Veterinary Parasitology 203 (2014) 127–138
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Immune response to Haemonchus contortus and Haemonchus placei in sheep and its role on parasite specificity Michelle C. Santos, Jorge K. Xavier, Mônica R.V. Amarante, César C. Bassetto, Alessandro F.T. Amarante ∗ UNESP – Universidade Estadual Paulista, Departamento de Parasitologia, Instituto de Biociências, Caixa Postal 510, CEP: 18618-000 Botucatu, SP, Brazil
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Article history: Received 29 October 2013 Received in revised form 19 February 2014 Accepted 21 February 2014
Keywords: Epidemiology Infection Inflammatory cells Nematode Resistance Ruminant
a b s t r a c t Two trials were conducted to determine the prepatent and the patent period of Haemonchus contortus and Haemonchus placei in Santa Ines crossbred sheep and to determine whether serial infections with both species confer protection against homologous or heterologous challenge. To evaluate the prepatent and patent periods of infection, five lambs received a single infection with 4000 H. contortus-infective larvae (L3), and another five received a single infection with 4000 H. placei L3. H. contortus presented patency earlier than H. placei. Animals infected with both species shed a large number of eggs in the faeces for several months with the highest counts, with means higher than 3000 eggs per gram of faeces (EPG) between 24 and 106 days and between 38 and 73 days post infection with H. contortus and H. placei, respectively. H. contortus eggs were detected in the faeces for a minimum of 302 days and a maximum of 538 days post infection, while the H. placei patent period lasted from 288 to 364 days. In the second trial, one group of lambs (n = 12) was serially infected 12 times (three times per week for four weeks) with 500 L3 of H. placei and then challenged with either H. placei (n = 6) or with H. contortus (n = 6). The lambs in the second group (n = 12) were serially infected 12 times with 500 L3 of H. contortus and then challenged with H. contortus (n = 6) or with H. placei (n = 6), and a third group of lambs was single challenged with H. placei (n = 6), H. contortus (n = 6), or remained uninfected throughout the trial period (control group, n = 6). Animals serially infected with H. placei and then challenged with the same species presented the most intense immune response with the highest levels of antiparasitic immunoglobulin and number of inflammatory cells in the abomasal mucosa. As a result, this group had the lowest rate of parasite establishment (2.68% of the 4000 L3 given), but this phenomenon did not occur in animals single challenged with H. placei, in which the rate of establishment was relatively high (25.3%), confirming that the protective immune response to H. placei develops only when animals are repeatedly infected with this species. However, when the animals were previously serially infected with H. placei and then challenged with H. contortus, no evidence of significant protection was observed (establishment of 19.18%). The results of the trials showed an important role played by the immune response on parasite–host specificity. © 2014 Elsevier B.V. All rights reserved.
∗ Corresponding author. Tel.: +55 14 3880 0523. E-mail address: [email protected] (A.F.T. Amarante). http://dx.doi.org/10.1016/j.vetpar.2014.02.048 0304-4017/© 2014 Elsevier B.V. All rights reserved.
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1. Introduction Haemonchus species are among the most important parasites of domestic ruminants in tropical and subtropical areas worldwide. Haemonchus are blood-sucking parasites that cause a decrease in animal performance and, in extreme cases, death, particularly in young animals and periparturient females, which are more susceptible to infections. Haemonchus species have complex histories with respect to host and geographic associations. According to Hoberg et al. (2004), biogeography and host distribution appear to be compatible with an African origin for Haemonchus, basal diversification driven by colonisation among grazing and browsing antelopes with limited cospeciation and, subsequently, a complex history of hostswitching to Caprinae, Bovinae, Camelidae, Giraffidae, and among other pecorans involving both core and satellite associations. The influence of domestic ruminants on the now cosmopolitan distributions of several Haemonchus species is also likely. Only the species associated with domesticated Caprini and Bovini, such as Haemonchus contortus, Haemonchus placei and Haemonchus similis, which are commonly found in domestic ruminants and frequently occur in sympatry, have distributions in the Nearctic and Neotropic areas and occur in satellite association among cervids and camelids. In several areas of the world, there is strong evidence that H. placei and H. similis are well adapted to parasitism in cattle and that H. contortus is well adapted to small ruminants. In Brazil, even when cattle and sheep share pastures, cross infections are rarely observed in the field (Amarante et al., 1997; Rocha et al., 2008; Brasil et al., 2012). Similarly, in the tropics of the Caribbean islands (French West Indies), H. similis is the major parasite in bovines, and H. contortus is the most prevalent parasite in small ruminants (Giudici et al., 1999). In the savannah of North Côte d’Ivoire, Africa, four domestic ruminant hosts (zebu-cattle, taurine-cattle, sheep and goats) share the same pastures throughout the year, and H. contortus is also the primary species in sheep and goats. However, approximately 10% of the worms recovered from goats belong to the H. placei species. In cattle, H. contortus was very rare; however, H. placei was the dominant species in both zebu and taurine cattle; nevertheless, the proportion of H. similis was higher in zebu than in taurine cattle (Achi et al., 2003). However, this phenomenon does not occur in several areas of the world where Haemonchus species appear to present more general behaviour regarding host specificity. Under field conditions, in the Sahelian areas of Mauritania, West Africa, the most striking finding was the role played by small ruminants in the survival strategy of H. placei under adverse arid climatic conditions. For example, of total H. placei, 56% were found in sheep; 34% in goats, and only 10% in zebu cattle (Jacquiet et al., 1998). In the USA, a H. contortus population very well adapted to cattle was observed in mixed infection with H. placei (Gasbarre et al., 2009). Therefore, the importance of the cross-infection in the maintenance of the different species in a delimited geographical area appears to be variable,
and it is a consequence of the adaptation of the parasite to survive in adverse conditions. Depending on the environmental conditions and availability of different species of ruminants sharing pastures, the Haemonchus species may exhibit more generalist or more specialist behaviour concerning host specificity. Regarding geographical distribution, there is no record of H. placei and H. similis occurrence in regions with long and cold winters. In contrast, H. contortus displays increasing importance in temperate climate countries. For example, this species was found in 37% of the flocks in Sweden, even at latitudes approximating those of the Polar Circle (Lindqvist et al., 2001). H. contortus exhibits considerable ecological and biological plasticity to overcome unfavourable conditions, both in the external and host environments. The parasites may have either become more cold tolerant to the development and survival of the freeliving stages and/or may have developed special survival mechanisms of the parasitic stages within the host to ensure between-year survival (reviewed by Waller et al., 2004). Therefore, this trial aimed to study the prepatent and patent periods of H. contortus and H. placei after a single infection in sheep. We also investigated whether serial infections with both species confer protection against homologous or heterologous challenges. 