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Protostrongylid infection in meat sheep from Northwestern Spain: Prevalence and risk factors
Veterinary Parasitology 178 (2011) 108–114
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Protostrongylid infection in meat sheep from Northwestern Spain: Prevalence and risk factors ˜ S. Cienfuegos, R. Panadero, C.M. López ∗ , G. Fernández, M. Vina, ˜ L. Vázquez, P. Díaz, J. Pato, N. Lago, V. Dacal, P. Díez-Banos, P. Morrondo Parasitología y Enfermedades Parasitarias, Departamento de Patología Animal: Sanidad Animal, Facultad de Veterinaria, Universidad de Santiago de Compostela, Avda Carballo Calero s/n, 27002 Lugo, Spain
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Article history: Received 15 July 2010 Received in revised form 20 December 2010 Accepted 22 December 2010 Keywords: Ovine Protostrongylid Risk factors Northwestern Spain Prevalence
a b s t r a c t 2093 Faecal samples from 74 commercial meat ovine flocks were collected and examined by the Baermann–Wetzel method for protostrongylid infection. The risk of being infected by lungworms was evaluated with a data mining classification tree (CHAID), and the intensity of infection with a general linear model (GLM). 242 out of 2093 faecal samples examined were positive for protostrongylid infection (11.6%; 95% CI 10.2–12.9). Only two species were found, Muellerius capillaris (97.9%) and Neostrongylus linearis (5.4%). 50 out of 74 farms presented at least one animal shedding protostrongylid larvae in faeces. All of them held animals infected by M. capillaris and seven presented mixed infections with N. linearis. Average larval output in infected sheep was 11.9 (SD 30.91). This study showed that protostrongylid prevalence in sheep for meat production was determined mainly by a positive interaction with Dictyocaulus filaria infection; other factors that have influenced over protostrongylid infection were age, introducing external animals in the flocks, mixed management with goats and animal density in pastures. Treatment effects on prevalence were only observed in flocks that did not introduce ewes. The lowest protostrongylid prevalence has been reported in flocks without D. filaria infection and without contact with goats. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In Galicia (North-west of Spain) there are around 24,000 sheep flocks with a mean of 11.5 animals, contributing to the economic and environmental sustainability of the whole farm. In those farms sheep often share pastures with cattle and goats. In this region traditionally sheep have been used as an alternative to herbicides for suppressing weeds from rangeland and forest, especially in mountainous areas. However, in last years the sector has been improving as a result of the creation in 1994 of the Galician Ovine and Caprine breeder Association (OVICA)
∗ Corresponding author. Tel.: +34 982822109; fax: +34 982822001. E-mail addresses: [email protected], [email protected] (C.M. López). URL: http://www.clopezsandez.es (C.M. López). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.12.038
devoted to develop the sheep industry in the region. At present there is over 400 flocks that have more than 100 animals (90,000 sheep and goats), being dedicated almost exclusively to meat production. In the NW of Spain small ruminants are mainly maintained in a semiextensive husbandry system consisting of a mixture of grazing and indoor-housing. In this region, mild temperatures and environmental humidity are optimal for the survival of many important livestock parasites, such as small lungworms (Protostrongylidae) and the grazing system may facilitate the infection by those parasites. Protostrongylids (Nematoda: Strongylida) are heteroxenous parasites that infect terrestrial molluscs as intermediate host. Ovine infections are commonly asymptomatic, but heavy infections cause decreased carcass weights, increased levels of mortality and impaired pulmonary gas exchange (Valero et al., 1992; Berrag and Cabaret, 1996).
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Studies conducted in the last decade in two different localities of Galicia (Díez et al., 1994, 1995) determined that Neostrongylus linearis was the protostrongylid with the highest prevalence and intensity of infection in sheep, followed by Muellerius capillaris and Cystocaulus ocreatus. However, there are no reports on the prevalence and factors associated with the infection involving the entire region. Most of the studies over factors associated with protostrongylid infection in sheep were aimed mainly to treatment effect or intermediate host influence. Only a few have introduced other factors, like irrigating pastures (Uriarte et al., 1985), the effect of sheep–goat mixed flocks (Mangeon and Cabaret, 1987; Regassa et al., 2010) and management practices (Cabaret et al., 1989; Alemu et al., 2006). Although these parasites are widely prevalent, their infections are neglected by farmers and clinicians. Moreover, in this region, endoparasite control practices in commercial ovine farms include a systematic single dose treatment in spring and/or autumn intended to control gastrointestinal nematode infection. Nevertheless, the effect that this systematic treatment is being on protostrongylid populations is unknown. Kloosterman et al. (1989, 1990) have shown that primary infection with gastrointestinal nematodes increased the establishment of Dictyocaulus viviparus in cattle. Recent papers have detected similar immune responses in sheep infected by gastrointestinal nematodes and Dictyocaulus filaria (French et al., 2009), but the interaction between the different lungworm infections in sheep has not been studied yet. The main objective of this study was to establish the prevalence and larval output of protostrongylids in flocks from northwestern Spain, to identify the species involved and, in a second term, to detect and classify determinant factors on sheep protostrongylid infection.
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2.2. Animals and flocks surveyed Between March 2007 and January 2009, 74 commercial meat ovine flocks with a total of 16,122 sheep of which 2,093 were sampled for diagnostic of protostrongylid and D. filaria infection. The sample size per flock was calculated trying to fulfill in 95% confidence interval a 90% precision, considering 15% prevalence; animals were randomly selected. All the animals were mixed breed. The flocks were distributed over the entire region and all of them were joined to OVICA. 51 out of 74 were purely sheep flocks and 23 were mixed sheep/goat farms with an average of 18.3 goats/farm. A questionnaire comprising data about the management system and animal information was completed in every farm during sampling. All farms used anthelmintic treatment to control gastrointestinal nematodes. Lambs are always fed by their dams until weaning (2–3 months).
