Identification of the Bartonella autotransporter CFA as a protective antigen and hypervariable target of neutralizing antibodies in mice.

The bacterial genus Bartonella comprises numerous emerging pathogens that cause a broad spectrum of disease manifestations in humans. The targets and mechanisms of the anti-Bartonella immune defense are ill-defined and bacterial immune evasion strategies remain elusive. We found that experimentally infected mice resolved Bartonella infection by mounting antibody responses that neutralized the bacteria, preventing their attachment to erythrocytes and suppressing bacteremia independent of complement or Fc receptors. Bartonella-neutralizing antibody responses were rapidly induced and depended on CD40 signaling but not on affinity maturation. We cloned neutralizing monoclonal antibodies (mAbs) and by mass spectrometry identified the bacterial autotransporter CFA (CAMP-like factor autotransporter) as a neutralizing antibody target. Vaccination against CFA suppressed Bartonella bacteremia, validating CFA as a protective antigen. We mapped Bartonella-neutralizing mAb binding to a domain in CFA that we found is hypervariable in both human and mouse pathogenic strains, indicating mutational antibody evasion at the Bartonella subspecies level. These insights into Bartonella immunity and immune evasion provide a conceptual framework for vaccine development, identifying important challenges in this endeavor.

'We should continue to recommend year-round flea protection for cats and dogs'.

Ian Wright argues that although there are environmental concerns regarding 'blanket' flea treatment of cats and dogs, without this measure pet owners would be at risk of potentially developing bartonellosis.

Subtractive proteomics and immunoinformatics approaches to explore Bartonella bacilliformis proteome (virulence factors) to design B and T cell multi-epitope subunit vaccine.

Bartonella bacilliformis a gram-negative facultative aerobe responsible for the Carrion's disease widely distributed in Ecuador, Peru, and Colombia with a high mortality rate when no specific treatment is received. B bacilliformis is transmitted by Sand fly (Lutzomyia verrucarum) to healthy individuals. Immunoinformatic and subtractive proteomics approaches were employed in this study to prioritize the best candidates for vaccine designing. These approaches resulted in five vaccine candidates, flagellar biosynthetic protein (Uniprot ID: A1UTU1), heme exporter protein C (UniProt ID: A1UU82), Cytochrome c-type biogenesis protein (Uniprot ID: A1URZ7), Hemin ABC transporter (Uniprot ID: A1US20) and Phosphatidate cytidylyltransferase (Uniprot ID: A1USE3). The mentioned proteins are antigenic and essential for pathogen survival. A range of immune-informatics tools was applied for the prediction of B and T cell epitopes for the vaccine candidate proteins. In-silico vaccine was constructed using carefully evaluated epitopes and consequently modeled for docking with human Toll-like receptor 4. TLR-4 agonist 50S ribosomal protein L7/L12 (UniproKB ID; P9WHE3) was linked to the vaccine as an adjuvant to boost immune response towards the vaccine. For stability evaluation of the vaccine-TLR-4 docked complex, MD simulations were performed. The final vaccine was back-translated and cloned in Eschericia coli to attain the maximal expression of the vaccine protein. The maximal expression was ensured, and the CAI score of 0.96 was reported. The current vaccine requires future experimental validation to confirm its effectiveness. The vaccine developed will be helpful to protect against B bacilliformis associated infections.

[DESIGN AND EVALUATION OF A MULTIEPITOPIC PROTEIN AS A CANDIDATE FOR A CARRION DISEASE VACCINE]. / DISEÑO Y EVALUACIÓN DE UNA PROTEÍNA MULTIEPITÓPICA COMO CANDIDATA PARA VACUNA CONTRA LA ENFERMEDAD DE CARRIÓN.

