Sebastian E. Carrasco

Bartonella endocarditis: A pathology shared by animal reservoirs and patients

Bartonellae were first recognized to cause endocarditis in humans in 1993 when cases caused by Bartonella quintana, B. elizabethae, and B. henselae were reported. Since the first isolation of Bartonella vinsonii subspecies berkhoffii from a dog with endocarditis, this organism has emerged as an important pathogen in dogs and an emerging pathogen in people. Subsequently, four types of B. vinsonii subsp. berkhoffii have been described, all of which have been associated with endocarditis in dogs. A limited number of dog endocarditis cases have also been associated with B. clarridgeiae, B. washoensis, B. quintana, and B. rochalimae. The second canine B. clarridgeiae endocarditis case is presented. The clinical and pathological characteristics of Bartonella endocarditis in dogs are similar to disease observed in humans, more often affecting the aortic valve, presenting with highly vegetative lesions with accompanying calcification, and in most instances high antibody titers. Pathological features in dogs include a combination of fibrosis, mineralization, endothelial proliferation, and neovascularization with variable inflammation. Endocarditis has also been described in animal species, which are the natural reservoir of specific Bartonella species, once thought to be solely healthy carriers of these pathogens. A few Bartonella endocarditis cases, including B. henselae, have been reported in cats in the USA and Australia. The second case of B. henselae type Houston I identified in the USA is presented. Furthermore, two cases of B. bovis endocarditis were recently described in adult cows from France. Finally, on‐going investigation of valvular endocarditis in free‐ranging Alaskan sea otters suggests the involvement of Bartonella species.

Manganese and zinc regulate virulence determinants in borrelia burgdorferi

ABSTRACT Borrelia burgdorferi, the causative agent of Lyme disease, must adapt to two diverse niches, an arthropod vector and a mammalian host. RpoS, an alternative sigma factor, plays a central role in spirochetal adaptation to the mammalian host by governing expression of many genes important for mammalian infection. B. burgdorferi is known to be unique in metal utilization, and little is known of the role of biologically available metals in B. burgdorferi. Here, we identified two transition metal ions, manganese (Mn2+) and zinc (Zn2+), that influenced regulation of RpoS. The intracellular Mn2+ level fluctuated approximately 20-fold under different conditions and inversely correlated with levels of RpoS and the major virulence factor OspC. Furthermore, an increase in intracellular Mn2+ repressed temperature-dependent induction of RpoS and OspC; this repression was overcome by an excess of Zn2+. Conversely, a decrease of intracellular Mn2+ by deletion of the Mn2+ transporter gene, bmtA, resulted in elevated levels of RpoS and OspC. Mn2+ affected RpoS through BosR, a Fur family homolog that is required for rpoS expression: elevated intracellular Mn2+ levels greatly reduced the level of BosR protein but not the level of bosR mRNA. Thus, Mn2+ and Zn2+ appeared to be important in modulation of the RpoS pathway that is essential to the life cycle of the Lyme disease spirochete. This finding supports the emerging notion that transition metals such as Mn2+ and Zn2+ play a critical role in regulation of virulence in bacteria.

Novel Bartonella infection in northern and southern sea otters (Enhydra lutris kenyoni and Enhydra lutris nereis)

Since 2002, vegetative valvular endocarditis (VVE), septicemia and meningoencephalitis have contributed to an Unusual Mortality Event (UME) of northern sea otters in southcentral Alaska. Streptococcal organisms were commonly isolated from vegetative lesions and organs from these sea otters. Bartonella infection has also been associated with bacteremia and VVE in terrestrial mammals, but little is known regarding its pathogenic significance in marine mammals. Our study evaluated whether Streptococcus bovis/equinus (SB/E) and Bartonella infections were associated with UME-related disease characterized by VVE and septicemia in Alaskan sea otter carcasses recovered 2004-2008. These bacteria were also evaluated in southern sea otters in California. Streptococcus bovis/equinus were cultured from 45% (23/51) of northern sea otter heart valves, and biochemical testing and sequencing identified these isolates as Streptococcus infantarius subsp. coli. One-third of sea otter hearts were co-infected with Bartonella spp. Our analysis demonstrated that SB/E was strongly associated with UME-related disease in northern sea otters (P<0.001). While Bartonella infection was also detected in 45% (23/51) and 10% (3/30) of heart valves of northern and southern sea otters examined, respectively, it was not associated with disease. Phylogenetic analysis of the Bartonella ITS region allowed detection of two Bartonella species, one novel species closely related to Bartonella spp. JM-1, B. washoensis and Candidatus B. volans and another molecularly identical to B. henselae. Our findings help to elucidate the role of pathogens in northern sea otter mortalities during this UME and suggested that Bartonella spp. is common in sea otters from Alaska and California.

