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| Author(s): |
Michelle Cardoso dos Santos
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Botucatu. 2017-04-25. |
| Institution: | Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu |
| Defense date: | 2017-02-21 |
| Advisor: | Alessandro Francisco Talamini do Amarante |
| Abstract | |
Two trials were performed from 2013 until 2015 in Suffolk sheep breed, these studies aimed to evaluate the dynamics of mixed infection by Haemonchus contortus and Haemonchus placei in sheep, as well as interspecific competition and mating with the hybrid production. Initially, one worm-free donor lamb received orally, in a single dose, artificial infection with 25,000 infective larvae (L3) of H. contortus, laboratory isolate SpHco2, while the donor calf was infected orally, in a single dose, with 25,000 of H. placei, laboratory isolate SpHpl1. In the first trial, performed in 2013, worm-free lambs kept indoors received initially 2,000 L3 of H. placei on day zero and, 11 days later the same animals were infected with 2,000 L3 of H. contortus (parental group, n=6). Individual fecal cultures from parental animals were performed and were used to L3 production, which was used to the infection of the animals from F1 group (first filial generation), groups F1-42 DPI (n=6) and F1-84 DPI (n=6), which allowed the hybrid production. All animals from groups: parental, F1-42 DPI and F1-84 DPI were euthanized 50, 42 and 84 days post-infection (DPI), respectively, for the identification and quantification of the parasites in order to determine the interspecific competition and hybridization. Six animals remained as a non-infected control. On the second trial, developed in 2014 and 2015, worm-free lambs were infected with 4,000 L3 of H. placei (n=16) or 4,000 L3 of H. contortus (n=16). These animals were initially kept indoors to confirm the establishment of the infections and then the animals were allocated to the same pastures, which were shared for 357 days. To evaluate the progression of the infection, two animals for each experimental group were euthanized in February 2014 and the subsequent euthanasia occurred every three months, with three animals from each experimental group, except on the last euthanasia, in February 2015 in which the remaining five animals from each group were euthanized. Worm-free tracer animals were introduced in the pasture every three months to evaluate the pasture contamination and nematode species present. In both studies, the animals were weighed and blood and feces samples were collected on the same day to laboratory analysis. In both trials, the animals were euthanized for the parasites recovery and identification. In the first trial, in the feedlot, there was observed the establishment of the infection for both species, with high fecal egg counts (FEC). The parasites recovered were identified by morphological and molecular techniques, proving the success of the infection. Polymerase chain reaction (PCR) reactions with the oligonucleotide (primers) species-specific showed the presence of H. contortus and H. placei in all experimental groups (groups parental, F1-42 DPI and F1-84 DPI) and eight hybrids (3.6% of the total of parasites evaluated), which were recovered from F1-42 DPI and F1-84 DPI groups. In the second trial, there was also the establishment of the infection for both species, with high FEC and low packed cell volume levels in the initial collections. Measurements of the L3 as well as morphological and molecular evaluation of the adult specimens, of the experimental and tracer sheep demonstrate the elimination of the H. placei over time. Two helminths were identified as hybrids using the same species-specific primers. On the last experiment, the mutation for levamisole resistance gene was evaluated by conventional PCR. These primers provided amplification only in H. contortus specimens. Of 20 adult specimens of H. contortus from the donor lamb 85% (n=17) showed homozygous resistant and 15% (n=3), heterozygous. From experimental animals described in trial 1 were evaluated 142 H. contortus specimens and 97.6% (n=123) were homozygous resistant and 2.4% (n=3) as heterozygous. In conclusion, the establishment of mixed infections by H. contortus and H. placei in sheep occurs, and the interspecific mating can produce F1 with hybrids offspring. Nonetheless, when sheep infected with H. contortus and H. placei shared the same pastures, interspecific competition promotes the extinction of H. placei (parasite from cattle) with the presence of only H. contortus, specie that has the sheep as preferred host. In addition, the primers pairs to levamisole resistance did not present amplification bands with DNA samples of H. placei - however, the SpHco2 laboratory isolate (H. contortus) showed molecular resistance to levamisole. (AU) | |
| FAPESP's process: | 12/23941-2 - Interaction between Haemonchus contortus and Haemonchus placei infections in sheep |
| Grantee: | Michelle Cardoso dos Santos |
| Support Opportunities: | Scholarships in Brazil - Doctorate |