2. Materials and methods Experiments 1 and 2 were conducted using 54 lambs with an initial mean body weight of 21.3 kg, and these lambs were obtained from crosses of purebred Santa Ines sires with crossbred Santa Ines ewes. The lambs were acquired just after weaning (at 2–3 months of age) from a commercial farm located in Bofete, State of São Paulo, and were maintained in pens with a concrete floor in the facilities for small ruminants of the University. Despite being raised indoors since birth at the farm of origin, the animals were shedding in average 597 nematode eggs per gram of faeces (range 0–5600) and faecal cultures demonstrated infection by Haemonchus spp. (87%) and Trichostrongylus spp. (13%). The animals were treated with albendazole (15 mg/kg, Valbazen® , Pfizer) and levamisole (10 mg/kg, Ripercol® , Fort Dodge) orally for three consecutive days, followed by treatment with trichlorfon (100 mg/kg, Neguvon® – Bayer) as a single dose. Then, a series of faecal examinations was performed to confirm the elimination of infection by nematodes. 2.1. The production of infective larvae of Haemonchus placei and Haemonchus contortus The H. placei and H. contortus infective-larvae (L3) that were used to infect donor animals were preserved in liquid nitrogen and were both levamisole-susceptible. H. placei was isolated from a bovine naturally infected with gastrointestinal nematodes in 2005 and was used to infect donor calves in two different occasions, the last one in 2009. The infective larvae produced in 2009 were used to infect the donor calves of the present study. The H. contortus isolate was kindly donated by Dr. F.A.M. Echevarria
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in 2001. Infective larvae produced from housed sheep was kept frozen since that year until to be used in the present trial. Both Haemonchus species had been previously identified based on the morphology of the spicules of adult males (Achi et al., 2003). Two male lambs, approximately six months-old, were infected with H. contortus and used as donors for the production of L3 of this species; while, two male calves, eight months-old, were the H. placei donors. The donor animals were maintained indoors, and before being infected, they received albendazole (15 mg/kg, Valbazen® , Pfizer) and levamisole (10 mg/kg, Ripercol® , Fort Dodge) orally for three consecutive days to eliminate any natural nematode infection. Their worm-free status was confirmed before the infection by a series of faecal examinations. They were kept in separate pens to avoid cross-contamination and, during the trial, they had free access to tap water and grass hay (Cynodon dactylon cv. Tifton 85) purchased from a farm with no ruminants, avoiding risks of food contamination by nematode-infective larvae. Each donor lamb was artificially infected, orally, with 10,000 L3 of H. contortus in a single dose, while each donor calf received 20,000 L3 of H. placei. Faecal cultures from the faeces of these donor animals (calves and sheep) were performed separately for the production of L3. All infective larvae were stored in distilled water and were less than 30 days-old when used to infect the trial lambs.
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2.2. Experiments The following trials were approved and conducted in accordance with the experimental protocol approved by the local Ethical Committee (protocol number 274-CEEA). 2.2.1. Trial 1 To evaluate the prepatent and the patent periods of infection, five lambs received a single infection with 4000 H. contortus L3 and another five were infected with 4000 H. placei L3. Faecal examinations were performed daily starting 14 days after the infection, with samples taken from each animal. During the patent period of infection, exams were performed weekly, and when no eggs were detected in faecal examinations for two consecutive weeks, this period was considered completed for each animal. At this point, the animal was sacrificed, the abomasum contents were collected, and all worms presented were preserved and enumerated. Throughout the trial, two worm-free tracer lambs were kept together with the experimental animals (one with each group) to verify any risk of environmental contamination with nematode-infective larvae. 2.2.2. Trial 2 The summary of the experimental design and the infection protocol used are presented in Fig. 1. The distribution of the lambs in the experimental groups was based on their
A: Experimental Design
Serial infections (Si) with Haemonchus placei (Hp) or Haemonchus contortus (Hc) SiHp (n=12)
Challenge (Ch) SiHp+ChHp (n=6) SiHp (n=12) SiHp+ChHc (n=6)
SiHc+ChHc (n=6) SiHc (n=12)
SiHc (n=12) SiHc+ChHp (n=6)
Control+ChHp (n=6) Control (n=18)
Control (n=18)
Control+ChHc (n=6) Control (n=6)
B: Timeline Anthelmintic End of Si Treatment
Start of Si
Challenge with 4000 L3 (single dose)
Si with 500 L3 (3X/week)
Day 0
Challenge
Sacrifice of all animals (worm recovery)
Day 25
Day 32
Day 35
Day 66
Fig. 1. Experimental design: One group of lambs was serially infected (Si) 12 times (three times per week; on monday, wednesday and friday), for four weeks, with 500 L3 of H. placei (Hp) and then challenged (Ch) with H. placei (subgroup SiHp + ChHp) or with H. contortus (subgroup SiHp + ChHc); lambs of a second group were serially infected 12 times with 500 L3 of H. contortus (Hc) and then challenged with Hc (subgroup SiHc + ChHc) or with H. placei (subgroup SiHc + ChHp); and the last group was single challenged with H. placei (subgroup control + ChHp), H. contortus (subgroup control + ChHc), or remained uninfected throughout the trial (control group). n = number of animals per group.
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body weight. The serial infections (Si) were administered over a period of four weeks. Seven days after the last infection (day 32), all animals were treated with levamisole (10 mg/kg, Ripercol® , Fort Dodge). Faecal examinations confirmed that the infections were eliminated from all animals after the anthelmintic treatment. Three days after treatment, the animals were artificially challenged with single doses of 4000 L3 of either H. placei or H. contortus, except the animals from the Control group (Fig. 1). The challenge was performed on the same day that the animals belonging to Trial 1 were infected using the same pool of infective larvae.
volume was brought up to 1 L with saline solution. In trial 1, all abomasum content was examined, whereas in Trial 2, the content was homogenised and divided in two containers of 500 ml each. One of the containers was immediately frozen for preservation of parasites, and 5% formalin was added to the other container. Worm identification and counting procedures were performed on the frozen material (50% of the content) as described by Ueno and Gonc¸alves, 1998, and the worms were stored in ethanol 70◦ . In Trial 2, if no worms were found in the frozen material, the 5% formalin fixed material was also carefully examined for worms.
2.3. Diet and health management
2.6. PCR for the identification of the species
The experimental animals remained indoors, and the concrete floor was washed every other day to minimise the risk of parasitic infection. The animals received decoquinate (Deccox® – Alpharma) according to the manufacturer’s recommendations throughout the trial period to prevent coccidiosis. The lambs were vaccinated against clostridiosis (Sintoxan T Polivalente® , Merial S.A.) at the beginning of the experiment. In trial 2, the lambs were fed ground hay, Tifton 85 (Cynodon dactilon. cv. Tifton 85), and a commercial concentrate (Suplementa Ovino Campo® – Presence) at a ratio of 60:40 (concentrate:hay). The total diet, corresponding to 4.5% of their body weight, was calculated to provide an average weight gain of 200 g/day (NRC, 2007), and it contained 12.8% crude protein, dry matter basis. In trial 1, the sheep had free access to the same hay and received 200 g of concentrate daily. In both trials, the animals had free access to tap water and mineral salt (Presensefós® – Presence).
