Introduction
Apart from the west of the United States of America and central-southeast Canada, the arid zones of northwestern Mexico are the home of desert bighorn sheep (DBS; Ovis canadensis). However, populations of this species have been significantly reduced in the past years due to the widespread illegal hunting of trophy males.
According to Mexican legislation, DBS require special protection (SEMARNAT, 2010). Thus, introducing this species into new habitats of northern Mexico for their propagation entails reducing diseases that could play any role in population expansion. It has been speculated that one of the decimating factors leading to the reduction of the population of DBS was the occurrence of diseases and parasites of domesticated sheep. Old reports have characterized the gastrointestinal nematodes of this species in Oregon (Kistner et al., Reference Kistner, Matlock, Wyse and Mason1977), Montana (Becklund & Senger, Reference Becklund and Senger1967) and British Columbia (Demarchi et al., Reference Demarchi, Hartwig and Demarchi2000). More recently, it has been reported that Strongyloides spp. are absent in DBS in New Mexico (Barone et al., Reference Barone, Wit, Hoberg, Gilleard and Zarlenga2020). In general, these reports indicate the presence of a wide variety of helminths in the abomasum, small intestine, caecum and large intestine. However, the population of these nematodes appeared to have little or no effect on the well-being of these DBS, as animals examined were in good physical condition. Characterization of the helminths population of DBS is crucial because it could alert wildlife biologists and veterinarians to the potential transmission of gastrointestinal parasites from livestock to DBS populations or, conversely, whether DBS populations are potential reservoirs of gastrointestinal nematodes that could affect farm animals.
Information on gastrointestinal parasitism in DBS in arid environments is fragmentary and scarce mainly because of the almost inaccessibility of these animals, as they live on steep slopes of mountains, with rocky soils, with hard approachability which protects them from their predators. In Mexico, the specific gastrointestinal helminth load of DBS is not known with certainty nor the seasonal variation of these internal parasites. Therefore, the present study aimed to investigate the seasonal prevalence of gastrointestinal helminth parasites of DBS in a conservation unit for wildlife management in northern Mexico. An additional objective was to assess the effect of season of the year on the species diversity of gastrointestinal nematodes in these DBS.
Materials and methods
Study area
The study was carried out in the Unit for Conservation of Wildlife ‘San Juan’ located at 26°50ʹ19.13ʺN and 101°2ʹ31ʺW (fig. 1) with an average altitude of 609 meters above sea level. The vegetation is semi-arid rangeland with temperatures ranging from −9 to 42 °C. Annual rainfall ranges from 410 to 505 mm, with most rain occurring from May to October. Three sites were selected based on watering points and feed bunks. These sites were cliffs on the mountain slopes. At the beginning of the study, there were 74 DBS including juvenile, adult females, young males, and adult rams, with approximately 60% males and 40% females.
Fig. 1. The geographical description of the study site.
Faecal collection procedure
Twenty-five fresh faecal samples (clumps) from each of the three study sites were collected from the natural habitat of DBS, with a total of 75 samples per season of the year. Faecal samples were collected on 18–21 January (winter), 17–19 April (spring), 20–23 July (summer) and 19–22 September 2019. Fresh faeces were collected from the ground within 1 km radius around the watering point in the morning (6–10 am) and afternoons (6–8:30 pm). A few hours before faeces collection, dry faeces corresponding to defecations of several days or weeks ago were removed to facilitate the gathering of fresh faecal samples (within an hour of excretion).
During the sampling day, groups of DBS were located with binoculars when they approached the drinking troughs. As soon as the animals left the watering point, fresh and shiny samples were collected around the water point along the trail these animals went through. In addition, it was observed that when the DBS ran, they defecated, which allowed making additional collections of fresh faeces. The faeces (20–25 grams per sample) were collected manually within an hour of excretion, with the use of latex gloves and a utility knife to obtain clean samples, eliminating soil residues and organic matter, then they were placed in previously labelled ziplock bags. To reduce the probability of mixing pellet groups from more than one individual, only contiguous pellets were collected, excluding pellets scattered more than about 10 cm from the group's centre (Harris et al., Reference Harris, Winnie, Amish, Beja-Pereira, Godinho, Costa and Luikart2010). Finally, these samples were placed in a cooler for transportation to the laboratory. Upon returning to the laboratory, the analysis was carried out the next collection day for four seasons in 2019. Given that the study was performed in a 5500-ha enclosure with complete segregation of other ungulates, the collected faecal samples were only from DBS.
