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Development and evaluation of a PCR-based assay for detection of Haemobartonella felis in cats and differentiation of H. felis from related bacteria by restriction fragment length polymorphism analysis.

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set of H. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA of Eperythrozoon suis, Mycoplasma genitalium, and Bartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI and MnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felis infection in cats.

Anaplasmataceae↗

Prevalence of selected infectious disease agents in cats from Arizona.

The objective of this study was to use polymerase chain reaction (PCR) assays to determine the prevalence of Ehrlichia species, Anaplasma phagocytophilum, Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' and Bartonella species from feral and relinquished cats in Phoenix and Nogales, Arizona. DNA from one or more of the organisms was amplified from 31 of 112 blood samples (27.7%). DNA consistent with Bartonella clarridgeiae 15 (13.4%), Bartonella henselae 14 (12.5%), 'Candidatus M haemominutum' 9 (8.0%), and M haemofelis 5 (4.5%) were detected. DNA of Ehrlichia species, Neorickettsia risticii, or A phagocytophilum was not amplified. Failure to amplify DNA of A phagocytophilum may relate to the absence of appropriate tick vectors. Failure to amplify Ehrlichia species DNA suggests that cats were not exposed, exposed but not infected, or infected but the DNA was not detected by the PCR assay used in this study. The Bartonella species and hemoplasma results suggest flea control should be maintained.

Anaplasma phagocytophilum↗

Rats of the genus Rattus are reservoir hosts for pathogenic Bartonella species: an Old World origin for a New World disease?

Bartonella species were isolated from the blood of 63 of 325 Rattus norvegicus and 11 of 92 Rattus rattus from 13 sites in the United States and Portugal. Infection in both Rattus species ranged from 0% (e.g., 0/87) to approximately 60% (e.g., 35/62). A 337-bp fragment of the citrate synthase (gltA) gene amplified by polymerase chain reaction was sequenced from all 74 isolates. Isolates from R. norvegicus were most similar to Bartonella elizabethae, isolated previously from a patient with endocarditis (93%-100% sequence similarity), followed by Bartonella grahamii and other Bartonella species isolated from Old World rodents (Clethrionomys species, Mus musculus, and Rattus species). These data suggest that Rattus species are a reservoir host for pathogenic Bartonella species and are consistent with a hypothesized Old World origin for Bartonella species recovered from Rattus species introduced into the Americas.

Animals↗

Aortic valve endocarditis in a dog due to Bartonella clarridgeiae.

We report the first documented case of endocarditis associated with Bartonella clarridgeiae in any species. B. clarridgeiae was identified as a possible etiological agent of human cat scratch disease. Infective vegetative valvular aortic endocarditis was diagnosed in a 2.5-year-old male neutered boxer. Historically, the dog had been diagnosed with a systolic murmur at 16 months of age and underwent balloon valvuloplasty for severe valvular aortic stenosis. Six months later, the dog was brought to a veterinary hospital with an acute third-degree atrioventricular block and was diagnosed with infective endocarditis. The dog died of cardiopulmonary arrest prior to pacemaker implantation. Necropsy confirmed severe aortic vegetative endocarditis. Blood culture grew a fastidious, gram-negative organism 8 days after being plated. Phenotypic and genotypic characterization of the isolate, including partial sequencing of the citrate synthase (gltA) and 16S rRNA genes indicated that this organism was B. clarridgeiae. DNA extraction from the deformed aortic valve and the healthy pulmonic valve revealed the presence of B. clarridgeiae DNA only from the diseased valve. No Borrelia burgdorferi or Ehrlichia sp. DNA could be identified. Using indirect immunofluorescence tests, the dog was seropositive for B. clarridgeiae and had antibodies against Ehrlichia phagocytophila but not against Ehrlichia canis, Ehrlichia ewingii, B. burgdorferi, or Coxiella burnetii.

Animals↗

Indoleamine 2,3-dioxygenase-expressing dendritic cells form suppurative granulomas following Listeria monocytogenes infection.