To confirm the species identity, genomic DNA was extracted from each Haemonchus specimens that was still found in the experimental animals of Trial 1 at the end of the study. Additionally, genomic DNA control samples were extracted from cattle and sheep blood and also from H. contortus and H. placei specimens kept in the laboratory. DNA extraction was performed using QIAamp® DNA Mini Kit (QIAGEN, GmbH, Hilden, Germany) according to the manufacturer’s instructions. DNA samples were PCR amplified with the primer pairs 75 and 86 described by Zarlenga et al., 1994. The GeneAmp® PCR System 9700 thermocycler (Applied Biosystems, Foster City, CA, USA) was used to perform the PCR reactions. Amplification was performed in a 10 l reaction containing 10 mM Tris–HCl, 1.5 mM MgCl2 , 50 mM KCl (pH 8.0), 100 M each dNTP, 20–50 ng genomic DNA and 0.5 U Taq polymerase (Invitrogen, Carlsbad, CA, USA). The cycling conditions were as follows: 95 ◦ C for 5 min followed by 35 cycles of 95 ◦ C for 1 min, 57 ◦ C for 1 min and 72 ◦ C for 2 min followed by 10 min at 72 ◦ C and 4 ◦ C to finalise. The PCR products were electrophoresed on a 2% agarose gel in 1% TAE buffer containing ethidium bromide and photographed under UV light using a Sony Cyber-shot DSC-HX1 camera (Sony Electronics, San Diego, CA, USA).
2.4. Faecal examination Faecal samples were collected directly from the rectum of animals for faecal egg counting (FEC) determination using a modified McMaster technique (Ueno and Gonc¸alves, 1998) in which each nematode egg counted represented 100 eggs per gram (EPG). In Trial 1, if no eggs were detected using the McMaster technique, a more sensitive Willis flotation method (sensitivity 0.05) with the highest mean (11 ± 3 cells per mm2 ) recorded in the SiHp + ChHp subgroup (Fig. 10C). 4. Discussion A single infection with 4000 L3 of either H. placei or H. contortus resulted in a relatively high parasite establishment rate in young sheep with an estimate rate of 25.7% and 22.9%, respectively (subgroups ChHp and ChHc, Trial 2). In addition, animals infected with both species shed a large number of eggs in the faeces for several months with the highest FECs (means > 3000 EPG) between 24–106 days
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Fig. 10. Mean number of mucosal mast cells (A), eosinophils (B) and globule leukocytes (C) per mm2 of the abomasal mucosa of lambs serially infected (Si) three times per week, from day 0 to day 25, with 500 L3 of either Haemonchus contortus (SiHc) or Haemonchus placei (SiHp) or kept as non-infected control. On day 32* all animals received anthelmintic treatment (A). On day 35** (B) animals were challenged (Ch) with 4000 L3 of either H. contortus (subgroups SiHp + ChHc, SiHc + ChHc and control + ChHc) or H. placei (subgroups SiHp + ChHp, SiHc + ChHp and control + ChHp). One control subgroup remained uninfected during the trial. Significant differences (P < 0.05) between the groups are indicated by different letters. Bars: standard error.
and 38–73 days post H. contortus and H. placei infection, respectively (Trial 1). Two sheep infected with H. contortus shed eggs for more than one year, with a maximum of 538 days (approximately one year and a half). Similarly, two distinct isolates of adult H. contortus worms were able to survive and maintain their ability to produce viable eggs up to 350 days after infection. One of the isolates lived in arid environments in Africa (Jacquiet et al., 1995), and the other isolate was from the McMaster Laboratory in Australia (Adams and Beh, 1981). These findings explain the parasite maintenance even in environments subjected to long periods of adverse weather conditions, such as in semi-arid regions.
The major difference between species was in the duration of the prepatent period of infection, which was shorter after H. contortus infection. The delayed patency and lower egg production may be also evidence of an immune response to H. placei during primary exposures. Nevertheless, Angulo-Cubillán et al., 2010 reported differences in the prepatent period of infection influenced by the origin of the H. contortus isolate. For example, the prepatent periods for the MSD and MRI isolates were similar to the periods observed in the present trial (average: 21.3 and 21.7 days, respectively), whereas for the Aran 99 isolate, the period was significantly longer (average: 28.1 days). A similar prepatent period of H. placei was reported by Riggs, 2001 who observed patency 25–32 days post H. placei infection in sheep. Animals serially infected with H. placei and then challenged with the same species showed the most intense immune response with the highest levels of anti-parasitic immunoglobulin and inflammatory cell numbers in the abomasal mucosa. Consequently, this group had the lowest rate of parasite establishment. Interestingly, this pattern did not occur in animals challenged once with H. placei (subgroup ChHp), in which the rate of establishment was relatively high (25.3% of the 4000 L3), demonstrating that the protective immune response to H. placei only develops when animals are repeatedly infected with this species. Importantly, in this study, we used antigens from H. contortus in the ELISA, and the high levels of IgG and IgA in the plasma in animals infected with H. placei demonstrated that both Haemonchus species share common or similar antigens. This phenomenon was demonstrated by the vaccination of calves with intestinal membrane glycoproteins from H. contortus that conferred substantial protection against H. placei, both in terms of reduced egg output and adult worm numbers (Bassetto et al., 2011). However, when animals were previously serially infected with H. placei and then challenged with the other species, H. contortus, no evidence of significant protection was observed; therefore, this lack of cross-protection was unexpected. In comparison with H. placei, the serial infection with H. contortus conferred reduced protection against the homologous challenge infection. The relatively poor acquired-immunity induced by H. contortus may reflect an adaptation by the parasite to evade the host immunity (Adams and Beh, 1981). Potentially, significant protection against H. contortus only develops when animals ingest L3s for a longer period of time. Bricarello et al., 2005 observed that the progressive decline in FEC of Santa Ines lambs, indicating the development of the immune response, started only nine weeks after beginning the serial infections with 300 H. contortus L3 three times weekly and that the degree of resistance was greatly influenced by the protein content of the diet. In addition lambs of the Santa Ines breed displayed higher level of resistance in comparison to those of Ile de France breed. The animals used in the present trial had been exposed to nematode infection at the farm of origin. Before the beginning of the trial, they were maintained for seven weeks free of parasites after the anthelmintic treatment. This previous contact with the gastrointestinal nematode could have conferred partial protection against the
