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Lungworm infection and ovine visna–maedi: Real risk factor or a confounding variable?
Small Ruminant Research 111 (2013) 157–161
Contents lists available at SciVerse ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Lungworm infection and ovine visna–maedi: Real risk factor or a confounding variable? ˜ R. Panadero, P. Díaz, P. Díez-Banos, ˜ C.M. López ∗,1 , N. Lago, M. Vina, P. Morrondo, G. Fernández Departamento de Patología Animal (Grupo INVESAGA), Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain
a r t i c l e
i n f o
Article history: Received 29 March 2012 Received in revised form 14 September 2012 Accepted 15 September 2012 Available online 4 October 2012 Keywords: Visna–maedi Prevalence Ovine Lungworm Protostrongylid
a b s t r a c t Serum and fecal samples (n = 2081) were taken from 74 commercial meat ovine flocks in Galicia (NW Spain) in order to establish the influence of lungworm infection with protostrongylids and/or Dictyocaulus filaria on visna–maedi (VM) seropositivity. VM was tested by an indirect ELISA test and lungworm larvae were detected by the modified Baermann–Wetzel technique. In addition to lungworm infection (protostrongylids and D. filaria), flock, management and individual factors were included in univariate and multivariate statistical tests. Overall VM seroprevalence was 18.9% and the prevalence by protostrongylids and D. filaria was 11.6% and 10.7%, respectively. Protostrongylid infection was considered as a risk factor for VM with univariate statistical analysis (1.49 significant OR), but neglected in a logistic multivariate regression analysis when the age of the animals was introduced as variable. As VM, protostrongylid infection is a chronic disease with a cumulative pattern over time, so that these two infections are closely related to the age of the animal. A Mantel–Haenszel procedure was applied and lungworm infection was detected as a confounding variable with no real effect on visna–maedi infection. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Visna–maedi (VM) is a worldwide disease caused by small ruminant lentiviruses (SRLV), a subgroup of the Retroviridae family, which causes chronic and multisystem disease in sheep (Narayan et al., 1982). SRLV do not constitute a risk to humans (Peterhans et al., 2004), but their importance is related to high economic losses due to an increase in mortality rates and a decrease in weight at lamb weaning and adult culling age (Arsenault et al., 2003).
∗ Corresponding author at: Departamento de Patología Animal (Grupo INVESAGA), Facultad de Veterinaria, Universidad de Santiago de Compostela, Avda Carballo Calero, s/n, 27002 Lugo, Spain. Tel.: +34 982822109; fax: +34 982822001. E-mail addresses: [email protected], [email protected] (C.M. López). 1 http://www.clopezsandez.es. 0921-4488/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.smallrumres.2012.09.010
VM is characterized by a long incubation period and symptoms rarely occur in sheep younger than two years of age (Narayan and Cork, 1985). These symptoms include lung fibrosis with a decrease in the total alveolar surface and an increase in the distance between the luminal and vascular sides of the alveoli (Pepin et al., 1998). There is also a decrease in the compliance of the lung tissue, which manifests itself as a kind of strained breathing; this pulmonary form is known as ‘maedi’ in Iceland, where it was first recognized and described. The most important routes by which Visna–maedi virus (VMV) is transmitted are the lactogenic one – from an infected ewe to its offspring via colostrum and milk (Pritchard and Dawson, 2000) – and horizontal transmission through respiratory fluids, mainly under high density conditions (Berriatua et al., 2003). There are also epidemiological evidences that horizontal transmission between lambs can occur, related to the variable of housing-time
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prior to weaning (Lago et al., 2012). Other routes of VMV transmission, like transplacental and seminal, have limited epidemiological importance (Blacklaws et al., 2004; Peterhans et al., 2004). Ovine lungworms are protostrongylids and Dictyocaulus filaria, being the first one the most frequent in ovine flocks. Protostrongylids are heteroxenous parasites that infect terrestrial molluscs as intermediate host. Ovine infections are commonly asymptomatic, but heavy infections cause decreased carcass weight, increased levels of mortality and impaired pulmonary gas exchange (Berrag and Cabaret, 1996; López et al., 2010). Lesions in lungs have been described as “granuloma-like worm nodules”, diffuse