Relative resistance of Menz and Washera sheep breeds to artificial infection with Haemonchus contortus in the highlands of Ethiopia

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1 Relative resistance of and sheep breeds to artificial infection with Haemonchus contortus in the highlands of Ethiopia Tesfaye Getachew, Biruk Alemu, Johann Sölkner, Solomon Gizaw, Aynalem Haile, Shenkute Gosheme & David Russell Notter Tropical Animal Health and Production ISSN Volume 47 Number 5 Trop Anim Health Prod (2015) 47: DOI /s

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3 Trop Anim Health Prod (2015) 47: DOI /s REGULAR ARTICLES Relative resistance of and sheep breeds to artificial infection with Haemonchus contortus in the highlands of Ethiopia Tesfaye Getachew 1,2 & Biruk Alemu 2 & Johann Sölkner 1 & Solomon Gizaw 3 & Aynalem Haile 4 & Shenkute Gosheme 2 & David Russell Notter 5 Received: 8 August 2014 /Accepted: 31 March 2015 /Published online: 14 April 2015 # Springer Science+Business Media Dordrecht 2015 Abstract The aim of this study was to evaluate the relative resistance of and sheep breeds to artificial infection with Haemonchus contortus. The challenge trial was conducted at the Debre Berhan Agricultural Research Center in Ethiopia. A total of 39 (=21, =18) lambs of about 6 months old were used for the trial. All lambs were initially treated against internal parasite using albendazole and Fasinex to free them from internal parasites and kept indoors. H. contortus third-stage larvae (L3) were prepared according to standard procedure from adult female parasite collected from abattoirs and recovered using the Baerman technique. Approximately 5000 infective larvae were inoculated to the experimental lambs at about 5 weeks after deworming. Fecal egg count (FEC), packed cell volume (PCV), FAffa MAlan CHArt (FAMACHA) score, lamb body weight, and survival of lambs were recorded at 28, 35, and 42 days after challenge. Breed effect was not significant (P>0.05), whereas time of measurement and the interaction of breed and time had significant (P<0.05) effects on FEC and PCV. At 28 days after * Tesfaye Getachew tesfayegecho@yahoo.com Department of Sustainable Agricultural Systems, Division of Livestock Sciences, University of Natural Resources and Life Sciences, Gregor Mendel Strasse 33, A-1180 Vienna, Austria Debre Berhan Agricultural Research Center, P.O.Box 112, Debre Berhan, Ethiopia International Livestock Research Institute (ILRI), Addis Ababa, Ethiopia International Center for Agricultural Research in the Dry Areas (ICARDA), Addis Ababa, Ethiopia Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, USA challenge, sheep had lower FEC than sheep, whereas at days 35 and 42, the sheep had lower FEC than lambs. The PCV decreased significantly throughout the post-challenge period. Despite their parasite burden, sheep were able to maintain live weight during the 42 days of challenge. Chi-square tests for breed differences in lamb survival were not significant at any time (28, 35, and 42 days after challenge). Lower FEC at 28 days and delayed rise in FEC after infection in lamb would allow in delaying anthelmintic treatment. In addition to maintain body weights during the infection period, sheep of both breeds exhibited substantial variability in PCV and FEC, suggesting opportunity to consider parasite resistance in selection program. Keywords Challenge trial. FAMACHA. Fecal egg count. Packed cell volume. Repeated measures Introduction Gastrointestinal nematodes, mainly the bloodsucking parasite Haemonchus contortus, can cause severe anemia, resulting in reduced feed intake, growth rate, weight loss, and even death in pasture-based sheep and goat production (Notter et al. 2003; Troell et al. 2005; Bambou et al. 2013). Anthelmintic treatment is frequently used as control of gastrointestinal nematodes. However, inadequate reduction in fecal egg count (FEC) after anthelmintic treatment has been reported (Coles 2003; Kamaraj et al. 2011; Falzon et al. 2013; Zanzani et al., 2014) as a result of frequent and incorrect use of chemotherapeutics like insufficient drug, incorrect administration, and wrong calibration of drenching gun. The development of drug resistance along with residual effect of the drugs rising cost of anthelmintic pushes to search alternative strategies. Developing good grazing and deworming management

