Francisella tularensis infection in dogs: 88 cases (2014-2016).

OBJECTIVE: To characterize the epidemiology, clinical signs, and treatment of dogs with Francisella tularensis infection in New Mexico. ANIMALS: 87 dogs in which 88 cases of tularemia (1 dog had 2 distinct cases) were confirmed by the New Mexico Department of Health Scientific Laboratory Division from 2014 through 2016 and for which medical records were available. PROCEDURES: Dogs were confirmed to have tularemia if they had a 4-fold or greater increase in anti-F tularensis antibody titer between acute and convalescent serum samples or F tularensis had been isolated from a clinical or necropsy specimen. Epidemiological, clinical, and treatment information were collected from the dogs' medical records and summarized. RESULTS: All 88 cases of tularemia were confirmed by paired serologic titers; the first (acute) serologic test result was negative for 84 (95%) cases. The most common reported exposure to F tularensis was wild rodent or rabbit contact (53/88 [60%]). Dogs had a median number of 3 clinical signs at initial evaluation; lethargy (81/88 [92%]), pyrexia (80/88 [91%]), anorexia (67/88 [76%]), and lymphadenopathy (18/88 [20%]) were most common. For 32 (36%) cases, the dog was hospitalized; all hospitalized dogs survived. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs with F tularensis infection often had nonspecific clinical signs and developed moderate to severe illness, sometimes requiring hospitalization. Veterinarians examining dogs from tularemia-enzootic areas should be aware of the epidemiology and clinical signs of tularemia, inquire about potential exposures, and discuss prevention methods with owners, including reducing exposure to reservoir hosts and promptly seeking care for ill animals.

Yersinia pestis infection in dogs: 62 cases (2003-2011).

OBJECTIVE: To describe the epidemiology, clinical signs, and treatment practices in dogs with Yersinia pestis infection in New Mexico. DESIGN: Retrospective case series. ANIMALS: 62 dogs with plague in New Mexico. PROCEDURES: Confirmed case animals had isolation of Yersinia pestis from a clinical specimen, a positive direct fluorescent antibody test result, or a minimum 4-fold change between acute and convalescent serum antibody titers with clinically compatible illness. Retrospective review of cases of laboratory-confirmed plague from 2003 to 2011 was performed with a standardized chart abstraction form. Epidemiologic, clinical, and treatment data were evaluated. RESULTS: 62 confirmed cases of canine plague were identified from 2003 to 2011. Most cases (85%) were confirmed by serologic titers alone or in conjunction with other testing methods. Clinical signs included fever (100%), lethargy (97%), anorexia (77%), lymphadenopathy (23%), vomiting (13%), diarrhea (8%), and abscesses (2%). Most case animals (73%) were treated with multiple antimicrobials. Sixty (97%) case animals survived; of the 2 nonsurvivors, one was euthanized and another died. Potential sources of exposure to Y pestis included hunting, rodent or rabbit exposure, and residence in rural areas. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that dogs with exposure to Y pestis can develop moderate to severe illness or die as a result of infection. Veterinarians practicing in and examining animals from the western United States need to be familiar with the epidemiology of plague and query owners about potential plague exposures when consistent clinical signs are present. Veterinarians are often the first to recognize signs of plague among sentinel populations and have the opportunity to intervene and prevent zoonotic disease transmission.

Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p).

Vacuolar ATPases (V-ATPases) are important for many cellular processes, as they regulate pH by pumping cytosolic protons into intracellular organelles. The cytoplasm is acidified when V-ATPase is inhibited; thus we conducted a high-throughput screen of a chemical library to search for compounds that acidify the yeast cytosol in vivo using pHluorin-based flow cytometry. Two inhibitors, alexidine dihydrochloride (EC(50) = 39 µM) and thonzonium bromide (EC(50) = 69 µM), prevented ATP-dependent proton transport in purified vacuolar membranes. They acidified the yeast cytosol and caused pH-sensitive growth defects typical of V-ATPase mutants (vma phenotype). At concentrations greater than 10 µM the inhibitors were cytotoxic, even at the permissive pH (pH 5.0). Membrane fractions treated with alexidine dihydrochloride and thonzonium bromide fully retained concanamycin A-sensitive ATPase activity despite the fact that proton translocation was inhibited by 80-90%, indicating that V-ATPases were uncoupled. Mutant V-ATPase membranes lacking residues 362-407 of the tether of Vph1p subunit a of V(0) were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V(1)V(0) complexes and that alexidine dihydrochloride uncouples the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, showing further specificity for V-ATPases. Thus, a new class of V-ATPase inhibitors (uncouplers), which are not simply ionophores, provided new insights into the enzyme mechanism and original evidence supporting the hypothesis that V-ATPases may not be optimally coupled in vivo. The consequences of uncoupling V-ATPases in vivo as potential drug targets are discussed.

Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry.

Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was monitored by high-throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where vacuolar proton-translocating ATPases (V-ATPases) are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise, V-ATPase-deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short- and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen a small chemical library of structurally diverse compounds to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond that anticipated for pH changes alone in the mutant cells). Once confirmed by dose-response assays (EC(50)=26 microM), we verified V-ATPase inhibition by disulfiram in secondary assays that measured ATP hydrolysis in vacuolar membranes. The inhibitory action of disulfiram against V-ATPase pumps revealed a novel effect previously unknown for this compound. Because V-ATPases are highly conserved, new inhibitors identified could be used as research and therapeutic tools in cancer, viral infections, and other diseases where V-ATPases are involved.

Reduced susceptibility to praziquantel among naturally occurring Kenyan isolates of Schistosoma mansoni.

BACKGROUND: The near exclusive use of praziquantel (PZQ) for treatment of human schistosomiasis has raised concerns about the possible emergence of drug-resistant schistosomes. METHODOLOGY/PRINCIPAL FINDINGS: We measured susceptibility to PZQ of isolates of Schistosoma mansoni obtained from patients from Kisumu, Kenya continuously exposed to infection as a consequence of their occupations as car washers or sand harvesters. We used a) an in vitro assay with miracidia, b) an in vivo assay targeting adult worms in mice and c) an in vitro assay targeting adult schistosomes perfused from mice. In the miracidia assay, in which miracidia from human patients were exposed to PZQ in vitro, reduced susceptibility was associated with previous treatment of the patient with PZQ. One isolate ("KCW") that was less susceptible to PZQ and had been derived from a patient who had never fully cured despite multiple treatments was studied further. In an in vivo assay of adult worms, the KCW isolate was significantly less susceptible to PZQ than two other isolates from natural infections in Kenya and two lab-reared strains of S. mansoni. The in vitro adult assay, based on measuring length changes of adults following exposure to and recovery from PZQ, confirmed that the KCW isolate was less susceptible to PZQ than the other isolates tested. A sub-isolate of KCW maintained separately and tested after three years was susceptible to PZQ, indicative that the trait of reduced sensitivity could be lost if selection was not maintained. CONCLUSIONS/SIGNIFICANCE: Isolates of S. mansoni from some patients in Kisumu have lower susceptibility to PZQ, including one from a patient who was never fully cured after repeated rounds of treatment administered over several years. As use of PZQ continues, continued selection for worms with diminished susceptibility is possible, and the probability of emergence of resistance will increase as large reservoirs of untreated worms diminish. The potential for rapid emergence of resistance should be an important consideration of treatment programs.

The tether connecting cytosolic (N terminus) and membrane (C terminus) domains of yeast V-ATPase subunit a (Vph1) is required for assembly of V0 subunit d.