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experimental infections. However, the relatively high establishment rate displayed by the groups that received only the single infection demonstrated that the animals were susceptible to infection. In Australia, young sheep raised on a naturally contaminated pasture exhibited resistance to incoming H. contortus infection from as early as 4 months of age, but this resistance was completely lost when they were treated with anthelmintic (Barger, 1988). Our results and from Australia suggest that the immune response against H. contortus infection also requires a continuous challenge to prevent the significant establishment of incoming larvae. H. contortus in sheep produces a long chronic infection and immunity is not easily elicited other than after prolonged repeated infection. In contrast, a bovine strain of H. placei not adapted to sheep is unable to evade the immune mechanisms of lambs, which are able to recognise the invasion by the “strange” species with a rapid activation of an effective immune response during serial infections. Helminth infections and the corresponding host immune response are products of a prolonged dynamic co-evolution between the host and its parasite. Regarding the success of the host-parasite relationship, Dineen, 1963 postulated that the selective pressure provided by the contemporary immune response is likely to be more precise and may favour the survival of only those variants of the metazoan parasite presenting a sufficiently reduced antigenic disparity with the host. The degree of antigenic disparity may be dependent on the evolutionary selection of genetic components of both the host and the parasite. In addition, a minimum or threshold level of antigenic information would be necessary for the stimulation of the immunological response. The role of the immunological response in the “adapted” host–parasite relationship is to control the parasitic burden, rather than to cause complete elimination of the infection. Le Jambre, 1983 suggested that H. contortus use a host-mediated response in sheep to limit their competitor H. placei and that the resulting exclusion and dislodgement of H. placei act as a major pre-mating barrier to species hybridisation. He observed that established H. contortus excluded the establishment of H. placei; however, established infections of adult H. placei did not exclude H. contortus. Furthermore, the exclusion or dislodgement of H. placei was abrogated by injecting the host with dexamethasone (Le Jambre, 1983). Differences in the host specificity of Haemonchus species are apparently influenced also by the environment. In wet tropical areas, where the environmental conditions favour high degrees of infection due to favourable conditions for the development and survival of the free living stages on pastures, cross infections are uncommon, as observed in Brazil (Santiago et al., 1975; Amarante et al., 1997, Rocha et al., 2008; Brasil et al., 2012), Australia (Le Jambre, 1983), French West Indies (Giudici et al., 1999, 2012; d’Alexis et al., 2012, 2012), and the North Côte d’Ivoire (Achi et al., 2003). This aspect is partially explained by the results obtained in the present study, which demonstrated that sheep repeatedly infected with H. placei acquire strong resistance against this parasite. In semi-arid environments with a low rate of infection, like Mauritania (Jacquiet et al., 1998), a threshold level of antigenic stimulation may not
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be reached and, in part, would explain hosts harbouring mixed infections. After a single infection, both H. contortus and H. placei may survive over one year inside of a host sheep. In contrast, a strong immune response to H. placei occurs in lambs after serial infections, which may explain its absence in areas grazed only by small ruminants. This biological aspect of host specificity can be exploited as an option for the prophylaxis of gastrointestinal nematode infections though the use of grazing strategies employing different species of ruminants (Fernandes et al., 2004; Mahieu, 2013); however, field studies will be necessary to better evaluate the interaction between infections by both species in ruminants. Acknowledgments The authors are grateful for the technical assistance provided by Maria Érika Picharillo, José H. Neves, and Nadino Carvalho. Michelle C. Santos (Grant number 2011/037066), Jorge K. Xavier (Grant number 2011/09779-5) and César C. Bassetto (Grant number 2010/18678-5) received financial support from Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Alessandro F. T. Amarante received support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). References Achi, Y.L., Zinsstag, J., Yao, K., Yeo, N., Dorchies, P., Jacquiet, P., 2003. Host specificity of Haemonchus spp. for domestic ruminants in the savanna in northern Ivory Coast. Vet. Parasitol. 116, 151–158. Adams, D.B., Beh, K.J., 1981. Immunity acquired by sheep from an experimental infection with Haemonchus contortus. Int. J. Parasitol. 11, 381–386. Amarante, A.F.T., Bangola Junior, J., Amarante, M.R.V., Barbosa, M.A., 1997. Host specificity of sheep and cattle nematodes in São Paulo state, Brazil. Vet. Parasitol. 73, 89–104. Amarante, A.F.T., Susin, I., Rocha, R.A., Silva, M.B., Mendes, C.Q., Pires, A.V., 2009. Resistance of Santa Ines and crossbred ewes to naturally acquired gastrointestinal nematode infections. Vet. Parasitol 165, 273–280. Angulo-Cubillán, F.J., García-Coiradas, L., Alunda, J.M., Cuquerella, M., De La Fuente, C., 2010. Biological characterization and pathogenicity of three Haemonchus contortus isolates in primary infections in lambs. Vet. Parasitol. 171, 99–105. Barger, I.A., 1988. Resistance of young lambs to Haemonchus contortus infection, and its loss following anthelmintic treatment. Int. J. Parasitol. 18, 1107–1109. Bassetto, C.C., Silva, B.F., Nelands, G.F.J., Smith, W.D., Amarante, A.F.T., 2011. Protection of calves against Haemonchus placei and Haemonchus contortus after immunization with gut membrane proteins from H. contortus. Parasite Immunol. 33, 377–381. Brasil, B.S.A.F., Nunes, R.L., Bastinetto, E., Drummond, M.G., Carvalho, D.C., Leite, R.C., Molento, M.B., Oliveira, D.A.A., 2012. Genetic diversity patterns of Haemonchus placei and Haemonchus contortus populations isolated from domestic ruminants in Brazil. Int. J. Parasitol. 42, 468–479. Bricarello, P.A., Amarante, A.F.T., Rocha, R.A., Cabral Filho, S.L., Huntley, J.F., Houdijk, J.G.M., Abdalla, A.L., Gennari, S.M., 2005. Influence of dietary protein supply on resistance to experimental infections with Haemonchus contortus in Ile de France and Santa Ines lambs. Vet. Parasitol. 134, 99–109. Dawkins, H.J.S., Windon, R.G., Eagleason, G.K., 1989. Eosinophil responses in sheep select for high and low responsiveness to Trichostrongylus colubriformis. Int. J. Parasitol. 19, 199–205. d’Alexis, S., Mahieu, M., Jackson, F., Boval, M., 2012. Cross-infection between tropical goats and heifers with Haemonchus contortus. Vet. Parasitol. 184, 384–386. Dineen, J.K., 1963. Immunological aspects of parasitism. Nature 197, 268–269.
