 goats with 8 goats tested and one being seropositive and one being questionable. Interestingly, these two goats were tested by rRT-PCR panBTV and were negative. Thus, for this farm we cannot exclude the possibility of misclassification. However, these misclassifications are non-differential and thus bias the results towards no association and are therefore not critical for the identified risk factors (Dohoo et al., 2003). Theoretically, there is a chance that some of the goats tested serologically only were positive for antibodies due to the exposure to BTV-8 instead of TOV, given that ELISA tests are not specific for the latter. But this chance is remote as the presence of BTV in TI in 2008 and 2009 is highly unlikely: a serological survey was conducted in 2003 and testing of sentinel cattle had been implemented in Switzerland, and notably in TI, already since 2003 due to the presence of BTV in the south of Italy (Cagienard et al., 2006). The active surveillance in cattle and sheep, in collaboration with the increasing disease awareness following the outbreak of BTV-8 in Europe, allowed to demonstrate freedom from BTV in Switzerland until the first BTV-8 outbreak in late autumn 2007 (Hofmann et al., 2008b; Schwermer et al., 2008; Stuber et al., 2009). Under the intensive national surveillance in the years 2008–2011 no cases of BTV have been found in TI. Retrospective testing of goat serum revealed a high proportion of seropositive goats already ten years ago in
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Ticino, while Bluetongue was absent in that region (Chaignat et al., 2010). In the north of Italy surveillance for BTV was very intensive since 2003, and in 2007 a BTV-8 infection was secluded to a limited area (Giovannini et al., 2008). Prior to this study, there were hints that the epidemiology of TOV shows some divergences from that of other BTV serotypes. The discovery of the virus in goats only is indicative for the mayor role of this species in the epidemiology of TOV. Generally, goats are often not clinically affected by BTV infections (Gerbier et al., 2008). The regional presence of BTV was sometimes discovered in healthy goats first. For example, the BTV/KM strain, belonging to BTV-2, was isolated and discovered in 2003 in healthy goats in Taiwan. This was the first report of BTV on this island (Ting et al., 2005). In 2010, BTV-12 was also found in clinically healthy ruminants in this region (Lee et al., 2010). According to the results obtained for the variables investigated in our screening study, transmission of TOV can be explained best by variables describing exposure to goats, sheep and wild animals. But it needs to be stressed that our variables do not implicitly include direct animalsto-animal contact, and it is likely that they are only associated with, and thus indicative for, an unknown risk modifying factor not measured in the study. Especially, the relevance of the factor ‘‘alpine pasture’’ on the factors describing exposure to goats and other species needs to be considered, especially as from the observation with the goatlings it became obvious that transmission occurs during the alpine pasture. As the animals were tested negative for TOV prior to alpine pasture time and positive afterwards, it is highly probable that they get infected on the pasture and not vertically from their dams. However, it should be considered that mating takes place usually during alpine pasture, and venereal and congenital transmission of BTV can occur in ruminants (Anon, 2008). In general, the results indicated that exposure to goats and red deer increases the likelihood of TOV seropositive goats, whereas the exposure to sheep reduces this likelihood. For ‘‘exposure of the goats to red deer’’ the physical closeness of this contact is not given. A majority of all farmers mentioned ‘‘exposure of the goats to red deer’’. This may be explained by the fact that at the time of our study the goat breeders had problems with a parasitical disease caused by Elaphostrongylus cervi, transmitted apparently from red deer to goats with a high mortality in goats (Pusterla et al., 1998). This problem was mentioned several times by owners during the interview. A preventive factor included in the final model was ‘‘exposure to sheep from other farms’’. Additionally, all other variables including ‘‘sheep’’ showed a preventive effect and for ‘‘number of sheep on the goat farm’’ the effect increased with the number of sheep. As sheep seem to be not susceptible for TOV in the field, the effect may be best explained by a ‘‘dilution effect’’, that can be effective independent from transmission ways. On mixed farms, contact between goats is less likely and/or the vector has less chance to feed on a goat. For other BTV serotypes, it is well known that clinical symptoms occur principally in some sheep breeds and some species of deer, while other susceptible species seroconvert without disease (Mellor