4 962 Trop Anim Health Prod (2015) 47: practices and using breeds having better resistance to parasite were suggested by many researchers (Baker et al. 2003; Gaba et al. 2012). The ability of sheep to resist helminth parasites varies substantially among and within breeds and is controlled by several genes (Amarante et al. 2005; Periasamy et al. 2014). Resistance to parasite is a heritable trait, and this offers the opportunity to include such traits in selective breeding programs. Tropical sheep breeds are the result of many generations of human and natural selection predominantly for survival under the prevailing feed scarcity, disease challenges, and low level of management rather than for high levels of production. Thus, their better resistance to withstand parasite infections might have been developed as these flocks coevolved with internal parasite infection. Better resistance of tropical sheep breeds to internal parasites is well documented. For instance, resistance to gastrointestinal parasite is well documented in Kenyan Small East African goat (Baker et al. 1998), US hair sheep (Burke and Miller 2002), Kenyan Red Maasai sheep (Baker et al. 2003), Ethiopian sheep (Haile et al. 2002), Pakistan Lohi sheep (Saddiqi et al. 2010), and Spanish Canaria hair sheep (González et al. 2011). Although breed variation has been documented in parasite resistance, little is known of the genetic basis. The rapid growing knowledge on gene and genomics of livestock can be used to characterize genes associated with parasite resistance. Assessing the relative resistance of breeds using challenge trial should be considered as a way towards mapping of genes controlling internal parasite. and are common Ethiopian sheep breeds. sheep produce meat and coarse wool, are adapted to the cool Ethiopian highlands, and are tolerant of drought, seasonal variation in feed availability, and endo-parasite infection (Haile et al. 2002; Gizaw et al. 2008). sheep are found in the northwestern highlands of Ethiopia. The breed is relatively large, moderately prolific, and generally well adapted to the prevailing conditions in the region (Gizaw et al. 2008; Taye et al. 2010). Its resistance to internal parasite has not been studied. The aim of this paper is therefore to compare and breeds for their relative ability to resist Haemonchus infection. Materials and methods Study area The challenge trial was conducted at the Debre Berhan Agricultural Research Center (DBARC) in Ethiopia from December 2011 to January The research center is located at latitude 9 36 N, longitude E, and altitude of 2780 m and has a long rainy season between June and September and a short rainy season between February and April. Based on the meteorological data collected at DBARC from 2000 to 2013, the annual rainfall of the area is about 1029 mm. The average monthly min temp is 6.7 C and ranged from 3.9 C in November to 8.9 C in August, while the average monthly max temp is 20 C and ranged from 18.2 C in August to 21.9 C in June. The mean relative humidity is 60 %. H. contortus is among the gastrointestinal parasite in the area (Tembely 1998). During the experimental period (December 2011 to January 2012), the minimum average temperature, maximum average temperature, and total amount of rainfall were 4.4 C, 18.7 C, and 5.1 mm, respectively. Animal management (n=21) and (n=18) lambs were used in a parasite challenge trial, and the number of females was nine in both breeds. Experimental lambs were bought from farmers in their respective areas, brought to the research center, and managed similarly for about 30 days to acclimatize the station environment. Lambs were collected from a total of 15 and 11 farmers for and sheep, respectively. Lambs were approximately 6 months old at the start of the study. Before the beginning of the challenge trial, all lambs were treated with albendazole and Fasinex to remove internal parasites. The efficacy of the anthelmintics was checked at 10 days after deworming. During the experimental period, all lambs were penned on a cement floor and provided with hay and commercial concentrate feed to prevent re-infection. The concentrate feed had 21 % crude protein and 14 MJ/kg metabolizable energy on dry matter basis. Larvae preparation and inoculation Adult H. contortus parasite was collected from a nearby abattoir. Female worms were separated from males by grossly witnessing the blood-filled intestine spirally coiled around white ovary giving an appearance of barber s pole worm. The female worms were washed and crushed using mortar and pestle to liberate eggs. Filtration was made to avoid debris, and eggs were collected by sedimenting them using slow centrifugation at 100 rpm for 2 min. A culture of third-stage larvae (L3) was prepared and recovered using the Baerman technique (Hansen and Perry 1994). The number of infective L3 in one drop of aliquot was determined using a microscope; 1 ml was equivalent to 20 drops of aliquot. The amount of aliquot needed to provide 5000 infective larvae was then calculated and inoculated into the experimental lambs at about 5 weeks after deworming. Data collection Fecal egg counts (FEC), packed cell volumes (PCV), FAffa MAlan CHArt (FAMACHA) scores, and live weights were recorded at 0, 28, 35, and 42 days after administration of L3.