lesions and emphysema (Sauerlander, 1988). The role of lung nematodes in the induction of VM disease has been controversial within the scientific community. Whereas Mornex et al. (1994) have not found any effect of simultaneous parasitic infection of lungworms on the development of VM disease, Giangaspero et al. (1993) suggest a synergy between lung parasitic infection and VM seropositivity. Moreover, in the review by Straub (2004), regular prophylactic treatment with anthelmintic drugs reduces the risk of VMV transmission, which seems to indicate that lungworm infections foster the spread of the virus in sheep. Recently, in a cross-sectional study carried out in Galicia by Lago et al. (2012), individual (24.8%) and flock VMV seroprevalence (52.6%) were assessed, as well as the main risk factors, such as flock size, sheep–goat contact, type of lamb housing prior to weaning and age. However, lungworm infection was not included in that study. Previous studies demonstrated that lungworm individual prevalence in sheep in Galicia has decreased over the last decades from 33.9% (Díez et al., 1995) to 11.6% (López et al., 2011); however, flock prevalence is still very high (67.6%; López et al., 2011). As there is no effective treatment or vaccines against VMV, the measures adopted to control this chronic viral infection could have no effect if lungworm infection, also chronic, insidious and generalized at flock level, facilitates VMV transmission. So lungworm effect over VM disease must be clarified. The main objective of this study is to establish whether lung infection with protostrongylid and/or D. filaria acts as a predisposing factor for sheep Visna–maedi infection.
older than 6 months of age (Cutlip et al., 1988). Fecal samples were collected directly from the rectum with plastic gloves and kept at 4 ◦ C until analyzed the same day with the Baermann–Wetzel technique (López et al., 2011) for the detection of protostrongylid and D. filaria first stage larvae (L1). Blood samples were collected from the jugular vein and the sera obtained were tested for anti-VMV antibodies with a commercial enzyme-linked immunosorbent assay (Pourquier VM/CAEV, Indirect ELISA/Screening, France) following the manufactured instructions. The sensitivity and specificity of the assay were 98.0% and 97.4%, respectively (Toft et al., 2007). Sheep handling and sampling protocol were designed to provoke minimum animal stress and in accordance with European Council Directive 86/609/EEC, amended by the Directive 2003/65/EC, and with Spanish derived legislation (R.D. 223/1988, R.D. 1201/2005). A questionnaire comprising management and animal information was completed at every farm during sampling. The aim of the questionnaire was to investigate the potential risk factors for VMV seropositivity (Lago et al., 2012). All farms used anthelmintic treatment against gastrointestinal nematodes. 2.2. Factors considered and statistical analysis The factors detected as VM predictors by Lago et al. (2012) were grouped and categorized to be analyzed along with lungworm infections in order to study the effect of all these factors on VMV seroprevalence. • Housing until weaning: 0 (no) and 1 (yes). • Flock size: categorized at 33.3 and 66.6 percentiles 1 (<96 animals), 2 (96–194), and 3 (>194). • Age groups: 1 (<13 months-old), 2 (13–48), and 3 (>48). • Sheep–goat contact: 0 (purely sheep flock or presence of at least one goat on the farm but without contact) and 1 (presence of at least one goat on the farm with both species hold together in pastures and/or in the stables). • Protostrongylid nematode infection: 0 (no) and 1 (yes). • D. filaria infection: 0 (no) and 1 (yes). • Protostrongylid nematode larvae fecal counts. • D. filaria larvae fecal counts. Protostrongylid and D. filaria larvae fecal counts were previously logtransformed (log(x + 1)) to introduce them as continuous variables in the logistic regression analysis. The associations between lungworm and VMV infection were studied individually with a Chi-square test, with the cc() function included in the epicalc package (Chongsuvivatwong, 2011) for R statistical package (R v.2.14.0; R Development Core Team, 2011). cc() function produces odds ratio, calculated with the exact method, 95% confidence interval, Chisquared and Fisher’s exact tests. Unconditional logistic regression analysis (glm() R function) and Mantel–Haentszel procedure (mhor() function of epicalc package for R; Chongsuvivatwong, 2011) were used to detect potential confounding factors. Akaike Information Criterion (AIC) was applied in the logistic regression with step() function; a backward direction was used in the stepwise procedure.
