Diagnosis of psoroptic sheep scab with an improved enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of specific antibodies against crude Psoroptes antigen. The diagnostic sensitivity was 93.7% in 191 sheep with clinical signs associated with mange. These animals originated from 29 flocks in which psoroptic mites were detected. All of 59 sheep infested with Psoroptes ovis were seropositive. Additionally, in 49% of 70 clinically unaffected sheep originating from P. ovis-infested flocks, specific antibodies could be detected, suggesting that asymptomatic infestations can be diagnosed by serology. The specificity of the ELISA was 96.5% as determined with 254 sheep originating from 44 flocks without clinical mange. Cross-reactivity in a low range was detected with selected sera of sheep with clinical chorioptic or forage mite infestations. Four sheep seroconverted 2 weeks after experimental P. ovis infestation, i.e. 2 weeks before clinical signs became obvious. After successful doramectin treatment of 14 sheep with naturally acquired P. ovis infestation, the ELISA values declined slowly but remained positive in seven cases beyond 17 weeks.     

Temporal pattern of isotype-specific antibody responses in primary and challenge infestations of sheep with Psoroptes ovis--the sheep scab mite

In sheep Psoroptes ovis provokes an allergic dermatitis with significant P. ovis antigen-specific IgE responses. The kinetics of the IgE response to primary and challenge infestations of P. ovis were reported earlier [Parasite Immunol. 22 (2000) 407]. The present study examines IgG, IgM and IgA responses to primary and challenge infestations of P. ovis and the profile of antigens/allergens reacting with IgG and IgE antibodies. Antigen-specific enzyme-linked immunosorbent assays (ELISAs) demonstrate that primary infestations elicited significant increases in levels of IgG and IgM but not IgA antibodies. IgG and IgM responses to primary and challenge infestations were not significantly different. Western blots of reduced P. ovis proteins indicate that IgG antibodies reacted with five major antigens following primary infestation and only three of these after challenge infestation. IgE antibodies bound to three major and five minor allergens after primary infestation and two additional minor allergens after challenge infestation. Immunodominant antigens >100 and <15 kDa and allergens >100 kDa were most consistent in stimulating substantial IgG and IgE antibody responses, respectively. These antigens/allergens may be exploited in immunodiagnosis and modulation of the host immune response.     

Clinical development and serological antibody responses in sheep and rabbits experimentally infested with Psoroptes ovis and Psoroptes cuniculi

Psoroptes ovis of sheep origin, and Psoroptes cuniculi of rabbit origin were used in experimental infestations. In experiment I, groups of four rabbits and four sheep were infested with 50-100 mites of each isolate on the skin of the back (skin infestation, SI) or in the external auditory canal (aural infestation, AI). In rabbits, SI and AI with P. cuniculi and AI with P. ovis induced in all animals typical ear lesions and pronounced antibody reactions to P. cuniculi antigens in ELISA. After SI of rabbits with P. ovis no clinical signs were detected, no mites could be reisolated and no specific antibodies were detected. In sheep, P. ovis SI induced mange whereas AI did not induce typical clinical signs and mites could not be reisolated. In both these animal groups, ELISA revealed pronounced and comparable specific antibody reactions. After SI and AI with P. cuniculi no clinical symptoms were observed and no mites could be reisolated. Nevertheless, low levels of specific antibody were detected. In experiment II, clinical progression and antibody reactions to P. ovis SI in naive sheep were compared with sheep previously exposed to P. ovis or P. cuniculi. In both pre-exposed groups of animals, clinical signs appeared within 2 days after challenge infestation and three days earlier than in primarily infested sheep. Subsequently, no obvious difference in the clinical progression was observed between the three groups of animals. The results of this study document antigenetic crossreactivity of the two morphologically and genetically distinguishable Psoroptes species but differences in their biological behaviour and virulence which both are of epidemiological and taxonomic relevance.     

