Use of enzyme-labelled protein G assay for the detection of anti Borrelia burgdorferi antibodies in wild animal sera

A modified ELISA was developed for the detection of anti-Borrelia burgdorferi (Bb) IgG antibodies in wild animal sera based on an Enzyme-Labelled-protein G Assay (ELGA). Microplates were coated with an extract of Bb sensu stricto strain (SV1) as antigen. Specific antibodies of the serum samples were detected by a peroxidase-labelled-protein G. Using comparative immunodiagnosis by means of a passive hemagglutination test (HA), ELGA was tested on 82 roe-deer blood samples. A correlation was found between the two methods (r = 0,66). Good reproducibility of titers was observed by ELGA technique. A minimal cross-reactivity was discovered with Leptospira. ELGA could facilitate the recognition of specific antibodies in collections of wild animal sera.Abbreviations MA microscopic agglutination - OD optical density - ELGA enzyme labelled protein G assay - HA passive hemagglutination - BSA bovine serum albumin - PBS phosphate buffered saline - Bb Borrelia burgdorferi     

Lyme disease in Italy: Isolation of Borrelia burgdorferi from a patient

A strain of Borrelia burgdorferi was isolated from a 48-year-old female patient suffering from a chronic form of polyarthritis. Significant titers of specific anti-Borrelia antibodies were not found. This is the first report of isolation of Borrelia burgdorferi from a patient in Italy.     

A population-based longitudinal study on the incidence and disease burden of gastroenteritis and Campylobacter and Salmonella infection in four regions of the Netherlands

The aim of this study was to estimate the incidence of gastroenteritis and Campylobacterand Salmonellainfection in the Dutch population, the disease burden, and the percentage of patients with gastroenteritis that consults a general practitioner. A sample of 6243 persons was invited to participate in the study, i.e. completing a questionnaire and submitting stool samples. The follow-up period was 17 weeks. In total, 2206 persons participated (= 35%), contributing 660 person years. The incidence (standardised by age and gender) of first episodes of gastroenteritis was 45 per 100 person years. Among patients with gastroenteritis, Salmonellaand Campylobacterwere cultured in 1.6 and 4.5%, respectively. The standardised incidence of first Campylobacterinfections was 9 per 100 person years, of first Salmonellainfections 4 per 100 person years. For 22% of the episodes of gastroenteritis, a general practitioner was consulted (either by phone or by practice visit). For 52% of the episodes, medicine were used. For 34% of the episodes, absence from school was reported and for 15%, absence from work was reported. Despite of possible biases, we can conclude that the incidence of gastroenteritis is very high and causes considerable use of medication, consultation of general practitioners and absence from work and school.     

Distribution and prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks of canton Ticino (Switzerland)

Free-living Ixodes ricinus ticks were collected from 12 different sites of canton Ticino, south of the Alps (Switzerland). Each tick was examined for the presence of Borrelia burgdorferi sensu lato (sl), the etiologic agent of Lyme borreliosis using direct fluorescent antibody assay, and isolation of the bacteria. Borreliae were characterized by PCR followed by RFLP. The abundance and infection rates of I. ricinus ticks varied greatly between the areas. Two localities were found free of Borrelia. The prevalence of infected ticks ranged from 5 to 19%. Most ticks (96%) were found infected by <50 spirochetes. Three B. burgdorferi sl species were successfully isolated: B. garinii dominated, followed by B. lusitaniae and B. valaisiana. Additionally, a mixed infection with B. garinii and B. valaisiana was observed. The distribution of the various Borrelia species in the different areas was heterogeneous. This is the first report of the presence of B. lusitaniae in I. ricinus in Switzerland.     

