Introduction
The conservation, sustainable use and promotion of animal genetic resources (AnGR) have been a priority in Portuguese and European Union (EU) policies. Over a number of years, the EU and the Portuguese Ministry of Agriculture have worked to keep AnGR and developed actions in collaboration with breeders associations for the protection, conservation and improvement of animal genetic heritage. Autochthonous breeds are a very valuable genetic resource owing to their biodiversity and can be distinguished for their ability to adapt to very adverse environmental conditions, to poor feed availability and also for their tolerance to parasitic infections (Paim et al., Reference Paim, Da Silva, Martins, Borges, Lima, Cardoso, Esteves, Louvandini and McManus2013; McManus et al., Reference McManus, Paim, de Melo, Brasil and Paiva2014). Livestock farming is central to the sustainability of rural communities worldwide (Morgan et al., Reference Morgan, Charlier, Hendrickx, Biggeri, Catalan, von Samson-Himmelstjerna and Demeler2013), and a large part of the sheep production in Portugal, like in many other countries, is based on extensive systems (Montossi et al., Reference Montossi, Font-i-Furnols, del Campo, Julián, Brito and Sañudo2013), taking advantage of forage resources in less favourable areas that would otherwise not be used. Sheep population in Portugal was estimated to be approximately two million in 2016 (FAOSTAT, 2016). Portugal has a range of particularities, with an enormous variability of orography, soil, climate, land structure, social and cultural traditions, that allow for a considerable diversity of genetic resources, such as a significant number of autochthonous breeds, namely of sheep. The Churra Galega Mirandesa (CGM) sheep is a Portuguese autochthonous breed raised in the north-east of Portugal, well adapted to the geographic and climatic conditions, with a great aptitude for meat production, with wool being used mainly for handicraft and milk being consumed only by lambs.
CGM sheep graze most of the year, because of the climatic conditions with continental and Atlantic influences. Such sheep are raised in extensive systems, fed on cereal stubble and natural pastures (characterized mainly by annual grass species), and most of them are only brought into the paddocks during the night. Consequently, these sheep are extremely exposed to a wide range of gastrointestinal (GI) helminths and protozoa (Fox et al., Reference Fox, Marion, Davidson, White and Hutchings2013; Kumar et al., Reference Kumar, Rao, Varghese and Rathor2013; Rinaldi et al., Reference Rinaldi, Catelan and Musella2015a).
In European countries, a range of Strongylida (Teladorsagia circumcincta, Haemonchus contortus, Trichostrongylus spp., Nematodirus battus, Cooperia spp., Chabertia spp. and Oesophagostomum spp.) have already been identified in usually mixed-parasitic infections in sheep (Fox et al., Reference Fox, Marion, Davidson, White and Hutchings2012; Zajac & Conboy, Reference Zajac and Conboy2012; Bowman, Reference Bowman2014; Rinaldi et al., Reference Rinaldi, Hendrickx and Cringoli2015b), but ruminants also carry large numbers of protozoa species (Taylor, Reference Taylor2000; Chartier & Paraud, Reference Chartier and Paraud2012).
GI parasitism and the subsequent host immune response have important consequences for sheep production, associated with reduced nutrient utilization, growth rate and milk production (Arsenos et al., Reference Arsenos, Fortomaris, Papadopoulos, Kufidis, Stamataris and Zygoyiannis2007; Jacobson et al., Reference Jacobson, Pluske, Besier, Bell and Pethick2009; Rinaldi et al., Reference Rinaldi, Hendrickx and Cringoli2015b). The subclinical parasitic infections are responsible for significant economic losses in animal productivity (Morgan et al., Reference Morgan, Charlier, Hendrickx, Biggeri, Catalan, von Samson-Himmelstjerna and Demeler2013; Roeber et al., Reference Roeber, Jex and Gasser2013). This research focuses on the lack of well-established data on the prevalence and distribution of sheep GI parasites in the north-east of Portugal in general, and in the CGM sheep breed in particular. Therefore, this study was designed to assess the diversity, the prevalence and burden of GI parasites in this autochthonous Portuguese sheep breed. Moreover, we evaluated the predictability of some risk factors, such as grazing type, cohabitant animals and the parasite-control practices on the GI parasitic prevalence and burden.
