Bartonella infections in deer keds (Lipoptena cervi) and moose (Alces alces) in Norway.

Infections with Bartonella spp. have been recognized as emerging zoonotic diseases in humans. Large knowledge gaps exist, however, relating to reservoirs, vectors, and transmission of these bacteria. We describe identification by culture, PCR, and housekeeping gene sequencing of Bartonella spp. in fed, wingless deer keds (Lipoptena cervi), deer ked pupae, and blood samples collected from moose, Alces alces, sampled within the deer ked distribution range in Norway. Direct sequencing from moose blood sampled in a deer ked-free area also indicated Bartonella infection but at a much lower prevalence. The sequencing data suggested the presence of mixed infections involving two species of Bartonella within the deer ked range, while moose outside the range appeared to be infected with a single species. Bartonella were not detected or cultured from unfed winged deer keds. The results may indicate that long-term bacteremia in the moose represents a reservoir of infection and that L. cervi acts as a vector for the spread of infection of Bartonella spp. Further research is needed to evaluate the role of L. cervi in the transmission of Bartonella to animals and humans and the possible pathogenicity of these bacteria for humans and animals.

Migratory birds, ticks, and Bartonella.

Bartonella spp. infections are considered to be vector-borne zoonoses; ticks are suspected vectors of bartonellae. Migratory birds can disperse ticks infected with zoonotic pathogens such as Rickettsia and tick-borne encephalitis virus and possibly also Bartonella. Thus, in the present study 386 tick specimens collected in spring 2009 from migratory birds on the Mediterranean islands Capri and Antikythera were screened for Bartonella spp. RNA. One or more ticks were found on 2.7% of the birds. Most ticks were Hyalomma rufipes nymphs and larvae with mean infestation rates of 1.7 nymphs and 0.6 larvae per infested bird. Bartonella spp. RNA was not detected in any of the tick specimens.

Arsenic trioxide influences viral replication in target organs of coxsackievirus B3-infected mice.

New antiviral agents are urgently needed. Based on in vitro studies, arsenic trioxide (As2O3) seems to affect viral replication, although this has been studied only marginally in vivo. In this study the replication of coxsackievirus B3 (CVB3) was studied in Balb/c mice administered 1 mg As2O3/kg bw once daily during 7 days of infection and in Vero cells exposed for 3 or 5 days to 0.4, 2 or 4 µM As2O3. Viral RNA was measured by reverse transcription PCR (RT-PCR) (in vitro and in vivo) and arsenic concentration was measured by inductively coupled plasma-mass spectrometry (ICP-MS) (in vivo). In vivo, As2O3 decreased viral RNA in the brain on days 3 (by 81%; p < 0.05) and 7 (by 97%; p < 0.01) and in the pancreas on day 7 (by 75%; p < 0.05), two of the target organs of this infection. The results were confirmed in vitro, where As2O3 dose-dependently reduced viral RNA, with the effect being more pronounced in the surrounding culture medium than inside the infected cells, indicating an impaired virion release. Thus, As2O3 reduced CVB3 replication both in vitro and in vivo, indicating that As2O3 is a viable option in the pursuit of new therapeutic agents against viral infections.

Persistent Chlamydophila pneumoniae infection in thoracic aortic aneurysm and aortic dissection?

OBJECTIVES: Chlamydophila pneumoniae (C. pneumoniae) has been associated with atherosclerosis and abdominal aortic aneurysm and is probably disseminated by peripheral blood mononuclear cells (PBMC). Viable and metabolically active bacteria can be demonstrated by the presence of bacterial mRNA and on-going dissemination by the presence of bacteria in PBMC. The aim of this study was to determine the prevalence of C. pneumoniae DNA and mRNA in aortic biopsies and C. pneumoniae DNA in PBMC in thoracic aortic aneurysm and aortic dissection patients. DESIGN: Real-time PCR was used to detect C. pneumoniae DNA and mRNA in biopsies and C. pneumoniae DNA in PBMC. RESULTS: C. pneumoniae DNA was found in biopsies in 26% (6/23) of aneurysm patients and 11% (2/18) of dissection patients but in none of the forensic autopsy controls. C. pneumoniae mRNA was not found in any biopsy, and all PBMC were C. pneumoniae-negative. CONCLUSIONS: Presence of C. pneumoniae DNA but not mRNA in aortic biopsies and no evidence of C. pneumoniae in PBMC suggest that the infection in the aorta has passed into a state of persistence.