Seroprevalence of Borrelia burgdorferi in Romania

In order to assess the seroprevalence of Borrelia burgdorferi in Romania and to define associated risk factors, a cross-sectional, observational study was performed in 13 districts during 1999. Sera from healthy blood donors (1598) and from forestry workers (1048) were tested. A two-step testing strategy was used in which sera were tested for anti-B. burgdorferi antibodies by a commercially available passive hemagglutination assays (PHA). All PHA positive sera were then evaluated by Western blot IgG. Demographic data regarding age, sex, profession, work place/residence, duration of employment (forestry workers), animals in the environment, and tick bites history were collected using a questionnaire. Data obtained from serological study were matched with that obtained from the questionnaire. The seroprevalence of B. burgdorferi in blood donors was of 4.3% (range 1.4–8.7%) and 9.3% (range 2.8–31.7%) in forestry workers. Seroprevalence was higher in forestry workers with a tick bite history (10.7 vs. 4.3%, p < 0.05). The highest seroprevalence in blood donors (8.7%) was noted in Maramures, a northern district of the country, whereas in forestry workers the highest seroprevalence (31.7%) was observed in a western district (Arad), where a previous study in entomology has demonstrated the highest density of Ixodes ricinus ticks in Romania.     

Seroepidemiological survey forBorrelia burgdorferi (Lyme disease) in dogs from northwestern of Spain

A random epidemiological study was undertaken to estimate the prevalence of canine borreliosis (Lyme disease) in Castilla y León, the largest region in Spain. The presence of antibodies was determined by indirect immunofluorescence assay (IFA), using theBorrelia burgdorferi B31 strain as antigen. Sera from 308 dogs from 7 provinces in the region were tested. Of all the animals sampled, 37 (21%) were seropositive (titres of 1/64 or above). Almost all the provinces had seroprevalence of 20% or below, except for an important focus of borreliosis in the Zamora province (42.8%). Potential risk factors (age, sex, use, habitat, season, and presence of ticks on the animals) relating to the presence of antibodies were evaluated. Those dogs which had at some time had ticks were more often seropositive, at 24.2%, than those which had never had them, at only 6.2% (p<10^–n). No significant differences were discovered for the remaining factors studied. This work indicates that dogs in Castilla y Leon, even if in most cases they do not develop the disease, are exposed to the Lyme disease agent.     

The outer surface protein A (OspA) vaccine against Lyme disease: efficacy in the rhesus monkey

The efficacy of an outer surface protein A (OspA) vaccine in three different formulations was investigated in the rhesus monkey. The challenge infection was administered using Ixodes scapularis ticks that were infected with the B31 strain of Borrelia burgdorferi. Protection was assessed against both infection and disease, by a variety of procedures. Some of the animals were radically immune suppressed, as an attempt to reveal any putative low level infection in the vaccinated animals. The significant difference found between the spirochaetal infection rates of ticks that had fed on vaccinated vs. control monkeys, lack of seroconversion in the vaccinated animals, and the absence of spirochaetal DNA in the skin of vaccinated animals in the weeks following the challenge, indicate that vaccinated monkeys were protected against tick challenge. The post-mortem immunohistochemical and polymerase chain reaction analyses, however, suggest that these monkeys may have undergone a low-level infection that was transient.     

Prevalence of anti-Toxoplasma gondii antibodies in the population of the area of Parma (Italy)

Between 1987 and 1991, the prevalence of IgG antibodies toToxoplasma gondii was determined by ELISA in 28,247 serum samples belonging to 19,432 subjects of the area of Parma (Italy). The overall prevalence was 48.5%. The correlation of infection with age, performed on 420 sera, showed a significant increase of positivity until 30–40 approximately years. In consecutive sera obtained from 172 subjects, the IgG and IgM production was analyzed for about 8 months, and four different patterns were delineated which were comprehensive of the wide range of immunological responses toToxoplasma gondii exposure observed. Among pregnant females the prevalence ofanti-Toxoplasma gondii antibodies was 48.7%, and 5 cases of seroconversion during the pregnancy were observed (0.27%) from which two cases of congenital toxoplasmosis originated.     

The Ixodes ricinus tick as a vector of Borrelia burgdorferi in Slovenia

In many parts of Slovenia, the vegetation, climate and animals form an ideal natural environment for the persistence of different tick species. In this study, Ixodes ricinus ticks, the prevalent tick species in our country, are identified as the vectors of Borrelia burgdorferi in Slovenia. Ticks were collected by flagging between May and October 1990. All the ticks collected from different parts of Slovenia were I. ricinus and each one was examined individually. The ticks were tested to determine the presence of B. burgdorferi by indirect immunofluorescent assay. Among the 496 ticks examined for borreliae there were 85 adults and 411 nymphs. Twenty (23.5%) adults and 18 (4.4%) nymphs were found positive for Borreliae. This study discusses the importance of I. ricinus ticks in the role of Lyme borreliosis in Slovenia.     

