A simple random amplified polymorphic DNA genotyping method for field isolates of Dermatophilus congolensis.

Dermatophilus congolensis is the pathogenic actinomycete that causes dermatophilosis in cattle, lumpy wool in sheep and rain scald in horses. Phenotypic variation between isolates has previously been described, but its genetic basis, extent and importance have not been investigated. Standard DNA extraction methods are not always successful for D. congolensis due to its complex life cycle, one stage of which is encapsulated. Here we describe the development of rapid and reliable DNA extraction and random amplified polymorphic DNA (RAPD) methods that can be used for genotyping D. congolensis field isolates. Our results suggest that genotypic variation between isolates correlates with host species. Several DNA extraction methods and RAPD protocols were compared. An extraction method based on incubation of the bacterium in lysozyme, sodium dodecyl sulphate (SDS) and proteinase K treatments and phenolic extraction yielded high-quality DNA, which was used to optimize RAPD-polymerase chain reaction (PCR) protocols for two random primers. An alternative rapid, non-phenolic extraction method based on proteinase K treatment and thermal shock was selected for routine RAPD typing of isolates. DNA extracted from reference strains from cattle, sheep and horse using either method gave reproducible banding patterns with different DNA batches and different thermal cyclers. The rapid DNA extraction method and RAPD-PCR were applied to 38 D. congolensis field isolates. The band patterns of the field and type isolates correlated with host species but not with geographical location.

Tick-Theileria interactions in response to immune activation of the vector.

Watt, D. M., Walker, A. R., Lamza, K. A., and Ambrose, N. C. 2001. Tick-Theileria interactions in response to immune activation of the vector. Experimental Parasitology 97, 89-94. Immune mechanisms towards the haemoprotozoan parasite Theileria parva were investigated in their tick vector, Rhipicephalus appendiculatus. The exoskeletons of adult ticks were initially pierced with bacteria-coated, saline-coated, or sterile dry glass needles. Haemolymph was extracted from the ticks at 6, 24, 48, and 72 h postinjection and applied to bacterial plates to measure the growth inhibition effects. The inhibition zones were larger with all the injected groups compared to uninjected controls. The largest inhibition zones were seen 24 h after injection with bacteria-coated needles. An experiment was carried out to investigate whether antibacterial immune responses were relevant to the parasite/tick relationship and, if so, which parasite form was most vulnerable. R. appendiculatus nymphs were infected with T. parva by feeding on an infected calf and were then injected with needles on days 7, 13, 15, and 17 throughout their moult in an attempt to induce tick immune responses at the same time as different lifecycle forms of T. parva would be present. Salivary glands from the moulted adult ticks in the control and different treatment groups were dissected out and examined for the presence of T. parva sporoblasts. No difference in infection levels was seen in any of the treatment groups compared with the controls, suggesting that immune responses in R. appendiculatus, induced by bacterial injection, do not affect T. parva infections. The fecundity of injected ticks was compared with that of uninjected controls to ensure that the injection procedure itself was not detrimental to the ticks. Injected females had higher engorgement masses than controls but reduced levels of egg hatching.

Preliminary characterisation of extracellular serine proteases of Dermatophilus congolensis isolates from cattle, sheep and horses.

Dermatophilus congolensis is a filamentous branching actinomycete that causes dermatophilosis, an exudative dermatitis in ruminants. The pathogenesis of this disease is poorly understood and virulence factors of D. congolensis have not been characterised. Culture filtrate (CF) of 14 D. congolensis isolates from cattle, 15 from sheep and four from horses were examined for proteolytic activity using azocasein as a non-specific substrate. The isolates were from a variety of geographical locations. All the isolates examined produced extracellular proteolytic activity. CF from 24 and 48 h cultures and from first and third passages contained proteases. Proteolytic activity was greatest in neutral to alkaline pH (pH 7-10). CF of bovine isolates contained more proteolytic activity than that of ovine isolates. Furthermore, in substrate SDS-PAGE gels containing azocasein the number of proteolytic bands and their molecular weights in CF of bovine, ovine and equine isolates were different, giving distinctive band patterns for isolates from each host species. Three out of four bovine isolates from Antigua gave a fourth band pattern. Bands of equivalent molecular weights to the proteases could not be identified in silver stained SDS-PAGE gels of CF. Serine protease inhibitors had a concentration-dependent inhibitory effect on proteolytic activity in CF and inhibited activity of all proteolytic bands in substrate gels. With the exception of EDTA which had a variable-enhancing effect on activity, inhibitors of other classes of protease had no effect on activity. We conclude that D. congolensis produces a number of extracellular alkaline serine proteases, our results suggest the presence of host-specific variation between isolates and to a lesser extent between isolates from the same host species.

