Immune and haematological parameters of Blackbelly ewes infected with gastrointestinal nematodes

Rev Colomb Cienc Pecu 2017; 30:219-230 Revista Colombiana de Ciencias Pecuarias 219 Immune and haematological parameters of Blackbelly ewes infected with gastrointestinal nematodes¤ Parámetros inmunológicos y hematológicos en ovejas Blackbelly infectadas con nematodos gastrointestinales Parâmetros imunes e hematológicos de ovelhas Blackbelly infectadas por nematoides gastrintestinais Roberto González Garduño1*, IAZ, Dr; María E López Arellano2, MVZ, PhD; Magnolia M Conde Felipe3, MV, PhD; Pedro Mendoza de Gives2, MVZ, PhD; Liliana Aguilar Marcelino2, IAZ, Dr; Gabriela Jaso Díaz4, MVZ, MC. 1Unidad Regional Universitaria Sursureste, Universidad Autónoma Chapingo, Teapa, Tabasco, México. 2Centro Nacional de Investigación Disciplinaria en Parasitología Veterinaria, INIFAP, Jiutepec, Morelos, México. 3Facultad de Veterinaria. Universidad Las Palmas de Gran Canaria, Las Palmas, España. 4Colegio de Postgraduados-Campus Montecillo, Montecillo, Estado de México, México. (Received: May 10, 2016; accepted: December 6, 2016) doi: 10.17533/udea.rccp.v30n3a05 Abstract Background: It is necessary to identify phenotypic traits related to natural resistance against gastrointestinal nematodes (GIN) in order to know the host immunity status in productive ewes. Objetive: To determine haematological and immunological parameters (IgA and IgG) during pregnancy and lactation in Blackbelly ewes naturally infected with GIN. Methods: The number of eggs per gram (EPG), packed cell volume (% PCV), plasmatic protein (PP), and peripheral eosinophils were determined during eight months. In addition, sera and saliva samples were collected to establish IgG and IgA kinetics by indirect enzyme-linked immunosorbent assay (ELISA). Results: The results showed 2,592 ± 2,403 EPG and 22.2 ± 4.0% PCV during lactation and 595 ± 901 EPG and 25.1 ± 2.5% PCV during pregnancy. A higher percentage of Trichostrongylus colubriformis larvae were observed in pregnancy (84 to100%) than in lactation (36 to 44%). The IgA activity in serum samples showed a marked reduction (from 80 to 10%) during lambing for both Haemonchus contortus and T. colubriformis antigens. In saliva samples, IgA activity with regard to the standard decreased from 56% at 60 ¤ To cite this article: González R, López ME, Conde MM, Mendoza P, Aguilar L, Jaso G. Immune and haematological parameters of Blackbelly ewes infected with gastrointestinal nematodes. Rev Colomb Cienc Pecu 2017; 30:219-230. * Corresponding author: Roberto González Garduño. Unidad Regional Universitaria Sursureste, Universidad Autónoma Chapingo. Km 7.5 Carr. Teapa-Vicente Guerrero. Teapa, Tabasco, México. CP 86800. E-mail: [email protected] 220 Rev Colomb Cienc Pecu 2017; 30:219-230 González R et al. Gastrointestinal nematodes in ewes days to 30% at 45 days before lambing and remained low for 45 days during lactation (23 to 32% activity). The eosinophils numbers were 2.0 x 109 cells L-1 in pregnancy and remained low at 0.7 x 109 cells L-1 in lactation. Conclusion: The studied variables refl ect the breakdown of immunity against GIN in Blackbelly ewes before and after lambing. Keywords: antigens, Haemonchus contortus, immunity, parasites, sheep, Trichostrongylus colubriformis. Resumen Antecedentes: La identifi cación del fenotipo relacionado con la resistencia contra nematodos gastrointestinales (GIN) es necesaria para conocer la inmunidad del huésped en ovejas en producción. Objetivo: Determinar los parámetros hematológicos e inmunológicos (IgA e IgG) en gestación y lactancia en ovejas Blackbelly infectadas naturalmente con GIN. Métodos: Se determinó el número de huevos por gramo de heces (EPG), se identifi caron las larvas y se registró el porcentaje del volumen celular aglomerado (% PCV), proteína plasmática (PP) y eosinófi los periféricos