2.3. Examination of faecal samples Faecal samples were collected directly from the rectum with plastic gloves and kept at 4 ◦ C until being analyzed by the Baermann–Wetzel technique in the same day. Briefly, 10 g of faeces were left overnight with non woven filters (Filter-Lab, Filtros Anoia, S.A., Barcelona, Spain) in glass funnels with a 12 ml centrifuge tube at the end of a silicone tube. These tubes were centrifugated 10 min at 350G and the last milliliter containing the larval population was reserved and analyzed in Favati chambers. The different protostrongylid species were counted and identified by the characteristics of the posterior section of the larvae according to Boev (1975). If the number of larvae was less than 100, all of them were identified, and if higher than 100, at least 100 were identified.
2.4. Factors considered and statistical analysis 2. Materials and methods 2.1. Study area
The variables were grouped and categorized for statistical analysis as:
The study was undertaken in Galicia, region located in northwestern Spain, covering an area of 29,574 km2 (Latitude 42◦ 23 60N, Longitude 7◦ 4 W). The climate is Atlantic with mild temperatures and high precipitations. In this study the region was divided into three zones according to its orographic and bioclimatic conditions: a Coastal area situated from sea level to 200 m, with moderate precipitations (1300–1500 mm) and temperatures (14 ◦ C); a Central area at 200–650 m above sea level with low precipitations (900–1300 mm) and moderate temperatures (11.5 ◦ C); and a Mountainous area situated at 650–1285 m, with low temperatures (10 ◦ C) and high precipitations (>1500 mm). In Galicia small ruminants are mainly maintained in a semiextensive husbandry system; flocks are maintained in pastures near the barn where are kept during the night; in mountainous areas, due to topographic and climatic conditions, sheep are managed under extensively grazing system.
• Climatic area: 0 (Mountainous), 1 (Central), 2 (Coastal areas). • Sampling period: 1 (Spring), 2 (Summer), 3 (Autumn), 4 (Winter). • Husbandry system: 0 (extensive), 1 (semiextensive). • Housing until weaning: 0 (No), 1 (Yes). • Flock size: categorized at 33.3 and 66.6 percentiles 1 (<96 animals), 2 (96–194), 3 (>194). • Age groups: 1 (<13 months-old), 2 (13–48), 3 (>48). • Goat presence: 0 (purely sheep flock), 1 (presence of at least one goat on the farm). • Sheep–goat contact: 0 (purely sheep flock or presence of at least one goat on the farm but without contact), 1 (presence of at least one goat on the farm and both species are hold together in pastures and/or in the stables). • Introduction of new animals in the farm (last 5 years); two categories: ◦ 0 (No), 1 (yes).
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• • • • • • •
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◦ 0 (No), 1 (only male animals), 2 (male and female animals). Number of sheep introduced in the last 5 years, categorized at 50 percentile: 1 (0–9), 2 (>9). Treatment pattern: 0 (Spring), 1 (Autumn), 2 (Spring and Autumn). Drugs applied. 0 (macrocyclic lactones – ML or ML plus benzimidazoles – BZ), 1 (only BZ). Pasture rotation: 0 (No), 1 (Yes). Manure as fertilizer: 0 (No), 1 (Yes). Proximity to another flocks (<2 km): 0 (No), 1 (Yes). D. filaria infection: 0 (No), 1 (Yes).
The risk of being infected by lungworms taking into account these variables was evaluated with a data mining classification tree (Kass, 1980); particularly an exhaustive Chi-squared automatic interaction detector (exhaustive CHAID), whereby sheep were classified as positive (larvae in faeces) or negative (no larvae in faeces) in the categorical dependent variable. CHAID identifies variables that divide sheep in subgroups with distinct patterns of positive/negative ratio. The CHAID model provides a way to identify major factors. CHAID evaluates all of the values of a potential factor using as a criterion the significance of a statistical Chi-squared test, selecting the best predictor variable to form the first branch in a classification tree; successively splitting in data set makes increasingly homogeneous nodes in relation to the dependent variable. This process continues recursively until the classification tree is fully grown. The method is exhaustive because after a factor is entered in the analysis, it remains in the list of predictor variables for use in future calculations and there can be significant interaction with more predictors. Some of the factors studied present more than two categories and CHAID does not restrict the number of branches from each node to a predetermined number and could be split in more than two branches. For the examination of the intensity of infection an unifactorial association analysis (ANOVA) was first applied on the 242 positive animal counts and 50 positive farms to restrict the final number of factors under consideration. Factors with p values less than 0.1 were selected to build a linear model (GLM). For these analyses, the dependent variable – number of protostrongylid larvae eliminated per gram of faeces (lpg) and mean larvae outputs in flocks – had been previously log-transformed, Ln(lpg). In both cases Bonferroni post hoc test was used to detect the differences between pairs. All statistical analyses were performed with the statistical package SPSS for Windows 18.0 and SPSS Answer Tree, 3.1 (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Prevalence of protostrongylid infection and associated risk factors Individual prevalence: 242 out of 2093 faecal samples examined were positive for protostrongylid infection (11.6%; 95% CI 10.2–12.9). Only two species were found, M. capillaris that was identified in 97.9% of the positive fae-