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et al., 2008). Some strains of BTV, like the serotype 8, can induce severe disease in other species like cattle and camelids (Maclachlan et al., 2009). In our study the goat breed kept had no influence on the TOV status of the farm. In consequence, the epidemiological picture for TOV is different from other BTV in the respect of species specificity. Given the fact that sheep can be infected experimentally, this finding can be indicative for a transmission pathway between goats that excludes sheep. That ‘‘exposure to goats from other farms’’ is a risk factor is self-evident, as the likelihood for transmission increases with increased number of contacts between goats. Contact to goats from other farms takes most often place during alpine pasture. Together with the findings from the goatlings investigation, the evidence provided is that transmission of TOV takes place during alpine pasture. Thus, there are two substantial arguments or an animal-to-animal transmission (horizontally or vertically): (1) despite intensive surveillance TOV has been found only in goats in a region where it was present at high prevalence in the goat population for at least 10 years, and (2) the nature of the risk factors in the final model. For BTV8, the possibility of a transplacental transmission has been discussed as well as a direct transmission through the ingestion of contaminated placenta (Desmecht et al., 2008; Menzies et al., 2008; Worwa et al., 2009). For BTV-11 it has been described in elk (Stott et al., 1982). In general, for field strains of BTV, vertical or venereal transmission is considered a rare event (Maclachlan et al., 2009). Only Luedke (1985) describes the perpetuation of BTV through three generations in cattle in the absence of vector midges. Thus, to solve the remaining question if TOV is transmitted directly from animal to animal, further studies are needed. Despite the limited data set, this screening study was able to direct the focus of further research questions concerning the transmission pathway of TOV towards vertical transmission in goats and horizontal transmission on alpine pasture with or without the involvement of an insect vector. Acknowledgements The authors would like to thank all participating animal holders for their cooperation, the cantonal veterinary authorities for their help in providing data, especially Tullio Vanzetti, Mario Bazzani and Boris Ferrari; Barbara Thu¨r from the Institute of Virology and Immunoprophylaxis in Mittelha¨usern, Switzerland, and Orlando Petrini from the Cantonal Institute of Microbiology in Bellinzona, Switzerland, for the critical and helpful discussions. References Anon, 2008. Bluetongue. In: Terrestrial Animal Health Code, 14th ed. Office International des Epizooties, Paris (Chapter 8.3).
Cagienard, A., Thu¨r, B., Griot, C., Hamblin, C., Sta¨rk, K.D.C., 2006. No evidence of bluetongue virus in Switzerland. Vet. Microbiol. 116, 13–20. Chaignat, V., Worwa, G., Scherrer, N., Hilbe, M., Ehrensperger, F., Batten, C., Cortyen, M., Hofmann, M., Thuer, B., 2009. Toggenburg Orbivirus, a new bluetongue virus: initial detection, first observations in field and experimental infection of goats and sheep. Vet. Microbiol. 138, 11–19. Chaignat, V., Schwermer, H., Casati, S., Planzer, J., Worwa, G., Vanzetti, T., Batten, C., Hofmann, M.A., Thu¨r, B., 2010. Occurrence and spatial distribution of Toggenburg Orbivirus in Switzerland. Small Rumin. Res. 93 (2), 157–164. Desmecht, D., Bergh, R.V., Sartelet, A., Leclerc, M., Mignot, C., Misse, F., Sudraud, C., Berthemin, S., Jolly, S., Mousset, B., Linden, A., Coignoul, F., Cassart, D., 2008. Evidence for transplacental transmission of the current wild-type strain of bluetongue virus serotype 8 in cattle. Vet. Rec. 163, 50–52. Dohoo, I.R., Martin, W., Stryhm, H., 2003. Veterinary Epidemiologic Research. AVC Inc., University of Prince Edward Island, Charlottetown, pp. 220–222. Gerbier, G., Biteau-Coroller, F., Grillet, C., Parodi, J., Zientara, S., Baldet, T., Guis, H., Roger, F., 2008. Description of the outbreak of bluetongue in Corsica in 2003, and lessons for surveillance. Vet. Rec. 162, 173–176. Giovannini, A., Calistri, P., Conte, A., Goffredo, M., Paladini, C., Lelli, R., Caporale, V., 2008. Bluetongue virus serotype 8 in Europe in 20072008 and the risk of virus introduction and spread in Italy. In: Symposium on ‘‘Bluetongue in Europe back to the future’’, 7 June 2008, Brescia, Italy. Hofmann, M.A., Renzullo, S., Mader, M., Chaignat, V., Worwa, G., Thuer, B., 2008a. Genetic characterization of Toggenburg orbivirus, a new bluetongue virus, from goats, Switzerland. Emerg. Infect. Dis. 14, 1855– 1861. Hofmann, M.A., Griot, C., Chaignat, V., Perler, L., Thu¨r, B., 2008b. Bluetongue disease reaches Switzerland. Schweiz. Arch. Tierheilkd. 