durante ocho meses. Además, se colectó suero y saliva para determinar la cinética de IgG e IgA por medio de un ensayo inmuno-enzimático (ELISA) indirecto. Resultados: Los resultados mostraron 2.592 ± 2.403 EPG y 22,2 ± 4,0% PCV durante la lactancia y 595 ± 901 EPG y 25,1 ± 2,5% PCV durante la gestación. Se observó un mayor porcentaje de larvas de Trichostrongylus colubriformis en gestación (84 a 100%) que en lactancia (36 a 44%). La actividad de la IgA en las muestras de suero mostró una marcada reducción después del parto para ambos antígenos de Haemonchus contortus y T. colubriformis (80 a 10%). En saliva, la actividad de la IgA disminuyó de 56 a 30% del día 60 al 45 antes del parto y se mantuvo baja en los primeros 45 días de la lactancia (actividad de 23 a 32%). El número de eosinófi los fue de 2,0 x 109 células L-1 durante la gestación, y se redujo a 0,7 x 109 células L-1 en la lactancia. Conclusión: Las variables estudiadas refl ejan la ruptura de la inmunidad contra GIN en ovejas Blackbelly antes y después del parto. Palabras clave: antígenos, Haemonchus contortus, inmunidad, ovinos, parásitos, Trichostrongylus colubriformis. Resumo Antecedentes: A identifi cação de traços fenotípicos relacionados à resistência natural contra nematóides gastrintestinais (GIN) é necessária para saber a imunidade do hospedeiro em ovelhas produtivas. Objetivo: Determinar parâmetros hematológicos e imunológicos (IgA e IgG) em períodos de gestação e lactação em ovelhas Blackbelly naturalmente infectados com GIN. Métodos: O número de ovos por grama (EPG), volume empacotado de células (% PCV), proteína plasmática (PP) e eosinófi los periféricos foram determinados durante oito meses. Além disso, as amostras de soro e saliva foram recolhidas para determinar a cinética de IgG e IgA por ELISA indireto. Resultados: Os resultados mostraram 2.592 ± 2.403 EPG e 22,2 ± 4,0% PCV durante a lactação e 595 ± 901 EPG e 25,1 ± 2,5% PCV durante a gravidez. A percentagem mais elevada de larvas de Trichostrongylus colubriformis foi observada na gravidez (84 a 100%) do que na lactação (36 a 44%). A atividade de IgA em amostras de soro mostrou uma redução acentuada (80 a 10%) durante o parto nos antígenos de Haemonchus contortus e T. colubriformis. Em amostras de saliva, a atividade de IgA diminuiu de 56 a 30% do dia 60 ao 45 antes do parto e permaneceu baixa por 45 dias durante a lactação (atividade de 23 a 32%). O número de eosinófi los foi de 2,0 x 109 células L-1 na gravidez e manteve-se baixo, com 0,7 x 109 células L-1 na lactação. Conclusaõ: As variáveis estudadas refl etem a quebra da imunidade contra GIN em ovelhas Blackbelly antes e depois do parto. Palavras chave: antigénios, Haemonchus contortus, imunidade, ovinos, parasitas, Trichostrongylus colubriformis. Introduction Tropical climate conditions have a strong infl uence on the high prevalence of gastro-intestinal nematodes (GIN), which are highly pathogenic parasites in grazing sheep. Some nematode species such as Haemonchus contortus and Trichostrongylus colubriformis are the main problem in endemic areas because they present anthelmintic resistance (CruzRojo et al., 2012; Macarthur et al., 2013). Selective breeding of resistant sheep able to regulate nematode 221 Rev Colomb Cienc Pecu 2017; 30:219-230 González R et al. Gastrointestinal nematodes in ewes infection could be a sustainable method for GIN control (Shakya et al., 2011; Shaw et al., 2013). It becomes necessary to identify the phenotypic traits related to natural resistance against GIN in order to obtain information related to host immunity in productive ewes. In sheep, low immune responses against GIN are frequently observed around parturition, and identifying resistant ewes is particularly important in animal selection (Kahn et al., 2003; Rocha et al., 2004). Number of eggs per gram of faeces (EPG) has been the main phenotypic trait used to