Development and verification of an enzyme-linked immunosorbent assay for the quantification of toxoid A and toxoid B from Clostridioides difficile.

Clostridioides difficile (C. difficile) is the most common cause of nosocomial antibiotic associated diarrhoea. The incidence of C. difficile infection (CDI) has been rising worldwide over the last 20 years with consequent rises in morbidity, mortality and healthcare costs, although the incidence has fallen in the UK over the last few years. Confirmation of diagnosis and early intervention are critical to the management of CDI. The standard treatment for CDI is the administration of antibiotics. However, vaccination has been recognized as the most cost-effective treatment for the prevention and possible long-term protection against CDI episode. There are several promising vaccine candidates in various stages of development. Many of these vaccines have displayed good efficacy for CDI under laboratory conditions or in clinical trials. With the emergence of vaccines against C. difficile, here we describe the development and verification of an Enzyme Linked Immunosorbent Assay (ELISA) that can be used for the quality control testing of candidate vaccines against C. difficile through the measurement of vaccine antigen content. Verification of the assay was performed by assessment of specificity, sensitivity, intermediate precision and relative accuracy. The ELISAs were specific for the toxoids being detected and the detection limit of the assay for toxoid A was 4.88 ng/mL and 3.91 ng/mL for toxoid B. The geometric coefficients of variation for intermediate precision did not exceed 25% and relative accuracy was within 77-130%. We therefore conclude that the ELISA described here is sufficiently sensitive, specific, precise and accurate for use for the quality control testing of candidate C. difficile vaccines.

Characterisation of adsorbed anthrax vaccine by two-dimensional gel electrophoresis.

The current UK anthrax vaccine is an alum precipitate prepared from static culture filtrate of the avirulent, unencapsulated Sterne strain of Bacillus anthracis. Protective antigen (PA) is regarded as the major immunogen in the vaccine and production conditions are intended to maximize the PA content. However, the precise composition of the vaccine is unknown and there are concerns that the observed side effects of vaccination may be caused by residual enzymatically active toxin components. Two-dimensional gel electrophoresis (2DGE) was used to define the protein components of the current UK anthrax vaccine. Consistency of composition was assessed by examining batches spanning 14 years of vaccine production. The reproducibility of the 2DGE technique was assessed by repeated analysis of selected vaccine batches. For two recently produced batches, between 86.7 and 88.8% of the spots could be matched. However, for one older batch, reproducibility of the spot pattern was considerably less, with a mean similarity of 53.4%. This difference may be explained by a change in production or because of decay during storage. Variation between the recently produced batches ranged from 72.9 to 84.3%, whereas the similarity between these and old batches was comparatively low at between 30 and 59%. Our results demonstrate that, as expected, the major antigen present in the vaccine is PA. The 83 and 63 kDa species are dominant but there are numerous lower molecular weight fragments resulting from proteolytic cleavage. In addition, we have established the presence of the toxin components, oedema factor and lethal factor, and S-layer proteins, EA1 and SAP. Mass spectrometry has also enabled us to identify several bacterial cell-derived proteins present in the vaccine, including PA, enolase, fructose-bisphosphate aldolase, nucleoside diphosphate kinase and a 60 kDa heat shock protein. The use of proteomics can provide useful information on the antigenic make up of this vaccine and the consistency of vaccine production.

Analysis of host responses of guinea pigs during Helicobacter pylori infection.

Host responses of guinea pigs infected with Helicobacter pylori were investigated. Passaged H. pylori colonised the stomach for up to 13 weeks after infection, but after 1 month the number of bacteria fell sharply. Specific antibodies, predominantly of the IgG2 subtype, were present from week 3 onwards. Antibodies to urease A and flagella were abundant. Severe inflammation of the gastric mucosa and damage to the stomach epithelium was seen. Infiltrates of mononuclear cells and eosinophils were found near the parietal glands. As infection progressed, inflammation and tissue damage became more localised and more variable between individual animals. These parameters can be used as markers for colonisation of the stomach by H. pylori.

Selection of putative immunogenic peptides by molecular modelling of the urease of Helicobacter pylori.

Computational methods such as molecular modelling are becoming an increasingly useful means of rationalising experimental data and creating a hypothesis that can suggest new experiments. In this report we discuss the application of molecular modelling methods to aid the selection of feasible peptide epitopes of the urease enzyme from Helicobacter pylori, an important vaccine candidate. Surface exposure was chosen as a criterion for the selection of three peptides which each had different levels of accessibility according to the 3D model. Antibodies raised against these peptides were analysed for their immunoreactivity with the holo enzyme. Only one anti-peptide antibody showed good reactivity with the urease. Our findings emphasise that surface exposure of peptide is not the only important criterion for the selection of immunogenic peptides.