Comparison of western blot and microimmunofluorescence as tools for lyme disease seroepidemiology

Sera from a population of 212 farmers of the Puy de Dôme (Midlands of France) who are in close contact with Ixodes ricinus, the vector of Lyme disease in Europe, and sera from 100 urban blood donors from the nearly city of Clermont Ferrand were examined by microimmunofluorescence (MIF) for antibodies reacting with Borrelia burgdorferi. The results showed a higher seroprevalence of IgG > 1/100 in farmers (25%) than in blood donors (10%). Using western blot with antibody at a 1/200 dilution, and regarding sera as being positive if they contained IgG reactive with more than 5 bands, 93/212 farmers (44%) and 20% of blood donors were positive. Reactions with specific protein bands (94, 73, 30 and 21 KDa) by western blot were demonstrated in 83/212 sera from farmers (39%) and 16% of blood donors. Both methods showed a higher seroprevalence in the farmer population. Western blot is a sensitive and specific test for seroepidemiology but, in highly endemic areas, it is not diagnostic for active Lyme borrelliosis.     

Occurrence of Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii in the Ixodes ricinus ticks from Eastern Slovakia

A total of 2816 unfed adults nymphs of Ixodes ricinus ticks were collected from vegetation in Koice (Eastern Slovakia) from 1994 to 1997. Prevalence of Borrelia burgdorferi s. 1. in I. ricinus ticks, detected by dark field microscopy, varied and depended upon the year and the habitat of the collected ticks. The lowest prevalence was observed in 1994 (4.8%). During 1995 it increased to 17.2% and during the next two years decreased to 15.5% and 14.2%. The rate of infection varied from 2.1 to 23.3% within 10 examined habitats of the Koice area. A different value of relative density of ticks was observed in various habitats. It ranged from 9–212 ticks per collecting hour within one flagged area (600m²) which is 1.5–35.5 ticks per 100m². Eight isolates were obtained from the infected ticks. Electrophoresis and immunoblotting with 6 monoclonal antibodies were used for the identification of Borrelia strains. Three tick isolates were identified as B. burgdorferi s. s. and the other three isolates were found to be B. garinii. One strain reacted as a mixed culture of B. burgdorferi s. s., and B. garinii. The strain originated from the Vihorlat Mountains habitat and was detected by PCR-SSCP as B. burgdorferi s. s. with a small amount of B. afzelii. The obtained results emphasize the epidemiological importance not only of B. garinii and B. afzelii but also of B. burgdorferi s. s. in Central Europe.     

Prevalence and diversity of Lyme borreliosis bacteria in marine birds

A potential role of seabirds in spreading Lyme disease (LB) spirochetes over large spatial scales was suggested more than 10 years ago when Borrelia garinii was observed in marine birds of both hemispheres. Since then, there have been few studies examining the diversity of Borrelia spp. circulating in seabirds, or the potential interaction between terrestrial and marine disease cycles. To explore these aspects, we tested 402 Ixodes uriae ticks collected from five colonial seabird species by amplification of the flaB gene. Both the average prevalence (26.0%+/-3.9) and diversity of LB spirochetes was high. Phylogenetic analyses grouped marine isolates in two main clades: one associated with B. garinii and another with B. lusitaniae, a genospecies typically associated with lizards. One sequence also clustered most closely with B. burgdorferi sensu stricto. Prevalence in ticks varied both among seabird species within colonies and among colonies. However, there was no clear association between different Borrelia isolates and a given seabird host species. Our findings indicate that LB spirochetes circulating in the marine system are more diverse than previously described and support the hypothesis that seabirds may be an important component in the global epidemiology and evolution of Lyme disease. Future work should help determine the extent to which isolates are shared between marine and terrestrial systems.     

