Gelas Khanakah

Isolation and polymerase chain reaction typing of Borrelia afzelii from a skin lesion in a seronegative patient with generalized ulcerating bullous lichen sclerosus et atrophicus

A 64‐year‐old woman presented with bullous and ulcerating lichen sclerosus et atrophicus (LSA) on the neck, trunk, genital and perigenital area and the extremities. Histology of lesional skin showed the typical manifestations of LSA; in one of the biopsies spirochaetes were detected by silver staining. Despite treatment with four courses of ceftriaxone with or without methylprednisone for up to 20 days, progression of LSA was only stopped for a maximum of 1 year. Spirochaetes were isolated from skin cultures obtained from enlarging LSA lesions. These spirochaetes were identified as Borrelia afzelii by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and polymerase chain reaction (PCR) analyses. However, serology for B. burgdorferi sensu lato was repeatedly negative. After one further 28‐day course of ceftriaxone the lesions stopped expanding and sclerosis of the skin was diminished. At this time cultures for spirochaetes and PCR of lesional skin for B. afzelii DNA remained negative. These findings suggest a pathogenetic role for B. afzelii in the development of LSA and a beneficial effect of appropriate antibiotic treatment.

Impact of a pertussis booster vaccination program in adolescents and adults on the epidemiology of pertussis in Austria

Background: A resurgence of pertussis has been observed in several countries; however, inconsistent data are available for Europe. In Austria, routine pertussis vaccination for babies is administered at 3, 4, and 5 months, and in the second year of life. Since 2002, regular boosters for all persons >6 years of age (including adults) are recommended. This study was undertaken to analyze epidemiologic trends of laboratory-reported pertussis to evaluate current vaccination strategy in Austria. Methods: Epidemiologic surveillance of laboratory-reported pertussis was conducted from January 1, 2000, to December 31, 2005. Infection was confirmed by positive serology, by positive culture of Bordetella pertussis, or by detection of sequences of the pertussis toxin gene by real-time polymerase chain reaction (RT-PCR). Data were assessed by age, hospitalization rate, seasonality, and incidence rate. Results: During the observation period 4395 reported cases of pertussis were eligible for analysis. The mean annual incidence increased from 6.4 per 100,000 population in 2000 to 11.1 cases per 100,000 population in 2005. Incidence rates were highest among children less than 1 year of age. Decreasing rates were observed for children and adolescents <16 years of age, whereas increasing rates were detected for persons 16 years of age and older. The mean age of reported pertussis cases increased from 30 years (±25.9 SD) in 2000 to approximately 44 years (±23.7 SD) in 2005. Hospitalization rates were highest in infants <6 months (86%) and lowest in those 10 to <50 years of age (17%), followed by an increase to 80% in persons 85 years of age and older. In general, no seasonal occurrence of disease was apparent. Conclusions: Pertussis incidence remains high among adults implying that coverage rates regarding booster vaccinations for adolescents and adults still are too low. Reinforced application of the current booster strategy is needed.

Species, developmental stage and infection with microbial pathogens of engorged ticks removed from dogs and questing ticks

Research into tick‐borne diseases implies vector sampling and the detection and identification of microbial pathogens. Ticks were collected simultaneously from dogs that had been exposed to tick bites and by flagging the ground in the area in which the dogs had been exposed. In total, 200 ticks were sampled, of which 104 came from dogs and 96 were collected by flagging. These ticks were subsequently examined for DNA of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia spp. and Babesia canis. A mixed sample of adult ticks and nymphs of Ixodes ricinus (Ixodida: Ixodidae) and Haemaphysalis concinna (Ixodida: Ixodidae) was obtained by flagging. Female I. ricinus and adult Dermacentor reticulatus (Ixodida: Ixodidae) ticks dominated the engorged ticks removed from dogs. Rickettsia spp. were detected in 17.0% of the examined ticks, A. phagocytophilum in 3.5%, B. canis in 1.5%, and B. burgdorferi s.l. in 16.0%. Ticks with multiple infections were found only among the flagging sample. The ticks removed from the dogs included 22 infected ticks, whereas the flagging sample included 44 infected ticks. The results showed that the method for collecting ticks influences the species composition of the sample and enables the detection of a different pattern of pathogens. Sampling strategies should be taken into consideration when interpreting studies on tick‐borne pathogens.

