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The aim of this study was to assess the suitability of PCR and ELISA as diagnostic method in young sheep naturally infested by Oestrus ovis larvae. The experiment was carried out from December 2020 to April 2021 with 39 lambs divided into two groups: infested (n = 26) and control treated group (n = 13). The infested group did not receive treatment against oestrosis, and the control group was treated with closantel (10 mg/kg orally) every 28 days in order to keep the animals as free as possible of O. ovis infestation. The clinical signs varied among animals regardless of the number of recovered larvae of each lamb, however, the thick mucus and mucopurulent nasal discharge scores were less frequent in lambs from treated group. There was no correlation between the nasal discharge score and the number of O. ovis recovered larvae (R² = 0.012, P = 0.165). Three control treated animals only presented first instar larvae (L1) (1 - 4 larvae/animal) which were smaller than L1 found in the lambs of the infested group. Ninety-two percent of the lambs from infested group (24/26) were parasitized by O. ovis with number ranging from 1 to 54 larvae per animal. A gradual increase in plasma IgG (anti-antigen of O. ovis larvae) levels of animals from infested group after the third week of the trial was observed, whereas the control lambs had low levels of IgG until the end of the experiment. The PCR had low sensitivity (26 %) and high specificity (100 %), and it presented poor agreement (k = 0.177) with the larvae detection after the lamb slaughter. The oestrosis clinical signs were not related to larvae infestation intensity and ELISA showed a greater advantage over the PCR technique in identifying animals that are carrying O. ovis., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The sheep nasal bots Oestrus ovis is parasite of the nasal cavities and sinuses of small ruminants causing oestrosis, one of the most frequent parasitic diseases in sheep and goats. The widely use of ivermectin and closantel by the sheep breeders in the treatment and prophylaxis of gastrointestinal nematodes resulted in widespread cases of anthelmintic resistance. However, there is no report about cases of O. ovis with drug-resistance. In this study, we evaluated the prophylactics and therapeutic effects of both antiparasitics in sheep with O. ovis natural infestation. The trial was carried out from early December 2019 to March 2020, with 30 crossbred males lambs allocated into three groups of 10 animals each: control (without treatment), treated with ivermectin (0.2 mg/kg subcutaneously) and treated with closantel (10 mg/kg orally). The animals were kept together grazing the same pasture area. The treatment groups were drenched in two occasions 70 days apart: on 5th December 2019 and on 13th February 2020. On 19th March 2020, all lambs were slaughtered. The lamb heads were removed and sectioned along their longitudinal and sagittal axis to search for larvae. Recovered O. ovis larvae were counted and identified according to their developmental stage (L1, L2, and L3). Seven of the control lambs were infested with O. ovis larvae ranging from six to 17 larvae (11.6 mean infestation intensity). All recovered larvae from control group were intact and active. Three animals treated with ivermectin had O. ovis larvae (1-3 larvae), however they were dead and in degeneration. The animals treated with closantel did not have any larvae. The clinical suggestive signs of oestrosis were scarce over the experimental period. The averages of daily weight gain were similar ( p > 0.05) among groups. Closantel and ivermectin had high efficacy against oestrosis and O. ovis parasitism did not hinder the performance of lambs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bello, Lins, Albuquerque, Ferreira, Amarante and Amarante.)

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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study aimed to evaluate the simultaneous infections of Haemonchus contortus and Haemonchus placei in sheep, as well as the production of hybrids. A parental group of lambs (n = 6) were mix-infected with 2000 infective larvae (L3) of H. placei and 2000 L3 of H. contortus. Faecal samples were taken from each of these six lambs to produce the first generation of L3 (F1-L3) in individual cultures. These F1-L3 were used to infect 12 lambs; six of them were euthanized at 42 days (Group F1-42) and six at 84 days (Group F1-84) post infection. Polymerase chain reaction (PCR) analysis, using species-specific primer pairs, was the gold standard method for identification of Haemonchus adult species and hybrids. The establishment rate of both species was similar in the parental group: 51.7% H. contortus and 48.3% H. placei. Of the 219 adult specimens from groups F1-42 and F1-84 analysed by PCR, eight (3.65%) were hybrids, 111 were H. contortus and 100 were H. placei. The morphological evaluation of the F1-L3 from the parental group showed a predominance of larvae with H. contortus size (51.5%) in comparison with H. placei (42.8%). In the second generation of L3 (F2-L3) produced by the F1-lambs, larvae with H. contortus morphology predominated, with 81.5% in the F1-42 group and 84.0% in the F1-84 group. In conclusion, an artificial mixed infection by H. contortus and H. placei was established in lambs and resulted in the production of a small number of hybrids among their offspring.

The occurrence and intensity of helminth infections were evaluated in sheep from pastures shared with cattle. In 2015 and 2016, young male sheep acquired in Santana do Livramento, Rio Grande do Sul, Brazil, were finished in integrated crop-livestock system. We selected the 12 sheep that showed the highest number of nematode eggs per gram of faeces to search for worms in the gastrointestinal tract. Haemonchus contortus and Trichostrongylus colubriformis were the major parasites. H. contortus presented mean intensities of 1,159 and 257 worms in 2015 and 2016, respectively. T. colubriformis displayed mean intensities of 4,149 and 2,427 worms in 2015 and 2016, respectively. Of the 127 male specimens of Haemonchus spp. analysed by Polymerase Chain Reaction (PCR), 125 were H. contortus, one Haemonchus placei and one hybrid. Other species detected were Cooperia punctata , Cooperia pectinata, Cooperia spatulata, Cooperia curticei, Ostertagia ostertagi, Teladorsagia circumcincta , Trichostrongylus axei, Nematodirus spathiger , and Trichuris ovis. Twenty lambs presented cysts of Taenia hydatigena in the liver and mesentery. One lamb presented Coenurus cerebralis, the larval stage of Taenia multiceps, in the brain. In conclusion, sheep from pasture shared with cattle presented a high diversity of nematode species. H. contortus and H. placei co-infection occur with consequent hybridization.

Haemonchus contortus and Haemonchus placei are among the major parasites of small ruminants and cattle. Although infection with these nematodes is host-specific, with H. placei predominating in cattle and H. contortus in sheep, cross-infections are observed in areas where both parasites are sympatric, and hybrid offspring can occur. Therefore, a fast and precise method is required for differentiating the parasites. Identification based on spicule morphometry is the most common technique for differentiating Haemonchus species. However, because these measurements overlap between species, morphological analysis is insufficient for differentiating between helminth species. In this work, we present a reliable, conventional polymerase chain reaction (PCR)-based method that uses two species-specific primer pairs to differentiate between H. contortus and H. placei specimens and their hybrids. Each primer pair produces one single and distinct amplification band for each species, which enables the detection of hybrid specimens. These primer pairs were validated by testing eight different populations of H. contortus, H. placei and hybrids.

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)