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Is there competition between Haemonchus contortus and Haemonchus placei in a pasture grazed by only sheep?
Veterinary Parasitology 279 (2020) 109054
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Research paper
Is there competition between Haemonchus contortus and Haemonchus placei in a pasture grazed by only sheep?
T
Michelle C. dos Santos, Mônica R.V. Amarante, Alessandro F.T. Amarante* Universidade Estadual Paulista (UNESP), Instituto de Biociências, Botucatu, SP, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Cross-infection Gastrointestinal nematodes Specificity Ovine Grazing
This study aimed to evaluate the dynamics of Haemonchus contortus and Haemonchus placei infections and hybridization between these species in grazing sheep without contact with cattle. On January 14, 2014, sixteen young sheep were infected with 4000 infective H. placei third-stage larvae L3; 11 days later, another group n = 16 was infected with 4000 H. contortus L3. The establishment rates of H. contortus and H. placei L3 were, on average, 61.6 % and 56.8 %, respectively, in the permanent sheep. After the establishment of patent infections, all permanent sheep were allocated together in the same clean pasture where they grazed for the next 12 months. Euthanasia of a sample of the permanent sheep was performed every three months: in May, August, November and February. Two weeks before the sheep were removed for euthanasia, 2 worm-free tracer sheep were introduced to the pasture to evaluate the larval population in the field. The tracer sheep grazed alongside the permanent sheep for 2 weeks. Then, they were housed indoors for 20 days; at the end of this period, they were euthanized. Parasites were recovered from the permanent and tracer sheep and identified using morphological and molecular techniques. A total of 432 worms (from permanent and tracer animals) were analyzed by PCR using species-specific primer pairs. Of these specimens, only two (0.46 %) male worms were identified as hybrids: one was recovered from a permanent animal euthanized in August and the other from a tracer sheep that grazed in May. The last detection of adult H. placei worms occurred in sheep euthanized in May (approximately 3.5 months after the beginning of the grazing period). The morphological evaluation of the L3 produced in fecal cultures showed that H. placei were progressively replaced by H. contortus populations starting in March. The last trace of H. placei L3 was found in August, when a small percentage (0.5 %) of infective larvae with H. placei morphology was identified in a fecal culture. In conclusion, hybridization between H. contortus and H. placei can occur in the field during coinfection. It was demonstrated that H. placei established successfully in artificially infected worm-free sheep; however, with concomitant natural reinfection with H. contortus, the H. placei population showed a rapid decrease and was eliminated within a few months in an environment without cattle.
1. Introduction Haemonchus spp. are blood-sucking parasites of the abomasum of ruminants that can cause severe anemia, resulting in morbidity and eventually potentially mortality. Among several species, Haemonchus contortus is considered to be the major parasite in small ruminants. In wet tropical and subtropical environments, haemonchosis in small ruminants occurs year round (Wilmsen et al., 2014); however, it is also an important disease in regions with a temperate climate, occurring even near latitudes approximating those of the Polar Circle (Lindqvist et al., 2001). Haemonchus placei is another important parasite for which cattle are preferential hosts. It apparently has a more restricted geographical
⁎
distribution than H. contortus; to the best of our knowledge, it has never been described in cattle from European countries or New Zealand. Its free-living stages possibly require environments with higher temperatures than those necessary for H. contortus development. For instance, in New South Wales, Australia, H. placei is common in beef cattle in the humid subtropical climate of the North Coast, while it is uncommon in the Tablelands, where the temperatures are relatively cool (Smeal et al., 1977). Nevertheless, H. placei has apparently expanded its range and has been recently reported in cattle in temperate southern Western Australia (Jabbar et al., 2014). It has also been reported as an important parasite of bison in Canada (Avramenko et al., 2018). With global warming, the trend of a long-term increase in the average temperature suggests that H. placei may expand its distribution and increase its
Corresponding author. E-mail address: [email protected] (A.F.T. Amarante).
https://doi.org/10.1016/j.vetpar.2020.109054 Received 17 October 2019; Received in revised form 3 February 2020; Accepted 6 February 2020 0304-4017/ © 2020 Elsevier B.V. All rights reserved.
Veterinary Parasitology 279 (2020) 109054
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monthly precipitation is highest (261 mm) in January and lowest (38 mm) in August. The average monthly temperature ranges from 23.2 °C in February to 17.1 °C in July (Escobedo et al., 2011).
importance for the cattle industry. H. contortus is considered to be a generalist nematode, with parasite transmission normally occurring between populations of domestic and wild ruminants (Cerutti et al., 2010; Shen et al., 2017; Winter et al., 2018). In locations where H. placei is absent, H. contortus has been frequently found in cattle. In some regions of New Zealand where H. contortus is common in sheep, it also appears to occur regularly in young cattle (Waghorn et al., 2019). Similarly, in the Tablelands, Australia, where most of the properties contain sheep, H. contortus is occasionally found in cattle (Smeal et al., 1977). There have also been occasional reports of H. contortus in cattle in the UK (Hogg et al., 2010) and in Belgium (Agneessens et al., 2000). Information about H. placei occurrence in ruminant species other than cattle is scarce. However, infection has been reported in sheep and goats that share a pasture with cattle (Amarante et al., 1997; Jacquiet et al., 1998; Almeida et al., 2018). Following artificial infection, H. placei was able to established in sheep and goats (Santos et al., 2014a; Reiniger et al., 2017; Santos et al., 2019a), and H. contortus was able to establish in young cattle (Bassetto et al., 2011; Fávero et al., 2016). In grazing areas where both species are sympatric, H. placei is the dominant species in cattle, while H. contortus is the dominant species in sheep and goats. Cross-infection has been reported in these areas but apparently does not cause morbidity or economic losses (Amarante et al., 1997; Jacquiet et al., 1998; Achi et al., 2003). In a recent study (Avramenko et al., 2017), H. placei was present in cattle at all 26 farms evaluated in São Paulo state, Brazil, while H. contortus was present in only three and at low percentages. Similarly, in the mid-southern United States, H. placei was present in 35 of 38 cattle farms evaluated, while H. contortus was present at only a few farms in low percentages (Avramenko et al., 2017). In that study, however, there was no mention of the presence or absence of small ruminants on the farms, which could indicate whether sheep and/or goats could have been the source of the H. contortus detected in the cattle. The frequent use of anthelmintics for haemonchosis prophylaxis has resulted in the appearance of resistant populations of both H. contortus and H. placei (Neves et al., 2014; Albuquerque et al., 2017). For this reason, alternative methods of control, such as the use of the integrated grazing of small ruminants and cattle, are necessary to maintain sustainable ruminant production. Improved utilization of pastures occurs when different species of ruminants share a pasture because they exhibit complementary selective feeding behaviors (d’Alexis et al., 2014). In addition, due to the parasite-host specificity of the major gastrointestinal nematode (GIN) species, grazing management approaches combining small ruminants and cattle can be employed to produce “clean pastures” to aid in GIN infection prophylaxis (Bailey et al., 2009; Mahieu and Aumont, 2009). As a result, the mixed grazing of cattle and sheep may lead to an increase in meat production per hectare (d’Alexis et al., 2014). The present trial was designed to evaluate the interaction between these two Haemonchus species in sheep initially infected with either H. placei or H. contortus that were grazed in the same pasture. The main objective was to evaluate the capacity of a H. placei population to compete with a H. contortus population to persist in sheep in the absence of cattle.
2.1. Production of infective larvae of Haemonchus placei and Haemonchus contortus The production of infective third-stage larvae (L3) of H. placei (SpHpl1 laboratory isolate) and H. contortus (SpHco2 laboratory isolate) was described by Santos et al. (2017). In brief, H. contortus L3 were used to infect a donor sheep, while H. placei L3 were used to infect a donor calf. Fecal cultures were performed separately for the production of L3. Then, each isolate was maintained in two male Suffolk lambs. These animals were kept indoors, and 4 weeks before infection, they received monepantel (2.5 mg/kg, Zolvix®, Novartis Animal Health) in a single dose to eliminate any natural strongyle infection. After confirmation of worm-free status, the two sheep received 4000 L3 of H. contortus (SpHco2), and the other two were infected with 4000 L3 of H. placei (SpHpl1). They were kept in separate pens to avoid cross-contamination, and during the trial, they had free access to tap water and hay (Cynodon dactylon cv. Tifton 85) purchased from a farm with no ruminants to avoid the risk of food contamination with infective nematode larvae. Fecal cultures from samples from each of the sheep were performed separately for the production of the L3 used to infect the experimental sheep. 2.2. Experimental design The trial design is summarized in Fig. 1. The trial was carried out with 32 male Suffolk lambs. All of the lambs were acquired from a commercial farm located in Paranapanema, São Paulo state, Brazil, just after weaning (2–3 months of age) and had an average body weight of 19 kg. Initially, the lambs were placed in pens with concrete floors in a facility for small ruminants at the University, and all animals were vaccinated against clostridiosis (Sintoxan Polivalente T®, Merial, Brazil) and received treatment with a single dose of monepantel (2.5 mg/kg, Zolvix®, Novartis Animal Health). After treatment, the determination of their worm-free status was based on a series of fecal examinations. The lambs were randomly allocated to two groups and were artificially infected orally with a single dose of 4000 L3 of either H. contortus or H. placei. The H. placei group (n = 16) was infected four weeks after monepantel treatment on January 14, 2014 (day 0), and the H. contortus group (n = 16) was infected 11 days later, on January 25, 2014. This period between the infections was established to allow both species to attain patency at the same time (Riggs, 2001; Santos et al., 2014b). All animals were kept indoors, and 35 days after H. placei infection (February 18, 2014), they were allocated together in the same pasture containing Brachiaria decumbens and Cynodon spp. (0.6 ha), where they remained until the end of the trial. For this reason, we called them “permanent lambs”. The pasture was considered worm free because it had not been grazed by ruminants for 18 months. Therefore, environmental contamination and subsequent reinfections were derived from Haemonchus eggs shed by experimentally infected sheep. The animals had free access to water and mineral salt. They were also fed daily with concentrate (Suplementa Ovino Campo® - Presence, with 17 % crude protein (CP)) in an amount corresponding to 1.5 % of their body weight until May. During the winter and beginning of spring, hay (Cynodon dactylon cv. Tifton 85 with 8 % CP) was offered, and after this period, the animals fed exclusively on the forage available in the paddock. This management technique allowed the sheep to experience moderate bodyweight gain: they weighed an average ( ± standard deviation) of 27.7 ± 2.72 kg at the start of the trial on January 14, 2014, and they weighed 55.1 ± 8.17 kg at the final sampling on February 10, 2015. Permanent lambs were not treated with anthelmintics during the trial.
