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Efficacy of a long-acting formulation of ivermectin against Psoroptes ovis (Hering, 1838) on cattle.

A study was conducted in cattle experimentally infested with Psoroptes ovis to compare the prophylactic control against P. ovis provided by a long-acting injectable formulation of ivermectin to that of a commercially available injectable formulation of doramectin. Thirty Holstein steers were used. Animals were allocated by restricted randomization based on Day 0 body weight, forming six replicates of five animals each. Within each replicate, one animal was randomly allocated to one of the following treatment groups, with ivermectin and doramectin administered subcutaneously where indicated: (1) untreated controls; (2) ivermectin long-acting injectable (LAI) 630 mcg/kg, 56 days before challenge; (3) ivermectin LAI 630 mcg/kg, 42 days before challenge; (4) ivermectin LAI 630 mcg/kg, 35 days before challenge; or (5) doramectin 200 mcg/kg, 35 days before challenge. Animals were housed in individual pens 1 week prior to treatment. All animals were experimentally infested with P. ovis mites in the area between the shoulders, on the same day. Live mites were counted in scrapings from mange lesions at 2 sites on each animal 14, 21 and 28 days after challenge. Live mites were found in 33, 67 and 83% of the untreated controls on each respective evaluation. No P. ovis mites were found in steers treated with ivermectin LAI. Those animals showed lower (P < 0.05) mite counts than untreated controls on evaluations conducted 21 and 28 days after challenge. These results indicate that the ivermectin long-acting injectable formulation prevents induced infestations by P. ovis for at least 56 days after treatment. Doramectin injectable formulation, used at 200 mcg/kg, did not have a prophylactic effect 35 days after treatment.

Animals↗

Ultrastructure of the alimentary canal of the sheep scab mite, Psoroptes ovis (Acari: Psoroptidae).

The mite Psoroptes ovis causes sheep scab disease in flocks throughout much of the world. A serious impediment to the development of novel control measures for this mite is our inability to produce in vitro colonies of the mite. Here, we describe the alimentary canal of the mite with the particular aim of determining what it feeds on, as part of a longer term goal, to develop in vitro culture techniques for P. ovis. The alimentary canal of P. ovis consists of a cuticle-lined foregut and hindgut separated by a microvilli-lined midgut. The foregut is divided into a pre-oral cavity and a muscular pharynx and oesophagus. The midgut consists of three ultrastructurally discrete areas: a stomach with bi-lobed ventriculi, a colon and a post-colon. The stomach is composed of two cell types. The most common cells (Type 1) are either cuboidal or squamous depending on the degree of gut distension and possess short microvilli, a single basally located nucleus, extensive rough endoplasmic reticulum and other components suggesting active secretion. The less common cells (Type 2), possess an extensive apical network of tubules and basally food vacuoles suggesting intracellular digestion. These cells extend into the gut lumen, become free-floating and degenerate. The colon and post-colon are composed of cells similar to Type 1 stomach cells but the post-colon epithelium possesses significantly longer microvilli. Cells from these areas have not been observed to leave the surrounding epithelium and enter the gut lumen, but it is suggested that significant absorption occurs in these two areas. Faecal pellets, often containing a significant number of bacteria, leave the digestive system through the cuticle-lined anal atrium.

Animals↗

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 naïve 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.

Amino Acid Sequence↗

The proteinases of Psoroptes ovis, the sheep scab mite--their diversity and substrate specificity.

The sheep scab mite, Psoroptes ovis, causes severe dermatitis in infected sheep with severe welfare and production implications. The dermatitis has the characteristics of an immediate hypersensitivity type reaction which, by analogy to other mite species, including the house dust mites (Dermatophagoides spp.), is likely to be invoked by a variety of allergens including mite-derived proteinases. Here, the proteinases in P. ovis extracts were characterised using substrate gel analysis, inhibitor sensitivity and their ability to degrade a variety of potential natural protein substrates. These analyses showed that mites contain several proteinases which could be differentiated on the basis of molecular size and inhibitor sensitivity with cysteine, metalloproteinases and aspartyl proteinases predominating. These proteinases degraded collagen and fibronectin, possibly indicative of a role in lesion initiation, they degraded several blood proteins, a property which may aid mite feeding and they degraded immunoglobulin G, possibly aiding immuno-evasion. Because proteinases, particularly the cysteine class, are demonstrably allergenic in other mite infestations, these proteinases clearly merit further immunological and biochemical definition.