150, 49–56. Hofmann, M.A., Renzullo, S., Planzer, J., Mader, M., Chaignat, V., Thuer, B., 2010. Detection of Toggenburg Orbivirus by a segment 2-specific quantitative RT-PCR. J. Virol. Methods 165, 325–329. Lee, F., Ting, L.J., Jong, M.H., Chang, W.M., Wang, F.I., 2010. Subclinical bluetongue virus infection in domestic ruminants in Taiwan. Vet. Microbiol. 142, 225–231. Luedke, A.J., 1985. Effect of bluetongue virus on reproduction in sheep and cattle. Prog. Clin. Biol. Res. 178, 71–78. Maclachlan, N.J., Drew, C.P., Darpel, K.E., Worwa, G., 2009. The pathology and pathogenesis of bluetongue. J. Comp. Pathol. 141, 1–16. Mellor, P.S., Carpenter, S., Harrup, L., Baylis, M., Mertens, P.P., 2008. Bluetongue in Europe and the Mediterranean Basin: history of occurrence prior to 2006. Prev. Vet. Med. 87, 4–20. Menzies, F.D., McCullough, S.J., McKeown, I.M., Forster, J.L., Jess, S., Batten, C., Murchie, A.K., Gloster, J., Fallows, J.G., Pelgrim, W., Mellor, P.S., Oura, C.A., 2008. Evidence for transplacental and contact transmission of bluetongue virus in cattle. Vet. Rec. 163, 203–209. Pusterla, N., Hertzberg, H., Viglezio, M., Vanzetti, T., Braun, U., 1998. The occurrence of lumbar paralysis in goats and the appearance of Elaphostrongylus cervi in red deer in the canton Ticino. Schweiz. Arch. Tierheilkd. 140, 76–82. Schwermer, H., Chaignat, V., Thu¨r, B., Hadorn, D., Scha¨rrer, S., Schaffner, F., Breidenbach, E., 2008. The monitoring program of bluetongue disease in Switzerland. Schweiz. Arch. Tierheilkd. 150, 129–132. Stott, J.L., Lauerman, L.H., Luedke, A.J., 1982. Bluetongue virus in pregnant elk and their calves. Am. J. Vet. Res. 43, 423–428. Stuber, B., Ochs, H., Tschuor, A., Zanolari, P., Danuser, J., Perler, L., 2009. Livestock owners with specifically increased disease awareness for Bluetongue: A new approach to disease surveillance. Schweiz. Arch. Tierheilkd. 151, 317–321. Ting, L.J., Lee, M.S., Huang, T.S., Huang, C.C., Kuo, S.T., Lee, F., Jong, M.H., Shiau, J.R., Lin, S.Y., 2005. Identification of bluetongue virus in goats in Taiwan. Vet. Rec. 156, 52. Worwa, G., Hilbe, M., Ehrensperger, F., Chaignat, V., Hofmann, M.A., Griot, C., Maclachlan, N.J., Thuer, B., 2009. Experimental transplacental infection of sheep with bluetongue virus serotype 8. Vet. Rec. 164, 499–500.
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Putative risk factors for infections with Toggenburg Orbivirus in goat herds in Southern Switzerland (Canton of Ticino) Antonella Reber a, Lothar Kreienbrock b, Simona Casati c, Vale´rie Chaignat d, Heinzpeter Schwermer e,* a
Veterinary Public Health Institute, Schwarzenburgstrasse 155, 3003 Liebefeld, Switzerland Institute for Biometry, Epidemiology and Information Processing, WHO-Collaborating Centre for Research and Training in Veterinary Public Health, University of Veterinary Medicine, Bu¨nteweg 2, D-30559 Hannover, Federal Republic of Germany c Cantonal Institute of Microbiology, Via Mirasole 22A, 6500 Bellinzona, Switzerland d Institute of Virology and Immunoprophylaxis, Sensemattstrasse 293, 3147 Mittelha¨usern, Switzerland e Swiss Federal Veterinary Office, Schwarzenburgstrasse 155, 3003 Liebefeld, Switzerland b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 January 2011 Received in revised form 9 May 2012 Accepted 18 May 2012
Toggenburg Orbivirus (TOV), only detected in goats, has been described as a member of the Bluetongue virus (BTV) serogroup. The transmission pathway of the virus seems different from other Bluetongue viruses (BTVs). The objective of this study was to explore risk factors, especially the influence of alpine pasture and the presence of other livestock species, for the presence of TOV infected goats on farms. Between February 2008 and September 2009, blood samples were collected and analyzed for TOV and hereupon a total of 60 goat farm owners (37 TOV-positive and 23 TOV-negative holdings) were interviewed. Additionally, goatlings were tested for TOV by rRT-PCR prior and after alpine pasture in 2009. These goatlings were positive for TOV only after the alpine pasture. The final logistic regression model included: ‘‘exposure to goats from other farms’’ (OR = 10.12, p = 0.007), ‘‘exposure of the goats to red deer’’ (OR = 4.79, p = 0.04) and ‘‘exposure to sheep from other farms’’ (OR = 0.05, p = 0.002). These variables do not implicitly include direct contact, and the findings are only vaguely indicative for a contact-driven transmission. Furthermore, it is likely that they are only associated with, and thus indicative for, an unknown risk factor associated with alpine pasture not measured in the study. The results of this screening study do not indicate iatrogenic transmission pathways as a main transmission mode and stimulate the formulation of hypotheses on the origin, the transmission pathway and other host species for TOV. Especially, the involvement of an insect vector in transmission on alpine pasture and the relevance of vertical transmission are to be clarified. ß 2012 Elsevier B.V. All rights reserved.
