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Introduction
Tropical climate conditions have a strong influence 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 (Cruz- Rojo et al., 2012; Macarthur et al., 2013). Selective breeding of resistant sheep able to regulate nematode 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 confirm 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 offers 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 influence 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), classified 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-five Blackbelly ewes (2.5 years old) in their second gestation were selected from a flock. 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 fifteen ewes per hectare. Lactating ewes were supplemented with 250 g of a mixed feed (sorghum, corn, soy, and ground sugar cane).Amodel of accelerated lambing was implemented in the flock (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. Identification of the prevalent nematode species was carried out in adults and larvae were recovered in faecal cultures (Thientpont et al., 1986) and identified 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 fluoride (PMSF, Sigma-Aldrich, St Louis Missouri, 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 confirmed 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:
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 effect of j-esim animal in the i-esim physiological stage αj(i) ~ N (0, σ2 a).
τk(i) = is the fixed effect of the time (-150, -120,-105, -90, -75, -60, -45, -30, -15, 15, 30, 45, 60, 75, 90).
ρτik = is the combined effect of physiological stage and time.
εijk ~ N (0, σ2 e) = 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).
Figure 2 Percentage of packed cell volume (PCV, %) and plasmatic protein (PP) of Blackbelly ewes during pregnancy and lactation.
Three main species of GIN were identified as H. contortus, T. colubriformis, and Cooperia curticei. The T. colubriformis population was the major species identified 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 identified (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 differences 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 (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.
Table 1 Percentage of nematode larvae obtained from copro-cultures of naturally infected Blackbelly ewes during pregnancy and lactation under tropical conditions.
*Strongyloides papillosus appeared in copro-cultures of all samples.
The number of eosinophils remained high (2.0 x 109 cell L-1) from the beginning of the pregnancy period and decreased significantly 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). Differences 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 differences 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 effect 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
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. Different letters on each variable indicate statistical differences (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 non- supplemented and supplemented Merino ewes in the same physiological stages (Kahn et al., 2003). Because pregnancy involves natural factors such as the foetal fluid, 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).
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. Different letters on each variable indicate statistical differences (p<0.05).
Figure 5 Circulating eosinophils and total leukocytes during the pregnancy and lactation periods of Blackbelly ewes. Different letters on each variable indicate statistical differences (p<0.05).
Table 2 Correlation coefficients of % PCV, EPG, plasmatic protein, and eosinophils during pregnancy and lactation periods in Blackbelly ewes.
Above the diagonal, coefficients correspond to pregnancy. Below the diagonal, coefficients correspond to lactation. *Significant (p<0.05), **Highly significant (p<0.01), NS: Not significant (p>0.05). EPG: Nematode eggs per gram of faeces. PCV: Packed cell volume (%)
Table 3 Correlation coefficients of IgA or IgG in serum and saliva during the pregnancy and lactation periods in Blackbelly ewes.
Above the diagonal, coefficients correspond to pregnancy; below the diagonal, coefficients correspond to lactation. *Significant (p<0.05), **Highly significant (p<0.01), NS: Not significant (p>0.05). HWEL: Hot water extract larval antigen for saliva. CWA: Crude worm antigen for serum.
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 first 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 specific IgA (Bowdridge et al., 2013).
Increased production of specific 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 reflective 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 confirm 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.
However, the values in serum and saliva showed no high correlation coefficients because the response in the two fluids occurs with different intensity over time. This behaviour has been observed because IgA has local rather than peripheral action (Prada-Jiménez et al., 2014).
A high correlation coefficient (r = 0.83) between CWAto both species (H. contortus and T. colubriformis) was found, but the correlation value were lower than found in saliva (r = 0.95), so CWA could also be used in general diagnostics against GIN, because trends between the two species are similar. However, with CWA in serum, it is possible to detect some individual differences in Ag in both nematode species.
Salivary IgA represents a possible means for detecting ewes with high resistance to GIN, especially during lactation. At this stage, it should be important to detect the ewes that have the ability to maintain high immunity levels despite the PPR. The IgA titres can be measured in blood, nasal secretions, and saliva because IgA is secreted in the gastrointestinal mucus and distributed to mucosal secretions through the blood (Prada-Jiménez et al., 2014). Considering our results, it is possible to determine basal levels one month before gestation to 45 days post-partum, whereas the peri-partum levels of salivary IgA are reduced as part of PPR.
According to Henderson et al. (2006) and Macarthur et al. (2013), evidence exists for a general immune response of the peri-parturient ewe beginning on day -29, as measured by a low number of eosinophil cells. In contrast, antibody titres in gut tissue of infected ewes remain elevated (Houdijk et al., 2005). In the same sense, the present study showed a low number of eosinophils in pregnant ewes one month before lambing, but a reduction in IgA levels occurred two months before partum in serum samples and 45 days in saliva. In addition, the recovery of the IgA levels was approximately 45 days post-partum when the IgA titres in serum and saliva occurred. However, the highest amounts of eosinophils were noted 90 days post-partum at the end of lactation period.
The increased number in EPG from day -29 to the lambing date was associated with the low number of eosinophils. Possibly, this type of response prior to parturition is a leading indicator of the general reduction of immunity associated with the peri- parturient physiology (Macarthur et al., 2013). During the final stages of pregnancy and across the lambing period, systemic immunity remained depressed as shown by the lower levels of circulating eosinophils and antibody titres. In particular, blood eosinophils concentrations drop precipitously across the span of the lambing period (Beasley et al., 2010). The number of eosinophils found at the beginning of lactation in the present study (0.7 x 109 L-1) exhibited a tendency similar to those indicated by Beasley et al. (2010) in Merino ewes, whereas the counts in the Blackbelly ewes in this study were higher than other unlike so reports in sheep (Cardia et al., 2011), but similar values were recorded for the same breed in another study (Tibbo et al., 2005).
In conclusion, IgA in saliva and serum reflects the breakdown of immunity before and after lambing. The reduction in IgA levels occurred two months before partum in the serum and 45 days in saliva. The recovery of immunity occurred close to 45 days post-partum when IgA titres in serum and saliva occurred.