Evaluation by enzyme-linked immunosorbent assay of local and systemic production of milk immunoglobulins to surface exopolysaccharide antigen in cows with staphylococcal mastitis

An enzyme-linked immunosorbent assay (ELISA) was developed to evaluate milk immunoglobulin levels to surface exopolysaccharide antigen of Staphylococcus aureus in cows with staphylococcal mastitis. Quarter milk samples were obtained from 24 lactating dairy cows on two occasions, one month apart. Cows were classified as S. aureus-positive (S. aureus cultured from at least one quarter on both sample dates) or S. aureus-negative. Individual quarter samples were tested for IgA (representing local synthesis) and IgG1 (primarily of systemic origin) specific for staphylococcal surface exopolysaccharide antigen. No significant differences were found for specific IgA or IgG1 between S. aureus-positive and S. aureus-negative cows, nor between infected and non-infected quarters of S. aureus-positive cows. The data indicate that, in cows with staphylococcal mastitis, milk immunoglobulins specific for exopolysaccharide antigen are not significantly increased by either the systemic or the local immune response.     

Cutaneous hypersensitivity reactions to Psoroptes ovis and Der p 1 in sheep previously infested with P. ovis--the sheep scab mite

We have previously shown that infestation with Psoroptes ovis induces an IgE response and intense tissue eosinophilia, typical of a Type I hypersensitivity response [Parasite Immunol. 22 (2000) 407]. Intradermal tests (IDSTs) suggest that there are also delayed and Arthus-type responses to this parasite. In order to study the nature of ovine cutaneous reactions to P. ovis, na?ve controls and experimentally infested sheep (n = 5) were challenged intradermally with mite antigen. Challenge elicited immediate (P < 0.001) and delayed (P < 0.005) wheal reactions in sensitised sheep. At 6 (P < 0.02) and 30 h (P < 0.001) the predominant infiltrating cells were eosinophils. To explore the role of circulating antibodies, na?ve sheep (n = 5) were subjected to Prausnitz-Kustner (PK) tests. These elicited immediate (P < 0.02) but not delayed wheal reactions. At 6 h eosinophils (P < 0.001) dominated the infiltrate. These results suggest that P. ovis allergens provoke an IgE-dependent immediate and late phase response and a cell-mediated eosinophil-rich delayed-type hypersensitivity response (ER-DTH).     

Characterisation of lesional infiltrates of dendritic cells and T cell subtypes during primary infestation of sheep with Psoroptes ovis, the sheep scab mite

Earlier studies of cattle and sheep have demonstrated that Psoroptes ovis infestations provoke an intense immunoinflammatory response dominated by eosinophils accompanied by a substantial infiltrate of lymphocytes. However, the kinetics of the lymphocyte response and the subtypes involved have not been characterised. We employed two groups of sheep to investigate the early (1-21 days) and later (21-63 days) infiltration of lymphocyte subpopulations and dendritic cells in primary infestations of sheep with P. ovis. Immunohistochemistry indicated that by 4 days after infestation numbers of CD4+ and CD45RA+ cells in lesional skin had increased significantly (P<0.03 and P<0.005, respectively) and that a significant increase in gammadelta T cells and dendritic cells (CD1b+) had occurred by 8 days (P<0.02 and P<0.01, respectively). Numbers of lymphocyte and dendritic cells declined from 49 to 63 days after infestation. Our observations suggest that mite-derived products exert a profound influence on the early recruitment of lymphocytes that may significantly influence the genesis of the adaptive immune response.     

Parasitism by Oestrus ovis: influence of sheep breed and nematode infections

Previous studies showed that Santa Ines (SI) hair sheep were more resistant to gastrointestinal nematode infections (GIN) than Ile de France (IF) sheep. The present experiment aimed to evaluate if that reported resistance difference against GIN also occurred against Oestrus ovis infestation and also to evaluate the influence of O. ovis infestation on the gastrointestinal nematodes (GIN) infections. SI (n=12) and IF (n=12) young male lambs were weaned at 2 months of age and moved to a paddock (0.3 ha) with Brachiaria decumbens grass, where they also received concentrate ration. The animals were kept together during the experimental period (September to early December 2009). Fecal and blood samples were taken from all animals every 2 weeks and body weight and nasal discharge score (oestrosis clinic signs) were recorded on the same occasion. In early December 2009, all lambs were sacrificed and O. ovis larvae and GIN were recovered, counted and identified according to the larval stage. All animals were infested by different larval instars of O. ovis without any statistical difference between breeds (P>0.05). The SI lambs had an average of 24.8 larvae, and the intensity of infection ranged between 14 and 39 larvae, while the IF lambs showed an average of 23.5 larvae with the minimum and maximum from 11 to 36 larvae, respectively. SI lambs presented the lowest nematode fecal egg counts (FECs) and the lowest mean numbers of Haemonchus contortus, Trichostrongylus colubriformis and Strongyloides papillosus, however, there was no significant differences between group means (P>0.05). Inverse relationship between numbers of O. ovis larvae and gastrointestinal nematodes was observed in both breeds. SI sheep showed a significant increase in blood eosinophils and total IgE serum levels and these variables were negatively correlated with nematode FEC. A negative correlation was observed between total IgE serum level and H. contortus burden in both breeds. In conclusion, there was no breed difference regarding O. ovis infestation and in each breed, animals with more nasal bot fly larvae tended to display smaller worm burden.     