OBJECTIVES.: To design and assess a multiepitopic protein as a candidate for a vaccine against Carrion disease. MATERIALS AND METHODS.: Using bioinformatics tools, epitopes of external membrane proteins were selected and a multiepitopic protein was designed. The multiepitopic protein gene was subcloned into the expression plasmid pET28b and transformed into E. coli BL21 pLys. The multiepitopic protein was expressed using isopropyl-ß-D-1-thiogalactopyranoside and purified using resin. This purified protein was used to immunize BALB/c mice obtaining polyclonal antibodies. In vitro invasion assays were conducted using a strain of Bartonella bacilliformis (B. bacilliformis) in human red blood cells. RESULTS.: The multiepitopic protein M1 presents preserved epitopes between isolates of B. bacilliformis with are non-toxic, and not homologous to human and surface proteins. Immunized mice presented IgG antibody levels capable of reducing in vitro the rate of invasion of B. bacilliformis into human red blood cells. CONCLUSIONS.: Multiepitopic protein M1 may serve as a candidate for a Carrion disease vaccine; however, more studies are needed to characterize the use of this antigen as a vaccine.

OBJETIVOS.: Diseñar y evaluar una proteína multiepítope como candidato a vacuna contra la enfermedad de Carrión. MATERIALES Y MÉTODOS.: Mediante herramientas bioinformáticas se seleccionó epítopes de proteínas de membrana externa y se diseñó una proteína multiepítope. El gen de la proteína multiepítope fue subclonado en el plásmido de expresión pET28b y transformado en E. coli BL21 pLys. La proteína multiepítope fue expresada usando isopropil-ß-D-1-tiogalactopiranósido y purificada usando resina. Esta proteína purificada fue utilizada para inmunizar ratones BALB/c y se obtuvo anticuerpos policlonales. Se realizaron ensayos de invasión in vitro usando una cepa de Bartonella bacilliformis (B. bacilliformis) a eritrocitos humanos. RESULTADOS.: La proteína multiepítope M1 presenta epítopes conservados entre aislamientos de B. bacilliformis, no tóxicos, no homólogos a proteínas humanas y superficiales. Los ratones inmunizados presentaron niveles de anticuerpos IgG capaces de reducir in vitro la tasa de invasión de B. bacilliformis a eritrocitos humanos. CONCLUSIONES.: La proteína multiepítope M1 podría servir como candidato a vacuna contra la enfermedad de Carrión; sin embargo, se requiere de más estudios para caracterizar el uso de este antígeno como vacuna.

Diseño y evaluación de una proteína multiepitópica como candidata para vacuna contra la enfermedad de carrión / Design and evaluation of a multiepitopic protein as a candidate for a carrion disease vaccine

RESUMEN Objetivos. Diseñar y evaluar una proteína multiepítope como candidato a vacuna contra la enfermedad de Carrión. Materiales y métodos. Mediante herramientas bioinformáticas se seleccionó epítopes de proteínas de membrana externa y se diseñó una proteína multiepítope. El gen de la proteína multiepítope fue subclonado en el plásmido de expresión pET28b y transformado en E. coli BL21 pLys. La proteína multiepítope fue expresada usando isopropil-β-D-1-tiogalactopiranósido y purificada usando resina. Esta proteína purificada fue utilizada para inmunizar ratones BALB/c y se obtuvo anticuerpos policlonales. Se realizaron ensayos de invasión in vitro usando una cepa de Bartonella bacilliformis (B. bacilliformis) a eritrocitos humanos. Resultados. La proteína multiepítope M1 presenta epítopes conservados entre aislamientos de B. bacilliformis, no tóxicos, no homólogos a proteínas humanas y superficiales. Los ratones inmunizados presentaron niveles de anticuerpos IgG capaces de reducir in vitro la tasa de invasión de B. bacilliformis a eritrocitos humanos. Conclusiones. La proteína multiepítope M1 podría servir como candidato a vacuna contra la enfermedad de Carrión; sin embargo, se requiere de más estudios para caracterizar el uso de este antígeno como vacuna.

ABSTRACT Objectives. To design and assess a multiepitopic protein as a candidate for a vaccine against Carrion disease. Materials and Methods. Using bioinformatics tools, epitopes of external membrane proteins were selected and a multiepitopic protein was designed. The multiepitopic protein gene was subcloned into the expression plasmid pET28b and transformed into E. coli BL21 pLys. The multiepitopic protein was expressed using isopropyl-β-D-1-thiogalactopyranoside and purified using resin. This purified protein was used to immunize BALB/c mice obtaining polyclonal antibodies. In vitro invasion assays were conducted using a strain of Bartonella bacilliformis (B. bacilliformis) in human red blood cells. Results. The multiepitopic protein M1 presents preserved epitopes between isolates of B. bacilliformis with are non-toxic, and not homologous to human and surface proteins. Immunized mice presented IgG antibody levels capable of reducing in vitro the rate of invasion of B. bacilliformis into human red blood cells. Conclusions. Multiepitopic protein M1 may serve as a candidate for a Carrion disease vaccine; however, more studies are needed to characterize the use of this antigen as a vaccine.