Presence of Chlamydophila pneumoniae DNA but not mRNA in stenotic aortic heart valves.

BACKGROUND: The pathogenesis of aortic valve stenosis may involve inflammation and we have previously demonstrated Chlamydophila pneumoniae (C. pneumoniae) DNA in stenotic aortic heart valves. Dissemination of these bacteria is probably mediated by alveolar macrophages. Bacterial DNA alone does not indicate whether the bacteria are viable and replicating. This study aimed to investigate the presence of C. pneumoniae mRNA, a marker of replicating bacteria, and C. pneumoniae DNA in stenotic aortic valves and the prevalence of C. pneumoniae in peripheral blood mononuclear cells (PBMC). METHODS: DNA was extracted from heart valves and PBMC and mRNA from heart valves from 76 patients undergoing aortic valve replacement surgery. C. pneumoniae DNA and mRNA were measured by real-time PCR targeting the ompA gene. RESULTS: C. pneumoniae DNA was demonstrated in 22% of heart valves and in 5% of PBMC. C. pneumoniae mRNA was not detected in any valve. Patients positive for C. pneumoniae in the valve underwent coronary artery by-pass grafting more often (p=0.01) and suffered from angina pectoris (p=0.02) and arterial hypertension (p=0.03) more often than patients negative for C. pneumoniae in the valve. CONCLUSIONS: These findings support a role for C. pneumoniae in the pathogenesis of aortic valve stenosis and indicate that the bacteria disseminate from the respiratory tract long before the patients were in need of surgery and that the valve infection thereafter entered into a persistent and non-replicative state. Moreover, patients positive for C. pneumoniae in the valve more often needed by-pass grafting because of more advanced coronary disease.

Thoracic aortic aneurysm patients with Chlamydophila pneumoniae infection showed a shift in trace element levels in serum and diseased aortic tissue.

Few studies have been performed on trace elements in tissues and serum in cardiovascular disease and none in aortic aneurysm. In this study the concentrations of 10 trace elements were determined in serum and aneurysmatic aortic tissue from 23 patients undergoing thoracic surgery. Macroscopically, normal thoracic aortic tissue specimens from 10 forensic autopsies and serum from 23 healthy blood donors served as controls. DNA from the intracellular respiratory pathogen Chlamydophila pneumoniae (C. pneumoniae), which may be involved in the pathogenesis of atherosclerosis, was found in 26% (6/23) of the patients but in none of the controls. The serum copper/zinc ratio, a well-known marker of ongoing infection and/or inflammation, was higher (26%, p<0.001) in aneurysm patients. C. pneumoniae requires iron for its growth. In our aneurysm patients iron was higher in serum (by 54%, p<0.001) and aneurysmal tissue (by 60%, p<0.001). Although calcium was lower in patient sera (by 8%, p<0.001), it tended to be higher (by 20%, ns) in aneurysmatic tissue. In addition, mercury concentrations in serum and aneurysmatic tissue were positively correlated (r=0.51, p<0.05). Moreover, C. pneumoniae-positive aneurysmatic tissues had lower concentrations of manganese (46%, p<0.05) and zinc (26%, ns) but a higher concentration of mercury (50%, p<0.05) than C. pneumoniae-negative aneurysmatic tissues. In conclusion, aneurysm patients showed a shift in trace element levels in serum and in the diseased part of the aorta, the pattern being partly different in C. pneumoniae-positive compared with C. pneumoniae-negative patients. The results are compatible with active infection and/or inflammation, possibly initiated by C. pneumoniae.

First known case of Bartonella quintana endocarditis in Sweden.

In this report, we present the first known case of Bartonella endocarditis in Sweden. IgG antibody titres to Bartonella spp. were elevated but blood cultures remained negative. Sequencing of a gltA fragment from DNA extracted from heart valve tissue specimens revealed sequence homology with B. quintana.