2. Materials and methods The trial was approved and conducted in accordance with the experimental protocol approved by the local ethical committee (Protocol Number 449-CEEA-UNESP, Institute of Biosciences, Brazil). The experiment was conducted in the experimental area of the University (22° 53′ 16′' S, 48° 29′ 57′' W; altitude 840 m above sea level) located in Botucatu, São Paulo state, Brazil. According to the Köppen classification system, the region’s predominant climate type is Cwa (humid subtropical), which is characterized by warm, rainy summers and dry winters (Alvares et al., 2014). The average accumulated 2
Veterinary Parasitology 279 (2020) 109054
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Fig. 1. Experimental design presenting information about the permanent sheep and the worm-free tracer lambs introduced into the pasture to graze with the flock.
2.5.1. Production and identification of third-stage larvae (L3) Composite fecal cultures for the production of L3 were separately prepared every two weeks for each group of permanent sheep, with an equal volume of feces from each animal according to the descriptions of Ueno and Gonçalves (1998). Differentiation of H. placei and H. contortus L3 was based on the measurement of the sheath tail (distance between the tip of the larval tail and the end of the sheath tail) of the larvae using standard light microscopy (Leica DM LS2, Germany) with an ocular micrometer (Zeiss®, Germany). The sheath tail is consistently shorter in H. contortus than in H. placei (van Wyk et al., 2004; Santos et al., 2014b). Throughout the trial, measurements were taken from 200 L3 from each group on each sampling day. In some fecal cultures that produced fewer than 200 Haemonchus L3, all larvae present were measured. The measurements of L3 obtained from the initial infection with L3 and of L3 obtained from the first fecal culture after infection (18 February 2014) (Table 1) were used to infer H. contortus and H. placei infection dynamics in the permanent lambs. We assumed that L3 that measured < 86 μm were H. contortus, and those that measured > 87 μm were H. placei, whereas intermediate values between 86 and 87 μm were considered to be unsuitable for species differentiation due to the overlap between H. contortus and H. placei measurements (Table 1).
2.3. Euthanasia of permanent lambs To evaluate the establishment of the worms, two permanent lambs from each experimental group were euthanized on February 24, 2014, which was 30 days postinfection (DPI) with H. contortus and 41 DPI with H. placei, to recover, identify and quantify the worms. Subsequently, three animals from each group were euthanized every three months (May, August and November) to recover, identify and quantify the worms (Fig. 1). On the last sampling day of the following year (February 2015), the five remaining animals in each group were euthanized.
2.4. Tracer lambs To better evaluate the L3 population in the pasture, worm-free tracer sheep were placed in the pasture 14 days before the necropsy date of the permanent sheep. Two tracer animals were introduced to the pasture, where they grazed together with the permanent animals for two weeks on four occasions (May, August, November and February) (Fig. 1). Then, the tracer animals were housed indoors for 20 days, and at the end of this period, they were euthanized for worm recovery, quantification, and identification.
2.5.2. Worm counts The animals were fasted for 12 h before being euthanized. The abomasum was opened along the greater curvature, and its content was placed in a graduated beaker and washed with saline solution so that the parasites adhering to the mucosa detached from the organ. The volume was brought up to 1 L with saline solution, and the content was homogenized and divided into two containers of 500 mL each. The containers were immediately frozen for the preservation of parasites. The mucosal layers of all abomasums were soaked in saline solution at 38 °C for 4 h. All digested material was collected and frozen.
2.5. Laboratory analyses Every 14 days, blood and fecal samples were obtained from each permanent sheep to evaluate the packed cell volume (PCV) and fecal egg count (FEC), respectively. The feces were collected directly from the rectum of animals for egg counting using a modified McMaster technique, in which each nematode egg counted represented 100 eggs per gram (EPG) (Ueno and Gonçalves, 1998).
Table 1 Measurement in μm of the sheath tail (distance between the tip of the larval tail and the end of the sheath tail) of infective H. placei and H. contortus larvae (L3). Description of the larvae
Species
n
Minimum-maximum
Mean ± SD
(1) L3 used to infect the permanent lambs
H. H. H. H.
100 100 200 200
60.3–79.4 85.7–104.8 63.8–86.2 86.2–111.7
66.0 ± 4.57 95.1 ± 3.55 72.1 ± 6.35 97.4 ± 4.67
(2) L3 from the first evaluation of the permanent lambs
contortus placei contortus placei
(1) L3 produced in fecal cultures from samples from donor lambs. (2) L3 produced in fecal cultures from samples taken from each group of permanent lambs on February 18, 2014. 3
Veterinary Parasitology 279 (2020) 109054
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presented the following sheath tail length measures: H. contortus had a mean ± SD of 72.1 ± 6.35 μm, and H. placei had a mean ± SD of 97.4 ± 4.67 μm.
Worm counts and identification procedures were performed with 500 mL of one of the containers plus all the content of the digested material. If no worms were found in those samples, the remaining content (500 mL) was also carefully examined for worms. The juvenile forms of the parasites were morphologically identified, and the sex determination of adults was carried out by observing the presence or absence of a copulatory bursa (Ueno and Gonçalves, 1998). For Haemonchus species morphological evaluation, 10 adult male worms were randomly chosen from each permanent sheep and from each tracer lamb for body, spicule and hook length assessment, as previously described by Santos et al. (2014b). In brief, the worms were placed on a glass slide and measured with a ruler. Then, with an insulin syringe needle, the worms were cut near the copulatory bursa to measure the spicules and hooks, while the anterior region of the body of the parasite was stored in a 1.5 mL Eppendorf tube. Each tube contained 150 μL of ultrapure water and was immediately frozen for subsequent DNA extraction.
3.2. Dynamics of infection during grazing (permanent animals) Infected lambs were grazed together in the same clean pasture for the first time on February 18. This coincided with a peak in FECs: 7063 ± 4998 EPG were detected in the group infected with H. contortus, and 6394 ± 3388 EPG were detected in the group infected with H. placei (Fig. 2). The parasitism caused a reduction in PCV values; on average, in the H. contortus group, the PCV was 36 % ( ± 3.16) on January 14, 11 days before the infection, and declined to 24 % ( ± 2.83) at 59 DPI on March 25, while in the H. placei group, the PCV was 35 % ( ± 2.92) on the day of infection (January 14) and declined to 23 % ( ± 3.41) at 70 DPI (March 25). Decrease in FECs in May and June (Fig. 2) were associated with increases in PCV values. On June 3, the mean PCV was 31 % ( ± 6.0) and 34 % ( ± 2.4) in the H. contortus and H. placei groups, respectively. As expected, the group initially infected with H. placei began the trial with larvae consistent with H. placei measurements. However, in March, it was possible to observe L3 with H. contortus measurements (4 %), although the H. placei larval percentage (93 %) was still high (Fig. 2). In the following month, April, at 85 DPI, H. contortus larvae predominated (95 %). In this group, a small percentage (0.25 %) of L3 with H. placei measurements was recorded for the last time in August. The group initially infected with H. contortus presented, from March to August, some L3 with H. placei measurements (above 87 μm) but in low percentages, with a maximum of 2 % in June (Fig. 2).
2.5.3. Molecular evaluation These analyses aimed to identify the Haemonchus species and evaluate the occurrence of hybridization between H. placei and H. contortus. When possible, 10 adult male worms from each permanent sheep were collected for the measurement of spicules and DNA extraction. When an animal hosted fewer than 10 adult male worms, females and/or juveniles were used to obtain a total of 10 worms per sheep for DNA extraction. In the case of the tracer sheep, in addition to the 10 male worms used for the morphometric assessment, DNA samples were also extracted from an additional 10 males; thus, a total of 20 adult male worms were collected for molecular analysis from each tracer lamb. Genomic DNA was extracted from each Haemonchus specimen separately using a QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Bovine and sheep blood DNA samples were also extracted for use as controls in the PCR analyses. The species-specific primer pair for the identification of H. contortus (HcBotuF1/R2) resulted in an amplification band of 260 base pairs (bp), and the species-specific primer pair for the identification of H. placei (HpBotuF/R) resulted in an amplification band of 459 bp (Amarante et al., 2017). 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, California, USA).