Reservoirs of Staphylococcus aureus in meat sheep and dairy cattle

The objective of the study was to investigate reservoirs and transmission of S. aureus in ewes and lambs in 3 meat sheep flocks. Repeated sampling of milk, teat skin, nasal- and vaginal mucous membranes was performed and samples were analysed for S. aureus. For comparison, samples were also collected from cows and young heifers in 3 dairy cattle herds. Selected isolates were compared by pulsed-field gel electrophoresis (PFGE). S. aureus was detected in 8 (1.5%) of 520 milk samples from ewes and in 38 (6.4%) of 588 milk samples from cows. From body site swabs, S. aureus was found in 394 (32.6%) of 1208 samples from sheep and in 67 (16.0%) of 420 samples from cattle. The proportion of S. aureus-positive nasal swabs from ewes and cows were 56.7% and 13.9%, respectively. From lambs, 58.2% of the nasal swabs were S. aureus-positive. In each flock, one S. aureus pulsotype predominated. Identical S. aureus pulsotypes were found in milk and from body sites. Paired S. aureus isolates from the nasal cavity of (i) ewes and their lambs, (ii) twins and (iii) from repeated swabs of individual ewes were compared by PFGE, and in the majority of cases the two isolates were identical. The results contribute new knowledge indicating frequent transmission of S. aureus between the dam and her lambs and within animals in a flock. In contrast to cattle, S. aureus is frequently present in the nose of sheep which may represent the primary reservoir of S. aureus in sheep flocks.     

Effects of the scab mite Psoroptes ovis on the haematology and live mass of Merino and Dorper sheep

Five Merino and five Dorper sheep were artificially infested with the sheep scab mite Psoroptes ovis and the effect of infestation on their haematology, serum protein levels and live mass recorded for a period of 14 weeks. The reaction of the Merino sheep to infestation was more severe than that of the Dorper sheep. Haematological values fluctuated within the normal range during the assessment period. The mean haemoglobin concentration of the Merino sheep declined until antiparastic treatment was administered 10 weeks after infestation, after which it gradually increased. The lymphocyte counts of both breeds of sheep declined from 2 weeks to 10 weeks post-infestation, but increased after treatment, while the highest eosinophil counts were recorded in the Merino sheep at the height of the acute disease 8-10 weeks post-infestation. Serum albumin values for both breeds and serum globulin values for the Merino sheep were higher than normal during the entire 14-week observation period. A decrease in serum albumin and an increase in serum globulin concentration occurred at the height of infestation in both breeds. The mean live mass of a second group of five infested Merino sheep decreased by 6.4 kg over a 16-week period compared to a gain of 4.56 kg for five infested Dorper sheep.     

Variation among Merino sheep in susceptibilty to lice (Bovicola ovis) and association with susceptibility to trichostrongylid gastrointestinal parasites.