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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.

Allergens↗

A simple pump-assisted method for collecting live, undamaged Psoroptes ovis from sheep using circulating saline.

The availability of large numbers of undamaged sheep scab mites, Psoroptes ovis, would be beneficial for discovery screening and development trials. There are several reported procedures for removing scab mites from sheep but they have limitations. To overcome this, a simple but versatile method employing the use of pumped saline was developed to remove all stages of the P. ovis mite from sheep. The method takes no more than 2 min to remove mites from the selected site with relative ease and is not affected by the condition of the fleece or lesion. The number of mites removed with the new method was 5-10 times more than detected by visual examination. These mites were undamaged and survived off-host for up to 16 days. The robust, portable equipment is easy to use under field conditions, making this method suitable for use as a diagnostic tool for early detection and monitoring of scab mites thus providing opportunities for development of novel alternative control strategies.

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Efficacy of pour-on and injectable formulations of moxidectin and ivermectin in cattle naturally infected with Psoroptes ovis: parasitological, clinical and serological data.

On the basis of Psoroptes ovis counts performed on day -7, 32 animals were randomly allocated to a control group of five animals or to four groups comprising six or seven animals which were treated, respectively, with pour-on ivermectin (IPO), injectable ivermectin (II), pour-on moxidectin (MPO) and injectable moxidectin (IM). Living mites were counted in skin scrapings on days 0, 7, 14, 28, 42 and 56 post-treatment (PT). Lesions were recorded on a standardized map on days 0 and 56 PT. Antibody kinetics were studied using ELISA on serially diluted sera. The antibody titres were expressed as the dilution giving the positive/negative cut-off. Until their treatment on day 28, the control animals remained parasitologically positive and their antibody titres increased. In treated groups, all living mite counts were negative on days 28 and 42 PT but some animals were still infected on days 7 and 14 PT. On day 56, living P. ovis were found in one animal of the IPO group. An equation of regression describing the antibody decrease was calculated with each individual data set. In most of the treated animals, the coefficient of determination R2, which describes the closeness of fit to the linear model, was above 0.9. The linear model could not be applied (low R2) to the antibody kinetics of four animals: the day 56 positive animal and its two neighbours in the IPO group and one animal from the MPO group. In the treated groups, the differences between the numbers of infected animals, the mean daily weight gains or the mean antibody titres were not statistically significant. Mean daily weight gains of the treated groups were higher than in control animals.

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Evaluation of the therapeutic and protective efficacy of doramectin against psoroptic scabies in cattle.

Three studies were conducted to evaluate the therapeutic and protective efficacy of doramectin when given by injection at a dose of 200 micrograms/kg against induced Psoroptes otis infestations of cattle. The first study investigated therapeutic efficacy. Mite infestations were established on 15 test animals held in stanchions by transfer of material from infested donor calves. Test animals were then allotted on the basis of mite counts to a treatment group (10 animals) which received doramectin and a control group (5 animals) which received saline. Skin scrapings were collected for mite counts on the day before treatment and on days 7, 14, 21 and 28 after treatment. Efficacy assessed on the basis of the proportion of animals cured by day 28 was 100%. The second study was designed to determine the duration of protective efficacy. Forty-eight scabies-free heifers were allotted to a treated group of 32 which received doramectin, and a control group of 16 which remained untreated. These treatment groups were each divided into eight subgroups. Commencing on treatment day and continuing at weekly intervals for 7 weeks, a subgroup of animals from each treatment was placed in stanchions and challenged by transfer of material from infested donor calves. Skin scrapings for mite counts were collected 7 and 14 days later. Infestations were successfully established on all untreated control calves. Doramectin prevented the establishment of infestation for three weeks and significantly (P < 0.05) reduced infestation levels for an additional two weeks. The third study established the duration of residual protection conferred by doramectin and ivermectin under contact transmission. Ninety-six scabies-free heifers were divided into two equal treatment groups. Animals in one group received doramectin and animals in the other group received ivermectin at its recommended dose of 200 micrograms/kg by subcutaneous injection. Each treatment group was then divided into eight subgroups of six animals. Commencing on treatment day and continuing at weekly intervals for 7 weeks a subgroup of animals from each treatment was exposed to purposely infested seeder animals for one week. Three animals from each treatment subgroup were then placed in individual stanchions in which grooming was prevented and the other three were placed together in a pen where normal grooming behavior was permitted. Skin scrapings for mite counts were collected at weekly intervals for up to 4 weeks. Doramectin provided complete protection against infestation for five weeks compared to four weeks for ivermectin. These periods were not influenced by grooming behavior.