5 Trop Anim Health Prod (2015) 47: Fecal samples were collected from the rectum and used to estimate the eggs per gram (EPG) of feces using the modified McMaster method with saturated sodium chloride as the flotation fluid. A sensitivity of 100 eggs per gram of feces was used. Blood samples were taken from the jugular vein on the days of fecal sampling to measure PCV. FAMACHA eye scores ranged from 1 to 5, with 1 considered normal (red color) and 5 highly anemic (pale color). The FAMACHA system can be used to select replacement animals that are resistant and resilient to H. contortus (Burke and Miller 2008). Riley and Van Wyk (2009) also suggested using FAMACHA score as an alternative to the analysis of hematocrit and fecal worm egg counts. Data analysis Changes in FEC, PCV, and body weight were analyzed with repeated measures using the PROC MIXED of SAS 9.2 (2009). The final model included fixed effects of breed, measurement time (the repeated factor), and all two-way interaction and random effect of animal. For the body weight analysis, initial weight was fitted as covariate. The effect of sex was considered in a preliminary analysis but was removed from the final model because sex effects and the interaction of sex with breed and time were not significant. Different covariance structures for the repeated effect were compared based on Akaike s information criteria (AIC), the corrected AIC, and the Bayesian information criteria (BIC). In all cases, an unstructured covariance structure was superior and was chosen for the final model. Each variance and covariance among the repeated observations was therefore estimated uniquely from the data and used to derive correlations among the repeated observations. PROC UNIVARIATE in SAS (SAS 2009) was used to evaluate the distribution of FEC and to detect outliers. Each observation was expressed as a residual deviation from the model and was therefore corrected for breed effects within each measurement time. Those residual values were used to evaluate the distribution of FEC and detect outliers. The FEC were not normally distributed and were therefore transformed as lnfec=ln(fec+100) in order to normalize the data. Resulting least squares means (LSM) were back transformed for presentation as EXP(LSM)-100. Standard errors of backtransformed means were estimated from standard errors of LSM (standard errors (SE)) by multiplying the observed SE by the back-transformed mean. Back-transformation of SE is based on the assumption that the standard error following transformation is approximately equal to the coefficient of variation of the back-transformed LSM. Furthermore, two extreme values were detected based on residual deviations of transformed FEC and were removed from the analysis. The distribution of FAMACHA score was summarized and visualized within and across breeds at each measurement time using stacked charts from Microsoft EXCEL. PROC FREQ of SAS 9.2 (SAS 2009) was used to analyze FAMACHA data to detect associations with breeds and measurement times. The standard chi-square test is not appropriate if more than 20 % of the cells have fewer than five observations, which was often the case in this experiment. Thus, Fisher s exact chi-square statistic was used for testing and was produced using the EXACT option in PROC FREQ in order to get the exact probability value when some cells have fewer than five expected observations. PROC MIXED of SAS was used to generate least squares means for FAMACHA scores. Lamb survival during the trial was analyzed using PROC GENMOD in SAS 9.2 (2009) assuming a binomial distribution and logit link function. Those lambs which showed higher risk of death were treated and excluded from the trial to save their life. Lamb records were coded as 1 for lambs that survived and 0 for lambs that did not survive (excluded from the experiment). Results and discussions Live weight and survival Least squares means for live weight at 0, 28, 35, and 42 days after challenge (Fig. 1) revealed a significant (P<0.05) breed effect on live weight, but effects of measurement time and measurement time breed interaction were not significant. lambs were heavier (P<0.05) than sheep at the beginning of the experiment and maintain that superiority throughout the experiment. sheep maintained their live weight during the 42 days of parasite challenge, whereas sheep lost about 1 kg during the parasite challenge given that they were on the same plain of nutrition and managed under similar indoor environment. lambs of similar ages and weights maintained on the station registered no loss or gain of weight over this period, which suggested that Body weight (kg) Days a er challenging Fig. 1 Least squares means of body weight of lambs during the infection period