Parasitological measurements
Nematode/coccidia faecal egg counts were determined using the McMaster technique. Four grams of faeces were thoroughly ground up and mixed with 56 mL of sugar solution. Samples were stirred continuously for 20 min until a homogeneous mixture was obtained. These mixtures were filtered through a sieve with 0.18-mm mesh and placed into a new beaker. The resulting filtrate was used to fill the two counting chambers of the McMaster (0.5 mL per chamber). Eggs of nematodes were allowed to float for 5 min and were searched using a microscope with 10× magnification. The number of eggs counted in the two chambers was multiplied by 50 to obtain the value of eggs per gram of faeces (EPG).
Also, duplicate faecal cultures were done for each sample within season to obtain nematode infective larvae for taxonomic identification (Van Wyk et al., Reference Van Wyk, Cabaret and Michael2004). First, faecal samples were macerated by adding water to get pasty faeces; then, 20 g were placed into a 10-cm Petri dish, which was placed inside another 15-cm Petri dish with water. The Petri dishes were placed in an incubator at a constant controlled temperature of 27 °C and 78% humidity for 11 days. On the 12th day of incubation, water samples from the Petri dish's surface were taken with a pipette, and a drop was placed on a slide, positioning a coverslip on the slide. Samples were observed under the microscope using 100× magnification to identify helminths or coccidian based on their size, oral cavity and type of sheath tail.
Statistical analyses
The EPG counts were analysed using the MIXED procedure of SAS (SAS Institute, Inc., Cary, NC). Frequencies were determined using PROC FREQ of SAS. EPG were transformed to log10 (EPG + 25) to correct the heterogeneity of variance and obtain a normal distribution approximation of data. The unit of the study was the sample over a one-year period. The model included the fixed effect of season, the fixed effect of site, the season × sampling site interaction and the random effect of samples within season. Means of variables analysed were compared using the probability of a statistical difference (PDIFF option of SAS).
The effect of season on the percentage of nematode species and coccidia was analysed using the GENMOD procedure of SAS with a linear logit model. If significant differences were found among seasons, the SAS LSMEAN/DIFF procedure was used to compare the means. Statistical differences were considered significant at P < 0.05.
Results and discussion
A limitation of this study is that it was not possible to collect faeces from animals of different stages of growth or pregnancy, and it is well known that gastrointestinal parasites are aggregated within host populations, namely young wild sheep (Festa-Bianchet, Reference Festa-Bianchet1989; Craig et al., Reference Craig, Pilkington and Pemberton2006, Reference Craig, Jones, Pilkington and Pemberton2009) and ewes during the periparturient period (Rose Vineer et al., Reference Rose Vineer, Baber, White and Morgan2019). Thus, we could not know how nematode eggs were aggregated across hosts within this DBS population. Additionally, although faeces can be collected easily, assignment to individuals is impossible under the current experimental conditions. It was assumed that faecal samples were from different animals each sampling season, and thus, by presumably using only faeces from different hosts at each time point, pseudoreplication was avoided.
Without accounting for differences in age and gender, the overall mean faecal egg count for the study was 395 ± 296, with a prevalence of 100%. Approximately one-fifth of all samples experienced loads of at least 500 EPG, considered ‘moderate’ shedders in sheep guidelines (Chagas et al., Reference Chagas, Oliveira, Esteves, De Oliveira, Giglioti, Giglioti, de O Carvalho, Ferrezini and Schiavone2008). This low occurrence of nematode eggs in the faecal samples is not likely to adversely affect free-ranging DBS. These results are lower than results of faecal egg counts from goats reared in zones close to the present study area, where EPG ranged from 418 to 884 (Olivas-Salazar et al., Reference Olivas-Salazar, Estrada-Angulo, Mellado, Aguilar-Caballero, Castro-Pérez, Gutiérrez-Blanco and Ruiz-Zárate2018). Likewise, Mellado et al. (Reference Mellado, González, García and García2004) reported EPG values up to 1600 in goats grazing a similar type of vegetation in northern Mexico than that of the DBS in the present study. One important reason for the low EPG registered in DBS in the present study was the good plane of nutrition of these animals, due to feed supplementation during the dry season, as good nutritional condition confers higher resistance to nematode infections (Coop & Kyriazakis, Reference Coop and Kyriazakis2001; Bakunzi et al., Reference Bakunzi, Mogapi, Motsei, Nyirenda, Ndou and Mwanza2018). Also, the steep and rough characteristics of the habitat of these animals impede the interaction with small ruminants and consequently transmission of helminths between species.
