Luminescent-serologic analysis of the antigenic interrelationship between R. canada and classic representatives of rickettsiae of the typhus group

The results of studying the antigenic relationships of R. canada, a new Rickettsia species, and classical Rickettsia species of the typhus group are presented. The study was carried out by luminescent serological analysis with the use of corpuscular antigens and the live infectious agent cultures. R. canada and Rickettsiae of the typhus group were similar in their antigenic structures; this, however, could be revealed only in the study of the live cultures of the infectious agents. The study of corpuscular antigens revealed unilateral relationship: R. prowazeki antigen could be detected with homologous and heterologous sera, R. canada antigen with homologous serum only. In the CFT and the agglutination test corpuscular R. canada antigen reacted with homologous and heterologous sera. The study of the live cultures of the infectious agents revealed that different R. prowazeki and R. typhi strains vary in the degree of their similarity to R. canada.     

Importance of humoral immunity indices in determining the Rickettsia prowazekii carrier state]

Experiments showed the possibility of making indirect conclusions concerning rickettsial carrier state by the method of determination of complement-fixing antibodies to R. prowazeki in the blood serum. Though not indicative of carrier state in individual animals, these antibodies, their dynamics and titers gave the evidence of group carrier state in cotton rats in respect of the causative agent of typhus. The number of animals carrying R. prowazeki increased with the rise of antibody titers. Negative seroconversion indicated the elimination of the causative agent from the body of the animal. The experimental results were confirmed by the data on the dynamics of the immunological structure of population, as well as by information contained in the literature on this problem.     

Experimental forms of infection and serological analysis of the antigenic structure of Rickettsia canada

Experimental forms of Rickettsia canada infection were characterized and serological analysis of the antigenic structure of R. canada was carried out. According to its pathogenicity for experimental animals, R. canada can be characterized as a poorly virulent species of rickettsiae, similar to R. prowazekii (for guinea pigs). The complement-fixing, haemagglutinating and agglutinating antigens of R. canada are fairly similar to those of the typhus group rickettsiae. The region of antigenic activity common to or identical in R. canada and the typhus group rickettsiae, is larger in R. canada than in the typhus group rickettsiae. R. canada has common antigens with Proteus OX19. R. canada has active toxic substances similar to those of R. prowazekii which, however, are detectable only with sera of Brill's disease convalescents. The position of R. canada in the taxonomy of rickettsiae is discussed.     

Staining of rickettsial antigens for microagglutination reaction.

Staining of antigens of R. prowazeki, R. canada and R. conori with hematoxylin-Harris for use in MAR was developed. Optimal results were obtained when rickettsial antigens were stained at 4 degrees C for 18 hours. Comparison of MAR with CFR has shown that MAR using stained rickettsial antigens is specific and as sensitive as CFR.     

Antigenic relatedness between glycoproteins of human respiratory syncytial virus subgroups A and B: evaluation of the contributions of F and G glycoproteins to immunity.

The degree of antigenic relatedness between human respiratory syncytial virus (RSV) subgroups A and B was estimated from antibody responses induced in cotton rats by respiratory tract infection with RSV. Glycoprotein-specific enzyme-linked immunosorbent assays of antibody responses induced by RSV infection demonstrated that the F glycoproteins of subgroups A and B were antigenically closely related (relatedness, R approximately 50%), whereas the G glycoproteins were only distantly related (R approximately 5%). Intermediate levels of antigenic relatedness (R approximately 25%) were seen in neutralizing antibodies from cotton rats infected with RSV of the two subgroups. Immunity against the F glycoprotein of subgroup A, induced by vaccinia-A2-F, conferred a high level of protection which was of comparable magnitude against challenge by RSV of either subgroup. In comparison, immunity against the G glycoprotein of subgroup A, induced by vaccinia-A2-G, conferred less complete, but significant, protection. Importantly, in vaccinia-A2-G-immunized animals, suppression of homologous challenge virus replication was significantly greater (13-fold) than that observed for the heterologous virus.     