6 964 Trop Anim Health Prod (2015) 47: Table 1 Survival of and sheep at 28, 35, and 42 days after infection with H. contortus Breed Number of lambs Survival (%) the parasite challenge did not affect the growth of lambs. Lamb survival during the challenging period is presented in Table 1. Chi-square tests for survival found no significant breed effect at any measurement time in all durations (28, 35, and 42 days after challenging). However, better survival was observed for sheep at 28 days and for sheep at 42 days. Lambs that survived to the end of the experiment had final mean±standard deviation (sd) PCVof 20.0±5.4 %. For lambs that did not survive, PCV at the measurement time immediately before death were very low, with a mean±sd (range) of 9.9±1.4 % (8 to 12 %). This result suggests that a PCV threshold of 12 % might be used to identify lambs with high risk of mortality. These lambs should be treated and removed from the experiment to reduce their risk of death. Fecal egg count and packed cell volume 28 days 35 days 42 days (0.54) 60.1 (0.44) 60.5 (0.45) (0.78) 61.4 (0.49) 55.8 (0.49) Values in parenthesis are standard errors Means, standard deviations, coefficients of variation, and minimum and maximum values of FEC and PCV at each measurement time are presented in Table 2. Immediately before infection, the mean±sd (range) PCV was 31.9±2.71 % (26.5 to 36.0) for sheep and 31.9±2.64 % (26.5 to 38.0) for sheep. PCV value decreased markedly through day 42. There was substantial variation within breeds in FEC and PCV as indicated by the observed standard deviations and ranges (Table 2). In both the and breeds, some lambs were able to maintain acceptable PCV and relatively low FEC throughout the experiment. sheep exhibited less variation in FEC at 28 days but considerably greater variation in FEC at 35 and 42 days. The presence of substantial within-breed variation in both FEC and PCV indicates potential for genetic improvement of parasite resistance in these two local sheep breeds. Back-transformed least squares means for FEC and PCVof and sheep during the challenge trial are presented in Table 3. FECs were zero for all lambs at the beginning of the trail, so the anthelmintic treatment was effective. Effects of measurement time and the measurement time breed interaction were significant for the log-transformed FEC (lnfec), but the overall breed effect was not significant (P>0.05). Lamb risk of death due to infection during the study was substantial, with less than 60 % of the lambs surviving to 42 days. For this reason, least squares means for backtransformed FEC at different times after infection are presented in Fig. 2a for all lambs entering the study and in Fig. 2b for only those animals that survived (n=13 for and n=10 for ) to the end of the experiment. Excluding lambs that did not survive from the analysis (Fig. 2b) highly reduced LSM for FEC for sheep (46 %) at 28 days than for sheep (34 %), which was consistent with very high FEC at this time in lambs that did not survive until the end Table 2 Means, standard deviations, and minimum and maximum values for packed cell volume (PCV) and fecal egg count (FEC) during the challenge trial Variables Mean Standard deviation CV % Minimum Maximum FEC FEC , FEC , FEC , , PCV PCV PCV PCV FEC FEC28 13, , , FEC FEC PCV PCV PCV PCV PCVt and FECt packed cell volume and fecal egg count at t days after infection, respectively

7 Trop Anim Health Prod (2015) 47: Table 3 Least squares means (LSM) and standard errors (SE) of backtransformed fecal egg count (FEC) and packed cell volume (PCV) before and after challenge with H. contortus Breed and time FEC PCV LSM SE LSM SE Breed NS NS Time * * a a b b c c d 1.3 Breed*time * * _ _ _ _ _ _ _ _ Means for measurement times within a column with different letters differ (P<0.05) NS non-significant *Significant at P<0.05 of the study. The breed time interaction, however, was significant (P<0.05) after removal of lambs that did not survive and was therefore not a result of early death losses in lambs with very high parasite loads. When compared to sheep, sheep thus had higher