Denise M. Imai

Ineffectiveness of Tigecycline against Persistent Borrelia burgdorferi

ABSTRACT The effectiveness of a new first-in-class antibiotic, tigecycline (glycylcycline), was evaluated during the early dissemination (1 week), early immune (3 weeks), or late persistent (4 months) phases of Borrelia burgdorferi infection in C3H mice. Mice were treated with high or low doses of tigecycline, saline (negative-effect controls), or a previously published regimen of ceftriaxone (positive-effect controls). Infection status was assessed at 3 months after treatment by culture, quantitative ospA real-time PCR, and subcutaneous transplantation of joint and heart tissue into SCID mice. Tissues from all saline-treated mice were culture and ospA PCR positive, tissues from all antibiotic-treated mice were culture negative, and some of the tissues from most of the mice treated with antibiotics were ospA PCR positive, although the DNA marker load was markedly decreased compared to that in saline-treated mice. Antibiotic treatment during the early stage of infection appeared to be more effective than treatment that began during later stages of infection. The viability of noncultivable spirochetes in antibiotic-treated mice (demonstrable by PCR) was confirmed by transplantation of tissue allografts from treated mice into SCID mice, with dissemination of spirochetal DNA to multiple recipient tissues, and by xenodiagnosis, including acquisition by ticks, transmission by ticks to SCID mice, and survival through molting into nymphs and then into adults. Furthermore, PCR-positive heart base tissue from antibiotic-treated mice revealed RNA transcription of several B. burgdorferi genes. These results extended previous studies with ceftriaxone, indicating that antibiotic treatment is unable to clear persisting spirochetes, which remain viable and infectious, but are nondividing or slowly dividing.

Resurgence of Persisting Non-Cultivable Borrelia burgdorferi following Antibiotic Treatment in Mice

The agent of Lyme borreliosis, Borrelia burgdorferi, evades host immunity and establishes persistent infections in its varied mammalian hosts. This persistent biology may pose challenges to effective antibiotic treatment. Experimental studies in dogs, mice, and non-human primates have found persistence of B. burgdorferi DNA following treatment with a variety of antibiotics, but persisting spirochetes are non-cultivable. Persistence of B. burgdorferi DNA has been documented in humans following treatment, but the significance remains unknown. The present study utilized a ceftriaxone treatment regimen in the C3H mouse model that resulted in persistence of non-cultivable B. burgdorferi in order to determine their long-term fate, and to examine their effects on the host. Results confirmed previous studies, in which B. burgdorferi could not be cultured from tissues, but low copy numbers of B. burgdorferi flaB DNA were detectable in tissues at 2, 4 and 8 months after completion of treatment, and the rate of PCR-positive tissues appeared to progressively decline over time. However, there was resurgence of spirochete flaB DNA in multiple tissues at 12 months, with flaB DNA copy levels nearly equivalent to those found in saline-treated mice. Despite the continued non-cultivable state, RNA transcription of multiple B. burgdorferi genes was detected in host tissues, flaB DNA was acquired by xenodiagnostic ticks, and spirochetal forms could be visualized within ticks and mouse tissues by immunofluorescence and immunohistochemistry, respectively. A number of host cytokines were up- or down-regulated in tissues of both saline- and antibiotic-treated mice in the absence of histopathology, indicating host response to the presence of non-cultivable, despite the lack of inflammation in tissues.

Lyme Neuroborreliosis in 2 Horses

Lyme neuroborreliosis—characterized as chronic, necrosuppurative to nonsuppurative, perivascular to diffuse meningoradiculoneuritis—was diagnosed in 2 horses with progressive neurologic disease. In 1 horse, Borrelia burgdorferi sensu stricto was identified by polymerase chain reaction amplification of B burgdorferi sensu stricto–specific gene targets (ospA, ospC, flaB, dbpA, arp). Highest spirochetal burdens were in tissues with inflammation, including spinal cord, muscle, and joint capsule. Sequence analysis of ospA, ospC, and flaB revealed 99.9% sequence identity to the respective genes in B burgdorferi strain 297, an isolate from a human case of neuroborreliosis. In both horses, spirochetes were visualized in affected tissues with Steiner silver impregnation and by immunohistochemistry, predominantly within the dense collagenous tissue of the dura mater and leptomeninges.