Prevalence of Borrelia burgdorferi (sensu lato) in Ixodes ricinus (L.) populations in France, according to a phytoecological zoning of the territory

Ixodes ricinus is considered as the main vector of Lyme Borreliosis in France. The aim of our investigations was to obtain a comprehensive view of the spatial risk linked to the distribution of the species in our country. Previous studies [1] have provided strong evidence that the species populations are widely distributed, so the objective of the present work was to ascertain the bacteriological infection of the tick by the agent of the zoonosis (Borrelia burgdorferi, sensu lato), over the French territory, whatever the ecological conditions may be. For this purpose, we kept the same framework as that used in our acarological investigation, a phytoecological zoning of the territory into 54 geographically separate and ecologically distinct units distributed into three climatic zones. Batches of ticks, picked up in these different phytoecological units (only two thirds of which were sampled), were submitted for bacteriological investigation. A total of 4,673 ticks were examined, individually, for the presence of Borellia burgdorferi, by immunofluorescence. Percentages of infection according to the various stages of Ixodes ricinus free stages, collected by flagging, were as follows: 4.95% in 3,247 nymphs, 11.2% in 699 males, 12.5% in 727 females. Larvae were ignored. Practically all the tested units harboured the bacterium. The percentage of tick samples (25 ticks or more) absolutely free of Borrelia, wherever they came from, is very low (not exceeding 10 percent of the sampled forests). Our study confirms the assessment of a widespread distribution of the zoonosis in France which was, until now, exclusively based on an approximate distribution of limited human cases observed in the country.     

Limited role of rodents as reservoirs of Borrelia burgdorferi sensu lato in Ireland

Despite the presence of Borrelia burgdorferi s.l.-infected Ixodes ricinus ticks in a west of Ireland location, very few small rodents from the same habitats were infected. Most of the infected ticks contained B. garinii or B. valaisiana, which implicates birds as the important reservoir hosts in this location.     

Epidemiological characteristics of dogs with Lyme borreliosis in the province of Soria (Spain)

From the point of view of the human disease, dogs are the most important animal reservoir of Lyme borreliosis; therefore, they are used as sentinel animals. In order to know the epidemiological characteristics of dogs with antibodies against Borrelia burgdorferi, 146 canine sera samples collected during 1993–94 have been studied. The antibody detection was made by an indirect immunofluorescence method and confirmed by a haemagglutination test. Seventeen dogs (11.6%) were seropositive, of which seven were hunting dogs, three were shepherd dogs, five were domestic pets, one was a watchdog and one was a stray dog. Seven dogs had longhair phenotype, 2 medium length type, 4 hard-hair and the other 4 were short-haired. Twelve seropositive dogs were males and five were females. All seropositive dogs were more than 1 year old. The dogs with greater seroprevalence were those that harboured ticks more often than the rest. Spring and summer were the seasons when more ticks were observed. The seroprevalence found in dogs was similar to that previously obtained in humans in this area. This finding gave evidence to an intimate association between human and canine seroprevalence.     

Tick bite and Lyme borreliosis risk at a recreational site in England

The risk of tick bite and Lyme borreliosis in a forested area in England with public access was studied over a two-year period. Tick infestation levels were high with more than 1000 members of the public reporting for tick removal at a local clinic. Most of the attached ticks were nymphs (82%) and distinct differences in anatomical sites of attachment were observed in children and adults. Children sustained nymphal bites to the head, neck and axilla region much more frequently than adults (48 vs. 10%), whereas adults were bitten on the lower legs more frequently than children (46 vs. 9%). The vegetation was heavily infested with ticks and high numbers were particularly associated with areas used by deer. The average density of nymphs collected from the vegetation was 14.1 per 10 m²(range 5.1–43.6). Infection rates of these nymphs determined by PCR and indirect IFA ranged from 5.2–17.0%, and the genospeciesBorrelia valaisianaand B. gariniiwere detected, suggesting that birds may be important reservoir hosts in this area. It is estimated that, at the level of tick challenge observed here, at least 50 persons per year may be bitten by infected ticks at this site. However, no cases of Lyme borreliosis have been reported through the clinic follow-up procedure, and sera from 19 forest workers were negative for antibody to B. burgdorferisensu lato. Despite the high challenge from tick bites, this particular recreational forest site poses a low risk of infection to the general public, and prophylactic antibiotic treatment or serological testing following a bite is not justified.     