Effectiveness of a 10% imidacloprid/4.5% flumethrin polymer matrix collar in reducing the risk of Bartonella spp. infection in privately owned cats.

BACKGROUND: Bartonella henselae, Bartonella clarridgeiae and the rare Bartonella koehlerae are zoonotic pathogens, with cats being regarded as the main reservoir hosts. The spread of the infection among cats occurs mainly via fleas and specific preventive measures need to be implemented. The effectiveness of a 10% imidacloprid/4.5% flumethrin polymer matrix collar (Seresto®, Bayer Animal Health), registered to prevent flea and tick infestations, in reducing the risk of Bartonella spp. infection in privately owned cats, was assessed in a prospective longitudinal study. METHODS: In March-May 2015 [Day 0 (D0)], 204 privately-owned cats from the Aeolian Islands (Sicily) were collared (G1, n = 104) or left as controls (G2, n = 100). The bacteraemia of Bartonella spp. was assessed at enrolment (D0) and study closure (D360) by PCR and DNA sequencing both prior to and after an enrichment step, using Bartonella alpha proteobacteria growth medium (BAPGM). RESULTS: A total of 152 cats completed the study with 3 in G1 and 10 in G2 being positive for Bartonella spp. Bartonella henselae genotype I ZF1 (1.35%) and genotype II Fizz/Cal-1 (6.76%) as well as B. clarridgeiae (5.41%) were detected in cats of G2. Bartonella clarridgeiae was the only species detected in G1. Based on the yearly crude incidence of Bartonella spp. infection (i.e. 3.85% in G1 and 13.51% in G2; P = 0.03) the Seresto® collar achieved a preventative efficacy of 71.54%. The incidence of Bartonella spp. infection was more frequent in flea-infested cats (6/33, 18.18%) than in uninfested ones (7/112, 5.88%) (P = 0.036). CONCLUSIONS: Cats living in the Aeolian Islands are exposed to B. henselae and B. clarridgeiae. The Seresto® collar provided significant risk reduction against Bartonella spp. infection in outdoor cats under field conditions. Such a preventative tool could be a key contribution for decreasing the risk of Bartonella spp. infection in cats and thus ultimately to humans.

First molecular detection and characterization of zoonotic Bartonella species in fleas infesting domestic animals in Tunisia.

BACKGROUND: Bartonellosis is an emerging vector-borne disease caused by different intracellular bacteria of the genus Bartonella (Rhizobiales: Bartonellaceae) that is transmitted primarily by blood-sucking arthropods such as sandflies, ticks and fleas. In Tunisia, there are no data available identifying the vectors of Bartonella spp. In our research, we used molecular methods to detect and characterize Bartonella species circulating in fleas collected from domestic animals in several of the country's bioclimatic areas. RESULTS: A total of 2178 fleas were collected from 5 cats, 27 dogs, 34 sheep, and 41 goats at 22 sites located in Tunisia's five bioclimatic zones. The fleas were identified as: 1803 Ctenocephalides felis (83%) (Siphonaptera: Pulicidae), 266 C. canis (12%) and 109 Pulex irritans (5%) (Siphonaptera: Pulicidae). Using conventional PCR, we screened the fleas for the presence of Bartonella spp., targeting the citrate synthase gene (gltA). Bartonella DNA was detected in 14% (121/866) of the tested flea pools [estimated infection rate (EIR) per 2 specimens: 0.072, 95% confidence interval (CI): 0.060-0.086]. The Bartonella infection rate per pool was broken down as follows: 55% (65/118; EIR per 2 specimens: 0.329, 95% CI: 0.262-0.402) in C. canis; 23.5% (8/34; EIR per 2 specimens: 0.125, 95% CI: 0.055-0.233) in P. irritans and 6.7% (48/714; EIR per 2 specimens: 0.032, 95% CI: 0.025-0.045) in C. felis. Infection rates, which varied significantly by bioclimatic zone (P < 0.0001), were highest in the humid areas. By sequencing, targeting the gltA gene and the 16S-23S rRNA Intergenic Spacer Regions (ITS), we identified three Bartonella zoonotic species: B. elizabethae, B. henselae, B. clarridgeiae, as well as uncharacterized Bartonella genotypes. CONCLUSIONS: To the best of our knowledge, this is the first time that fleas in Tunisia have been shown to carry zoonotic species of Bartonella. The dog flea, Ctenocephalides canis, should be considered the main potential vector of Bartonella. Our study not only provides new information about this vector, but also offers a public health update: medical practitioners and farmers in Tunisia should be apprised of the presence of Bartonella in fleas and implement preventive measures.