Trace element balance is changed in infected organs during acute Chlamydophila pneumoniae infection in mice.

Most infectious diseases are accompanied by changed levels of several trace elements in the blood. However, sequential changes in trace elements in tissues harbouring bacterial infections have not been studied. In the present study the respiratory pathogen Chlamydophila pneumoniae (C. pneumoniae), adapted to C57BL/6J mice, was used to study whether the balance of trace elements is changed in infected organs. Bacteria were quantitatively measured by real-time PCR in the blood, lungs, liver, aorta, and heart on days 2, 5, and 8 of the infection. Concentrations of 13 trace elements were measured in the liver, heart, and serum by inductively coupled plasma mass-spectrometry (ICP-MS). Infected mice developed expected clinical signs of disease and bacteria were found in lungs, liver, and heart on all days. The number of bacteria peaked on day 2 in the heart and on day 5 in the liver. The copper/zinc (Cu/Zn) ratio in serum increased as a response to the infection. Cu increased in the liver but did not change in the heart. Iron (Fe) in serum decreased progressively, whereas in the heart it tended to increase, and in the liver it progressively increased. C. pneumoniae may thus cause a changed trace element balance in target tissues of infection that may be pivotal for bacterial growth.

High Bartonella spp. seroprevalence in a Swedish homeless population but no evidence of trench fever.

Blood samples and epidemiological data were collected from 50 homeless patients in central Stockholm, Sweden. Sera were analysed for antibodies to B. henselae, B. quintana, B. elizabethae and B. grahamii. Whole blood was cultured and used as substrate for a newly developed quantitative real time polymerase chain reaction (QPCR) specifically targeting Bartonella spp. DNA. 61 matched blood donor sera were used as controls. Homeless patients were significantly more often seropositive to Bartonella spp. than controls (OR 7.58 (3.30-17.39), p<0.05). Reactivity to the B. elizabethae antigen was dominating, although the difference between patients and controls was most significant in seroreactivity to the B. henselae antigen. There was no evidence of an ongoing B. quintana epidemic. The absence of louse infestation could explain the lack of B. quintana bacteraemia and the failure to amplify Bartonella DNA.

Chlamydophila pneumonia: Specific mRNA in aorta ascendens in patients undergoing coronary artery by-pass grafting.

The objective of this prospective study was to investigate if Chlamydophila pneumoniae (Cp)-specific DNA and mRNA are present in tissue samples from the wall of aorta ascendens in patients undergoing by-pass surgery for coronary artery disease (CAD) that includes stable angina pectoris (SAP, 25 patients) and acute coronary syndrome (ACS, 19 patients). Viable Cp was detected in 8/44 (18%) patients using reversed transcriptase PCR (RT-PCR) against bacterial mRNA with detection of cDNA using real-time PCR against the MOMP gene. Cp DNA was detected by nested PCR in 22/44 (50%) patients and by real-time PCR in 13/44 (30%) patients. In total, 24/44 (55%) patients were positive for Cp nucleic acid in any PCR. Antibodies to Cp were detected in 13/24 (54%) Cp PCR-positive and in 15/20 (75%) Cp PCR-negative patients. Nested PCR was run on throat swabs from all patients. No significant differences were noted between SAP and ACS patients regarding PCR results or serology. It has been suggested that Cp may be a 'silent passenger' picked up by the atherosclerotic plaque. Our findings of viable and metabolically active bacteria in aortic tissue add further support to the hypothesis that Cp may have an active role in the pathogenesis of atherosclerosis.

Laboratory diagnosis of sexually transmitted infections in estonia in 2001-2002: shortcomings with impact on diagnostic quality and surveillance.

