Ahnak-knockout mice show susceptibility to Bartonella henselae infection because of CD4+ T cell inactivation and decreased cytokine secretion.

The present study evaluated the role of AHNAK in Bartonella henselae infection. Mice were intraperitoneally inoculated with 2 × 108 colony-forming units of B. henselae Houston-1 on day 0 and subsequently on day 10. Blood and tissue samples of the mice were collected 8 days after the final B. henselae injection. B. henselae infection in the liver of Ahnak-knockout and wild-type mice was confirmed by performing polymerase chain reaction, with Bartonella adhesion A as a marker. The proportion of B. henselaeinfected cells increased in the liver of the Ahnak-knockout mice. Granulomatous lesions, inflammatory cytokine levels, and liver enzyme levels were also higher in the liver of the Ahnak-knockout mice than in the liver of the wild-type mice, indicating that Ahnak deletion accelerated B. henselae infection. The proportion of CD4＋interferon-γ (IFN-γ)＋ and CD4＋interleukin (IL)-4＋ cells was significantly lower in the B. henselae-infected Ahnak-knockout mice than in the B. henselae-infected wild-type mice. In vitro stimulation with B. henselae significantly increased IFN-γ and IL-4 secretion in the splenocytes obtained from the B. henselae-infected wild-type mice, but did not increase IFN-γ and IL-4 secretion in the splenocytes obtained from the B. henselae-infected Ahnak-KO mice. In contrast, IL-1α, IL-1ß, IL-6, IL-10, RANTES, and tumor necrosis factor-α secretion was significantly elevated in the splenocytes obtained from both B. henselae-infected wild-type and Ahnak-knockout mice. These results indicate that Ahnak deletion promotes B. henselae infection. Impaired IFN-γ and IL-4 secretion in the Ahnak-knockout mice suggests the impairment of Th1 and Th2 immunity in these mice. [BMB Reports 2019; 52(4): 289-294].

Identification of a novel Afipia species isolated from an Indian flying fox.

An old world fruit bat Pteropus giganteus, held in captivity and suffering from necrosis of its wing digits, failed to respond to antibiotic therapy and succumbed to the infection. Samples submitted to the National Centre for Foreign Animal Disease were tested for viral infection. Vero E6 cells exhibited minor but unique cytopathic effects on second blind passage, and full CPE by passage four. Utilizing an unbiased random amplification technique from cell culture supernatant, we identified a bacterium belonging to the Bradyrhizobiaceae. Purification of cell culture supernatant on TY media revealed a slow growing bacterial isolate. In this study using electron microscopy, 16S rRNA gene analysis and whole genome sequencing, we identify a novel bacterial species associated with the site of infection belonging to the genus Afipia. This genus of bacteria is very diverse, with only a limited number of species characterized. Afipia felis, previously described as the etiological agent to cause cat scratch disease, and Afipia septicemium, most recently shown to cause disease in humans, highlight the potential for members of this genus to form a branch of opportunistic pathogens within the Bradyrhizobiaceae. Increased utilization of next generation sequencing and genomics will aid in classifying additional members of this intriguing bacterial genera.

Impairment of circulating endothelial progenitors in Down syndrome.

BACKGROUND: Pathological angiogenesis represents a critical issue in the progression of many diseases. Down syndrome is postulated to be a systemic anti-angiogenesis disease model, possibly due to increased expression of anti-angiogenic regulators on chromosome 21. The aim of our study was to elucidate some features of circulating endothelial progenitor cells in the context of this syndrome. METHODS: Circulating endothelial progenitors of Down syndrome affected individuals were isolated, in vitro cultured and analyzed by confocal and transmission electron microscopy. ELISA was performed to measure SDF-1α plasma levels in Down syndrome and euploid individuals. Moreover, qRT-PCR was used to quantify expression levels of CXCL12 gene and of its receptor in progenitor cells. The functional impairment of Down progenitors was evaluated through their susceptibility to hydroperoxide-induced oxidative stress with BODIPY assay and the major vulnerability to the infection with human pathogens. The differential expression of crucial genes in Down progenitor cells was evaluated by microarray analysis. RESULTS: We detected a marked decrease of progenitors' number in young Down individuals compared to euploid, cell size increase and some major detrimental morphological changes. Moreover, Down syndrome patients also exhibited decreased SDF-1α plasma levels and their progenitors had a reduced expression of SDF-1α encoding gene and of its membrane receptor. We further demonstrated that their progenitor cells are more susceptible to hydroperoxide-induced oxidative stress and infection with Bartonella henselae. Further, we observed that most of the differentially expressed genes belong to angiogenesis, immune response and inflammation pathways, and that infected progenitors with trisomy 21 have a more pronounced perturbation of immune response genes than infected euploid cells. CONCLUSIONS: Our data provide evidences for a reduced number and altered morphology of endothelial progenitor cells in Down syndrome, also showing the higher susceptibility to oxidative stress and to pathogen infection compared to euploid cells, thereby confirming the angiogenesis and immune response deficit observed in Down syndrome individuals.

