Analysis of the stability of the reference genes GAPDH, SDHA and RPL-19 in sheep from a semi-arid region infected by gastrointestinal nematodes

Abstract Analyzing the stability of reference genes already described as universal is an important methodology to lead gene expression analysis because different studies have shown that the expression of universal reference genes may vary between experimental treatments. In this sense, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Succinate dehydrogenase complex subunit A (SDHA) and Ribosomal Protein L-19 (RPL-19) reference genes (already described in other studies with sheep from different regions, breeds and infectious agents or in organisms evolutionarily close to sheep) were investigated in the abomasum, small and large intestines of resistant and susceptible crossbred sheep groups to gastrointestinal nematode infections in the Semi-arid region in Northeast of Brazil. The animals were naturally infected to determine the resistance or susceptibility status by counting eggs per gram (EPG) of feces from the gastrointestinal tract after 33 weeks of observations of infection evolution. Relative gene expression was performed by RT-qPCR methodology using Sybr green and relative gene expression stability was tested by different software programs such as REST, BestKeeper, geNorm and Normfinder. Our results showed the susceptible animals had increase in egg counts per gram of feces than resistant animals (p < 0.001), and both groups showed a mixed infection by nematodes of the genus Haemonchus, Trichostrongylus, Oesophagostomum and Trichuris. Furthermore, we show the importance of analyzing different genes in different software programs and the importance to choose ideal reference genes. In this sense, GAPDH was the most stable gene in the abomasum, whereas SDHA was the most stable in the small and large intestines. In addition, we discuss about variables which can interfere in relative expression such as breed, species, climate and tissue. However, utilizing other reference genes already described in other studies with the same and different variables should be performed

The effect of Brachiaria brizantha cultivars on host-parasite-environment interactions in sheep naturally infected with gastrointestinal nematodes.

The objective was to evaluate the effect of different cultivars of Brachiaria brizantha on the dynamics and concentration of the larval stages of gastrointestinal nematodes in the soil and forage strata, as well as their effects on the performance of naturally infected lambs. Overall, 48 90-day-old lambs with an initial weight of 19.04 ± 0.96 kg were observed. Moreover, a randomised block factorial design with four cultivars of Brachiaria brizantha (Marandu, Xaraés, Piatã and Paiaguás grasses) under intermittent stocking (with a pre-grazing canopy height of 40 cm and post-grazing canopy height of 20 cm) for two grazing cycles was used. The following variables were analysed: faecal egg counting, faecal culture, mean corpuscular volume, FAMACHA© score, weight and body condition score, the recovery of larvae from pasture and soil samples, nutritional value and the production and structural components of forage. Lambs grazing Marandu grass demonstrated the highest level of nematode infection (P < 0.05). However, the nutritional value did not differ between cultivars. Marandu grass had the highest pasture density (P < 0.05), while Paiaguás grass had the highest percentage of dead material (P < 0.05). The various genera of gastrointestinal nematodes found in the faecal cultures, regardless of the cultivars, include Haemonchus (92.01%), Trichostrongylus (4.55%), Strongyloides (3.06%) and Oesophagostomum (0.37%). Lambs grazing Xaraés grass had the lowest body weight (P < 0.05). Furthermore, larvae concentrations were highest in Marandu and Paiaguás pastures; infective Haemonchus and Trichostrongylus larvae were recovered from pasture and soil samples. The different cultivars of Brachiaria brizantha produce diverse and relevant microclimatic conditions to contaminate soil, pastures and animals. Animal performance was not compromised despite the Marandu and Paiaguás cultivars having the highest levels of contamination and infection. Based on parasitological aspects, the Brachiaria brizantha cultivars Xaraés and Piatã are recommended for grass-based sheep production systems over the other cultivars since they contribute to the reduction of larval contamination and infection