Animals↗

The relationship between parasite counts, lesions, antibody titres and daily weight gains in Psoroptes ovis infested cattle.

Psoroptes ovis counts, extent of lesions (clinical index: ClinI), daily weight gains (DWGs) and anti-P. cuniculi antibody titres in ELISA were recorded during seven therapeutic field trials. Relationship between these different data were studied. The differences between the mean DWG of treated and untreated control animals of the different trials ranged from 39 to 1206 g/day. Data were pooled for statistical analysis and the influence of trial conditions (management of the herd, farm, treatment) was extracted from each individual data by subtracting, from the DWG of each animal, the mean DWG of its trial. Such data were called daily weight gain over the trial mean (DWG/TM). Multiple regression of DWG/TM, calculated over the period between the clinical examinations in control animals (n = 40), on clinical indices and sex demonstrated a significant DWG/TM reduction per percentage of affected body surface according to the following equation: DWG/TM (in g/day)= 135-22 ClinI(0)-13 ClinI(28/35)-70 (if male) (R2 adjusted = 0.39), where ClinI(0) and ClinI(28/35) are the clinical indices recorded respectively at the beginning (day 0) and at the end (day 28 or 35) of the experimental period during which the animals were left untreated. A nonsignificant relationship (R2 adjusted = 0.07) was found between the antibody titres on day 28 or 35 and the individual DWG/TM This would suggest that serology could be used to estimate the prevalence of the disease on a local or national basis but not to calculate its economic impact.

Animals↗

Efficacy of ivermectin in a controlled release formulation against Psoroptes ovis (Hering, 1838) gervais, 1841 (Acari: Psoroptidae) on sheep.

Three trials including 42 sheep were conducted in Brazil or Germany to evaluate the therapeutic (two trials) and prophylactic (one trial) efficacy of an ivermectin controlled release capsule (CRC) against Psoroptes ovis infestation. In one therapeutic trial naturally infested sheep were used while in the other trials infestations were experimentally induced. In each trial half of the animals were treated on Day 0 with one ivermectin controlled release capsule that delivers ivermectin at a rate of 1.6 mg/day for approximately 100 days, that is 20 mcg/kg/day to a 80 kg animal, while the other half remained untreated. In both therapeutic trials mites were counted in skin scrapings and their presence was recorded at predilection sites one day before treatment and at weekly intervals from Day 7 to Day 56. In the trial conducted to evaluate the prophylactic efficacy the sheep were experimentally infested with P. ovis 21 and 28 days post-treatment and mites were counted and recorded at predilection sites on Days 42, 49 and 56. The ivermectin controlled release capsule was completely effective in eliminating the P. ovis mites within 28 days of administration and it prevented the establishment of an infestation of P. ovis induced 21 and 28 days after administration.

Animals↗

The construction of a cDNA expression library for the sheep scab mite Psoroptes ovis.

The need for alternative control strategies for sheep scab is critical. One approach is to develop vaccines based on 'concealed' antigens derived from Psoroptes ovis. This strategy requires the identification and characterisation of potential target antigens, which has been hampered by the problem of limited biological material for isolation of protein antigens. To aid the discovery of P. ovis antigens and to provide a resource for generating recombinant protein, we constructed a P. ovis cDNA expression library, using total RNA isolated from 250 mg of mixed-stage P. ovis and the Clontech SMART cDNA synthesis kit. The presence of P. ovis-specific sequences was confirmed using PCR amplification and sequencing of actin. The sequences of cDNA inserts from six random clones included one with high homology to the Dermatophagoides pteronyssinus (house dust mite) antigen p Dp15. This is a glutathione S-transferase known to be an important house dust mite antigen. We conclude that this library will be a useful tool for the identification of potential target antigens for the immunological control of P. ovis and to further our understanding of the pathology of sheep scab.