determine GIN resistance and is a reference to establish the relationship between nematode burden and immune system response. Protective immunity to GIN dependens on innate and acquired immune factors such as eosinophilia and immunoglobulin (Ig) A and G, which could help confi rm the host resistance against GIN (Alba-Hurtado and Muñoz-Guzmán, 2012; Bishop, 2012). For instance, the high association between IgA response and T. circumcincta infection observed by Strain et al. (2002) suggests the role of this antibody (Ab) as a possible biomarker for resistance to GIN infections. Sheep challenged with GIN were able to increase IgA levels against carbohydrate larval surface antigen (CarLA; Harrison et al., 2003a; 2003b). The widespread presence of CarLA in various nematode species has been suggested as a biomarker that off ers practical, rapid, and easy diagnostic possibilities for identifying GIN resistance in sheep (Shaw et al., 2012). In order to conduct selection of resistant ewes, physiological aspects such as lactation and gestation shoul be evaluated because they can affect the development of the host immune system against GIN (Beasley et al., 2010). The aim of the present study was to determine the infl uence of physiological stage in the variability of parasitological, haematological and immunological parameters of Blackbelly ewes naturally infected with gastrointestinal nematodes in tropical conditions. Material and methods Ethical considerations This project was approved by the Research Institute of Animal Science at Universidad Autónoma Chapingo (approval number 145503001, from March 2014). The study was conducted in Salto de Agua (Chiapas, Mexico), classifi ed as a tropical wet climate (Aw) and located at 17°34′ N and 92°29′ W at 85 m.a.s.l.. The mean temperature in 2013 and 2014 was 26.6 °C, with 3,298 mm of rainfall (Kottek et al., 2006). Experimental design Twenty-fi ve Blackbelly ewes (2.5 years old) in their second gestation were selected from a fl ock. Ewes grazed through the study period; therefore, they were naturally infected with GIN. Faecal samples, blood, saliva, and live weight were taken every 2 weeks through the study (December 2014 to July 2015). The ewes were maintained in rotational grazing in paddocks of star grass (Cynodon plectostachyus) and humidicola grass (Brachiaria humidicola) at a stocking density of fi fteen ewes per hectare. Lactating ewes were supplemented with 250 g of a mixed feed (sorghum, corn, soy, and ground sugar cane). A model of accelerated lambing was implemented in the fl ock (González et al., 2010). The model considered three lambing seasons (April, August, and December 2013-2014). Blood samples were collected fortnightly to measure haematological traits. The % PCV was determined by the micro-haematocrit method and plasmatic protein was expressed in g dL-1. In addition, leukocytes were counted and classified morphologically as neutrophils, eosinophils, basophils, monocytes, and lymphocytes. Saliva samples were collected fortnightly according to the technique cited by Shaw et al. (2012). Faecal samples were collected at 15-day intervals for 8 months. The number of EPG was determined by the McMaster technique. Identifi cation of the prevalent nematode species was carried out in adults and larvae were recovered in faecal cultures (Thientpont et al., 1986) and identifi ed by morphometric characterization (Van Wyk and Mayhew, 2013). Crude worm antigen (CWA) was extracted from adult species of H. contortus (n = 50) and T. colubriformis (n = 200). Nematodes were collected from the abomasum and intestine of infected donors. Tissues were extracted by grinding nematodes in a mortar with 10% 1 mM phenylmethylsulphonyl fl uoride (PMSF, Sigma-Aldrich, St Louis Missouri, 222 Rev Colomb Cienc Pecu 2017; 30:219-230 González R et al. Gastrointestinal nematodes in ewes USA). Samples were collected