D. Lacasta

Management and environmental factors related to benzimidazole resistance in sheep nematodes in Northeast Spain

A survey to determine the level of parasite resistance to benzimidazoles (BZ) under field conditions was performed on 107 commercial sheep farms located in the Aragon region of northeast Spain. Resistance was measured using the discriminant dose, a simplified form of the in vitro egg hatch assay (EHA). Taking into account the spatial structure of the data, a multivariate approach was applied to management and environmental variables as well as to their relationships with BZ resistance levels compiled from each flock. Results estimated that 11% of flocks had resistant parasite populations, although we suspected the presence of BZ-resistant parasite strains in 98% of the sample. Resistance levels were more similar among the nearest flocks, suggesting a contagious spatial distribution of resistance (i.e., resistance at neighbouring farms was not independent from one another). Management variables such as frequency of deworming, grazing in private pastures and underdosing were positively related to resistance levels, whereas only the use of BZ was negatively related to resistance levels, likely because BZ were replaced by other anthelmintics in flocks where reduced BZ efficacy was suspected. In addition to climatic conditions and seasonality, land use was an environmental variable associated with observed BZ resistance levels. Generally, resistance was highest in cooler and wetter areas but was lower in flocks sampled during January-March compared to flocks sampled in April-June or October-December. Variation partitioning procedures showed that the variation of resistance explained by the effect of environmental variables was higher than management variables. The effects of both variable groups, however, highly overlapped with the spatial structure of resistant levels, which suggests that a considerable amount of the effects attributable to both variable groups may be actually due to the spatial distribution of resistance. The resistance variation explained by the spatial component suggested that other uncontrolled factors acting at short spatial scale (e.g., common management and environmental variables; the importation of resistant strains and their posterior spread across neighbouring flocks; the selection history of the worms carried out by historical management events previous to this survey; and genetic, physiological or both types of parasite population variation) could yield this contagious spatial structure of BZ resistance. Although further research is needed, both seasonal variation and the dependence of resistance levels among neighbouring flocks should be taken into account in the design of future research or observational resistance programmes to minimise spatial and temporal pseudo-replication. Thus, research would avoid biased estimations of resistance prevalence or of its relationship with putative factors.

Natural border disease virus infection in feedlot lambs

Border disease is caused by border disease virus (BDV, a pestivirus from the family Flaviviridae ) infection in sheep and goats (Vantsis and others 1976). BDV infection causes sizeable economic losses in sheep production around the world. In Spain, serological surveys have found 100 per cent flocks and 8–93 per cent sheep seropositive, respectively (Valdazo-Gonzalez and others 2006). BD is considered a congenital disease, but infections in healthy animals at all age groups may also occur. These are named acute infections and are characterised by transitory leucopaenia and fever associated with viraemia (Nettleton and others 1998). The aim of this study was to evaluate the impact of these acute infections in lambs in a commercial feedlot. A longitudinal observational study in a feedlot located in Aragon (Spain) was carried out. Lambs were supplied to the feedlot at minimum 10 kg bodyweight (BW) (45 days old), housed in groups of 250–300 per pen and slaughtered at 25–35 kg BW (about 45 days later). Thirty-six male lambs were randomly selected, ear-tagged, weighed, clinically evaluated and sampled on days: 0 (day lambs entered the feedlot), 14, 27 and 41. From each lamb, whole blood in anticoagulant solution and serum samples for haematological, virological and serological studies were taken. Haematological analysis with an electronic counter …

Variability of the egg hatch assay to survey benzimidazole resistance in nematodes of small ruminants under field conditions

The egg hatch assay (EHA) is one of the main in vitro methods for detection of benzimidazole resistance in nematode parasites of small ruminants. However, although the EHA has been standardised at the laboratory level, the diagnostic performance of this method has not been fully characterised for field screenings. In the present work, monthly variation of benzimidazole resistance estimated by EHA was surveyed over two years in three sheep flocks and in one goat and an additional sheep flock sharing the same pastures. Resistance was measured by calculating both the effective dose of thiabendazole (TBZ) that inhibited hatching of ≥50% of parasite eggs (ED₅₀) and the proportion (Pdd) of eggs hatching at a discriminating dose of 0.1 μg/ml TBZ. Pdd exhibited higher variability than ED₅₀, in agreement with the higher sensitivity of Pdd to changes in resistance levels. Both resistance parameters, however, were highly correlated, and their variation was similarly related to the same factors. Resistance levels differed among sheep flocks, and the resistance level of the goat flock was higher than that measured for the sheep flock sharing the same pasture. Moreover, monthly variation of resistance in goats did not mirror that recorded in sheep. Resistance levels varied seasonally, with the highest values recorded in the spring, and they were inversely related to the number of days that samples were stored under anaerobic conditions. In addition, they were directly associated with the relative abundance of Teladorsagia spp. but inversely related to the relative abundance of Haemonchus spp. After controlling for the effects of these identified factors for variation, inter-monthly sampling variation due to unknown factors was the main source of variability, accounting for more than 60-70% of variance for both resistance parameters and yielding absolute estimation errors higher than 0.06 for ED₅₀ or 0.2 for Pdd when resistance was estimated from a single sampling. Optimum sample size, estimated from variance components, suggested that at least 4-5 samplings would be needed to halve this absolute error, whereas additional samplings would slightly increase precision but at the cost of substantially increasing sampling effort. More research is needed to identify the main factors involved in this inter-sampling variation to standardise the implementation of EHA under field conditions.