Material and methods
Study area
The study was conducted between September and December 2016 and included sheep flocks located in the north-east of Portugal. For this purpose, we joined the association of the breeders of CGM sheep (ACOM), the officially recognized entity for the CGM sheep breed herd-book management, and visited their CGM sheep flocks. The definition of the sample was, therefore, of convenience sampling. According to the ACOM, the CGM includes 6763 females and 170 males approximately, but the number has been decreasing and the breed has been classified as an endangered autochthonous breed (DGAV, 2013). The traditional sheep production system is extensive and most flocks are maintained outside, within fenced areas for sheep grazing, and are only brought into the paddocks during the winter and cold days. Sheep graze on natural pastures, and it is common for flocks to graze in community pastures. The flocks were sampled from four municipalities of the Bragança district (Miranda do Douro, Vimioso, Mogadouro and Bragança) (fig. 1), which have similar climatic conditions with continental and Atlantic influences, characterized by cold winters and warm summers.
Fig. 1. Localization of the flocks sampled in the study area.
Faecal sample collection and analysis
The sample size expected for this study was determined by using a confidence level (CL) of 95%, an error (D) of 5% and an expected prevalence (P) of 20%, by calculating Z2(P(1–P))/D2, where the value of the standard normal distribution (Z) is Z = 1.96 for CL 95%. So, the required number of sampled sheep was of 246 or more.
The geographical coordinates and elevation data of farm locations were registered. Fresh faecal samples were collected from sheep older than nine months of age, directly from the rectal ampoule/rectum or immediately after defecation. The samples were stored in individual plastic bags, and labelled with animal identification number, age, sex, day/month of collection and farm location, and transported and maintained under 4°C until the arrival at the laboratory. Each sample was first examined macroscopically for the possible detection of proglottids.
All individual samples were analysed using three methods: flotation in saturated sodium chloride solution (Willis), natural sedimentation technique for qualitative assessment and the McMaster method (quantitative) (Zajac & Conboy, Reference Zajac and Conboy2012). In terms of the burden of infection, they were classified as low, moderate and high when the number of eggs/oocysts per gram of faeces (EPG/OPG) was up to 1000, between 1001 and 2000, and above 2001, respectively (Soulsby, Reference Soulsby1988).
Data collection
The design of the questionnaire followed a literature review about sheep breeding and management, which was subsequently tested and reviewed with regard to the terms used. This resulted in a final questionnaire composed of 13 questions, related to flock size, cohabitant animals, food management and deworming practices and procedures, number of treatments/year and date of last treatment. Body condition (Bc) was recorded according to Ducanson (Reference Ducanson2012), based on five scores.
The questionnaire was administered to every farm at the same time as faecal samples were collected.
Statistical analysis
The prevalence (P) was calculated by P = n/N, where n = number of samples positive to different species of GI parasites and N = total number of samples analysed. Analyses were conducted using SAS® (SAS Institute, 2004). The association between the prevalence of the various parasites and independent variables, like municipality, month of collection, grazing type, deworming, cohabitation with other animals (cattle, goats, donkeys, horses and dogs), Bc and the total number of animals on the farm (the latter two were considered as co-variables) were evaluated using the Chi-square test (χ2). The CL was held at 95% and 99%; P < 0.05 and P < 0.01 was set for significance.