3.3. Worm burden and morphological and molecular evaluation of Haemonchus To illustrate the results of the molecular analysis, Fig. 3 shows the PCR products obtained from 3 worms of each Haemonchus species obtained from different permanent sheep. Fig. 4 shows the products obtained from two male hybrids that were amplified with both speciesspecific primer pairs. One worm hybrid was found in a tracer lamb (n. 22) that was grazed in May and another in a permanent sheep (n. 25) that was euthanized in August. The morphometry of adult males is presented in Fig. 5; the body length, spicule and hook measurements of H. placei were larger than those of H. contortus (P < 0.001). 3.3.1. Permanent lambs The highest worm burdens were observed at the first euthanasia in February; there were 2203 and 2728 worms at 30 DPI in two lambs from the H. contortus group and 2600 and 1940 worms at 41 DPI in two lambs from the H. placei group (Table 2). These results demonstrate that artificial infection with 4000 L3 resulted in a high degree of establishment in both species. Necropsies on the other euthanasia dates revealed distinct individual variation in the number of recovered worms. For example, on the second euthanasia date in May, two animals showed a high total worm burden: one animal initially infected with H. contortus (5767 worms, 117 DPI) and another animal initially infected with H. placei (2503 worms, 128 DPI). In contrast, one of the sheep did not present worms (Table 2). In general, over the experimental period, the worm burden in both of the experimental groups decreased, except on the last date of euthanasia (11 February 2015, 382 DPI), when one animal showed a high total worm burden of 18,755 worms. Of the 272 worms analyzed by PCR, only 21 (7.72 %) were H. placei, with amplification bands of 459 bp. These worms were recovered exclusively in February and May (2014) from sheep that initially underwent artificial infection with H. placei (Table 2). Of the remaining, 250 (91.91 %) were identified as H. contortus (with PCR products of 260 bp), and only one male worm was identified as a hybrid (0.37 %), showing amplification bands of both 260 and 459 bp (Fig. 4). This hybrid worm
2.6. Statistical analyses The results are presented descriptively. Data on adult worm measurements were analyzed with unpaired t-tests at a significance level of 1 %. 3. Results 3.1. Measurements of L3 and establishment of infection The L3 used to infect the permanent lambs presented the following sheath tail length measurements: H. contortus had a mean ± standard deviation (SD) of 66.0 ± 4.57 μm, and H. placei had a mean ± SD of 95.1 ± 3.55 μm (Table 1). To confirm the establishment and identity of the species, two permanent lambs from each group were euthanized on February 24. On average, 61.6 % and 56.8 % of H. contortus and H. placei L3 had established, respectively. The examination of adult male worms by PCR confirmed the species identity (Table 2). Additionally, the infective larvae presented the morphological characteristics of their respective species (Fig. 2) in the first evaluation of L3 from composite fecal cultures from samples collected from each group on February 18, 2014. The L3, which were derived from only the artificial infection, 4
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Table 2 Worm burden and identification of Haemonchus worms (Haemonchus contortus, Hc; Haemonchus placei, Hp) by PCR. Permanent lambs were artificially infected with 4000 L3 of H. placei (Group Hp, n = 16) on January 14 (2014) or with H. contortus (Group Hc, n = 16) on January 25, 2014. Sheep shared the same pasture for 357 days starting on February 18, 2014 and were exposed to reinfection by both Haemonchus species. Euthanasia date
Group
ID
Adults
Feb 24, 2014
Hc
21 29 18 20 11 13 23 8 16 26 15 25 28 2 3 14 5 19 27 6 10 12 1 7 9 17 31 4 22 24 30 32
Males 956 1262 1251 2173 1 1200 1 0 765 11 274 40 541 0 510 196 4 42 231 1272 4 0 0 189 11 8766 0 255 15 190 18 2
Hp May 22, 2014
Hc
Hp
Aug 23, 2014
Hc
Hp
Nov 19, 2014
Hc
Hp
Feb 11, 2015
Hc
Hp
Females 1237 1466 1319 1656 0 2329 0 0 1390 18 300 38 962 2 854 241 13 57 461 1376 30 0 0 224 178 9949 0 1141 44 482 93 34
Juveniles
Total worms
PCR*
10 0 30 40 10 2238 0 0 348 4 171 61 54 0 30 73 4 16 2 44 3 7 4 120 6 40 1 214 1 1 96 17
2203 2728 2600 1940 11 5767 1 0 2503 33 745 139 1557 2 1394 510 21 115 694 2692 37 7 4 533 195 18755 1 1610 60 673 207 53
Hp 0 0 10 10 0 0 0 – 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Hc 10 10 0 0 9 10 1 – 9 10 10 9 10 2 10 10 10 10 10 10 10 6 3 10 10 10 1 10 10 10 10 10
Hybrid 0 0 0 0 0 0 0 – 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
ID: Sheep identification number. * When possible, 10 adult male worms were individually identified per animal. Some sheep had fewer than 10 worms.
with H. placei, the H. placei population was gradually replaced by H. contortus and became apparently extinct from the experimental site by the end of the trial. Several factors may have caused the disappearance of H. placei, including the role of the immune response in the hostparasite relationship. In comparison with those infected with H. contortus, a strong immune response was observed in sheep repeatedly infected with H. placei, limiting the establishment of this species (Santos et al., 2014b). In the present trial, the permanent sheep were constantly reinfected with L3 during grazing, which might have induced the development of an intense immune response against H. placei, with a consequent reduction in the establishment of this species. Similarly, in experimentally infected sheep, Le Jambre (1983) observed an 80 % decrease in H. placei after the sheep were inoculated with H. contortus, demonstrating the ability of H. contortus to exclude and dislodge H. placei from the sheep. In sheep and cattle that share pastures in São Paulo state, it has also been demonstrated that Haemonchus presents high host specificity (Amarante et al., 1997; Silva et al., 2015). Interspecific crossbreeding and the production of hybrids has been reported in mixed infections (natural or artificial) of H. contortus and H. placei (Bremner, 1955; Le Jambre, 1979, 1981; Brasil et al., 2012; Chaudhry et al., 2015; Santos et al., 2019b). A total of 432 worms (from permanent and tracer animals) were analyzed by PCR using speciesspecific primer pairs. Of these, only two (0.46 %) male worms were identified as hybrids. These results are in accordance with the low percentage of hybrids observed in field studies by Brasil et al. (2012) and Chaudhry et al. (2015) and when sheep undergo simultaneous artificial infections with both species (Santos et al., 2019b). The low production of hybrids might be due to the existence of a mating barrier between the species. Such a barrier has been reported even among
was recovered from a sheep initially infected with H. contortus and euthanized in August 2014 (Table 2). 3.3.2. Tracer animals Tracer sheep always acquired Haemonchus infection (Table 3), demonstrating the presence of Haemonchus L3 in the pasture throughout the trial. From the 160 specimens recovered from the tracer sheep and evaluated by PCR analyses, 140 (87.5 %) were identified as H. contortus, 19 (11.9 %) were identified as H. placei, and only one male worm (0.6 %) was identified as a hybrid, with amplification bands of both 260 and 459 bp (Table 3, Fig. 4). Only tracer animals that grazed in May acquired H. placei infection. 4. Discussion Both H. placei and H. contortus isolates had a high establishment rate in the permanent lambs following artificial infection. This result is in agreement with the findings of a previous trial with the same isolates that showed a peak of 6380 EPG occurring 45 days after a single inoculation with 4000 L3 of H. placei in young sheep, followed by a progressive decline, but sheep were still shedding eggs in their feces 304 DPI (Santos et al., 2014b). Such results demonstrate that sheep are a suitable host for H. placei. For this reason, the quick “disappearance” of the H. placei population was unexpected in the present study. In the present trial, the last trace of H. placei was found in August, approximately seven months after the experimental infection, when we found a small percentage (0.5 %) of infective larvae with H. placei morphology in the fecal culture. Thus, in the group initially infected 5
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Fig. 4. For each DNA sample, PCRs were performed separately for each primer pair: (A) PCR results for the species-specific primers HcBotuF1/R2 for Haemonchus contortus; and (B) PCR results for the species-specific primers HpBotuF/R for Haemonchus placei. Lanes 1 and 9–100 bp molecular markers (GE Healthcare); lanes 2 and 3 - bovine and ovine DNA samples, respectively (hosts); lane 4 - H. contortus DNA sample (control); lane 5 - H. placei DNA sample (control); lane 6 –male hybrid DNA sample from the worm from the tracer lamb n. 22 (grazing period: May 2014); lane 7 –male hybrid DNA sample from the worm from the permanent sheep n. 25 (euthanized in August 2014) and lane 8 - reagents without DNA.
different strains of H. contortus, such as MHco3(ISE), MHco4(WRS) and MHc10(CAVR), which originated on different continents. Coinfecting strains interbreed to some extent, but there are significant mating barriers between MHco10(CAVR) and the other two strains (Sargison et al., 2019). Nevertheless, in artificial backcrossing, male and female hybrids can be semisterile, possibly perpetuating a low level of reproductive efficiency within the population (Le Jambre, 1981). Therefore, further studies are necessary to evaluate the possible role of hybrid production in the introgression of genes from the H. placei to the H. contortus population and vice versa. The results of the morphological analysis of the sheath tail of infective larvae and spicule morphometrics matched those of the PCR analysis. H. placei presented larger of body length, spicule and hook measurements than H. contortus, in agreement with other studies (Lichtenfels et al., 1994; Santos et al., 2014b, Santos et al., 2019b). Our results showed that the H. placei population declined over a relatively short time period, while H. contortus increased and dominated the parasitic abomasal fauna of the sheep. Therefore, management systems using cattle and sheep to produce clean pastures could be a useful tool for the prevention of haemonchosis, corroborating previous studies (Fernandes et al., 2004; d’Alexis et al., 2012; Brito et al., 2013). In conclusion, it was demonstrated that H. placei established successfully in artificially infected worm-free sheep; however, under concomitant natural reinfection with H. contortus, the H. placei population experienced a rapid decrease and was eliminated from an environment without cattle. Additionally, during H. contortus and H. placei coinfection, a small proportion of hybrids were produced.
Fig. 2. Mean eggs per gram of feces and species composition of the lambs artificially infected with 4000 L3 of Haemonchus placei (Group H. placei; n = 16) on January 14 or with Haemonchus contortus (Group H. contortus; n = 16) on January 25. The sheep shared the same pasture for 357 days (from February 18, 2014, until February 10, 2015) and were exposed to reinfection by both Haemonchus species. Species composition based on the identification of thirdstage larvae (L3) produced in fecal cultures. Measurement of the sheath tails: H. contortus < 86 μm; H. placei > 87 μm; and intermediate measurement between 86 μm and 87 μm.
Declaration of Competing Interest The authors declare no conflict of interest.
Fig. 3. For each DNA sample, PCRs were performed separately for each primer pair: (A) PCR results for the species-specific primers HcBotuF1/R2 for Haemonchus contortus; and (B) PCR results for the species-specific primers HpBotuF/R for Haemonchus placei. Lane 1–100 bp molecular markers (GE Healthcare); Lanes 2 and 3 –bovine and ovine DNA, respectively (hosts); Lane 4 –H. contortus DNA sample (control); Lane 5 - H. placei DNA sample (control); Lanes 6, 8 and 11 –H. contortus DNA samples from different permanent sheep; Lanes 9, 10 and 12 –H. placei DNA samples from different permanent sheep; and Lanes 7 and 13 –reagents without DNA.