Detection of Borrelia burgdorferi sensu stricto by reverse line blot in the joints of Dutch patients with Lyme arthritis.

OBJECTIVE: To analyze the presence of Borrelia burgdorferi sensu lato in synovial samples from the knee joint of patients with Lyme arthritis by polymerase chain reaction, and to differentiate the species by reverse line blot (RLB). METHODS: Synovial fluid (SF) and synovial tissue (ST) samples were obtained from patients with Lyme arthritis (n = 4) and from patients with various other forms of arthritis (n = 9). DNA extracted from synovial samples was amplified by using, as a target, the spacer region between the 5S and 23S ribosomal RNA genes of B. burgdorferi sensu lato. Subsequently, 4 species-specific DNA probes were used in the RLB for specific hybridization. RESULTS: DNA from B. burgdorferi sensu stricto DNA was detected in the SF and ST from 3 patients with Lyme arthritis. B. burgdorferi sensu lato DNA was not detected in the synovial samples from 9 control patients. CONCLUSION: The relationship between different species of B. burgdorferi sensu lato and arthritis can be studied using direct analysis of extracted DNA from joint samples. This method can be used to study the association between particular clinical manifestations of Lyme disease and different species of B. burgdorferi sensu lato.

Sexual transmission of Borrelia garinii by male Ixodes persulcatus ticks (Acari, Ixodidae).

We investigated the transmission of Borrelia garinii and Borrelia afzelii between male and female Ixodes persulcatus ticks. For this purpose the infection rate of partners from tick couples was determined by polymerase chain reaction and reverse line blot. In couples, where the male tick was infected with B. garinii, four out of nine female partners carried B. garinii. In eight couples, male ticks had a dual infection of B. afzelii and B. garinii and three female partners were infected by Borrelia spirochetes. Two female ticks carried B. garinii, and one female tick had a dual infection. No evidence for transmission of B. afzelii from male to female ticks was found among seven couples. In 45 couples where the female tick was infected, not one male tick carried spirochetes. The difference in the B. garinii infection rate between male and female ticks among these couples is highly significant. Our data suggest that transmission of B. garinii from male ticks to female ticks does occur. Sexual transmission of this pathogen may play an important role in the maintenance of B. garinii in I. persulcatus.

Detection and identification of Ehrlichia, Borrelia burgdorferi sensu lato, and Bartonella species in Dutch Ixodes ricinus ticks.

A sensitive and specific PCR hybridization assay was developed for the simultaneous detection and identification of Ehrlichia and Borrelia burgdorferi sensu lato. In separate assays the 16S rRNA gene of Ehrlichia species and the 23S-5S rRNA spacer region of B. burgdorferi sensu lato were amplified and labeled by PCR. These PCR products were used in a reverse line blot hybridization assay in which oligonucleotide probes are covalently linked to a membrane in parallel lines. Hybridization of the samples with the oligonucleotide probes on this membrane enabled the simultaneous detection and identification of Ehrlichia, B. burgdorferi, and Bartonella species in 40 different samples. The application of the assay to DNA extracts from 121 Ixodes ricinus ticks collected from roe deer demonstrated that 45% of these ticks carried Ehrlichia DNA. More than half of these positive ticks carried species with 16S rRNA gene sequences closely related to those of E. phagocytophila and the human granulocytic ehrlichiosis agent. The majority of the other positive ticks were infected with a newly identified Ehrlichia-like species. In addition, 13% of the ticks were infected with one or more B. burgdorferi genospecies. In more than 70% of the ticks 16S rRNA gene sequences for Bartonella species or other species closely related to Bartonella were found. In five of the ticks both Ehrlichia and B. burgdorferi species were detected.

Presence and distribution of Borrelia burgdorferi sensu lato species in internal organs and skin of naturally infected symptomatic and asymptomatic dogs, as detected by polymerase chain reaction.