Humoral Immune Response in Dogs Naturally Infected with Borrelia burgdorferi Sensu Lato and in Dogs after Immunization with a Borrelia Vaccine

ABSTRACT Lyme arthritis in dogs can be induced under experimental and natural conditions. However, the veterinary relevance of canine borreliosis is still under extensive investigation. The prevalence of symptoms is clearly low, although the risk of tick exposure is high. Current research focuses on case definitions, methods for diagnosing clinical disease in dogs, and discrimination between an immune response to a natural infection and an immune response to vaccination. In this experimental study, 23 dogs raised under tick-free conditions were allocated to two groups. The 11 dogs in the first group were vaccinated with a commercial borrelia vaccine and subsequently developed detectable antibody titers. The 12 dogs in the second group were walked on two consecutive days in an area where ticks were endemic. On day 5 after exposure, engorged ticks were removed from the 12 dogs and were analyzed for Borrelia DNA by a real-time PCR assay. Blood samples were taken before exposure/vaccination and at defined time points thereafter. Antibody responses were evaluated using an immunofluorescence antibody test (IFAT) and Western blotting. Seven dogs from which Borrelia-positive ticks were removed seroconverted and developed individual immune responses. Blood and urine samples taken from the tick-exposed group at weeks 1 and 3 for real-time PCR analysis and culture were always negative for bacterial DNA. In conclusion, despite serological evidence of infection/immunization, no clinical signs of disease were observed. The antibody patterns in a single Western blot did not permit differentiation between the different antigen sources (vaccine versus natural infection). However, repeated Western blot analyses may be useful for the confirmation of infection or vaccination status, since the time courses of the levels of specific antibodies seem to be different.

Seasonal variations in detecting Borrelia burgdorferi sensu lato in rodents from north eastern Austria