Keywords: Switzerland Bluetongue virus Goat Questionnaire Logistic regression Transmission Toggenburg Orbivirus
1. Introduction The Bluetongue virus (BTV) serogroup holds some of the most important pathogens of the genus Orbivirus. Recently, a new member of the Bluetongue virus (BTV) serogroup named Toggenburg Orbivirus (TOV) has been
* Corresponding author. Tel.: +41 31 323 30 53; fax: +41 31 323 95 43. E-mail address: [email protected] (H. Schwermer). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.05.027
described in goats from Switzerland (Hofmann et al., 2008a; Chaignat et al., 2009). The index cases were detected on two distinct goat holdings north of the Alps, in the regions of Solothurn and Toggenburg (Hofmann et al., 2008b; Chaignat et al., 2009). Adult goats, in the field and after experimental TOV infection, only occasionally show clinical symptoms or gross pathological alterations. However, it could be demonstrated that the virus is transmissible to and replicates in goats. In contrast to adult goats, several stillborn and weak born kids were reported on the index
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farms. Several facts indicate a vertical transmission on these farms (Chaignat et al., 2009). Although it was possible to infect sheep experimentally, no evidence of an infection process outside the goat population could be found in the field despite extensive testing in cattle, sheep or other domestic ruminants (Chaignat et al., 2010). These field observations revealed no indication of clinical disease, but a seroprevalence rate of 4% in sheep on farms with high seroprevalence of TOV in goats (Chaignat et al., 2010). Following the discovery of the new virus, a serological prevalence study for TOV in the Swiss goat population was conducted to gain insight into the spatial distribution of the virus. Although TOV seems to be of minor importance as a pathogen in the field, even in the absence of clinical disease TOV interferes with the diagnostics for BTV in goats and thus the distribution of TOV is of general interest. The national study showed that cantons north of the Alps were free, cantons in the Alps had very few cases, but in the Canton of Ticino (TI), south of the Alps, an exceptionally high prevalence of around 60% on the farm level and 49% on the animal level was observed (Chaignat et al., 2010). In TI, the positive farms were distributed over the whole canton. However, cases were more abundant in areas with summer alpine pasture (Chaignat et al., 2010). Evidence for the long presence of TOV in goats in TI was found after testing serum stored in the national serum bank, collected in 1998: twenty percent of the tested samples were positive in BT ELISA (Chaignat et al., 2010). Given the negative results of the surveillance for BT in this region (Cagienard et al., 2006), and given TOV is serologically indistinguishable from other BTV, the most likely reason for these results are TOV infections. Additionally, it is known that BT disease is either rare or not recognized in many regions with enzootic BTV infection (Maclachlan et al., 2009). Thus, it is most likely that TOV stayed undiscovered for a long period of time. Given the recent detection and identification, but albeit high regional and within-herd prevalence only in goats even more than ten years ago, questions about the origin and transmission pathway of the virus raised. Before the beginning of our study, different hypotheses on the epidemiology and transmission pathway of TOV had already been discussed. Because of the high prevalence in that region, we focused and carried out our study in TI. The purpose of this cross-sectional screening study was to identify putative risk factors for the presence of TOV infected goats on the farm, to unravel particular epidemiological features of this virus and to appraise the hypothesized transmission pathways. The results should direct further investigations on the epidemiology of this virus and should support future studies rather than being finally conclusive. 2. Materials and methods 2.1. Definition of the outcome variable (case definition) The unit of analysis was the goat farm. A goat farm was declared to be a case farm, if at least one goat was tested seropositive. Otherwise the farm was declared and
identified as negative. All samples were taken by official veterinarians and selected randomly. If there were up to five goats on the farm, samples were taken from all animals. On farms with more than five and less than 15 goats, at least five samples were taken. On farms with at least 15 goats, 8–75 goats were tested serologically. The higher number of tested animals refers to farms with high seroprevalence, where we tried to identify freshly infected animals for virus isolation. With an assumed sensitivity of 95% of the ELISA and a design prevalence on the farm of 40%, these figures correspond to probabilities between 95% and 99% to detect at least one positive goat in herds of 15, respectively, more than 15, goats (Freecalc 2.0, Australian Centre for International Agricultural Research, Canberra, Australia). 2.2. Serological analysis All blood samples were tested at the Institute of Virology and Immunoprophylaxis (IVI, Mittelha¨usern, Switzerland) or at the Cantonal Institute of Microbiology (Bellinzona, Switzerland). The testing procedure was conducted as described by Chaignat et al. (2010). Briefly, the diagnostic kit in use was Bluetongue virus Antibody Test Kit (VMRD, Pullman, WA, USA) (BT ELISA VMRD) and positive sera were confirmed with the ID.Screen Blue Tongue Competition ELISA Kit (ID.VET, Montpellier, France) and the double recognition ELISA INGEZIM BTV DR1 (INGENASA, Madrid, Spain) (BT ELISA INGEZIM). If weak positive results were obtained using BT ELISA VMRD, Blue Tongue Competitive ELISA Kit (B.D.S.L., Irvine, UK) (BT ELISA BDSL) was used as confirmation tests. The tests were used according to the manufacturer’s instructions. No TOV specific serology assay is available but as TI did not have any BTV infection up to the study period and did not vaccinate goats against BTV, seropositivity is a decisive hint for TOV infection. Additionally, in 81 seropositive samples from seven farms with high seroprevalence, TOV genome could be detected by a TOV rRT-PCR and confirmed by sequencing. Viral RNA was detected using RT-qPCR-panBTV and a first version of RT-qPCR-TOV (Hofmann et al., 2010). 2.3. Study population Farms that had a testing result for TOV in 2008 and 2009 were included into the sampling frame. The study population comprised farms sampled in the 2008 annual Caprine Artheritis Encephalitis (CAE) survey, a crosssectional survey (sample of 2008). For that survey, the herds were chosen randomly from the database of the AGIS (Swiss agricultural information system; Federal Office for Agriculture). In 2008, serum samples were collected between February and March, prior to the vaccination of cattle and sheep against BTV-8. Five samples per herd were tested with BT ELISA. In the 2009 sampling, farms with seropositive goats were sampled again. Goats and, if present, sheep and cattle were sampled. On these farms, five to ten samples from yet untested goats and additional five to ten samples from unvaccinated sheep and cattle were collected.