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Mahieu, M., 2013. Effects of stocking rates on gastrointestinal nematode infection levels in a goat/cattle rotational stocking system. Vet. Parasitol. 198, 136–144. Riggs, N.L., 2001. Experimental cross-infections of Haemonchus placei (Place, 1893) in sheep and cattle. Vet. Parasitol. 94, 191–197. Rocha, R.A., Bresciani, T.F.M., Barros, L.H., Fernandes, M.B., Amarante, A.F.T., 2008. Sheep and cattle alternately: nematode parasitism and pasture descontamination. Small. Rumin. Res. 75, 135–143. Santiago, M.A.M., Costa, U.C., Benevenga, S.F., 1975. Estudo comparativo da prevalência de helmintos em ovinos e bovinos criados na mesma pastagem. Pesq. Agrop. Bras. 10, 51–58. Silva, B.F., Bassetto, C.C., Amarante, A.F.T., 2012. Immune responses in sheep naturally infected with Oestrus ovis (Diptera: Oestridae) and gastrointestinal nematodes. Vet. Parasitol. 190, 120–126. Ueno, H., Gonc¸alves, P.C., 1998. Manual para diagnóstico das helmintoses de ruminantes, 4rd ed. Japan International Cooperation Agency, Tokyo, 149 pp. Waller, P.J., Rudby-Martin, L., Ljungströmc, B.L., Rydzik, A., 2004. The epidemiology of abomasal nematodes of sheep in Sweden, with particular reference to over-winter survival strategies. Vet. Parasitol. 122, 207–220. Zarlenga, D.S., Stringfellow, F., Nobary, M., Lichtenfels, J.R., 1994. Cloning and characterization of ribosomal RNA genes from three species of Haemonchus (Nematoda: Trichostrongyloidea) and identification of PCR primers for rapid differentiation. Exp. Parasitol. 78, 28–36.
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Immune response to Haemonchus contortus and Haemonchus placei in sheep and its role on parasite specificity Michelle C. Santos, Jorge K. Xavier, Mônica R.V. Amarante, César C. Bassetto, Alessandro F.T. Amarante ∗ UNESP – Universidade Estadual Paulista, Departamento de Parasitologia, Instituto de Biociências, Caixa Postal 510, CEP: 18618-000 Botucatu, SP, Brazil
a r t i c l e
i n f o
Article history: Received 29 October 2013 Received in revised form 19 February 2014 Accepted 21 February 2014
Keywords: Epidemiology Infection Inflammatory cells Nematode Resistance Ruminant
a b s t r a c t Two trials were conducted to determine the prepatent and the patent period of Haemonchus contortus and Haemonchus placei in Santa Ines crossbred sheep and to determine whether serial infections with both species confer protection against homologous or heterologous challenge. To evaluate the prepatent and patent periods of infection, five lambs received a single infection with 4000 H. contortus-infective larvae (L3), and another five received a single infection with 4000 H. placei L3. H. contortus presented patency earlier than H. placei. Animals infected with both species shed a large number of eggs in the faeces for several months with the highest counts, with means higher than 3000 eggs per gram of faeces (EPG) between 24 and 106 days and between 38 and 73 days post infection with H. contortus and H. placei, respectively. H. contortus eggs were detected in the faeces for a minimum of 302 days and a maximum of 538 days post infection, while the H. placei patent period lasted from 288 to 364 days. In the second trial, one group of lambs (n = 12) was serially infected 12 times (three times per week for four weeks) with 500 L3 of H. placei and then challenged with either H. placei (n = 6) or with H. contortus (n = 6). The lambs in the second group (n = 12) were serially infected 12 times with 500 L3 of H. contortus and then challenged with H. contortus (n = 6) or with H. placei (n = 6), and a third group of lambs was single challenged with H. placei (n = 6), H. contortus (n = 6), or remained uninfected throughout the trial period (control group, n = 6). Animals serially infected with H. placei and then challenged with the same species presented the most intense immune response with the highest levels of antiparasitic immunoglobulin and number of inflammatory cells in the abomasal mucosa. As a result, this group had the lowest rate of parasite establishment (2.68% of the 4000 L3 given), but this phenomenon did not occur in animals single challenged with H. placei, in which the rate of establishment was relatively high (25.3%), confirming that the protective immune response to H. placei develops only when animals are repeatedly infected with this species. However, when the animals were previously serially infected with H. placei and then challenged with H. contortus, no evidence of significant protection was observed (establishment of 19.18%). The results of the trials showed an important role played by the immune response on parasite–host specificity. © 2014 Elsevier B.V. All rights reserved.
∗ Corresponding author. Tel.: +55 14 3880 0523. E-mail address: [email protected] (A.F.T. Amarante). http://dx.doi.org/10.1016/j.vetpar.2014.02.048 0304-4017/© 2014 Elsevier B.V. All rights reserved.
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1. Introduction Haemonchus species are among the most important parasites of domestic ruminants in tropical and subtropical areas worldwide. Haemonchus are blood-sucking parasites that cause a decrease in animal performance and, in extreme cases, death, particularly in young animals and periparturient females, which are more susceptible to infections. Haemonchus species have complex histories with respect to host and geographic associations. According to Hoberg et al. (2004), biogeography and host distribution appear to be compatible with an African origin for Haemonchus, basal diversification driven by colonisation among grazing and browsing antelopes with limited cospeciation and, subsequently, a complex history of hostswitching to Caprinae, Bovinae, Camelidae, Giraffidae, and among other pecorans involving both core and satellite associations. The influence of domestic ruminants on the now cosmopolitan distributions of several Haemonchus species is also likely. Only the species associated with domesticated Caprini and Bovini, such as Haemonchus contortus, Haemonchus placei and Haemonchus similis, which are commonly found in domestic ruminants and frequently occur in sympatry, have distributions in the Nearctic and Neotropic areas and occur in satellite association among cervids and camelids. In several areas of the world, there is strong evidence that H. placei and H. similis are well adapted to parasitism in cattle and that H. contortus is well adapted to small ruminants. In Brazil, even when cattle and sheep share pastures, cross infections are rarely observed in the field (Amarante et al., 1997; Rocha et al., 2008; Brasil et al., 2012). Similarly, in the tropics of the Caribbean islands (French West Indies), H. similis is the major parasite in bovines, and H. contortus is the most prevalent parasite in small ruminants (Giudici et al., 1999). In the savannah of North Côte d’Ivoire, Africa, four domestic ruminant hosts (zebu-cattle, taurine-cattle, sheep and goats) share the same pastures throughout the year, and H. contortus is also the primary species in sheep and goats. However, approximately 10% of the worms recovered from goats belong to the H. placei species. In cattle, H. contortus was very rare; however, H. placei was the dominant species in both zebu and taurine cattle; nevertheless, the proportion of H. similis was higher in zebu than in taurine cattle (Achi et al., 2003). However, this phenomenon does not occur in several areas of the world where Haemonchus species appear to present more general behaviour regarding host specificity. Under field conditions, in the Sahelian areas of Mauritania, West Africa, the most striking finding was the role played by small ruminants in the survival strategy of H. placei under adverse arid climatic conditions. For example, of total H. placei, 56% were found in sheep; 34% in goats, and only 10% in zebu cattle (Jacquiet et al., 1998). In the USA, a H. contortus population very well adapted to cattle was observed in mixed infection with H. placei (Gasbarre et al., 2009). Therefore, the importance of the cross-infection in the maintenance of the different species in a delimited geographical area appears to be variable,