Acknowledgments The authors are grateful for the technical assistance provided by Adriana M. D. Anaya, César C. Bassetto, José H. Neves, Gabriela F. Caetano, Maria Regina L. Silva, Nadino Carvalho and Natália B. Pinto. Michelle C. Santos received financial support from the Sao Paulo Research Foundation (FAPESP, Grant Number 2012/23941-2 and 6
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Fig. 5. Morphometrics of male adult worms of Haemonchus contortus (n = 209) and H. placei (n = 21). PCR analysis was used as the gold standard for Haemonchus spp. identification. The ends of the box are the upper and lower quartiles; the median is marked by a vertical line inside the box; and the two lines outside the box extend to the highest and lowest observations. For all variables, there was a significant difference between Haemonchus species (P < 0.001).
Table 3 Worm burden of the tracer lambs and identification of Haemonchus worms (Haemonchus contortus, Hc; Haemonchus placei, Hp) by PCR. Tracers grazed together with the permanent sheep for 14 days. After this period of grazing, the tracer animals were kept indoors for 20 days, and at the end of this period, they were euthanized for the recovery, quantification and identification of the parasites. Grazing period
May 8–22, 2014 Aug 8–22, 2014 Nov 4–20, 2014 Jan 27 to Feb 10, 2015
Tracer
Adults
Juveniles
Males
Females
9 22 51 62 42 25
223 342 73 413 715 848
345 336 80 534 1057 1026
51 28 11 0 0 166
45
449
580
0
Total
contortus and Haemonchus placei in sheep from Santana do Livramento, Brazil. Rev. Bras. Parasitol. Vet. 27, 280–288. Alvares, C.A., Stape, J.L., Sentelhas, P.C., Gonçalves, J.L.M., Sparovek, G., 2014. Köppen’s climate classification map for Brazil. Meteorol. Z. 22, 711–728. Amarante, A.F.T., Bangola Junior, J., Amarante, M.R.V., Barbosa, M.A., 1997. Host specificity of sheep and cattle nematodes in Sao Paulo state, Brazil. Vet. Parasitol. 73, 89–104. Amarante, M.R.V., Santos, M.C., Bassetto, C.C., Amarante, A.F.T., 2017. PCR primers for straightforward differentiation of Haemonchus contortus, Haemonchus placei and its hybrids. J. Helmintol. 91, 757–761. Avramenko, R.W., Redman, E.M., Lewis, R., Bichuette, M.A., Palmeira, B.M., Yazwinski, T.A., Gilleard, J.S., 2017. The use of nemabiome metabarcoding to explore gastrointestinal nematode species diversity and anthelmintic treatment effectiveness in beef calves. Int. J. Parasitol. 47, 893–902. Avramenko, R.W., Bras, A., Redman, E.M., Woodbury, M.R., Wagner, B., Shury, T., Liccioli, S., Windeyer, M.C., Gilleard, J.S., 2018. High species diversity of trichostrongyle parasite communities within and between Western Canadian commercial and conservation bison herds revealed by nemabiome metabarcoding. Parasit. Vectors 11, 299. https://doi.org/10.1186/s13071-018-2880-y. Bailey, J.N., Walkden-Brown, S.W., Kahn, L.P., 2009. Comparison of strategies to provide lambing paddocks of low gastro-intestinal nematode infectivity in a summer rainfall region of Australia. Vet. Parasitol. 161, 218–231. Bassetto, C.C., Silva, B.F., Newlands, 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, L.R., Bastianetto, 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, 469–479. Bremner, K.C., 1955. Cytological studies on the specific distinctness of the ovine and bovine “strains” of the nematodeHaemonchus contortus (Rudolphi) Cobb (Nematoda: Trichostrongylidae). Aust. J. Zool. 3, 312–323. Brito, D.L., Dallago, B.S.L., Louvandini, H., Santos, V.R.V., Torres, S.E.F.A., Gomes, E.F., Amarante, A.F.T., Melo, C.B., McManus, C.M., 2013. Effect of alternate and simultaneous grazing on endoparasite infection in sheep and cattle. Rev. Bras. Parasitol. Vet. 22, 485–494. Cerutti, M.C., Citterio, C.V., Bazzocchi, C., Epis, S., D’Amelio, S., Ferrari, N., Lanfranchi, P., 2010. Genetic variability of Haemonchus contortus(Nematoda: Trichostrongyloidea) in alpine ruminant host species. J. Helminthol. 84, 276–283. Chaudhry, U., Redman, E.M., Abbas, M., Muthusamy, R., Ashraf, K., Gilleard, J.S., 2015. Genetic evidence for diagnosticon between Haemonchus contortus and Haemonchus placei in natural field populations and its implications for interspecies transmission of anthelmintic resistance. Int. J. Parasitol. 45, 149–159. 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. d’Alexis, S., Sauvant, D., Boval, M., 2014. Mixed grazing systems of sheep and cattle to improve liveweight gain: a quantitative review. J. Agric. Sci. 152, 655–666. Escobedo, J.F., Gomes, E.N., Oliveira, A.P., Soares, J., 2011. Ratios of UV, PAR and NIR
PCR* Hp
Hc
Hybrid
619 706 164 947 1772 2040
5 14 0 0 0 0
15 5 40 20 20 20
0 1 0 0 0 0
1029
0
20
0
On August 8, a tracer lamb died due to an undetermined cause, and only one lamb (Number 51) was euthanized. For this reason, 40 adult male worms were used from this tracer. * Twenty adult male worms were individually identified by PCR per animal.
2015/12900-1). Alessandro F. T. Amarante is the recipient of a fellowship from CNPq (305187/2017-1). This study was supported by the São Paulo Research Foundation (FAPESP - Grant Number 2014/023056). 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. Agneessens, J., Claerebout, E., Dorny, P., Borgsteede, F.H., Vercruysse, J., 2000. Nematode parasitism in adult dairy cows in Belgium. Vet. Parasitol. 90, 83–92. Albuquerque, A.C.A., Bassetto, C.C., Almeida, F.A., Amarante, A.F.T., 2017. Development of Haemonchus contortus resistance in sheep under suppressive or targeted selective treatment with monepantel. Vet. Parasitol. 246, 112–117. Almeida, F.A., Bassetto, C.C., Amarante, M.R.V., Albuquerque, A.C.A., Starling, R.Z.C., Amarante, A.F.T., 2018. Helminth infections and hybridization between Haemonchus
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Soares, V.E., Lopes, W.D.Z., Costa, A.J., Oliveira, G.P., 2019a. Viability of Haemonchus placei parasitism in experimentally infected young goats. Vet. Parasitol. 271, 64–67. Santos, M.C., Amarante, M.R.V., Amarante, A.F.T., 2019b. Establishment of co-infection and hybridization of Haemonchus contortus and Haemonchus placei in sheep. J. Helminthol. 93, 697–703. Santos, M.C., Redman, E., Amarante, M.R.V., Gilleard, J.S., Amarante, A.F.T., 2017. A panel of microsatellite markers to discriminate and study interactions between Haemonchus contortus and Haemonchus placei. Vet. Parasitol. 244, 71–75. Santos, M.C., Xavier, J.K., Amarante, M.R.V., Bassetto, C.C., Amarante, A.F.T., 2014a. Immune response to Haemonchus contortus and Haemonchus placei in sheep and its role on parasite specificity. Vet. Parasitol. 203, 127–138. Santos, M.C., Amarante, M.R.V., Silva, M.R.L., Amarante, A.F.T., 2014b. Differentiation of Haemonchus placei from Haemonchus contortus by PCR and by morphometrics of adult parasites and third stage larvae. Rev. Bras. Parasitol. Vet. 23, 495–500. Sargison, N.D., Redman, E., Morrison, A.A., Bartley, D.J., Jackson, F., Hoberg, E., Gilleard, J.S., 2019. Mating barriers between genetically divergent strains of the parasitic nematode Haemonchus contortus suggest incipient speciation. Int. J. Parasitol. 49, 531–540. Shen, D.D., Wang, J.F., Zhang, D.Y., Peng, Z.W., Yang, T.Y., Wang, Z.D., Bowman, D.D., Hou, Z.J., Liu, Z.S., 2017. Genetic diversity of Haemonchus contortus isolated from sympatric wild blue sheep (Pseudois nayaur) and sheep in Helan Mountains, China. Parasit. Vectors 10, 437. https://doi.org/10.1186/s13071-017-2377-0. Silva, M.R.L., Amarante, M.R., Bresciani, K.D., Amarante, A.F.T., 2015. Host-specificity and morphometrics of femaleHaemonchus contortus, H. placei and H. similis (Nematoda: Trichostrongylidae) in cattle and sheep from shared pastures in São Paulo State. Brazil. J. Helminthol. 9, 302–306. Smeal, M.G., Hotson, I.K., Mylrea, P.J., Jackson, A.R., Campbell, N.J., Kirton, H.C., 1977. Studies on nematode infections of beef cattle in New South Wales. Aust. Vet. J. 53, 566–573. Ueno, H., Gonçalves, P.C., 1998. Manual Para Diagnóstico Das Helmintoses De Ruminantes, 4 ed. Japan International Cooperation Agency, Tokyo 143 p. van Wyk, J.A., Cabaret, J., Michael, L.M., 2004. Morphological identification of nematode larvae of small ruminants and cattle simplified. Vet. Parasitol. 119, 277–306. Waghorn, T.S., Bouchet, C., Bekelaar, K., Leathwick, D.M., 2019. Nematode parasites in young cattle: what role for unexpected species? N. Z. Vet. J. 67, 40–45. Wilmsen, M.O., Silva, B.F., Bassetto, C.C., Amarante, A.F.T., 2014. Gastrointestinal nematode infections in sheep raised in Botucatu, state of São Paulo, Brazil. Rev. Bras. Parasitol. Vet. 23, 348–354. Winter, J., Rehbein, S., Joachim, A., 2018. Transmission of helminths between species of ruminants in Austria appears more likely to occur than generally assumed. Front. Vet. Sci. 5, 30. https://doi.org/10.3389/fvets.2018.00030.