Initially, frequencies were analysed through the PROC FREQ of SAS (SAS Institute, 2004). Subsequently, the probability of presence/absence of the various parasites expressing variability (strongyle, Nematodirus spp., Eimeria spp., Moniezia benedeni, Dicrocoelium spp. and Trichuris spp.) was analysed by logistic regression through PROC LOGISTIC of SAS (SAS Institute, 2004). Strongyle, Nematodirus spp. and Eimeria spp. were subjected to analysis of variance through PROC GLM of SAS (SAS Institute, 2004) and the least square mean (LSM) was estimated. The χ2 and analysis of variance one-way tests were used respectively to compare the prevalence and the mean intensity of the different GI parasites infections.
Results
Questionnaires
Cooperation with the sheep farmers was satisfactory and all of them participated in the questionnaire. A total of 49 replies to the questionnaires were received, one for each flock sampled. Most farms (48.9%) have less than 130 sheep and only two (4.1%) do not have other cohabiting animals; dogs are almost ubiquitous (93.9%) and donkeys are very common (24.5%). The vast majority of flocks (81.6%) graze on community pastures. Most farmers (75.5%) report deworming (table 1).
Table 1. Characterization of flock size, other animal cohabitation, grazing time, feeding and deworming frequency.
a Sheep feeding supplements are defined as food with a lower amount of fibre and more energy and/or protein for sheep.
Parasite prevalence
A total of 512 faecal samples from 49 flocks were analysed and in 97.1% (497/512) one or more GI parasites have been identified. The overall prevalence of the parasites found and the results per parasite for each municipality are presented in table 2. Nematoda, Cestoda, Trematoda and Protozoan parasites have been identified. Strongyle-type eggs (85.4%) were the most prevalent parasites. Miranda do Douro and Vimioso were those where a greater diversity of parasites was found, namely Dicrocoelium spp. Fasciola hepatica was only identified in Miranda do Douro.
Table 2. Number and percentage of the different parasites found by municipality.
The infection burden based on faecal eggs count (FEC) ranged from 50 to 6250 EPG for helminths and from 50 to 17550 OPG for protozoa. The mean, range of EPG and level of infection per sample are presented in table 3.
Table 3. Percentage of low, moderate and high burden of infection, and mean, standard deviation and range of EPG/OPG.
SE, standard error; SD, standard deviation.
In relation to the burden of the GI parasites, the study revealed that the mean EPG was notably low for all species of GI parasites observed.
Analysis of association of parasite prevalence and environmental factors
The association between each GI parasite and the factors studied is presented in table 4. χ² shows the degree of association with independent factors, such as municipality, month of collection, type of pasture, deworming, cohabitation with animals (cattle, goats, donkeys, horses and dogs), Bc and flock size.
Table 4. Multivariate analysis of parasitic infections and potential risk factors.
**Significant for P < 0.01; *significant for P < 0.05; ns: non-significant (P > 0.05). Bc, body condition.
In most of the GI species found, there was no difference in prevalence according to the variables shown in table 4, but when analysing the different variables that interfere with GI parasites infections, it was observed that the month of sampling, grazing time, deworming, the presence of cohabitant animals (like dogs) and Bc were the variables that exerted significant influences on the occurrence of some GI parasites.
Statistical differences in the results obtained were found between the periods of grazing. Table 4 shows that there were no significant differences in distribution between the municipalities in the study area and flock size (P > 0.05).
When sheep graze during all day, the risk of the infection by Nematodirus spp. is 1.5 higher (odds ratio (OR) = 1.515, P < 0.05). Non-dewormed sheep are 1.6 times (OR = 1.594, P < 0.05) more likely to have Nematodirus spp. infection. Grazing time is also associated with occurrence of Trichuris spp.: when grazing occurs during some hours in the early morning and late afternoon, the risk of infection with Trichuris spp. is 2.2 times higher (OR = 2.183, P < 0.05).
In the case of Dicrocoelium spp., when grazing during all day, animals are 2.2 times more likely to be infected by Dicrocoelium spp. (OR = 2.164, P < 0.01). The month of sampling was significantly associated with the occurrence of M. benedeni (P < 0.05) and Dicrocoelium spp. (P < 0.01). The risk of occurrence of M. benedeni and Dicrocoelium spp. is 5.9 and 3.9 times higher, respectively, in October.