3. Results
2. Materials and methods
3.1. Individual effect of lungworm infections
2.1. Study area: animals and flocks surveyed
Out of the 2081 serum samples extracted, 394 were seropositive to VMV (18.9%); 242 animals shed protostrongylid nematode (11.6%; mean 11.9 SD 30.91) and 223 D. filaria L1 (10.7%: mean 8.5 SD 41.89) in fecal samples. VMV seroprevalence was higher (60 out of 242; 24.8%) in sheep positive to protostrongylid infection than in protostrongylid negative animals (334 out of 1839; 18.2%). This difference was statistically significant with a Chi-squared algorithm (P = 0.013; Table 1). In the case of D. filaria, positive sheep also showed higher VMV seroprevalence (51 out of 223; 22.9%) than negative animals (343 out of 1858; 18.5%), but on this occasion difference was not significant (P = 0.112). Table 1 shows the effect of protostrongylid and
The study was undertaken in Galicia, a region located in Northwestern Spain (latitude 42◦ 30 0 N, longitude 8◦ 6 0 W), covering an area of 29,574 km2 . In Galicia, small ruminants are mainly maintained in a semi extensive husbandry system; flocks are kept in pastures near the barn where they are sheltered during the night. In mountainous areas, due to topographic and climatic conditions, sheep spend longer periods of time grazing in more distant meadows in comparison with other areas of the region. Between March 2007 and January 2009, a total of 2081 animals from 74 commercial meat ovine flocks were surveyed for VM and lungworm infection. Sample size was calculated in every farm with a 95% confidence interval (C.I.) and 90% precision, considering a theoretical 15% lungworm prevalence. Animals were randomly selected. To avoid detecting passively acquired VM colostral antibodies, all animals tested were
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Table 1 Results of Chi-squared test, odds-ratio and 95% C.I. of protostrongylid and D. filaria infection over VMV seropositivity in sheep (cc() function of epicalc package). Chi-squared Protostrongylid infection D. filaria infection *
6.13 2.52
P
OR *
0.013 0.112
95% C.I.
1.49 1.31
1.06–2.05 0.92–1.84
Significative difference (P < 0.05).
Table 2 Logistic regression (glm() function; R statistical package). Age are not included in the analysis. Z value Housing until weaning Flock size: small (1–95) Flock size: medium (96–194) Flock size: large (<194) Goat contact Protostrongylid infection a * ***
P
OR
95% C.I.
0.018*
1.43
1.06–1.92
<0.001*** <0.001*** <0.001*** 0.053
3.77 0.62 1.50 1.26
2.65–5.37 0.48–0.80 1.26–1.80 0.99–1.59
2.371 a
7.392 −3.583 4.491 1.932
Reference category. Significative difference (P < 0.05). Significative difference (P < 0.001).
Table 3 Logistic regression (glm() function; R statistical package). Adjusted significance and odds-ratio for all the factors studied. Z value Housing until weaning Age: 0–12 months-old Age: 13–48 Age: >49 Flock size: small (1–95) Flock size: medium (96–194) Flock size: large (<194) Goat contact Protostrongylid infection a * ***
2.093
P
OR
95% C.I.
0.036*
1.38
1.02–1.87
<0.001*** <0.001***
4.91 0.58
3.09–7.80 0.42–0.79
<0.001*** 0.001*** <0.001*** 0.365
3.88 0.65 1.83 1.11
2.72–5.53 0.50–0.85 1.41–2.37 0.87–1.42
a
6.750 −3.479 a
7.491 −4.521 4.563 0.905
Reference category. Significance at P < 0.05 Significance at P < 0.001.
Dictyocaulus infection over VMV seroprevalence, the OR with 95% confidence interval (C.I.) and statistical significance.
