Influence of Pathological Conditions Caused by Gastrointestinal Parasites Infection on Pregnant Ewe s Behavior

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1 Acta Scientiae Veterinariae, : RESEARCH ARTICLE Pub ISSN Influence of Pathological Conditions Caused by Gastrointestinal Parasites Infection on Pregnant Ewe s Behavior Angelica Risso 1, Julcemar Dias Kessler 1, Vanessa Souza Soriano 1, Maria Luisa Appendino Nunes 1, Gustavo Machado 2, Anaiara Langaro 1, Rafaella Rossetto 1, Tais Zuffo 1, Matheus Dallago 1, Patric Castro 1, Andréia Volpato 1, Rhayana Kharyna Grosskopf 1 & Aleksandro Schafer Da Silva 1 ABSTRACT Background: Infection by helminthes is a main problem affecting sheep, causing significant economic losses. Among these parasites that may affect sheep, Haemonchus contortus, a hematophagous parasite caused of anemia. The anemia due to parasitosis causes animal apathy, the main goal of most studies is to show how anemia affects animal performance, with little research on the relationship of animal behavior and helminthosis. Therefore, the objective of this study was to evaluate the effect of anemia caused by helminthes on the behavior of pregnant ewes before and after antihelminthic treatment. Material, Methods & Results: Pregnant ewes (n = 18) Lacaune breed from the same herd in Southern of Brazil were classified into three groups of six animals each according to their degree of anemia based on the Famacha method (F) and hematocrit counts (H) in addition to fecal examination on day 0 of the study. The groups were formed as follow: Group A (level 1-2 (F), (H) 30%), Group B (level 3 (F); (H) among 21-29%), and Group C (level 4-5 (F); (H) 20%). Famacha method, hematological analysis and fecal examination were repeated at the end of the experiment on day 18. Behavioral assessment was done concomitantly by two methods: direct temporal observation with real-time registration and continuous observation with conspicuous registration for each event performed by the animal. Animal behavior was observed during six periods (P1 to P6) six h a day (08:00-10:00 AM, 12:00-02:00 PM, and from 04:00-06:00 PM), being tree-point periods prior (P1, P2 and P3) and three after the antihelminthic treatment (P4, P5 and P6). The animals from the Group C had higher eggs per gram (EPG) counts in faces when compared to other groups. The animals from the Group B showed higher ratios of the variable lying ruminating (1.74) when compared to the Group A. Animals from the Group C showed higher ratio for the variable ruminating standing (1.38), lying idle (4.75) and standing idle (1.34), compared to females from the Group A. In conspicuous method, it was noticed that animals from the Group B showed lower ratio of the variable drinking water (0.87) when compared to the Group A. The Group C showed higher ratios of the variable eating (1.47), when compared to the Group A. Discussion: Behavioral changes mostly of the time may be the first indication of disease, since there is a direct link between behavior and animal health. A study in order to identify the effects of H. contortus experimental infection on lambs and observed that there is an effect on age, behavior, and growth rate, but the degree of parasite infection did not influence the pattern of animal behavior, different from our experimental study. In our study the anemic animals from the Group C where seen eating and drinking more frequently but they were also standing and lying idle for longer periods of time. These behaviors may be related to the need of blood replacement due to anemia faced after helminthic infection. In addition, studies describe that gastrointestinal parasitism altered animal grazing behavior with parasitized animals becoming more selective when avoiding contaminated pasture, taking smaller bites at reduced bite rates compared with non-infected animals. Based on our results, we were able to conclude that the degrees of anemia, and therefore the helminthosis, play important influence on the behavior of pregnant ewes. Keywords: anemia, ethology, Famacha, hematocrit. Received: 23 December 2014 Accepted: 24 May 2015 Published: 26 June Departamento de Zootecnia, Universidade do Estado de Santa Catarina (UDESC), Chapecó, SC, Brazil. 2 Laboratório de Epidemiologia Veterinária (EPILAB), Faculdade de Veterinária (FAVET), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil. CORRESPONDENCE: A.S. Da Silva [aleksandro_ss@yahoo.com.br - Tel. +55 (49) ]. Departamento de Zootecnia, Centro de Educação Superior do Oeste (UDESC), Prédio Zootecnia, Bairro Santo Antônio. CEP Chapecó, SC, Brazil. 1