Bc seemed to have a significant association with the prevalence of Eimeria spp. (P < 0.01) (table 5): Eimeria spp. was higher in sheep with poor Bc and increasing Bc was significantly associated with decreased odds of Eimeria spp. infection.
Table 5. Results of linear regression analysis for strongyle, Nematodirus spp. and Eimeria spp. between deworming and body condition (Bc).
ns, non-significant (P > 0.05); *significant for P < 0.05; **significant for P < 0.01. Number obs., number of observations; r 2, coefficient of determination.
The strongyle and Nematodirus spp. FEC were significantly influenced by deworming (P < 0.05) and Bc (P < 0.01).
The LSM of FEC in strongyle and Nematodirus spp. shows significant differences between deworming and non-deworming.
The LSM of FEC in dewormed sheep is lower (231.78 ± 27.41) than in non-dewormed sheep (361.09 ± 51.24). No significant variation was seen between deworming and Eimeria spp. The Bc of sheep is influenced by the burden of strongyle, Nematodirus spp. and Eimeria spp.
For each plus one in Bc, the amount of strongyle, Nematodirus spp. and Eimeria spp. decreases −152.20 ± 41.45, −10.39 ± 2.71 and −295.34 ± 92.31, respectively.
Discussion
The current study is the first GI parasites survey in CGM sheep breed in their original location and aim to document the prevalence, diversity and burden of helminth and protozoa in those flocks.
The results of the survey clearly evidence a high prevalence of GI helminths. The diversity found in this survey could be higher if we had carried out coprocultures to distinguish different strongyle genera or species eggs. These parasites, alone or in association, can be responsible for negative effects on sheep health and production (Bowman, Reference Bowman2014).
The observed strongyle-type eggs prevalence rates did vary between the municipalities. Different studies performed in the north-west of Spain, in a region situated close to the one considered in our study, with similar geographic elevation and climatic conditions, showed a higher prevalence of strongyle and F. hepatica has been sporadically found (Pedreira et al., Reference Pedreira, Paz-Silva and Sánchez-Andrade2006; Martínez-Valladares et al., Reference Martínez-Valladares, Robles-Pérez, Martínez-Pérez, Cordero-Pérez, Famularo, Fernández-Pato, González-Lanza, Castañón-Ordóñez and Rojo-Vázquez2013; Atlija et al., Reference Atlija, Prada, Gutiérrez-Gil, Rojo-Vázquez, Stear, Arranz and Martínez-Valladares2016).
Strongyle and Eimeria spp. were the most common parasites found, similarly to what has been observed in previous studies, not only in Europe (Torina et al., Reference Torina, Dara, Marino, Sparagano, Vitale, Reale and Caracappa2004; Skirnisson, Reference Skirnisson2007; Idris et al., Reference Idris, Moors, Sohnrey and Gauly2012; Kantzoura, Reference Kantzoura2012; Rinaldi et al., Reference Rinaldi, Catelan and Musella2015a) but also in other continents (Kumar et al., Reference Kumar, Jakhar, Singh, Potliya, Kumar and Pal2015; Tramboo et al., Reference Tramboo, Shahardar, Allaie, Wani and Bushra2015; Kelemework et al., Reference Kelemework, Tilahun, Benalfew and Getachew2016; Sultan et al., Reference Sultan, Elmonir and Hegazy2016). Concerning Eimeria spp. prevalence, Chartier and Paraud (Reference Chartier and Paraud2012) assert that these protozoan species are more frequent in young animals, but in the present study the high prevalence was detected in adult sheep. Sheep Bc was correlated with the presence of Eimeria spp., which is understandable, as Chartier and Paraud (Reference Chartier and Paraud2012) have found that these parasites cause great economic losses because of diarrhoea.