3.2. Adjusted results with logistic regression and Mantel–Haenszel techniques All factors included in this study apart from those referred to lungworm infection were known to be significant (Lago et al., 2012) in the VMV infection. Protostrongylid infection was near to significance when age was not introduced as a factor in logistic regression analysis (Table 2), but it is rejected (P > 0.1) by the algorithm when age is included (Table 3). If Akaike Information Criterion (AIC) is applied with a backward direction in the stepwise, all the factors related to lungworm infection (protostrongylid and D. filaria positiveness and larvae output) are rejected (P > 0.1) by the statistical analysis. This statistical test shows both the adjusted odds ratios and signification for all factors and indicates clearly that lungworm infection is acting as a confounding factor, related with age. In order to demonstrate the individual association and the confounding effect of protostrongylid infection, Mantel–Haenszel technique was applied (Table 4). Protostrongylid infection is not related with VMV
seroprevalence in the combined effects (Mantel–Haenszel 2 = 1.95; P = 0.163) or stratified by age; none of the oddsratio shown in Table 4 was significant with respect to protostrongylid infection. 4. Discussion Prevalence of VMV was higher in sheep infected with protostrongylid nematodes than in non-infected ones. This paired relationship was statistically significant, as previously reported by other authors (Giangaspero et al., 1993). However, De Boer et al. (1979) have not found evidence of the influence of the protostrongylid Muellerius capillaris larvae on VMV transmission and Mornex et al. (1994) assessed that lesions observed in lungs of sheep infected by
Table 4 Odds ratio of protostrongylid infection -stratified by age- over VMV seroprevalence calculated with Mantel–Haenszel technique (mhor() function of epicalc package; R statistical package). OR Age 1 (6–12 months) Age 2 (13–48 months) Age 3 (49 months or more) M–H combined
2.48 1.21 1.25 1.26
95% C.I. 0.05–20.71 0.60–2.29 0.84–1.85 0.91–1.73
P value 0.371 0.512 0.270 0.163
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VMV were not altered by simultaneous infection by protostrongylid nematodes. Logistic regression allowed us to detect the possible confounding effect of all factors considered, introducing all of them into one mathematical model. The possible relation between VMV seropositivity and protostrongylid infection was rejected as non-significative by the statistical analysis when the age of the animal was introduced as a variable. Age is related with protostrongylid infections (Cabaret et al., 1978; Regassa et al., 2010) and M. capillaris prevalence increased with the age of the host (López et al., 2011); VM is also a chronic disease (Simard and Morley, 1991; Arsenault et al., 2003), and prevalence increases with the age of the animals. The association of the two chronic infections with animal age generates an artificial positive relation as individual statistical algorithm has shown. Multivariate techniques indicate that protostrongylid infection in sheep is only a confounding variable in VMV seropositivity. However, this conclusion is in contrast with Straub (2004), who affirmed that regular prophylactic treatments with anthelmintic drugs reduce the risk of VMV transmission, because lungworm infection would favor the spread of the virus. However, this effect could also be caused by other parasites different from lungworms. This study did not cover all of the parasite species affected by anthelmintic treatments, so new studies with a wider approach to helminth populations in sheep must be done to elucidate the effect of parasites on VM seropositivity. The effect of anthelmintic treatment over sheep VM seropositivity could also be explained by the immunostimulation caused by drugs like levamisole (Lanusse et al., 2009). A non-specific immunostimulating effect has been detected in different mammalian species treated with levamisole, for instance in neonatal calves (Mohri et al., 2005). In the case that levamisole was part of the treatments described by Straub (2004), an immunostimulation caused directly by the drug could underlie the variable “treatment”. Anthelmintic treatment was not included as a factor in this study because all the flocks were systematically dewormed in spring and/or autumn, so there was not a control group. On the other hand, the most frequently employed antiparasitic drugs in ovine in Galicia are albendazole or albendazole + macrocyclic lactone, and levamisole is not used to treat ovine flocks in this region (López et al., 2011). 5. Conclusions This study has proved that protostrongylid infection in sheep acts as a confounding factor in VMV transmission; an anthelmintic treatment targeted to protostrongylid nematode infection should not have any effect on VM control in sheep. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
Acknowledgements The authors thank to OVICA (Galician Association of Ovine and Caprine Breeders) and the veterinarians of the ADSG ACIVO for their collaboration in the realization of this study. This work was supported by the research project PGIDIT06RAG26101PR.