Control of pathogens by formation of abscesses and granulomas is a major strategy of the innate immune system, especially when effector mechanisms of adaptive immunity are insufficient. We show in human listeriosis that DCs expressing indoleamine 2,3-dioxygenase (IDO), together with macrophages, are major cellular components of suppurative granulomas in vivo. Induction of IDO by DCs is a cell-autonomous response to Listeria monocytogenes infection and was also observed in other granulomatous infections with intracellular bacteria, such as Bartonella henselae. Reporting on our use of the clinically applied anti-TNF-alpha antibody infliximab, we further demonstrate in vitro that IDO induction is TNF-alpha dependent. Repression of IDO therefore might result in exacerbation of granulomatous diseases observed during anti-TNF-alpha therapy. These findings place IDO(+) DCs not only at the intersection of innate and adaptive immunity but also at the forefront of bacterial containment in granulomatous infections.

Antigens, CD↗

Cloning, characterization, and expression of Bartonella henselae p26.

In order to identify immunoreactive Bartonella henselae proteins, B. henselae antiserum from an experimentally infected cat was used to screen a B. henselae genomic DNA expression library. One immunoreactive phage clone contained a gene (p26) with significant nucleotide identity with orthologs in brucellae, bartonellae, and several plant-associated bacteria. p26 gene sequences from four B. henselae strains, one B. koehlerae strain, and one B. clarridgeiae strain were cloned. Comparative nucleotide sequence analysis showed that p26 is a potential marker for molecular diagnosis of infection, as well as for identification to species level and genotyping of Bartonella sp. isolates. Alignment of the predicted amino acid sequences illustrated conserved putative protein features including a hydrophobic transmembrane region, a peptide cleavage site, and four dominant antigenic sites. Expression of p26 in Escherichia coli produced two proteins (26 and 27.5 kDa), both of which were reactive with feline anti-B. henselae antisera. Furthermore, murine hyperimmune serum raised against either recombinant protein reacted with both proteins. No reactivity to either recombinant protein was detected in nonimmune serum, and reactivity persisted as long as 20 weeks for one cat. The p26 protein product is an immunodominant antigen that is expressed during infection in cats as a preprotein and is subsequently cleaved to form mature P26.

Amino Acid Sequence↗

[Ecological aspects of human pathogens Ehrlichia and Bartonella].

The review presents data on the natural circulation of Bartonellas and Erlichias with the participation of lice, fleas, mosquitoes, Ixodes ticks, as well as rodents, large wild mammals, and domestic animals. It outlines the human infection routes that are mainly associated with the attack of infected carriers and with the contacts with different warm-blooded animals. The risk of bartonellosis spread is evidenced by the data showing that 12.3% of clothes lice collected from homeless individuals in Moscow turned out to be infected. In a focus of erlichiasis, the infection rates of Ambliomma americanum and Ixodes scapularis were 4.9 and as high as 20%, respectively; 100% of white-tailed deers (Odocoileus virginianus) were seropositive. However, many issues associated with the preservation of these natural vectors, with the route of their transmission to human beings and animals are to be studied.

Animals↗

The Etest for antimicrobial susceptibility testing of Bartonella henselae.

The in-vitro susceptibility of 10 isolates of Bartonella henselae was assessed using the Etest. The organisms, one reference human strain and nine feline isolates, were grown on chocolate agar and the Etests read at days 5, 8 and 11. Six antibiotics, erythromycin, azithromycin, doxycycline, ciprofloxacin, rifampicin and vancomycin were evaluated. The results correlated well with published results using agar dilution. The results confirmed the high in-vitro susceptibility of B. henselae to erythromycin, azithromycin, doxycycline and rifampicin and to a lesser extent ciprofloxacin. The majority of isolates were resistant to vancomycin. Although in-vitro results of B. henselae susceptibility testing may not necessarily correlate with clinical response, the Etest may be a simpler way for laboratories to monitor for the development of resistance particularly in the setting of relapsing infection.

Animals↗

Interaction of pathogens with the endothelium.

