Major differences in the larval anatomy of the digestive and excretory systems of three Oestridae species revealed by micro-CT.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro-computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a 'glandular band' by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio-opaque concretions. Moreover, the anatomy of O. ovis third-instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a 'band' and anterior Malpighian tubules narrowly uniform throughout their entire length.

Internal morphological changes during metamorphosis in the sheep nasal bot fly, Oestrus ovis.

During the larval stage, oestrid flies (Diptera: Oestridae) are obligate parasites, whereas during the adult stage they are free-living and do not feed. Like other cyclorrhaphous flies, oestrids undergo metamorphosis inside an opaque puparium, formed by the contracted and hardened cuticle of the third-instar larva. The present study documents the internal morphological changes taking place during metamorphosis of the sheep nasal bot fly, Oestrus ovis L., using non-invasive, micro-CT-based virtual histology and provides quantitative data of volumetric changes in specific organs. Virtual histological sections allowed visualisation of the progression and completion of the apolyses, which delimit the different intra-puparial stages, and the connection to the tracheal system of a large gas bubble, which plays an essential role during early metamorphosis. Overall, our results show that the sequence of morphological and volumetric changes in tissues and organs is similar to those found in other cyclorrhaphous flies, but they also reveal developmental differences that result in an adult vestigial digestive tract. Future studies could develop non-invasive, reliable methods for aging the intra-puparial forms of different oestrid species of veterinary importance, based on both qualitative and quantitative markers, thus improving our knowledge of their development and the efficiency of control strategies.

Prevalence of dental and mandibular disorders in culled sheep in Spain.

At the slaughterhouse, 1465 culled sheep heads were studied in order to identify disorders of the mandibular cheek teeth. Of these, 227 (15.5%) had evidence of osteomyelitis. The lesional profile showed that the mandible was affected in a similar proportion on both sides (46.7% on the right side vs 50.7% on the left side), mainly in the middle region (55.3%) and with most of the lesions closed without fistulisation (89.4%). In addition, swelling was palpable, with an increase in thickness in the area of the affected body (2.65 ± 0.065 cm vs 1.74 ± 0.030 cm). In 78.6% of the animals, the regional lymph nodes were enlarged. Concerning the mandibular cheek teeth, more than half of the animals lacked at least one tooth (57.0%), with the first premolar being the most frequently missing tooth (34.8%) and the third molar the least (8.2%). The impaction of food around the teeth was very common with the posterior molars being more frequently affected. In the knowledge of the authors, this is the first study that analyses the prevalence of mandibular molar and premolar disorders in sheep, and these are revealed as an important condition affecting culled sheep.

Micro-computed tomography visualization of the vestigial alimentary canal in adult oestrid flies.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage as they acquire all necessary nutrients during the parasitic larval stage. The adult mouthparts and digestive tract are therefore frequently vestigial; however, morphological data on the alimentary canal in adult oestrid flies are scarce and a proper visualization of this organ system within the adult body is lacking. The present work visualizes the morphology of the alimentary canal in adults of two oestrid species, Oestrus ovis L. and Hypoderma lineatum (de Villiers), with the use of non-invasive micro-computed tomography (micro-CT) and compares it with the highly developed alimentary canal of the blow fly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae). Both O. ovis and H. lineatum adults showed significant reductions of the cardia and the diameter of the digestive tract, an absence of the helicoidal portion of the midgut typical of other cyclorrhaphous flies, and a lack of crop and salivary glands. Given the current interest in the alimentary canal in adult dipterans in biomedical and developmental biology studies, further understanding of the morphology and development of this organ system in adult oestrids may provide valuable new insights in several areas of research.

Technical Note: A new device for cervical insemination of sheep - design and field test.

Deep semen deposition, avoiding retrograde flow, lesions and stress, has proved to be very important in the success of sheep AI. The objective of the present study was to develop a new, suitable anti-retrograde flow device for sheep cervical AI (DARIO) that enables deep deposition of semen into the cervix without any modifications to the procedures currently used, and to compare the fertility, fecundity, and prolificacy rates between DARIO and a traditional catheter. Field tests were performed on 16 farms actively participating in the non-profit National Association of Rasa Aragonesa Breeders´ genetic selection scheme and where sheep management was similar. A total of 242 AI lots were considered, including 1,299 ewes; 126 lots (662 ewes) were inseminated using DARIO, and 116 lots (637 ewes) using a traditional commercially-available catheter (control group). Several factors affecting AI results were included in the model for mean comparison between DARIO and control groups (farm and ram as random factors; catheter, year and photoperiod as fixed effects; catheter × photoperiod interaction). The type of catheter had a significant effect on fertility ( < 0.01) and fecundity rates ( < 0.01) but no significant effect was detected on the prolificacy rate ( = 0.45). For fertility rate (percentage of ewes lambing after AI), means ± SE for DARIO and control groups were 59.44 ± 2.13% and 49.60 ± 2.48%, respectively; for fecundity rates, means ± SE for DARIO and control groups were 0.99 ± 0.04 and 0.82 ± 0.05 lambs/inseminated ewe, respectively, and, for prolificacy rates, means ± SE for DARIO and control groups were 1.68 ± 0.04 and 1.63 ± 0.04 lambs/ewe lambing, respectively. Fertility rate was greater in the decreasing photoperiod ( = 0.01). Significant effects were found for both year ( < 0.05) and farm ( < 0.01) on fertility, fecundity, and prolificacy rates. Neither ram nor catheter × photoperiod showed any significant effects on the variables investigated ( > 0.05). Overall, the use of DARIO instead of the traditional commercially-available catheter increased both fertility and fecundity rates; the marginal mean differences were 9.05 pregnant ewes per 100 inseminated and 0.15 lambs per inseminated ewe, respectively.