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Contents lists available at SciVerse ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Lungworm infection and ovine visna–maedi: Real risk factor or a confounding variable? ˜ R. Panadero, P. Díaz, P. Díez-Banos, ˜ C.M. López ∗,1 , N. Lago, M. Vina, P. Morrondo, G. Fernández Departamento de Patología Animal (Grupo INVESAGA), Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain
a r t i c l e
i n f o
Article history: Received 29 March 2012 Received in revised form 14 September 2012 Accepted 15 September 2012 Available online 4 October 2012 Keywords: Visna–maedi Prevalence Ovine Lungworm Protostrongylid
a b s t r a c t Serum and fecal samples (n = 2081) were taken from 74 commercial meat ovine flocks in Galicia (NW Spain) in order to establish the influence of lungworm infection with protostrongylids and/or Dictyocaulus filaria on visna–maedi (VM) seropositivity. VM was tested by an indirect ELISA test and lungworm larvae were detected by the modified Baermann–Wetzel technique. In addition to lungworm infection (protostrongylids and D. filaria), flock, management and individual factors were included in univariate and multivariate statistical tests. Overall VM seroprevalence was 18.9% and the prevalence by protostrongylids and D. filaria was 11.6% and 10.7%, respectively. Protostrongylid infection was considered as a risk factor for VM with univariate statistical analysis (1.49 significant OR), but neglected in a logistic multivariate regression analysis when the age of the animals was introduced as variable. As VM, protostrongylid infection is a chronic disease with a cumulative pattern over time, so that these two infections are closely related to the age of the animal. A Mantel–Haenszel procedure was applied and lungworm infection was detected as a confounding variable with no real effect on visna–maedi infection. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Visna–maedi (VM) is a worldwide disease caused by small ruminant lentiviruses (SRLV), a subgroup of the Retroviridae family, which causes chronic and multisystem disease in sheep (Narayan et al., 1982). SRLV do not constitute a risk to humans (Peterhans et al., 2004), but their importance is related to high economic losses due to an increase in mortality rates and a decrease in weight at lamb weaning and adult culling age (Arsenault et al., 2003).
∗ Corresponding author at: Departamento de Patología Animal (Grupo INVESAGA), Facultad de Veterinaria, Universidad de Santiago de Compostela, Avda Carballo Calero, s/n, 27002 Lugo, Spain. Tel.: +34 982822109; fax: +34 982822001. E-mail addresses: [email protected], [email protected] (C.M. López). 1 http://www.clopezsandez.es. 0921-4488/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.smallrumres.2012.09.010
VM is characterized by a long incubation period and symptoms rarely occur in sheep younger than two years of age (Narayan and Cork, 1985). These symptoms include lung fibrosis with a decrease in the total alveolar surface and an increase in the distance between the luminal and vascular sides of the alveoli (Pepin et al., 1998). There is also a decrease in the compliance of the lung tissue, which manifests itself as a kind of strained breathing; this pulmonary form is known as ‘maedi’ in Iceland, where it was first recognized and described. The most important routes by which Visna–maedi virus (VMV) is transmitted are the lactogenic one – from an infected ewe to its offspring via colostrum and milk (Pritchard and Dawson, 2000) – and horizontal transmission through respiratory fluids, mainly under high density conditions (Berriatua et al., 2003). There are also epidemiological evidences that horizontal transmission between lambs can occur, related to the variable of housing-time
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prior to weaning (Lago et al., 2012). Other routes of VMV transmission, like transplacental and seminal, have limited epidemiological importance (Blacklaws et al., 2004; Peterhans et al., 2004). Ovine lungworms are protostrongylids and Dictyocaulus filaria, being the first one the most frequent in ovine flocks. Protostrongylids are heteroxenous parasites that infect terrestrial molluscs as intermediate host. Ovine infections are commonly asymptomatic, but heavy infections cause decreased carcass weight, increased levels of mortality and impaired pulmonary gas exchange (Berrag and Cabaret, 1996; López et al., 2010). Lesions in lungs have been described as “granuloma-like worm nodules”, diffuse lesions and emphysema (Sauerlander, 1988). The role of lung