The endothelium lines the inner surface of the vessel wall establishing a multifunctional, semi-permeable cellular barrier at blood-tissue interface. The large total surface of the endothelium is exposed to pathogens, pathogen-derived products as well as to agents of the activated host defense during an inflammatory reaction. The endothelium is not only specifically targeted by important infective agents like Rickettsiae (1) or Bartonella (2), it is involved in virtually most, if not all, acute inflammatory responses. Pathogens attack the endothelium by a wide variety of strategies, as different as activation of preformed receptor-mediated pathways in the endothelium, release of pore-forming exotoxins or intracellular replication and chronic parasitism. These pathophysiological forces affect the endothelial phenotype, resulting in endothelial barrier dysfunction, increased leukocyte-endothelial interaction, mediator release, and procoagulant activity. Moreover, endothelial responses retroact on the invading pathogen as well as on the host defense resulting in a complex and dynamic interaction. Endothelial activation contributes considerably to inflammation and resulting clinical characteristics. In this context the endothelium is not just a passive victim, it rather aggravates the ongoing struggle with the pathogen. In this review we focus on some important mechanisms of the cellular microbiology of endothelial infection by bacteria and viruses.

Animals↗

Bartonella interactions with endothelial cells and erythrocytes.

Bartonella species are emerging human pathogens responsible for a wide range of clinical manifestations, including Carrion's disease, trench fever, cat-scratch disease, bacillary angiomatosis-peliosis, endocarditis and bacteraemia. During infection of their human or animal reservoir host(s), these arthropod-borne pathogens typically invade and persistently colonize mature erythrocytes. However, in both reservoir and incidentally infected hosts, endothelial cells are target cells for bartonellae. Endothelial interactions involve a unique mode of cellular invasion, the activation of a proinflammatory phenotype and the formation of vasoproliferative tumours. Based on the establishment of bacterial genetics and appropriate infection models, recent work has begun to elucidate the cell and molecular biology of these unusual pathogen-host cell interactions.

Animals↗

Cosegregation of a novel Bartonella species with Borrelia burgdorferi and Babesia microti in Peromyscus leucopus.

During surveillance for various tickborne pathogens in the upper Midwest during the summer and early fall of 1995, a Bartonella-like agent was detected in the blood of mice that were concurrently infected with Borrelia burgdorferi or Babesia microti (or both). The organism was isolated in pure culture after inoculation of blood from wild-caught mice into C.B-17 scid/scid mice. Phylogenetic analysis of the 16S rRNA and the citrate synthase genes showed that the novel Bartonella species and a Bartonella isolate from a mouse captured on Martha's Vineyard, Massachusetts, were closely related to each other and secondarily related to Bartonella grahamii and Bartonella vinsonii. Further analysis of Peromyscus leucopus blood and tissue samples demonstrated that the novel Bartonella species was exclusively found in conjunction with B. burgdorferi and B. microti. Patent coinfection with these agents may be relatively frequent in naturally infected mice.

Animals↗

Culture-negative Bartonella endocarditis in a patient with renal failure: the value of molecular methods in diagnosis.

Members of the genus Bartonella are increasingly recognised as a cause of culture-negative endocarditis, particularly in those patients with underlying risk factors (e.g., homelessness and alcoholism (B. quintana) or valvulopathy and cat ownership (B. henselae). The aortic and mitral-valves are most commonly involved. Here, a case is reported of culture-negative right-sided endocarditis, without any of the above risk factors, due to Bartonella sp. in a 69-year-old man who presented with acute renal failure. The diagnosis was made using a broad-range 16S rRNA polymerase chain reaction (PCR) technique and direct automated sequencing on a peripheral blood sample, which was subsequently confirmed serologically. A review of the literature on Bartonella endocarditis is also presented. Molecular laboratory methods using peripheral blood or blood cultures may be very useful in the diagnosis of causal agents in culture-negative endocarditis and add further support to the recently inclusion of molecular (PCR) diagnosis, as a major Duke's criterion, for the diagnosis of infective endocarditis.

Acute Kidney Injury↗

A nested-PCR with an Internal Amplification Control for the detection and differentiation of Bartonella henselae and B. clarridgeiae: an examination of cats in Trinidad.