by centrifugation at 20,000 x g for 20 min at 4 °C. The supernatant, containing CWA, was collected and stored at -20 °C. In addition, hot water extraction of L3 (HWEL) was performed for surface antigens from exsheathed larvae of H. contortus and T. colubriformis (Harrison et al., 2008). Protein concentration of both antigenic preparations was determined with the method by Bradford (1976) and confi rmed by SDS-PAGE. Sera and saliva samples were analysed using indirect ELISA against CWA and HWEL antigens from H. contortus and T. colubriformis, respectively. Briefl y, antigenic products were diluted to 2.5 μg mL-1 in carbonated buff er (pH 9.6), distributed into 96-well plates (NUNC MaxiSorb, Denmark) and incubated overnight at 4 °C. The wells were washed three times with PBST (0.1 M phosphate, 0.14 M sodium chloride, pH 7.2 and 0.05% Tween 20). Non-specifi c binding sites were blocked by incubation at 37 °C with 0.5% skimmed milk in PBST for 1 h. Duplicate serum samples diluted 1:100 (IgA and IgG) in PBST and saliva samples diluted at 1:20 (IgA) were incubated for 1 h at 37 °C. The plates were washed with PBST before the addition of a horseradish peroxidase-conjugated rabbit anti-sheep IgA and IgG (Bethyl Laboratories, Montgomery, AL, USA) diluted at 1:5,000 in PBST and incubated for 45 min at 37 °C. Then, 50 μL of tetramethylbenzidine (TMB, Sigma Aldrich, St Louis Missouri, USA) substrate solution was added and allowed to incubate for 15 min at RT. The reaction was stopped with 50 μL 1 M H2SO4 and optical densities (OD) were determined with a microplate absorbance reader (Imark, Bio-Rad, Mexico, DF) at 450 nm for IgA and IgG. A pool of serum of ewes with high level of IgA against H. contortus and T. colubriformis served as positive control to standardize values between plates. A negative control and three wells for each antigen without serum (blank) were included on each plate. Therefore, the blank absorbance was subtracted from the samples to correct for nonspecifi c binding (Ramírez-Restrepo et al., 2010). The activity of IgG was expressed as the percentage of the positive standard serum using the formula by Cardoso et al. (2013). Statistical analyses The data obtained per ewe around parturition were re-arranged from the calendar date to the day relative to each sheep parturition date. After this, dates were grouped in fi fteen days to analyse the eff ect of time. All data were analysed using SAS software release 9.2. (SAS, 2004). In addition, all variables were log-transformed (except PCV) to approximate a normal distribution. Repeated measures were used to test EPG, packed cell volume (% PCV), peripheral eosinophils, and Ig activity (Williams et al., 2010). The model used was: Yijk =m + ρi + αj(i) + τk + ρτik + εijk Where Yijk = is the FEC, PCV, or DWG. μ = is the mean. ρi = is the fixed effect of physiological stage (gestation and lactation). αj(i) = is the random eff ect of j-esim animal in the i-esim physiological stage αj(i) ~ N (0, σ2a). τk(i) = is the fi xed eff ect of the time (-150, -120, -105, -90, -75, -60, -45, -30, -15, 15, 30, 45, 60, 75, 90). ρτik = is the combined eff ect of physiological stage and time. εijk ~ N (0, σ2e) = is the residual error. A Pearson correlation analysis was used to determine the relationship between indicators of immunity and the other parameters. Results Ewes had higher EPG (2,592 ± 2,403) during lactation compared with the gestation period (595 ± 901; Figure 1). The PCV was approximately 25.1 ± 2.5% during pregnancy and this parameter was markedly reduced to 20 ± 1.3 and 19.2 ± 3.3% at 30 and 45 days after lambing. In addition, plasmatic protein was 6.5 ± 0.5 g dL-1 during pregnancy, and decreased to 5.7 ± 0.4 g dL-1 30 days post-partum. Body weight increased 17% (from 30.1 to 36.2 Kg) in the last two months of pregnancy, and lambing reduced this value in the same proportion (Figure 2). 