Jaagsiekte Sheep Retrovirus Can Reach Peyer's Patches and Mesenteric Lymph Nodes of Lambs Nursed by Infected Mothers.

Ovine pulmonary adenocarcinoma (OPA) is a contagious lung cancer of sheep caused by jaagsiekte sheep retrovirus (JSRV). It is generally accepted that transmission by the respiratory route occurs under natural conditions. However recent studies strongly indicate that JSRV can also be transmitted to lambs perinatally via colostrum and milk (C/M). The aim of this work was to confirm that C/M can transmit JSRV infection to lambs under natural conditions and investigate the initial events associated with this transmission route. We have analyzed the presence of JSRV in C/M samples from 22 naturally infected, asymptomatic ewes throughout a lactation period, and in various tissues collected from a group of 36 of their lambs that were fed naturally. The lambs were euthanized at 12, 24, 48, and 72 hours and at 5 and 10 days after birth. We detected JSRV-provirus by PCR in the somatic C/M cells from 10/22 ewes (45.45%). The virus was also detected in 9/36 lambs (25%). JSRV-infected cells, with lymphoreticular-like morphology, were observed by immunohistochemistry (IHC) and in situ hybridization (ISH) in Peyer's patches (PP) from the small intestine of the youngest lambs and in mesenteric lymph nodes (MLN) from lambs older than 72 hours. The virus was also detected by PCR in white blood cells (WBC) in 2/36 lambs (5.5%). These results confirm colostral transmission of JSRV to lambs under natural conditions. Infected lymphoreticular cells contained in C/M appear to be involved. These cells can cross the intestinal barrier of newborn lambs, reach the MLN and enter into circulation.

Vaccination schedules in small ruminant farms.

Development and implementation of health management plans is the cornerstone of profitable farms; prevention of microbial diseases by means of vaccination is an integral part of such a plan. In every production type and management system in small ruminants, microbial diseases have a major significance, hence their proper control must be based in good health management practices, including use of effective and safe vaccines. Development of various types of vaccines is evolving very quickly in recent years and the improvement of new type of vaccines offers prospects. The article reviews and discusses vaccination programs and latest advances in development of vaccines against diseases that cause major economic losses in small ruminants. Specifically, vaccination schedules for the following diseases are reviewed: bacterial abortion (abortion associated with Brucella melitensis, Campylobacter spp., Chlamydophila abortus, Coxiella burnetii, Salmonella abortus ovis or Salmonella brandenburg), caseous lymphadenitis, clostridial diseases, colibacillosis, contagious echtyma, epididymitis caused by Brucella ovis, footrot, mammary diseases (contagious agalactia, mastitis), paratuberculosis and respiratory diseases (respiratory disease caused by Mannheimia haemolytica or other Pasteurellaceae).

Flow and clogging of a sheep herd passing through a bottleneck.

We present an experimental study of a flock passing through a narrow door. Video monitoring of daily routines in a farm has enabled us to collect a sizable amount of data. By measuring the time lapse between the passage of consecutive animals, some features of the flow regime can be assessed. A quantitative definition of clogging is demonstrated based on the passage time statistics. These display broad tails, which can be fitted by power laws with a relatively large exponent. On the other hand, the distribution of burst sizes robustly evidences exponential behavior. Finally, borrowing concepts from granular physics and statistical mechanics, we evaluate the effect of increasing the door size and the performance of an obstacle placed in front of it. The success of these techniques opens new possibilities regarding their eventual extension to the management of human crowds.

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.

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.

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.

Effect of addition of soybean trypsin inhibitor to colostrum on immunological status in goat kids.

The aim of the study was to evaluate the influence of soybean trypsin inhibitor (TI) on immunoglobulin G (IgG) serum levels and growth in neonatal goat kids. Twenty-four newborn kids were fed with natural colostrum (group A), and 24 kids received the same colostrum with 1 g of TI per litre (group B). Blood samples were obtained at birth and on days 1, 2 and 4 of life to analyze serum proteins, IgG and haematological parameters. There were no clinical signs of disease and no significant differences in body weight between the groups. Haematological parameters were not affected by treatment. The peak of serum IgG was reached at 24 h of life, but no effects of soybean TI was observed on serum IgG levels. The apparent efficiency of absorption of IgG was similar in both groups (group A 24.5% vs. group B 25.2%, p > 0.05). The addition of TI to colostrum did not change the concentration of serum proteins and their fractions in goat kids. The correlation between serum IgG and gamma-globulin was positive and significant (p < 0.01, r = 0.64) in group A, but not in group B (p > 0.05, r = 0.08), suggesting a negative influence of soybean TI on gamma-globulin absorption. These results show that addition of soybean TI to colostrum did not improve the performance or immunological status in goat kids.

Unilateral scrotal pyocele in ram caused by Staphylococcus capitis.

A massive unilateral scrotal pyocele caused by Staphylococcus capitis in a 6-year-old ram is reported. Ultrasound examination of the right hemiscrotum showed an irregular hyperechoic mass in an anechoic fluid. A dense exudate was collected from the scrotum for microbiological analysis. Grossly, there was an extensive greenish purulent exudate and a completely atrophied right testis. Coagulase-negative S. capitis was isolated in pure culture. To the authors' knowledge, this is the first report of genital infection by S. capitis in rams. This microorganism should be included in the differential diagnosis of ovine genital infections.

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.