OBJECTIVES: The objectives of this study were to comprehensively characterize the range, content, and performance of sexually transmitted infection (STI) testing services in Estonia during the period 2001 to 2002 and to determine if the observed diagnostic laboratory practices and methods adhered to international evidence-based recommendations. STUDY: Survey data, focusing on organization and performance characteristics of STI diagnostics services, were assessed using questionnaires, telephone interviews, and site visits to all responding facilities providing STI diagnostics services in Estonia. Guidelines of international evidence-based recommendations for STI testing were used as references. RESULTS: There were significant shortcomings in STI testing availability and practices. Among all participating laboratories diagnosing STIs, only a minority (n = 16, 28%) offered testing for the full minimum range of relevant STIs in Estonia, i.e., Treponema pallidum, Neisseria gonorrhoeae, Chlamydia trachomatis, and Trichomonas vaginalis. In addition, because testing methods used were not properly selected, confirmation of several infections in accordance with evidence-based requirements was not possible, which has an impact both on STI diagnostic quality and surveillance.

Bartonella spp. seroprevalence in healthy Swedish blood donors.

Serum samples were collected from healthy blood donors in 5 regions in Sweden in 1999, i.e. from the local Blood Centres (collecting facilities) in Boden, Jönköping, Lund, Skövde, and Uppsala. In total, 498 serum samples (63% males, 37% females) were received and tested by immunofluorescence assay for antibodies against B. elizabethae, B. grahamii, B. henselae (Houston-1), B. henselae (Marseille), B. quintana, and B. vinsonii subsp. vinsonii. An overall Bartonella spp. seroprevalence of 16.1% was found, with a predominance of immunoreactivity to B. elizabethae, at 14.1%; B. grahamii, 2.6%; B. henselae (Houston-1), 1.2%; B. henselae (Marseille), 1.8%; B. quintana, 0.2%; and B. vinsonii subsp. vinsonii, 0.0%. Univariate and multivariate analyses of epidemiological and demographical information revealed an increased rate of B. elizabethae seropositivity in blood donors working outdoors, being out in the wild a minimum of once a week, hunting moose, having cat contact, and travelling to Eastern Europe. Living in the southern region of Sweden (Lund area) was associated with decreased seropositivity to B. elizabethae.

Bartonella spp. antibodies in forensic samples from Swedish heroin addicts.

A high frequency of Bartonella elizabethae seropositivity (39%) was recorded among intravenous heroin addicts in Stockholm, Sweden, who died from a lethal injection. Some of the B. elizabethae-seropositive individuals also had antibodies to B. henselae Houston-1, B. grahamii, and B. quintana, but none had antibodies to B. henselae Marseille or B. vinsonii subsp. vinsonii. Hepatitis was a frequent finding but no case had peliosis hepatitis. There was no case of endocarditis, but in three persons active subacute-to-chronic myocarditis was found; two of these cases were Bartonella-positive and HIV-negative.

A study on forensic samples of Bartonella spp antibodies in Swedish intravenous heroin addicts.

Infection with Bartonella, an emerging bacterial pathogen which often affects immunodeficient patients, has been reported in Sweden over the past few years, with a high seroprevalence of B. elizabethae. A high prevalence of antibodies against B. elizabethae has also been found in urban intravenous drug users in the USA. Using immunofluorescence, we retrospectively examined serum samples taken at autopsy from 59 Swedish intravenous drug addicts from the Stockholm area for evidence of antibodies against 6 pathogenic strains of Bartonella. The 59 addicts died following heroin injection during the years 1987-1992 and include 24 individuals (41%) who were additionally HIV-positive. An overall seropositivity rate for Bartonella spp. of 39% (23/59) was found with the following antigenic reactivities: B. elizabethae, 39% (23/59); B. grahamii, 3% (2/59); B. henselae (Houston-1), 14% (8/59); and B. quintana, 3% (2/59). There were no positive reactions for B. henselae (Marseille) or B. vinsonii subsp. vinsonii. The Bartonella-seropositive cases included 11/23 (48%) individuals who were HIV-positive. Subacute to chronic myocarditis was seen in 2/11 microscopically investigated Bartonella-seropositive cases that were HIV-negative and in 1/14 seronegative cases. No cases of endocarditis or other common manifestations of Bartonella infection were found. An overall Bartonella seropositivity of 21% (9/44) was observed in control forensic autopsy samples.