cal samples (11.3%; 95% CI 10.0–12.6) and N. linearis, only identified in 13 samples (0.6%; 95% CI 0.3–0.9). The CHAID algorithm stratified variables that play an important role in protostrongylid presence and indicated (Fig. 1) that sheep not infected with D. filaria presented lower protostrongylid prevalence (node 1) than those infected (node 2). No other factor was detected influencing animals positive to D. filaria, but in the node of sheep negative to Dictyocaulus, sheep age was pointed out as a determining factor; sheep until 48 months showed lower prevalence (5.8%, node 4) than older animals (14.7%, node 3). The prevalence inside the younger sheep node was determined by sheep–goat contact, with a lower prevalence in animals that were not in contact with goats (4%; node 7) compared with sheep in contact with goats (12.5%; node 8). On the other hand, prevalence in older animals was determined by introducing external animals in the flock; protostrongylid prevalence in farms that introduce ewes from other flocks was much higher (20.8%; node 6) than that in farms that did not introduce other animals or introduce only males (11.4%; node 5). Finally, prevalence in old animals in farms that did not introduce ewes was only determined by the type of drugs applied in usual gastrointestinal nematode treatment, identified by the CHAID algorithm as a determining factor; node composed by animals treated only with BZ (node 9) showed higher prevalence than that treated with ML alone or ML plus BZ (node 10). Farm prevalence: 50 out of 74 farms presented at least one animal shedding protostrongylid larvae in faeces (67.6%; 95% CI 56.9–78.3). All of them presented animals infected by M. capillaris and seven presented mixed infections with N. linearis (9.5%; 95% CI 2.8–16.2). The CHAID algorithm indicated that introducing new animals from another flocks was the most important determining factor for prevalence at flock level (Fig. 2), as the prevalence in farms that introduced animals from other flocks was almost twice than that in farms that did not purchase new animals. 3.2. Intensity of infection by protostrongylids Individual: Average larval output in infected sheep was 11.9 (SD 30.91). All the different factors studied were first considered separately using ANOVA; Dictyocaulus infection (F = 9.529; p = 0.002), housing until weaning (F = 8.511; p = 0.004), manure as fertilizer (F = 5.541; p = 0.019), husbandry system (F = 3.259; p = 0.072) and drugs applied (F = 3.010; p = 0.084) shown a statistical significance under 0.1. Interacting analysis (GLM) between those factors indicated that the most influential factor over intensity of infection was housing until weaning (F = 13.695; p < 0.000) and Dictyocaulus infection (F = 4.391; p = 0.037), with no interaction between them (F = 0.044; p = 0.835). Animals in flocks from the beginning showed lower protostrongylid larvae mean output (7.6; SD 23.01) than those housed until weaning (30.6; SD 49.45); animals infected with D. filaria showed also higher mean output (24.8; SD 54.31) than those not infected (8.0; SD 17.47). No other factor were significant in GLM model.
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Fig. 1. Classification tree by exhaustive CHAID stratifying risk-factors for protostrongylid infection.
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Fig. 2. Classification tree by exhaustive CHAID showing determining factor for farm prevalence.
Farms: Dictyocaulus infection (F = 10.053; p = 0.003), manure fertilizer (F = 4.066; p = 0.053) and husbandry system (F = 2.943; p = 0.097) showed statistical significance under 0.1 in ANOVA considering separately all factors. In the interacting analysis (GLM) with all those factors, only D. filaria were significant on intensity of infection level (F = 7.998; p = 0.007); farms with at least one animal positive to D. filaria presented higher mean larvae output (10.6; SD 14.36) than farms without infected sheep (3.6; SD 8.45). 4. Discussion The prevalence by protostrongylid nematodes recorded in this study was lower than the 33.9% obtained by Díez et al. (1995) in the same region. It was also much lower than the 78.8% observed by Morrondo et al. (1990) in León (adjacent area with drier weather conditions); and by Uriarte et al. (1985) in Zaragoza (55.2% in irrigated and 37.9% in non-irrigated pastures). Moreover, most of the data obtained in sheep from Europe and North of Africa (Cabaret, 1986; Alemu et al., 2006) have shown higher protostrongylid prevalence (50–80%) than that obtained in this survey. Only two different species of Protostrongylidae were identified in this study, M. capillaris and N. linearis. Most of the positive animals were infected with M. capillaris (97.9%), whereas only 13 animals were infected by N. lin-
earis (5.4%). Previous studies carried out in the last decade in Galicia found N. linearis as the most prevalent species, followed by M. capillaris and C. ocreatus (Díez et al., 1994, 1995). It seems that generalized anthelmintic treatment to control gastrointestinal nematodes in commercial flocks has reduced not only the prevalence of protostrongylid infection but also selected M. capillaris among the protostrongylid genera, reducing considerably N. linearis populations and eliminating completely C. ocreatus on this territory. According to different authors (Bliss and Greiner, 1985; McCraw and Menzies, 1986; Helle, 1986; Díez et al., 1995; Rehbein and Visser, 2002; López et al., 2010) most anthelmintic treatments have shown an important lack of efficacy against protostrongylid lungworms, and in particular against M. capillaris. Some of those authors found that Muellerius larvae reappeared in faecal samples in less than 60 days, even in animals under strict isolation. This reappearance might be explained by immature Muellerius forms, not affected by anthelmintic treatment, developing to maturity after destruction of the original adult population (McCraw and Menzies, 1986, 1988) or by the protection conferred by altered tissue surrounding the lungworm that is more pronounced than in other protostrongylid species (Rehbein and Visser, 2002). For this reason, specific treatment for protostrongylid nematode infections would require higher dosage than those for