components to global solar radiation measured at Botucatu site in Brazil. Renew. Energy 36, 169–178. Fávero, F.C., Buzzulini, C., Cruz, B.C., Felippelli, G., Maciel, W.G., Salatta, B., Teixeira, W.F.P., Soares, V.E., Oliveira, G.P., Lopes, W.D.Z., Costa, A.J., 2016. Experimental infection of calves with Haemonchus placei and Haemonchus contortus: assessment of parasitological parameters. Vet. Parasitol. 217, 25–28. Fernandes, L.H., Seno, M.C.Z., Amarante, A.F.T., Souza, H., Belluzzo, C.E.C., 2004. Efeito do pastejo rotacionado e alternado com bovinos adultos no controle da verminose em ovelhas. Arq. Bras. Med. Vet. Zoot. 56, 733–740. Hogg, R., Whitaker, K., Collins, R., Holmes, P., Mitchell, S., Anscombe, J., Redman, L., Gilleard, J., 2010. Haemonchosis in large ruminants in the UK. Vet. Rec. 166, 373–374. Jabbar, A., Cotter, J., Lyon, J., Koehler, A.V., Gasser, R.B., Besier, B., 2014. Unexpected occurrence of Haemonchus placei in cattle in southern Western Australia. Infect. Genet. Evol. 21, 252–258. Jacquiet, P., Cabaret, J., Thiam, E., Cheikh, D., 1998. Host range and the maintenance of Haemonchus spp. in an adverse arid climate. Int. J. Parasitol. 28, 253–261. Le Jambre, L.F., 1979. Hybridization studies of Haemonchus contortus(Rudolphi, 1803) andH. placei (Place, 1893) (Nematoda: Trichostrongylidae). Int. J. Parasitol. 9, 455–463. Le Jambre, L.F., 1981. Hybridization of Australian Haemonchus placei (Place, 1893), Haemonchus contortus cayugensis (Das e Whitlock, 1960) and Haemonchus contortus (Rudolphi, 1803) from Louisiana. Int. J. Parasitol. 11, 323–330. Le Jambre, L.F., 1983. Pre-mating barriers in hybrid to species hybridization in Haemonchus. Int. J. Parasitol. 13, 365–370. Lichtenfels, J.R., Pilitt, P.A., Hoberg, E.P., 1994. New morphological characters for identifying individual specimens of Haemonchus spp. (Nematoda: Trichostrongyloidea) and a key to species in ruminants of North America. J. Parasitol. 80, 107–119. Lindqvist, Å., Ljungström, B.-L., Nilsson, O., Waller, P.J., 2001. The dynamics, prevalence and impact of nematode infections in organically raised sheep in Sweden. Acta Vet. Scand. 42, 377–389. Mahieu, M., Aumont, G., 2009. Effects of sheep and cattle alternate grazing on sheep parasitism and production. Trop. Anim. Health Prod. 41, 229–239. Neves, J.H., Carvalho, N., Rinaldi, L., Cringoli, G., Amarante, A.F.T., 2014. Diagnosis of anthelmintic resistance in cattle in Brazil: a comparison of different methodologies. Vet. Parasitol. 206, 216–226. Reiniger, R.C.P., Dias de Castro, L.L., Benavides, M.V., Berne, M.E.A., 2017. Can Haemonchus placei-primary infected naïve lambs withstand Haemonchus contortus infections? Res. Vet. Sci. 114, 136–142. Riggs, N.L., 2001. Experimental cross-infections of Haemonchus placei (Place, 1893) in sheep and cattle. Vet. Parasitol. 94, 191–197. Santos, I.B., Maciel, W.G., Felippelli, G., Toscano, J.H.B., Cruz, B.C., Chagas, A.C.S.,
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Research paper
Is there competition between Haemonchus contortus and Haemonchus placei in a pasture grazed by only sheep?
T
Michelle C. dos Santos, Mônica R.V. Amarante, Alessandro F.T. Amarante* Universidade Estadual Paulista (UNESP), Instituto de Biociências, Botucatu, SP, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Cross-infection Gastrointestinal nematodes Specificity Ovine Grazing
This study aimed to evaluate the dynamics of Haemonchus contortus and Haemonchus placei infections and hybridization between these species in grazing sheep without contact with cattle. On January 14, 2014, sixteen young sheep were infected with 4000 infective H. placei third-stage larvae L3; 11 days later, another group n = 16 was infected with 4000 H. contortus L3. The establishment rates of H. contortus and H. placei L3 were, on average, 61.6 % and 56.8 %, respectively, in the permanent sheep. After the establishment of patent infections, all permanent sheep were allocated together in the same clean pasture where they grazed for the next 12 months. Euthanasia of a sample of the permanent sheep was performed every three months: in May, August, November and February. Two weeks before the sheep were removed for euthanasia, 2 worm-free tracer sheep were introduced to the pasture to evaluate the larval population in the field. The tracer sheep grazed alongside the permanent sheep for 2 weeks. Then, they were housed indoors for 20 days; at the end of this period, they were euthanized. Parasites were recovered from the permanent and tracer sheep and identified using morphological and molecular techniques. A total of 432 worms (from permanent and tracer animals) were analyzed by PCR using species-specific primer pairs. Of these specimens, only two (0.46 %) male worms were identified as hybrids: one was recovered from a permanent animal euthanized in August and the other from a tracer sheep that grazed in May. The last detection of adult H. placei worms occurred in sheep euthanized in May (approximately 3.5 months after the beginning of the grazing period). The morphological evaluation of the L3 produced in fecal cultures showed that H. placei were progressively replaced by H. contortus populations starting in March. The last trace of H. placei L3 was found in August, when a small percentage (0.5 %) of infective larvae with H. placei morphology was identified in a fecal culture. In conclusion, hybridization between H. contortus and H. placei can occur in the field during coinfection. It was demonstrated that H. placei established successfully in artificially infected worm-free sheep; however, with concomitant natural reinfection with H. contortus, the H. placei population showed a rapid decrease and was eliminated within a few months in an environment without cattle.
1. Introduction Haemonchus spp. are blood-sucking parasites of the abomasum of ruminants that can cause severe anemia, resulting in morbidity and eventually potentially mortality. Among several species, Haemonchus contortus is considered to be the major parasite in small ruminants. In wet tropical and subtropical environments, haemonchosis in small ruminants occurs year round (Wilmsen et al., 2014); however, it is also an important disease in regions with a temperate climate, occurring even near latitudes approximating those of the Polar Circle (Lindqvist et al., 2001). Haemonchus placei is another important parasite for which cattle are preferential hosts. It apparently has a more restricted geographical
⁎
distribution than H. contortus; to the best of our knowledge, it has never been described in cattle from European countries or New Zealand. Its free-living stages possibly require environments with higher temperatures than those necessary for H. contortus development. For instance, in New South Wales, Australia, H. placei is common in beef cattle in the humid subtropical climate of the North Coast, while it is uncommon in the Tablelands, where the temperatures are relatively cool (Smeal et al., 1977). Nevertheless, H. placei has apparently expanded its range and has been recently reported in cattle in temperate southern Western Australia (Jabbar et al., 2014). It has also been reported as an important parasite of bison in Canada (Avramenko et al., 2018). With global warming, the trend of a long-term increase in the average temperature suggests that H. placei may expand its distribution and increase its
Corresponding author. E-mail address: [email protected] (A.F.T. Amarante).
https://doi.org/10.1016/j.vetpar.2020.109054 Received 17 October 2019; Received in revised form 3 February 2020; Accepted 6 February 2020 0304-4017/ © 2020 Elsevier B.V. All rights reserved.
Veterinary Parasitology 279 (2020) 109054
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monthly precipitation is highest (261 mm) in January and lowest (38 mm) in August. The average monthly temperature ranges from 23.2 °C in February to 17.1 °C in July (Escobedo et al., 2011).
importance for the cattle industry. H. contortus is considered to be a generalist nematode, with parasite transmission normally occurring between populations of domestic and wild ruminants (Cerutti et al., 2010; Shen et al., 2017; Winter et al., 2018). In locations where H. placei is absent, H. contortus has been frequently found in cattle. In some regions of New Zealand where H. contortus is common in sheep, it also appears to occur regularly in young cattle (Waghorn et al., 2019). Similarly, in the Tablelands, Australia, where most of the properties contain sheep, H. contortus is occasionally found in cattle (Smeal et al., 1977). There have also been occasional reports of H. contortus in cattle in the UK (Hogg et al., 2010) and in Belgium (Agneessens et al., 2000). Information about H. placei occurrence in ruminant species other than cattle is scarce. However, infection has been reported in sheep and goats that share a pasture with cattle (Amarante et al., 1997; Jacquiet et al., 1998; Almeida et al., 2018). Following artificial infection, H. placei was able to established in sheep and goats (Santos et al., 2014a; Reiniger et al., 2017; Santos et al., 2019a), and H. contortus was able to establish in young cattle (Bassetto et al., 2011; Fávero et al., 2016). In grazing areas where both species are sympatric, H. placei is the dominant species in cattle, while H. contortus is the dominant species in sheep and goats. Cross-infection has been reported in these areas but apparently does not cause morbidity or economic losses (Amarante et al., 1997; Jacquiet et al., 1998; Achi et al., 2003). In a recent study (Avramenko et al., 2017), H. placei was present in cattle at all 26 farms evaluated in São Paulo state, Brazil, while H. contortus was present in only three and at low percentages. Similarly, in the mid-southern United States, H. placei was present in 35 of 38 cattle farms evaluated, while H. contortus was present at only a few farms in low percentages (Avramenko et al., 2017). In that study, however, there was no mention of the presence or absence of small ruminants on the farms, which could indicate whether sheep and/or goats could have been the source of the H. contortus detected in the cattle. The frequent use of anthelmintics for haemonchosis prophylaxis has resulted in the appearance of resistant populations of both H. contortus and H. placei (Neves et al., 2014; Albuquerque et al., 2017). For this reason, alternative methods of control, such as the use of the integrated grazing of small ruminants and cattle, are necessary to maintain sustainable ruminant production. Improved utilization of pastures occurs when different species of ruminants share a pasture because they exhibit complementary selective feeding behaviors (d’Alexis et al., 2014). In addition, due to the parasite-host specificity of the major gastrointestinal nematode (GIN) species, grazing management approaches combining small ruminants and cattle can be employed to produce “clean pastures” to aid in GIN infection prophylaxis (Bailey et al., 2009; Mahieu and Aumont, 2009). As a result, the mixed grazing of cattle and sheep may lead to an increase in meat production per hectare (d’Alexis et al., 2014). The present trial was designed to evaluate the interaction between these two Haemonchus species in sheep initially infected with either H. placei or H. contortus that were grazed in the same pasture. The main objective was to evaluate the capacity of a H. placei population to compete with a H. contortus population to persist in sheep in the absence of cattle.
