Unfavorable genetic correlations between fecal egg count and milk production traits in the French blond-faced Manech dairy sheep breed.

BACKGROUND: Genetic selection has proven to be a successful strategy for the sustainable control of gastrointestinal parasitism in sheep. However, little is known on the relationship between resistance to parasites and production traits in dairy breeds. In this study, we estimated the heritabilities and genetic correlations for resistance to parasites and milk production traits in the blond-faced Manech breed. The resistance to parasites of 951 rams from the selection scheme was measured through fecal egg counts (FEC) at 30 days post-infection under experimental conditions. Six milk production traits [milk yield (MY), fat yield (FY), protein yield (PY), fat content (FC), protein content (PC) and somatic cell score (LSCS)], were used in this study and were collected on 140,127 dairy ewes in first lactation, as part of the official milk recording. These ewes were related to the 951 rams (65% of the ewes were daughters of the rams). RESULTS: Fecal egg counts at the end of the first and second infections were moderately heritable (0.19 and 0.37, respectively) and highly correlated (0.93). Heritabilities were moderate for milk yields (ranging from 0.24 to 0.29 for MY, FY and PY) and high for FC (0.35) and PC (0.48). MY was negatively correlated with FC and PC (- 0.39 and - 0.45, respectively). FEC at the end of the second infection were positively correlated with MY, FY and PY (0.28, 0.29 and 0.24, respectively with standard errors of ~ 0.10). These slightly unfavorable correlations indicate that the animals with a high production potential are genetically more susceptible to gastrointestinal parasite infections. A low negative correlation (- 0.17) was also found between FEC after the second infection and LSCS, which suggests that there is a small genetic antagonism between resistance to gastrointestinal parasites and resistance to mastitis, which is another important health trait in dairy sheep. CONCLUSIONS: Our results indicate an unfavorable but low genetic relationship between resistance to gastrointestinal parasites and milk production traits in the blond-faced Manech breed. These results will help the breeders' association make decisions about how to include resistance to parasites in the selection objective.

Asymptomatic Cryptosporidium infections in ewes and lambs are a source of environmental contamination with zoonotic genotypes of Cryptosporidium parvum.

Protozoan parasites of the Cryptosporidium genus cause severe cryptosporidiosis in newborn lambs. However, asymptomatic infections also occur frequently in lambs and ewes. In sheep, the most commonly detected Cryptosporidium species are C. ubiquitum, C. xiaoi and C. parvum. Due to a lack of relevant information about such infections in France, we investigated the situation on five dairy sheep farms in the Pyrénées-Atlantiques Department in south-western France in December 2017. Individual fecal samples were collected from 79 female lambs (5-17 days old) and their mothers (72 ewes). Oocysts were screened using Heine staining before and after Bailenger concentrations. Cryptosporidium species identification and genotyping were performed using real-time PCR and gp60 gene sequencing. No cases of clinical cryptosporidiosis were observed in the 79 lambs. Microscopically, Cryptosporidium spp. oocysts were observed in only one lamb on one farm (prevalence 1.3%) and one ewe on another farm (prevalence 1.4%). By contrast, Cryptosporidium spp. DNA was detected in 17 ewes (prevalence ranging from 10.5% to 50% depending on the farm) and in 36 lambs (prevalence ranging from 0% to 77.8% depending on the farm). Only zoonotic Cryptosporidium parvum IId and IIa genotypes were identified when genotyping was possible. Cryptosporidium ubiquitum and C. xiaoi were detected on one and three farms, respectively. We conclude that healthy young lambs and their mothers during the peripartum period could be a source of environmental contamination with oocysts.

TITLE: Les infections asymptomatiques par Cryptosporidium chez les brebis et les agneaux sont une source de contamination environnementale par les génotypes zoonotiques de Cryptosporidium parvum. ABSTRACT: Les parasites protozoaires du genre Cryptosporidium provoquent une cryptosporidiose sévère chez les agneaux nouveau-nés. Cependant, des infections asymptomatiques surviennent aussi fréquemment chez les agneaux et les brebis. Chez les ovins, les espèces de Cryptosporidium les plus couramment détectées sont C. ubiquitum, C. xiaoi et C. parvum. En raison d'un manque d'informations pertinentes sur ces infections en France, nous avons enquêté sur la situation de cinq élevages ovins laitiers des Pyrénées-Atlantiques en décembre 2017. Des échantillons fécaux individuels ont été collectés sur 79 agnelles (5 à 17 jours) et leurs mères (72 brebis). Les oocystes ont été criblés en utilisant une coloration Heine avant et après concentration par la technique de Bailenger. L'identification et le génotypage des espèces de Cryptosporidium ont été réalisés à l'aide de la PCR en temps réel et du séquençage du gène gp60. Aucun cas de cryptosporidiose clinique n'a été observé chez les 79 agneaux. Au microscope, les oocystes de Cryptosporidium spp. n'ont été observés que chez un agneau dans une ferme (prévalence 1,3 %) et chez une brebis dans une autre ferme (prévalence 1,4 %). En revanche, de l'ADN de Cryptosporidium spp. a été détecté chez 17 brebis (prévalence allant de 10,5 % à 50 % selon les fermes) et chez 36 agneaux (prévalence variant de 0 % à 77,8 % selon les fermes). Seuls les génotypes zoonotiques de Cryptosporidium parvum IId et IIa ont été identifiés lorsque le génotypage était possible. Cryptosporidium ubiquitum et C. xiaoi ont été détectés respectivement dans une et trois fermes. Nous concluons que les jeunes agneaux en bonne santé et leurs mères, autour de l'agnelage, pourraient être une source de contamination environnementale par les oocystes.

