Poxviruses and the passive quest for novel hosts.

Poxviruses are famous, or infamous, as agents of disease introduced into novel host species and between populations of the same species. This discussion concerns selected examples of poxviruses associated with vertebrate infections, i.e., the Chordopoxvirus subfamily of the family Poxviridae. Brief note is made of examples of members of the genera Leporipoxvirus and Parapoxvirus-like agents that have been recognized to have significant trans-host species impact. The remaining bulk of the discussion involves examples of members of the genus Orthopoxvirus, which are known to be (have been) involved with human disease, and their zoonotic origins.

Vaccines for emerging infections.

Emerging infectious diseases represent a grave threat to animal and human populations in terms of their impact on global health, agriculture and the economy. Vaccines developed for emerging infections in animals can protect animal health and prevent transmission of zoonotic diseases to humans. Examples in this paper illustrate how industry and public health can collaborate to develop a vaccine to prevent an emerging disease in horses (West Nile virus vaccine), how poultry vaccination can protect animals and prevent transmission to people (avian influenza vaccine), how regulatory changes can pave the way for vaccines that will control the carrier state in animals and thus prevent infection in humans (Bartonella henselae vaccine in cats) and how novel technologies could be applied to vaccinate wildlife reservoir species for rabies. Stemming from the realisation that zoonotic diseases are the predominant source of human emerging infectious diseases, it behoves academic, public health, and animal health agencies to consider creative constructive approaches to combat serious public health challenges. Vaccination of vector/reservoir species, when efficacious vaccines are available, offers significant advantages to combating zoonotic human disease.

Extensive lesions of monkeypox in a prairie dog (Cynomys sp).

Monkeypox with extensive lesions was diagnosed in a prairie dog that was involved in a recent human outbreak of monkeypox in the Midwestern United States. Gross lesions included oral ulcers, pulmonary consolidation, enlarged cervical and thoracic lymph nodes, and multifocal, small, white umbilicated plaques in the gastrointestinal wall. Microscopic lesions were extensive in the lungs and consisted of fibrinonecrotic bronchopneumonia with vasculitis and poorly defined eosinophilic intracytoplasmic inclusion bodies in cells thought to be alveolar epithelial cells, histiocytes, and fibroblasts. Multifocal necrotizing lesions, often accompanied by myxedema, were also present in most of the other examined organs. Aggregates of pox viral particles were observed within lesions by transmission electron microscopy. Monkeypox virus infection was confirmed by real-time polymerase chain reaction and virus culture at the Centers for Disease Control and Prevention. This report highlights the difficulties of rapid diagnosis of exotic or emerging diseases and further substantiates the prairie dog as an animal model of monkeypox.

In vitro susceptibilities of Bartonella and Rickettsia spp. to fluoroquinolone antibiotics as determined by immunofluorescent antibody analysis of infected Vero cell monolayers.

The in vitro susceptibilities of Bartonella and Rickettsia spp. to different concentrations of ciprofloxacin, levofloxacin, ofloxacin and sparfloxacin in Vero cell cultures, were determined by enumeration of immunofluorescent-stained bacilli. After incubation in a CO(2)-enriched atmosphere, inocula were replaced and tested with media containing 12 different concentrations of each antibiotic in replicate for each species and the monolayers were re-incubated. Growth status was determined by evaluation of immunofluorescent staining bacilli. Effective inhibitory antibiotic dilution endpoints were determined by counting Bartonella- and Rickettsia-specific fluorescent foci across a range of antibiotic dilutions with an epi-fluorescent microscope, and were compared with an antibiotic-negative control. Based upon the use of C(max):MIC and AUC:MIC data, levofloxacin exhibited activity against Bartonella elizabethae and B. quintana.

Association of Bartonella species and Coxiella burnetii infection with coronary artery disease.

