A comparison of extracted proteins of isolates of Dermatophilus congolensis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting.

Antigenic diversity within a collection of 18 isolates of Dermatophilus congolensis from different Continents was examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting with sera from cattle with clinical dermatophilosis using whole cell extracts obtained by three methods and one extract of extracellular products of D. congolensis. One of the methods involving the release of a lysostaphin-solubilized protein (LSP) of whole cells of D. congolensis revealed a number of discrete and easily-identifiable bands in SDS-PAGE which were found suitable for characterizing protein patterns and was, therefore, subsequently used for a comparative analysis of the proteins of all the D. congolensis isolates. Six electropherotypes (ET) of D. congolensis were identified among the 18 isolates using the protein profiles based on the presence of four protein bands at Molecular weights (MW) 62, 28, 17.4 and 16.4 kDa. The ETs were found among isolates from different animal species and from different sources with ET1 consisting of three bovine and two equine isolates; ET2, two bovine and three ovine isolates; ET3, two bovine isolates; ET4, two bovine isolates; ET5, one bovine and one ovine isolates and ET6, two bovine isolates. Immunoblotting of the extracts of D. congolensis isolates with sera from cattle with clinical dermatophilosis infection demonstrated protein bands of MW ranging from 9 kDa to 188 kDa. Sera from chronic dermatophilosis infection demonstrated a 28 kDa protein which was immunodominant in the LSP extracts of all the 18 isolates of D. congolensis tested while sera from mild infections demonstrated mainly the 62 kDa protein in the same extracts. However, many protein bands were demonstrated in surface membrane (TSMP) and extracellular protein extracts with sera from only mildly infected animals. The protein patterns observed in all isolates of D. congolensis revealed global antigenic similarities and distinct differences among isolates which could not be associated with either geographic, climatic or host factors. Also sera from infected animals from endemic regions of dermatophilosis could not differentiate isolates of D. congolensis. This suggests the possibility that such sera must have come from animals that had been infected by a multitude of D. congolensis strains present in the herd environment and strains an animal could have come across during the 'ritual' annual cross-country migration of the cattle herds.     

Antibodies to malignant catarrhal fever virus antigens in the sera of normal and naturally infected cattle in Kenya

Six different serological tests were used to examine Kenyan cattle sera for antibodies to the herpesvirus of malignant catarrhal fever. Significantly higher levels of indirect immunofluorescent antibody to early and late virus antigens and of complement fixing antibody were found in the sera of 13 naturally infected cattle than in 482 sera collected from four different groups of normal cattle. Virus neutralising and immunoprecipitating antibodies were also found in some infected cattle sera but not in normal cattle sera. Many non-specific reactions occurred using counterimmunoelectrophoresis. These preliminary results indicate that the serological diagnosis of wildebeest-associated malignant catarrhal fever may be possible.     

The heterogenicity of Cowdria ruminantium stocks: cross-immunity and serology in sheep and pathogenicity to mice

Ten stocks of Cowdria ruminantium (Ball 3, Breed, Comoro, Germishuys, Kümm, Kwanyanga, Mali, Mara, Nonile and Welgevonden) were compared from a cross-immunity, serological and mouse pathogenicity point of view. They were found to differ in varying degrees. Except for the Ball 3, Comoro and Germishuys stocks that were similar but not identical, there was no pattern in the antigenic diversity of the 10 stocks. The Welgevonden stock emerged as the stock that elicits an immunity against most of the South African stocks. The inability of the reference Ball 3 stock to protect sheep against no fewer than 6 other stocks questions the advisability of retaining this stock as the vaccine stock. The antigenic diversity of the 10 stocks could not be correlated with the antibody levels detected with the indirect fluorescent antibody test, since the sera against all 10 stocks reacted positively to the Kümm stock antigen and the variation in titres was not stock-related.     