Electrophoretic and antigenic characterisation of Dermatophilus congolensis extracellular products.

Dermatophilus congolensis is the causative agent of bovine dermatophilosis and lumpy wool in sheep. Two field isolates of D. congolensis, one each from a cow in Ghana and a sheep in Scotland, were cultured for 24-72 h in a synthetic medium based on RPMI-1640. Culture filtrates were examined by SDS-PAGE and considered to contain extracellular products released by growing hyphae and filaments. Electrophoretic profiles of culture filtrates of the two isolates contained common bands and bands that were unique to each isolate. The composition of extracellular products altered with increasing culture periods indicating that specific products were released at different stages of growth. Culture filtrate prepared in the presence of serine protease and metalloprotease inhibitors contained more and better defined bands than that prepared without protease inhibitors indicating the presence of proteases in culture filtrates. Western blot analysis of extracellular products using a panel of sera showed that the two isolates from different host species and distant geographical locations contained cross-reactive antigens. Natural and experimental infections stimulated antibody responses to antigens in culture filtrates, sera from animals that were disease free but in-contact with dermatophilosis-infected animals also contained antibodies to extracellular antigens. The antigens recognised by most sera had molecular weights of 200 kDa in the bovine isolate, 170 kDa in the ovine isolate and 67, 27 and 52-55 kDa in both isolates. The number of antigenic bands of both isolates was positively correlated with the intensity of challenge and the severity of infection: antibodies in sera from disease-free cattle in Ghana recognised more antigens than sera from disease-free sheep in Scotland and more antigens were recognised by sera from chronically-infected Ghanaian cattle than by sera from experimentally-infected calves and sheep. The latter developed antibodies to antigens of 27 and 24 kDa during the course of infection. The electrophoretic profiles of extracellular products of D. congolensis are less complex than those of other structures of the bacterium yet they exhibit differences between the two isolates. Extracellular products contain antigens recognised by sera from naturally exposed and experimentally-infected animals that may be involved in immunity to D. congolensis or immunopathogenesis of dermatophilosis.

The pathogenesis of dermatophilosis.

Pathogen, host and environmental factors must be considered in order to understand the pathogenesis of dermatophilosis. A frequently cited sequence of events involves physical damage to the skin, bacterial multiplication in the epidermis, repeated cycles of invasion by hyphae, infiltration by neutrophils and exudate, regeneration of epidermis and reinvasion. This paper is concerned with pathogen driven mechanisms involved in the origin and development of Dermatophilus congolensis infections. Primary infections of calves under controlled conditions at clipped, cleaned, defatted sites result in characteristic dermatophilosis crusts, illustrating that D. congolensis itself is pathogenic. Calves infected in this way develop protective immune responses to subsequent infections. In contrast first and second infections of calves simultaneously infested with Amblyomma variegatum result in more severe lesions that take significantly longer to heal than lesions on tick free calves. Immunity to D. congolensis is isolate specific, however the antigens that elicit immunity have not been defined, they might include pathogenic or virulence factors. Hyphae are the life-cycle stage most closely associated with the living epidermis. During in vitro growth hyphae secrete antigens and proteolytic enzymes into culture medium. Proteolytic activity has been linked to virulence of D. congolensis. The characteristics of proteases released into culture medium varies between isolates. This raises the possibility that proteases, with as yet undefined functions, act as pathogenic and virulence factors, that they are the targets for protective immune responses and that A. variegatum infestation interferes with host immune responses that normally inhibit their activity.

Lymphocyte proliferative responses and the occurrence of dermatophilosis in cattle naturally infested with Amblyomma variegatum.