2.1. Production of infective larvae of Haemonchus placei and Haemonchus contortus The production of infective third-stage larvae (L3) of H. placei (SpHpl1 laboratory isolate) and H. contortus (SpHco2 laboratory isolate) was described by Santos et al. (2017). In brief, H. contortus L3 were used to infect a donor sheep, while H. placei L3 were used to infect a donor calf. Fecal cultures were performed separately for the production of L3. Then, each isolate was maintained in two male Suffolk lambs. These animals were kept indoors, and 4 weeks before infection, they received monepantel (2.5 mg/kg, Zolvix®, Novartis Animal Health) in a single dose to eliminate any natural strongyle infection. After confirmation of worm-free status, the two sheep received 4000 L3 of H. contortus (SpHco2), and the other two were infected with 4000 L3 of H. placei (SpHpl1). They were kept in separate pens to avoid cross-contamination, and during the trial, they had free access to tap water and hay (Cynodon dactylon cv. Tifton 85) purchased from a farm with no ruminants to avoid the risk of food contamination with infective nematode larvae. Fecal cultures from samples from each of the sheep were performed separately for the production of the L3 used to infect the experimental sheep. 2.2. Experimental design The trial design is summarized in Fig. 1. The trial was carried out with 32 male Suffolk lambs. All of the lambs were acquired from a commercial farm located in Paranapanema, São Paulo state, Brazil, just after weaning (2–3 months of age) and had an average body weight of 19 kg. Initially, the lambs were placed in pens with concrete floors in a facility for small ruminants at the University, and all animals were vaccinated against clostridiosis (Sintoxan Polivalente T®, Merial, Brazil) and received treatment with a single dose of monepantel (2.5 mg/kg, Zolvix®, Novartis Animal Health). After treatment, the determination of their worm-free status was based on a series of fecal examinations. The lambs were randomly allocated to two groups and were artificially infected orally with a single dose of 4000 L3 of either H. contortus or H. placei. The H. placei group (n = 16) was infected four weeks after monepantel treatment on January 14, 2014 (day 0), and the H. contortus group (n = 16) was infected 11 days later, on January 25, 2014. This period between the infections was established to allow both species to attain patency at the same time (Riggs, 2001; Santos et al., 2014b). All animals were kept indoors, and 35 days after H. placei infection (February 18, 2014), they were allocated together in the same pasture containing Brachiaria decumbens and Cynodon spp. (0.6 ha), where they remained until the end of the trial. For this reason, we called them “permanent lambs”. The pasture was considered worm free because it had not been grazed by ruminants for 18 months. Therefore, environmental contamination and subsequent reinfections were derived from Haemonchus eggs shed by experimentally infected sheep. The animals had free access to water and mineral salt. They were also fed daily with concentrate (Suplementa Ovino Campo® - Presence, with 17 % crude protein (CP)) in an amount corresponding to 1.5 % of their body weight until May. During the winter and beginning of spring, hay (Cynodon dactylon cv. Tifton 85 with 8 % CP) was offered, and after this period, the animals fed exclusively on the forage available in the paddock. This management technique allowed the sheep to experience moderate bodyweight gain: they weighed an average ( ± standard deviation) of 27.7 ± 2.72 kg at the start of the trial on January 14, 2014, and they weighed 55.1 ± 8.17 kg at the final sampling on February 10, 2015. Permanent lambs were not treated with anthelmintics during the trial.
2. Materials and methods The trial was approved and conducted in accordance with the experimental protocol approved by the local ethical committee (Protocol Number 449-CEEA-UNESP, Institute of Biosciences, Brazil). The experiment was conducted in the experimental area of the University (22° 53′ 16′' S, 48° 29′ 57′' W; altitude 840 m above sea level) located in Botucatu, São Paulo state, Brazil. According to the Köppen classification system, the region’s predominant climate type is Cwa (humid subtropical), which is characterized by warm, rainy summers and dry winters (Alvares et al., 2014). The average accumulated 2
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Fig. 1. Experimental design presenting information about the permanent sheep and the worm-free tracer lambs introduced into the pasture to graze with the flock.
2.5.1. Production and identification of third-stage larvae (L3) Composite fecal cultures for the production of L3 were separately prepared every two weeks for each group of permanent sheep, with an equal volume of feces from each animal according to the descriptions of Ueno and Gonçalves (1998). Differentiation of H. placei and H. contortus L3 was based on the measurement of the sheath tail (distance between the tip of the larval tail and the end of the sheath tail) of the larvae using standard light microscopy (Leica DM LS2, Germany) with an ocular micrometer (Zeiss®, Germany). The sheath tail is consistently shorter in H. contortus than in H. placei (van Wyk et al., 2004; Santos et al., 2014b). Throughout the trial, measurements were taken from 200 L3 from each group on each sampling day. In some fecal cultures that produced fewer than 200 Haemonchus L3, all larvae present were measured. The measurements of L3 obtained from the initial infection with L3 and of L3 obtained from the first fecal culture after infection (18 February 2014) (Table 1) were used to infer H. contortus and H. placei infection dynamics in the permanent lambs. We assumed that L3 that measured < 86 μm were H. contortus, and those that measured > 87 μm were H. placei, whereas intermediate values between 86 and 87 μm were considered to be unsuitable for species differentiation due to the overlap between H. contortus and H. placei measurements (Table 1).
2.3. Euthanasia of permanent lambs To evaluate the establishment of the worms, two permanent lambs from each experimental group were euthanized on February 24, 2014, which was 30 days postinfection (DPI) with H. contortus and 41 DPI with H. placei, to recover, identify and quantify the worms. Subsequently, three animals from each group were euthanized every three months (May, August and November) to recover, identify and quantify the worms (Fig. 1). On the last sampling day of the following year (February 2015), the five remaining animals in each group were euthanized.
2.4. Tracer lambs To better evaluate the L3 population in the pasture, worm-free tracer sheep were placed in the pasture 14 days before the necropsy date of the permanent sheep. Two tracer animals were introduced to the pasture, where they grazed together with the permanent animals for two weeks on four occasions (May, August, November and February) (Fig. 1). Then, the tracer animals were housed indoors for 20 days, and at the end of this period, they were euthanized for worm recovery, quantification, and identification.
2.5.2. Worm counts The animals were fasted for 12 h before being euthanized. The abomasum was opened along the greater curvature, and its content was placed in a graduated beaker and washed with saline solution so that the parasites adhering to the mucosa detached from the organ. The volume was brought up to 1 L with saline solution, and the content was homogenized and divided into two containers of 500 mL each. The containers were immediately frozen for the preservation of parasites. The mucosal layers of all abomasums were soaked in saline solution at 38 °C for 4 h. All digested material was collected and frozen.
2.5. Laboratory analyses Every 14 days, blood and fecal samples were obtained from each permanent sheep to evaluate the packed cell volume (PCV) and fecal egg count (FEC), respectively. The feces were collected directly from the rectum of animals for egg counting using a modified McMaster technique, in which each nematode egg counted represented 100 eggs per gram (EPG) (Ueno and Gonçalves, 1998).
Table 1 Measurement in μm of the sheath tail (distance between the tip of the larval tail and the end of the sheath tail) of infective H. placei and H. contortus larvae (L3). Description of the larvae
Species
n
Minimum-maximum
Mean ± SD
(1) L3 used to infect the permanent lambs
H. H. H. H.
100 100 200 200
60.3–79.4 85.7–104.8 63.8–86.2 86.2–111.7
66.0 ± 4.57 95.1 ± 3.55 72.1 ± 6.35 97.4 ± 4.67
(2) L3 from the first evaluation of the permanent lambs
contortus placei contortus placei
(1) L3 produced in fecal cultures from samples from donor lambs. (2) L3 produced in fecal cultures from samples taken from each group of permanent lambs on February 18, 2014. 3
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presented the following sheath tail length measures: H. contortus had a mean ± SD of 72.1 ± 6.35 μm, and H. placei had a mean ± SD of 97.4 ± 4.67 μm.
Worm counts and identification procedures were performed with 500 mL of one of the containers plus all the content of the digested material. If no worms were found in those samples, the remaining content (500 mL) was also carefully examined for worms. The juvenile forms of the parasites were morphologically identified, and the sex determination of adults was carried out by observing the presence or absence of a copulatory bursa (Ueno and Gonçalves, 1998). For Haemonchus species morphological evaluation, 10 adult male worms were randomly chosen from each permanent sheep and from each tracer lamb for body, spicule and hook length assessment, as previously described by Santos et al. (2014b). In brief, the worms were placed on a glass slide and measured with a ruler. Then, with an insulin syringe needle, the worms were cut near the copulatory bursa to measure the spicules and hooks, while the anterior region of the body of the parasite was stored in a 1.5 mL Eppendorf tube. Each tube contained 150 μL of ultrapure water and was immediately frozen for subsequent DNA extraction.