Comparison of three enzyme-linked immunosorbant assays with the indirect immunofluorescent antibody test for the diagnosis of canine infection with Ehrlichia canis

The aim of this study was to compare three different enzyme-linked immunosorbant assays (recombinant major antigenic protein 2 (rMAP2)-ELISA, the Immunocomb (Biogal, Israel) and the Snap 3Dx assay (IDEXX Laboratories Inc., USA)) with the indirect immunofluorescent antibody test in detecting anti-Ehrlichia canis immunoglobulin-G (IgG) antibodies. Samples tested were collected from dogs suspected to be naturally infected with E. canis and from experimentally infected dogs.When qualitative results (positive/negative) were compared, there was an overall agreement of 81% (54/67) between the indirect immunofluorescence antibody (IFA) test and the rMAP2-ELISA. An overall agreement of 94% (63/67) was found between the IFA test and the Immunocomb, and an overall agreement of 91% (61/67) was found between the IFA test and the Snap 3Dx assay. In 50 of 67 (74.6%) samples tested, complete agreement in the qualitative results was found in all four tests. Sixteen of 17 samples with disagreement in the qualitative results were found to have IFA titers of 1:320 or less. The sensitivities and specificities of the tests were found to be 0.71 and 0.85 for the rMAP2-ELISA, 0.86 and 0.98 for the Immunocomb, and 0.71 and 1.00 for the Snap 3Dx assay.The tests performed in this study were found to be highly specific in detecting E. canis antibodies. Their sensitivity was found to be low with sera having IFA titers of < or =1:320, while high with sera having titers greater than 1:320. Repeating the serological tests 1-2 weeks after the first antibody assay may overcome the sensitivity problem with titers of < or =1:320.     

Inflammatory cell and immune function in Merino sheep with chronic dermatophilosis

Components of inflammatory and immunological responses were compared in 17 Merino sheep with chronic dermatophilosis (Group 1) and 15 Merino sheep that had recovered from the disease (Group 2). The functions studied included: (i) total and differential white cell counts; (ii) phagocytic function and intracellular killing by neutrophils; (iii) humoral immune response to T-dependent and T-independent antigens and to Dermatophilus congolensis. (iv) lymphocyte blastogenic responses to phytohaemagglutinin; (v) bovine serum albumen and D. congolensis antigens; (vi) quantification of T-lymphocyte subsets in skin lesions resulting after re-infection with D. congolensis zoospores. After all lesions were treated and the sheep were shorn, both groups of sheep were re-infected with D. congolensis. Both groups had similar infection rate, severity of lesions and rate of resolution after re-infection. The Group 2 sheep had significantly higher primary and secondary antibody responses to killed Brucella abortus cells than Group 1 sheep, but Group 1 sheep had higher levels of specific D. congolensis antibody throughout the trial. Neutrophils from Group 1 sheep showed a higher phagocytic rate for D. congolensis zoospores than Group 2 sheep when the zoospores were opsonised by sera from the Group 1 sheep, but there was no difference in their ability to kill ingested zoospores. Although there were some differences between the groups in the proportion of lymphocytes in lesions that reacted with monoclonal antibodies to T4, T8 and T19-19 lymphocyte markers at various times after re-infection, the sheep in Group 2 consistently had higher levels of lymphocytes reacting to a monoclonal antibody for the T6 lymphocyte antigen in skin biopsies collected 9, 15 and 21 days post-inoculation (p.i.) than did sheep in Group 1. Group 2 sheep also had higher levels of epidermal cells with immunohistochemical properties of Langerhans cells at lesion sites 15 and 21 days p.i.     

Enzyme-linked immunosorbent assays for detecting antibody to Rickettsia australis in sera of various animal species

New endemic areas of spotted fever-like rickettsial disease have been found in south-eastern Australia (Gippsland, Victoria and Flinders Island, Tasmania). The rickettsia responsible is currently unknown although it may be Rickettsia australis. To investigate serological evidence of rickettsial exposure in various wild animal species, a competitive ELISA was developed which detected antibodies to R. australis. It was based on inhibition of an indirect ELISA detecting antibody to R. australis in guinea pig sera. Pre- and post-infection sera from 2 dogs, 2 rabbits, 5 mice and 6 rats, experimentally infected with R. australis, were tested by competitive ELISA. The results showed that all pre-infection sera were negative and all post-infection sera positive for antibody to R. australis. To test the utility of the competitive ELISA for detecting natural rickettsial infection in non-laboratory animals, 51 dog sera, negative for rickettsial antibody by immunofluorescence (IF) and 20 IF positive dog sera (collected from various locations on the east coast of Australia) were tested. Compared to the IF test the competitive ELISA was 90% sensitive and 96% specific. This new test has potential for detecting antibody to R. australis in the sera of different wild animal species.     