Plaque Assay of Rickettsiae in a Mammalian Cell Line

Clear-cut and repeatable plaque assays were obtained for three rickettsiae of the spotted fever group (Rickettsia rickettsi, R. conori, and R. montana) in Vero cells used in a manner similar to that for arboviruses. In addition, three typhus group agents (R. typhi, R. canada, R. prowazeki) induced plaques in these cells. In preliminary tests Coxiella burneti (Nine Mile strain) failed to produce plaques. Comparable results were obtained in plastic flasks and plastic culture trays incubated in ambient air with or without addition of N-2-hydroxyethyl-piperazine-N'-2-ethanesulfinic acid buffer. Larger and more well defined R. rickettsi plaques were produced when cultures were overlaid with Leibovitz (L15) medium than with either medium 199 or Eagle medium. Phosphate-buffered saline containing bovine plasma albumin (fraction V), in contrast to brain heart infusion broth, as a diluent for preparing inocula consistently permitted development of larger and more numerous plaques with three agents: R. rickettsi, R. conori, and R. montana. When R. rickettsi and R. typhi were assayed in parallel in primary chicken embryo cultures and Vero cells, comparable results were obtained, but with R. canada results in Vero cells were superior. In contrast, R. prowazeki produced inconsistent results in Vero cells.     

Detection of antibodies to Rickketsia prowazekii by using the antigen neutralization test]

The authors studied a possibility of using the antigen neutralization test with dry immunoglobulin typhus erythrocytic diagnostic agent for the purpose of detection of Rickettsia prowazeki antibodies. Blood sera of 315 healthy persons, 24 patients with sporadic typhus, and 18 laboratory animals immunized with R. sibirica and R. burneti, as well as with Proteus OX19 were examined. The results obtained pointed to the high specificity and sensitivity of the given serological test. A possibility of its use for antibody detection both in the typhus patients and in persons who sustained this infection in the past was demonstrated. In difference from the complement fixation test it permits to study anticomplementary sera.     

Antigenic relations of Rickettsia prowazekii and Rickettsia canada, established in the study of sera of patients with Brill's disease

Sera of patients with Brill's disease and of healthy persons with spotted fever in their past history were examined in the complement fixation reaction (CFR) to determine antigenic relations between R. prowazekii and R. canada. R. canada was found to have common antigenic determinants with R. prowazekii and R. mooseri. However, the antigenic determinants of R. canada differed from those of the mentioned rickettsiae. The titres of complement-fixing antibodies in the sera of patients with Brill's disease with the antigen of R. mooseri were lower than the titres with the homologous antigen within the range of 1-2 twofold dilutions of the serum. However, the oscillations of the titres with the antigen of R. canada in the study of the same sera were expressed in 1-5 twofold dilutions. In serological identification of canada rickettsiosis, antigens of rickettsiae of the spotted fever group should invariably be included in the investigation of the sera.     

Response to ivermectin treatment of parasitic stages of Haemonchus contortus resistant or susceptible to ivermectin.

Two groups of 33 helminth-naive lambs were infected with 5,000 L3 of an ivermectin-resistant or -susceptible strain of Haemonchus contortus (groups R and S). On days 6, 10, 16, and 21 postinfection, 5 animals from each group were chosen at random and orally treated with 0.2 mg/kg of ivermectin. On each occasion, 2 randomly selected lambs from each group were also killed to determine the number and stage of development of the worms present at the time of treatment. These necropsies revealed that by day 6 early and late fourth-stage larvae were present, whereas on day 10 the early fifth stage had been reached; by days 16 and 21 all worms had reached the adult stage. Necropsies on day 28 postinfection revealed that although animals treated at day 6 had 26.3% fewer worms than the controls, there was no significant difference (P greater than 0.05) between worm burdens from any of the animals infected with the R strain and treated at different times after infection when compared with the untreated controls. With ivermectin significant reductions were obtained in the worm burdens of the animals infected with the susceptible strain; these were reduced by 96% when treatment was given on day 6 against fourth-stage larvae and 98.9% when the drug was given on day 21 against adult stages. From these results it is clear that resistance to ivermectin in this strain of H. contortus is present as early as the fourth larval stage.     