Skrjabinema spp. is a rare parasite in sheep (Zajac & Conboy, Reference Zajac and Conboy2012). Amarante (Reference Amarante2014) described it in Brazil, where it has been found in animals that share grass or cohabit with goats, as we confirmed to happen in this study. Moreover, to our knowledge, this is the first report of Skrjabinema spp. in Portugal.
The prevalence of Trichuris spp. was 6.4% and has been already reported in other European countries (Torina et al., Reference Torina, Dara, Marino, Sparagano, Vitale, Reale and Caracappa2004; Pedreira et al., Reference Pedreira, Paz-Silva and Sánchez-Andrade2006; Kantzoura, Reference Kantzoura2012). The prevalence and burden of this parasite is considerably higher than those found in neighbouring provinces in Spain, with the same or similar environmental conditions (Pedreira et al., Reference Pedreira, Paz-Silva and Sánchez-Andrade2006).
The livestock husbandry systems and management practices can have a major influence on the transmission of that infection to a susceptible host population. Tariq et al. (Reference Tariq, Chishti, Ahmad and Shawl2008) reported that faecal egg, pasture larval and worm counts were highest in communitary grazing sheep, as it still happens in this Portuguese region.
Two Cestoda species, Moniezia expansa and M. benedeni, commonly occur in ruminants (Strobel et al., Reference Strobel, de Ponte, Knoppe and Bhushan2013; Diop et al., Reference Diop, Yanagida, Hailemariam, Menkir, Nakao, Sako, Ba and Ito2015) and were found in 0.8% and 2.3%, respectively. While M. expansa was the most frequent Cestoda in some studies (Bashtar et al., Reference Bashtar, Hassanein, Abdel-Ghaffar, Al-Rasheid, Hassan, Mehlhorn, Al-Mahdi, Morsy and Al-Ghamdi2011; Nguyen et al., Reference Nguyen, Le, Nguyen, Nguyen and Vu-Khac2012), worldwide, other researchers only report M. benedeni (Moazeni & Nili, Reference Moazeni and Nili2004). October was significantly associated with increased prevalence of M. benedeni. This may be explained on the basis of parasites from the genera Moniezia being seasonal: peaks of infection occur in periods of greater activity of intermediate hosts, namely in spring and autumn (Taylor et al., Reference Taylor, Coop and Wall2007; Bashtar et al., Reference Bashtar, Hassanein, Abdel-Ghaffar, Al-Rasheid, Hassan, Mehlhorn, Al-Mahdi, Morsy and Al-Ghamdi2011; Bowman, Reference Bowman2014).
The present study seems to suggest that the climate may have an impact in the occurrence of fasciolosis and dicrocoeliasis. Fasciola hepatica was only identified in Miranda do Douro, a municipality that falls within the catchment area of the River Douro, and flocks were located in villages with irrigated areas or near the Douro River, which is essential for the development of the intermediate hosts of Fasciola spp. Martínez-Valladares et al. (Reference Martínez-Valladares, Robles-Pérez, Martínez-Pérez, Cordero-Pérez, Famularo, Fernández-Pato, González-Lanza, Castañón-Ordóñez and Rojo-Vázquez2013) have observed that F. hepatica occurrence was correlated significantly with the climate data.
The prevalence of Dicrocoelium spp. found is higher than that found in other European countries (Kantzoura, Reference Kantzoura2012; Taylor, Reference Taylor2012). Studies on geographic distribution and frequency rate of intermediate hosts are necessary for the control of the disease in animals and for the prevention of human infection. Those human infections are rare (Moure et al., Reference Moure, Zarzuela and Espasa2016), but can occur (Ella & Mohammad, Reference Ella and Mohammad2015). As Dicrocoelium spp. and Fasciola spp. are zoonotic, sheep deworming is suggested. Sheep farmers and veterinarians need to be properly educated about the risks of zoonosis and about the importance of regular parasitological analysis.