Mean EPG excreted by DBS showed seasonal variation with the highest (P < 0.05) values recorded in winter and autumn (table 1) and the lowest in spring and summer. The rainy season in this zone includes autumn, and therefore, it confirms that rainfall is one of the most epizootiological factors that affect helminth egg and larvae development associated with sheep (Brahma et al., Reference Brahma, Jas, Das and Ghosh2018; Dafur et al., Reference Dafur, Mbap, Tok and Okoh2021b). This finding is similar to that of other studies where EPG increases in the rainy season in sheep maintained on rangelands (Khajuria et al., Reference Khajuria, Katoch, Yadav, Godara, Gupta and Singh2013; Jansen et al., Reference Jansen, Nyangiwe, Yawa, Dastile, Mabhece, Muchenje and Mpendulo2020). Faecal output of nematode eggs did not differ among sampling sites corroborating the low intensity of infection in DBS regardless of different feeding zones of this ecosystem. There was not a significant season × grazing site interaction.
Table 1. The effect of season and grazing site on mean nematode faecal egg counts (eggs per gram of faeces) of desert bighorn sheep (Ovis canadensis) in northern Mexico.
a, b For the same column and variable difference, superscript letters indicate statistical significance (P < 0.05).
The present study revealed that DBS were infested with various gastrointestinal helminths (table 2). Many of these helminths have been found in bighorn sheep in Oregon (Kistner et al., Reference Kistner, Matlock, Wyse and Mason1977), Montana (Becklund & Senger, Reference Becklund and Senger1967) and British Columbia (Demarchi et al., Reference Demarchi, Hartwig and Demarchi2000). However, studies from Canada and the United States of America show a wider variety of nematodes in bighorn sheep (e.g., Ostertagia spp., Marshallagia marshalli, Skrjabinema ovis, among others) than that observed in the present study. The much drier and hotter characteristics of the zone of the present study seem to explain these differences. Additionally, there is an inextricable association between plants consumed by artiodactyls and parasites (Waller et al., Reference Waller, Bernes, Thamsborg, Sukura, Richter, Ingebrigtsen and Höglund2001), and pastures of drier ecosystems may impede the survival of gastrointestinal parasites found in colder and more humid habitats. Different sympatric wild ungulates in different ecosystems may also explain these differences.
Table 2. The effect of season on percentage of nematodes and coccidia of desert bighorn sheep (Ovis canadensis) in northern Mexico.
a, b, c For the same raw difference, superscript letters indicate statistical significance (P < 0.05).
Strongyloides spp. was the most common finding in faeces collected and the percentage of faecal samples containing this nematode differed (P < 0.05) between seasons (table 2). Other authors have observed similar findings (Kandasamy et al., Reference Kandasamy, Rajapakse and Rajakaruna2013; Bansal et al., Reference Bansal, Agrawal and Haque2015; Poddar et al., Reference Poddar, Begum, Alim, Dey, Hossain and Labony2017), who reported a high prevalence rate of Strongyloides spp. in grazing sheep. Haemonnchus contortus, the most pathogenic parasitic nematode affecting sheep herds worldwide (Harder, Reference Harder2016; Besier et al., Reference Besier, Kahn, Sargison and Van Wyk2016), was only observed in winter with a low prevalence in faecal samples collected. This is contrary to other studies in sheep where H. contortus has been the most prevalent helminth (Sharma et al., Reference Sharma, Agrawal, Mandal, Nigam and Bushan2009; Nuraddis et al., Reference Nuraddis, Mulugeta, Mihreteab and Sisay2014; Tariq & Lateef, Reference Tariq and Lateef2017). The low prevalence of H. contortus seems to be due to the long periods of drought in the study sites. These climatic conditions reduced the requirement for moisture to develop the free-living stages of this nematode (Swarnkar & Singh, Reference Swarnkar and Singh2020).