Long-term effects of short-term provision of protein-enriched diets on resistance to nematode infection, and live-weight gain and wool growth in sheep.

Weaner sheep that had been hand-fed on diets containing increasing concentrations of protein for a 9-week period (when uninfected, or infected with Haemonchus contortus) were studied during the next 69 weeks when put on to pasture as a single, unsupplemented flock. During the 9-week period, groups of 12 sheep (six infected, six uninfected) were offered one of five iso-energetic (9.0 MJ kg(-1)) diets containing 10, 13, 16, 19 or 22% crude protein. All sheep were treated with anthelmintic at the end of the 9 weeks and then put out to pasture for 69 weeks, where they were all subject to the same environmental variables including nematode larval challenge. During the grazing period, animals that had previously received the higher protein diets consistently had higher live-weight gain and wool production, higher antibody responses to both H. contortus and Trichostrongylus colubriformis antigenic challenge in vitro, and lower faecal nematode egg counts than did the lambs previously offered the lower protein diets. Faecal egg counts of the grazing sheep that had been artificially infected with H. contortus while being hand-fed were similar to those of the uninfected sheep and there was no interaction between previous infection and dietary protein concentration. We conclude that short periods of enhanced post-weaning nutrition can have long-term and perhaps life-long effects on production.     

Murine and epidemic typhus rickettsiae: how close is their relationship?

Typhus fever has occurred globally as epidemic and endemic disorders. In 1910, Brill reported a typhus-like illness which Zinsser and others determined to be recurrent epidemic typhus fever. Maxcy, in 1926, proposed rodents and fleas as reservoir and vector, respectively, of endemic typhus, which Dyer confirmed in 1930. Animals experimentally infected with epidemic typhus (Rickettsia prowazeki) are immune to murine typhus (Rickettsia typhi) and vice versa. Similar solid cross-immunity exists for humans. The two diseases are clinically similar in pathologic and serologic reactions. Human epidemic typhus presumably involved a man-louse-man cycle without an animal reservoir. This concept is now questioned. Antibodies to R. prowazeki have been reported in livestock in Africa, rats in Manila, and from flying squirrels and humans in the United States. R. prowazeki was recovered from blood specimens of goats, sheep, from ixodid ticks, louse, and flea-ectoparasites of flying squirrels, and tissues of flying squirrels. More than 20 cases of squirrel-related acute epidemic typhus have been reported in the United States. R. prowazeki has not been recovered from human cases. Chemical studies of R. prowazeki and R. typhi show genetic similarities but differences in genome size and degree of hybridization suggest that interconversions between the two agents do not occur rapidly in nature. It is proposed that, with time, their relatedness will become even closer.     

Discovery of novel Streptococcus pneumoniae antigens by screening a whole-genome lambda-display library

Streptococcus pneumoniae is a causative agent of otitis media, pneumonia, meningitis and sepsis in humans. For the development of effective vaccines able to prevent pneumococcal infection, characterization of bacterial antigens involved in host immune response is crucial. In order to identify pneumococcal proteins recognized by host antibody response, we created an S. pneumoniae D39 genome library, displayed on lambda bacteriophage. The screening of such a library, with sera either from infected individuals or mice immunized with the S. pneumoniae D39 strain, allowed identification of phage clones carrying S. pneumoniae B-cell epitopes. Epitope-containing fragments within the families of the histidine-triad proteins (PhtE, PhtD), the choline-binding proteins (PspA, CbpD) and zinc metalloproteinase B (ZmpB) were identified. Moreover, library screening also allowed the isolation of phage clones carrying three distinct antigenic regions of a hypothetical pneumococcal protein, encoded by the ORF spr0075 in the R6 strain genome sequence. In this work, Spr0075 is first identified as an expressed S. pneumoniae gene product, having an antigenic function during infection.     