Coronary artery disease is an inflammatory condition associated with several infections. We prospectively evaluated 155 consecutive patients undergoing coronary angiography for evidence of Bartonella species and Coxiella burnetii infection. All Bartonella cultures were found to be negative. Multivariable logistic regression analysis that controlled for potential confounding factors revealed no association between coronary artery disease and seropositivity to Bartonella henselae (odds ratio [OR], 0.852; 95% confidence interval [CI], 0.293-2.476), Bartonella quintana (OR, 0.425; 95% CI, 0.127-1.479), C. burnetii phase 1 (OR, undefined), and C. burnetii phase 2 (OR, 0.731; 95% CI, 0.199-2.680). The geometric mean titer (GMT) for C. burnetii phase 1 assay was slightly higher in persons with coronary artery disease than in those without such disease (P<.02). B. henselae, B. quintana, and C. burnetii seropositivity was not strongly associated with coronary artery disease. On the basis of GMTs, C. burnetii infection may have a modest association with coronary artery disease.

Serodiagnosis of Bartonella bacilliformis infection by indirect fluorescence antibody assay: test development and application to a population in an area of bartonellosis endemicity.

Bartonella bacilliformis causes bartonellosis, a potentially life-threatening emerging infectious disease seen in the Andes Mountains of South America. There are no generally accepted serologic tests to confirm the disease. We developed an indirect fluorescence antibody (IFA) test for the detection of antibodies to B. bacilliformis and then tested its performance as an aid in the diagnosis of acute bartonellosis. The IFA is 82% sensitive in detecting B. bacilliformis antibodies in acute-phase blood samples of laboratory-confirmed bartonellosis patients. When used to examine convalescent-phase sera, the IFA is positive in 93% of bartonellosis cases. The positive predictive value of the test is 89% in an area of Peru where B. bacilliformis is endemic and where the point prevalence of infection is 45%.

Bartonella henselae, B. quintana, and B. bacilliformis: historical pathogens of emerging significance.

Bartonella species were virtually unrecognized as modern pathogens of humans until the last decade. However, identification of Bartonella species as the agents of cat-scratch disease, bacillary angiomatosis, urban trench fever, and possible novel presentations of Carrion's disease has left little doubt of the emerging medical importance of this genus of organisms. The three primary human pathogenic bartonellae, Bartonella bacilliformis (Carrion's disease), B. henselae (cat-scratch disease), and B. quintana (trench fever), present noteworthy comparisons in the epidemiology, natural history, pathology, and host-microbe interaction that this review will briefly explore.

Differentiation of pathogenic Bartonella species by infrequent restriction site PCR.

Infrequent restriction site PCR (IRS-PCR) is a recently described DNA fingerprinting technique based on selective amplification of restriction endonuclease-cleaved fragments. Bartonella isolates associated with human disease and related nonhuman isolates were analyzed by IRS-PCR genomic fingerprinting. Preparation of DNA templates began with double digestion using three different restriction endonuclease combinations. Combinations included the frequently cutting endonuclease HhaI in conjunction with an infrequently cutting endonuclease, EagI, SmaI, or XbaI. Digestion was followed by ligation of oligonucleotide adapters designed with ends complementary to the restriction endonuclease sites. Amplification of fragments flanked with an EagI, SmaI, or XbaI site in combination with an HhaI site produced a series of different-sized amplicons resolvable into patterns by polyacrylamide gel electrophoresis (PAGE). The pattern complexity was varied by the addition of selective nucleotides to the 3' ends of the EagI-, SmaI-, or XbaI-specific primers. Amplicons were also generated with fluorescently labeled primers and were subsequently resolved and detected by capillary electrophoresis. Analysis by traditional slab PAGE and capillary electrophoresis provided suitable resolution of patterns produced with the enzyme combinations EagI-HhaI and SmaI-HhaI. However, the combination of XbaI-HhaI produced too many fragments for sufficient resolution by traditional PAGE, thus requiring the better resolving properties of capillary electrophoresis. Due to the flexibility in modulating the pattern complexity and electrophoresis methods, these techniques allow for a high level of experimental optimization. The results provide evidence of the discriminatory power, ease of use, and flexibility of the IRS-PCR method as it applies to the identification of human-pathogenic Bartonella species.

