Humoral antibody response to glutaraldehydetreated antigens of Dermatophilus congolensis

Summary

Glutaraldehyde‐treated whole cell antigens (GA.WcA) of Dermatophilus congolensis induced in guinea pigs immunological memory in contrast to cell wall antigens treated similarly (GA.CwA). However, GA.WcA could not induce a secondary response in animals primed with untreated WcA while GA.CwA on the other hand did stimulate a secondary response in animals primed with untreated CwA. Primary antibody production was induced by both GA.CwA and untreated CwA to a similar level in their respective hosts but it was the secondary response that was found similar in response to GA.WcA and untreated WcA. However, both untreated WcA and CwA induced primary and secondary antibody production in their respective hosts though these responses were considerably higher in guinea pigs given untreated CwA. This study showed that both untreated and GA‐treated antigens of D. congolensis are capable of stimulating antibody production in guinea pigs but they differ in their levels of stimulation.     

Humoral antibody response to glutaraldehyde-treated antigens of Dermatophilus congolensis

Glutaraldehyde-treated whole cell antigens (GA.WcA) of Dermatophilus congolensis induced in guinea pigs immunological memory in contrast to cell wall antigens treated similarly (GA.CwA). However, GA.WcA could not induce a secondary response in animals primed with untreated WcA while GA.CwA on the other hand did stimulate a secondary response in animals primed with untreated CwA. Primary antibody production was induced by both GA.CwA and untreated CwA to a similar level in their respective hosts but it was the secondary response that was found similar in response to GA.WcA and untreated WcA. However, both untreated WcA and CwA induced primary and secondary antibody production in their respective hosts though these responses were considerably higher in guinea pigs given untreated CwA. This study showed that both untreated and GA-treated antigens of D. congolensis are capable of stimulating antibody production in guinea pigs but they differ in their levels of stimulation.     

Use of a monoclonal antibody in the diagnosis of infection by Dermatophilus congolensis

A monoclonal antibody (McAb) to Dermatophilus congolensis was produced from murine hybridoma cultures and purified by affinity chromatography. Species specificity was demonstrated using indirect immunofluorescent staining; the McAb was shown to react with 10 D congolensis isolates but not with 10 Nocardia species isolates, a Rhodococcus and a Streptomyces species isolate. The McAb was used to demonstrate D congolensis in clinical material from confirmed bovine and ovine cases and presumptive equine cases of dermatophilosis by indirect immunofluorescent staining.     

Dermatophilus congolensis Infection in Goats in Tanzania

When goats in Eastern Tanzania were screened for skin diseases, Dermatophilus congolensis was isolated from the skin lesions in 8 of 484 animals examined. In one severely affected case, the disease was also characterized by histological studies (Gram stain, Giemsa stain and routine HE studies) and electron microscopy. The histological picture was characterized by hyperkeratosis, parakeratosis, acanthosis, folliculitis and an inflammatory cellular reaction involving the epidermis. Gram stain and Giemsa stain revealed longitudinal and transverse branching filaments in the deeper layers of the epidermis. Ultrastructural studies demonstrated D. congolensis in various morphological forms, ranging from filamentous to tuber-shaped structures, mixed with numerous coccoid bodies of variable size. In some instances, the organisms were geometrically arranged in parallel rows of beading and were present in and among the degenerated epithelial cells. Several host cells showed degenerative changes. Ticks present on the goats were Amblyomma variegatum, Rhipicephalus evertsi, Rhipicephalus pravus and Boophilus sp. The clinical signs, pathological lesions, diagnosis, epidemiology and pathogenesis of the disease are discussed.     

Hemolytic interactions of Dermatophilus congolensis.

The strains of Dermatophilus congolensis grew on blood agar with washed sheep erythrocytes with marked total hemolysis. In testing for hemolytic interactions they gave a significant synergistic effect of a characteristic shape with Rhodococcus equi and Streptococcus agalactiae, whereas with Staphylococcus aureus producing beta hemolysin and with Staphylococcus aureus producing delta hemolysin a simultaneous synergistic as well as antagonistic effect were observed. First of all a conspicuous inhibition of in the beta hemolysin zone began and then the hemolytic effect of D. congolensis was enhanced. A similar double reaction was also observed with Listeria ivanovii. With delta hemolysin there was an inhibition of the hemolytic effect of D. congolensis and at the same time a synergistic effect could be observed. Also D. congolensis gave a weak synergistic effect with Micrococcus lylae and Listeria monocytogenes, and a further weak antagonistic effect with alpha hemolysin of Staphylococcus aureus, Staphylococcus hyicus, Staphylococcus chromogenes and Micrococcus luteus. No interaction of D. congolensis was established with Corynebacterium pseudotuberculosis.     