A. Reber et al. / Veterinary Microbiology 160 (2012) 29–34
In total, test results for TOV from 95 farms were available, with 57 farms having at least one seropositive goat. With a statistical power analysis with two independent proportions using the statistical software PASS 2008 (http:// www.ncss.com/pass.html) we determined that a sample size of 60 farms has to be provided (sample size group 1 = 30, sample size group 2 = 30) to detect an odds ratio of 4 with a power of approximately 80% (target alpha at 5%). As we were interested in factors having a robust effect on the outcome, we decided to conduct the study albeit the low number of data sets. The 60 farms were selected randomly. Additionally, as preliminary results indicated that infection might took place on alpine pasture, 36 goatlings originating from three TOV-affected holdings (holding A [in-herd seroprevalence 42%], B [23%] and C [38%]) were tested for TOV by rRT-PCR prior and after alpine pasture in 2009. These animals were born during the vector-free period in January and February 2009 and were not vaccinated against BTV-8. 2.4. Questionnaire and data collection For the collection of concomitant data, one out of three interviewers filled out a questionnaire in Italian during a telephone interview with the owner. The gathered information was: stable location, minimum and maximum altitude of the farm pasture; the number and breed of goats, sheep, cattle, other ruminants and equines on the farm; introduction of animals in the previous three years, with date, species and origin; frequency of exposure to animals from other farms with species, period and motive; frequency of exposure to wild animals with species, period and kind of exposure; detailed information on vaccination history; the name of the veterinarian. Additionally, we also asked if there had been sanitary problems or special observations in the last three years. The interview took approximately ten minutes. Many farmers had problems to
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answer the question about vaccination and often did not recollect whether it was a vaccination or another injection, or the name of the vaccine. 2.5. Statistical analysis For general data description mean, median, and coefficient of variation were calculated for the continuous variables and frequency tables were set up for the categorical variables. Calculation of odds ratio for the categorical variables and further (risk factor) analyses were only performed, if each category comprised at least five units. As the number of observations was small and the distributions of continuous variables were clumps at low numbers with an extreme skewed distribution, the continuous variables were categorized with their median to avoid a loss of power within the analyses. Because of the uncertainty on the data for vaccination, we simplified this factor and made a binary categorization into general presence or absence of vaccination on the farm. A basic set of variables was suitable for further analysis, i.e. ‘‘size of the goat farm’’ (farm_size) with a median of 20 and categorized in two sizes, ‘‘number of sheep on the goat farm’’ (sheep_size) with a median of 30 and three categories, ‘‘alpine pasture’’, ‘‘vaccinated’’, ‘‘purchase of goats in the last 3 years’’, ‘‘exposure of the goats to red deer’’, ‘‘exposure to sheep from other farms’’ and ‘‘exposure to goats from other farms’’ (Table 1). Model selection for the risk factor analyses was conducted by an automated forward selection approach using an entry p-value of 25% and a staying p-value of 15%. These analyses were improved by systematically mixing backward and forward selection to check for model sensitivity, if no different final model was found. All statistical analyses were conducted with SAS 9.2 (http://www.sas.com) with the use of PROC LOGISTIC for performing ordinary logistic regression analyses.