2 INTRODUCTION Infection by helminthes is a main problem affecting sheep, causing significant economic losses, especially when associated with poor nutrition, bad management and inefficient antihelminthic treatments [11]. Among these parasites that may affect sheep, Haemonchus contortus stands out as the most prevalent and pathogenic one throughout the Brazilian territory. Because it is a hematophagous parasite, a single animal even with a moderate load of parasites may show anemia shortly after infection [2]. Furthermore, infected animals may present with hypoproteinemia, apathy, and darkened feces with lower stool consistency, submandibular edema (mumps), loss of appetite, weight loss due to lower food intake and, consequently, increased water intake, probably in order to compensate its loss of body weight [16]. Prophylaxis and proper management may be the most effective way to prevent all this damage caused by helminthes [11], in addition to the possibility to slow down parasitic resistance. Even though Vaz [16] showed that anemia due to parasitosis causes animal apathy, the main goal of most studies is to show how anemia affects animal performance, with little research on the relationship of animal behavior and helminthosis. Schichowski et al. [14] conducted a study in order to identify the effects of H. contortus experimental infection on lambs and observed that there is an effect on age, behavior, and growth rate, but the degree of parasite infection did not influence the pattern of animal behavior. In this sense, the objective of this study was to evaluate the effect of anemia due to helminthosis in the behavior of pregnant ewes before and after antihelminthic treatment. MATERIALS AND METHODS This study was conducted in a commercial dairy sheep farm located in Chapecó, Santa Catarina State, Southern Brazil. Pregnant ewes (n = 18) with approximately two months of pregnancy checked by ultrasound, Lacaune breed were classified into three categories of six animals each according to the Famacha method (color of ocular mucosa) [15], parasitological examination (feces), and blood analysis (hematology) as follow: Group A (level 1-2 (F), (H) 30%), Group B (level 3 (F); (H) between 21-29%), and Group C (level 4-5 (F); (H) 2 20%). All animals were housed with free access to water and feed in the same pen (7.9 x 3.1 m) on a well-closed barn to avoid external influences. Fecal samples were collected directly from the rectum on day 0 (the beginning of the experiment) and 18 (the end of the experiment) for egg counting (EPG). One coproculture was performed for each group to identify types of helminthes. The total erythrocyte count, hemoglobin, and total leukocyte count were performed at the beginning and at the end of the experiment using a semi-automatic counter (Celm 530 ) 1. Hematocrit was performed according to the methodology described by Feldman et al. [4]. For all analysis, the blood was collected from the jugular vein using vacutainer, and stored in tubes with anticoagulant (EDTA). Behavioral assessment was done simultaneous by two methods with the objective to evaluate the behavior pattern of each group, and occasionally, to identify the occurrence of specific behaviors. On both methods, behavior was observed during six periods (P1 to P6) six h per day (08:00-10:00 AM, 12:00-02:00 PM and 04:00-06:00 PM), being three periods before antihelminthic treatment (P1, P2, and P3) and three after the antihelminthic treatment (P4, P5, and P6). The animals were identified by different numbers sprayed with ink of different colors on the left side of the body, in order to easily identify individual behavior. For behavior evaluation of different groups at different periods, the focal method was used, with instantaneous recording every 15 min (Table 1) by two well-trained observers. The occurrence of any behavior such as drinking or eating was recorded after continuous observation. For this last method, all animals were observed continuously on all six periods, for only one person by conspicuous method as described by Broom and Fraser [1]. For conspicuous method, the registration of a behavior only was done if it lasted at least 30 s (drinking or eating). The antihelminthic treatment was performed on day 6 of the study. The antiparasitic used was levamisole (Ripercol ) 2 at a single dose of 12 mg kg -1, since it is a drug without side effects to pregnant ewes in the dosage recommended by the manufacturer. At the end of the experiment (day 18) fecal and blood examination along with the Famacha