Eimeria ovinoidalis was recovered from faeces of bighorn sheep, although the percentage of faecal samples with this protozoa was low and was only recovered in winter. This was surprising since Eimeria spp. are prevalent in small ruminants (Chartier & Paraud, Reference Chartier and Paraud2012; Souza et al., Reference Souza, Cruz, Teixeira Neto, Albuquerque, Melo and Tapia2015) and this intestinal parasite is one of the most significant threats for sheep production (Carrau et al., Reference Carrau, Silva, Pérez, Failing, Mantínez-Carrasco, Macías, Taubert, Hermosilla and Ruiz2018). The low parasitism by Eimeria spp. appears to be due to the reduced population of DBS in the present study, as high population density contributes to the propagation of coccidia species (Sharma et al., Reference Sharma, Paul, Rout, Mandal, Bhusan, Sharma and Kushwah2017; Oliveira de Macedo et al., Reference Oliveira de Macedo, Bezerra-Santos, Lopez de Mendonça, Alves, Ramos and de Carvalho2020).
The percentage of positive faecal samples to Nematodirus spp. is presented in table 2. The highest percentage of DBS infested with this helminth was in spring (dry season) and the lowest in summer (beginning of the rainy season). These results are not in line with other studies where this gastrointestinal parasite is more abundant in the rainy season and decreases during the dry season (Dafur et al., Reference Dafur, Mbap and Dafur2021a). The low percentage of animals affected with this intestine nematode is consistent with observations in grazing sheep in native pastures (Sissay et al., Reference Sissay, Uggla and Waller2007; Papri et al., Reference Papri, Chatlod and Avasthe2017). As described in studies in Africa and Asia (Nuraddis et al., Reference Nuraddis, Mulugeta, Mihreteab and Sisay2014; Khalafalla et al., Reference Khalafalla, Elseify and Elbahy2011), Chabertia spp. was not a critical intestinal nematode in the present study (table 2). Also, in the current study, the percentage of faecal samples positive to Cooperia spp. infection was low, which agrees with various studies in sheep in Asia (Naem & Gorgani, Reference Naem and Gorgani2013; Raza et al., Reference Raza, Younas and Schlecht2014) and Africa (Amadi et al., Reference Amadi, Avoaja and Essien2012; Blackie, Reference Blackie2014). In the present study, all species of gastrointestinal parasites showed significant seasonal presence in faecal samples, indicating that climatic conditions played an important role in the dispersion and survival of helminths and coccidia.
Conclusions
These results reveal a seasonal variation of gastrointestinal egg parasites shedding in DBS with a marked increase of faecal eggs counts during the winter and autumn seasons, Strongyloides spp. being the most prevalent helminth throughout the year. Most faecal samples showed that animals had a low or mild helminths’ load, which indicates that DBS under natural conditions in this semi-arid zone of northern Mexico harboured low gastrointestinal parasite loads that do not pose immediate threats to the herd health and animal welfare. This knowledge will benefit the conservation management of this species subject to special protection and help design proper management activities.
Acknowledgements
The authors thank the owner of the San Juan ranch, Monclova, Mexico, for access to his property and logistic support to carry out this investigation.
Financial support
This research was supported by a grant from the Autonomous Agrarian University Antonio Narro (03001-2258).
Conflicts of interest
None.
Ethical standards
The Autonomous Agrarian University Antonio Narro Institutional Animal Care and Use Committee approved all actions connected with cows used for this study (protocol number 3001-2114).
Author's contribution
Data adquisicion: J.E. García, F.A. Rodriguez-Huerta. Study design and drafted the manuscript: M. Mellado, E.A. Lozano. Analysed the results: J.E. García, M. Mellado. Revised the manuscript and reviewed the pertinent literature: J. Encina. All authors read and approved the final version of the manuscript.