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.     

Serological reactivity of duck sera to three electrophoretically characterized extracts of Aspergillus

Three different antigens, whole culture, cell sap shaker culture and culture filtrate extracts of Aspergillus were characterized by crossed immunoelectrophoresis. Thirteen to fifteen precipitin lines were produced with their homologous antisera. Application of these extracts to the assay of wild duck sera showed that the whole culture extract was the most sensitive of the three antigens in detecting antibody. Countercurrent immunoelectrophoresis, used with a modified buffer, produced results comparable in sensitivity to immunodiffusion in detecting avian antibody.     

Comparison of a modified counterimmunoelectrophoresis test and a microimmunodiffusion test for detection of pseudorabies virus antibodies in porcine sera.

The correlation of a modified counterimmunoelectrophoresis (CIE) test and a microimmunodiffusion test for detecting pseudorabies virus antibodies in porcine sera was investigated, using as reference a standard virus neutralization test. The counterimmunoelectrophoresis test exhibited a sensitivity comparable to the microimmunodiffusion test but was not as sensitive as the virus neutralization test. The best feature of the modified counterimmunoelectrophoresis test is that it is a rapid test. It provides an alternative to currently used diagnostic tests for detection of pseudorabies virus antibodies in sera from field reared and experimentally reared swine exposed to pseudorabies virus.     

Detection of Dermatophilus congolensis antibody in the milk of streptothricosis infected cows

Passive haemagglutination and agar gel diffusion tests were used to detect specific antibody to Dermatophilus congolensis antigens in serum and milk of eight streptothricosis infected Friesian milking cows. All the sera and milk samples showed the presence of antibody but titres were higher in sera. Precipitating antibodies were detected only in three sera. A possible implication of this finding is discussed with respect to passive immunity in the young calves from infected dams.     

重组M蛋白-乳胶凝集试验检测PRRS病毒血清抗体的研究

利用纯化的 PRRS病毒重组 M蛋白致敏乳胶制成乳胶抗原 ,成功地建立了一种检测 PRRS病毒血清抗体的乳胶凝集试验 (L AT)诊断方法。用制备的乳胶 M抗原分别检测猪瘟、猪伪狂犬病、猪细小病毒病、猪弓形体病、猪衣原体病、猪乙型脑炎阳性血清 ,结果均为阴性 ,无交叉反应 ,说明建立的 L AT方法具有良好的特异性。用建立的乳胶凝集试验方法与国外IDEXX公司 PRRS病毒抗体检测试剂盒同时对 76份猪血清样本进行检测 ,结果表明建立的 L AT方法的特异性和敏感性均为 95 % ,两种方法的总符合率为 87% ,检出率基本一致。研究结果表明 L AT方法具有操作简便、快速、敏感性高、特异性强、价格低廉且可用于现场检测等优点 ,是一种适合基层兽医单位用于 PRRS病毒血清抗体检测的新方法     

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.     

The prevalence of antibodies of Brucella abortus, Dermatophilus congolensis and bovine leukaemia virus in Nigerian slaughter cattle

In a pilot survey to compare the relative prevalence of three diseases in apparently healthy White Fulani Zebu (WFZ) cattle slaughtered in Nigeria, sera from 80 randomly selected animals with no significant gross lesions on ante mortem and post mortem inspection were examined for antibodies to Brucella abortus, Dermatophilus congolensis and bovine leukaemia virus. Of the samples screened, 5.0, 8.8 and 2.0% showed serological evidence for brucellosis, cutaneous streptothricosis and bovine leukosis respectively.     