Interactions between Chlamydia pneumoniae and trace elements: a possible link to aortic valve sclerosis.

An association between Chlamydia pneumoniae and atherosclerotic cardiovascular diseases has been suggested. However, other factors may interact in the pathogenesis of valve sclerosis. Therefore, trace elements important for C. pneumoniae growth and host defense and markers of C. pneumoniae infection were studied in sclerotic valves and serum. Forty-six patients undergoing surgical valve replacement due to advanced aortic sclerosis were prospectively studied. Valves from 15 forensic cases with no heart valve disease and plasma from 46 healthy volunteers served as controls. C. pneumoniae was detected in 16/46 (34.8 %) sclerotic valves and in 0/15 forensic controls. IgG and IgA antibodies to C. pneumoniae were present in 54.3% and 26.1 % patients, respectively. In the patients' valves, iron, magnesium, and zinc each correlated to calcium, a marker of the histopathological severity of disease. Patients showed 10- to 70-fold increases of these trace elements in valves and an increased copper/zinc ratio in serum. In a majority of aortic sclerosis patients, one of several markers of C. pneumoniae infection were detected and all patients had a disturbed trace element balance in valves and serum suggestive of active immune process and infection. The pattern of trace element changes was essentially similar regardless of positive makers of C. pneumoniae, suggesting a similar etiopathogenesis in both subgroups. The 20-fold increase in iron, essential for C. pneumoniae growth, in sclerotic valves suggests a new possible link to this infection in aortic sclerosis.

Prevalence of antibodies to Bartonella henselae, B. elizabethae and B. quintana in Swedish domestic cats.

Sera from 292 cats were analyzed by means of indirect immunofluorescence for antibodies to Bartonella henselae, B. quintana and B. elizabethae. The sera were sent to the Swedish National Institute of Veterinary Medicine for health monitoring and were tested retrospectively for antibodies to Bartonella. The most prevalent antibodies (25%) reacted with the B. elizabethae antigen. Cats with such antibodies were older than those without antibodies. The prevalence of antibodies to B. elizabethae was higher in the south of Sweden than in the north, with the highest prevalence (46%) being found in cats living in the Stockholm region. There was no difference in sex distribution. A low prevalence (1%) of antibodies to B. henselae was found and no sera reacted with B. quintana. The high prevalence of antibodies to B. elizabethae is consistent with previous findings in Swedish patients. The small number of cats with B. henselae antibodies observed in this study could be due to the cold climate and the low occurrence of cat fleas in Sweden.

Chlamydia pneumoniae in patients undergoing surgery for thoracic aortic disease.

OBJECTIVE: To investigate if Chlamydia pneumoniae is present in the wall of the thoracic aorta in patients operated on for aneurysm or aortic dissection. DESIGN: Consecutive patients undergoing surgery for thoracic aortic aneurysm (TAA, 32 patients) and for aortic dissection (6 patients) were included in this prospective study. Tissue samples from the aorta were analysed for the presence of C. pneumoniae by polymerase chain reaction (PCR), histopathology, immunohistochemistry and in one aortic tissue sample C. pneumoniae was verified by electron microscopy and immunogold labelling technique. Cultured Hep 2 cells infected with C. pneumoniae were used as a positive control for electron microscopy. Sera for microimmunofluorescence were obtained in 36/38 and throat swabs for C. pneumoniae PCR in 17/38 patients. RESULTS: Chlamydia pneumoniae was detected by PCR in 4 of 32 TAA tissue samples (12%) and in 0 of 6 patients operated on for aortic dissection. Chlamydia pneumoniae inclusion bodies in one of the PCR positive tissue samples were verified by electron microscopy. IgG antibodies to C. pneumoniae were present in 17/31 (55%) and IgA in 15/31 (48%) of the TAA patients and in none of five tested patients with dissection. None of the tested throat swabs was positive. CONCLUSION: In this study we report the presence of C. pneumoniae by PCR and electron microscopy in the wall of TAA. A high prevalence of serum IgA antibodies to C. pneumoniae was found in TAA patients. In contrast no signs of C. pneumoniae were detected in patients with thoracic aortic dissection.