Table 1 Univariate statistics of factors associated with the infection of goats with Toggenburg Orbivirus (TOV) in the canton of Ticino. OR, odds ratio; CI, confidence interval. Factor description
Category
Number of sheep on the goat farm
0 1–30 >30
TOV-negative n (%)
TOV-positive n (%)
OR
p-Value
95% CI
9 (27.27) 5 (38.46) 9 (64.29)
24 (72.73) 8 (61.54) 5 (35.71)
1 0.6 0.21
0.46 0.02
0.16–2.33 0.06–0.79
Vaccinated (any vaccination)
No Yes
20 (44.44) 3 (20)
25 (55.56) 12(80)
1 3.20
0.10
0.79–12.91
Alpine pasture
<500 m >500 m
7 (58.33) 16 33.33)
5 (41.67) 32 (66.67)
1 2.80
0.12
0.77–10.22
Size of the goat farm
1–20 >20
18 (54.55) 5 (18.52)
15 (45.45) 22 (81.48)
1 5.28
0.006
1.61–17.33
Exposure of the goats to red deer
No Yes
10 (62.5) 11 (26.83)
6 (37.5) 30 (73.17)
1 4.68
0.009
1.47–14.92
Purchase of goats in the past 3 years
No Yes
16 (50) 6 (26.09
16 (50) 17 (73.91)
1 2.41
0.13
0.78–7.48
Exposure to goats from other farms
No Yes
16 (59.26) 7 (21.21)
11 (40.74) 26 (78.79)
1 5.40
0.004
1.74–16.79
Exposure to sheep from other farms
No Yes
13 (28.89 10 (66.67)
32 (71.11 5 (33.33)
1 0.20
0.01
0.06–0.71
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3. Results From the 60 goat farms tested, 37 farms were positive by the serological blood test and 23 were negative. Nera Verzasca (28 farms), the Chamois-coloured (15 farms) and Saanen (11 farms) were the mostly often kept breeds. In addition, most breeders also had crossbreeds (36 farms). Rare breeds were Tibetan goat, Peacock goat, Stieffelgeiss goat, Boer-goat, Mountain goat und Grisons. We could observe small (20; 20 = the median of number of goats on the farm) and also big goat farms (>20) with goats only. On most of the mixed farms, the number of goats on the farm and the number of sheep on the goat farm were inversely proportional, e.g. in large flocks of goats, the number of sheep on the farm was small, and in small goat flocks the number of sheep was large. None of our 60 goat farms had small ruminants imported in the last three years. Many farmers reported exposure of their goats to wild animals. The species observed were the Western European red deer (Cervus elaphus elaphus) (41 farms), roe deer (Capreolus capreolus) (34 farms), chamois (Rupicapra rupicapra) (29 farms), wild boar (Sus scrofa) (16 farms) and fox (Vulpes vulpes) (10 farms). The type and closeness of the exposure varied from ‘‘occasionally seen on the pasture’’ to ‘‘regularly staying together with the goats’’. Only 16 farms reported to have had their goats vaccinated. The used vaccines were OVILIS1 HEPTAVAC ad us. vet. against enterotoxemia (12 farms), ECTHYBEL1 ad us. vet. against Ecthyma contagiosum (2 farms) and FOOTVAX1 ad us. vet. against foot rot (2 farms). Fifty-one goat farms had a veterinarian. Generally, only one or two farmers mentioned to have the same veterinarian, and the three veterinarians mentioned by more farmers served both on positive and negative farms. Thus, we could not find any connection between positive farms and a specific veterinarian. The distribution and respective numbers of the eight variables suitable for statistical analysis are given in Table 1. Sixty-five percent of the positive farms, but only 39% of the negative farms, had no sheep. The preventive effect of
‘‘sheep size’’ increased with the categories of higher number of sheep on the farm. Additionally, exposure to sheep from other farms also showed a preventive effect. Most farms reported to have sent their goats on alpine pasture; 69% for negative farms and 80% for positive farms. Half of the negative farms reported exposure to red deer, but 93% of positive farms. The proportion of small farms among the negative farms (78%) doubled that of positive farms (40%). Purchase of goats or exposure to goats from other farms increased the risk of a farm to be positive. The final model included the following factors: ‘‘exposure to goats from other farms’’ with an odds ratio (OR) of 10.12 (p = 0.0073), ‘‘exposure of the goats to red deer’’ with an OR of 4.793 (p = 0.0446) and ‘‘exposure to sheep from other farms’’ with an OR of 0.047 (p = 0.0022) (Table 2). Within the selection process odds ratios from the multi-factorial model show huge differences to nonadjusted values, indicating the confounding process within the variables selected. But the comparison of the odds ratios on all steps of the selection process and with the odds ratios from the full model of all variables under study showed less variation. In the final model, the two factors ‘‘exposure to goats from other farms’’ and ‘‘exposure to sheep from other farms’’ seemed to be more influential than ‘‘exposure of the goats to red deer’’. All 36 goatlings were rRT-PCR-negative for TOV before they were sent to alpine pasture. In autumn 2009, after the alpine pasture, 8 out of 13 were positive on farm A, 7 out of 13 on farm B (both farms drove their goats on the same alp) and 2 out of 10 on farm C. 4. Discussion This study aimed to clarify some hypotheses of the infection pathway of TOV and direct further investigations. Thus, within the study we sifted the putative risk factors for the presence of goats seropositive for TOV in herds in TI. Major results were to exclude iatrogenic infection and transmission by contaminated vaccines or mechanical
Table 2 Logistic regression models of factors associated with the infection of goats with Toggenburg Orbivirus (TOV) in the canton of Ticino. Single factors, full model and forward model selection (AIC, Akaike information criterion). Factor description