In vitro susceptibilities of Rickettsia and Bartonella spp. to 14-hydroxy-clarithromycin as determined by immunofluorescent antibody analysis of infected vero cell monolayers.

The in vitro susceptibilities of Rickettsia akari, Rickettsia conorii, Rickettsia prowazekii, Rickettsia rickettsii, Bartonella elizabethae, Bartonella henselae and Bartonella quintana to different concentrations of clarithromycin, 14-hydroxy-clarithromycin (the primary metabolite of clarithromycin) and tetracycline in Vero cell cultures, were determined by enumeration of immunofluorescently-stained bacilli. The extent of antibiotic-induced inhibition of foci was recorded for each dilution of antibiotic and compared with an antibiotic-negative control. Based upon MIC data, clarithromycin alone is highly active against all three Bartonella spp., R. akari and R. prowazekii, while 14-hydroxy-clarithromycin is active against R. conorii, R. prowazekii and R. rickettsii. Further testing is warranted in animal models and human clinical trials, to examine the activity of both clarithromycin and its primary metabolite and to define further the role of clarithromycin in therapy, particularly of infections caused by obligate intracellular bacteria such as Rickettsia and Bartonella spp.

An outbreak of acute bartonellosis (Oroya fever) in the Urubamba region of Peru, 1998.

During May 1998, we conducted a case-control study of 357 participants from 60 households during an outbreak of acute bartonellosis in the Urubamba Valley, Peru, a region not previously considered endemic for this disease. Blood and insect specimens were collected and environmental assessments were done. Case-patients (n = 22) were defined by fever, anemia, and intra-erythrocytic coccobacilli seen in thin smears. Most case-patients were children (median age = 6.5 years). Case-patients more frequently reported sand fly bites than individuals of neighboring households (odds ratio [OR] = 5.8, 95% confidence interval [CI] = 1.2-39.2), or members from randomly selected households > or = 5 km away (OR = 8.5, 95% CI = 1.7-57.9). Bartonella bacilliformis isolated from blood was confirmed by nucleotide sequencing (citrate synthase [g/tA], 338 basepairs). Using bacterial isolation (n = 141) as the standard, sensitivity, specificity, and positive predictive value of thin smears were 36%, 96%, and 44%, respectively. Patients with clinical syndromes compatible with bartonellosis should be treated with appropriate antibiotics regardless of thin-smear results.

Characterization of Bartonella henselae-specific immunity in BALB/c mice.

BALB/c mice were inoculated with Bartonella henselae by both systemic and mucosal routes. Culture analysis of tissues from mice infected intraperitoneally with a high dose of B. henselae yielded positive results 24 hr after infection. However, culture analysis of blood taken between 6 hr and 7 days after infection from groups receiving live B. henselae were negative. Following intraperitoneal infection, B. henselae was detected by polymerase chain reaction in liver and mesenteric lymph nodes by 6 hr and up to 7 days after infection in liver, kidney and spleen tissue. Enzyme-linked immunosorbent assay (ELISA) of serum samples collected as early as 13 days after infection indicated humoral immune responses to B. henselae. Specific humoral responses remained through week 6. Analysis of faecal samples revealed induction of B. henselae-specific immunoglobulin A by day 28 after infection. In addition, B. henselae-specific cellular responses were indicated by a positive delayed-type hypersensitivity and a T helper 1 (Th1) (CD4+ T cell)-type cytokine response following in vitro stimulation of splenocytes. The significance and implications of these data in relation to B. henselae infections are discussed.

Evaluation of intraspecies genetic variation within the 60 kDa heat-shock protein gene (groEL) of Bartonella species.