Protein supply influences the nutritional penalty associated with the development of immunity in lambs infected with Trichostrongylus colubriformis

The influence of dietary protein supply on the nutritional penalty associated with the acquisition phase of the immune response to gastrointestinal nematodes in lambs was investigated. Groups of lambs were offered either a low-protein diet (L; 62 g metabolizable protein (MP)/kg dry matter (DM)) or high-protein diet (H; 95 g MP/kg DM) while being either infected with the equivalent of 2.000 L3 Trichostrongylus colubriformis/day (IF), similarly infected and concurrently immuno-suppressed with methylprednisolone acetate (ISIF), immuno-suppressed only (IS) or kept as uninfected controls (C). Body composition of all animals was measured on days -8 and 76 of infection using X-ray computed tomography. Temporal changes in serum phosphate and serum albumin concentrations, which provided an indicator of pathological damage, in addition to patterns of total daily nematode egg excretion and comparative worm burdens at slaughter indicated that a protective immune response was developed in H-IF, but not L-IF, H-ISIF or L-ISIF groups. Compared to their respective non-infected controls, the gross efficiency of use of metabolizable energy (ME) for net energy (NE) deposition in the carcass was reduced by 0.23 in H-IF (P < 0.05), 0.13 in H-ISIF (P > 0.05), 0.49 in L-IF (P < 0.01) and 0.23 in L-ISIF (P > 0.05). It is concluded that the reduction in ME utilization and reduced performance, which can be attributed to the immunological response, are lessened in animals offered a high-protein diet. Furthermore, evidence is presented to indicate a possible association between T. colubriformis L3 IgA antibody production and loss of performance in lambs infected with this nematode.     

Serological Investigation of Granulocytic Ehrlichia Infection in Sheep in Norway

Serum samples of 749 sheep from 75 sheep flocks in Norway, i.e. 361 lambs (6 to 7 months old) and 388 adults (>1.5 year), were analysed for antibodies to Ehrlichia equi. Ten animals from each flock were examined. Seropositive animals were found along the coast of southern Norway from Vestfold to S?r-Tr?ndelag (as far north as 63°38'N). Seropositive sheep were not found in southeast, east or northern Norway. Thirty-two flocks were seropositive, although tick-borne fever had only been diagnosed earlier in half of these. In 78% of the seropositive flocks, more than 80% of the sheep were seropositive. A total of 35.7 % and 36.3 % of lambs and adults were found seropositive, respectively. However, the overall seroprevalence among animals that had been grazing on Ixodes pastures were 0.80 for the lambs and 0.84 for the adults. Mean antibody titres (± SD) (log₁₀) in seropositive lambs and adults were 2.59 (± 0.449) and 2.70 (± 0.481), respectively. No significant differences in either seroprevalence or mean antibody titre between sheep of different ages were obtained in this study. Based on antibodies 94% of sheep flocks on Ixodes pastures were infected with a granulocytic Ehrlichia infection. The association between seropositive flocks and Ixodes infested pasture shows a very high degree of agreement (p < 0.00001). The present study indicates that granulocytic Ehrlichia infection in sheep is underdiagnosed in Norway.     

Passive immune protection by herpes simplex virus-specific monoclonal antibodies and monoclonal antibody-resistant mutants altered in pathogenicity.