Quantitation of hemorrhagic enteritis virus antigen and antibody using enzyme-linked immunosorbent assays

Enzyme-linked immunosorbent assays (ELISAs) were developed to quantitate hemorrhagic enteritis virus (HEV) antibodies in turkey sera and HEV antigens in tissue extracts. These assays were more sensitive than the commonly used agar-gel precipitin tests in detecting antigen and antibody. The antibody-ELISA was used to monitor the presence and decline of passive antibodies in turkey poults and the seroconversion of turkeys infected with HEV. The antigen-ELISA was carried out using a monoclonal antibody; this test was used to quantitate HEV antigen in experimentally infected turkeys in a time-sequence experiment. Both ELISAs measured a strong antigenic relationship between an avirulent strain (HEV-A) and a virulent strain (HEV-V).     

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.     

Study of antigenic heterogeneity among Actinobacillus pleuropneumoniae serotype 7 strains

Actinobacillus pleuropneumoniae serotype 7 strains were studied for their antigenic heterogeneity using rabbit polyclonal hyperimmune sera against all the known twelve reference strains of A. pleuropneumoniae and a battery of different serological tests such as coagglutination (COA), immunodiffusion (ID), indirect hemagglutination (IHA), counterimmunoelectrophoresis (CIE), rapid dot-ELISA (RDE), serum soft-agar (SSA) and growth agglutination (GA). Reference serotype 7 strain (WF83) showed cross-reactivity with reference serotype 1B strain but not with other serotypes. Field serotype 7 strains showed cross-reactivities with serotypes 1A, 1B, 4, 9, 10, and 11 in COA, ID, and CIE tests, but not in IHA test. Two field strains of serotype 7 (90-3182 and 86-1411) which appeared to be different from the typical serotype 7 strains were selected for further antigenic characterization by SDS-PAGE, Western blot, and Tricine SDS-PAGE assays, and identified as serotypes 1 and 7, respectively. For serotyping atypical strains, it is suggested to use Western blot assay as a confirmatory test to identify serotype-specific capsular and somatic antigens.     

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.     

An adhesion-hemadsorption inhibition test for the detection of serum antibody to Mycoplasma gallisepticum

A simple adhesion-hemadsorption inhibition (AHAI) test was developed for the detection of antibodies to Mycoplasma gallisepticum in the chicken sera. The AHAI antibody was detected simultaneously with HI antibody from sera of chickens intratracheally inoculated with viable cells of M. gallisepticum. A good correlation between HI and AHAI antibody titers was obtained with 382 (84.7%) of 451 sera from chickens reared on farms spontaneously contaminated with M. gallisepticum, whereas the remainder, 69 sera, was positive for HI but negative for AHAI test. It was not apparent whether the latters exhibited a non-specific reaction or the discrepancy was due to the lower sensitivity of AHAI reaction. The AHAI test does not require a great amount of antigen, special reagents or instruments, or pre-absorption treatment of test sera, and, therefore, it may serve as a simple serological test for detecting antibodies to M. gallisepticum.     

Cellular immune responses of the rat to experimental infection with Dermatophilus congolensis

The host cell-mediated immune response was examined following experimentally-induced infection of rats with Dermatophilus congolensis, the causal agent of the skin disease dermatophilosis. Mononuclear cells (MC) isolated from Wistar rats 10 days following the induction of a third infection underwent a strong and specific proliferative response, as assessed by a [3H]thymidine incorporation assay, when cultured with various concentrations of inactivated D. congolensis cocci. Using specific monoclonal antibodies in an indirect fluorescent antibody test, this in vitro response was found to be characterised by a large expansion of the W3/25 (T-helper phenotype) population to form 56% of the total. Finally, the primed and stimulated MC were assessed for their ability to produce factors capable of inhibiting macrophage migration. The culture supernatants of D. congolensis-stimulated MC from infected rats caused significant migration inhibition of normal rat peritoneal exudate cells, whilst the supernatants of similarly-stimulated MC from naive rats failed to cause significant inhibition. The results show that a MC subpopulation becomes primed following experimentally-induced infection with D. congolensis and becomes activated after subsequent, in vitro, exposure.     

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.