223 Rev Colomb Cienc Pecu 2017; 30:219-230 González R et al. Gastrointestinal nematodes in ewes Figure 1. Faecal egg count in Blackbelly ewes naturally infected during pregnancy and lactation. Figure 2. Percentage of packed cell volume (PCV, %) and plasmatic protein (PP) of Blackbelly ewes during pregnancy and lactation. Nematode eggs per gram of faeces (EPG) Plasmatic protein g dL-1 Packed cell volume (PCV, %) Physiological stage and sampling day Pregnancy Pregnancy Lactation Lactation EPG Physiological stage and sampling day Three main species of GIN were identifi ed as H. contortus, T. colubriformis, and Cooperia curticei. The T. colubriformis population was the major species identifi ed during pregnancy, followed by H. contortus. During lactation, H. contortus was the dominant nematode species. Other nematodes, such as Strongyloides papillosus and Oesophagostomum columbianum, were also identifi ed (Table 1). Kinetics of IgA decreased during pregnancy until the lambing period and reached the lowest levels between 5.71 ± 1.53 to 7.2 ± 1.62% compared with the standard, for H. contortus CWA, and 10.29 ± 2.72 to 12.60 ± 2.79% compared with standard for T. colubriformis CWA (Figure 3a). The IgA kinetics changed 30 days after parturition when the levels began to increase, as shown in Figure 3a. The IgG anti-CWA levels were erratic, and no diff erences were observed between pregnancy and lactation (p> 0.05; Figure 3b). The IgA response using HWEL Ag decreased significantly 45 days before the lambing period 224 Rev Colomb Cienc Pecu 2017; 30:219-230 González R et al. Gastrointestinal nematodes in ewes (30.6 ± 5.1% respect to standard) and remained low during the 45 days postpartum (23 to 37%). Subsequently, IgA increased at 60 to 75 days (64 to 70% respect to standard). The response of IgA against H. contortus and T. colubriformis HWEL was similar, as shown in Figure 4. The number of eosinophils remained high (2.0 x 109 cell L-1) from the beginning of the pregnancy period and decreased signifi cantly 45 days before lambing (p<0.01) respect to the lowest levels, which occurred virtually throughout the lactation period (0.7 x 109 cell L-1). The number of total leukocytes remained high during pregnancy (15.7 x 109 cell L-1) and a decrease was observed from early lactation to 60 days post-lambing to a low level (12.2 x 109 cell L-1; Figure 5). Diff erences were found between -45 days before lambing and 60 days post-partum (p<0.01). The other PMN cells did not show important changes. A negative correlation occurred between EPG and PCV (Table 2). While EPG increased, PCV decreased by 48% during lactation. In pregnancy, the correlation was also negative but was only 38% (p<0.01). The same behaviour was observed between EPG and PP during lactation. An important and positive correlation was determined between plasmatic protein concentration and PCV in the lactation period (r = 0.53). A slight negative correlation was observed between number of eosinophils and the EPG only in gestation (r = -0.28). In addition, in the lactation period, positive correlations were observed in IgA between the H. contortus HWEL Ag and the CWA from H. contortus in serum samples (r = 0.45). For the HWEL, the correlation between H. contortus and T. colubriformis was high (r = 0.95 to 0.98) in both pregnancy and lactation. Furthermore, high values of IgA were found in the H. contortus and T. colubriformis antigens (r = 0.83 to 0.88; Table 3). Discussion The haematological and immune responses of Blackbelly ewes showed diff erences between the pregnancy and lactation periods. The nematode infection caused by H. contortus and T. colubriformis was observed three weeks prior to parturition, when there was an acute increase in the number of excreted EPG. At approximately the peri-parturient time, the increased number of EPG observed in late pregnancy and beginning