Tissues from Dutch family dogs symptomatic for borreliosis according to established criteria and from infected but asymptomatic dogs were tested for Borrelia burgdorferi sensu lato DNA using a polymerase chain reaction. Subsequently, B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana were identified by hybridization. Symptomatic dogs showed a higher prevalence of Borrelia in liver samples (9 of 15) than asymptomatic dogs (9 of 43) p = 0.0049. Overall, B. garinii was the most prevalent species and occurred together with up to three other species in on liver sample. B. burgdorferi sensu stricto however, was predominantly detected in samples of synovial membranes, skin, cerebrospinal fluid, bladder, heart, and bone marrow. Nine out of 10 symptomatic dogs with a very high antibody titre were positive for Borrelia DNA by PCR in one or more of these tissues. We conclude that dissemination in naturally infected European dogs occurs and that the two most prevalent species, B. burgdorferi sensu stricto and B. garinii, differ in their tropism.

A serological study of cohorts of young dogs, naturally exposed to Ixodes ricinus ticks, indicates seasonal reinfection by Borrelia burgdorferi sensu lato.

Thirty-three family dogs were monitored for antibodies to Borrelia burgdorferi sensu lato over a 3-year period. Serum samples were collected before and during the season of high tick activity. Antibody levels were measured with an ELISA based on whole-cell antigens and an ELISA with a purified recombinant flagellin (r410). Antibody levels measured with the whole-cell ELISA increased after the first exposure to ticks. Following the first seasonal period of tick quiescence, antibody levels decreased, and subsequently increased again in the second tick season. Thereafter whole-cell ELISA titres persisted at moderate levels and did not decrease between tick seasons. The recombinant flagellin ELISA did not show a strong response in the first tick season, but did in the second tick season and levels of antibodies continued to fluctuate thereafter. We conclude that most dogs in this study developed an antibody response against Borrelia burgdorferi sensu lato after their first tick infestation and were thereafter repeatedly immunologically stimulated, probably reinfected, during the consecutive tick seasons.

Molecular detection of pathogen DNA in ticks (Acari: Ixodidae): a review.

Ticks play an important role in human and veterinary medicine, in particular due to their ability to transmit a wide spectrum of pathogenic micro-organisms of protozoal, rickettsial, bacterial and viral origin. Pathogens in ticks can be identified by conventional methods such as indirect immunofluorescence, isolation in cell culture or by using histological staining techniques. However, the advent of the polymerase chain reaction (PCR) has resulted in tremendous improvements in the specific and sensitive detection of pathogen DNA in ticks. In this paper, literature on DNA extraction methods, PCR protocols, primers and probes, which are in use for the successful detection and identification of pathogens in ticks, are critically reviewed. Some recommendations are also given towards the end of this review.

Identification of four Borrelia burgdorferi sensu lato species in Ixodes ricinus ticks collected from Dutch dogs.

The prevalence of Borrelia species infection was determined by polymerase chain reaction (PCR) in 138 ticks collected from dogs which were walked regularly in the wooded areas near the city of Eindhoven, the Netherlands. The PCR amplified the spacer region between the 5S and 23 S rRNA genes, and the Borrelia species was identified by hybridization with specific probes. Borrelia burgdorferi sensu lato was present in 20 of 138 (14.5%) ticks. Four species were identified: B. burgdorferi sensu stricto (n = 8), B. afzelii (n = 4), B. garinii (n = 2), and B. valaisiana (n = 2). One PCR product was non-typeable. Three ticks contained more than one species, all including B. burgdoferi sensu stricto, and one tick even contained four species. There was a significant difference (P < 0.05) in prevalence of B. burgdorferi sensu stricto between non-engorged ticks (either questing or attached) and semi-engorged ticks, 12% (10 of 85) and 2% (1 of 53), respectively.

Clinical features of Lyme borreliosis in the middle Urals and distribution of Borrelia burgdorferi sensu lato species in local Ixodes persulcatus ticks.

From 1991 to 1993, we investigated the clinical features of Lyme borreliosis (LB) in 864 patients from the Sverdlovsk region (population 4.5 million) in the middle Urals. Ixodes persulcatus ticks were collected in the vicinity of Yekaterinburg to determine the presence of Borrelia burgdorferi sensu lato species. From 1991 to 1993, the number of patients with LB increased from 91 to 320 and 453, respectively. Nearly all LB patients (97%) recalled a tick bite and the first signs of LB developed between May and August. Erythema migrans (EM) was seen in 820 patients (94.9%) and fever was common (44.6%). Neuroborreliosis, mainly radiculoneuritis, was found in 154 patients (17.8%), secondary erythema was seen in 53 patients, and Lyme arthritis (LA) was diagnosed in 35 patients. Carditis was rare and acordermatitis chronica atrophicans (ACA) was not seen. Only 44 patients developed one or more symptoms of LB without a preceding EM. Few patients were seropositive for tick-borne encaphalitis. Borrelial DNA was detected in 67% of I. persulcatus ticks. Infected ticks carried predominantly Borrelia garinii or Borrelia afzelii, mixed infections of both species were common. Borrelia valaisiana was detected once, and B. burgdorferi sensu stricto was not found. Although the course of LB in the middle Urals in comparable to that of European LB, several discrepancies were noted. LB patients often recall tick bites, fever is common, LA is mild and ACA is absent.