A phylogenetic investigation was done on the members of the genus Bartonella, based on the DNA sequence analysis of the groEL gene, which encodes the 60 kDa heat-shock protein GroEL. Nucleotide sequence data were determined for a near full-length fragment (1368 bp) of the groEL gene of the established Bartonella species and used to infer intraspecies phylogenetic relationships. Phylogenetic trees were inferred from multiple sequence alignments by using both distance and parsimony methods, which demonstrated an architecture composed of six well-supported lineages. The results are consistent with relationships deduced from recent sequence analysis studies based upon citrate synthase (gItA) and previously observed genotypic and phenotypic characteristics; however, they showed greater statistical support at the intragenus level. This suggests that groEL may be a more robust tool for phylogenetic analysis of Bartonella lineages.

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.

Experimental infection of cotton rats with three naturally occurring Bartonella species.

The kinetics of infection and humoral immune response of laboratory-bred cotton rats (Sigmodon hispidus) challenged with three Bartonella spp. recovered from the blood of naturally infected cotton rats captured in Georgia (USA) are described. Bartonella spp. infection, as determined by bacteremia, occurred in all 18 cotton rats inoculated with live Bartonella of each species at either a low dose, 10(3) colony-forming units (CFU's), or high dose, 10(7) CFU. Cotton rats inoculated with lower doses of Bartonella spp. developed higher bacteremia that persisted for longer periods than in those inoculated with high doses. Peak bacteremia varied among Bartonella spp, ranging from 10(4) to 10(6) CFUs per 1.0 ml of blood. Antibody measured by immunofluorescence assays using species-specific antigens indicated more rapidly rising and higher antibody titers in cotton rats challenged with high doses vs. low doses and with inactivated bacteria vs. live bacteria. Each group of rats produced high IgG titers to the homologous challenge antigen; low or unmeasurable cross-reactivity was detected to heterologous Bartonella antigens. Exposure of cotton rats to a specific Bartonella sp. resulted in protection, as measured by detectable bacteremia, in eight of nine animals challenged with the same Bartonella sp. used initially; no evidence of resistance to secondary challenge with different Bartonella spp. was obtained. Cross-protection between Bartonella spp., isolated from the same rodent species, may not occur.

Evaluation of human seroreactivity to Bartonella species in Sweden.

Among the species that compose the expanding genus Bartonella, thus far only B. henselae and B. quintana have reportedly been isolated from humans in Europe. To evaluate the prevalence of Bartonella infection in Sweden, we conducted a retrospective serological examination of 126 human serum samples. These samples were analyzed for antibodies to B. henselae, B. quintana, and B. elizabethae. Serum samples from 100 blood donors, who spanned the ages of 20 to 60 and had no apparent clinical signs of illness, were also studied as a control group. An immunoglobulin G indirect fluorescence antibody assay revealed 4 and 8.3% Bartonella positivity rates for the blood donor and patient group, respectively, when a cutoff titer of >/=64 was chosen. Among the blood donors, four were seropositive to B. elizabethae; one of these also had concordant positive titer to B. henselae. In the patient group, 14 serum samples were positive against Bartonella spp. These serum specimens represented nine patients. In three of these seropositive patients, paired serum samples displayed a fourfold increase in antibody titer to at least one of the three antigens. These three patients are discussed. In this report we also present a case study of a 60-year-old Swedish male with fatal myocarditis. Postmortem serological analysis revealed a high titer against B. elizabethae. PCR and nucleotide sequencing of the myocardial tissue from this patient, and of liver tissue from one of the other three patients, showed sequences similar to B. quintana. The age, geographical origin, animal contacts, and serological response pattern to the different Bartonella antigens differed among the four patients. This study substantiates the presence of Bartonella spp. in Sweden, documents the seroreactivity to three Bartonella antigens in Swedish patients, and reports the first two cases of B. quintana-like infections in Sweden.

Prevalence of Bartonella henselae and Bartonella clarridgeiae in an urban Indonesian cat population.