of lactation periods agrees with PCV reduction, lower eosinophils count, and decrease of IgA. The peri-parturient rise (PPR) is a well-known phenomenon, and has been observed in several wool breeds relating to the eff ect of female sex hormones (Kahn et al., 2003; Williams et al., 2010). At the same time, when EPG counts increased the % PCV was reduced, especially during lactation, as a result of the increased susceptibility of females to GIN –particularly blood-sucking. The PCV reduction from Table 1. Percentage of nematode larvae obtained from copro-cultures of naturally infected Blackbelly ewes during pregnancy and lactation under tropical conditions. Physiological status Sampling period Species Haemonchus contortus Cooperia curticei Trichostrongylus colubriformis Oesophagostomum columbianum Pregnancy -120 -90 0.0 0.0 100.0 0.0 -90 -60 0.5 0.0 84.8 14.8 -60 -30 5.1 18.7 66.1 10.1 -30 -1 4.1 24.3 69.5 2.1 Lactation 1 30 42.7 12.7 43.7 0.9 30 60 57.0 4.4 36.1 2.5 60 90 51.5 0.0 48.5 0.0 *Strongyloides papillosus appeared in copro-cultures of all samples. 225 Rev Colomb Cienc Pecu 2017; 30:219-230 González R et al. Gastrointestinal nematodes in ewes Figure 3. Dynamics of IgA and IgG activity against Haemonchus contortus and Trichostrongylus colubriformis in serum during pregnancy and lactation of Blackbelly ewes under tropical conditions. Diff erent letters on each variable indicate statistical diff erences (p<0.05). 27% to less than 20% is considered an important health parameter in susceptible sheep because the infection can cause death if an animal is not treated in a timely manner (Macarthur et al., 2013; González-Garduño et al., 2014). In addition, the low % PCV during lactation was associated with an increase in the number of H. contortus larvae in cultures, as shown in Table 3. During lactation, the percentage of H. contortus larvae increased from 4 to 40% in the coprocultures. Differences observed between larvae percentage during pregnancy and lactation might be attributed to the host response against nematode species. For instance, resistant ewes better resisted infection with T. colubriformis than infection with T. circumcincta (Williams et al., 2010). In normal physiological processes the weight of Blackbelly ewes change during pregnancy with a positive gain of 5 Kg in the last two months before lambing. Similar results were found in nonsupplemented and supplemented Merino ewes in the same physiological stages (Kahn et al., 2003). Because IgA activity (% from standard) IgG activity (% from the standard) Physiological stage and sampling day Physiological stage and sampling day IgA Haemonchus contortus IgA Trichostongylus colubriformis IgG Haemonchus contortus IgG Trichostongylus colubriformis Pregnancy Pregnancy Lactation Lactation 226 Rev Colomb Cienc Pecu 2017; 30:219-230 González R et al. Gastrointestinal nematodes in ewes Table 2. Correlation coeffi cients of % PCV, EPG, plasmatic protein, and eosinophils during pregnancy and lactation periods in Blackbelly ewes. Lactation Pregnancy EPG % PCV Plasmatic protein Eosinophils EPG 1-0.38** -0.22 NS -0.28* PCV -0.48** 1 0.28* 0.09 NS Plasmatic protein -0.40** 0.53** 1 0.40** Eosinophils -0.04 NS -0.03 NS 0.07 NS 1 Above the diagonal, coeffi cients correspond to pregnancy. Below the diagonal, coeffi cients correspond to lactation. *Signifi cant (p<0.05), **Highly signifi cant (p<0.01), NS: Not signifi cant (p>0.05). EPG: Nematode eggs per gram of faeces. PCV: Packed cell volume (%). Figure 4. IgA activity in saliva with HWEL antigen of Trichostrongylus colubriformis (TcHWEL) and Haemonchus contortus (HcHWEL) during the pregnancy and lactation periods of Blackbelly ewes under tropical conditions. HWEL: Hot water extract larval antigen. Diff erent letters on each variable indicate statistical diff erences (p<0.05). Figure 5. Circulating eosinophils and total leukocytes