Rate of infection of Ixodes ricinus ticks with Borrelia burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii and group VS116 in an endemic focus of Lyme disease in Italy.

A study to evaluate the natural rate of infection of Ixodes ricinus with Borrelia burgdorferi sensu lato was carried out in an endemic focus of Lyme disease in the Trieste area in northern Italy. Two-hundred and twenty-seven ticks collected in ten different stations were tested individually for the presence of the spirochetes using polymerase chain reaction techniques able to identify both Borrelia burgdorferi sensu lato and the four genospecies (Borrelia burgdorferi sensu stricto, Borrelia garinii. Borrelia afzelii and group VS116). Multiple infection of individual ticks was found. The infection rate ranged from 0-70%. Infection of Ixodes ricinus with Borrelia burgdorferi group VS116 was found for the first time in Italy in both a high and a low endemic focus of Lyme disease.

Epidemiologic, ecologic and clinical characteristics of Lyme borrelliosis in northwest Croatia.

Investigated were the epidemiologic, ecologic and clinical characteristics of Lyme borreliosis in northwest Croatia. In a seroepidemiologic study, human sera were analyzed by indirect immunofluorescent assay (IFA), and ten out of 134 serum samples were positive for B. burgdorferi antibodies. In a seroepizootiologic study, wildlife and domestic animals were tested by inhibition ELISA. Antibodies to B. burgdorferi were found in nine out of 42 roe deer sera, and in three out of nine hare sera. Sera of wild boars (n = 10), cattle (n = 103), and dogs (n = 13) were negative for antibodies to B. burgdorferi. The presence of Borrelia burgdorferi sensu lato was assessed in ixodes ricinus ticks in the Lyme borreliosis endemic region of northwest Croatia. Ticks (n = 123) were collected at five-different locations and analyzed by polymerase chain reaction (PCR). Borrelia burgdorferi sensu lato DNA was detected in 56 out of 124 ticks (45%). Four genomic groups were identified by genotyping: B. afzelii (n = 26), B. garinii (n = 5), VS116 group (n = 5), and Borrelia burgdorferi sensu stricto (n = 1). Mixed infections of B. afzelii with VS116 group (n = 10), and B. afzelii with Borrelia burgdorferi sensu stricto (n = 1) were also detected. Eight ticks contained B. burgdorferi sensu lato that could not be typed, indicating the possible existence of a specific genomic group of B. burgdorferi sensu lato in northwest Croatia. Sex distribution of Lyme borreliosis patients in northwest Croatia showed a slight preponderance of the female gender and prevalence of the working active age range of 20 to 50 years. The persons who periodically visit the landscape, are most commonly affected, whereas those with an increased risk of tick bites are considerably less frequently involved. The most frequent clinical manifestation of Lyme borreliosis in northwest Croatia is erythema migrans with 46%, followed by neurologic manifestations, particularly peripheral neuritis, with 32%. The rest of clinical manifestations in stage II and III of Lyme borreliosis are very rarely recorded. The presence of B. afzelii and B. garinii in the highest percentage is in agreement with the local occurrence of cutaneous and neurologic manifestations of Lyme borreliosis. The presence of VS116 group in ticks from northwest Croatia in this and other studies in some European countries may indicate that VS116 group is well established in the European ixodes ricinus ticks. The role of the VS116 group in the etiology of Lyme borreliosis remains to be clarified.

Identification of Borrelia burgdorferi sensu lato species in Europe.

Characterisation at the species level of 142 Borrelia isolates obtained from ticks, humans and rodents in Western Europe was carried out and their geographical distribution was described. Borrelia garinii was the predominant species representing 44% of the isolates and B. afzelii and B. burgdorferi sensu stricto constituted 27% and 19% of isolates respectively. B. valaisiana, (formerly group VS116) constituted 10.5% of isolates. Some differences in the Borrelia species distribution were observed from one country to another, possibly linked to different sources of samples. In the human samples, which were mostly collected in Austria, B. afzelii was preferentially isolated from skin and B. garinii from CSF. B. afzelii was consistently isolated from rodents captured in Switzerland, but one isolate of B. garinii was obtained from a rodent in Austria. B. garinii was by far the most abundant species isolated from Ixodes ricinus ticks in all studied countries. B. valaisiana was isolated from I. ricinus ticks collected from vegetation and from I. ricinus engorged on birds.