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gastrointestinal nematodes (Richard and Cabaret, 1992) or repeated treatments (McCraw and Menzies, 1986). The co-infection with D. filaria was identified as the most determining factor for ovine protostrongylid infection; prevalence in animals with a concomitant infection by D. filaria was higher than in those not infected. Taking into consideration that both lungworms have different transmission routes it is possible that positive interactions were immunologically mediated so that the presence of one species could facilitate the subsequent establishment of the other. Kloosterman et al. (1989, 1990) found positive interactions between lungworms in calves after previous infections with gastrointestinal nematodes and French et al. (2009) have detected that D. filaria infection generates similar immune response in ovine than Teladorsagia circumcincta infection in abomasal mucosa, so Dictyocaulus infection could provoke a lowered immunity response as described Kloosterman et al. (1989) for gastrointestinal nematodes that could facilitate sheep protostrongylid infection. However, further studies are necessary to elucidate this aspect. The second factor determined for protostrongylid prevalence was the age of the sheep. Age is a well-known influential factor in protostrongylid infections (Cabaret et al., 1978; Alemu et al., 2006; Regassa et al., 2010); prevalence increased with the age of the host. In our study sheep prevalence was two times and a half higher in old sheep (>4 year-old) than in young aniamls (<49 months), confirming that M. capillaris infection tends to be cumulative over time. In young sheep with Protostrongylidae pure infections the prevalence was influenced by the contact with goats. Some studies have assessed that goats are more susceptible to protostrongylid infections than sheep, presenting higher prevalence (Mangeon and Cabaret, 1987; Alemu et al., 2006) and intensity of infection (Mangeon and Cabaret, 1987; Berrag and Urquhart, 1996). The prevalence by protostrongylids found by Cienfuegos et al. (2009) in goats in Galicia (78.6%) and infection intensity (283.2 ± 782.5) were much higher than those found in sheep. Those results confirm the epidemiological implication of goats as an important source of pasture contamination (Berrag and Urquhart, 1996). On the other hand, protostrongylid prevalence in old group not infected with D. filaria was influenced by introducing ewes in the flock, with almost the double (20.8%) in protostrongylid prevalence that farms that did not introduced animals or introduced only males (11.4%). Most of the farms introduce males to avoid consanguinity, but the number is small and the effect is limited in time affecting only the prevalence, but not larval output. On the contrary, ewes are introduced in higher number than males, sometimes even whole flocks, so the effect is greater. Like sheep–goat contact (Berrag and Urquhart, 1996), it could be a source of pasture contamination to flocks free of protostrongylids. Another factor influencing sheep protostrongylid infection in flocks that did not introduce animals or introduce only males was the type of drugs used in these flocks; those animals treated with macrocyclic lactones (alone or combined with BZs) showed a lower prevalence than those treated only with BZs. Recent investigations in sheep infected with protostrongylids (Papadopoulos et al.,
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2004; Geurden and Vercruysse, 2007) have obtained better results with MLs than with BZs. Being completely effective or not, treatment with ML cut down larval output and the intermediate-host infection level, reducing the final prevalence in sheep population. For the intensity of infection, the influential factors were housing until weaning and Dictyocaulus infection. The intensity of infection (11.9 lpg) was slightly higher than that obtained in previous studies in this region (4.69 lpg; Díez et al., 1995), but lower than those obtained in other Spanish regions (232 lpg; Uriarte et al., 1985) and in general in Europe and North Africa (50–350 lpg; Cabaret, 1986). The most important factor influencing flock prevalence was the introduction of new animals from another flocks. Prevalence was significantly higher in farms that had introduced males and/or females from other flocks. However, for the flock infection intensity, farms with the greatest output were those that had animals infected with D. filaria. This study showed that protostrongylid prevalence in sheep under semi-extensive system for meat production is determined mainly by a positive interaction with D. filaria infection, mixed management with goats or introducing external animals in the flocks. Treatment effect was only observed in old animals in flocks free of D. filaria infection. The lowest protostrongylid prevalence has been reported in flocks free of D. filaria, with no contact with goats and that had not introduced external animals. Moreover, the presence of protostrongylid nematodes in flocks should be considered to apply a correct anthelmintic treatment in sheep (higher dose and/or repeated treatment) to reduce prevalence and avoid M. capillaris selection in small ruminants; specific treatments are therefore essential to control these parasites. 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. References Alemu, S., Leykun, E.G., Ayelet, G., Zeleke, A., 2006. Study on small ruminant lungworms in northeastern Ethiopia. Vet. Parasitol. 142, 330–335. Berrag, B., Cabaret, J., 1996. Impaired pulmonary gas exchange in ewes naturally infected by small lungworms. Int. J. Parasitol. 26, 1397–1400. Berrag, B., Urquhart, G.M., 1996. Epidemiological aspects of lungworm infections of goats in Morocco. Vet. Parasitol. 61, 81–95. Bliss, E.L., Greiner, E.C., 1985. Efficacy of fenbendazole and ambendazole against Muellerius capillaris in dairy goats. Am. J. Vet. Res. 46, 1923–1925. Boev, S.N., 1975. Principles of nematodology. In: Ryzhikov, M.M. (Ed.), XXV Protostrongilidy. Moscow, Izdatel’stovo Nauka, p. 648 pp. Cabaret, J., 1986. Repartition geographique des protostrongylides des ovins Frequence et importance de cette parasitose pulmonaire en Europe et en Afrique du Nord. Epidémiol. Santé Anim. 10, 61–72. Cabaret, J., Anjorand, N., Leclerc, C., 1989. Parasitic risk factors on pastures of French dairy goat farms. Small Rumin. Res. 2, 69–78. Cabaret, J., Dakkak, A., Alahkam, L., 1978. Considerations sur l’elimination des larves L1 de protostrongylines dans les feces des ovins nature de la distribution, influence de l’age de l’animal et des traitments anthelminthiques. Ann. Soc. Belge Méd. Trop. 58, 309–314.