Carrion's disease: an eradicable illness?

Carrion's disease is a neglected tropical disease caused by Bartonella bacilliformis, a vector-borne pathogen restricted to the Andean valleys of Peru, Ecuador and Colombia. Carrion's disease is a biphasic illness; in the acute phase the case-fatality rate can be as high as 88 %, related to high parasitemia, arriving to almost all erythrocytes, and secondary bacterial infections close related with the development of transient immunosuppression in the earlier illness phases. In addition, there are an undefined number of asymptomatic carriers that are reservoirs of the etiological agent of Carrion's disease in endemic areas, they make take into account due to they are the perpetuators of this disease. The actual scenario of Carrion's disease, in which the illness is arriving to new areas, due to the expansion of the vector's distribution, suggests that now may be a crucial time to design a strategy focusing on its elimination.

[Experiences in the prevention and control of Carrión's disease in Peru]. / Experiencias en la prevención y control de la enfermedad de Carrión en el Perú

Carrion's disease, the iconic disease in Peruvian medicine has been found in the mountains of Ecuador, Colombia and the Andean valleys of Peru. In the 1990s, the phenomenon of El Niño was associated with significantly increased risk of disease in Ancash, Cajamarca and Cusco. In Cusco in 1998 there was an acute phase epidemic in various Andean provinces and the jungle area. Between 2001 and 2005 the disease has spread or reactivated in different regions such as Ancash, Cajamarca, Amazonas, Piura, Cusco, La Libertad, Puno, and Ayacucho. In 2004 a major outbreak of the disease in all of Peru was presented, reporting more than 11 164 cases, and therefore diverse strategies based vector susceptibility studies was applied, lowering significantly the number of cases.

Experiencias en la prevención y control de la enfermedad de Carrión en el Perú / Experiences in the prevention and control of Carrión's disease in Peru

La enfermedad de Carrión, enfermedad emblemática de la medicina peruana, ha sido descrita en la sierra de Ecuador, Colombia y valles interandinos del Perú. En la década de 1990, el fenómeno de El Niño fue asociado con incremento significativo del riesgo de enfermedad en Ancash, Cajamarca y Cusco. Justamente en Cusco en 1998 se produjo una importante epidemia de la fase aguda en diversas provincias andinas y la zona selvática. Posteriormente entre 2001 y 2005 la enfermedad se ha expandido o reactivado en diversas regiones como Ancash, Cajamarca, Amazonas, Piura, Cusco, La Libertad, Puno, Ayacucho. El 2004 se presentó un rebrote importante de la enfermedad en todo el Perú, reportándose más de 11 164 casos, por ello se aplicaron diversas estrategias de control basados en estudios de susceptibilidad de vectores, disminuyendo en forma significativa el número de casos.

Carrion’s disease, the iconic disease in Peruvian medicine has been found in the mountains of Ecuador, Colombia and the Andean valleys of Peru. In the 1990s, the phenomenon of El Niño was associated with significantly increased risk of disease in Ancash, Cajamarca and Cusco. In Cusco in 1998 there was an acute phase epidemic in various Andean provinces and the jungle area. Between 2001 and 2005 the disease has spread or reactivated in different regions such as Ancash, Cajamarca, Amazonas, Piura, Cusco, La Libertad, Puno, and Ayacucho. In 2004 a major outbreak of the disease in all of Peru was presented, reporting more than 11 164 cases, and therefore diverse strategies based vector susceptibility studies was applied, lowering significantly the number of cases.