Amino Acid Sequence↗

Simulation analysis of the population dynamics of the mite, Psoroptes ovis, infesting sheep.

The pattern of population growth of the ectoparasitic mite, Psoroptes ovis (Acari: Psoroptidae), on its ovine host is considered through the development of a Leslie matrix-based, simulation model. The model is parameterised using experimental data in conjunction with reanalysis of published data. The model shows that on sheep P. ovis populations grow at a rate of approximately 11% per day and the population doubles every 6.3 days. Additional rates of adult mortality, in excess of 50% per day, need to be imposed to prevent population growth. The predictions of the model are tested by comparison of the expected numbers of mites with the numbers recorded in lesions either on naturally infested sheep where the date of infestation can be estimated or on one artificially infested animal, where the initial number of mites and date of infestation are known precisely. In both cases the observed number of mites in lesions relate closely to the numbers expected from the simulations. The model simulations do not support the concept of a 'lag' phase as distinct from the 'growth' phase in the changing pattern of mite abundance on an infested sheep and suggests that the observed pattern of growth is a natural function of an exponential increase in numbers. The development of such models and their use in explaining the demographic processes which drive mite population dynamics are discussed.

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Hydrolytic enzymes of Psoroptes cuniculi (Delafond).

Phosphatases, C4 and C8 esterases, leucine and valine aminopeptidases, N-acetyl-beta-glucosaminidase, beta-glucosidase, beta-galactosidase and beta-glucuronidase were detected in extracts of the parasitic mite Psoroptes cuniculi. Lipase, trypsin-like and chymotrypsin-like activities were not present. Haemoglobin was hydrolysed by a detergent-soluble fraction of the mite extracts with a maximum hydrolysis between pH 3 and 5. Acid proteinase activity was greater against haemoglobin than bovine serum albumin. Inhibitors of cysteine, serine and metallo-proteinases failed to inhibit the hydrolysis of H-Pro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH while pepstatin A inhibited its hydrolysis in a dose-dependent manner (IC50 8.02 x 10(-11) M (+/- 0.30 x 10(-11). Thermal inactivation of the proteolytic activity followed an exponential decay pattern. Typical K(m) and Vmax values were 7.2 x 10(-5) (+/- 0.7 x 10(-5) M-1 and 1.13 x 10(-3) (+/- 0.05 x 10(-3) OD unit-1 min-1 respectively. Acid proteinase activity eluted from a size exclusion column in a single, major peak representing a molecular weight range of 21-24.5 kDa. The major endoproteinase of P. cuniculi therefore appears to be a cathepsin D-like aspartic proteinase.

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Microfloral diversity of cultured and wild strains of Psoroptes ovis infesting sheep.

PCR amplification of 16S rDNA was used to determine the diversity of bacteria associated with 3 strains of sheep scab mite, Psoroptes ovis. Eight species of bacteria were identified by phylogenetic analysis of the PCR product sequences. Seven of these species are previously unreported in association with sheep scab mites. Five species were matched to Serratia marcesens, Proponibacteium acnes, Phyllobacterium rubiacearum, Pantoea agglomerans, Curacaobacter baltica, whereas the remaining 3 sequences matched unclassified sequences belonging to the gamma proteobacteria, pseudomonads and streptococci. Bacterial diversity of the in vivo cultured strain was very low and did not match the diversity of the 2 wild collected isolates. The diversity of the bacteria in relation to the disease of sheep scab and the possible importance of these bacteria in the diet of the mites are discussed.

Animals↗

Expressed sequence tag survey of gene expression in the scab mite Psoroptes ovis--allergens, proteases and free-radical scavengers.