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Protostrongylid infection in meat sheep from Northwestern Spain: Prevalence and risk factors ˜ S. Cienfuegos, R. Panadero, C.M. López ∗ , G. Fernández, M. Vina, ˜ L. Vázquez, P. Díaz, J. Pato, N. Lago, V. Dacal, P. Díez-Banos, P. Morrondo Parasitología y Enfermedades Parasitarias, Departamento de Patología Animal: Sanidad Animal, Facultad de Veterinaria, Universidad de Santiago de Compostela, Avda Carballo Calero s/n, 27002 Lugo, Spain
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Article history: Received 15 July 2010 Received in revised form 20 December 2010 Accepted 22 December 2010 Keywords: Ovine Protostrongylid Risk factors Northwestern Spain Prevalence
a b s t r a c t 2093 Faecal samples from 74 commercial meat ovine flocks were collected and examined by the Baermann–Wetzel method for protostrongylid infection. The risk of being infected by lungworms was evaluated with a data mining classification tree (CHAID), and the intensity of infection with a general linear model (GLM). 242 out of 2093 faecal samples examined were positive for protostrongylid infection (11.6%; 95% CI 10.2–12.9). Only two species were found, Muellerius capillaris (97.9%) and Neostrongylus linearis (5.4%). 50 out of 74 farms presented at least one animal shedding protostrongylid larvae in faeces. All of them held animals infected by M. capillaris and seven presented mixed infections with N. linearis. Average larval output in infected sheep was 11.9 (SD 30.91). This study showed that protostrongylid prevalence in sheep for meat production was determined mainly by a positive interaction with Dictyocaulus filaria infection; other factors that have influenced over protostrongylid infection were age, introducing external animals in the flocks, mixed management with goats and animal density in pastures. Treatment effects on prevalence were only observed in flocks that did not introduce ewes. The lowest protostrongylid prevalence has been reported in flocks without D. filaria infection and without contact with goats. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In Galicia (North-west of Spain) there are around 24,000 sheep flocks with a mean of 11.5 animals, contributing to the economic and environmental sustainability of the whole farm. In those farms sheep often share pastures with cattle and goats. In this region traditionally sheep have been used as an alternative to herbicides for suppressing weeds from rangeland and forest, especially in mountainous areas. However, in last years the sector has been improving as a result of the creation in 1994 of the Galician Ovine and Caprine breeder Association (OVICA)
∗ Corresponding author. Tel.: +34 982822109; fax: +34 982822001. E-mail addresses: [email protected], [email protected] (C.M. López). URL: http://www.clopezsandez.es (C.M. López). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.12.038
devoted to develop the sheep industry in the region. At present there is over 400 flocks that have more than 100 animals (90,000 sheep and goats), being dedicated almost exclusively to meat production. In the NW of Spain small ruminants are mainly maintained in a semiextensive husbandry system consisting of a mixture of grazing and indoor-housing. In this region, mild temperatures and environmental humidity are optimal for the survival of many important livestock parasites, such as small lungworms (Protostrongylidae) and the grazing system may facilitate the infection by those parasites. Protostrongylids (Nematoda: Strongylida) are heteroxenous parasites that infect terrestrial molluscs as intermediate host. Ovine infections are commonly asymptomatic, but heavy infections cause decreased carcass weights, increased levels of mortality and impaired pulmonary gas exchange (Valero et al., 1992; Berrag and Cabaret, 1996).
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Studies conducted in the last decade in two different localities of Galicia (Díez et al., 1994, 1995) determined that Neostrongylus linearis was the protostrongylid with the highest prevalence and intensity of infection in sheep, followed by Muellerius capillaris and Cystocaulus ocreatus. However, there are no reports on the prevalence and factors associated with the infection involving the entire region. Most of the studies over factors associated with protostrongylid infection in sheep were aimed mainly to treatment effect or intermediate host influence. Only a few have introduced other factors, like irrigating pastures (Uriarte et al., 1985), the effect of sheep–goat mixed flocks (Mangeon and Cabaret, 1987; Regassa et al., 2010) and management practices (Cabaret et al., 1989; Alemu et al., 2006). Although these parasites are widely prevalent, their infections are neglected by farmers and clinicians. Moreover, in this region, endoparasite control practices in commercial ovine farms include a systematic single dose treatment in spring and/or autumn intended to control gastrointestinal nematode infection. Nevertheless, the effect that this systematic treatment is being on protostrongylid populations is unknown. Kloosterman et al. (1989, 1990) have shown that primary infection with gastrointestinal nematodes increased the establishment of Dictyocaulus viviparus in cattle. Recent papers have detected similar immune responses in sheep infected by gastrointestinal nematodes and Dictyocaulus filaria (French et al., 2009), but the interaction between the different lungworm infections in sheep has not been studied yet. The main objective of this study was to establish the prevalence and larval output of protostrongylids in flocks from northwestern Spain, to identify the species involved and, in a second term, to detect and classify determinant factors on sheep protostrongylid infection.
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2.2. Animals and flocks surveyed Between March 2007 and January 2009, 74 commercial meat ovine flocks with a total of 16,122 sheep of which 2,093 were sampled for diagnostic of protostrongylid and D. filaria infection. The sample size per flock was calculated trying to fulfill in 95% confidence interval a 90% precision, considering 15% prevalence; animals were randomly selected. All the animals were mixed breed. The flocks were distributed over the entire region and all of them were joined to OVICA. 51 out of 74 were purely sheep flocks and 23 were mixed sheep/goat farms with an average of 18.3 goats/farm. A questionnaire comprising data about the management system and animal information was completed in every farm during sampling. All farms used anthelmintic treatment to control gastrointestinal nematodes. Lambs are always fed by their dams until weaning (2–3 months).