Sheep of two bloodlines of Merino were artificially infested with equal numbers of lice (Bovicola ovis) and the resulting louse populations were monitored over the following 20 months. The sheep were shorn 6 and 17 months after infestation and, for analysis, the louse counts considered in 3 years separated by shearings. Nematode faecal egg counts (FECs) were assessed on faecal samples collected on five occasions, three times following natural challenge and twice after artificial challenge with 40,000 trichostrongyloid larvae (84% Trichostrongylus vitrinus). In addition, blood samples were collected and measured for B. ovis-specific immunoglobulins (predominantly IgG), B. ovis-specific IgE and serum total IgE. Bloodlines differed significantly in the size of louse populations at the end of year 2, FEC after both natural and artificial challenge and in serum levels of all three antibodies (p<0.05). There were also large variations in louse counts and FEC among sheep within bloodlines. Louse counts at inspections after louse populations had been allowed to build up were highly repeatable, both between and within years. However, correlations with counts at inspections soon after initial infestation and following shearing were lower. FEC after natural challenge was correlated with louse counts in year 2 (r=0.45, p<0.01) and year 3 (r=0.38, p<0.05), but the correlation with counts in year 1 was not significant (r=0.25, p>0.05). FEC following artificial challenge was significantly correlated with louse counts in year 3 (r=0.36, p<0.05), but not in year 2 (r=0.25, p>0.05) or year 1 (r=0.04, p>0.05). Louse counts in the 3 years were significantly correlated with anti-B. ovis antibody concentration (r=0.60, 0.48, 0.36), but not with levels of either anti-B. ovis or total serum IgE.These results suggest that sheep with greater resistance to gastrointestinal parasites also tend to be less susceptible to lice. Whether this is due to interaction of the effects of the parasites or to correlation in underlying resistance mechanisms requires clarification.     

Lipid ingestion from sheep epidermis by Psoroptes ovis (Acari: Psoroptidae)

Skin biopsies from three groups of sheep infested with Psoroptes ovis were cryofixed in liquid nitrogen to preserve outer epidermis with its lipid. From one group of five sheep (Group A), biopsies were taken from relatively healthy skin near the edge of scab lesions where mites had congregated. Frozen vertical sections from these biopsies were stained with Oil Red O and haematoxylin before mounting. Red lipid globules were plentiful within the body cavity of sectioned mites, but ingested lipid could not be distinguished from endogenous mite body stores by this technique. In a second group of five sheep (Group B), enclosed lumbar skin areas were coloured red with the lipophilic stains Oil Red O or Sudan IV. These enclosed coloured skin areas were inoculated with P. ovis and sampled by skin biopsy 1 or 4 days later. Cryofixed biopsies were cut into vertical frozen sections and mounted without staining for examination. Red-coloured lipid within mites, matching red-coloured lipid on outer epidermis, was evidence for the epidermal origin of P. ovis ingesta in the early stages of an infestation. From two other sheep (Group C), cryofixed biopsies were examined by scanning electron microscope and mites were seen with mouthparts embedded in abraded outer epidermis, but the precise depth of epidermal penetration was not determined. A light microscope survey of 3198 frozen vertical skin sections from the first 10 sheep (Groups A and B) showed that inner stratum corneum of the epidermis was the deepest penetration recorded for gnathosomes of P. ovis cryofixed in situ by liquid nitrogen. No structure of P. ovis was identified in dermal tissues.     

Treatment and control of sheep scab (Psoroptes ovis) with ivermectin under field conditions in South Africa.

Four trials including 11,266 sheep were conducted in South Africa to evaluate the efficacy of the systemic parasiticide ivermectin against field outbreaks of sheep scab (Psoroptes ovis) when two doses of approximately 200 micrograms/kg were administered subcutaneously seven days apart (days 0 and 7). As sheep scab is a notifiable disease in South Africa, it was not possible to include an untreated control group. The prevalence of clinically affected animals in the four treated flocks varied from 0.4 per cent to 99 per cent before the two treatments. After the treatments, there were no signs of active clinical infection in any of the sheep between days 28 and 30, or at subsequent examinations. P ovis mites were recovered from scrapings from 114 of 127 indicator sheep before the treatment but no mites were recovered from them between days 28 and 30 or 42 and 58 after the treatments.     

Host preference of the sheep scab mite,Psoroptes ovis

Sheep scab mites, Psoroptes ovis, collected from a Merino donor sheep, were used to infest Merino and Dorper sheep, and Angora and Boer goats. Mites were placed on the sheep on 1 or 2 occasions and on 5 occasions on the goats. All the animals were examined at regular intervals for the presence of scab lesions and living mites. Both sheep breeds developed lesions, but those on the Merino sheep were always larger than those on the Dorper sheep at the same intervals after infestation. None of the goats developed lesions or showed signs of irritation, or harboured any mites.     