Isolation of Dermatophilus congolensis from a cat

Dermatophilus congolensis was isolated from a cat with dermatitis. The isolate was sensitive to oxytetracyclin, streptomycin and penicillin but resistant to ampicillin, amoxicillin, gentamycin and cefoperazone.     

Extent of skin penetration byDermatophilus congolensis in bovine streptothricosis

Summary Hyphae ofDermatophilus congolensis were noted in the upper epidermis especially in the stratum corneum and sometimes reaching the basement membrane (dermo-epidermal junction). Hyphae and coccoid forms were also seen in hair follicle sheaths and sebaceous glands which formed the main avenues for dermal invasion. Dermatophilus organisms were seen in the papillary layer of the dermis in areas where the basement membrane of the epidermis had disintegrated but none were seen in areas where the basement membrane was intact.

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Sumario Hifas deDermatophilus congolensis fueron vistas en la epidermis superior, especialmente, en el estrato córneo y algunas veces se encontraron en la membrana basal (union dermo-epidermo). Las hifas y las formas cocoides fueron tambien vistas en las vainas de los folículos pilósos y glándulas sebáceas, las cuales formaban las mas grandes “avenidas” para la invasión dermal. Los organismos Dermatophilus fueron vistos en la capa papilar de la dermis en areas en donde la membrana basal de la epidermis se habia desintegrado, pero, ningun organismo fue visto en areas en donde la membrana basal estaba intacta.

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Résumé Les hyphes deDermatophilus congolensis sont trouvées dans la couche supérieure de l'épiderme, en particulier dans lestratum corneum, et quelquefois atteignent la membrane basale (frontière dermo-épidermique). Des hyphes et des formes cocco?des sont vues aussi dans la gaine des follicules pileux et dans les glandes sébacées qui constituent les principales voies d'invasion du derme. Ces mêmes éléments microbiens s'observent aussi dans la couche papillaire du derme, dans les zones ou la membrane basale de l'épiderme a été détruite; par contre on ne les trouve jamais dans les zones où cette membrane est restée intacte.

     

Serodiagnosis of Dermatophilus congolensis infection by counterimmunoelectrophoresis

Sixty-one sera from animals that had contact with Dermatophilus congolensis were examined by comparing three serological methods; counterimmunoelectrophoresis, passive haemagglutination, and agar gel diffusion, and by using four different antigenic extracts of D congolensis. The counterimmunoelectrophoresis was the most satisfactory of the methods having been found to be specific and sensitive, easy to perform and suitable for screening large numbers of samples. It was also found to have a higher antibody detection rate (82.2 per cent) than the other methods thus making it suitable for seroepidemiological surveys. It was found to be capable of detecting multiple antibodies and also revealed dissimilarities among the different antigenic extracts. The cellular antigens of D congolensis were found to detect antibody in more sera than the extracellular antigen; the cell wall extract proved to be the most satisfactory of all, detecting antibody from the largest number of sera compared to the other extracts in all the three serological tests.     

Strain variation in Dermatophilus congolensis demonstrated by cross-protection studies

Cross-protection studies were conducted with vaccines prepared from two isolates of Dermatophilus congolensis (designated strain 1 and strain 2). The vaccines were prepared as either heat-inactivated, washed, formalized filamentous phase bacterium, mixed with alum as an adjuvant, and inoculated intramuscularly (type A vaccine) or sedimented live filaments inoculated intradermally (type B vaccine). The vaccinated sheep were challenged with D. congolensis zoospores of one or other strain. Challenge sites were observed for the presence and severity of lesions. Serum antibody levels to D. congolensis were monitored after vaccination and challenge. Type A and B vaccines from both strains produced some reduction in the severity of lesions when sheep were challenged with strain 1 but not with strain 2. Unvaccinated control sheep developed more severe and persistent lesions when challenged with strain 2 than controls challenged with strain 1. Serum antibody levels to the type B vaccine prepared from strain 1 were significantly higher (P less than 0.05) than antibody levels to type B vaccine from strain 2. These findings showed there was significant variation in virulence and antigenicity between these two isolates of D. congolensis.     

Serum and skin surface antibody responses in merino sheep given three successive inoculations with Dermatophilus congolensis

Three antigens prepared from different phases of the life cycle of Dermatophilus congolensis were used in an enzyme-linked immunosorbent assay to measure serum and skin surface antibody responses in sheep after a first, second and third inoculation with D. congolensis. After the first inoculation, a strong antibody response to the flagella, filament and soluble antigens was detected after 7-21 days in the sera from sheep that were regularly biopsied; the antibody response at the skin surface was detected 28-42 days after inoculation, when the lesions were resolving. Strong anamnestic responses were detected in the serum of sheep that were biopsied and some of the nonbiopsied sheep after the second and third inoculations, but the skin surface antibody response at these times was variable.     