2.3. Examination of faecal samples Faecal samples were collected directly from the rectum with plastic gloves and kept at 4 ◦ C until being analyzed by the Baermann–Wetzel technique in the same day. Briefly, 10 g of faeces were left overnight with non woven filters (Filter-Lab, Filtros Anoia, S.A., Barcelona, Spain) in glass funnels with a 12 ml centrifuge tube at the end of a silicone tube. These tubes were centrifugated 10 min at 350G and the last milliliter containing the larval population was reserved and analyzed in Favati chambers. The different protostrongylid species were counted and identified by the characteristics of the posterior section of the larvae according to Boev (1975). If the number of larvae was less than 100, all of them were identified, and if higher than 100, at least 100 were identified.
2.4. Factors considered and statistical analysis 2. Materials and methods 2.1. Study area
The variables were grouped and categorized for statistical analysis as:
The study was undertaken in Galicia, region located in northwestern Spain, covering an area of 29,574 km2 (Latitude 42◦ 23 60N, Longitude 7◦ 4 W). The climate is Atlantic with mild temperatures and high precipitations. In this study the region was divided into three zones according to its orographic and bioclimatic conditions: a Coastal area situated from sea level to 200 m, with moderate precipitations (1300–1500 mm) and temperatures (14 ◦ C); a Central area at 200–650 m above sea level with low precipitations (900–1300 mm) and moderate temperatures (11.5 ◦ C); and a Mountainous area situated at 650–1285 m, with low temperatures (10 ◦ C) and high precipitations (>1500 mm). In Galicia small ruminants are mainly maintained in a semiextensive husbandry system; flocks are maintained in pastures near the barn where are kept during the night; in mountainous areas, due to topographic and climatic conditions, sheep are managed under extensively grazing system.
• Climatic area: 0 (Mountainous), 1 (Central), 2 (Coastal areas). • Sampling period: 1 (Spring), 2 (Summer), 3 (Autumn), 4 (Winter). • Husbandry system: 0 (extensive), 1 (semiextensive). • Housing until weaning: 0 (No), 1 (Yes). • Flock size: categorized at 33.3 and 66.6 percentiles 1 (<96 animals), 2 (96–194), 3 (>194). • Age groups: 1 (<13 months-old), 2 (13–48), 3 (>48). • Goat presence: 0 (purely sheep flock), 1 (presence of at least one goat on the farm). • Sheep–goat contact: 0 (purely sheep flock or presence of at least one goat on the farm but without contact), 1 (presence of at least one goat on the farm and both species are hold together in pastures and/or in the stables). • Introduction of new animals in the farm (last 5 years); two categories: ◦ 0 (No), 1 (yes).
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• • • • • • •
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◦ 0 (No), 1 (only male animals), 2 (male and female animals). Number of sheep introduced in the last 5 years, categorized at 50 percentile: 1 (0–9), 2 (>9). Treatment pattern: 0 (Spring), 1 (Autumn), 2 (Spring and Autumn). Drugs applied. 0 (macrocyclic lactones – ML or ML plus benzimidazoles – BZ), 1 (only BZ). Pasture rotation: 0 (No), 1 (Yes). Manure as fertilizer: 0 (No), 1 (Yes). Proximity to another flocks (<2 km): 0 (No), 1 (Yes). D. filaria infection: 0 (No), 1 (Yes).
The risk of being infected by lungworms taking into account these variables was evaluated with a data mining classification tree (Kass, 1980); particularly an exhaustive Chi-squared automatic interaction detector (exhaustive CHAID), whereby sheep were classified as positive (larvae in faeces) or negative (no larvae in faeces) in the categorical dependent variable. CHAID identifies variables that divide sheep in subgroups with distinct patterns of positive/negative ratio. The CHAID model provides a way to identify major factors. CHAID evaluates all of the values of a potential factor using as a criterion the significance of a statistical Chi-squared test, selecting the best predictor variable to form the first branch in a classification tree; successively splitting in data set makes increasingly homogeneous nodes in relation to the dependent variable. This process continues recursively until the classification tree is fully grown. The method is exhaustive because after a factor is entered in the analysis, it remains in the list of predictor variables for use in future calculations and there can be significant interaction with more predictors. Some of the factors studied present more than two categories and CHAID does not restrict the number of branches from each node to a predetermined number and could be split in more than two branches. For the examination of the intensity of infection an unifactorial association analysis (ANOVA) was first applied on the 242 positive animal counts and 50 positive farms to restrict the final number of factors under consideration. Factors with p values less than 0.1 were selected to build a linear model (GLM). For these analyses, the dependent variable – number of protostrongylid larvae eliminated per gram of faeces (lpg) and mean larvae outputs in flocks – had been previously log-transformed, Ln(lpg). In both cases Bonferroni post hoc test was used to detect the differences between pairs. All statistical analyses were performed with the statistical package SPSS for Windows 18.0 and SPSS Answer Tree, 3.1 (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Prevalence of protostrongylid infection and associated risk factors Individual prevalence: 242 out of 2093 faecal samples examined were positive for protostrongylid infection (11.6%; 95% CI 10.2–12.9). Only two species were found, M. capillaris that was identified in 97.9% of the positive fae-