FEC at 28 days after challenge (P<0.05) but lower FEC at 35 and 42 days (P<0.05). This result indicated that sheep were initially more susceptible but were also capable of reducing FEC later in the study. By contrast, FEC for sheep continued to increase throughout the study. Differences among animals in FEC were generally consistent across measurement times; correlations among lnfec at 28 and 35, 28 and 42, and 35 and 42 days were 0.55, 0.78, and 0.81, respectively, confirming the reliability of the result. Similar to this study, interaction of time and breed and the absence of breed effect were observed for Dorper sheep breed when compared with other hair sheep breeds in the USA (Burke and Miller 2002). Previously, resistant breeds have been reported to require few or no anthelmintic treatments (Matika et al. 2003). Lower FEC at 28 days in sheep and the delayed raise in FEC observed in this study could help to decrease the frequency of anthelmintic treatment in this breed. High values of FEC in sheep at 28 days showed its susceptibility at the beginning, and dramatic reduction in subsequent time might be associated with other factors like reduction of the fecundity of the worm as their size could be affected due to higher worm burden (Rowe et al. 2008). Relative better resistance of sheep compared to their crosses with exotic Awassi sheep (Tibbo 2006) and to another indigenous sheep breed called Horro (Haile et al., 2002; Rege et al., 2002) in maintaining their PCV and controlling fecal EPG and worm burden. The relative reduction of FEC at the later days of treatment in sheep is also interesting. In contrast to this study, clear variation among breed in FEC was also observed in many other breeds (Gauly et al. 2002; Vanimisetti et al. 2004a; González et al. 2008, 2011; Saddiqi et al. 2010). There was no difference between breeds (P>0.05) in PCV, but the effects of measurement time and measurement time breed interaction were significant (P<0.05) (Fig. 3). The initial PCV was 31.9 % for both breeds, decreased throughout the infection period, and was below 18 % by 42 days after infection. The decreases in PCV during the infection period were consistent with the high values observed for FEC. Similar patterns of PCV reduction were reported by Vanimisetti et al. (2004a), Saddiqi et al. (2010), Bambou et al. (2011), and Babar et al. (2013) in different sheep breeds. The rate of decline in PCV between 28 and 42 days was slightly less for the sheep, despite their more rapid increases in FEC after 28 days and may further indicate relative resistance and resilience to the parasite in this breed. Differences in PCV Fig. 2 Least squares means of back-transformed fecal egg count at different times after infection considering all observation (a) and only those animals that survived to the end of experiment (b) Back transformed fecal egg count (g) Days a er infec on a Back transformed fecal egg count (g) Days a er infec on b

8 966 Trop Anim Health Prod (2015) 47: % 30 80% PCV (%) among animals were very consistent during the challenge trial, with correlations between PCV at 28 and 35, 28 and 42, and 35 and 42 days of 0.95, 0.94, and 0.96, respectively. By contrast, correlations of PCV at day 0 with subsequent values for PCV during infection were low, ranging from 0.07 to FAMACHA score day Days a er infec on Fig. 3 Least squares means of packed cell volume at different times after infection Frequencies of FAMACHA scores by breed within each measurement time and overall frequencies for the two breeds across times are presented in Figs. 4 and 5, respectively. Fisher s exact chi-square test revealed that there was no breed effect on FAMACHA score at any measurement time (P>0.05). Least squares means of FAMACHA score of Frequency 60% 40% 20% 0% Days a er challenge (days) FAMACHA 1 FAMACHA 2 FAMACHA 3 FAMACHA 4 FAMACHA 5 Fig. 5 Frequencies of FAMACHA scores (%) at each measurement time and lambs from the mixed-model analysis (Fig. 6) documented a significant effect (P<0.01) of measurement time on FAMACHA score and confirmed that the breed effect and the measurement time breed interaction were not significant (P>0.05). The increase in FAMACHA score after infection observed in both breeds generally corresponds to the increase FEC and decrease PCV due to infection in this study. Thus, it might be used as a tool to identify animals for treatment. Burke and miller (2008) andburkeetal.