and it is a consequence of the adaptation of the parasite to survive in adverse conditions. Depending on the environmental conditions and availability of different species of ruminants sharing pastures, the Haemonchus species may exhibit more generalist or more specialist behaviour concerning host specificity. Regarding geographical distribution, there is no record of H. placei and H. similis occurrence in regions with long and cold winters. In contrast, H. contortus displays increasing importance in temperate climate countries. For example, this species was found in 37% of the flocks in Sweden, even at latitudes approximating those of the Polar Circle (Lindqvist et al., 2001). H. contortus exhibits considerable ecological and biological plasticity to overcome unfavourable conditions, both in the external and host environments. The parasites may have either become more cold tolerant to the development and survival of the freeliving stages and/or may have developed special survival mechanisms of the parasitic stages within the host to ensure between-year survival (reviewed by Waller et al., 2004). Therefore, this trial aimed to study the prepatent and patent periods of H. contortus and H. placei after a single infection in sheep. We also investigated whether serial infections with both species confer protection against homologous or heterologous challenges. 2. Materials and methods Experiments 1 and 2 were conducted using 54 lambs with an initial mean body weight of 21.3 kg, and these lambs were obtained from crosses of purebred Santa Ines sires with crossbred Santa Ines ewes. The lambs were acquired just after weaning (at 2–3 months of age) from a commercial farm located in Bofete, State of São Paulo, and were maintained in pens with a concrete floor in the facilities for small ruminants of the University. Despite being raised indoors since birth at the farm of origin, the animals were shedding in average 597 nematode eggs per gram of faeces (range 0–5600) and faecal cultures demonstrated infection by Haemonchus spp. (87%) and Trichostrongylus spp. (13%). The animals were treated with albendazole (15 mg/kg, Valbazen® , Pfizer) and levamisole (10 mg/kg, Ripercol® , Fort Dodge) orally for three consecutive days, followed by treatment with trichlorfon (100 mg/kg, Neguvon® – Bayer) as a single dose. Then, a series of faecal examinations was performed to confirm the elimination of infection by nematodes. 2.1. The production of infective larvae of Haemonchus placei and Haemonchus contortus The H. placei and H. contortus infective-larvae (L3) that were used to infect donor animals were preserved in liquid nitrogen and were both levamisole-susceptible. H. placei was isolated from a bovine naturally infected with gastrointestinal nematodes in 2005 and was used to infect donor calves in two different occasions, the last one in 2009. The infective larvae produced in 2009 were used to infect the donor calves of the present study. The H. contortus isolate was kindly donated by Dr. F.A.M. Echevarria
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in 2001. Infective larvae produced from housed sheep was kept frozen since that year until to be used in the present trial. Both Haemonchus species had been previously identified based on the morphology of the spicules of adult males (Achi et al., 2003). Two male lambs, approximately six months-old, were infected with H. contortus and used as donors for the production of L3 of this species; while, two male calves, eight months-old, were the H. placei donors. The donor animals were maintained indoors, and before being infected, they received albendazole (15 mg/kg, Valbazen® , Pfizer) and levamisole (10 mg/kg, Ripercol® , Fort Dodge) orally for three consecutive days to eliminate any natural nematode infection. Their worm-free status was confirmed before the infection by a series of faecal examinations. They were kept in separate pens to avoid cross-contamination and, during the trial, they had free access to tap water and grass hay (Cynodon dactylon cv. Tifton 85) purchased from a farm with no ruminants, avoiding risks of food contamination by nematode-infective larvae. Each donor lamb was artificially infected, orally, with 10,000 L3 of H. contortus in a single dose, while each donor calf received 20,000 L3 of H. placei. Faecal cultures from the faeces of these donor animals (calves and sheep) were performed separately for the production of L3. All infective larvae were stored in distilled water and were less than 30 days-old when used to infect the trial lambs.
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2.2. Experiments The following trials were approved and conducted in accordance with the experimental protocol approved by the local Ethical Committee (protocol number 274-CEEA). 2.2.1. Trial 1 To evaluate the prepatent and the patent periods of infection, five lambs received a single infection with 4000 H. contortus L3 and another five were infected with 4000 H. placei L3. Faecal examinations were performed daily starting 14 days after the infection, with samples taken from each animal. During the patent period of infection, exams were performed weekly, and when no eggs were detected in faecal examinations for two consecutive weeks, this period was considered completed for each animal. At this point, the animal was sacrificed, the abomasum contents were collected, and all worms presented were preserved and enumerated. Throughout the trial, two worm-free tracer lambs were kept together with the experimental animals (one with each group) to verify any risk of environmental contamination with nematode-infective larvae. 2.2.2. Trial 2 The summary of the experimental design and the infection protocol used are presented in Fig. 1. The distribution of the lambs in the experimental groups was based on their
A: Experimental Design
Serial infections (Si) with Haemonchus placei (Hp) or Haemonchus contortus (Hc) SiHp (n=12)
Challenge (Ch) SiHp+ChHp (n=6) SiHp (n=12) SiHp+ChHc (n=6)
SiHc+ChHc (n=6) SiHc (n=12)
SiHc (n=12) SiHc+ChHp (n=6)
Control+ChHp (n=6) Control (n=18)
Control (n=18)
Control+ChHc (n=6) Control (n=6)
B: Timeline Anthelmintic End of Si Treatment
Start of Si
Challenge with 4000 L3 (single dose)
Si with 500 L3 (3X/week)
Day 0
Challenge
Sacrifice of all animals (worm recovery)
Day 25
Day 32
Day 35
Day 66
Fig. 1. Experimental design: One group of lambs was serially infected (Si) 12 times (three times per week; on monday, wednesday and friday), for four weeks, with 500 L3 of H. placei (Hp) and then challenged (Ch) with H. placei (subgroup SiHp + ChHp) or with H. contortus (subgroup SiHp + ChHc); lambs of a second group were serially infected 12 times with 500 L3 of H. contortus (Hc) and then challenged with Hc (subgroup SiHc + ChHc) or with H. placei (subgroup SiHc + ChHp); and the last group was single challenged with H. placei (subgroup control + ChHp), H. contortus (subgroup control + ChHc), or remained uninfected throughout the trial (control group). n = number of animals per group.
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body weight. The serial infections (Si) were administered over a period of four weeks. Seven days after the last infection (day 32), all animals were treated with levamisole (10 mg/kg, Ripercol® , Fort Dodge). Faecal examinations confirmed that the infections were eliminated from all animals after the anthelmintic treatment. Three days after treatment, the animals were artificially challenged with single doses of 4000 L3 of either H. placei or H. contortus, except the animals from the Control group (Fig. 1). The challenge was performed on the same day that the animals belonging to Trial 1 were infected using the same pool of infective larvae.
volume was brought up to 1 L with saline solution. In trial 1, all abomasum content was examined, whereas in Trial 2, the content was homogenised and divided in two containers of 500 ml each. One of the containers was immediately frozen for preservation of parasites, and 5% formalin was added to the other container. Worm identification and counting procedures were performed on the frozen material (50% of the content) as described by Ueno and Gonc¸alves, 1998, and the worms were stored in ethanol 70◦ . In Trial 2, if no worms were found in the frozen material, the 5% formalin fixed material was also carefully examined for worms.