3 method were performed to check animal recovery after treatment. Firstly, a descriptive analysis of the data was conducted in order to check for frequencies of the variables. The variables related to each group were considered independent from other groups and were analyzed descriptively. The following are the analyzed variables: EPG (eggs per gram of feces), hemogram (total erythrocyte count, hemoglobin, and total leucocytes). All variables were checked for normality by Shapiro-Wilk test and for homoscedasticity by Levene test, in the case of violation of the above assumptions required for ANOVA test, data were transformed into Log(x). We also used mixed model to verify differences between the beginning (day 0) and at the end of the experiment (day 18), in order to verify changes on measured variables due to treatment. A post hoc test Tukey HSD was used considering P < 0.05 as significant. For the behavioral study, a bivariate analysis (Pearson s chi-square test) for both methods of observation was performed. Firstly, for the evaluation based on instantaneous records, we have checked the association between dependent variable-polynomial taking into account that this variable has eight levels: eating (E), drinking (D), walking (W), lying ruminating (LR), standing ruminating (SR), Animal laying (AL), standing idle (SI) and other (OT) [Table 1]; and independent variables: date of the observation, hour of the observation, drug antihelminthic used, and the animal observed. For the conspicuous method, the occurrence of drinking and eating were recorded to verify the association between dependent variables (number of times the animal drank water and/or ingested food) with independent variables (date of the observation, hour of the observation, drug antihelminthic used and the animal observed). It was considered statistically significant when P < For the calculations of risk ratios, a confidence interval of 95% was considered. All analyses were done on R software, v (R Development Core Team). Thus, it was estimated the ratio of each independent variable on the occurrence of behavior alteration. The ratio for each alteration was estimated simultaneously, avoiding the use of multiple statistical tests producing proportional estimations among variables, which allowed direct comparison to the baseline variable. 3 RESULTS Regarding the results for mixed factorial analysis of variance between the beginning of the experiment (day 0) and the end point of the experiment (day 18), it was found neither statistical difference between nor within groups for all variables measured: EPG and blood count (hematocrit, erythrocyte, hemoglobin and total leukocytes). For the groups B and C, the hematocrit values, erythrocyte number, and hemoglobin concentration were lower than reference values for ewes [4], which characterizes anemia. Based on MCV values (mean corpuscular volume) and MCHC (mean corpuscular hemoglobin concentration) that may indicate the type of anemia, we were able to see that the Group B had normocytic and normochromic anemia, and animals from the Group C had macrocytic and normochromic anemia. The total leucocyte counts did not differ between groups, however those from the Groups A and B (day 0) had higher numbers than the reference values [4], which indicates a leukocytosis due to parasitism. Hematological variables and EPG results can be seen in Table 2. The behavior results from the conspicuous method are presented in Table 3. The animals from the Group B showed higher ratios of the variable lying ruminating (1.74) when compared to the Group A. Animals from the Group C showed higher ratio for the variable ruminating standing (1.38), lying idle (4.75) and standing idle (1.34), compared to females from the Group A. The behavior results from the conspicuous method are presented in Table 4. It was noticed that animals from the Group B showed lower ratio of the variable drinking water (0.87) when compared to the Group A. The Group C showed higher ratios of the variable eating (1.47), when compared to the Group A. The animals from the Group C had higher EPG counts when compared to other groups (P 0.05; Table 3). On the coproculture performed on day 0 it was found mainly H. contortus, however Trichostrongylus spp. was the most prevalent parasite at the end of the experiment (Figure 1).

4 Figure 1. Coproculture results of sheep in the beginning of the experiment (day 0) and after treatment at the end of the experiment (day 18). The animals were infected by Trichostrongylus spp. [clear bar] and Haemonchus spp. [dark bar]. Table 1. Ethogram for pregnant ewes in open shed. Behavior Behavior description Eating (E) Animal eating from feeders Drinking (D) Animal drinking water from drinkers Walking (W) Animal moving around the bay without displaying rumination Lying rumination (LR) Animal with chewing movements rumination for at least 30 s; at laying bases Standing rumination (SR) Animal standing with chewing movements of rumination for at least 30 s Animal laying (AL) Animal laying without rumination and without walking Standing idle (SI) Animal standing without rumination and without walking Other (OT) Animals showing negative social behavior (halter directed to other animals); animal licking or biting objects or the bay DISCUSSION In the present study, we found that anemia caused by parasitism of helminthes altered