Chronic Proliferative Rhinitis associated with Salmonella enterica subspecies diarizonae serovar 61:k:1, 5, (7) in Sheep in Spain

A chronic proliferative rhinitis in sheep associated with Salmonella enterica subspecies diarizonae serovar 61:k:1, 5, (7) is described. Ten adult sheep from eight traditionally managed Rasa Aragonesa flocks located in Aragon (Spain) were studied. Clinical signs began with bilateral thick mucus nasal discharge, wheezing and snoring and progressed to partial or complete obstruction of the nostrils. Necropsy examination revealed swollen ventral nasal turbinates with a roughened mucosal surface partially covered by small polyps. Histopathology revealed chronic proliferative rhinitis with a predominant population of neutrophils that infiltrated the mucosal epithelium. Plasma cells and macrophages were present in the lamina propria. Organisms expressing Salmonella antigen immunohistochemically were detected within epithelial cells. Salmonella enterica subspecies diarizonae serovar 61:k:1, 5, (7) was isolated as a sole microorganism from nasal swabs taken from five animals. The implication of finding this bacterium in various diseases in sheep and its role as a potential zoonosis are discussed.

A case of ovine female XY sex reversal syndrome not related to anomalies in the sex-determining region Y (SRY).

The human XY gonadal sex reversal is a rare disease. Primary amenorrhea, lack of secondary sexual characteristics and the possible existence of streak gonads are the most common signs of this syndrome. Five cases of this syndrome have been described in the last 42 years in bovines. However, to the knowledge of the authors, no similar descriptions are available in sheep. In the present report, we are presenting the case of a 4 year old ewe 2n = 54,XY showing primary sterility and total absence of masculinization signs. The promoter and the complete coding segment of the sex determining region Y (SRY) were sequenced and found to be absolutely normal. The presence of the testis-specific protein gene (TSPY) was also verified by means of PCR. Necropsy findings include short vagina, lack of uterus and complete absence of masculine remains. Streak gonads concordant with previous descriptions in humans were also found.

Ovine Heritable Arthrogryposis Multiplex Congenita with Probable Lethal Autosomal Recessive Mutation

A novel congenital syndrome in lambs is described that is characterized by: (1) kyphoscoliosis (severe curvature, hunching and twisting of the thoracic spine, with associated abnormalities of the ribs and sternum), (2) distal arthrogryposis of the carpal and tarsal joints, and (3) cleft hard and soft palate or palatoschisis (a median fissure of the palate). Male and female lambs are affected equally and in multiple births there may be only one affected lamb. The affected lambs are born full term, but die shortly after birth because they are unable to start breathing. Affected lambs have slightly reduced bodyweight (due to low muscle mass) compared with normal newborn lambs of the same flock. The syndrome is remarkably similar to the recently described bovine heritable arthrogryposis multiplex congenita in the Angus breed. Genetic analysis of the flock by means of DNA microsatellite markers, together with study of the reproductive management of the flock, allowed us to discount an environmental aetiology (e.g. intoxications or infections). In contrast, evidence supports an autosomal recessive inherited disease for which the name ovine heritable arthrogryposis multiplex congenita is proposed.

Persistent efficacy of a long acting injectable formulation of moxidectin against natural infestations of the sheep nasal bot (Oestrus ovis) in Spain