cal samples (11.3%; 95% CI 10.0–12.6) and N. linearis, only identified in 13 samples (0.6%; 95% CI 0.3–0.9). The CHAID algorithm stratified variables that play an important role in protostrongylid presence and indicated (Fig. 1) that sheep not infected with D. filaria presented lower protostrongylid prevalence (node 1) than those infected (node 2). No other factor was detected influencing animals positive to D. filaria, but in the node of sheep negative to Dictyocaulus, sheep age was pointed out as a determining factor; sheep until 48 months showed lower prevalence (5.8%, node 4) than older animals (14.7%, node 3). The prevalence inside the younger sheep node was determined by sheep–goat contact, with a lower prevalence in animals that were not in contact with goats (4%; node 7) compared with sheep in contact with goats (12.5%; node 8). On the other hand, prevalence in older animals was determined by introducing external animals in the flock; protostrongylid prevalence in farms that introduce ewes from other flocks was much higher (20.8%; node 6) than that in farms that did not introduce other animals or introduce only males (11.4%; node 5). Finally, prevalence in old animals in farms that did not introduce ewes was only determined by the type of drugs applied in usual gastrointestinal nematode treatment, identified by the CHAID algorithm as a determining factor; node composed by animals treated only with BZ (node 9) showed higher prevalence than that treated with ML alone or ML plus BZ (node 10). Farm prevalence: 50 out of 74 farms presented at least one animal shedding protostrongylid larvae in faeces (67.6%; 95% CI 56.9–78.3). All of them presented animals infected by M. capillaris and seven presented mixed infections with N. linearis (9.5%; 95% CI 2.8–16.2). The CHAID algorithm indicated that introducing new animals from another flocks was the most important determining factor for prevalence at flock level (Fig. 2), as the prevalence in farms that introduced animals from other flocks was almost twice than that in farms that did not purchase new animals. 3.2. Intensity of infection by protostrongylids Individual: Average larval output in infected sheep was 11.9 (SD 30.91). All the different factors studied were first considered separately using ANOVA; Dictyocaulus infection (F = 9.529; p = 0.002), housing until weaning (F = 8.511; p = 0.004), manure as fertilizer (F = 5.541; p = 0.019), husbandry system (F = 3.259; p = 0.072) and drugs applied (F = 3.010; p = 0.084) shown a statistical significance under 0.1. Interacting analysis (GLM) between those factors indicated that the most influential factor over intensity of infection was housing until weaning (F = 13.695; p < 0.000) and Dictyocaulus infection (F = 4.391; p = 0.037), with no interaction between them (F = 0.044; p = 0.835). Animals in flocks from the beginning showed lower protostrongylid larvae mean output (7.6; SD 23.01) than those housed until weaning (30.6; SD 49.45); animals infected with D. filaria showed also higher mean output (24.8; SD 54.31) than those not infected (8.0; SD 17.47). No other factor were significant in GLM model.
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Fig. 1. Classification tree by exhaustive CHAID stratifying risk-factors for protostrongylid infection.
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Fig. 2. Classification tree by exhaustive CHAID showing determining factor for farm prevalence.
Farms: Dictyocaulus infection (F = 10.053; p = 0.003), manure fertilizer (F = 4.066; p = 0.053) and husbandry system (F = 2.943; p = 0.097) showed statistical significance under 0.1 in ANOVA considering separately all factors. In the interacting analysis (GLM) with all those factors, only D. filaria were significant on intensity of infection level (F = 7.998; p = 0.007); farms with at least one animal positive to D. filaria presented higher mean larvae output (10.6; SD 14.36) than farms without infected sheep (3.6; SD 8.45). 4. Discussion The prevalence by protostrongylid nematodes recorded in this study was lower than the 33.9% obtained by Díez et al. (1995) in the same region. It was also much lower than the 78.8% observed by Morrondo et al. (1990) in León (adjacent area with drier weather conditions); and by Uriarte et al. (1985) in Zaragoza (55.2% in irrigated and 37.9% in non-irrigated pastures). Moreover, most of the data obtained in sheep from Europe and North of Africa (Cabaret, 1986; Alemu et al., 2006) have shown higher protostrongylid prevalence (50–80%) than that obtained in this survey. Only two different species of Protostrongylidae were identified in this study, M. capillaris and N. linearis. Most of the positive animals were infected with M. capillaris (97.9%), whereas only 13 animals were infected by N. lin-
earis (5.4%). Previous studies carried out in the last decade in Galicia found N. linearis as the most prevalent species, followed by M. capillaris and C. ocreatus (Díez et al., 1994, 1995). It seems that generalized anthelmintic treatment to control gastrointestinal nematodes in commercial flocks has reduced not only the prevalence of protostrongylid infection but also selected M. capillaris among the protostrongylid genera, reducing considerably N. linearis populations and eliminating completely C. ocreatus on this territory. According to different authors (Bliss and Greiner, 1985; McCraw and Menzies, 1986; Helle, 1986; Díez et al., 1995; Rehbein and Visser, 2002; López et al., 2010) most anthelmintic treatments have shown an important lack of efficacy against protostrongylid lungworms, and in particular against M. capillaris. Some of those authors found that Muellerius larvae reappeared in faecal samples in less than 60 days, even in animals under strict isolation. This reappearance might be explained by immature Muellerius forms, not affected by anthelmintic treatment, developing to maturity after destruction of the original adult population (McCraw and Menzies, 1986, 1988) or by the protection conferred by altered tissue surrounding the lungworm that is more pronounced than in other protostrongylid species (Rehbein and Visser, 2002). For this reason, specific treatment for protostrongylid nematode infections would require higher dosage than those for