ZusammenfassungÖsterreich ist ein bekanntes Endemiegebiet der Lyme-Borreliose. Um die saisonalen Schwankungen in Nagerpopulationen zu erfassen, die Wirte der Erreger dieser Spirochätose sind, wurden Nager im Nordosten Österreichs gesammelt und auf ihre Durchseuchung mit Borrelien untersucht. Während eines Untersuchungsjahres wurden alle 6 Wochen Lebendfallen in jeweils einem von drei Fangorten (Hohenau, Ernstbrunn, Wien/Wienerwald), welche die Hauptmerkmale der Habitate für kleine Nager im Nordosten Österreichs aufweisen, ausgelegt. Die Nager wurden identifiziert und Herz, Harnblase und Hirn unter weitgehend aseptischen Bedingungen entnommen. Diese Organe wurden für die Anzüchtung von Borrelien weiterverarbeitet. Proben vom Herzmuskel wurden zusätzlich für den molekularen Nachweis von Borrelien mittels Real-Time Polymerase-Ketten-Reaktion verwendet. Dazuwurden Borrelien-Universalprimer sowie Spezies-spezi-fische Primer eingesetzt. Insgesamt wurden 938 Mäuse gefangen, überwiegend Apodemus flavicollis (44%), gefolgt von Clethrionomys glareolus (35%), Microtus arvalis (9%), A. sylvaticus (7%) und Mus musculus (6%). Die Gesamtzahl der gefangenen Tiere variierte deutlich zwischen den einzelnen Fangorten (Hohenau, Ernstbrunn, Wienerwald entsprechend 10:9:2), wie auch die Verteilung der verschiedenen Nagerarten. Die Anzüchtung von Borrelienstämmen gelang nur von 65 (7%) Tieren und häufiger von der Harnblase als vom Herzmuskel, nur einmal von einer Hirnprobe. Mittels PCR wurden Borrelien in 223 Herzmuskelproben nachgewiesen (24%), am häufigsten von A. flavicollis (43%) und C. glareolus (38%). B. afzelii wurde am häufigsten identifiziert, gefolgt von B. burgdorferi sensu stricto und B. garinii sowie Mischinfektionen von B. afzelii mit B. burgdorferi sensu stricto. B. garinii wurde am häufigsten in den Proben von A. sylvaticus nachgewiesen (ca 20%). Mit den verwendeten Methoden gelang es in ca. 3% der PCR-positiven Proben nicht, eine Genospezies zu ermitteln. Die Nagerarten A. flavicollis, A. sylvaticus, M. arvalis und C. glareolus wurden als Reservoire von B. afzelii, B. garinii und B. burgdorferi sensu stricto bestätigt. Bemerkennswert ist die hohe Präsenz von B. garinii in Herzmuskelproben von A. sylvaticus.SummaryAustria is well known as an endemic area of Lyme borreliosis. To assess the annual variation of rodent populations that may host agents of Lyme borreliosis we collected rodents in northeastern Austria. Life traps were set out every six weeks during a year consecutively in one each of the three different zones (Hohenau, Ernstbrunn, Vienna Woods) that cover the main habitat characteristics of small mammals in northeastern Austria. Rodents were collected and identified. Samples of heart, urine bladder and brain were removed under aseptic conditions for cultivation of borrelia. Samples of heart muscle were additionally used for molecular detection of borrelia by Real-Time polymerase chain reaction. PCR was performed with borrelia universal primers and with species-specific primers. 938 mice were caught, most frequently Apodemus flavicollis (44%), followed by Clethrionomys glareolus (35%), Microtus arvalis (9%), A. sylvaticus (7%) and Mus musculus (6%). Significant differences were seen in the total number of catch per area (Hohenau, Ernstbrunn, Vienna Woods equal 10:9:2) and in the distribution of the various rodent species in the respective areas. Borrelia strains were grown from only 65 (7%) animals, and more frequently isolated from bladder wall than from heart muscle, and only once from brain. Heart specimens of 223 animals were positive by borrelia PCR (24%), most frequently of the rodent species A. flavicollis (43%) and C. glareolus (38%). Borrelia afzelii was most frequently identified, followed by B. burgdorferi sensu stricto, B. garinii and by mixed infection of B. afzelii with B. burgdorferi sensu stricto. B. garinii was most frequently detected in heart samples of A. sylvaticus (about 20%). In about 3% of PCR positive samples the identification of one of the three mentioned genospecies of borrelia could not be ascertained with the test panel used. The results confirm the rodent species A. flavicollis, A. sylvaticus, M. arvalis and C. glareolus as reservoir animals for B. afzelii, B. garinii and B. burgdorferi sensu stricto, agents of Lyme borreliosis. Notable is the salient presence of B. garinii in heart specimens of A. sylvaticus.

Horses and Borrelia: immunoblot patterns with five Borrelia burgdorferi sensu lato strains and sera from horses of various stud farms in Austria and from the Spanish Riding School in Vienna.

Grazing animals are continuously exposed to tick bites. Consequently, one may expect that horses will become infected with the various pathogens carried by ticks including Borrelia burgdorferi sensu lato. Whether horses may develop clinical disease due to this pathogen is controversially discussed. We were interested to learn about the infection of horses with Borrelia burgdorferi sensu lato within one season by studying the dynamics of the humoral immune response in paired blood samples. The majority of horses examined were Lipizzaner from the stud farm in Piber/Steiermark, and from the Spanish Riding School in Vienna. Smaller groups of animals of different breeds were from stud farms in Kärnten, Niederösterreich, Salzburg and Steiermark. Clinical status and medical history were obtained and blood was drawn at the beginning of the highest tick activity and nine months later in 1998. Immunoblot technique (Western blot) was used in order to determine the dynamics in the immune response patterns. As antigens served the genospecies Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii, Borrelia lusitaniae, and Borrelia valaisiana. 309 horses (age median 7 years, range 1/12 to 33 years) were seen at the first round. 186 of these animals (60.2%; median age 6 years, range 4/12 to 33 years) were re-examined in the second round. All animals were in normal health condition during both rounds of examination and blood sampling. Analysis of the immunoblot patterns was based on in-house-, Pko-, Pka2-, Pbi-, and European Union Concerted Action on Lyme Borreliosis (EUCALB) 2 & 3-criteria; analyses revealed a variety of positive results with different strains and criteria. Positive immunoblot results with 186 paired samples and B. afzelii as antigen, for example, ranged from 52 to about 91% in the first, and 53 to 93% in the second round. The age dependency analyses showed that the first infection with B. burgdorferi sensu lato occurs in the first year. Re-infection is characterised by appearance of additional bands. Continuously tick-exposed horses show a stable pattern of bands whilst in unexposed horses the number of bands decreases with age. In this study horses became repeatedly infected with B. burgdorferi sensu lato but, apparently, developed only rarely, if at all, clinical diseases. The infectious agent is predominantly B. afzelii. Antibodies to other borrelia genospecies are predominantly due to cross reactivity.