(2007) suggested using FAMACHA system to identify superior sires for parasite resilience/resistance and to identify anemic sheep. However, the result did not correspond to the breed variation across time observed for FEC and PCV in this study. The very large breed measurement time interaction observed in this study indicates a substantial difference between and sheep in their response to parasite Fig. 4 Frequencies of FAMA CHA scores (%) by breed immediately before challenge (a) and at 28 days (b), 35 days (c), and 42 days (d) after challenge Frequency 100% 80% 60% 40% 20% a Frequency 100% 80% 60% 40% 20% b 0% FAMACHA 1 FAMACHA 2 0% FAMACHA 3 FAMACHA 4 FAMACHA 5 100% 80% c 100% 80% d Frequency 60% 40% 20% Frequency 60% 40% 20% 0% 0% FAMACHA 2 FAMACHA 3 FAMACHA 4 FAMACHA 5 FAMACHA 4 FAMACHA 5

9 Trop Anim Health Prod (2015) 47: FAMACHA score Days a er infec on Fig. 6 Least squares means of FAMACHA scores for and lambs during the infection period infection. The lambs were initially highly susceptible, with rapid increases in FEC to high levels by 28 days after infection. However, lambs subsequently expressed reduced FEC levels, suggesting mobilization of immune effectors to limit long-term effects of infection. In contrast, lambs exhibited only modest increases in FEC to 28 days, suggesting higher initial resistance to infection. However, FEC continued to increase in this breed, in marked contrast to the pattern of change in FEC observed in lambs. The PCVs declined rapidly to 28 days and continued to decline, although at a less rapid rate, to 42 days post-challenge. Declines in PCV were similar between the two breeds. The lambs thus did not have lower PCV at 28 days or exhibit improvements, or less rapid declines, in PCV after 28 days, despite the observed reductions in FEC. This result suggests that the observed reductions in FEC in lambs after 28 days were not associated with reduced symptoms of parasitism, expressed either as PCV, FAMACHA scores, or mortality levels. The lambs may not have had sufficient time to recover by 42 days from initial high levels of infection observed at 28 days, or the lambs may have been more resilient to parasitism, maintaining comparable levels of PCV despite higher worm burdens. Vanimisetti et al. (2004b) reported that Dorper crossbred lambs often had higher FEC but did not differ in PCV when compared to Dorset crossbred and Katahdin lambs. When challenged in an artificial infection with approximately 5000 infective L3 of H. contortus, sheep had lower FEC at 28 days after infection but lambs had lower FEC at 35 and 42 days after infection. Lower FEC at 28 days, delayed rise in FEC after infection, and maintenance of body weights during the infection period in lambs suggest a somewhat higher level of resistance to H. contortus in this breed. lambs also showed more resilient to parasitism. Substantial variation in FEC and PCV was observed in both breeds, and correlations among repeated measures of FEC and PCV during the experimental period were high. These results indicate opportunity to consider parasite resistance and or resilience in selection program. This information is helpful for the future work using genomic information to identify genes responsible for parasite resistance. Acknowledgments This study is part of the joint FAO/IAEA Coordinated Research Project D supported by the Division of Nuclear Techniques in Food and Agriculture, International Atomic Energy Agency, Vienna, Austria. We gratefully acknowledge Debre Berhan Agricultural Research Center for the laboratory and barn space and the overall facilitation of the study. We are also grateful for the livestock research staff of Debre Berhan Agricultural Research Center involved in the project, particularly Dr. Hayat Seid and laboratory technician Tsege Mariam for their devoted work in sampling and laboratory procedures. Conflict of interest interests. References The authors declare that they have no conflicting Amarante, A.F.T., Bricarello, P.A., Huntley, J.F., Mazzolin, L.P., Gomes, J.C., Relationship of abomasal histology and parasite-specific immunoglobulin A with the resistance to Haemonchus contortus infection in three breeds of sheep, Veterinary Parasitology, 128, Babar, M.E., Hussain, T., Abdullah, M., Ali, A., Nadeem, A., Kamran, Z., Ali, M.M., Evolution of genetic resistance to Haeminchus contortus infection in Pakistani sheep breeds. 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