nematodes in the induction of VM disease has been controversial within the scientific community. Whereas Mornex et al. (1994) have not found any effect of simultaneous parasitic infection of lungworms on the development of VM disease, Giangaspero et al. (1993) suggest a synergy between lung parasitic infection and VM seropositivity. Moreover, in the review by Straub (2004), regular prophylactic treatment with anthelmintic drugs reduces the risk of VMV transmission, which seems to indicate that lungworm infections foster the spread of the virus in sheep. Recently, in a cross-sectional study carried out in Galicia by Lago et al. (2012), individual (24.8%) and flock VMV seroprevalence (52.6%) were assessed, as well as the main risk factors, such as flock size, sheep–goat contact, type of lamb housing prior to weaning and age. However, lungworm infection was not included in that study. Previous studies demonstrated that lungworm individual prevalence in sheep in Galicia has decreased over the last decades from 33.9% (Díez et al., 1995) to 11.6% (López et al., 2011); however, flock prevalence is still very high (67.6%; López et al., 2011). As there is no effective treatment or vaccines against VMV, the measures adopted to control this chronic viral infection could have no effect if lungworm infection, also chronic, insidious and generalized at flock level, facilitates VMV transmission. So lungworm effect over VM disease must be clarified. The main objective of this study is to establish whether lung infection with protostrongylid and/or D. filaria acts as a predisposing factor for sheep Visna–maedi infection.
older than 6 months of age (Cutlip et al., 1988). Fecal samples were collected directly from the rectum with plastic gloves and kept at 4 ◦ C until analyzed the same day with the Baermann–Wetzel technique (López et al., 2011) for the detection of protostrongylid and D. filaria first stage larvae (L1). Blood samples were collected from the jugular vein and the sera obtained were tested for anti-VMV antibodies with a commercial enzyme-linked immunosorbent assay (Pourquier VM/CAEV, Indirect ELISA/Screening, France) following the manufactured instructions. The sensitivity and specificity of the assay were 98.0% and 97.4%, respectively (Toft et al., 2007). Sheep handling and sampling protocol were designed to provoke minimum animal stress and in accordance with European Council Directive 86/609/EEC, amended by the Directive 2003/65/EC, and with Spanish derived legislation (R.D. 223/1988, R.D. 1201/2005). A questionnaire comprising management and animal information was completed at every farm during sampling. The aim of the questionnaire was to investigate the potential risk factors for VMV seropositivity (Lago et al., 2012). All farms used anthelmintic treatment against gastrointestinal nematodes. 2.2. Factors considered and statistical analysis The factors detected as VM predictors by Lago et al. (2012) were grouped and categorized to be analyzed along with lungworm infections in order to study the effect of all these factors on VMV seroprevalence. • Housing until weaning: 0 (no) and 1 (yes). • Flock size: categorized at 33.3 and 66.6 percentiles 1 (<96 animals), 2 (96–194), and 3 (>194). • Age groups: 1 (<13 months-old), 2 (13–48), and 3 (>48). • Sheep–goat contact: 0 (purely sheep flock or presence of at least one goat on the farm but without contact) and 1 (presence of at least one goat on the farm with both species hold together in pastures and/or in the stables). • Protostrongylid nematode infection: 0 (no) and 1 (yes). • D. filaria infection: 0 (no) and 1 (yes). • Protostrongylid nematode larvae fecal counts. • D. filaria larvae fecal counts. Protostrongylid and D. filaria larvae fecal counts were previously logtransformed (log(x + 1)) to introduce them as continuous variables in the logistic regression analysis. The associations between lungworm and VMV infection were studied individually with a Chi-square test, with the cc() function included in the epicalc package (Chongsuvivatwong, 2011) for R statistical package (R v.2.14.0; R Development Core Team, 2011). cc() function produces odds ratio, calculated with the exact method, 95% confidence interval, Chisquared and Fisher’s exact tests. Unconditional logistic regression analysis (glm() R function) and Mantel–Haentszel procedure (mhor() function of epicalc package for R; Chongsuvivatwong, 2011) were used to detect potential confounding factors. Akaike Information Criterion (AIC) was applied in the logistic regression with step() function; a backward direction was used in the stepwise procedure.