during the pregnancy and lactation periods of Blackbelly ewes. Diff erent letters on each variable indicate statistical diff erences (p<0.05). IgA activity (% from standard) Eosinophils (x 109 cell/L) Leukocytes (x 109 cell/L) Physiological stage and sampling day Physiological stage and time relative to lambing Pregnancy Lactation 227 Rev Colomb Cienc Pecu 2017; 30:219-230 González R et al. Gastrointestinal nematodes in ewes Table 3. Correlation coeffi cients of IgA or IgG in serum and saliva during the pregnancy and lactation periods in Blackbelly ewes. Lactation Pregnancy Haemonchus contortus antigen Trichostrongylus colubriformis antigen Serum Saliva Serum Saliva CWA Ag HWEL Ag CWA Ag HWEL Ag IgA IgG IgA IgA IgG IgA H. contortus CWA-IgA 10.47** 0.19NS 0.88** 0.41** 0.16NS CWA-IgG 0.33** 1 0.21NS 0.31* 0.95** 0.16NS HWEL-IgA 0.45* -0.12NS 1 0.42* -0.09NS 0.98** T. colubriformis CWA-IgA 0.83** 0.52** 0.24NS 1 0.48** 0.21NS CWA-IgG 0.38** 0.96** 0.20NS 0.33* 1 0.15NS HWEL-IgA 0.42* -0.17NS 0.95** 0.39* -0.12NS 1 Above the diagonal, coeffi cients correspond to pregnancy; below the diagonal, coeffi cients correspond to lactation. *Signifi cant (p<0.05), **Highly signifi cant (p<0.01), NS: Not signifi cant (p>0.05). HWEL: Hot water extract larval antigen for saliva. CWA: Crude worm antigen for serum. pregnancy involves natural factors such as the foetal fl uid, foetus and placenta, the pre-partum period is considered the time with the most severe nutrient pressure (Houdijk et al., 2005). Consequently, nutrition is the main strategy for developing mature immunity during late pregnancy and lactation against parasitic infections (Beasley et al., 2010). The weight change supports the theory that the peri-parturient relaxation in immunity is probably linked to the increasing needs of energy and protein for foetal growth and hormonal changes (Mahieu and Amount, 2007). The IgA might be an indicator of immunity regulation due to the reduction of Ab titters when pregnancy is progressing, and the lowest level occurred across the fi rst months of lactation; the opposite situation occurred with faecal egg counts (r = -0.23). These same results were observed in the study by Beasley et al. (2010) with under or overfed ewes, with total plasma Ab showing low titres in pregnant ewes regardless of the diet. The rapid recovery of systemic total Ab following early weaning suggests an important role in the modulation of the EPG count around parturient rise (Beasley et al., 2010). After the challenge of a nematode infection, hair-ewes had numerous and rapid humoral immune responses, including the ability to reduce the number of EPG and maintain significantly high titres of circulating specifi c IgA (Bowdridge et al., 2013). Increased production of specifi c IgA after infection is also an indicator of immune responsiveness according to the response to H. contortus (Amarante et al., 2005) and T. circumcinta (Martínez-Valladares et al., 2005; Henderson et al., 2006). Plasma IgA titres in early weaned ewes increased, becoming significantly higher to those measured in suckled ewes (Beasley et al., 2010). In pregnant sheep, the high concentration of IgA in sera samples in middle lactation may be more refl ective of recovery from infection (Bowdridge et al., 2013), as shown in Figure 3. The decrease in EPG number may be due to IgA protection mechanisms, which include larval immobilization (Harrison et al., 2003a) and suppression of the EPG number (Martínez-Valladares et al., 2005; McCoy et al., 2008). This together with cellular mechanisms, including eosinophils counts. In this study, ELISA was used to confi rm that HWEL, as well as CarLa, is a highly conserved carbohydrate Ag (Harrison 2003a). The antibody immune responses in saliva to larval H. contortus and T. colubriformis HWEL Ag were similar. So it could be used as a diagnostic test for infection with GIN.