the behavior patterns of pregnant ewes. Behavioral changes mostly of the time may be the first indication of disease, since there is a direct link between behavior and animal health [1]. The main behaviors that are significantly affected when animals have pathologies are the reduction on physical activities and appetite [1,7]. Increase on some cytokines of sick animals may lead to loss of appetite, which at the beginning saves animal energy, since there is no activity and energy spend in the digestive tract [1]. However, in our study the anemic animals from the Group C where seen eating and drinking more frequently but they were also standing and lying idle 4 for longer periods of time. These behaviors may be related to the need of blood replacement due to anemia faced after helminthic infection. The level of welfare of any sick animal is reduced when compared to a physiological healthy animal. In sheep, the use of behavior patterns to identify stress is a common practice [3,6]. However, very little is known about the effect of gastrointestinal disease on the degree of animal welfare. The behavior is one of the indicators of animal welfare, which means that the prediction of this variable alone is almost impossible. In this study, we noticed that anemic animals susceptible to the environmental changes, tried to overcome the disease by spending more time in idleness avoiding extra energy expenditure, and ingesting food and water

5 Table 2. Hematological variables [hematocrit (%), total erythrocytes (x10 6 µl), hemoglobin concentration (mg/dl), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC - %), total leucocytes (x10 3 µl)], and number of eggs per gram of feces (EPG) in pregnant ewes before and after antihelminthic treatment. Variable Treatment Group A Group B Group C P-value Hematocrit Erythrocyte Hemoglobin MCV MCHC Leucocyte Before 32.17±2.56 a 25.33±1.0 b 18.50±1.67 c <0.01 After 32.5±3.17 a 28.33±1.67 b 20.17±4.22 c <0.01 Before 8.77±0.4 a 7.28±0.67 b 4.51±0.97 c <0.01 After 8.71±0.73 a 7.37±0.47 b 4.58±1.37 c <0.01 Before 10.28±0.68 a 8.57±0.44 b 5.97±0.61 c <0.01 After 10.52±1.15 a 8.82±0.75 b 6.17±1.36 c <0.01 Before 36.6±2.4 a 34.8±2.1 a 41.0±2.2 b <0.05 After 37.3±3.7 a 38.4±1.8 a 43.9±2.4 b <0.01 Before 31.9±2.3 a 33.8±2.7 a 32.2±1.7 a >0.05 After 32.3±1.6 a 31.1±2.2 a 30.5±1.9 a >0.05 Before 14.55±6.27 a 12.65±3.95 a 10.43±1.41 a >0.05 After 10.57±2.56 a 13.72±3.98 a 11.25±3.22 a >0.05 Before ± a 500±536 a 9920±5944 b <0.05 EPG After ± a ± a ±1190 a >0.05 Note: Same letters superscript within the same line means no statistical difference among groups, at 5% significance. Total leukocyte and EPG data had no normal distribution, so it was transformed by logarithm transformation before analysis of variance. Table 3. Results from multinomial regression for punctual behavior analysis 1,2. Behavior Group A* Group B P-value Group C P-value Eating** Drinking ( ) ( ) 0.83 Walking ( ) ( ) 0.72 Lying rumination ( ) < ( ) 0.65 Standing rumination ( ) ( ) 0.02 Lying idle ( ) ( ) 0.01 Standing idle ( ) ( ) 0.04 Other ( ) ( ) 0.52 *Reference group. **Significance level. 1 This model was nested by day, time of the observation and animal analyzed for variance within variables. 2 The variables animal group formation inside each experimental group, and the quadrant were the animal was by the time of observation were not analyzed due to low variability. Table 4. Results from multinomial regression for conspicuous behavior analysis 1. Variable Group A* Group B P-value Group C P-value Drinking water ( ) ( ) 0.11 Eating ( ) ( ) *Reference group. 1 This model was nested by day, time of the observation and animal analyzed for variance within variables. 5

6 on larger amounts to recover their body condition. Although sheep rarely show combat behavior, sometimes it is possible to observe aggressive behavior especially when competing for food and bedding areas [3,9]. In this experiment, aggressive and abnormal behavior where counted as others (OT), since it occurred with low frequency and did not differ between other experiment groups. Behavioral studies allow us to identify animals that may be suspect of being infected by helminthes, but it cannot be used isolated, being necessary the association with other tests, like Famacha method and fecal analysis. In the farm, even under long distances, it is possible the identification of isolated animals, which may indicate abnormal behavior and possibly disease, since sheep naturally, shows gregarious behavior [5]. Although these changes on behavior were not identified on stabled animals, this study proves that there are many other possible behavioral changes that can assist farmers to identify diseased animals. According to the literature, gastrointestinal parasitism altered animal grazing behavior with parasitized animals becoming more