Model selection (AIC) Single risk factors
Full model (68.427)
Step 1 (74.604)
Step 2 (64.707)
Step 3 (62.454)
Step 4 (62.523)
Final model (62.454)
4.793 (0.0446)
5.180 (0.0411) 2.905 (0.1788) 12.046 (0.0057) 0.059 (0.0055)
4.793 (0.0446)
Odds ratio (p-value) Small sheep farms vs. no sheep Large sheep farms vs. no sheep Any vaccination: yes vs. no Alpine pasture: higher vs. lower 500 m Size of the goat farm: larger vs. smaller than 20 animals Exposure of the goats to red deer: yes vs. no Purchase of goats in the past 3 years: yes vs. no Exposure to goats from other farms: yes vs. no Exposure to sheep from other farms: yes vs. no
0.6 (0.46) 0.21 (0.02) 3.20 (0.10) 2.80 (0.12)
1.345 0.544 3.296 0.504
5.28 (0.006)
2.363 (0.2648)
4.68 (0.009)
6.850 (0.0853)
2.41 (0.13)
4.180 (0.1092)
5.40 (0.004)
14.829 (0.0107)
0.20 (0.01)
(0.7543) (0.5780) (0.2412) (0.5673)
0.068 (0.0258)
5.403 (0.0035)
13.344 (0.0016) 0.070 (0.0031)
10.120 0.0073) 0.047 (0.0022)
10.120 (0.0073) 0.047 (0.0022)
A. Reber et al. / Veterinary Microbiology 160 (2012) 29–34
vectors as main mode of infection. Furthermore, the evidence provided was that the transmission of the virus takes place on the alpine pasture. The results are not indicative for a vertical transmission. However, the study design had some weaknesses, mainly due to the limited time and resources. As the study population consisted of 60 farms only, the power to detect a risk factor was limited. Thus, only factors with a high OR could be identified. The many factors with too few observations in at least one category could not be analyzed as they did not fulfill the inclusion criterions for the univariate and multivariate analysis, e.g. veterinarians or breeds. Additionally, the interval between taking blood samples and collecting the questionnaires was two years. However, there was no indication that the situation of the investigated factors changed during that time. All contacted 60 owners answered the questionnaire (response rate 100%). However, it is disappointing that many farmers were not aware of the vaccination history of their goats. In Switzerland, all vaccinations and treatments must be recorded in the stable book. All three interviewers spoke Italian and were aware of the topic and the TOV status of the farms. Thus, interviewer bias cannot be excluded. Another weakness can be seen in the definition of a positive farm, as it is based on a sample from a herd and thus misclassification can occur, as a farm can spuriously be declared as negative. However, as the within herd prevalence in TI was 44%, and our on-farm samples were large enough to detect a 40% prevalence with a probability of at least 95%, a high proportion of farms declared false negative is unlikely. For positive farms, misclassification may have occurred due to a false positive test results or purchase of serologically positive goats. From three farms we observed one seropositive result only. However, two of these farms had only two goats, and only one had 60 goats with 8 goats tested and one being seropositive and one being questionable. Interestingly, these two goats were tested by rRT-PCR panBTV and were negative. Thus, for this farm we cannot exclude the possibility of misclassification. However, these misclassifications are non-differential and thus bias the results towards no association and are therefore not critical for the identified risk factors (Dohoo et al., 2003). Theoretically, there is a chance that some of the goats tested serologically only were positive for antibodies due to the exposure to BTV-8 instead of TOV, given that ELISA tests are not specific for the latter. But this chance is remote as the presence of BTV in TI in 2008 and 2009 is highly unlikely: a serological survey was conducted in 2003 and testing of sentinel cattle had been implemented in Switzerland, and notably in TI, already since 2003 due to the presence of BTV in the south of Italy (Cagienard et al., 2006). The active surveillance in cattle and sheep, in collaboration with the increasing disease awareness following the outbreak of BTV-8 in Europe, allowed to demonstrate freedom from BTV in Switzerland until the first BTV-8 outbreak in late autumn 2007 (Hofmann et al., 2008b; Schwermer et al., 2008; Stuber et al., 2009). Under the intensive national surveillance in the years 2008–2011 no cases of BTV have been found in TI. Retrospective testing of goat serum revealed a high proportion of seropositive goats already ten years ago in
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Ticino, while Bluetongue was absent in that region (Chaignat et al., 2010). In the north of Italy surveillance for BTV was very intensive since 2003, and in 2007 a BTV-8 infection was secluded to a limited area (Giovannini et al., 2008). Prior to this study, there were hints that the epidemiology of TOV shows some divergences from that of other BTV serotypes. The discovery of the virus in goats only is indicative for the mayor role of this species in the epidemiology of TOV. Generally, goats are often not clinically affected by BTV infections (Gerbier et al., 2008). The regional presence of BTV was sometimes discovered in healthy goats first. For example, the BTV/KM strain, belonging to BTV-2, was isolated and discovered in 2003 in healthy goats in Taiwan. This was the first report of BTV on this island (Ting et al., 2005). In 2010, BTV-12 