We studied evidence of Bartonella henselae and Bartonella clarridgeiae infection in 54 cats living in Jakarta, Indonesia. By using an indirect immunofluorescence assay, we found immunoglobulin G antibody to B. henselae in 40 of 74 cats (54%). The blood of 14 feral cats was cultured on rabbit blood agar plates for 28 days. Bartonella-like colonies were identified as B. henselae or B. clarridgeiae by using restriction fragment length polymorphism analysis and direct sequencing of the PCR amplicons. Of the cats sampled in the study, 6 of 14 (43%; all feral) were culture positive for B. henselae; 3 of 14 (21%; 2 feral and 1 pet) culture positive for B. clarridgeiae. This is the first report that documents B. henselae and B. clarridgeiae infections in Indonesian cats.

Characterization of human immunoglobulin (Ig) isotype and IgG subclass response to Bartonella henselae infection.

Serologic parameters of cat scratch disease (CSD) were evaluated by Western blot analysis. Sera from patients with serologically confirmed CSD antigen were screened for immunoglobulin (Ig) isotype-specific as well as IgG subclass-specific reactivity against Bartonella henselae whole-cell antigen. Bartonella-negative control sera were used to determine baseline antibody activity. Heterogeneous B. henselae-specific IgG reactivity with numerous protein bands, ranging from >150 to <17 kDa, was observed. Though individual banding patterns were variable, one approximately 83-kDa B. henselae protein (Bh83) was immunoreactive with all CSD sera tested, suggesting it is a conserved antigen during infection. Bh83 was not recognized by reference human antisera against Rickettsia rickettsii, Chlamydia group positive, Treponema pallidum, Orientia tsutsugamushi, Fransciscella tularensis, Ehrlichia chaffeensis, Mycoplasma pneumoniae, and Escherichia coli, although other cross-reactive proteins were evident. Significantly, CSD sera failed to recognize the 83-kDa protein when tested against Bartonella quintana antigen, though sera from B. quintana-infected patients did react to Bh83. This cross-reactivity suggests epitope conservation during infection with B. henselae or B. quintana. Western blot analysis further revealed similar banding patterns when B. henselae was reacted against the Ig isotypes IgG and IgG1 and both secretory and alpha chains of IgA. Neither IgM nor IgE reacted significantly to Bartonella antigen by our Western blot analysis. Dissection of the antibody response at the IgG subclass level indicated that prominent antigen recognition was limited to IgG1. These observations provide insight into induced immunity during CSD and provide evidence for conserved epitope expression during infection with B. henselae or B. quintana.

Prospective randomized double blind placebo-controlled evaluation of azithromycin for treatment of cat-scratch disease.

OBJECTIVE: To determine the efficacy of azithromycin in the treatment of patients with typical cat-scratch disease. DESIGN: Prospective, randomized, double blind, placebo-controlled clinical trial. SETTING: Large military medical center and its referring clinics. PATIENTS: Active duty military members and their dependents with laboratory-confirmed, clinically typical cat-scratch disease. INTERVENTION: Study participants assigned by randomization to treatment with oral azithromycin or placebo for 5 days. OUTCOME MEASURES: Lymph node volume was calculated using three dimensional ultrasonography at entry and at weekly intervals. The ultrasonographer was blinded to the treatment groups. Endpoint evaluations were predetermined as time in days to 80% resolution of the initial total lymph node volume. RESULTS: Demographic and clinical data showed that the azithromycin and placebo treatment groups were comparable at entry although the placebo group tended to be older. Eighty percent decrease of initial lymph node volume was documented in 7 of 14 azithromycin-treated patients compared with 1 of 15 placebo-treated controls during the first 30 days of observation (P = 0.026). After 30 days there was no significant difference in rate or degree of resolution between the two groups. CONCLUSIONS: Treatment of patients with typical cat-scratch disease with oral azithromycin for five days affords significant clinical benefit as measured by total decrease in lymph node volume within the first month of treatment.