3. Results
2. Materials and methods
3.1. Individual effect of lungworm infections
2.1. Study area: animals and flocks surveyed
Out of the 2081 serum samples extracted, 394 were seropositive to VMV (18.9%); 242 animals shed protostrongylid nematode (11.6%; mean 11.9 SD 30.91) and 223 D. filaria L1 (10.7%: mean 8.5 SD 41.89) in fecal samples. VMV seroprevalence was higher (60 out of 242; 24.8%) in sheep positive to protostrongylid infection than in protostrongylid negative animals (334 out of 1839; 18.2%). This difference was statistically significant with a Chi-squared algorithm (P = 0.013; Table 1). In the case of D. filaria, positive sheep also showed higher VMV seroprevalence (51 out of 223; 22.9%) than negative animals (343 out of 1858; 18.5%), but on this occasion difference was not significant (P = 0.112). Table 1 shows the effect of protostrongylid and
The study was undertaken in Galicia, a region located in Northwestern Spain (latitude 42◦ 30 0 N, longitude 8◦ 6 0 W), covering an area of 29,574 km2 . In Galicia, small ruminants are mainly maintained in a semi extensive husbandry system; flocks are kept in pastures near the barn where they are sheltered during the night. In mountainous areas, due to topographic and climatic conditions, sheep spend longer periods of time grazing in more distant meadows in comparison with other areas of the region. Between March 2007 and January 2009, a total of 2081 animals from 74 commercial meat ovine flocks were surveyed for VM and lungworm infection. Sample size was calculated in every farm with a 95% confidence interval (C.I.) and 90% precision, considering a theoretical 15% lungworm prevalence. Animals were randomly selected. To avoid detecting passively acquired VM colostral antibodies, all animals tested were
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Table 1 Results of Chi-squared test, odds-ratio and 95% C.I. of protostrongylid and D. filaria infection over VMV seropositivity in sheep (cc() function of epicalc package). Chi-squared Protostrongylid infection D. filaria infection *
6.13 2.52
P
OR *
0.013 0.112
95% C.I.
1.49 1.31
1.06–2.05 0.92–1.84
Significative difference (P < 0.05).
Table 2 Logistic regression (glm() function; R statistical package). Age are not included in the analysis. Z value Housing until weaning Flock size: small (1–95) Flock size: medium (96–194) Flock size: large (<194) Goat contact Protostrongylid infection a * ***
P
OR
95% C.I.
0.018*
1.43
1.06–1.92
<0.001*** <0.001*** <0.001*** 0.053
3.77 0.62 1.50 1.26
2.65–5.37 0.48–0.80 1.26–1.80 0.99–1.59
2.371 a
7.392 −3.583 4.491 1.932
Reference category. Significative difference (P < 0.05). Significative difference (P < 0.001).
Table 3 Logistic regression (glm() function; R statistical package). Adjusted significance and odds-ratio for all the factors studied. Z value Housing until weaning Age: 0–12 months-old Age: 13–48 Age: >49 Flock size: small (1–95) Flock size: medium (96–194) Flock size: large (<194) Goat contact Protostrongylid infection a * ***
2.093
P
OR
95% C.I.
0.036*
1.38
1.02–1.87
<0.001*** <0.001***
4.91 0.58
3.09–7.80 0.42–0.79
<0.001*** 0.001*** <0.001*** 0.365
3.88 0.65 1.83 1.11
2.72–5.53 0.50–0.85 1.41–2.37 0.87–1.42
a
6.750 −3.479 a
7.491 −4.521 4.563 0.905
Reference category. Significance at P < 0.05 Significance at P < 0.001.
Dictyocaulus infection over VMV seroprevalence, the OR with 95% confidence interval (C.I.) and statistical significance.
