Thesis (Ph. D.)--Georgia State University, 2008. / Title from file title page. Barbara Baumstark, committee chair; Phang-Cheng Tai, Zehava Eichenbaum, committee members. Electronic text (251 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed July 14, 2008. Includes bibliographical references (p. 223-238).

Tübingen, Univ., Diss., 2004.

Objective: To study the presence of Bartonella bacilliformis in ticks collected from two wild mammals in Madre de Dios, Peru. Results: A total of 110 ticks were collected. Among the 43 Amblyomma spp. extracted from the 3 Tapirus terrestris only 3 were positive for B. bacilliformis. In addition, 12 out of the 67 Rhipicephalus (Boophilus) microplus obtained from the 3 Pecari tajacu were positive for B. bacilliformis. For the first time B. bacilliformis have been detected in arthropods other than Lutzomyia spp. Further studies are required to elucidate the possible role of ticks in the spread of South American Bartonellosis. / This work was supported by Cienciativa of CONCYTEC Peru, under the contract N° 164‑2016‑FONDECYT. Dr. Lescano is sponsored by the training grant D43 TW007393 awared by the Fogarty International Center of the US National Institutes of Health. / Revisión por pares / Revisión por pares

Human head and body lice are obligatory hematophagous ectoparasites that belong to a single species Pediculus humanus. Only body lice, however, are vectors of the infections gram-negative bacteria Bartonella quintana. Due to their near identical genomes, yet differential vector competency, head and body lice provide an ideal model system to study an insects ability to gain or lose vector competency. Using our in vitro louse rearing system, we have infected both head and body lice with a blood containing B. quintana in order to detect differences in B. quintana proliferation between head and body lice as well as transcriptional regulation of immune-related genes. B. quintana proliferates rapidly in body lice after 6 days post-infection, but declines in head lice after 4 days post-infection, possibly explaining, in part, the differential vector competence between the two insects. A transcriptome analysis using whole lice followed by qPCR verification of head and body lice immune-related genes was then conducted using uninfected, versus B. quintana infected lice to identify potential genes involved in vector competence. The immune-related genes Defensin 1, Fibrinogen-related protein and Spaetzle, were differentially regulated between head and body lice and were identified as potential targets for future research. Previously studied immune-related genes, PGRP, Defensin 1 and Defensin 2 transcription levels were also assessed in body louse midgut using qPCR following B. quintana infection. In this case, B. quintana infection did not result in significant effects on the transcript levels of these genes in midgut tissue. Overall transcriptional profiles of head and body lice genomes were notably different, including difference in the expression of 18.3 % of immune related genes, a finding that strongly supports the contention that immune system differences between head and body lice are the primary reason for difference in vector capacity.

Esta tesis se encuentra en proceso de registro como patente. / Antecedentes: La Enfermedad de Carrión es una patología importante que requiere un diagnóstico precoz para su manejo a fin de disminuir la morbimortalidad de la misma. Objetivo: Desarrollar una prueba inmunocromatográfica para la detección rápida de Bartonella bacilliformis usando anticuerpos policlonales de conejo etiquetados con oro coloidal. Metodología: Los anticuerpos policlonales contra Bartonella bacilliformis fueron obtenidos mediante la inmunización de conejos con la proteína GroEL de Bartonella bacilliformis. Se utilizó oro coloidal de 40 nm para la conjugación el anticuerpo primario obtenido. La sensibilidad analítica se evaluó con diferentes concentraciones de B. bacilliformis incluidas en un rango de 1x101 UFC/mL a 1x105 UFC/mL. La especificidad analítica o reacción cruzada se analizó con microorganismo de diferentes especies, incluyendo Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus viridans, Chlamydophila pneumoniae, Escherichia coli y Candida spp. Resultados: Las concentraciones óptimas del anticuerpo de captura y el anticuerpo de revestimiento (línea T) fueron de 2 mg/mL y 0.2 mg/mL respectivamente. Se determinó que la sensibilidad analítica de la prueba se encuentra en un rango de 1x102 UFC/mL y 1x101 UFC/mL. No se evidenciaron reacciones cruzadas con los microorganismos evaluados. Se determinó que el tiempo de corrida óptimo para el registro de resultados es de 20 minutos y el volumen mínimo requerido de la muestra analizada fue de 50 µL. Conclusión: Se desarrolló la primera prueba inmunocromatográfica para la detección rápida, sensible y específica de Bartonella bacilliformis utilizando anticuerpos policlonales contra la proteína GroEL, la cual deberá ser validada mediante estudios posteriores. / Background: Carrión's disease is an important disease that requires a timely diagnosis and management to reduce its morbimortality. Objective: Develop a lateral flow assay for the rapid detection of Bartonella bacilliformis using colloidal gold-labeled rabbit polyclonal antibodies. Materials and methods: Polyclonal antibodies against Bartonella bacilliformis were produced by the immunization of rabbits with GroEL protein purified from Bartonella bacilliformis. Colloidal gold of 40 nm was used for the conjugation process with the rabbit polyclonal antibodies. The analytical sensitivity was evaluated by testing solutions of B. bacilliformis with different concentrations ranging between 1 x 101 to 1 x 105 CFU/mL. Analytical specificity was determined by testing cross-reactivity with microorganisms from different species, including Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus viridans, Chlamydophila pneumoniae, Escherichia coli and Candida spp. Results: The optimal concentrations for the capture antibody and the coating antibody (T-line) were 2 mg / mL and 0.4 mg / mL, respectively. The analytical sensitivity was determined to be between 1x102 CFU/mL and 1x101 CFU/mL. There were no cross-reactions observed with the groups of bacteria used in this study. We determined that the flowing time and volume required for an optimum signal to be generated was 20 minutes and 50 µL, respectively. Conclusions: We developed the first gold-based lateral flow assay for the rapid, sensitive and specific detection of Bartonella bacilliformis using polyclonal antibodies against the protein GroEL, which needs to be validated in future studies. / Tesis