Prevalence and clustering of louse infestation in Queensland sheep flocks.

Information provided by wool growers in Queensland, Australia between 1995 and 1997 was used to assess the prevalence and spatial distribution of louse (Bovicola ovis) infestation in sheep flocks. The estimated prevalence of louse-infested flocks was 40% (95% confidence interval, 35-46%). Although the prevalence of infestation was higher in western regions (41-50%) compared to the south region of Queensland (31%), the difference was not statistically significant (P > 0.05). Significant (P = 0.02) clustering of infested flocks was detected in the south region where two foci were apparent. We conclude that Queensland sheep flocks have a moderate prevalence of louse infestation, and that clustering of infestation is not strong. The control of lice is an industry-wide issue that needs to be addressed by most wool growers in Queensland.     

The rate of spread of sheep scab within small groups of Merino and Dorper sheep

A single Merino sheep, artificially infested with the sheep scab mite, Psoroptes ovis, and a similarly infested Dorper sheep were placed with 9 uninfested Merino or 9 uninfested Dorper sheep respectively during winter and the rate of spread of infestation on the uninfested sheep observed. The same procedure was repeated in summer. It took 14 and 8 weeks respectively in winter before all sheep in the 2 groups displayed lesions of sheep scab, whereas in summer it took 10 and 12 weeks before all sheep had lesions.     

Expression and characterisation of a Psoroptes ovis glutathione S-transferase

The astigmatid mite Psoroptes ovis is the causative agent of sheep scab, a highly contagious parasitic disease of sheep. Infection causes severe allergic dermatitis, resulting in damage to the fleece and hide, loss of condition and occasional mortality. Interest in the P. ovis allergens led us to characterise a glutathione S-transferase (GST) which displays homology to GST allergens isolated from the house dust mite, Dermatophagoides pteronyssinus and the cockroach, Blatella germanica. A cDNA encoding a mu-class GST from P. ovis was expressed in Escherichia coli and the recombinant protein purified for biochemical analysis. SDS-PAGE analysis indicated that the purified product was homogeneous and had an apparent molecular weight of 30 kDa. The recombinant GST (rGST) is active towards the substrate 1-chloro-2,4-dinitrobenzene (CDNB), whereas 1,2-dichloro-4-nitrobenzene (DCNB) is a poor substrate. The recombinant protein was also tested for recognition by IgE and IgG antibodies in serum from P. ovis na?ve and P. ovis infested sheep. Neither IgE nor IgG antibodies were detected to the rGST. Prausnitz--Küstner testing with rGST did not provoke a characteristic weal and flare response. Biopsies collected at the PK test sites were stained for eosinophils, neutrophils, mast cells and basophils. Neutrophil, mast cell and basophil counts were not significantly different to the controls. Eosinophil numbers were significantly higher than controls, but were not due to an IgE response.     

Temporal relationship between infestation with lice (Bovicola ovis Schrank) and the development of pruritic behaviour and fleece derangement in sheep

Pruritic behaviour and deranged fleece are often used as indicators of sheep louse infestation but the exact relationship between infestation and the observation of signs of pruritis was unclear. Two studies were conducted to examine this association. In the first, 24 castrate Merino sheep were randomly assigned to six pens in groups of four and the sheep in three pens infested with 10 lice each on the right mid-side. Louse numbers were counted, fleece derangement scored and pruritic behaviour assessed periodically on each sheep until 38 weeks after infestation. In the second study a single moderately infested sheep was paddocked for 15 weeks with 32 uninfested sheep and louse numbers and fleece derangement monitored for 41 weeks. In the pen studies, differences between infested and non-infested sheep in fleece derangement and pruritic behaviour first became significant (p<0.05) at 8 and 14 weeks, respectively and at louse densities of 0.06 and 0.27 per 10 cm wool part. Some sheep showed definite signs of deranged fleece as early as 5 weeks after initial infestation. In the paddock studies, it took 37 weeks until lice were detected on all sheep in the flock. The correlation between louse numbers and fleece derangement score first became significant (r=0.44 and p<0.05) at 9 weeks after introduction of the lousy sheep, reached a maximum of r=0.79 (p<0.001) at 22 weeks when 84% of sheep had lice detected and the mean louse density was 0.29 per part, and then declined to r=0.12 (n.s.) at 41 weeks when all sheep were infested and the mean louse density was 3.04 per part. It is concluded that fleece derangement is a powerful early indicator of the presence of lice and that sheep may exhibit signs of pruritis well before lice can be readily found by direct inspection. Fleece derangement may be useful as a basis for establishing economic thresholds for the application of long wool treatments in developing louse infestations but appears to be a poor indicator of louse numbers once the infestation is advanced.     

