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Bovine besnoitiosis caused by Besnoitia besnoiti is a chronic and debilitating disease. The most characteristic clinical signs of chronic besnoitiosis are visible tissue cysts in the scleral conjunctiva and the vagina, thickened skin and a generally poor body condition. However, many seropositive animals remain subclinically infected, and the role that these animals may play in spreading the disease is not known. The aim of the present study was to assess the intra-organ parasite distribution, the parasite load and the parasite-associated lesions in seropositive but subclinically infected animals. These animals were seropositive at the time of several consecutive samplings, had visible tissue cysts in the past and, at time of slaughter, had detectable specific anti-Besnoitia spp. antibody levels, but they did not show evident clinical signs at culling. Thus, histopathological, immunohistochemical and molecular analyses of several samples from the respiratory tract, reproductive tract, other internal organs and skin from six cows were performed. The tissue cysts were located primarily in the upper respiratory tract, i.e., in the rhinarium and larynx/pharynx (four cows), followed by the distal genital tract (vulva/vagina) and the skin of the neck (three and two cows, respectively, out of the four cows with cysts in the respiratory tract). We were unable to detect any parasites in the two remaining cows. Cysts were associated with a significant non-purulent inflammatory infiltrate consisting predominantly of T lymphocytes and activated monocytes/macrophages in two cows. The parasite burden, estimated by quantitative real-time PCR, was very low. It is noteworthy that the only animal that showed a recent increase in the antibody titre had the highest parasite burden and the most conspicuous inflammatory reaction against the cysts. In conclusion, although these cows no longer displayed any visible signs of besnoitiosis, they remained infected. Therefore, cows without visible signs of disease may still be able to transmit the parasite.

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The study was conducted to evaluate the effect of age at weaning and breed on the stress response of calves to weaning and their temperament. At calving, 14 Parda de Montañ a calves and 14 Pirenaica calves were randomly assigned to either early weaning (at 90 days) or traditional weaning (at 150 days) treatment. During nursing, calves were allowed to suckle their dams twice a day for 30 min. After weaning, calves were placed in an adjacent barn without access to their dams, where they remained for 7 days. On day 8 after weaning, they were transported to a feedlot where they received an intensive diet. Blood samples were taken 168 h before weaning (baseline) and 6, 24, 48 and 168 h after weaning for cortisol, fibrinogen and haematology analyses, and temperament was measured 90 and 180 days after weaning with the flight speed test. Cortisol concentration increased after weaning, irrespective of age at weaning. Early-weaned calves had a lower fibrinogen baseline and a greater increase in fibrinogen concentrations 48 h after weaning than traditionally weaned calves. Moreover, fibrinogen concentration returned to baseline values 168 h after weaning in traditionally weaned calves, whereas it remained high in early-weaned calves. Concerning breed effects, Pirenaica calves had higher cortisol concentration and fibrinogen increments after weaning than Parda de Montañ a calves. Slight alterations occurred after weaning in haematology, but all parameters returned to baseline values 168 h after weaning, with no significant effects of age at weaning or breed. Despite the absence of clinical signs, early-weaned calves of both breeds suffered marginal anaemia, according to haemoglobin values. Regardless of age at weaning, Pirenaica calves had greater reactivity to human presence than Parda de Montañ a calves, according to their higher flight speed values measured. Finally, early-weaned calves were lighter than traditionally weaned calves at weaning, but had similar weight gains in the feedlot. Consequently, they needed an additional 40 days to reach the target weight, irrespective of breed. Therefore, age at weaning had no major effects on the stress response to weaning or temperament, but early weaning increased the length of the feedlot period. On the other hand, Pirenaica calves were more reactive than Parda de Montañ a calves to the stress of weaning and human presence.