was also found in clinically healthy ruminants in this region (Lee et al., 2010). According to the results obtained for the variables investigated in our screening study, transmission of TOV can be explained best by variables describing exposure to goats, sheep and wild animals. But it needs to be stressed that our variables do not implicitly include direct animalsto-animal contact, and it is likely that they are only associated with, and thus indicative for, an unknown risk modifying factor not measured in the study. Especially, the relevance of the factor ‘‘alpine pasture’’ on the factors describing exposure to goats and other species needs to be considered, especially as from the observation with the goatlings it became obvious that transmission occurs during the alpine pasture. As the animals were tested negative for TOV prior to alpine pasture time and positive afterwards, it is highly probable that they get infected on the pasture and not vertically from their dams. However, it should be considered that mating takes place usually during alpine pasture, and venereal and congenital transmission of BTV can occur in ruminants (Anon, 2008). In general, the results indicated that exposure to goats and red deer increases the likelihood of TOV seropositive goats, whereas the exposure to sheep reduces this likelihood. For ‘‘exposure of the goats to red deer’’ the physical closeness of this contact is not given. A majority of all farmers mentioned ‘‘exposure of the goats to red deer’’. This may be explained by the fact that at the time of our study the goat breeders had problems with a parasitical disease caused by Elaphostrongylus cervi, transmitted apparently from red deer to goats with a high mortality in goats (Pusterla et al., 1998). This problem was mentioned several times by owners during the interview. A preventive factor included in the final model was ‘‘exposure to sheep from other farms’’. Additionally, all other variables including ‘‘sheep’’ showed a preventive effect and for ‘‘number of sheep on the goat farm’’ the effect increased with the number of sheep. As sheep seem to be not susceptible for TOV in the field, the effect may be best explained by a ‘‘dilution effect’’, that can be effective independent from transmission ways. On mixed farms, contact between goats is less likely and/or the vector has less chance to feed on a goat. For other BTV serotypes, it is well known that clinical symptoms occur principally in some sheep breeds and some species of deer, while other susceptible species seroconvert without disease (Mellor
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et al., 2008). Some strains of BTV, like the serotype 8, can induce severe disease in other species like cattle and camelids (Maclachlan et al., 2009). In our study the goat breed kept had no influence on the TOV status of the farm. In consequence, the epidemiological picture for TOV is different from other BTV in the respect of species specificity. Given the fact that sheep can be infected experimentally, this finding can be indicative for a transmission pathway between goats that excludes sheep. That ‘‘exposure to goats from other farms’’ is a risk factor is self-evident, as the likelihood for transmission increases with increased number of contacts between goats. Contact to goats from other farms takes most often place during alpine pasture. Together with the findings from the goatlings investigation, the evidence provided is that transmission of TOV takes place during alpine pasture. Thus, there are two substantial arguments or an animal-to-animal transmission (horizontally or vertically): (1) despite intensive surveillance TOV has been found only in goats in a region where it was present at high prevalence in the goat population for at least 10 years, and (2) the nature of the risk factors in the final model. For BTV8, the possibility of a transplacental transmission has been discussed as well as a direct transmission through the ingestion of contaminated placenta (Desmecht et al., 2008; Menzies et al., 2008; Worwa et al., 2009). For BTV-11 it has been described in elk (Stott et al., 1982). In general, for field strains of BTV, vertical or venereal transmission is considered a rare event (Maclachlan et al., 2009). Only Luedke (1985) describes the perpetuation of BTV through three generations in cattle in the absence of vector midges. Thus, to solve the remaining question if TOV is transmitted directly from animal to animal, further studies are needed. Despite the limited data set, this screening study was able to direct the focus of further research questions concerning the transmission pathway of TOV towards vertical transmission in goats and horizontal transmission on alpine pasture with or without the involvement of an insect vector. Acknowledgements The authors would like to thank all participating animal holders for their cooperation, the cantonal veterinary authorities for their help in providing data, especially Tullio Vanzetti, Mario Bazzani and Boris Ferrari; Barbara Thu¨r from the Institute of Virology and Immunoprophylaxis in Mittelha¨usern, Switzerland, and Orlando Petrini from the Cantonal Institute of Microbiology in Bellinzona, Switzerland, for the critical and helpful discussions. References Anon, 2008. Bluetongue. In: Terrestrial Animal Health Code, 14th ed. Office International des Epizooties, Paris (Chapter 8.3).
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