3.2. Dynamics of infection during grazing (permanent animals) Infected lambs were grazed together in the same clean pasture for the first time on February 18. This coincided with a peak in FECs: 7063 ± 4998 EPG were detected in the group infected with H. contortus, and 6394 ± 3388 EPG were detected in the group infected with H. placei (Fig. 2). The parasitism caused a reduction in PCV values; on average, in the H. contortus group, the PCV was 36 % ( ± 3.16) on January 14, 11 days before the infection, and declined to 24 % ( ± 2.83) at 59 DPI on March 25, while in the H. placei group, the PCV was 35 % ( ± 2.92) on the day of infection (January 14) and declined to 23 % ( ± 3.41) at 70 DPI (March 25). Decrease in FECs in May and June (Fig. 2) were associated with increases in PCV values. On June 3, the mean PCV was 31 % ( ± 6.0) and 34 % ( ± 2.4) in the H. contortus and H. placei groups, respectively. As expected, the group initially infected with H. placei began the trial with larvae consistent with H. placei measurements. However, in March, it was possible to observe L3 with H. contortus measurements (4 %), although the H. placei larval percentage (93 %) was still high (Fig. 2). In the following month, April, at 85 DPI, H. contortus larvae predominated (95 %). In this group, a small percentage (0.25 %) of L3 with H. placei measurements was recorded for the last time in August. The group initially infected with H. contortus presented, from March to August, some L3 with H. placei measurements (above 87 μm) but in low percentages, with a maximum of 2 % in June (Fig. 2).
2.5.3. Molecular evaluation These analyses aimed to identify the Haemonchus species and evaluate the occurrence of hybridization between H. placei and H. contortus. When possible, 10 adult male worms from each permanent sheep were collected for the measurement of spicules and DNA extraction. When an animal hosted fewer than 10 adult male worms, females and/or juveniles were used to obtain a total of 10 worms per sheep for DNA extraction. In the case of the tracer sheep, in addition to the 10 male worms used for the morphometric assessment, DNA samples were also extracted from an additional 10 males; thus, a total of 20 adult male worms were collected for molecular analysis from each tracer lamb. Genomic DNA was extracted from each Haemonchus specimen separately using a QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Bovine and sheep blood DNA samples were also extracted for use as controls in the PCR analyses. The species-specific primer pair for the identification of H. contortus (HcBotuF1/R2) resulted in an amplification band of 260 base pairs (bp), and the species-specific primer pair for the identification of H. placei (HpBotuF/R) resulted in an amplification band of 459 bp (Amarante et al., 2017). 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, California, USA).
3.3. Worm burden and morphological and molecular evaluation of Haemonchus To illustrate the results of the molecular analysis, Fig. 3 shows the PCR products obtained from 3 worms of each Haemonchus species obtained from different permanent sheep. Fig. 4 shows the products obtained from two male hybrids that were amplified with both speciesspecific primer pairs. One worm hybrid was found in a tracer lamb (n. 22) that was grazed in May and another in a permanent sheep (n. 25) that was euthanized in August. The morphometry of adult males is presented in Fig. 5; the body length, spicule and hook measurements of H. placei were larger than those of H. contortus (P < 0.001). 3.3.1. Permanent lambs The highest worm burdens were observed at the first euthanasia in February; there were 2203 and 2728 worms at 30 DPI in two lambs from the H. contortus group and 2600 and 1940 worms at 41 DPI in two lambs from the H. placei group (Table 2). These results demonstrate that artificial infection with 4000 L3 resulted in a high degree of establishment in both species. Necropsies on the other euthanasia dates revealed distinct individual variation in the number of recovered worms. For example, on the second euthanasia date in May, two animals showed a high total worm burden: one animal initially infected with H. contortus (5767 worms, 117 DPI) and another animal initially infected with H. placei (2503 worms, 128 DPI). In contrast, one of the sheep did not present worms (Table 2). In general, over the experimental period, the worm burden in both of the experimental groups decreased, except on the last date of euthanasia (11 February 2015, 382 DPI), when one animal showed a high total worm burden of 18,755 worms. Of the 272 worms analyzed by PCR, only 21 (7.72 %) were H. placei, with amplification bands of 459 bp. These worms were recovered exclusively in February and May (2014) from sheep that initially underwent artificial infection with H. placei (Table 2). Of the remaining, 250 (91.91 %) were identified as H. contortus (with PCR products of 260 bp), and only one male worm was identified as a hybrid (0.37 %), showing amplification bands of both 260 and 459 bp (Fig. 4). This hybrid worm
2.6. Statistical analyses The results are presented descriptively. Data on adult worm measurements were analyzed with unpaired t-tests at a significance level of 1 %. 3. Results 3.1. Measurements of L3 and establishment of infection The L3 used to infect the permanent lambs presented the following sheath tail length measurements: H. contortus had a mean ± standard deviation (SD) of 66.0 ± 4.57 μm, and H. placei had a mean ± SD of 95.1 ± 3.55 μm (Table 1). To confirm the establishment and identity of the species, two permanent lambs from each group were euthanized on February 24. On average, 61.6 % and 56.8 % of H. contortus and H. placei L3 had established, respectively. The examination of adult male worms by PCR confirmed the species identity (Table 2). Additionally, the infective larvae presented the morphological characteristics of their respective species (Fig. 2) in the first evaluation of L3 from composite fecal cultures from samples collected from each group on February 18, 2014. The L3, which were derived from only the artificial infection, 4
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Table 2 Worm burden and identification of Haemonchus worms (Haemonchus contortus, Hc; Haemonchus placei, Hp) by PCR. Permanent lambs were artificially infected with 4000 L3 of H. placei (Group Hp, n = 16) on January 14 (2014) or with H. contortus (Group Hc, n = 16) on January 25, 2014. Sheep shared the same pasture for 357 days starting on February 18, 2014 and were exposed to reinfection by both Haemonchus species. Euthanasia date
Group
ID
Adults
Feb 24, 2014
Hc
21 29 18 20 11 13 23 8 16 26 15 25 28 2 3 14 5 19 27 6 10 12 1 7 9 17 31 4 22 24 30 32
Males 956 1262 1251 2173 1 1200 1 0 765 11 274 40 541 0 510 196 4 42 231 1272 4 0 0 189 11 8766 0 255 15 190 18 2
Hp May 22, 2014
Hc
Hp
Aug 23, 2014
Hc
Hp
Nov 19, 2014
Hc
Hp
Feb 11, 2015
Hc
Hp
Females 1237 1466 1319 1656 0 2329 0 0 1390 18 300 38 962 2 854 241 13 57 461 1376 30 0 0 224 178 9949 0 1141 44 482 93 34
Juveniles
Total worms
PCR*
10 0 30 40 10 2238 0 0 348 4 171 61 54 0 30 73 4 16 2 44 3 7 4 120 6 40 1 214 1 1 96 17
2203 2728 2600 1940 11 5767 1 0 2503 33 745 139 1557 2 1394 510 21 115 694 2692 37 7 4 533 195 18755 1 1610 60 673 207 53
Hp 0 0 10 10 0 0 0 – 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Hc 10 10 0 0 9 10 1 – 9 10 10 9 10 2 10 10 10 10 10 10 10 6 3 10 10 10 1 10 10 10 10 10
Hybrid 0 0 0 0 0 0 0 – 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
ID: Sheep identification number. * When possible, 10 adult male worms were individually identified per animal. Some sheep had fewer than 10 worms.
with H. placei, the H. placei population was gradually replaced by H. contortus and became apparently extinct from the experimental site by the end of the trial. Several factors may have caused the disappearance of H. placei, including the role of the immune response in the hostparasite relationship. In comparison with those infected with H. contortus, a strong immune response was observed in sheep repeatedly infected with H. placei, limiting the establishment of this species (Santos et al., 2014b). In the present trial, the permanent sheep were constantly reinfected with L3 during grazing, which might have induced the development of an intense immune response against H. placei, with a consequent reduction in the establishment of this species. Similarly, in experimentally infected sheep, Le Jambre (1983) observed an 80 % decrease in H. placei after the sheep were inoculated with H. contortus, demonstrating the ability of H. contortus to exclude and dislodge H. placei from the sheep. In sheep and cattle that share pastures in São Paulo state, it has also been demonstrated that Haemonchus presents high host specificity (Amarante et al., 1997; Silva et al., 2015). Interspecific crossbreeding and the production of hybrids has been reported in mixed infections (natural or artificial) of H. contortus and H. placei (Bremner, 1955; Le Jambre, 1979, 1981; Brasil et al., 2012; Chaudhry et al., 2015; Santos et al., 2019b). A total of 432 worms (from permanent and tracer animals) were analyzed by PCR using speciesspecific primer pairs. Of these, only two (0.46 %) male worms were identified as hybrids. These results are in accordance with the low percentage of hybrids observed in field studies by Brasil et al. (2012) and Chaudhry et al. (2015) and when sheep undergo simultaneous artificial infections with both species (Santos et al., 2019b). The low production of hybrids might be due to the existence of a mating barrier between the species. Such a barrier has been reported even among
was recovered from a sheep initially infected with H. contortus and euthanized in August 2014 (Table 2). 3.3.2. Tracer animals Tracer sheep always acquired Haemonchus infection (Table 3), demonstrating the presence of Haemonchus L3 in the pasture throughout the trial. From the 160 specimens recovered from the tracer sheep and evaluated by PCR analyses, 140 (87.5 %) were identified as H. contortus, 19 (11.9 %) were identified as H. placei, and only one male worm (0.6 %) was identified as a hybrid, with amplification bands of both 260 and 459 bp (Table 3, Fig. 4). Only tracer animals that grazed in May acquired H. placei infection. 4. Discussion Both H. placei and H. contortus isolates had a high establishment rate in the permanent lambs following artificial infection. This result is in agreement with the findings of a previous trial with the same isolates that showed a peak of 6380 EPG occurring 45 days after a single inoculation with 4000 L3 of H. placei in young sheep, followed by a progressive decline, but sheep were still shedding eggs in their feces 304 DPI (Santos et al., 2014b). Such results demonstrate that sheep are a suitable host for H. placei. For this reason, the quick “disappearance” of the H. placei population was unexpected in the present study. In the present trial, the last trace of H. placei was found in August, approximately seven months after the experimental infection, when we found a small percentage (0.5 %) of infective larvae with H. placei morphology in the fecal culture. Thus, in the group initially infected 5
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Fig. 4. For each DNA sample, PCRs were performed separately for each primer pair: (A) PCR results for the species-specific primers HcBotuF1/R2 for Haemonchus contortus; and (B) PCR results for the species-specific primers HpBotuF/R for Haemonchus placei. Lanes 1 and 9–100 bp molecular markers (GE Healthcare); lanes 2 and 3 - bovine and ovine DNA samples, respectively (hosts); lane 4 - H. contortus DNA sample (control); lane 5 - H. placei DNA sample (control); lane 6 –male hybrid DNA sample from the worm from the tracer lamb n. 22 (grazing period: May 2014); lane 7 –male hybrid DNA sample from the worm from the permanent sheep n. 25 (euthanized in August 2014) and lane 8 - reagents without DNA.