Prolonged Bartonella henselae bacteremia caused by reinfection in cats.

We analyzed the genetic relatedness of blood culture isolates of Bartonella henselae from 2 cats of patients with cat-scratch disease at admission and after 12 months. Isolates from each cat at different times were clonally unrelated, which suggested reinfection by a second strain.

Differential detection of Bartonella species and strains in cat scratch disease diagnostics by polymerase chain reaction amplification of 16S ribosomal RNA gene.

Cat scratch disease (CSD) has been difficult to diagnose in animals because of the protracted clinical course of infection and the quiescent phases when the microbial culprit lies dormant. The causative agent in CSD appears to be multiple species and strains of Bartonella. Using polymerase chain reaction (PCR) techniques for amplification of highly variable regions of the 16S ribosomal RNA (rRNA) gene sequence, a very sensitive species- and strain-specific assay for CSD-causing Bartonella species was developed. PCR primers were designed to specifically amplify the 16S rRNA gene of Bartonella species but not of other microbial pathogens. This initial PCR was multiplexed with a universal primer set, based on conserved sequence regions in the 16S rRNA gene, that provides a 162-bp fragment in all species tested. Subsequently, 3 distinct nested PCR primer sets enabled the individual amplification and specific detection of Bartonella henselae type 1, B. henselae type II, and B. clarridgeae. Thus, this 2-step PCR procedure enabled the sensitive detection and identification of these species and the B. henselae genotype by exploiting minor sequences differences. Verification of these results were demonstrated with both sequencing and ligase chain reaction techniques. The diagnostic usefulness of this CSD test has been demonstrated by the analysis of specimens from control and infected cats. The diagnosis was confirmed and the specific B. henselae strain was correctly identified in peripheral blood specimens obtained from control and strain-specific CSD-infected cats. Such an accurate and sensitive diagnostic tool for the detection and identification of CSD causative agents should be a useful for the medical, veterinary, and scientific communities.

Evaluation and use of a nested polymerase chain reaction assay in cats experimentally infected with Bartonella henselae genotype I and Bartonella henselae genotype II.

Cats have been shown to be infected with Bartonella henselae genotype I, B. henselae genotype II, and B. clarridgeiae. Feline bartonellosis infections and the strains involved in these infections are important in both veterinary and human medicine. Nucleic acid amplification methods such as polymerase chain reaction (PCR) are being used in both research and diagnostics as tools for understanding many infectious diseases. Bartonella bacteremia in cats is detected by blood culture; however, because of the limitations of culture (delayed turnaround time and sensitivity limits), PCR may be a more efficient method for identifying infected cats. Three distinct PCR assays that could differentiate among B. henselae genotype I, B. henselae genotype II. and B. clarridgeiae were developed and used to detect as few as 3.2 organisms. Fourteen cats experimentally infected with B. henselae genotype I and B. henselae genotype II were followed by bacterial culture and PCR through the course of infection, including periods of primary and relapsing bacteremia. The PCR assay was positive in 11 of the 14 cats for periods of 1-9 weeks after culture became negative. Of the 223 blood specimens that were culture negative, the PCR assay was positive in 38 (17%) of the specimens. Two of the 14 cats developed relapsing bacteremia. The 2 B. henselae genotypes were amplified in the cats and the bacteremic phase of these infections as determined by PCR lasted for a longer period than previously determined by culture. Using laboratory assays such as PCR to understand the strains involved in feline bartonellosis and the course of the infection is important in the understanding of these zoonotic agents.

[Cat scratch disease. Clinical study of 26 patients. Role and importance of PCR]. / Maladie des griffes du chat. Etude clinique de 26 patients. Intérêt et place de la PCR.

PURPOSE: Cat scratch disease is a mild pathology but diagnosis often remain difficult. METHODS: A retrospective study has been conducted by the department of infectious diseases at the University hospital in Angers. Between January 1994 and October 1998, 26 observations were recorded providing the presence of three criteria out of four among the following: contact with a cat, clinical presentation and its favorable course, absence of any other cause noticed, and the presence of either a positive serology or a positive PCR, or the examination of a suggestive pathology. RESULTS: Fourteen men and 12 women were concerned. From a clinical point of view, the inoculation lesion was observed six times, all patients showed at least one adenopathy during their illness, 12 patients showed only an adenopathy without clinical signs. A surgical biopsy was carried out on nine patients and a diagnosis established. Bartonella henselae serology was done in all patients. Six presented a significant rate of IgG antibodies as early as the first dosage. A seroconversion was observed in four cases belatedly 1 to 2 months after the beginning of the symptomatology. The method's sensitivity was approximately 38%. A PCR search was accomplished in the pus obtained from a ganglionic puncture on 12 patients. It was positive seven times, which corresponds to a sensitivity of about 58%. In associating these two diagnostic criteria a sensitivity rate of nearly 92% was reached, the diagnosis not having been confirmed only in one case. The outcome proved to be favorable in all cases, with or without an antibiotic treatment. CONCLUSION: The association of serology and PCR in the pus permits a certain diagnosis in the majority of the cases and avoids the more aggressive biopsy.