Bartonella species infection in cats: ABCD guidelines on prevention and management.

OVERVIEW: Over 22 Bartonella species have been described in mammals, and Bartonella henselae is most common worldwide. Cats are the main reservoir for this bacterium. B henselae is the causative agent of cat scratch disease in man, a self-limiting regional lymphadenopathy, but also of other potentially fatal disorders in immunocompromised people. INFECTION: B henselae is naturally transmitted among cats by the flea Ctenocephalides felis felis, or by flea faeces. A cat scratch is the common mode of transmission of the organism to other animals, including humans. Blood transfusion also represents a risk. DISEASE SIGNS: Most cats naturally infected by B henselae do not show clinical signs but cardiac (endocarditis, myocarditis) or ocular (uveitis) signs may be found in sporadic cases. B vinsonii subspecies berkhoffii infection has reportedly caused lameness in a cat affected by recurrent osteomyelitis and polyarthritis. DIAGNOSIS: Isolation of the bacterium is the gold standard, but because of the high prevalence of infection in healthy cats in endemic areas, a positive culture (or polymerase chain reaction) is not confirmatory. Other compatible diagnoses must be ruled out and response to therapy gives a definitive diagnosis. Serology (IFAT or ELISA) is more useful for exclusion of the infection because of the low positive predictive value (39-46%) compared with the good negative predictive value (87-97%). Laboratory testing is required for blood donors. DISEASE MANAGEMENT: Treatment is recommended in the rare cases where Bartonella actually causes disease.

Bartonella bacilliformis: a systematic review of the literature to guide the research agenda for elimination.

BACKGROUND: Carrion's disease affects small Andean communities in Peru, Colombia and Ecuador and is characterized by two distinct disease manifestations: an abrupt acute bacteraemic illness (Oroya fever) and an indolent cutaneous eruptive condition (verruga Peruana). Case fatality rates of untreated acute disease can exceed 80% during outbreaks. Despite being an ancient disease that has affected populations since pre-Inca times, research in this area has been limited and diagnostic and treatment guidelines are based on very low evidence reports. The apparently limited geographical distribution and ecology of Bartonella bacilliformis may present an opportunity for disease elimination if a clear understanding of the epidemiology and optimal case and outbreak management can be gained. METHODS: All available databases were searched for English and Spanish language articles on Carrion's disease. In addition, experts in the field were consulted for recent un-published work and conference papers. The highest level evidence studies in the fields of diagnostics, treatment, vector control and epidemiology were critically reviewed and allocated a level of evidence, using the Oxford Centre for Evidence-Based Medicine (CEBM) guidelines. RESULTS: A total of 44 studies were considered to be of sufficient quality to be included in the analysis. The majority of these were level 4 or 5 (low quality) evidence and based on small sample sizes. Few studies had been carried out in endemic areas. CONCLUSIONS: Current approaches to the diagnosis and management of Carrion's disease are based on small retrospective or observational studies and expert opinion. Few studies take a public health perspective or examine vector control and prevention. High quality studies performed in endemic areas are required to define optimal diagnostic and treatment strategies.

Feline bartonellosis.

Bartonella infection is common among domestic cats, but the role of Bartonella species as feline pathogens requires further study. Most Bartonella species that infect cats are zoonotic. Cats are the mammalian reservoir and vector for Bartonella henselae, an important zoonotic agent. Cat fleas transmit Bartonella among cats, and cats with fleas are an important source of human B henselae infections. New information about Bartonella as feline pathogens has recently been published, and this article summarizes much of that information. Issues surrounding diagnosis and treatment of feline Bartonella infections are described, and prevention of zoonotic transmission of Bartonella is discussed.

Immunogenicity of Bartonella henselae P26 in cats.