The Early Dissemination Defect Attributed to Disruption of Decorin-Binding Proteins is Abolished in Chronic Murine Lyme Borreliosis

ABSTRACT The laboratory mouse model of Lyme disease has revealed that Borrelia burgdorferi differentially expresses numerous outer surface proteins that influence different stages of infection (tick-borne transmission, tissue colonization, dissemination, persistence, and tick acquisition). Deletion of two such outer surface proteins, decorin-binding proteins A and B (DbpA/B), has been documented to decrease infectivity, impede early dissemination, and, possibly, prevent persistence. In this study, DbpA/B-deficient spirochetes were confirmed to exhibit an early dissemination defect in immunocompetent, but not immunodeficient, mice, and the defect was found to resolve with chronicity. Development of disease (arthritis and carditis) was attenuated only in the early stage of infection with DbpA/B-deficient spirochetes in both types of mice. Persistence of the DbpA/B-deficient spirochetes occurred in both immunocompetent and immunodeficient mice in a manner indistinguishable from that of wild-type spirochetes. Dissemination through the lymphatic system was evaluated as an underlying mechanism for the early dissemination defect. At 12 h, 3 days, 7 days, and 14 days postinoculation, DbpA/B-deficient spirochetes were significantly less prevalent and in lower numbers in lymph nodes than wild-type spirochetes. However, in immunodeficient mice, deficiency of DbpA/B did not significantly decrease the prevalence or spirochete numbers in lymph nodes. Complementation of DbpA/B restored a wild-type phenotype. Thus, the results indicated that deficiency of DbpA/B allows the acquired immune response to restrict early dissemination of spirochetes, which appears to be at least partially mediated through the lymphatic system.

Dynamics of connective-tissue localization during chronic Borrelia burgdorferi infection

The etiologic agent of Lyme disease, Borrelia burgdorferi, localizes preferentially in the extracellular matrix during persistence. In chronically infected laboratory mice, there is a direct association between B. burgdorferi and the proteoglycan decorin, which suggests that decorin has a role in defining protective niches for persistent spirochetes. In this study, the tissue colocalization of B. burgdorferi with decorin and the dynamics of borrelial decorin tropism were evaluated during chronic infection. Spirochetes were found to colocalize absolutely with decorin, but not collagen I in chronically infected immunocompetent C3H mice. Passive immunization of infected C3H-scid mice with B. burgdorferi-specific immune serum resulted in the localization of spirochetes in decorin-rich microenvironments, with clearance of spirochetes from decorin-poor microenvironments. In passively immunized C3H-scid mice, tissue spirochete burdens were initially reduced, but increased over time as the B. burgdorferi-specific antibody levels waned. Concurrent repopulation of the previously cleared decorin-poor microenvironments was observed with the rising tissue spirochete burden and declining antibody titer. These findings indicate that the specificity of B. burgdorferi tissue localization during chronic infection is determined by decorin, driven by the borrelia-specific antibody response, and fluctuates with the antibody response.

The Influence of Dietary Fat Source on Life Span in Calorie Restricted Mice

Calorie restriction (CR) without malnutrition extends life span in several animal models. It has been proposed that a decrease in the amount of polyunsaturated fatty acids (PUFAs), and especially n-3 fatty acids, in membrane phospholipids may contribute to life span extension with CR. Phospholipid PUFAs are sensitive to dietary fatty acid composition, and thus, the purpose of this study was to determine the influence of dietary lipids on life span in CR mice. C57BL/6J mice were assigned to four groups (a 5% CR control group and three 40% CR groups) and fed diets with soybean oil (high in n-6 PUFAs), fish oil (high in n-3 PUFAs), or lard (high in saturated and monounsaturated fatty acids) as the primary lipid source. Life span was increased (p < .05) in all CR groups compared to the Control mice. Life span was also increased (p < .05) in the CR lard mice compared to animals consuming either the CR fish or soybean oil diets. These results indicate that dietary lipid composition can influence life span in mice on CR, and suggest that a diet containing a low proportion of PUFAs and high proportion of monounsaturated and saturated fats may maximize life span in animals maintained on CR.

Characterization of beta-hemolytic streptococci isolated from southern sea otters (Enhydra lutris nereis) stranded along the California coast.

The goal of this study was to characterize the beta-hemolytic streptococci cultured from southern sea otters (Enhydra lutris nereis) stranded off the coast of California (USA) and to verify identifications made using the Lancefield system. Lancefield serotyping and biochemical analysis alone was inadequate for isolate characterization. Final identification was based on sequence analysis of a portion of the 16s ribosomal RNA gene from 12 of the 35 total isolates. The majority of isolates (10 of 12; 83.3%) were Streptococcus phocae and reacted with Lancefield group G and F antisera or were less frequently untypeable. The remaining isolates belonging to Lancefield group G were identified as S. dysgalactiae subsp. equisimilis (2 of 12; 16.7%). This is the first report of S. phocae in southern sea otters and further evidence of S. phocae expressing cell surface antigens compatible with Lancefield group G typing.