Virus-neutralizing monoclonal antibodies specific for 13 different genetically defined epitopes of glycoproteins gC, gB, and gD of herpes simplex virus type 1, strain KOS-321, were compared for their ability to provide passive immunity to DBA-2 mice challenged intracranially. Protection was highly specific, since individual monoclonal antibodies failed to protect against infection with monoclonal antibody-resistant (mar) mutants altered in the single epitope recognized by the injected antibody. The dose-response kinetics of passive immunity paralleled the in vitro neutralization titers for each antibody. No correlation was observed between immune protection and antibody isotype or complement-dependent in vitro neutralization titers. This suggests that virus neutralization was not the protective mechanism. In general, antibodies reactive with epitopes of gC were protective at the lowest antibody doses, antibodies specific for gB were less efficient in providing immunity, and antibodies against gD were the least effective. mar mutants with single epitope changes in gC and multiple epitope changes in gB showed highly reduced pathogenicity, requiring up to 5 X 10(6) PFU to kill 50% of infected animals. These findings indicated that antigenic variation affects virus growth and spread in the central nervous system. Thus, mutations which affect antigenic structure also can alter virus pathogenicity. The alteration of these epitopes does not, however, appreciably reduce the development of resistance to infection. Infection of mice with these mutants or inoculation of mice with UV-inactivated, mutant-infected cells before challenge rendered the animals resistant to infection with wild-type herpes simplex virus type 1.     

Phenotypic analysis of host-parasite interactions in lambs infected with Teladorsagia circumcincta

Female Blackface lambs expected to exhibit genetic variability for resistance to gastrointestinal nematodes, were either exposed to continuous experimental infections of Teladorsagia circumcincta or were sham-dosed to monitor phenotypic responses to infection. As a measure of parasitism and host response, worm-eggs in faeces (faecal egg count, FEC) were counted over a 3-month period and worm burdens were ascertained at post-mortem. The host response to the infection was also measured by differential counts of white blood cells, anti-T. circumcincta IgA antibody levels and body weight. Results suggest that nematode abundance (mean number of parasites per host) and prevalence (proportion of infected animals) were maximal shortly after the beginning of infection (21 days p.i.) when virtually all the infected animals were shedding worm eggs. Increasing anti-T. circumcincta IgA antibody and eosinophil concentrations were associated with a reduction in total numbers of adult worms and an increase in the frequency of early L4s. The data also suggest that genetic selection for an enhanced anti-T. circumcincta IgA response might complement selection based on a reduced FEC as a strategy to select for resistance to gastrointestinal nematodes.     

Reduced Weight Gain Due to Subclinical Anaplasma phagocytophilum (Formerly Ehrlichia phagocytophila) Infection

Tick-borne fever (TBF) is caused by the rickettsia Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila) and is a common disease in sheep in areas of Norway infested by Ixodes ricinus ticks. TBF can cause both direct and indirect losses to sheep kept on tick-infested pastures. In the present work we studied a sheep flock of 26 ewes and 50 lambs on pasture from May until September. No cases of TBF had earlier been observed on this pasture. Blood samples from lambs with a reduced weekly weight gain were collected and analysed for A. phagocytophilum infection by blood smear examination. In addition, at the end of the study, sera from all lambs were analysed by an indirect fluorescent antibody assay (IFA) to determine the antibody titre to E. equi. No clinical signs of tick-borne infections were observed, except in one lamb. However, 30 (60%) of the lambs grazing on this pasture became infected with A. phagocytophilum, and the infected lambs had a reduced weight gain (mean) of 3.8 kg compared with the uninfected lambs. The present study indicates that A. phagocytophilum infection may be widespread and contribute to considerable productivity losses even on pastures with no apparent tick infestation. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Effect of Two Different Oxytetracycline Treatments in Experimental Ehrlichia phagocytophila Infected Lambs