BACKGROUND: Bartonella species are bacterial blood parasites of animals capable of causing disease in both animals and man. Cat-Scratch Disease (CSD) in humans is caused mainly by Bartonella henselae and is acquired from the cat, which serves as a reservoir for the bacteria. A second species, B. clarridgeiae is also implicated in the disease. Diagnosis of Bartonellosis by culture requires a week or more of incubation on enriched media containing blood, and recovery is often complicated by faster growing contaminating bacteria and fungi. PCR has been explored as an alternative to culture for both the detection and species identification of Bartonella, however sensitivity problems have been reported and false negative reactions due to blood inhibitors have not generally been addressed in test design. METHODS: A novel, nested-PCR was designed for the detection of Bartonella henselae and B. clarridgeiae based on the strategy of targeting species-specific size differences in the 16S-23S rDNA intergenic regions. An Internal Amplification Control was used for detecting PCR inhibition. The nested-PCR was utilized in a study on 103 blood samples from pet and stray cats in Trinidad. RESULTS: None of the samples were positive by primary PCR, but the Nested-PCR detected Bartonella in 32/103 (31%) cats where 16 were infected with only B. henselae, 13 with only B. clarridgeiae and 3 with both species. Of 22 stray cats housed at an animal shelter, 13 (59%) were positive for either or both species, supporting the reported increased incidence of Bartonella among feral cats. CONCLUSION: The usefulness of a single PCR for the detection of Bartonella henselae and B. clarridgeiae in the blood of cats is questionable. A nested-PCR offers increased sensitivity over a primary PCR and should be evaluated with currently used methods for the routine detection and speciation of Bartonella henselae and B. clarridgeiae. In Trinidad, B. henselae and B. clarridgeiae are the predominant species in cats and infection appears highest with stray cats, however B. clarridgeiae may be present at levels similar to that of B. henselae in the pet population.

Animals↗

Bacillary angiomatosis.

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AIDS-Related Opportunistic Infections↗

Cat scratch disease and acute rejection after pediatric renal transplantation.

Cat scratch disease (CSD) can lead to unexplained fever, generalized lymphadenopathy and organomegaly in immunocompetent individuals. CSD has rarely been reported in immunocompromised transplant recipients, where its clinical features would mimic the more common post-transplant lymphoproliferative disease (PTLD). We report three cases of CSD seen recently in children who had received prior kidney transplants. The three children were between 7 and 9 yr old, and had received kidney transplants 2-4 yr prior, with stable renal function. In each case, there was unexplained fever with either lymphadenopathy or organomegaly. The diagnosis of CSD was suggested by a history of new cats being introduced into each household and confirmed in all cases by the serological presence of a significant titer (> 1 : 64) of IgM antibodies to Bartonella henselae. Tests for other bacterial infections, cytomegalovirus and Epstein-Barr virus infections were negative. All the patients showed a clinical improvement with anti-microbial therapy. In patients A and B, the CSD was associated with an acute rejection episode shortly after diagnosis. The rejection episodes were reversed by intravenous steroid pulse therapy. Only four cases of CSD have been previously reported following solid organ transplantation. Acute rejection following CSD has not been previously reported. CSD should be included in the differential diagnosis of fever in the post-transplant setting, especially where PTLD is suspected.

Animals↗

[Rochalimaea henselae, Afipia felis and cat-scratch disease].

Several years ago, Rochalimaea henselae has emerged as an agent of bacillary angiomatosis, bacillary peliosis and recurrent septicaemia that generally occur in patients infected with human immunodeficiency virus. An aetiologic role in cat scratch disease is also suspected widely on the basis of a serologic survey. Its slow growth and its culture requirement explain that this pathogen, a gram-negative bacterium, could not be isolated until 1990. Moreover, blood and tissue samples request lysis and crushing for recovering by culture. The clinical, histological, microbiological and pathogenic aspects of these infections are described and discussed.

AIDS-Related Opportunistic Infections↗

Bacillary angiomatosis.

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AIDS-Related Opportunistic Infections↗