V-ATPases are molecular motors that reversibly disassemble in vivo. Anchored in the membrane is subunit a. Subunit a has a movable N terminus that switches positions during disassembly and reassembly. Deletions were made at residues securing the N terminus of subunit a (yeast isoform Vph1) to its membrane-bound C-terminal domain in order to understand the role of this conserved region for V-ATPase function. Shrinking of the tether made cells pH-sensitive (vma phenotype) because assembly of V(0) subunit d was harmed. Subunit d did not co-immunoprecipitate with subunit a and the c-ring. Cells contained pools of V(1) and V(0)(-d) that failed to form V(1)V(0), and very low levels of V-ATPase subunits were found at the membrane. Although subunit d expression was stable and at wild-type levels, growth defects were rescued by exogenous VMA6 (subunit d). Stable V(1)V(0) assembled after yeast cells were co-transformed with VMA6 and mutant VPH1. Tether-less V(1)V(0) was delivered to the vacuole and active. It retained 63-71% of the wild-type activity and was responsive to glucose. Tether-less V(1)V(0) disassembled and reassembled after brief glucose depletion and readdition. The N terminus retained binding to V(1) subunits and the C terminus to phosphofructokinase. Thus, no major structural change was generated at the N and C termini of subunit a. We concluded that early steps of V(0) assembly and trafficking were likely impaired by shorter tethers and rescued by VMA6.

Evolution of the opossum major histocompatibility complex: evidence for diverse alternative splice patterns and low polymorphism among class I genes.

The opossum major histocompatibility complex (MHC) shares a similar organization with that of non-mammals while containing a diverse set of class I genes more like that of eutherian (placental) mammals. There are 11 class I loci in the opossum MHC region, seven of which are known to be transcribed. The previously described Monodelphis domestica (Modo)-UA1 and Modo-UG display characteristics consistent with their being classical and non-classical class I genes, respectively. Here we describe the characteristics of the remaining five transcribed class I loci (Modo-UE, -UK, -UI, -UJ and -UM). All five genes have peptide-binding grooves with low or no polymorphism, contain unpaired cysteines with the potential to produce homodimer formation and display genomic organizational features that would be unusual for classical class I loci. In addition, Modo-UJ and -UM were expressed in alternatively spliced mRNA forms, including a potentially soluble isoform of Modo-UJ. Thus, the MHC region of the opossum contains a single class I gene that is clearly classical and six other class I genes each with its own unique characteristics that probably perform roles other than or in addition to antigen presentation.

Towards an understanding of the mechanism of action of praziquantel.

Although praziquantel (PZQ) has been used to treat schistosomiasis for over 20 years its mechanism of action remains unknown. We have developed an assay based on the transcriptional response of Schistosoma mansoni PR-1 to heat shock to confirm that while 6-week post-infection (p.i.) schistosomes are sensitive to PZQ, 4-week p.i. schistosomes are not. Further, we have used this assay to demonstrate that in mice this sensitivity develops between days 37 and 40 p.i. When PZQ is linked to the fluorophore BODIPY to aid microscopic visualization, it appears to enter the cells of intact 4 and 6-week p.i. schistosomes as well as mammalian NIH 3T3 cells with ease suggesting that the differential effects of PZQ is not based on cell exclusion. A transcriptomal analysis of gene expression between 4 and 6 weeks p.i. revealed 607 up-regulated candidate genes whose products are potential PZQ targets. A comparison of this gene list with that of genes expressed by PZQ sensitive miracidia reduced this target list to 247 genes, including a number involved in aerobic metabolism and cytosolic calcium regulation. Finally, we also report the effect of an in vitro sub-lethal exposure of PZQ on the transcriptome of S. mansoni PR-1. Annotation of genes differentially regulated by PZQ exposure suggests that schistosomes may undergo a transcriptomic response similar to that observed during oxidative stress.

Reconstructing an ancestral mammalian immune supercomplex from a marsupial major histocompatibility complex.

The first sequenced marsupial genome promises to reveal unparalleled insights into mammalian evolution. We have used the Monodelphis domestica (gray short-tailed opossum) sequence to construct the first map of a marsupial major histocompatibility complex (MHC). The MHC is the most gene-dense region of the mammalian genome and is critical to immunity and reproductive success. The marsupial MHC bridges the phylogenetic gap between the complex MHC of eutherian mammals and the minimal essential MHC of birds. Here we show that the opossum MHC is gene dense and complex, as in humans, but shares more organizational features with non-mammals. The Class I genes have amplified within the Class II region, resulting in a unique Class I/II region. We present a model of the organization of the MHC in ancestral mammals and its elaboration during mammalian evolution. The opossum genome, together with other extant genomes, reveals the existence of an ancestral "immune supercomplex" that contained genes of both types of natural killer receptors together with antigen processing genes and MHC genes.