Real-time PCR on skin biopsies for super-spreaders' detection in bovine besnoitiosis.

BACKGROUND: Bovine besnoitiosis, an emerging disease in Europe that can be transmitted by vectors, is caused by the apicomplexan Besnoitia besnoiti. Bovine besnoitiosis is difficult to control due to the complexity of its diagnosis in the acute stage of the disease, poor treatment success and chronically asymptomatic cattle acting as parasite reservoirs. When serological prevalence is low, detection and specific culling of seropositive cattle is feasible; however, economic considerations preclude this approach when serological prevalence is high. The aims of this study were to evaluate the accuracy of detection of super-spreaders in highly infected herds and to test their selective elimination as a new control strategy for bovine besnoitiosis. METHODS: Previous real-time PCR analyses performed on skin tissues from 160 asymptomatic animals sampled at slaughterhouses showed that the tail base was the best location to evaluate the dermal parasite DNA load. All seropositive animals (n = 518) from eight dairy or beef cattle farms facing a high serological prevalence of besnoitiosis were sampled at the tail base and their skin sample analysed by real-time PCR. A recommendation of rapid and selective culling of super-spreaders was formulated and provided to the cattle breeders. Subsequent serological monitoring of naïve animals was used to evaluate the interest of this control strategy over time. RESULTS: Among the 518 seropositive animals, a low proportion of individuals (14.5%) showed Cq values below 36, 17.8% had doubtful results (36 < Cq ≤ 40) and 67.8% had negative PCR results. These proportions were grossly similar on the eight farms, regardless of their production type (beef or dairy cattle), size, geographical location or history of besnoitiosis. Within two weeks of the biopsy, the rapid culling of super-spreaders was implemented on only three farms. The numbers of newly infected animals were lower on these farms compared to those where super-spreaders were maintained in the herd. CONCLUSIONS: Real-time PCR analyses performed on skin biopsies of seropositive cattle showed huge individual variabilities in parasite DNA load. The rapid culling of individuals considered as super-spreaders seems to be a new and encouraging strategy for bovine besnoitiosis control.

Attractiveness and Specificity of Different Polyethylene Blue Screens on Stomoxys calcitrans (Diptera: Muscidae).

Stomoxys calcitrans is considered as a major pest of livestock worldwide. Insecticides have been extensively used to control this pest but resistance to these chemical compounds is now reported in many countries. Therefore, a more sustainable and efficient control is needed. Seven different types of blue screens, with reflectances around 460 nm, were tested during summer 2016 in southwestern France to evaluate their attractiveness and their specificity for stable flies. Height of the screen and orientation (east or west) of a blue screen were also considered. High levels of S. calcitrans captures were recorded during this study (from 141 to 7301 individuals per blue screen and per day) whereas the numbers of tabanids and pollinator insects remained extremely low (less than 10 individuals per screen and per day). No significant difference in attractiveness has been shown between the different types of blue screens. The lower half of the blue screens caught significantly more stable flies (70%) than the higher half (30%). The "east" side of the screen attracted 60% of stable flies but this was not significantly different from the west side. These results are highlighting the interest in these blue polyethylene screens for controlling stable flies in cattle farms, in comparison with more expensive blue fabrics.

First report of multiple resistance to eprinomectin and benzimidazole in Haemonchus contortus on a dairy goat farm in France.