Aerobic oral and rectal bacteria of free-ranging steller sea lion pups and juveniles (Eumetopias Jubatus) in Alaska

Bacteriologic cultures from oral, rectal, and lesion samples from free-ranging Steller sea lion (SSL, Eumetopias jubatus) pups and juveniles in Alaska (2001–2005) were examined to determine frequency of infection by a specific subset of common and pathogenic aerobic bacteria. Associations between isolated bacteria and age, sex, body condition, location, and sampling season were investigated. Salmonella spp. isolates were further evaluated to determine spatial clustering (n=48) and to identify serovars (n=13) and antimicrobial susceptibility patterns (n=11). We sampled 356 SSL pups (n=272) and juveniles (n=84), and identified 988 isolates of 13 bacterial genera of specific interest. Pasteurella spp. (43.8%), beta-hemolytic Streptococcus spp. (30.6%), and Mannheimia spp. (18.2%) were the most commonly isolated oral bacteria (n=499 isolates), whereas Escherichia coli (47.6%), beta-hemolytic E. coli (32.4%), Salmonella spp. (10.4%), and Campylobacter spp. (7.8%) were the most frequently isolated rectal bacteria (n=460 isolates). Salmonella was most commonly found in pups from western stocks and in samples collected during fall/winter seasons. A significant Salmonella cluster was detected at the Perry Island haulout. Five serovars were isolated: Enteritidis, Infantis, Newport, Reading, and Stanley. Pulsed-field gel electrophoresis provided evidence that Salmonella isolates were most likely being maintained within the SSL population in Alaska.

Bartonella spp. Exposure in Northern and Southern Sea Otters in Alaska and California

Since 2002, an increased number of northern sea otters (Enhydra lutris kenyoni) from southcentral Alaska have been reported to be dying due to endocarditis and/or septicemia with infection by Streptococcus infantarius subsp. coli. Bartonella spp. DNA was also detected in northern sea otters as part of mortality investigations during this unusual mortality event (UME) in Kachemak Bay, Alaska. To evaluate the extent of exposure to Bartonella spp. in sea otters, sera collected from necropsied and live-captured northern sea otters, as well as necropsied southern sea otters (Enhydra lutris nereis) unaffected by the UME, were analyzed using an immunofluorescent antibody assay. Antibodies against Bartonella spp. were detected in sera from 50% of necropsied and 34% of presumed healthy, live-captured northern sea otters and in 16% of necropsied southern sea otters. The majority of sea otters with reactive sera were seropositive for B. washoensis, with antibody titers ranging from 1:64 to 1:256. Bartonella spp. antibodies were especially common in adult northern sea otters, both free-living (49%) and necropsied (62%). Adult stranded northern sea otters that died from infectious causes, such as opportunistic bacterial infections, were 27 times more likely to be Bartonella seropositive than adult stranded northern sea otters that died from noninfectious causes (p<0.001; 95% confidence interval 2.62-269.4). Because Bartonella spp. antibodies were detected in necropsied northern sea otters from southcentral (44%) and southwestern (86%) stocks of Alaska, as well as in necropsied southern sea otters (16%) in southcentral California, we concluded that Bartonella spp. exposure is widely distributed among sea otter populations in the Eastern Pacific, providing context for investigating future disease outbreaks and monitoring of Bartonella infections for sea otter management and conservation.

Toll-like receptor 3 (TLR3) promotes the resolution of Chlamydia muridarum genital tract infection in congenic C57BL/6N mice

Chlamydia trachomatis urogenital serovars primarily replicate in epithelial cells lining the reproductive tract. Epithelial cells recognize Chlamydia through cell surface and cytosolic receptors, and/or endosomal innate receptors such as Toll-like receptors (TLRs). Activation of these receptors triggers both innate and adaptive immune mechanisms that are required for chlamydial clearance, but are also responsible for the immunopathology in the reproductive tract. We previously demonstrated that Chlamydia muridarum (Cm) induces IFN-β in oviduct epithelial cells (OE) in a TLR3-dependent manner, and that the synthesis of several cytokines and chemokines are diminished in Cm-challenged OE derived from TLR3-/- 129S1 mice. Furthermore, our in vitro studies showed that Cm replication in TLR3-/- OE is more efficient than in wild-type OE. Because TLR3 modulates the release inflammatory mediators involved in host defense during Cm infection, we hypothesized that TLR3 plays a protective role against Cm-induced genital tract pathology in congenic C57BL/6N mice. Using the Cm mouse model for human Chlamydia genital tract infections, we demonstrated that TLR3-/- mice had increased Cm shedding during early and mid-stage genital infection. In early stage infection, TLR3-/- mice showed a diminished synthesis of IFN-β, IL-1β, and IL-6, but enhanced production of IL-10, TNF-α, and IFN-γ. In mid-stage infection, TLR3-/- mice exhibited significantly enhanced lymphocytic endometritis and salpingitis than wild-type mice. These lymphocytes were predominantly scattered along the endometrial stroma and the associated smooth muscle, and the lamina propria supporting the oviducts. Surprisingly, our data show that CD4+ T-cells are significantly enhanced in the genital tract TLR3-/- mice during mid-stage Chlamydial infection. In late-stage infections, both mouse strains developed hydrosalpinx; however, the extent of hydrosalpinx was more severe in TLR3-/- mice. Together, these data suggest that TLR3 promotes the clearance of Cm during early and mid-stages of genital tract infection, and that loss of TLR3 is detrimental in the development hydrosalpinx.