Borrelia burgdorferi in rodents (Apodemus flavicollis andA. sylvaticus): Duration and enhancement of infectivity forIxodes ricinus ticks

Ixodes ricinus is an important vector ofBorrelia burgdorferi in Europe, and small rodents (Apodemus flavicollis, A. sylvaticus andClethrionomys glareolus) are important sources for infecting ticks. In this study, we examined their reservoir role by studying the duration of their infectivity for ticks.A. flavicollis andA. sylvaticus mice captured in nature were exposed to uninfectedI. ricinus larvae at different times after their capture: 10 days, and 2, 7, 11, 14 and 40 months. Ticks were examined for spirochaetes after moulting using direct immunofluorescence. All animals remained infective for ticks their life long but the efficiency of transmission from hosts to ticks varied from one individual to the other, presenting a three-fold variation (26.5% to 81.4%). Rodents continously exposed to successive infestations by larvalI. ricinus ticks over a period of one month showed an enhancement of infectivity for larval ticks during this period.     

Clinical and laboratory evidence of the importance of the tick D. marginatus as a vector of B. burgdorferi in some areas of sporadic Lyme disease in Bulgaria

56 adult ticks D. marginatus and 38 adult ticks H. punctata were sampled by the flagging method, transferred to the laboratory, dissected and tested for infection with Lyme disease spirochetes. 3 (7.9%) of the adult H. punctata and 2 (3.57%) of the adult D. marginatus were infected with B. burgdorferi (Bb). This report also presents the case of a patient, who developed Lyme disease symptoms after he had been bitten by a D. marginatus tick. The clinical diagnosis was confirmed by serological testing and by a biopsy, taken from the patient's skin lesion. The skin biopsy was examined under an electron microscope and Bb was found in the sections of the deeper strata of the dermis in two structural forms: (a) cylindrical bodies (protoplasm cylinder) with circular ends covered with a three-layered membrane; (b) granules, situated among the collagenous fibres either closely adhered to them or covered with a membrane. The result of the study demonstrates that in single cases in some ecosystems, ticks D. marginatus might be implicated in Bb transmission to humans as secondary vectors.     

Detection of Escherichia coli O157:H7 and other intestinal pathogens in patients with diarrhoeal disease

Six hundred and six faeces specimens from patients with diarrhoea, aged 0–>60 and living in the area of Fano (Italy), were examined for Escherichia coli 0157:H7, Salmonella, Shigellaand thermophilic Campylobacterspp. E. coli 0157:H7 was not found, confirming the limited circulation of this bacterium in our country. On the other hand, Salmonellae were the most frequent pathogens (5.6%), followed by Campylobacters (2.3%) and Shigellae (0.3%).     

The occurrence and sources of Campylobacter spp., Salmonella enterica and Escherichia coli O157:H7 in the Salmon River, British Columbia, Canada

In this study, we wished to assess the prevalence and determine the sources of three zoonotic bacterial pathogens (Salmonella, Campylobacter, and Escherichia coli O157:H7) in the Salmon River watershed in southwestern British Columbia. Surface water, sewage, and animal faecal samples were collected from the watershed. Selective bacterial culture and PCR techniques were used to isolate these three pathogens and indicator bacteria from these samples and characterize them. Campylobacter was the most prevalent pathogen in all samples, followed by Salmonella, and E. coli O157:H7. E. coli O157:H7 and Salmonella isolation rates from water, as well as faecal coliform densities correlated positively with precipitation, while Campylobacter isolation rates correlated negatively with precipitation. Analysis of DNA extracted from water samples for the presence of Bacteroides host-species markers, and comparisons of C. jejuni flaA-RFLP types and Salmonella serovars from faecal and water samples provided evidence that human sewage and specific domestic and wild animal species were sources of these pathogens; however, in most cases the source could not be determined or more than one source was possible. The frequent isolation of these zoonotic pathogens in the Salmon River highlights the risks to human health associated with intentional and unintentional consumption of untreated surface waters.