different strains of H. contortus, such as MHco3(ISE), MHco4(WRS) and MHc10(CAVR), which originated on different continents. Coinfecting strains interbreed to some extent, but there are significant mating barriers between MHco10(CAVR) and the other two strains (Sargison et al., 2019). Nevertheless, in artificial backcrossing, male and female hybrids can be semisterile, possibly perpetuating a low level of reproductive efficiency within the population (Le Jambre, 1981). Therefore, further studies are necessary to evaluate the possible role of hybrid production in the introgression of genes from the H. placei to the H. contortus population and vice versa. The results of the morphological analysis of the sheath tail of infective larvae and spicule morphometrics matched those of the PCR analysis. H. placei presented larger of body length, spicule and hook measurements than H. contortus, in agreement with other studies (Lichtenfels et al., 1994; Santos et al., 2014b, Santos et al., 2019b). Our results showed that the H. placei population declined over a relatively short time period, while H. contortus increased and dominated the parasitic abomasal fauna of the sheep. Therefore, management systems using cattle and sheep to produce clean pastures could be a useful tool for the prevention of haemonchosis, corroborating previous studies (Fernandes et al., 2004; d’Alexis et al., 2012; Brito et al., 2013). In conclusion, it was demonstrated that H. placei established successfully in artificially infected worm-free sheep; however, under concomitant natural reinfection with H. contortus, the H. placei population experienced a rapid decrease and was eliminated from an environment without cattle. Additionally, during H. contortus and H. placei coinfection, a small proportion of hybrids were produced.
Fig. 2. Mean eggs per gram of feces and species composition of the lambs artificially infected with 4000 L3 of Haemonchus placei (Group H. placei; n = 16) on January 14 or with Haemonchus contortus (Group H. contortus; n = 16) on January 25. The sheep shared the same pasture for 357 days (from February 18, 2014, until February 10, 2015) and were exposed to reinfection by both Haemonchus species. Species composition based on the identification of thirdstage larvae (L3) produced in fecal cultures. Measurement of the sheath tails: H. contortus < 86 μm; H. placei > 87 μm; and intermediate measurement between 86 μm and 87 μm.
Declaration of Competing Interest The authors declare no conflict of interest.
Fig. 3. For each DNA sample, PCRs were performed separately for each primer pair: (A) PCR results for the species-specific primers HcBotuF1/R2 for Haemonchus contortus; and (B) PCR results for the species-specific primers HpBotuF/R for Haemonchus placei. Lane 1–100 bp molecular markers (GE Healthcare); Lanes 2 and 3 –bovine and ovine DNA, respectively (hosts); Lane 4 –H. contortus DNA sample (control); Lane 5 - H. placei DNA sample (control); Lanes 6, 8 and 11 –H. contortus DNA samples from different permanent sheep; Lanes 9, 10 and 12 –H. placei DNA samples from different permanent sheep; and Lanes 7 and 13 –reagents without DNA.
Acknowledgments The authors are grateful for the technical assistance provided by Adriana M. D. Anaya, César C. Bassetto, José H. Neves, Gabriela F. Caetano, Maria Regina L. Silva, Nadino Carvalho and Natália B. Pinto. Michelle C. Santos received financial support from the Sao Paulo Research Foundation (FAPESP, Grant Number 2012/23941-2 and 6
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Fig. 5. Morphometrics of male adult worms of Haemonchus contortus (n = 209) and H. placei (n = 21). PCR analysis was used as the gold standard for Haemonchus spp. identification. The ends of the box are the upper and lower quartiles; the median is marked by a vertical line inside the box; and the two lines outside the box extend to the highest and lowest observations. For all variables, there was a significant difference between Haemonchus species (P < 0.001).
Table 3 Worm burden of the tracer lambs and identification of Haemonchus worms (Haemonchus contortus, Hc; Haemonchus placei, Hp) by PCR. Tracers grazed together with the permanent sheep for 14 days. After this period of grazing, the tracer animals were kept indoors for 20 days, and at the end of this period, they were euthanized for the recovery, quantification and identification of the parasites. Grazing period
May 8–22, 2014 Aug 8–22, 2014 Nov 4–20, 2014 Jan 27 to Feb 10, 2015
Tracer
Adults
Juveniles
Males
Females
9 22 51 62 42 25
223 342 73 413 715 848
345 336 80 534 1057 1026
51 28 11 0 0 166
45
449
580
0
Total
contortus and Haemonchus placei in sheep from Santana do Livramento, Brazil. Rev. Bras. Parasitol. Vet. 27, 280–288. Alvares, C.A., Stape, J.L., Sentelhas, P.C., Gonçalves, J.L.M., Sparovek, G., 2014. Köppen’s climate classification map for Brazil. Meteorol. Z. 22, 711–728. Amarante, A.F.T., Bangola Junior, J., Amarante, M.R.V., Barbosa, M.A., 1997. Host specificity of sheep and cattle nematodes in Sao Paulo state, Brazil. Vet. Parasitol. 73, 89–104. Amarante, M.R.V., Santos, M.C., Bassetto, C.C., Amarante, A.F.T., 2017. PCR primers for straightforward differentiation of Haemonchus contortus, Haemonchus placei and its hybrids. J. Helmintol. 91, 757–761. Avramenko, R.W., Redman, E.M., Lewis, R., Bichuette, M.A., Palmeira, B.M., Yazwinski, T.A., Gilleard, J.S., 2017. The use of nemabiome metabarcoding to explore gastrointestinal nematode species diversity and anthelmintic treatment effectiveness in beef calves. Int. J. Parasitol. 47, 893–902. Avramenko, R.W., Bras, A., Redman, E.M., Woodbury, M.R., Wagner, B., Shury, T., Liccioli, S., Windeyer, M.C., Gilleard, J.S., 2018. High species diversity of trichostrongyle parasite communities within and between Western Canadian commercial and conservation bison herds revealed by nemabiome metabarcoding. Parasit. Vectors 11, 299. https://doi.org/10.1186/s13071-018-2880-y. Bailey, J.N., Walkden-Brown, S.W., Kahn, L.P., 2009. Comparison of strategies to provide lambing paddocks of low gastro-intestinal nematode infectivity in a summer rainfall region of Australia. Vet. Parasitol. 161, 218–231. Bassetto, C.C., Silva, B.F., Newlands, 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, L.R., Bastianetto, 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, 469–479. Bremner, K.C., 1955. Cytological studies on the specific distinctness of the ovine and bovine “strains” of the nematodeHaemonchus contortus (Rudolphi) Cobb (Nematoda: Trichostrongylidae). Aust. J. Zool. 3, 312–323. Brito, D.L., Dallago, B.S.L., Louvandini, H., Santos, V.R.V., Torres, S.E.F.A., Gomes, E.F., Amarante, A.F.T., Melo, C.B., McManus, C.M., 2013. Effect of alternate and simultaneous grazing on endoparasite infection in sheep and cattle. Rev. Bras. Parasitol. Vet. 22, 485–494. Cerutti, M.C., Citterio, C.V., Bazzocchi, C., Epis, S., D’Amelio, S., Ferrari, N., Lanfranchi, P., 2010. Genetic variability of Haemonchus contortus(Nematoda: Trichostrongyloidea) in alpine ruminant host species. J. Helminthol. 84, 276–283. Chaudhry, U., Redman, E.M., Abbas, M., Muthusamy, R., Ashraf, K., Gilleard, J.S., 2015. Genetic evidence for diagnosticon between Haemonchus contortus and Haemonchus placei in natural field populations and its implications for interspecies transmission of anthelmintic resistance. Int. J. Parasitol. 45, 149–159. 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. d’Alexis, S., Sauvant, D., Boval, M., 2014. Mixed grazing systems of sheep and cattle to improve liveweight gain: a quantitative review. J. Agric. Sci. 152, 655–666. Escobedo, J.F., Gomes, E.N., Oliveira, A.P., Soares, J., 2011. Ratios of UV, PAR and NIR
PCR* Hp
Hc
Hybrid
619 706 164 947 1772 2040
5 14 0 0 0 0
15 5 40 20 20 20
0 1 0 0 0 0
1029
0
20
0
On August 8, a tracer lamb died due to an undetermined cause, and only one lamb (Number 51) was euthanized. For this reason, 40 adult male worms were used from this tracer. * Twenty adult male worms were individually identified by PCR per animal.
2015/12900-1). Alessandro F. T. Amarante is the recipient of a fellowship from CNPq (305187/2017-1). This study was supported by the São Paulo Research Foundation (FAPESP - Grant Number 2014/023056). 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. Agneessens, J., Claerebout, E., Dorny, P., Borgsteede, F.H., Vercruysse, J., 2000. Nematode parasitism in adult dairy cows in Belgium. Vet. Parasitol. 90, 83–92. Albuquerque, A.C.A., Bassetto, C.C., Almeida, F.A., Amarante, A.F.T., 2017. Development of Haemonchus contortus resistance in sheep under suppressive or targeted selective treatment with monepantel. Vet. Parasitol. 246, 112–117. Almeida, F.A., Bassetto, C.C., Amarante, M.R.V., Albuquerque, A.C.A., Starling, R.Z.C., Amarante, A.F.T., 2018. Helminth infections and hybridization between Haemonchus
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