Influence of arthritis-related protein (BBF01) on infectivity of Borrelia burgdorferi B31.

BackgroundLyme borreliosis, caused by tick-borne Borrelia burgdorferi, is a multi-phasic, multi-system disease in humans. Similar to humans, C3H mice develop arthritis and carditis, with resolution and periodic bouts of recurrence over the course of persistent infection. Borrelia burgdorferi arthritis-related protein (Arp/BBF01), a highly conserved protein among B. burgdorferi s.s. isolates, has been shown to be antigenic in humans with Lyme borreliosis, and a target for antibody-mediated disease resolution in the mouse model.ResultsA mutant strain of B. burgdorferi s.s. deficient of the arp gene and a complemented version of that mutant were created and examined for phenotypic effects in mice compared to wild-type B. burgdorferi. Deletion of arp did not abolish infectivity, but did result in a higher infectious dose compared to wild-type B. burgdorferi, which was restored by complementation. Spirochete burdens in tissues of C3H-scid mice were lower when infected with the arp mutant, compared to wild-type, but arthritis was equally severe. Spirochete burdens were also lower in C3H mice infected with the arp mutant, but disease was markedly reduced. Ticks that fed upon infected C3H mice were able to acquire infection with both wild-type and arp mutant spirochetes. Arp mutant spirochetes were marginally able to be transmitted to naïve hosts by infected ticks.ConclusionThese results indicated that deletion of BBF01/arp did not abrogate, but diminished infectivity and limited spirochete burdens in tissues of both immunocompetent and immunodeficient hosts, and attenuated, but did not abolish the ability of ticks to acquire or transmit infection.

Proteomic analysis of highly prevalent amyloid A amyloidosis endemic to endangered island foxes.

Amyloid A (AA) amyloidosis is a debilitating, often fatal, systemic amyloid disease associated with chronic inflammation and persistently elevated serum amyloid A (SAA). Elevated SAA is necessary but not sufficient to cause disease and the risk factors for AA amyloidosis remain poorly understood. Here we identify an extraordinarily high prevalence of AA amyloidosis (34%) in a genetically isolated population of island foxes (Urocyon littoralis) with concurrent chronic inflammatory diseases. Amyloid deposits were most common in kidney (76%), spleen (58%), oral cavity (45%), and vasculature (44%) and were composed of unbranching, 10 nm in diameter fibrils. Peptide sequencing by mass spectrometry revealed that SAA peptides were dominant in amyloid-laden kidney, together with high levels of apolipoprotein E, apolipoprotein A-IV, fibrinogen-α chain, and complement C3 and C4 (false discovery rate ≤0.05). Reassembled peptide sequences showed island fox SAA as an 111 amino acid protein, most similar to dog and artic fox, with 5 unique amino acid variants among carnivores. SAA peptides extended to the last two C-terminal amino acids in 5 of 9 samples, indicating that near full length SAA was often present in amyloid aggregates. These studies define a remarkably prevalent AA amyloidosis in island foxes with widespread systemic amyloid deposition, a unique SAA sequence, and the co-occurrence of AA with apolipoproteins.

Rhabditid nematode-associated ophthalmitis and meningoencephalomyelitis in captive Asian horned frogs (Megophrys montana).

Between 2006 and 2008, 4 captive Asian horned frogs (Megophrys montana) were diagnosed with ocular and neurologic disease associated with rhabditid nematodiasis. Mortality, either spontaneous or by humane euthanasia, was high (3/4, 75%). Gross and histologic findings included varying degrees of ulcerative keratitis, histiocytic uveitis and retinitis, meningoencephalomyelitis, and epidermal chromatophore (iridophore) hyperplasia with intralesional nematodes. Entry into the host was presumed to be by direct invasion of the skin and the cornea with migration through the optic nerve to the brain and spinal cord. One frog was diagnosed with rhabditid nematodiasis antemortem, and clinical signs and lesions in the frog did not progress after unilateral enucleation and anthelminthic treatment were completed. Gross and tissue morphology of the nematodes were consistent with the order Rhabditida. DNA was extracted separately from 2 individual nematodes that were isolated from frozen and ethanol-preserved eye and brain tissue. These DNA templates were used for polymerase chain reaction amplification and sequencing of nuclear 28S large subunit (LSU) and internal transcribed spacer (ITS) ribosomal DNA regions. Comparison of the LSU and ITS sequences to those deposited in GenBank revealed an exact match for Caenorhabditis elegans.