Abundant expression of chemokines in malignant and infective human lymphadenopathies.

Lymph nodes can be the primary target of infection or malignant transformation and may exhibit characteristic patterns of leukocyte infiltration analogous to those seen in inflammation of other tissues. Leukocyte migration to lymph nodes in vivo is a highly regulated, multi-step process that depends upon adhesion molecules and as yet, uncharacterized chemotactic signals. Chemokines are a key part of the orchestrated code of signals that directs leukocyte subsets to sites of inflammation or immune response. The potential role of these chemoattractants in selective trafficking of leukocyte subsets into lymph nodes was assessed by determining the expression of chemokines on a range of pathological and normal human lymph nodes and by evaluating the cellular composition of each lymph node. In situ hybridization using chemokine riboprobes and immunohistochemistry using specific antibodies were performed in order to correlate the mRNA and protein expression of the chemokines. The cellular source(s) of each chemokine was assessed by immunohistochemical staining of adjacent sections using antibodies directed against distinctive cellular markers. Substantial, but varied, expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, macrophage chemotactic protein (MCP)-1, eotaxin, and interleukin 8 (IL-8) were detected in the pathological lymph nodes by diverse cell types. Control lymph nodes showed expression only of RANTES, mainly by high endothelial venules. In all lymph nodes, except the nodes infiltrated with breast cancer, chemokine mRNA expression was highly concordant with the corresponding protein. In contrast with in vitro studies that have suggested discrete target cell specificity of chemokines, this study showed that with the possible exception of the neutrophil chemoattractant, IL-8, no chemokine appeared to be uniquely associated with the accumulation of a specific leukocyte subset. These data implicate chemokines in the recruitment of leukocytes to lymph nodes affected by diverse disease states.

Cat scratch disease: detection of Bartonella henselae DNA in archival biopsies from patients with clinically, serologically, and histologically defined disease.

Serological and epidemiological studies suggest that Bartonella henselae is the etiological agent of cat scratch disease. We designed a study to detect B. henselae in archival biopsies by polymerase chain reaction amplification of the 16S rRNA gene followed by Southern blot hybridization. Forty-two histologically defined cat scratch disease biopsies and eighteen controls were selected for blinded analysis. After testing, charts were reviewed for clinical, immunological, and microbial evidence of infection. Results were correlated with duration of illness and antimicrobial therapy. B. henselae DNA was identified in 27 of 42 (64%) histologically defined patients and 23 of 34 (68%) patients defined both clinically and histologically. There were no false positives (0 of 18). A small subset (n = 14) had cat scratch disease serological tests performed. B. henselae was identified in 8 of 10 serologically positive patients. Polymerase chain reaction detected 50% of our DNA-positive cases (most of these early in the clinical course). Southern blotting of amplicons both doubled sensitivity (detecting patients > 4 weeks into illness) and confirmed B. henselae as the causative species. Our study strongly associates B. henselae with cat scratch disease, suggesting that it may be the most likely etiological agent in the majority of patients with cat scratch disease.

[Cat scratch disease. Bartonella henselae antibodies and DNA detection in regional lymphadenopathy]. / Katzenkratzkrankheit. Bartonella-henselae-Antikörper und Nukleinsäurenachweis bei regionaler Lymphadenopathie.

HISTORY AND CLINICAL FINDINGS: Two hard, pressure-sensitive nodules developed in the lower jaw of a 22-year-old woman. After a dental cause had been excluded, she was treated for suspected tonsillitis with Ceftibuten. Erythrocyte sedimentation rate was increased to 18 mm in the first hour. There were no other significant biochemical findings and fine-needle biopsy of one of the nodules showed nonspecific inflammatory reaction. INVESTIGATIONS: Sonography revealed two lymph nodes, 7 and 22 mm in diameter. Suspected cat scratch disease was confirmed by immunofluorescence with Bartonella (Rochalimaea) henselae and quintana antigens. TREATMENT AND COURSE: After a course of Clarithromycin (250 mg twice daily) for 6 weeks the lymph nodes had shrunk and the overlying skin was thin and discoloured brown. One node was incised and drained and the material examined. Microbiology was negative, but DNA sequencing confirmed Bartonella henselae. As a consequence, Rifampicin was given for 2 months (600 mg daily). Wound healing was very slow and the scar had regressed little after 9 months.