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gastrointestinal nematodes (Richard and Cabaret, 1992) or repeated treatments (McCraw and Menzies, 1986). The co-infection with D. filaria was identified as the most determining factor for ovine protostrongylid infection; prevalence in animals with a concomitant infection by D. filaria was higher than in those not infected. Taking into consideration that both lungworms have different transmission routes it is possible that positive interactions were immunologically mediated so that the presence of one species could facilitate the subsequent establishment of the other. Kloosterman et al. (1989, 1990) found positive interactions between lungworms in calves after previous infections with gastrointestinal nematodes and French et al. (2009) have detected that D. filaria infection generates similar immune response in ovine than Teladorsagia circumcincta infection in abomasal mucosa, so Dictyocaulus infection could provoke a lowered immunity response as described Kloosterman et al. (1989) for gastrointestinal nematodes that could facilitate sheep protostrongylid infection. However, further studies are necessary to elucidate this aspect. The second factor determined for protostrongylid prevalence was the age of the sheep. Age is a well-known influential factor in protostrongylid infections (Cabaret et al., 1978; Alemu et al., 2006; Regassa et al., 2010); prevalence increased with the age of the host. In our study sheep prevalence was two times and a half higher in old sheep (>4 year-old) than in young aniamls (<49 months), confirming that M. capillaris infection tends to be cumulative over time. In young sheep with Protostrongylidae pure infections the prevalence was influenced by the contact with goats. Some studies have assessed that goats are more susceptible to protostrongylid infections than sheep, presenting higher prevalence (Mangeon and Cabaret, 1987; Alemu et al., 2006) and intensity of infection (Mangeon and Cabaret, 1987; Berrag and Urquhart, 1996). The prevalence by protostrongylids found by Cienfuegos et al. (2009) in goats in Galicia (78.6%) and infection intensity (283.2 ± 782.5) were much higher than those found in sheep. Those results confirm the epidemiological implication of goats as an important source of pasture contamination (Berrag and Urquhart, 1996). On the other hand, protostrongylid prevalence in old group not infected with D. filaria was influenced by introducing ewes in the flock, with almost the double (20.8%) in protostrongylid prevalence that farms that did not introduced animals or introduced only males (11.4%). Most of the farms introduce males to avoid consanguinity, but the number is small and the effect is limited in time affecting only the prevalence, but not larval output. On the contrary, ewes are introduced in higher number than males, sometimes even whole flocks, so the effect is greater. Like sheep–goat contact (Berrag and Urquhart, 1996), it could be a source of pasture contamination to flocks free of protostrongylids. Another factor influencing sheep protostrongylid infection in flocks that did not introduce animals or introduce only males was the type of drugs used in these flocks; those animals treated with macrocyclic lactones (alone or combined with BZs) showed a lower prevalence than those treated only with BZs. Recent investigations in sheep infected with protostrongylids (Papadopoulos et al.,
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2004; Geurden and Vercruysse, 2007) have obtained better results with MLs than with BZs. Being completely effective or not, treatment with ML cut down larval output and the intermediate-host infection level, reducing the final prevalence in sheep population. For the intensity of infection, the influential factors were housing until weaning and Dictyocaulus infection. The intensity of infection (11.9 lpg) was slightly higher than that obtained in previous studies in this region (4.69 lpg; Díez et al., 1995), but lower than those obtained in other Spanish regions (232 lpg; Uriarte et al., 1985) and in general in Europe and North Africa (50–350 lpg; Cabaret, 1986). The most important factor influencing flock prevalence was the introduction of new animals from another flocks. Prevalence was significantly higher in farms that had introduced males and/or females from other flocks. However, for the flock infection intensity, farms with the greatest output were those that had animals infected with D. filaria. This study showed that protostrongylid prevalence in sheep under semi-extensive system for meat production is determined mainly by a positive interaction with D. filaria infection, mixed management with goats or introducing external animals in the flocks. Treatment effect was only observed in old animals in flocks free of D. filaria infection. The lowest protostrongylid prevalence has been reported in flocks free of D. filaria, with no contact with goats and that had not introduced external animals. Moreover, the presence of protostrongylid nematodes in flocks should be considered to apply a correct anthelmintic treatment in sheep (higher dose and/or repeated treatment) to reduce prevalence and avoid M. capillaris selection in small ruminants; specific treatments are therefore essential to control these parasites. 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. References Alemu, S., Leykun, E.G., Ayelet, G., Zeleke, A., 2006. Study on small ruminant lungworms in northeastern Ethiopia. Vet. Parasitol. 142, 330–335. Berrag, B., Cabaret, J., 1996. Impaired pulmonary gas exchange in ewes naturally infected by small lungworms. Int. J. Parasitol. 26, 1397–1400. Berrag, B., Urquhart, G.M., 1996. Epidemiological aspects of lungworm infections of goats in Morocco. Vet. Parasitol. 61, 81–95. Bliss, E.L., Greiner, E.C., 1985. Efficacy of fenbendazole and ambendazole against Muellerius capillaris in dairy goats. Am. J. Vet. Res. 46, 1923–1925. Boev, S.N., 1975. Principles of nematodology. In: Ryzhikov, M.M. (Ed.), XXV Protostrongilidy. Moscow, Izdatel’stovo Nauka, p. 648 pp. Cabaret, J., 1986. Repartition geographique des protostrongylides des ovins Frequence et importance de cette parasitose pulmonaire en Europe et en Afrique du Nord. Epidémiol. Santé Anim. 10, 61–72. Cabaret, J., Anjorand, N., Leclerc, C., 1989. Parasitic risk factors on pastures of French dairy goat farms. Small Rumin. Res. 2, 69–78. Cabaret, J., Dakkak, A., Alahkam, L., 1978. Considerations sur l’elimination des larves L1 de protostrongylines dans les feces des ovins nature de la distribution, influence de l’age de l’animal et des traitments anthelminthiques. Ann. Soc. Belge Méd. Trop. 58, 309–314.
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