2.3. Diet and health management
2.6. PCR for the identification of the species
The experimental animals remained indoors, and the concrete floor was washed every other day to minimise the risk of parasitic infection. The animals received decoquinate (Deccox® – Alpharma) according to the manufacturer’s recommendations throughout the trial period to prevent coccidiosis. The lambs were vaccinated against clostridiosis (Sintoxan T Polivalente® , Merial S.A.) at the beginning of the experiment. In trial 2, the lambs were fed ground hay, Tifton 85 (Cynodon dactilon. cv. Tifton 85), and a commercial concentrate (Suplementa Ovino Campo® – Presence) at a ratio of 60:40 (concentrate:hay). The total diet, corresponding to 4.5% of their body weight, was calculated to provide an average weight gain of 200 g/day (NRC, 2007), and it contained 12.8% crude protein, dry matter basis. In trial 1, the sheep had free access to the same hay and received 200 g of concentrate daily. In both trials, the animals had free access to tap water and mineral salt (Presensefós® – Presence).
To confirm the species identity, genomic DNA was extracted from each Haemonchus specimens that was still found in the experimental animals of Trial 1 at the end of the study. Additionally, genomic DNA control samples were extracted from cattle and sheep blood and also from H. contortus and H. placei specimens kept in the laboratory. DNA extraction was performed using QIAamp® DNA Mini Kit (QIAGEN, GmbH, Hilden, Germany) according to the manufacturer’s instructions. DNA samples were PCR amplified with the primer pairs 75 and 86 described by Zarlenga et al., 1994. The GeneAmp® PCR System 9700 thermocycler (Applied Biosystems, Foster City, CA, USA) was used to perform the PCR reactions. Amplification was performed in a 10 l reaction containing 10 mM Tris–HCl, 1.5 mM MgCl2 , 50 mM KCl (pH 8.0), 100 M each dNTP, 20–50 ng genomic DNA and 0.5 U Taq polymerase (Invitrogen, Carlsbad, CA, USA). The cycling conditions were as follows: 95 ◦ C for 5 min followed by 35 cycles of 95 ◦ C for 1 min, 57 ◦ C for 1 min and 72 ◦ C for 2 min followed by 10 min at 72 ◦ C and 4 ◦ C to finalise. The PCR products were electrophoresed on a 2% agarose gel in 1% TAE buffer containing ethidium bromide and photographed under UV light using a Sony Cyber-shot DSC-HX1 camera (Sony Electronics, San Diego, CA, USA).
2.4. Faecal examination Faecal samples were collected directly from the rectum of animals for faecal egg counting (FEC) determination using a modified McMaster technique (Ueno and Gonc¸alves, 1998) in which each nematode egg counted represented 100 eggs per gram (EPG). In Trial 1, if no eggs were detected using the McMaster technique, a more sensitive Willis flotation method (sensitivity 0.05) with the highest mean (11 ± 3 cells per mm2 ) recorded in the SiHp + ChHp subgroup (Fig. 10C). 4. Discussion A single infection with 4000 L3 of either H. placei or H. contortus resulted in a relatively high parasite establishment rate in young sheep with an estimate rate of 25.7% and 22.9%, respectively (subgroups ChHp and ChHc, Trial 2). In addition, animals infected with both species shed a large number of eggs in the faeces for several months with the highest FECs (means > 3000 EPG) between 24–106 days
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Fig. 10. Mean number of mucosal mast cells (A), eosinophils (B) and globule leukocytes (C) per mm2 of the abomasal mucosa of lambs serially infected (Si) three times per week, from day 0 to day 25, with 500 L3 of either Haemonchus contortus (SiHc) or Haemonchus placei (SiHp) or kept as non-infected control. On day 32* all animals received anthelmintic treatment (A). On day 35** (B) animals were challenged (Ch) with 4000 L3 of either H. contortus (subgroups SiHp + ChHc, SiHc + ChHc and control + ChHc) or H. placei (subgroups SiHp + ChHp, SiHc + ChHp and control + ChHp). One control subgroup remained uninfected during the trial. Significant differences (P < 0.05) between the groups are indicated by different letters. Bars: standard error.
and 38–73 days post H. contortus and H. placei infection, respectively (Trial 1). Two sheep infected with H. contortus shed eggs for more than one year, with a maximum of 538 days (approximately one year and a half). Similarly, two distinct isolates of adult H. contortus worms were able to survive and maintain their ability to produce viable eggs up to 350 days after infection. One of the isolates lived in arid environments in Africa (Jacquiet et al., 1995), and the other isolate was from the McMaster Laboratory in Australia (Adams and Beh, 1981). These findings explain the parasite maintenance even in environments subjected to long periods of adverse weather conditions, such as in semi-arid regions.
The major difference between species was in the duration of the prepatent period of infection, which was shorter after H. contortus infection. The delayed patency and lower egg production may be also evidence of an immune response to H. placei during primary exposures. Nevertheless, Angulo-Cubillán et al., 2010 reported differences in the prepatent period of infection influenced by the origin of the H. contortus isolate. For example, the prepatent periods for the MSD and MRI isolates were similar to the periods observed in the present trial (average: 21.3 and 21.7 days, respectively), whereas for the Aran 99 isolate, the period was significantly longer (average: 28.1 days). A similar prepatent period of H. placei was reported by Riggs, 2001 who observed patency 25–32 days post H. placei infection in sheep. Animals serially infected with H. placei and then challenged with the same species showed the most intense immune response with the highest levels of anti-parasitic immunoglobulin and inflammatory cell numbers in the abomasal mucosa. Consequently, this group had the lowest rate of parasite establishment. Interestingly, this pattern did not occur in animals challenged once with H. placei (subgroup ChHp), in which the rate of establishment was relatively high (25.3% of the 4000 L3), demonstrating that the protective immune response to H. placei only develops when animals are repeatedly infected with this species. Importantly, in this study, we used antigens from H. contortus in the ELISA, and the high levels of IgG and IgA in the plasma in animals infected with H. placei demonstrated that both Haemonchus species share common or similar antigens. This phenomenon was demonstrated by the vaccination of calves with intestinal membrane glycoproteins from H. contortus that conferred substantial protection against H. placei, both in terms of reduced egg output and adult worm numbers (Bassetto et al., 2011). However, when animals were previously serially infected with H. placei and then challenged with the other species, H. contortus, no evidence of significant protection was observed; therefore, this lack of cross-protection was unexpected. In comparison with H. placei, the serial infection with H. contortus conferred reduced protection against the homologous challenge infection. The relatively poor acquired-immunity induced by H. contortus may reflect an adaptation by the parasite to evade the host immunity (Adams and Beh, 1981). Potentially, significant protection against H. contortus only develops when animals ingest L3s for a longer period of time. Bricarello et al., 2005 observed that the progressive decline in FEC of Santa Ines lambs, indicating the development of the immune response, started only nine weeks after beginning the serial infections with 300 H. contortus L3 three times weekly and that the degree of resistance was greatly influenced by the protein content of the diet. In addition lambs of the Santa Ines breed displayed higher level of resistance in comparison to those of Ile de France breed. The animals used in the present trial had been exposed to nematode infection at the farm of origin. Before the beginning of the trial, they were maintained for seven weeks free of parasites after the anthelmintic treatment. This previous contact with the gastrointestinal nematode could have conferred partial protection against the