Preliminary characterization of Borrelia burgdorferi CSF isolates

SummaryBorrelia burgdorferi was cultivated from three cerebrospinal fluid (CSF) samples of children (aged three and a half, four and a half and eight years) who were admitted to the hospital because of acute facial palsy, aseptic meningitis, and aseptic meningitis plus facial palsy. CSF was taken on day one in two cases and on day two in the remaining case after onset of symptoms. All three strains showed a very similar SDS-PAGE pattern, without an OspB and 20kD band. However, of nine monoclonal antibodies (Moab) raised againstB. burgdorferi B31, the Moab H5332 recognized two strains, one of them very weakly, and the flagella specific Moabs H9724, H605, and H6TS (less intensively) recognized all strains. This preliminary characterization reveals heterogeneity among CSFBorrelia isolates of cases from a very close geographic area.ZusammenfassungBorrelia burgdorferi wurde aus drei Liquorproben von Kindern (Alter: dreieinhalb, viereinhalb und acht Jahre) angezüchtet, welche aufgrund akuter Fazialisparese, aseptischer Meningitis und aseptischer Meningitis mit Fazialisparese ins Krankenhaus eingeliefert worden waren. Liquor wurde in zwei Fällen am ersten Tag, in einem am zweiten Tag nach Auftreten der Symptome gewonnen. Die drei Borrelien-Isolate waren in ihrem SDS-PAGE-Muster sehr ähnlich. Sie unterschieden sich jedoch in ihrer Reaktion mit neun monoklonalen Antikörpern (MoAb), die gegen den TypstammB. burgdorferi B31 entwickelt worden waren. MoAb H5332 erkannte zwei Stämme, einen davon in einer sehr schwachen Reaktion; die Flagella-spezifischen MoAbs H9724, H605 und H6TS, dieser weniger intensiv, erkannten alle Stämme. Diese vorläufige Charakterisierung zeigt Heterogenität unterB. burgdorferi-Liquor-Isolaten von Patienten aus der gleichen geografischen Region.

Perception of species-specific vocalizations in rats : role of the cholinergic septo-hippocampal pathway and aging

The effect of a chemical lesion of the cholinergic septo‐hippocampal pathway induced by ethylcholine aziridinium (AF64A) on brain potentials evoked by species‐specific vocalization containing informations of high biological relevance was studied in young adult (10 months) and aged (24 months) rats by means of neocortical electroencephalographic recordings. In control rats, the perception of a rats vocalization in a life endangering situation (lasting 0.8 s) initiated an evoked potential followed by a late positive slow wave (LPSW)‐complex and a direct current shift with a duration of up to 16 s. Four months after treatment with AF64A (2 nmol into each of the lateral ventricles), the main negative component of the initial acoustic evoked potential (peak latency of about 60 ms after stimulus onset) was reduced (P = 0.04) both in young adult and aged rats. Further changes included a decrease of the late positive wave amplitude in young adult rats (P = 0.001) and a shorter duration of the LPSW‐complex in aged rats (P = 0.03). AF64A induced also changes in specific components revealed by Principle Component Analysis, but only in the group of young rats. A decrease in the slow wave component (factor 1, 3000–4000 ms after stimulus onset ; P = 0.02) was observed. Age per se affected the late positive potential shifts as indicated by a shorter latency of the late positive wave (P = 0.03).