The proliferative response of lymphocytes from tick-infested Zebu type, N'Dama and Friesian cattle and acaricide-treated Zebu types and Friesians in concanavalin A (Con A) stimulated cultures was monitored regularly for periods ranging from 11 to 27 months. The numbers of ticks on the animals and the presence of dermatophilosis were also noted. The Friesian cattle carried most and the N'Dama fewest Amblyomma variegatum ticks. The tick-infested Friesians all developed severe clinical dermatophilosis within 5 months of becoming tick-infested. Dermatophilosis lesions on the tick-infested Zebu type and N'Dama cattle were less common and less severe especially in the N'Damas. The proliferative response of lymphocytes from tick-infested Friesians in Con A stimulated cultures fell to almost half that of the acaricide-treated Friesians soon after the former became tick-infested. The tick-infested Zebu types also developed a depressed response compared with the tick-free Zebu group over a 27 month study period. However, the responses of the N'Damas was similar to that of the tick-free Zebu types. The addition of autologous serum to Con A stimulated cultures of lymphocytes derived from the tick-infested Zebu types and N'Damas suppressed their proliferative response compared with that of similar cultures for the tick-free Zebu types.

Seasonal prevalence of ticks and their association with dermatophilosis in cattle on the Accra plains of Ghana.

The seasonal abundance of adult ticks on cattle and their association with dermatophilosis were investigated in five herds on the coastal plains of Ghana over a 26-month period. Four genera, Amblyomma, Boophilus, Rhipicephalus and Hyalomma were identified, A. variegatum being the predominant species occurring throughout the year with two peaks of infestation, one in April-May and the other in November. A significant positive correlation was revealed between A. variegatum and dermatophilosis in four of the five herds. Significant positive correlations were found between H. m. rufipes and dermatophilosis in two of the herds and between Rh. senegalensis and dermatophilosis in one herd. Negative correlations of statistical significance were observed between Boophilus species and dermatophilosis in three of the herds. Nevertheless, it was considered that A. variegatum was the most important tick factor involved in the pathogenesis of the disease.

Comparative effects of sexual assault on sexual functioning of child sexual abuse survivors and others.

This study quantitatively and qualitatively examined the effects of sexual assault on the sexual functioning of 37 sexually active women an average of 8.21 years postevent (Mdn = 4.08 years). More than 80% of the sample reported some sexual dysfunction with a partner as a result of the assault. Greatest impairment was reported by subjects who either had a history of child sexual abuse or had no prior sexual victimization before the current assault as compared with subjects who had prior sexual assaults. When data were examined by type of perpetrator, adverse effects were greatest for subjects assaulted by a health care professional. Qualitative analysis revealed that, for the total sample, greatest effects were in the area of adverse feeling states (part of desire dysfunction) as early response inhibitors, with subjects who had a history of child sexual abuse being the only group to report orgasmic dysfunction and guilt. There was no statistically significant difference in sexual dysfunction between subjects who filed civil suits and those who did not. Implications for treatment are discussed.

Further evidence for the protective role of sub-parietal cell membranous secretory product on the cuticle of a pentastomid arthropod parasite developing in its rodent intermediate host.

The pentastomid parasite Porocephalus crotali, develops to an infective stage within a granulomatous lesion in the tissues of rodent intermediate hosts. A conspicuous layer of sub-parietal cell (SPC) secretory product, which coats the intermoult cuticle up to a depth of 12 microns, is described. Around the first five nymphal instars this material consists of an amorphous matrix with distinctive electron-lucid lacunae, but that around later instars (six and seven), while retaining much of the original morphology, possesses a significant membranous component. Host effector cells, most notably eosinophils and macrophage/epithelioid cells, are frequently completely enveloped by SPC secretion but invariably appear unreactive to it. Host cells may penetrate to the outermost layer of the epicuticle but again but again cytotoxic activity is absent. During ecdysis, effector cells are recruited to the intercuticular space where widespread degranulation is evident. Some of this is specifically directed against the underside of the cast cuticle, but not against the newly exposed cuticle. Protracted degranulation eventually reduces the cast cuticle to fragments which are endocytosed by giant cells. 1 cm long infective (seventh-stage) nymphs, which retain the sixth stage cuticle as a protective sheath, are largely devoid of membranous secretion and these were dissected from cysts, washed, and surgically transplanted into the body cavities of naive and infected mice. Pronounced differences in the onset and intensity of the subsequent inflammatory response in the two categories of host indicate some form of specific recognition. In both groups of mice though, the cuticle is an eventual target for attack by effector cells, and parasites are killed. The protective function of SPC secretion is discussed.