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experimental infections. However, the relatively high establishment rate displayed by the groups that received only the single infection demonstrated that the animals were susceptible to infection. In Australia, young sheep raised on a naturally contaminated pasture exhibited resistance to incoming H. contortus infection from as early as 4 months of age, but this resistance was completely lost when they were treated with anthelmintic (Barger, 1988). Our results and from Australia suggest that the immune response against H. contortus infection also requires a continuous challenge to prevent the significant establishment of incoming larvae. H. contortus in sheep produces a long chronic infection and immunity is not easily elicited other than after prolonged repeated infection. In contrast, a bovine strain of H. placei not adapted to sheep is unable to evade the immune mechanisms of lambs, which are able to recognise the invasion by the “strange” species with a rapid activation of an effective immune response during serial infections. Helminth infections and the corresponding host immune response are products of a prolonged dynamic co-evolution between the host and its parasite. Regarding the success of the host-parasite relationship, Dineen, 1963 postulated that the selective pressure provided by the contemporary immune response is likely to be more precise and may favour the survival of only those variants of the metazoan parasite presenting a sufficiently reduced antigenic disparity with the host. The degree of antigenic disparity may be dependent on the evolutionary selection of genetic components of both the host and the parasite. In addition, a minimum or threshold level of antigenic information would be necessary for the stimulation of the immunological response. The role of the immunological response in the “adapted” host–parasite relationship is to control the parasitic burden, rather than to cause complete elimination of the infection. Le Jambre, 1983 suggested that H. contortus use a host-mediated response in sheep to limit their competitor H. placei and that the resulting exclusion and dislodgement of H. placei act as a major pre-mating barrier to species hybridisation. He observed that established H. contortus excluded the establishment of H. placei; however, established infections of adult H. placei did not exclude H. contortus. Furthermore, the exclusion or dislodgement of H. placei was abrogated by injecting the host with dexamethasone (Le Jambre, 1983). Differences in the host specificity of Haemonchus species are apparently influenced also by the environment. In wet tropical areas, where the environmental conditions favour high degrees of infection due to favourable conditions for the development and survival of the free living stages on pastures, cross infections are uncommon, as observed in Brazil (Santiago et al., 1975; Amarante et al., 1997, Rocha et al., 2008; Brasil et al., 2012), Australia (Le Jambre, 1983), French West Indies (Giudici et al., 1999, 2012; d’Alexis et al., 2012, 2012), and the North Côte d’Ivoire (Achi et al., 2003). This aspect is partially explained by the results obtained in the present study, which demonstrated that sheep repeatedly infected with H. placei acquire strong resistance against this parasite. In semi-arid environments with a low rate of infection, like Mauritania (Jacquiet et al., 1998), a threshold level of antigenic stimulation may not
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be reached and, in part, would explain hosts harbouring mixed infections. After a single infection, both H. contortus and H. placei may survive over one year inside of a host sheep. In contrast, a strong immune response to H. placei occurs in lambs after serial infections, which may explain its absence in areas grazed only by small ruminants. This biological aspect of host specificity can be exploited as an option for the prophylaxis of gastrointestinal nematode infections though the use of grazing strategies employing different species of ruminants (Fernandes et al., 2004; Mahieu, 2013); however, field studies will be necessary to better evaluate the interaction between infections by both species in ruminants. Acknowledgments The authors are grateful for the technical assistance provided by Maria Érika Picharillo, José H. Neves, and Nadino Carvalho. Michelle C. Santos (Grant number 2011/037066), Jorge K. Xavier (Grant number 2011/09779-5) and César C. Bassetto (Grant number 2010/18678-5) received financial support from Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Alessandro F. T. Amarante received support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). References Achi, Y.L., Zinsstag, J., Yao, K., Yeo, N., Dorchies, P., Jacquiet, P., 2003. Host specificity of Haemonchus spp. for domestic ruminants in the savanna in northern Ivory Coast. Vet. Parasitol. 116, 151–158. Adams, D.B., Beh, K.J., 1981. Immunity acquired by sheep from an experimental infection with Haemonchus contortus. Int. J. Parasitol. 11, 381–386. Amarante, A.F.T., Bangola Junior, J., Amarante, M.R.V., Barbosa, M.A., 1997. Host specificity of sheep and cattle nematodes in São Paulo state, Brazil. Vet. Parasitol. 73, 89–104. Amarante, A.F.T., Susin, I., Rocha, R.A., Silva, M.B., Mendes, C.Q., Pires, A.V., 2009. Resistance of Santa Ines and crossbred ewes to naturally acquired gastrointestinal nematode infections. Vet. Parasitol 165, 273–280. Angulo-Cubillán, F.J., García-Coiradas, L., Alunda, J.M., Cuquerella, M., De La Fuente, C., 2010. Biological characterization and pathogenicity of three Haemonchus contortus isolates in primary infections in lambs. Vet. Parasitol. 171, 99–105. Barger, I.A., 1988. Resistance of young lambs to Haemonchus contortus infection, and its loss following anthelmintic treatment. Int. J. Parasitol. 18, 1107–1109. Bassetto, C.C., Silva, B.F., Nelands, G.F.J., Smith, W.D., Amarante, A.F.T., 2011. Protection of calves against Haemonchus placei and Haemonchus contortus after immunization with gut membrane proteins from H. contortus. Parasite Immunol. 33, 377–381. Brasil, B.S.A.F., Nunes, R.L., Bastinetto, E., Drummond, M.G., Carvalho, D.C., Leite, R.C., Molento, M.B., Oliveira, D.A.A., 2012. Genetic diversity patterns of Haemonchus placei and Haemonchus contortus populations isolated from domestic ruminants in Brazil. Int. J. Parasitol. 42, 468–479. Bricarello, P.A., Amarante, A.F.T., Rocha, R.A., Cabral Filho, S.L., Huntley, J.F., Houdijk, J.G.M., Abdalla, A.L., Gennari, S.M., 2005. Influence of dietary protein supply on resistance to experimental infections with Haemonchus contortus in Ile de France and Santa Ines lambs. Vet. Parasitol. 134, 99–109. Dawkins, H.J.S., Windon, R.G., Eagleason, G.K., 1989. Eosinophil responses in sheep select for high and low responsiveness to Trichostrongylus colubriformis. Int. J. Parasitol. 19, 199–205. d’Alexis, S., Mahieu, M., Jackson, F., Boval, M., 2012. Cross-infection between tropical goats and heifers with Haemonchus contortus. Vet. Parasitol. 184, 384–386. Dineen, J.K., 1963. Immunological aspects of parasitism. Nature 197, 268–269.
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