Cat scratch disease (CSD) has an estimated prevalence of approximately 200,000 persons in the USA, and approximately 22,000 new cases occur annually. Cats are the natural carriers of Bartonella henselae, the agent for CSD. Zoonotic transmission of B. henselae can result in CSD in immunocompetent humans and bacillary angiomatosis in immunosuppressed humans. Infection in cats often goes undetected. Development of a vaccine to prevent feline infection is warranted to reduce the prevalence of infection in the feline population and to decrease the potential for zoonotic transmission. One of the immunoreactive proteins identified from our previous study was P26. In this study, we demonstrated that B. henselae recombinant P26 (rP26) was immunogenic in cats. Four cats immunized with rP26 and four control cats were challenged with B. henselae type I and blood samples were collected for culture, PCR, and serology. Immunization with rP26 did not provide protection against B. henselae infection in cats at the doses used in this study. However, p26 PCR proved to be more sensitive for detection of infection in cats compared to gltA PCR. Furthermore, ELISA using rP26 as the substrate was more sensitive than ELISA using B. henselae type I outer membrane proteins.

[Risk of infections among orienteers]. / Infeksjonsrisiko hos orienteringsløpere.

BACKGROUND: Research on orienteers is useful for assessing the risk of infections associated with physical activity in the forest. In this paper four types of infections are reviewed, and the efficacy of preventive initiatives is discussed. MATERIAL AND METHODS: The paper is based on literature retrieved from a non-systemic search in PubMed. RESULTS: Hepatitis B infection was more prevalent among orienteers before they were obliged to use protective clothing. In the 1980s, there was an increase of sudden unexpected death among young Swedish orienteers. Bartonella infection was later suggested as an underlying cause. No unexpected deaths have occurred among young orienteers after 1992 when specific advice was given regarding training and competitions. Orienteers do not seem to be affected by lyme borreliosis or tick-borne encephalitis (TBE) more often than others, but only two old studies have been performed. INTERPRETATION: Orienteers may be at risk of acquiring infection from lyme borreliosis and TBE in Norway in the future, as the incidence of these contagions is increasing. Norwegian medical personnel should consider TBE vaccination of orienteers and others who wander in areas with a high prevalence of infected ticks.

Bartonella quintana and Bartonella pediococcus Infection after Aortic Valve Replacement.

Bartonella quintana and Bartonella pediococcus infections are very rare causes of endocarditis. Urban trench fever with relapsing febrile illness, headache, leg pain, and endocarditis has now begun to be a more important cause of disease in socially disadvantaged persons. The diagnosis is difficult because the growth of B. quintana in blood culture takes 20-40 days. B. pediococcus may be an opportunistic pathogen in severely compromised hosts, although it has been described as a harmless bacterium. We describe a patient who developed bioprosthetic valve infection with B. quintana and B. pediococcus after valve replacement.

Effect of experimental ectoparasite control on bartonella infections in wild Richardson's ground squirrels.

The purpose of this study was to investigate the role of ectoparasites in transmitting Bartonella infections in wild Richardson's ground squirrels (Spermophilus richardsonii). Richardson's ground squirrels were trapped, examined for fleas, and tested for Bartonella bacteremia once monthly, at six sites, from April to September 2004. After the initial trapping session in April, burrows at three sites were treated with deltamethrin insecticide. Richardson's ground squirrels trapped on treated sites were less likely to have fleas and had fewer fleas than squirrels on control sites in all months following treatment. We found no difference in the prevalence of Bartonella infections on control and treated sites in May, immediately following treatment; however, significantly fewer squirrels were infected with Bartonella on treated sites in June and July. We conclude that ectoparasites are a main route of transmission for Bartonella infections in Richardson's ground squirrels.

Emerging or re-emerging bacterial zoonotic diseases: bartonellosis, leptospirosis, Lyme borreliosis, plague.

There are a whole series of emerging and re-emerging zoonotic diseases present in the Northern Hemisphere and the author describes four of them, namely, bartonellosis, leptospirosis, Lyme borreliosis and plague. Reasons for the emergence or re-emergence of such diseases are not clear, but factors such as human demographics, economic development and land use, international travel and commerce, and microbial adaptation, are thought to be involved. Control of emerging and re-emerging diseases has become a major challenge for the international community and it is important to disseminate information about diagnosis and control capabilities, particularly to people working in public health.