Identification of an alkaline ceramidase gene from Dermatophilus congolensis

A random amplified polymorphic DNA (RAPD) procedure was used to identify a specific 0.6 kb DNA fragment unique to Dermatophilus congolensis. This 0.6 kb fragment was evaluated as a specific DNA probe and used to design oligonucleotide primers for polymerase chain reaction (PCR) amplification. The nucleotide sequences adjacent to this DNA fragment were determined by inverse PCR allowing the identification of a 4.1 kb sequence. Analysis of this revealed a complete open reading frame (ORF) with a high similarity to an alkaline ceramidase from Pseudomonas aeruginosa. The molecular weight of the enzyme derived from the predicted amino acid sequence is 74,662 Da, its pI is 9.81. The predicted N-terminal sequence of the enzyme contains a signal sequence indicating that the enzyme is exported by the bacterium. Since ceramides have important protective and cell regulatory roles in the epidermis we suggest that this ceramidase may have a role in the pathogenesis of dermatophilosis. It is the first completely sequenced gene described for D. congolensis.     

盘羊刚果嗜皮菌的分离与鉴定

从疑似刚果嗜皮菌感染的盘羊皮肤病料中分离刚果嗜皮菌,并进行鉴定。将皮肤病料于28 ℃风干、粉碎,加无菌水浸泡15 d,静置,取上清加6%酵母膏液,60 ℃条件下30 min,混匀后连续10倍倍比稀释涂板,37 ℃培养48 h后挑取可疑菌落进行细菌形态学、培养特性、间接免疫酶标染色法鉴定,并根据GenBank上发表的刚果嗜皮菌16S rRNA序列设计特异性引物对分离株进行PCR扩增,得到与预期相符的606 bp基因片段,结果证实该盘羊分离株为刚果嗜皮菌。     

Dermatophilus congolensis bovine dermatophilosis in the French West Indies. III. A comparison between infected and non-infected cattle

The determination of the distinctive features between infected and uninfected cattle by Dermatophilus has been performed among 93 bovine stock in La Martinique, and 120 in La Guadeloupe and Saint-Martin Islands. Nearly half of the herds had been or were infected. Morbidity rates reach 33 per cent in La Martinique and 29 per cent in La Guadeloupe with a mortality ratio of 15 per cent and 5 per cent respectively. No frequency difference could be observed with respect to the season or the geographical area. Similarly, differences are little or nil as regards animal exposure toward aggressive factors such as sun, soil moisture, thorns, etc. On the other hand, the presence of Amblyomma variegatum is a determining factor considering the prevailing situation in La Martinique, where all breeds of cattle are sensitive to the disease. In this case, 63 per cent of the tick infested cattle develops dermatophilosis versus 2.5 per cent for the still uninfected animals. Conversely, in La Guadeloupe were Creole zebus are highly resistant (as a consequence of their natural selection), only 27 per cent of the infected stock suffers from this illness. In these two islands, regular dipping measures have proved to be beneficial in that it reduces the disease frequency. The study also reveals an underlying typology of the various rearing systems, thus allowing their classification as for their behaviour and response when meeting illness. In La Martinique, whatever they may be, quite none of them is infected in the tick free area. In the tick infested zones, the traditional small breeders rearing a sensitive cattle and who do not care with prophylactic rules, are mostly stricken, while more structured ones have succeeded in controlling the disease, owning to some appropriate measures together with a fair animal management. On the other hand, in La Guadeloupe where this tick is cosmopolitan, the small breeders who run a resistant Creole cattle, are not concerned, despite a rudimentary prophylactic behaviour. Conversely, the intensive rearing system, based upon improved imported breeds, meets great difficulties to master the infection.     

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.     

Cure of Dermatophilus congolensis infection in cattle by long-acting oxytetracycline

Under conditions simulating traditional husbandry, a single intramuscular dose (20 mg/kg) of long-acting oxytetracycline was efficacious in treating different grades of bovine dermatophilosis. There was complete healing in 26 out of 28 animals (93 per cent) within four weeks. By contrast, only four out of 11 animals treated with penicillin (70,000 iu/kg) plus streptomycin (70mg/kg) were apparently cured and three relapsed within one month. No spontaneous recoveries were observed among 18 untreated animals. In the group treated with oxytetracycline, two, in the penicillin plus streptomycin, seven, and in the untreated control group, 11 animals died of the disease.