Pour-on eprinomectin was recently registered for lactating small ruminants. Given the high prevalence of benzimidazole resistance in gastrointestinal nematodes in dairy goats, many farmers use eprinomectin exclusively to treat their animals. On a French dairy goat farm, a veterinary practitioner noted a poor response to two types of eprinomectin treatment (pour-on application and injectable formulation). Therefore, we evaluated the efficacy of both formulations of eprinomectin, as well as moxidectin and fenbendazole, using the fecal egg count reduction test (FECRT) according to the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines. Nematode species were identified at days 0 and post-treatment days 14 after bulk larval cultures, by morphology and real-time PCR. Plasma concentrations of eprinomectin were analyzed by high-performance liquid chromatography (HPLC) at post-treatment days 2 and 5 in the eprinomectin-treated groups. Egg count reductions were poor in animals treated with topical (-16.7%; 95% CI:[-237; 59]) or subcutaneous (21.5%; 95% CI:[-126; 73]) eprinomectin, and with fenbendazole (-5.8%; 95% CI:[-205; 63]). Haemonchus contortus was the main species identified by morphology and by real-time PCR before and after treatment. The plasma concentrations of eprinomectin were determined in all eprinomectin-treated animals and were above 2 ng/ml at post-treatment day 2, indicating that the lack of effect was not due to low exposure of the worms to the drug. Interestingly, moxidectin remained effective in all infected animals. This is the first report of multiple resistance to eprinomectin and benzimidazole in H. contortus on a French dairy goat farm with moxidectin as a relevant alternative.

Experimental infections of rabbits with proliferative and latent stages of Besnoitia besnoiti.

Cattle besnoitiosis due to Besnoitia besnoiti is spreading across Europe and is responsible for severe economic losses in newly infected herds. Experimentally speaking, rabbits have been found to be susceptible to this parasite. The adaptation of B. besnoiti to rabbits may offer a new, easier and cheaper model of investigation for this disease. This study compared the virulence between tachyzoites and bradyzoites of B. besnoiti in rabbits. Eighteen New Zealand rabbits were allocated into three groups of six animals each. The rabbits from the control (group C), "tachyzoite" (group T) and "bradyzoite" (group B) groups were subcutaneously injected in the right flank with 66 µg of ovalbumin, 6.10(6) tachyzoites (125th passage on Vero cells) and 6.10(6) bradyzoites (collected from a natural infected cow) of B. besnoiti, respectively. Clinical follow-up and blood sampling for serological survey and qPCR were performed during 10 weeks until euthanasia. Molecular and immunohistochemistry examination was achieved on 25 samples of tissue per rabbit. Seroconversion occurred in group T without any clinical signs. Rabbits of group B exhibited a febrile condition (temperature above 40 °C from day 8 to day 11 following injection) with positive qPCR in blood. Cysts of B. besnoiti were found on skin samples and organs of rabbits from group B in tissue explored with threshold cycle (Ct) values below 30. These results suggest a higher virulence of bradyzoites in rabbits than Vero cell-cultivated tachyzoites. The proposed model could be used to assess the in vivo effectiveness of vaccine or drugs against cattle besnoitiosis.

Functional investigation of a QTL affecting resistance to Haemonchus contortus in sheep.

This study reports a functional characterization of a limited segment (QTL) of sheep chromosome 12 associated with resistance to the abomasal nematode Haemonchus contortus. The first objective was to validate the identified QTL through the comparison of genetically susceptible (N) and resistant (R) sheep produced from Martinik × Romane back-cross sheep. The R and N genotype groups were then experimentally infected with 10 000 H. contortus larvae and measured for FEC (every three days from 18 to 30 days post-challenge), haematocrit, worm burden and fertility. Significant differences in FEC and haematocrit drop were found between R and N sheep. In addition, the female worms recovered from R sheep were less fecund. The second step of the characterization was to investigate functional mechanisms associated with the QTL, thanks to a gene expression analysis performed on the abomasal mucosa and the abomasal lymph node. The gene expression level of a candidate gene lying within the QTL region (PAPP-A2) was measured. In addition, putative interactions between the chromosome segment under study and the top ten differentially expressed genes between resistant MBB and susceptible RMN sheep highlighted in a previous microarray experiment were investigated. We found an induction of Th-2 related cytokine genes expression in the abomasal mucosa of R sheep. Down-regulation of the PAPP-A2 gene expression was observed between naïve and challenged sheep although no differential expression was recorded between challenged R and N sheep. The genotyping of this limited region should contribute to the ability to predict the intrinsic resistance level of sheep.

In vitro pre-exposure of Haemonchus contortus L3 to blood eosinophils reduces their establishment potential in sheep.