FEC has been a diagnostic tool to evaluate infections, but has low sensitivity (Sargison, Reference Sargison2013; Atlija et al., Reference Atlija, Prada, Gutiérrez-Gil, Rojo-Vázquez, Stear, Arranz and Martínez-Valladares2016). Mean EPG/OPG is commonly used as an indicator for the severity of an infection in a population. The degree of EPG/OPG in most samples was low, mirroring various reports in other regions worldwide (Tariq et al., Reference Tariq, Chishti, Ahmad and Shawl2008; Idris et al., Reference Idris, Moors, Sohnrey and Gauly2012; Rinaldi et al., Reference Rinaldi, Catelan and Musella2015a; Atlija et al., Reference Atlija, Prada, Gutiérrez-Gil, Rojo-Vázquez, Stear, Arranz and Martínez-Valladares2016) and in neighbouring regions in Spain with the same climatic conditions (Martínez-Valladares et al., Reference Martínez-Valladares, Robles-Pérez, Martínez-Pérez, Cordero-Pérez, Famularo, Fernández-Pato, González-Lanza, Castañón-Ordóñez and Rojo-Vázquez2013).
The animal Bc was also related with the EPG mean of strongyle-type and Nematodirus spp. Van-Wik et al. (Reference Van-Wik, Hoste, Kaplan and Besier2006) report the same conclusion in their study with H. contortus.
The association observed between Nematodirus spp., Trichuris spp., Dicrocoelium spp. and grazing time was expected and is related to the epidemiology of these parasites (Bowman, Reference Bowman2014). In essence, it is important to limit the intake of infectious stages with pasture management, such as turn-out time and the duration of the grazing period (Thamsborg et al., Reference Thamsborg, Roepstorff, Nejsum and Mejer2010). In relation to the Dicrocoelium spp. infection, it is important not to graze early in the morning or late in the afternoon, when there is the highest number of ants present on the herbage (Otranto & Traversa, Reference Otranto and Traversa2003).
The severe infection found in one flock in this survey may be associated with the conditions of hygiene and over density observed. Deniz (Reference Deniz2009) notes that coccidiosis in sheep is often associated with overcrowding and faecal contamination of drinking water or feed.
The fact that this study shows a low parasite burden in the CGM sheep breed can be explained by resistance and resilience to parasitism. Tariq et al. (Reference Tariq, Chishti, Ahmad and Shawl2008) and McManus et al. (Reference McManus, Paim, de Melo, Brasil and Paiva2014) reported with their surveys that the prevalence of GI parasites was higher in exotic breeds than in autochthonous ones.
In general, the overall prevalence of GI parasites in this study area still does not indicate GI parasites as a serious health problem; however, it is worth highlighting the importance of zoonotic parasites, especially among farmers and the human population. Effective veterinary care, routine epidemiological surveillance and enhanced educational campaigns on sheep parasitic zoonoses are key measures to increase productivity and reduce the public health risks associated with sheep farming in the north-east of Portugal.
The diversity of GI parasites and the consequences for sheep can become a serious threat to sheep production, especially in CGM sheep, an endangered autochthonous sheep breed. The constant surveillance and the ongoing refinement and development of parasite control strategies are crucial. These findings can be used to target high-risk farms with appropriate control measures against GI parasites of sheep in Portugal and other areas with similar climatic conditions. New studies should be conducted in the future to differentiate the GI nematodes involved, as well as Eimeria species.
Acknowledgements
Miguel Costa is a fellow from Global Public Health Doctoral programme “FCT fellowship PD/BD/135759/2018”. The authors would like to thank the staff of Associação de Criadores de Ovinos da Raça Churra Galega Mirandesa for their cooperation and for sharing their facilities during the collection of faecal samples in different areas. The authors would also like to thank Prof. Dr. Nuno Ereira Torres Ferreira for the English review of the manuscript. The authors are grateful to all of the farmers who collaborated in this study.
Conflicts of interest
None.