Cydectin(®) 2% LA Solution for Injection for Sheep (Pfizer Animal Health) is a long-acting (LA) formulation of moxidectin for the treatment and prevention of mixed infections of gastro-intestinal nematodes, respiratory nematodes and certain arthropod parasites in sheep. To evaluate the duration of persistent efficacy against nasal bots (Oestrus ovis), a natural exposure study was conducted in Spain during the summer of 2011. One hundred and twenty nasal bot-free, Rasa Aragonesa sheep were randomly allocated to eight groups of 15 animals each. On Day 0, four groups were treated at the recommended dose rate of 1 mg moxidectin/kg bodyweight. Four groups remained untreated as negative controls. All animals were held in nasal bot-proof housing except for exposure to natural challenge when one group of treated sheep and one of group of control animals were transferred to a local pasture at either 0-20, 20-40, 40-60, or 60-80 days after treatment. Following challenge, sheep were scored for clinical signs of bot infestation, necropsied and the heads sectioned for larval recovery. Nasal bot larvae were retrieved from 7 to 11 control sheep following each exposure period indicating that adult bots were active throughout the study. In the first challenge up to 20 days after treatment, when sheep were slaughtered immediately after exposure, the majority of larvae were first instar (L1) and only 3 of the 15 control sheep were infested with second instars (L2). There was 100% efficacy against L2 and 38.1% reduction in the number of live L1 in the treated sheep but mean counts were not significantly different between treatment and control groups (P ≥ 0.05). For the subsequent exposure periods 20-80 days after treatment (necropsies 7-9 days after challenge), 6-10 sheep were infested with L1 and 9-11 control sheep were infested with L2 and third instars (L3). There was negligible efficacy against L1, but treatment with moxidectin resulted in 100% control of L2 and L3. These results are consistent with the biology of nasal bots and control with a systemic agent, as the slower growing L1 have limited feeding and are therefore less susceptible to systemic parasiticides. The study demonstrated that the persistent efficacy of this long-acting injectable formulation of moxidectin protects against the development of active O. ovis infestations for at least 80 days after treatment.

Ovine congenital myotonia associated with a mutation in the muscle chloride channel gene

Congenital myotonia (CM) is characterised by a delay in muscular relaxation after sudden contractions. In a recent outbreak of ovine CM affecting 1% of new-born lambs in a Spanish flock of Rasa Aragonesa sheep, a comparative pathology approach was taken: because a mutation in the muscle chloride channel gene (CLCN1) was identified as responsible for CM in goats, the same gene was sequenced in the affected lambs. A non-synonymous single nucleotide variation (SNV) in the second exon of CLCN1 was associated with this pathology. Rams carrying this SNV heterozygously were thereafter identified and replaced by wild-type homozygous young males. No additional CM cases were detected in subsequent lambing seasons.

SQUAMOUS CELL CARCINOMA OF THE VAGINA AND CERVIX IN SHEEP - CASE REPORT

This report describes the clinical and histopathological characteristics of a squamous cell carcinoma infiltrating the cervix and the vaginal wall, producing reproductive symptoms and subnormal fertility in an adult ewe. Necropsy showed a large (15-cm-long) neoplastic mass infiltrating the vaginal wall and the cervix. Histopathological examination revealed atypical squamous epithelial cords invading the basal membrane and dermis, round anaplastic cells, focal areas of necrosis, keratinisation of isolated cells, and pronounced infiltration by mononuclear cells around the cords. No squamous cell carcinoma of such localisation has been reported from sheep before. In humans, this tumour is the most common gynaecological malignancy in the world.

Dissemination of intestinal pathogens between lambs and puppies in sheep farms

Abstract Objectives of the present work were (i) to confirm pathogens implicated in cases of diarrhoea in newborn and young lambs in sheep farms in Greece and (ii) to investigate a possible relation in dissemination of pathogens between lambs and dogs present in the farm. Work was carried out in 22 sheep farms, with (i) flock size over 150 animals, (ii) presence of clinical signs of diarrhoea in lambs in the flock and (iii) close and continuous contact and movement of shepherd dogs within the animal shed of each farm. Faecal sample collection from lambs was performed within 48h of onset of clinical signs and prior to administration of any antimicrobial or antiparasitic medication to lambs. Faecal samples were also collected from puppies in the farm. In total, samples were collected from 126 lambs and 58 puppies. Samples were processed by using established techniques for isolation of bacteria, detection of viruses and observation of protozoan oocycts. Escherichia coli isolates obtained during the study, were tested for antimicrobial resistance against a variety of antimicrobial agents. In total, 236 bacterial isolates were recovered from faecal samples of lambs and 165 isolates from faecal samples of puppies. E. coli was the most frequently isolated microorganism: 104 isolates from lambs and 109 isolates from puppies were recovered. Other bacteria isolated were Enterobacter spp., Proteus spp., Klebsiella spp., (lambs and puppies), Clostridium perfringens, Citrobacter freundi, Salmonella enterica subsp. diarizonae (only lambs) and Streptococcus spp. (only puppies). Group A Rotavirus was detected in samples from lambs (2.5%) and Parvovirus in samples from puppies (5%). Cryptosporidium spp. oocysts were observed in samples from lambs and puppies. This is the first report of isolation of S. enterica subsp. diarizonae and of detection of Rotavirus from lambs in Greece. Rates of E. coli isolates from puppies resistant to antimicrobial agents were, in general, smaller than respective rates in isolates from lambs. Two pairs of isolates from the same farm (one from a lamb and one from a puppy) with identical patterns of resistance to antimicrobial agents were detected, which provides some evidence in support of a hypothesis that members of each pair might possibly have been spread from one animal species to the other.