The effect of 2 different oxytetracycline treatments in acute E. phagocytophila infected lambs was investigated. Twenty 5-month-old lambs of the Dala and Rygja breeds were used. Ten lambs were inoculated intravenously with a stabilate of an ovine E. phagocytophila strain. On the third day of fever, 4 lambs were given long-acting oxytetracycline (Terramycin prolongatum vet^?, Pfizer) (20 mg/kg) intramuscularly and another 4 lambs were given short-acting oxytetracycline (Terramycin vet^?, Pfizer) (10 mg/kg) intravenously for 5 consecutive days. The lambs were examined for the presence of Ehrlichia infection by blood smear evaluation, polymerase chain reaction (PCR) and antibody titre against E. equi. One month after the last antibiotic treatment, 250 ml citrate blood from each of these lambs were inoculated into each of 10 susceptible lambs, which were observed during the following 6 weeks. The results indicate that oxytetracycline given in the acute stage of the infection may effectively teminate the development of fever, rickettsemia and weight reduction in E. phagocytophila infected lambs. No difference was observed between the 2 treatment groups. However, at least 3 of 8 antibiotic treated lambs (37.5%) were still infected with granulocytic Ehrlichia 3 months after treatment.     

Effect of immunosuppression on experimental Argentine hemorrhagic fever in guinea pigs.

Immunosuppression with cyclosporin A or cyclophosphamide had no apparent effect on the disease course of guinea pigs infected with a virulent strain of Junin virus. Immunosuppression of guinea pigs infected with an attenuated strain of Junin virus led to fulminating Argentine hemorrhagic fever. All immunosuppressed infected animals died. Virus distribution patterns in target organs, as determined by plaque assay and fluorescent antibody procedures, were similar to those from non-immunosuppressed animals infected with a virulent strain. Histopathological lesions in immunosuppressed guinea pigs infected with an attenuated strain of virus were similar to those in non-immunosuppressed guinea pigs infected with a virulent strain. Histological changes attributable to the immunosuppressive drug(s) were regularly observed. Immunosuppressed animals infected with attenuated Junin virus and non-immunosuppressed animals infected with virulent virus failed to develop antibody or responded at a minimal level. Virus-specific cytotoxic spleen cell activity, previously shown to be antibody dependent, failed to develop in the same animals. The presence of a competent immune response, probably serum antibody, determined whether Argentine hemorrhagic fever infection of the guinea pig was lethal or whether recovery ensued; no evidence for harmful effects of the immune response was obtained.     

Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro

A major unknown in human immunodeficiency virus (HIV-1) vaccine design is the efficacy of antibodies in preventing mucosal transmission of R5 viruses. These viruses, which use CCR5 as a coreceptor, appear to have a selective advantage in transmission of HIV-1 in humans. Hence R5 viruses predominate during primary infection and persist throughout the course of disease in most infected people. Vaginal challenge of macaques with chimeric simian/human immunodeficiency viruses (SHIV) is perhaps one of the best available animal models for human HIV-1 infection. Passive transfer studies are widely used to establish the conditions for antibody protection against viral challenge. Here we show that passive intravenous transfer of the human neutralizing monoclonal antibody b12 provides dose-dependent protection to macaques vaginally challenged with the R5 virus SHIV(162P4). Four of four monkeys given 25 mg of b12 per kg of body weight 6 h prior to challenge showed no evidence of viral infection (sterile protection). Two of four monkeys given 5 mg of b12/kg were similarly protected, whereas the other two showed significantly reduced and delayed plasma viremia compared to control animals. In contrast, all four monkeys treated with a dose of 1 mg/kg became infected with viremia levels close to those for control animals. Antibody b12 serum concentrations at the time of virus challenge corresponded to approximately 400 (25 mg/kg), 80 (5 mg/kg), and 16 (1 mg/kg) times the in vitro (90%) neutralization titers. Therefore, complete protection against mucosal challenge with an R5 SHIV required essentially complete neutralization of the infecting virus. This suggests that a vaccine based on antibody alone would need to sustain serum neutralizing antibody titers (90%) of the order of 1:400 to achieve sterile protection but that lower titers, around 1:100, could provide a significant benefit. The significance of such substerilizing neutralizing antibody titers in the context of a potent cellular immune response is an important area for further study.