3.2. Adjusted results with logistic regression and Mantel–Haenszel techniques All factors included in this study apart from those referred to lungworm infection were known to be significant (Lago et al., 2012) in the VMV infection. Protostrongylid infection was near to significance when age was not introduced as a factor in logistic regression analysis (Table 2), but it is rejected (P > 0.1) by the algorithm when age is included (Table 3). If Akaike Information Criterion (AIC) is applied with a backward direction in the stepwise, all the factors related to lungworm infection (protostrongylid and D. filaria positiveness and larvae output) are rejected (P > 0.1) by the statistical analysis. This statistical test shows both the adjusted odds ratios and signification for all factors and indicates clearly that lungworm infection is acting as a confounding factor, related with age. In order to demonstrate the individual association and the confounding effect of protostrongylid infection, Mantel–Haenszel technique was applied (Table 4). Protostrongylid infection is not related with VMV
seroprevalence in the combined effects (Mantel–Haenszel 2 = 1.95; P = 0.163) or stratified by age; none of the oddsratio shown in Table 4 was significant with respect to protostrongylid infection. 4. Discussion Prevalence of VMV was higher in sheep infected with protostrongylid nematodes than in non-infected ones. This paired relationship was statistically significant, as previously reported by other authors (Giangaspero et al., 1993). However, De Boer et al. (1979) have not found evidence of the influence of the protostrongylid Muellerius capillaris larvae on VMV transmission and Mornex et al. (1994) assessed that lesions observed in lungs of sheep infected by
Table 4 Odds ratio of protostrongylid infection -stratified by age- over VMV seroprevalence calculated with Mantel–Haenszel technique (mhor() function of epicalc package; R statistical package). OR Age 1 (6–12 months) Age 2 (13–48 months) Age 3 (49 months or more) M–H combined
2.48 1.21 1.25 1.26
95% C.I. 0.05–20.71 0.60–2.29 0.84–1.85 0.91–1.73
P value 0.371 0.512 0.270 0.163
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VMV were not altered by simultaneous infection by protostrongylid nematodes. Logistic regression allowed us to detect the possible confounding effect of all factors considered, introducing all of them into one mathematical model. The possible relation between VMV seropositivity and protostrongylid infection was rejected as non-significative by the statistical analysis when the age of the animal was introduced as a variable. Age is related with protostrongylid infections (Cabaret et al., 1978; Regassa et al., 2010) and M. capillaris prevalence increased with the age of the host (López et al., 2011); VM is also a chronic disease (Simard and Morley, 1991; Arsenault et al., 2003), and prevalence increases with the age of the animals. The association of the two chronic infections with animal age generates an artificial positive relation as individual statistical algorithm has shown. Multivariate techniques indicate that protostrongylid infection in sheep is only a confounding variable in VMV seropositivity. However, this conclusion is in contrast with Straub (2004), who affirmed that regular prophylactic treatments with anthelmintic drugs reduce the risk of VMV transmission, because lungworm infection would favor the spread of the virus. However, this effect could also be caused by other parasites different from lungworms. This study did not cover all of the parasite species affected by anthelmintic treatments, so new studies with a wider approach to helminth populations in sheep must be done to elucidate the effect of parasites on VM seropositivity. The effect of anthelmintic treatment over sheep VM seropositivity could also be explained by the immunostimulation caused by drugs like levamisole (Lanusse et al., 2009). A non-specific immunostimulating effect has been detected in different mammalian species treated with levamisole, for instance in neonatal calves (Mohri et al., 2005). In the case that levamisole was part of the treatments described by Straub (2004), an immunostimulation caused directly by the drug could underlie the variable “treatment”. Anthelmintic treatment was not included as a factor in this study because all the flocks were systematically dewormed in spring and/or autumn, so there was not a control group. On the other hand, the most frequently employed antiparasitic drugs in ovine in Galicia are albendazole or albendazole + macrocyclic lactone, and levamisole is not used to treat ovine flocks in this region (López et al., 2011). 5. Conclusions This study has proved that protostrongylid infection in sheep acts as a confounding factor in VMV transmission; an anthelmintic treatment targeted to protostrongylid nematode infection should not have any effect on VM control in sheep. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
Acknowledgements The authors thank to OVICA (Galician Association of Ovine and Caprine Breeders) and the veterinarians of the ADSG ACIVO for their collaboration in the realization of this study. This work was supported by the research project PGIDIT06RAG26101PR.
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