Light microscope observations of granulomatous reactions against developing Porocephalus crotali (Pentastomida: Porocephalida) in mouse and rat.

The development of granulomatous reactions against moulting nymphal pentastomids (Porocephalus crotali) in the tissues of rat and mouse intermediate hosts is described. Adipose tissue and lungs are favoured sites for encystment accounting for 70% of larvae. Six moults separate the primary larva from the final infective stage which first appears about 80 days post-infection (p.i.) and is fully infective by day 120. Larvae, and particularly their cast cuticles, are the foci of granulomatous reactions characterized by an intense eosinophilia. During ecdysis, large numbers of eosinophils permeate the entire lesion but, significantly, degranulation is limited to the underside of cast cuticles where the resultant debris is endocytosed by macrophage/epithelioid cells. A pronounced asymmetry in the granulomatous lesion, evident even in the earliest cysts, results from the accumulation of individual epithelioid granulomas associated with cuticle fragments close to the ventral side of the developing parasite; each is circumscribed by fibrosis. External to this region are extensive tracts of tissue composed of mature plasma cells. Particularly in rats, large numbers of partially degranulated mast cells (= globule leucocytes) also surround cuticle granulomas, and mast cell granules can accumulate within macrophages and fibroblasts. Inflammation slowly subsides once the infective stage is attained. This 1 cm-long larva resides in a thin, fibrotic, C-shaped cyst and can remain viable for years: uniquely this instar retains its last moulted cuticle as a protective sheath. Nymphal instars II-VI feed predominantly upon eosinophils but we do not yet know whether this requirement is obligate.

Fine structural aspects of secretory processes in a pentastomid arthropod parasite in its mouse and rattlesnake hosts.

The histology and development of three extensive glands in the porocephalid pentastomid Porocephalus crotali is described by light and electron microscopy, during growth of the parasite to an infective stage in the tissues of mouse; the infective stage in rattlesnake definitive hosts is also included. These glands elaborate excretory/secretory components which are channelled, via chitin-lined efferent ductules, on to the parasite cuticle. Hook and frontal glands are relatively compact, and within each gland ductules serving individual secretory lobules collect into common ducts which discharge over each of the four hooks, or at the anterior margin of the cephalothorax respectively. Subparietal gland cell lobules, composed of two large and two small secretory cells, are distributed under the cuticle and each is served by a single efferent ductule; these erupt over the entire cuticle. The large cells in subparietal glands secrete lamellate droplets which coat the cuticle with thin layers. Identical cells are found in hook and frontal glands, in addition to to three morphologically distinct types of protein secretory cell. Preliminary data on the composition and immunological properties of the various secretory products are presented.

Studies on the host/parasite interface during the development of a pentastomid arthropod parasite in rodent intermediate hosts, with observations on protective surface membranes.

The changing structure of the cuticle of the arthropod pentastomid parasite Porocephalus crotali, during growth to the infective stage in mouse and rattlesnake hosts, is described. The outermost cuticulin layer of the cuticle in instars II-VI is elevated to form a dense mat of epicuticular hairs. Since the VI larval cuticle is retained by the infective (VII) nymph as a protective sheath, effectively all stages in mice present a hairy surface to the host and this may constitute a physical barrier to inflammatory cells. The entire surface is overlain by a triple-track 'unit' membrane whose biophysical properties resemble those of a conventional plasma membrane, and there is evidence to suggest that this membrane is susceptible to immune attack. Under natural circumstances, epicuticular hairs entrap secretion, delivered to the cuticle via innumerable minute ducts which communicate with tegumental secretory cells termed subparietal cells (SPC). SPC synthesize lamellate droplets which unfold on the cuticle to constitute a layer of protective polymorphic vesicles. By contrast, infective nymphs in snakes possess a smooth cuticle and SPC membranous secretion is stacked over the entire surface, in sheets up to 20 deep. The function of the lipid and protein components of SPC secretion is discussed.