Different authors have reported that eosinophils are capable of immobilising infective larvae of different species of nematodes in vitro. However, classifying larvae as mobile or immobile is so subjective that it does not always mean all apparently immobile larvae are dead or those that are mobile are capable of surviving further immune responses if administered to their natural hosts. The objective of this experimental study was therefore to substantiate the role of eosinophils in the killing of Haemonchus contortus infective larvae by comparing the infectivity in sheep of larvae that had been incubated with eosinophil-enriched cell suspensions with control larvae. Since it was not possible to isolate pure eosinophils from sheep blood, we were compelled to evaluate the effects of other blood cells contaminating our eosinophil-enriched suspensions. Although eosinophils and neutrophils were the only cells found adherent to H. contortus infective larvae in vitro, induced eosinophils in the presence of immune serum were primarily responsible for the drastic reduction in larval motility compared to the minor effects of neutrophils and mononuclear cells. Corresponding reductions in faecal egg count and worm numbers were observed when the incubated larvae were transferred intra-abomasally to sheep. Interestingly, the proportion of larvae that failed to establish was much higher following incubation with induced eosinophils compared with other cells or with immune serum alone. Although this study did not address the in vivo role of eosinophils in sheep, the results strongly indicate that sheep blood eosinophils have a larval killing potential in vitro, and a larval mobility test alone may not fully explain the level of damage inflicted on the larvae.

Haemonchus contortus (Nematoda: Trichostrongylidae) infection in lambs elicits an unequivocal Th2 immune response.

Selection of resistant animals and host immunization have been proposed as alternative methods for the control of gastrointestinal nematode parasites. However, a better knowledge of the mechanisms involved in protective immunity against these parasites is required for the development of optimal strategies. In this study, 3 month old INRA 401 lambs (n = 81) were allocated into three groups: uninfected control, challenged either once (primary-infected animals) or twice (previously-infected animals) with 10,000 Haemonchus contortus L3. Uninfected control and challenged animals were sequentially sacrificed at 3, 7, 15 and 28 days post challenge. In both challenged groups, a clear Th2-oriented immune response was observed in the abomasal lymph node and mucosa. IL-4 and IL-13 mRNA over-expression, recruitment of eosinophils, mast cells and globule leukocytes and production of specific systemic IgG and mucosal IgA were observed earlier in previously-infected animals than in primary-infected ones. At 28 days post infection, no differences between intensities of these responses were observed between the challenged groups. Worm establishment rates were similar in previously-infected and primary-infected lambs. However, reductions of worm development, female fecundity and fecal egg output were observed in previously-infected sheep. We conclude that H. contortus infection in young INRA 401 lambs induced an unequivocal Th2 immune response resulting in the regulation of worm egg production without affecting their establishment.

Regulation of Oestrus ovis (Diptera: Oestridae) populations in previously exposed and naïve sheep.

Larvae of Oestrus ovis (Insecta: Diptera: Oestridae) are common parasites of nasal and sinus cavities of sheep and goats. During larval development, a specific immune reaction is initiated by the host with a humoral local and systemic response and the recruitment of eosinophils and mast cells in the upper airways mucosae. Nevertheless, the roles of these responses in the regulation of O. ovis larvae populations in sheep are not yet known. The aim of this study was to compare the establishment and the development of larvae as well as some inflammatory or immune parameters between different groups of half-sibling sheep: (i) a primed group experimentally infected twice before a challenge infection, (ii and iii) two groups infected once only and previously treated with a long-lasting corticoïd before the challenge (one group) or not (the other group). A fourth group of non-infected animals was added in the experimental design. The larval establishment rate was 23% in the corticoïd treated group compared to about 10% in the two other infected groups. Moreover, the larval development appeared more rapid in the corticoïd treated group than in the two other infected groups suggesting that the inflammatory response is involved in the regulation of O. ovis populations. By contrast, no differences in the establishment rates were shown in the primed group compared to the naïve group (without corticoïd treatment) despite evidence of higher eosinophilia, serum specific IgG, and immediate hypersensibility to excretory-secretory products of larvae. The specific lymphocyte proliferation was reduced in the primed group compared to the naïve one suggesting that an immuno-suppression occurs following repetitive O. ovis infections.

Cellular and humoral local immune responses in sheep experimentally infected with Oestrus ovis (Diptera: Oestridae).

Cellular and humoral local responses were investigated following repetitive artificial Oestrus ovis infections in lambs. The presence of larvae induced a huge local recruitment of either leucocytes (T and B lymphocytes, macrophages) or granulocytes (eosinophils, mast cells and globule leucocytes). This cellular response was more pronounced in the ethmoid and sinus (development sites of second and third instar larvae) than in the septum or turbinates where first instar larvae migrate. Infected lambs produced Oestrus ovis specific IgG and IgA antibodies in their mucus. This local humoral response was mainly directed against larval salivary gland antigens and not against larval digestive tract antigens. Compared to the control animals, the sinusal mucosa of infected animals was extremely thickened and the epithelium exhibited hyperplasia, metaplasia and eosinophilic exocytosis. The possible roles of these local immune responses in the regulation of O. ovis larvae populations in sheep are discussed.