Psoroptes ovis, the causative agent of sheep scab, is an important ectoparasitic mite infecting sheep, goats and cattle. Infection is characterized by an extensive dermatitis, scab formation and intense itching. Initial focal lesions spread outwards, coalesce and may extend over the whole body. The host response to infestation has all the characteristics of an immediate-type hypersensitivity reaction but the mite antigens and allergens which initiate this response are almost completely undefined. Here, 507 randomly selected cDNAs derived from a mixed population of P. ovis were sequenced and the resultant nucleotide sequences subjected to Cluster analysis and Blast searches. This analysis yielded 280 clusters of which 49 had > 1 sequence with 24 showing significant Blast X homology to another protein in the databases. There were 231 sequences which appeared on one occasion and 109 of these showed significant Blast X homology to other sequences in the databases. This analysis identified homologues of 9 different types of allergens which have been characterized in other allergic conditions such as responses to house dust mites. It also identified a number of cysteine proteases which may contribute to lesion development as well as several free-radical scavenging enzymes which may protect the mite from host immune effector responses.

Allergens↗

Molecular characterization, expression and localization of tropomyosin and paramyosin immunodominant allergens from sheep scab mites (Psoroptes ovis).

cDNAs encoding the immunodominant allergens tropomyosin and paramyosin were amplified from RNA extracted from the sheep scab mite Psoroptes ovis. The tropomyosin cDNA contained an open reading frame (ORF) of 852 bp which encoded a predicted protein with 98% and 97% identity to the house dust mite allergens Der f 10 and Der p 10 respectively. The complete paramyosin ORF generated by RT-PCR was 2625 bp in length and encoded an 875aa predicted protein of 102.6 kDa with 97%, 95% and 89% identity to the paramyosins of Dermatophagoides pteronyssinus (Der p 11), Sarcoptes scabiei and Blomia tropicalis (Blo t 11) respectively. Full length tropomyosin and truncated and full-length paramyosin were expressed as recombinant proteins. IgG and IgE in sera from sheep with a 6-week duration primary infestation of P. ovis did not detect either full-length or truncated recombinant paramyosin. IgG in both infested and naïve sheep sera detected recombinant tropomyosin, suggesting cross-reactivity to tropomyosin and to other invertebrate species to which the sheep may have been exposed. Staining with antibodies directed against tropomyosin and paramyosin was observed throughout sections of P. ovis. Staining was especially prevalent in the anterior sections of the mites, possibly associated with locomotory muscles in this region.

Allergens↗

Eukaryotic expression of recombinant Pso o 1, an allergen from Psoroptes ovis, and its localization in the mite.

A cDNA encoding the immunogen Pso o 1 from Psoroptes ovis was obtained by polymerase chain reaction (PCR) amplification. The amplicon contained the entire coding sequence for the prepro-enzyme in an open reading frame (ORF) of 966 bp. This gene encoded a predicted protein of 322 amino acids (aa) with 64% aa identity (80% similarity) to the major house dust mite faecal allergen Der f 1. The pro-enzyme form of Pso o 1 was expressed as a recombinant protein in the Pichia pastoris-eukaryotic expression system. Maturation of the recombinant pro-enzyme by autocatalytic activation was not observed, and such maturation could not be achieved using a number of techniques known to activate recombinant Der p 1 and Der f 1 expressed in the same system. Serum raised against recombinant Pso o 1 cross-reacted with mature Der p 1 and allowed Pso o 1 to be immunolocalized to the gut of P. ovis.

Allergens↗

Structure/activity relationship of some natural monoterpenes as acaricides against Psoroptes cuniculi.

The pharmacological activity of many essential oils on a large number of human and animal pathogens, as used in folk medicine, has been confirmed world-wide by several laboratory investigations. Unfortunately, the biological properties of essential oils can be extremely inconsistent because of the variability of their chemical composition. The acaricidal activities of some natural terpenoids, which are the main constitutents of several essential oils, were evaluated in vitro against the mange mite (Psoroptes cuniculi) of the rabbit, by direct contact and by inhalation. Because the test components represent different chemical classes (hydrocarbons, alcohols, and phenols, with free and esterified or etherified functional groups), it was also possible to discern in a preliminary fashion a correlation between chemical structure and acaricidal activity. The results obtained suggest that molecules possessing free alcoholic or phenolic groups showed the most potent acaricidal activity.

Animals↗