The prevalence of itchmite, Psorergates ovis, among sheep flocks with a history of fleece derangement

An investigation of sheep flocks in the main sheep raising areas of New South Wales showed that the itchmite Psorergates ovis was frequently associated with fleece derangement. In 26 of the 41 flocks examined, P. ovis was the only ectoparasite detected. P. ovis and the sheep body louse Damalinia ovis, were found in 5 flocks. No external parasites were found on sheep examined from the 10 remaining flocks. The type of fleece derangement most frequently recorded was rubbing which in some cases was combined with areas of chewed fleece. Among flocks, there were positive relations between the prevalence of fleece derangement and prevalence of itchmite or scurf and between itchmite count and mean scurf score. Within flocks, itchmite infested sheep or sheep with scurf had higher prevalences of fleece derangement than sheep on which no mites or no scurf were found. Itchmite infested sheep had a higher prevalence of scurf than those with no detectable mite infestation. There were no significant differences in itchmite populations or fleece derangement between untreated flocks and flocks treated with synthetic pyrethroids, organophosphates or arsenic and rotenone.     

Comparison of infection rates of Oestrus ovis between sheep and goats kept in mixed flocks

Oestrosis is a nasal myiasis of sheep and goats caused by larvae of the fly Oestrus ovis and can lead to severe clinical signs, which together with the disturbance caused by the adult fly may result into serious economic losses. Infection rates and larval burdens are always higher in sheep than in goats after either natural or artificial infestation. The aim of this study was to compare the host preference of the adult fly O. ovis between sheep and goats in mixed flocks, where they are kept together under the same husbandry conditions and hence, are very similarly exposed to the fly preference. Blood sera samples were collected from a total of 397 sheep and 335 goats, from 43 mixed flocks located at different regions of Greece. Antibodies specific to O. ovis IgG were measured by ELISA. A flock was considered positive when at least one individual was positive, i.e. showed a seropositivity of >or=20% in relation to positive control sera. A total of 193 (48.6%) sheep and 58 (17.9%) goats were found to be seropositive against O. ovis. Thirty-eight (88.4%) out of 43 flocks had at least one seropositive animal. The mean seroconversion against O. ovis in animals from the different flocks was 38.6% and 13.6% for sheep and goats, respectively, whereas the variance of infection within each flock was 0-100%. The mean seropositivity between sheep that were found to be positive or negative was 60.6% and 5.4%, respectively, whereas the corresponding values between goats were 35.2% and 5.2%, respectively. No significant difference in the seroconversion values was noted between flocks from the different areas (P=0.817), whereas a very significant difference was observed between animal species (P=0.001). However, there was no significant difference when seroconversion comparisons were made within samples of the same animals species, sheep or goats from different flocks of all the regions included in the study (P=0.695). The results of this study clearly demonstrate that O. ovis has a widespread distribution in Greece, and the seroprevalence is significantly higher in sheep than goats (P=0.001).     

绵羊痒螨病的免疫学研究进展

羊痒螨成虫的盐水和吐温萃取物对已经发展到活跃期的痒螨病绵羊有显著的保护效应,通过PCR扩增获得了羊痒螨的cDNA编码免疫原Pso01,试验证实绵羊痒螨保护性抗原的成分复杂。在该病诊断方面,应用改进的ELISA法诊断自然感染的羊痒螨病,其敏感性为93．7％,免疫组织化学研究表明,在感染后4d受损伤的皮肤中CD4^＋和CD45RA^＋细胞的数量显著增加,并且γδT细胞和树突状细胞的CD1b^＋显著增加持续了8d。随着绵羊瘁螨cRNA表达文库的建立,为应用现代分子生物学技术开发抗绵羊瘁螨病的疫苗积累了有价值的资料,从而为应用免疫学方法防控绵羊瘁螨病展示了光明的前景。