selective when avoiding contaminated pasture, taking smaller bites at reduced bite rates compared with non-infected animals [8]. The EPG is a very important test for herd monitoring and it is often used to verify the effectiveness of treatments associated with larval culture [13]. According to researchers, when an antihelminthic is capable to reduce EPG counts in more than 90% it can be considered efficient and can be selected for treatment of herd, but where the reduction is loss than 80%, this product is considered inefficient [12], as shown in the present study after treatment with levamisole. The larval culture showed that the resistance was linked to Trichostrongylus spp., since at day 18 we found that more than 95% of the eggs were from this parasite. These results are in accordance to Klauck et al. [10], which evaluated sheep herds in Santa Catarina State. Reinforcing that treatment was ineffective since we recorded the hematological parameters that characterize anemia on day 0 and 18, and no statistical differences were found. Briefly, pregnant ewes naturally infected by gastrointestinal parasites showed behavioral changes related to pathological pattern (anemia). These changes were more severe when the level of anemia was high. It must be pointed out that ewes with anemia showed the behavior of lying and standing idle more frequently, as already shown in the literature in cases of anemia. On the other hand, these animals were seen more often drinking and eating them healthy animals, presumably in an attempt to recover the clinical disease. MANUFACTURERS 1 Equipadora de Laboratórios Modernos. São Caetano do Sul, SP, Brazil. 2 Fort Dodge Saúde Animal Ldta. Campinas, SP, Brazil. Ethical approval. The experiment (protocol number ) was approved by the ethics committee on animal research at the Universidade do Estado de Santa Catarina (UDESC). Declaration of interest. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. REFERENCES 1 Broom D.M. & Frasier A.F Comportamento e bem-estar de animais domésticos. 4th edn. São Paulo: Manole, 452p. 2 De Souza G.A.F Avaliação do método Famacha como estratégia auxiliar no controle de helmintoses gastrintestinais de ovinos no semiárido da Paraíba, Brasil. 57f. Patos, PB. Dissertação (Mestrado em Zootecnia) - Programa de Pós-graduação em Zootecnia, Universidade Federal de Campina Grande. 3 Ewbank R Behavioral responses to stress in farm animals. In: Moberg G.P. (Ed.). Animal Stress. Bethesda: American Physiological Society, pp Feldman B.G., Zinkl J.G. & Jain N.C Schalm s Veterinary Hematology. 5th edn. Philadelphia: Lippincott Williams & Wilkins, 1344p. 5 Fernandes M.A.M., Barros C., De Paula E.F.E. & De Souza D.F Aprendendo a conhecer caprinos e ovinos. Disponível em: < -ovinos-parte-i-73854n.aspx>. [Acessado em 02/06/ 2014]. 6 Gougoulis D.A., Kyriazakis I. & Fthenakis G.C Diagnostic significance of behaviour changes of sheep: A selected review. Small Ruminant Research. 92(1):

7 7 Gregory N.G Physiological mechanisms causing sickness behavior and suffering in diseased animals. Animal Welfare. 7(1): Hutchings M.R., Kyriazakis I., Anderson D.H., Gordon I.J. & Coop R.L Behavioural strategies used by parasitized and non-parasitized sheep to avoid ingestion of gastro-intestinal nematodes associated with feces. Animal Science. 67(1): Keeling L.J. & Gonyou H.W Social behavior in farm animals. New York: CABI Publishing, 403p. 10 Klauck V., Pazinato R., Lopes L.S., Cucco D.C., De Lima H.L., Volpato A., Randavelli W.M., Stefani L.C.M. & Da Silva A.S Trichostrongylus and Haemonchus anthelmintic resistance in naturally infected sheep from southern Brazil. Anais da Academia Braseira de Ciências. 86(4): Lagares A.F.B.F Parasitoses de pequenos ruminantes na região da Cova da Beira. 115f. Lisboa, Portugal. Dissertação (Mestrado em Medicina Veterinária) - Programa de Pós-graduação em Medicina Veterinária, Universidade Técnica de Lisboa. 12 Niciura S.C.M., Veríssimo C.J. & Molento M.B TRCOF: teste de redução da contagem de ovos nas fezes. Disponível em: < [Acessado em 16/05/2014]. 13 Salgado J.A. & Moraes F.R Coprocultura: um exame importante no controle da verminose. Disponível em: < [Acessado em: 09/05/2014]. 14 Schichowski C., Moors E. & Gauly M Influence of weaning age and an experimental Haemonchus contortus infection on behaviour and growth rates of lambs. Applied Animal Behaviour Science. 125(1): Sotomaior C.S., Rosalinski-Moraes F., da Costa A.R., Maia D., Monteiro A.L. & van Wyk J.A Sensitivity and specificity of the FAMACHA( ) system in Suffolk sheep and crossbred Boer goats. Veterinary Parasitology. 190(1): Vaz C.M.S.L., Kalil R.G. & Selaive-Villarroel A Sistemas de criação de ovinos nos ambientes ecológicos do Sul e do Rio Grande Do Sul. Disponível em: < CriacaoOvinosAmbientesEcologicosSulRioGrandeSul/manejo.htm>. [Acessado em: 02/06/2014]