Is there competition between Haemonchus contortus and Haemonchus placei in a pasture grazed by only sheep?

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.

A panel of microsatellite markers to discriminate and study interactions between Haemonchus contortus and Haemonchus placei.

Haemonchus contortus and Haemonchus placei are two closely related economically important parasites of ruminants. Their close morphological similarity, common occurrence as co-infections and ability to hybridize makes definitive diagnosis and epidemiological studies in field populations challenging. In this paper, we describe the development of a panel of microsatellite markers that can be used to discriminate and study the genetics of these two parasite species in co-infections and mixed field populations. We have identified two additional microsatellites (Hp52 and Hp53), in addition to three previously reported microsatellites (Hcms3561, Hcms53265 and Hcms36) that have a discrete set of alleles between the two species. Multilocus genotyping of worms with this 5 marker panel from 3 geographically diverse H. placei isolates and 4 geographically diverse H. contortus populations allows unambiguous species assignment of individual worms. This panel of markers should provide a valuable resource in studying the biology and epidemiology of these important ruminant parasite species in the field.

Immune response to Haemonchus contortus and Haemonchus placei in sheep and its role on parasite specificity.

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 anti-parasitic 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.

Environmental factors influencing the transmission of Haemonchus contortus.

Infection with the gastrointestinal nematode Haemonchus contortus causes considerable losses in the sheep industry. In this study, we evaluated the effect that climate has on third-stage larvae (L3) of H. contortus in terms of their migration from sheep feces to Brachiaria decumbens grass, as well as their distribution among the forage plants. Fecal samples containing H. contortus L3 was deposited on the soil among the herbage at an initial height of 30 cm. Sample collection began 24h after contamination and was performed on alternate days over 13 days. The L3 were recovered and quantified in three strata (heights) of grass (0-10 cm, 10-20 cm and >20 cm) as well as in the remaining feces and a superficial layer of soil, collected from beneath the feces. In order to obtain results under different environmental conditions, fecal samples containing H. contortus L3 were deposited on pasture in January (summer), in April (autumn), and July (winter). In all of the periods, the L3 were able to migrate from the feces to the herbage. However, rains, accompanied by high relative humidity and high temperatures, apparently favored migration. The highest L3 recovery rate in the pasture was in the summer observation period, which had the highest number of days with measurable precipitation, high relative humidity (>68.2%), and the highest temperatures at the soil level (minimum and maximum means of 19°C and 42°C, respectively). Under those conditions, larvae began to reach the upper stratum of the grass (>20 cm) by 24h after the deposition of fecal matter, the number of larvae having reached that stratum peaking at seven days after deposition. In the autumn observation period, there was no rainfall in the first five days post-contamination. During that period, high numbers of larvae were found in the fecal samples demonstrating that feces can act as a reservoir of larvae in the absence of rain. Except for two days in the summer observation period, when most of the L3 were recovered from the tops of blades of grass, L3 where located predominantly at the base of the herbage. In conclusion, rainfall favors the migration of L3 from feces to herbage. In addition, larval migration up and along blades of grass can occur relatively rapidly when the temperature is high.