Bartonella bacilliformis, agente causal de la Enfermedad de Carrión transmitida por insectos flebotominos del género Lutzomyia y otros, es una endemia ancestral que afecta especialmente a la población más pobre de nuestros valles interandinos. En el Perú está presente en 12 de 25 departamentos. Es endémica en Ancash, Cajamarca, Amazonas, Cusco, La Libertad y Piura. Pocas son las investigaciones acerca de la susceptibilidad in vitro a antimicrobianos de Bartonella bacilliformis, no se cuenta con una prueba estandarizada de antibiograma para esta bacteria, como tampoco se conocen los mecanismos de resistencia ni las secuencias de los genes asociados a dicha resistencia. Por consiguiente, se ha planteado evaluar la resistencia antimicrobiana in vitro de cepas de Bartonella bacilliformis aisladas de zonas endémicas, mediante métodos convencionales y métodos moleculares, con la finalidad de conocer la resistencia a drogas de las cepas circulantes en las zonas endémicas. En el presente trabajo se obtuvieron muestras sanguíneas de 47 pacientes procedentes de Piura, Cusco y Ancash las cuales se sembraron en placas de agar sangre e incubaron a 30°C con 5% CO2. Se ha estandarizado un método de difusión por disco (para el antibiograma) y la prueba de Épsilon (para determinar la concentración mínima inhibitoria) para el estudio in vitro de la susceptibilidad antimicrobiana de B. bacilliformis, empleando los aislados clínicos y una cepa del Instituto Pasteur de Francia. Luego se procedió a extraer el DNA genómico, amplificar los genes mencionados, secuenciarlos y analizarlos mediante herramientas bioinformáticas. Se obtuvieron 3 cultivos positivos. Las cepas fueron sensibles a la ciprofloxacina, gentamicina, rifampicina, eritromicina, cloranfenicol, ceftriaxona y amoxicilina y resistentes al ácido nalidíxico. Del análisis de las secuencias aminoacídicas de las proteínas de GyrA, GyrB, ParC y ParE de B. bacilliformis, asociados a la resistencia de las quinolonas (ciprofloxacina, ácido nalidíxico) se concluye que presentan diferencias aminoacídicas de Ser por Ala, en comparación con las secuencias de las proteínas respectivas de E. coli K12 MG1655. Esta diferencia probablemente confiera resistencia al ácido nalidíxico pero no a la ciprofloxacina en B. bacilliformis. Se determinó que las QRDR de las proteínas GyrA, GyrB, ParC y ParE de B. bacilliformis están comprendidas entre los aminoácidos 74 a 124, 428 a 426, 67 a 118, y 473 a 530, respectivamente. Para el antibiótico rifampicina se determinó la RRDR en el gen rpoB comprendida entre los aminoácidos 521 a 547, y para el gen rplD de la proteína L4 relacionado con resistencia a eritromicina reportamos la ERDR nunca antes mencionado y comprende los aminoácidos 60 a 79, siendo el primer reporte para este gen en Bartonella bacilliformis. Finalmente en el modelamiento de las estructuras terciarias de las regiones determinantes de resistencia se apreciaron cambios en las posiciones de Ser por Ala, y estos cambios permiten una débil interacción con la droga. Al evaluar los perfiles de hidrofobicidad de los aminoácidos pertenecientes a la región determinante de resistencia, nos permitió inferir que los cambios de aminoácidos con propiedades diferentes como Ser por Ala contribuyen a la resistencia.