Detection of Borrelia burgdorferi sensu lato in ticks: immunofluorescence assay versus polymerase chain reaction.

Immunofluorescence (IFA) and polymerase chain reaction (PCR) were examined as methods for detecting Borrelia burgdorferi sensu lato spirochaetes in unfed Ixodes ricinus nymphs. Although similar results were produced in some cases, a great deal of variation occurred. Furthermore, in both the highly controlled initial laboratory study, involving 252 shared samples, and the study on field-collected ticks (n = 460), the IFA tended to detect more infected ticks than the PCR. The basis for these findings are as yet undetermined. The development of a quality assurance scheme is recommended so that laboratories can validate their methods and a preliminary feasibility study suggested that such a scheme is practical.

European interlaboratory comparison of Lyme borreliosis serology.

Serological testing for Lyme borreliosis was compared in 5 European reference laboratories with a total of 79 sera in order to determine variations in laboratory performance. A considerable range of methods were used and several laboratories employed 2 or 3 genomospecies of Borrelia burgdorferi sensu lato. No laboratory relied routinely on a single test and each weighted the significance of the findings of the various tests differently. A difference in strategy between laboratories in high and low prevalence areas was apparent in that laboratories in low prevalence areas emphasised specificity more than sensitivity and therefore produced fewer false positives, but also missed some cases. Overall agreement between the laboratories was poor and it was concluded that there is a need for a quality assurance scheme within Europe.

Tick-borne borrelioses pathogen identification in Ixodes ticks (Acarina, Ixodidae) collected in St. Petersburg and Kaliningrad Baltic regions of Russia.

Two isolated Baltic seashore populations of Ixodes ticks were studied as vectors of different Borrelia genospecies in Russia by using darkfield microscopy and modified polymerase chain reaction (PCR). In the Kalinigrad region (Kurish Spit, forests near the settlements of Lesnoye and Rybachy), 788 Ixodes ricinus (L.) adults and nymphs were collected by flagging and studied by darkfield microscopy during 1995-1996. There were 88 darkfield microscopy positive specimens (11.2%) of which 69 were also analyzed by PCR. Borrelia afzelii and B. garinii were found individually and together in ticks. In this region, on the Kurish Spit, 7 patients with tick borrelioses were observed: 2 in the Russian part of Spit and 5 in the Lithuanian part. A significant difference was found between Borrelia prevalence during the spring and fall peaks of tick abundance. Specimens that were darkfield microscopy positive prevailed in the fall (25.15%) in comparison with the spring peak (7.3%). The number of specimens with identified genospecies prevailed in the spring: 22 out of 35 versus 4 out of 31 in the fall. Among 29 PCR positive I. ricinus, 21 contained B. afzelii, 3 had B. garinii, and 2 had dual infection. In 1995, only B. afzelii infected specimens were observed. In the vicinity of St. Petersburg (the seashore of the northern Gulf of Finland, in forests near Lisy Nos, Morskaja) during 1992-1996, 31 patients with a tick-borne borrelioses were registered. We collected 487 Ixodes persulcatus Schulze by flagging and studied them by darkfield microscopy in 1995-1996 of which 144 ticks (29.6%) were darkfield microscopy positive. Sixty darkfield-positive specimens were analyzed by PCR, and in 88.3% of cases genospecies were identified. B. afzelii and B. garinii were identified individually and together in ticks. In 1995, I. persulcatus with dual infection prevailed with 11 out of 21 (52.4% positive), whereas in 1996, most I. persulcatus ticks contained B. garinii (81.2%). Dual infection was observed in 4 of 32 (12.5%) ticks. Dual infections in I. persulcatus females increased within the seasonal peak of tick activity as was observed in 1995 and in 1996. Many patients not only had erythema migrans, but also exhibited early neurological symptoms that coincided with the number of tick vectors that had dual infections in June, indicating that these patients were bitten by female ticks that had dual infections. A significant difference existed between levels of infection in I. ricinus and I. persulcatus, with all 3 types of Borrelia infection observed 2 times more often in I. persulcatus than in I. ricinus and dual infection occurred in I. persulcatus 3.7 times more often. It appeared that I. persulcatus is a much more dangerous vector of tick-borne borrelioses than I. ricinus.