Abstract We designed a 16S rRNA gene PCR-RFLP scheme to identify all currently described Bartonella spp. The 16S rRNA genes of all Bartonella spp. were in-silico analyzed in order to design a RFLP technique able to discriminate among different species. The restriction enzymes selected were MaeIII, MseI, Sau96I, BsaAI, DrdI, FokI, BssHII, BstUI, AluI, TspDTI and HphI which, according to a decision-making tree, facilitated the differentiation of all the currently described species of Bartonella.The technique was experimentally tested in different species of Bartonella, including human pathogenic B. bacilliformis and B. henselae with a 100% of concordance with the in-silico predicted patterns.This novel RFLP assay could be used to identify both human and non-human pathogenic Bartonella in diagnostic, phylogenetic and epidemiologic studies.

joruiz@clinic.ub.es / Introduction: Bartonella bacilliformis is the etiological agent of Carrion’s disease, which is a neglected disease linked to people in low-socioeconomic populations in Andean valleys. An outbreak of B. bacilliformis was reported in a rural area of the Peruvian Amazon region. The aim of this study was to characterize this outbreak using molecular techniques. Methodology: Fifty-three blood samples from patients diagnosed with Carrion’s disease were analyzed by molecular tools, using both a Bartonella-specific polymerase chain reaction (PCR) and an universal PCR, both based on 16S rRNA gene amplification. Additional water samples from the area were also analyzed. Results: Unexpectedly, the samples were positive only when the universal PCR was used. Although environmental contamination cannot be ruled out, the results showed that Sphingomonas faeni was the possible causative agent of this outbreak, and that water was the most feasible infection source. Conclusions: Diagnosis by clinical criteria or microscopy may lead to misdiagnosis. There is a need to include molecular tools in the routine diagnosis of febrile syndromes, including Carrion’s disease.

Ctenocephalides felis est une espèce de puce cosmopolite parasitant majoritairement les carnivores domestiques. Elle est vectrice de nombreux agents pathogènes zoonotiques dont les bactéries du genre Bartonella, bactéries intracellulaires facultatives. La compétence vectorielle de cette puce a été investiguée pour B. henselae, B. quintana, B. clarridgeiae, B. tribocorum et B. birtlesii. Dans ces conditions expérimentales, utilisant un système de gorgement sur membrane, ces espèces ont persisté pendant les trois jours d'une première étude, et pour B. henselae durant les 13 jours de survie des puces, dans une seconde étude. Les cinq espèces de bartonelles ont été retouvées dans les fèces. Pour ces cinq espèces, nos résultats montrent une absence de transmission verticale transovarienne chez la puce et suggèrent une possibilité de contamination horizontale. Nous proposons enfin un protocole original d'évaluation de l'efficacité d'un traitement antiparasitaire chez le chat, pour prévenir sa contamination par Bartonella spp. / Ctenocephalides felis is a cosmopolitan flea species mainly parasitizing pets, transmitting several pathogens of veterinary and zoonotic importance including the facultative intracellular bacteria of the genus Bartonella. The vector competence of this flea was investigated for B. henselae, B. quintana, B. clarridgeiae, B. tribocorum and B. birtlesii, using an artificial feeding system. In these experimental conditions, these bartonellae proved to persist for three days, in a first study, while B. henselae persisted for the 13 days of its life span, in a second study. All five bartonellae were excreted in the flea's faeces. On the whole, these five species were not transmitted transovarially in the fleas, though horizontal transmission was suggested. Furthermore, we propose an original protocol allowing the evaluation of the efficacy of ectoparasiticidal products against Bartonella spp. infection in cats.

MSc (Med), Virology, Faculty of Health Sciences, University of the Witwatersrand / Bartonella is a genus of fastidious bacteria responsible for a wide range of both symptomatic and asymptomatic infections. Bartonellae are often considered obligate pathogens where infection is concurrent with immunological suppression of the host. The objectives of this study were: to determine the prevalence of Bartonella infections in HIV-positive patients presenting for treatment at a Gauteng HIV-clinic, to determine the extent of bartonellae affecting the healthy population, to determine the seroprevalence of Bartonella henselae and Bartonella quintana antibodies in HIV-negative antenatal patient sera taken from various maternity units in Gauteng public hospitals, and to investigate cats, dogs, and rodents in Johannesburg for carriage of bartonellae. A total of 382 HIV-positive patients attending the HIV clinic and 42 clinically healthy volunteers agreed to participate. Three-hundred and forty-two residual sera from the national antenatal survey were selected and tested for IgG and IgM antibodies against Bartonella. There were 179 dogs, 98 cats and 124 rodents enrolled in this study. The seroprevalence for Bartonella in humans was carried out using IgG and IgM commercially available kits. HIV-positive patients were found to have 32% IgG and 14% IgM seroprevalence, whereas the healthy volunteers had a lower IgG (19%) and higher IgM seroprevalence than the HIV-positive counterparts. All blood samples were cultured, but only the cat and rodent specimens yielded isolates. These were sequenced for species identification. The cat isolates were 99 and 100% similar to B. henselae URBHLIE 9 previously isolated from a patient with endocarditis, and the rat isolates were 98 – 99% similar to either RN24BJ (candidus ‘B. thailandensis’) or RN28BJ, previously isolated from rodents in China. The PCR prevalences were: 22.5% in HIV-positive patients; 9.5% in clinically healthy volunteers; 23.5% in cats; 9% in dogs; and 25% in rodents. Findings of this study have important implications for HIV-positive patients