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Background: The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative., Methods: We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology., Results: Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor β signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory., Conclusions: Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor β signaling and hippocampal function., (Copyright © 2019 Society of Biological Psychiatry. All rights reserved.)

Supercharging electrospray ionization can be a powerful tool for increasing charge states in mass spectra and generating unfolded ion structures, yet key details of its mechanism remain unclear. The structures of highly extended protein ions and the mechanism of supercharging were investigated using ion mobility-mass spectrometry. Head-to-tail-linked polyubiquitins (Ubq 1-11 ) were used to determine size and charge state scaling laws for unfolded protein ions formed by supercharging while eliminating amino acid composition as a potential confounding factor. Collisional cross section was found to scale linearly with mass for these ions and several other monomeric proteins, and the maximum observed charge state for each analyte scales with mass in agreement with an analytical charge state scaling law for protein ions with highly extended structures that is supported by experimental gas-phase basicities. These results indicate that these highly unfolded ions can be considered quasi-one-dimensional, and collisional cross sections modeled with the Trajectory Method in Collidoscope show that these ions are significantly more extended than linear α-helices but less extended than straight chains. The effect of internal disulfide bonds on the extent of supercharging was probed using bovine serum albumin, β-lactoglobulin, and lysozyme, each of which contains multiple internal disulfide bonds. Reduction of the disulfide bonds led to a marked increase in charge state upon supercharging without significantly altering folding in solution. This evidence supports a supercharging mechanism in which these proteins unfold before or during evaporation of the electrospray droplet and ionization occurs by the Chain Ejection Model.

Ion mobility-mass spectrometry (IM-MS) can be a powerful tool for determining structural information about ions in the gas phase, from small covalent analytes to large, native-like or denatured proteins and complexes. For large biomolecular ions, which may have a wide variety of possible gas-phase conformations and multiple charge sites, quantitative, physically explicit modeling of collisional cross sections (CCSs) for comparison to IMS data can be challenging and time-consuming. We present a "trajectory method" (TM) based CCS calculator, named "Collidoscope," which utilizes parallel processing and optimized trajectory sampling, and implements both He and N 2 as collision gas options. Also included is a charge-placement algorithm for determining probable charge site configurations for protonated protein ions given an input geometry in pdb file format. Results from Collidoscope are compared with those from the current state-of-the-art CCS simulation suite, IMoS. Collidoscope CCSs are within 4% of IMoS values for ions with masses from ~18 Da to ~800 kDa. Collidoscope CCSs using X-ray crystal geometries are typically within a few percent of IM-MS experimental values for ions with mass up to ~3.5 kDa (melittin), and discrepancies for larger ions up to ~800 kDa (GroEL) are attributed in large part to changes in ion structure during and after the electrospray process. Due to its physically explicit modeling of scattering, computational efficiency, and accuracy, Collidoscope can be a valuable tool for IM-MS research, especially for large biomolecular ions. Graphical Abstract ᅟ.

By 1883 a Veterinary Division had been established within the United States Department of Agriculture, itself established in 1862. Federal concern about animal health in the U.S.A. emerged as early as 1865 when Congress adopted regulations aimed at controlling importation of livestock. It was not until 1884 that the Bureau of Animal Industry (BAI) was formally created by Act of Congress, and shortly after that the Zoological Laboratory was established and assigned responsibility for study of parasites and the diseases they produce in animals. Classically trained parasitologists working in USDA's BAI soon became internationally recognized for their contributions to basic research and development of programs for prevention and control of parasitic diseases. Leadership by a series of BAI-employed parasitologists led to the emergence of veterinary parasitology as a sub-discipline. Maurice C. Hall who served as president of both the American Society of Parasitologists and the American Veterinary Medical Association was a central figure in development of veterinary parasitology in the U.S.A., which flourished in his country and elsewhere today.

The Companion Animal Parasite Council hosted a meeting to identify quantifiable factors that can influence the prevalence of tick-borne disease agents among dogs in North America. This report summarizes the approach used and the factors identified for further analysis with mathematical models of canine exposure to tick-borne pathogens.

Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, is a tick-borne rickettsial pathogen that is infective to a wide range of mammals, including dogs and people. Amblyomma americanum, the lone star tick, is considered the primary vector of E. chaffeensis, but this pathogen has been detected in other tick species, including the brown dog tick, Rhipicephalus sanguineus. We hypothesized that the Arkansas strain of E. chaffeensis is infective to R. sanguineus, and used a novel PCR assay to test for acquisition of this pathogen by R. sanguineus and A. americanum ticks that were simultaneously fed on experimentally infected dogs. Although E. chaffeensis was not frequently detected in peripheral blood of these dogs, the pathogen was detected in both tick species and in canine lung, kidney, lymph node, bone marrow and frontal lobe samples. One dog (AFL) was maintained for several years, and ticks again acquired E. chaffeensis from this dog 566 days after intradermal inoculation with E. chaffeensis, but the pathogen was not detected in ticks fed on the same dog at 764 or 1086 days after the intradermal inoculation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: To investigate the simultaneous occurrence of more than 1 Clostridium difficile ribotype in patients' stool samples at the time of diagnostic testing., Methods: Stool samples submitted for diagnostic testing for the presence of toxigenic C. difficile were obtained for 102 unique patients. A total of 95 single colonies of C. difficile per stool sample were isolated on selective media, subcultured alongside negative (uninoculated) controls, and polymerase chain reaction (PCR) ribotyped using capillary gel electrophoresis., Results: Capillary-based PCR ribotyping was successful for 9,335 C. difficile isolates, yielding a median of 93 characterized isolates per stool sample (range, 69-95). More than 1 C. difficile ribotype was present in 16 of 102 (16%) C. difficile infection (CDI) cases; 2 of the 16 mixtures were composed of at least 3 ribotypes, while the remaining 14 were composed of at least 2., Conclusions: Deep sampling of patient stool samples coupled with capillary-based PCR ribotyping identified a high rate of mixed CDI cases compared with previous estimates. Studies seeking to quantify the clinical significance of particular C. difficile ribotypes should account for mixed cases of disease.

Capillary-based PCR ribotyping was used to quantify the presence/absence and relative abundance of 98 Clostridium difficile ribotypes from clinical cases of disease at health care institutions in six states of the United States. Regionally important ribotypes were identified, and institutions in close proximity did not necessarily share more ribotype diversity than institutions that were farther apart.

Background: Studies of Clostridium difficile outbreaks suggested that certain ribotypes (eg, 027 and 078) cause more severe disease than other ribotypes. A growing number of studies challenge the validity of this hypothesis., Methods: We conducted a cross-sectional study of C. difficile infection (CDI) to test whether ribotype predicted clinical severity when adjusted for the influence of other predictors. Toxigenic C. difficile isolates were cultured from stool samples, screened for genes encoding virulence factors by polymerase chain reaction (PCR) and ribotyped using high-throughput, fluorescent PCR ribotyping. We collected data for 15 covariates (microbiologic, epidemiologic, and laboratory variables) and determined their individual and cumulative influence on the association between C. difficile ribotype and severe disease. We then validated this influence using an independent data set., Results: A total of 34 severe CDI cases were identified among 310 independent cases of disease (11.0%). Eleven covariates, including C. difficile ribotype, were significant predictors of severe CDI in unadjusted analysis. However, the association between ribotypes 027 and 078 and severe CDI was not significant after adjustment for any of the other covariates. After full adjustment, severe cases were significantly predicted only by patients' white blood cell count and albumin level. This result was supported by analysis of a validation data set containing 433 independent CDI cases (45 severe cases; 10.4%)., Conclusions: Ribotype is not a significant predictor of severe CDI when adjusted for the influence of any other variables separately or in combination. White blood cell count and albumin level are the most clinically relevant predictors of severe CDI cases.

To clarify effects of diet and body weight on prostate cancer development, three studies were undertaken using the TRAMP mouse model of this disease. In the first experiment, obesity was induced by injection of gold thioglucose (GTG). Age of prostate tumor detection (~33 wk) and death (~43 wk) was not significantly different among the groups. In the second study, TRAMP-C2 cells were injected into syngeneic C57BL6 mice and tumor progression was evaluated in mice fed either high-fat or low-fat diets. The high fat fed mice had larger tumors than did the low-fat fed mice. In the third study, tumor development was followed in TRAMP mice fed a high fat diet from 6 weeks of age. There were no significant effects of body weight status or diet on tumor development among the groups. When the tumors were examined for the neuroendocrine marker synaptophysin, there was no correlation with either body weight or diet. However, there was a significant correlation of the expression of synaptophysin with earlier age to tumor detection and death. In summary, TRAMP-C2 cells grew faster when the mice were fed a high-fat diet. Further synaptophysin may be a marker of poor prognosis independent of weight and diet.

Novel Hepatozoon spp. sequences collected from previously unrecognized vertebrate hosts in North America were compared with documented Hepatozoon 18S rRNA sequences in an effort to examine phylogenetic relationships between the different Hepatozoon organisms found cycling in nature. An approximately 500-base pair fragment of 18S rDNA common to Hepatozoon spp. and some other apicomplexans was amplified and sequenced from the tissues or blood of 16 vertebrate host species from the southern United States, including 1 opossum (Didelphis virginiana), 2 bobcats (Lynx rufus), 1 domestic cat (Felis catus), 3 coyotes (Canis latrans), 1 gray fox (Urocyon cinereoargenteus), 4 raccoons (Procyon lotor), 1 pet boa constrictor (Boa constrictor imperator), 1 swamp rabbit (Sylvilagus aquaticus), 1 cottontail rabbit (Sylvilagus floridanus), 4 woodrats (Neotoma fuscipes and Neotoma micropus), 3 white-footed mice (Peromyscus leucopus), 8 cotton rats (Sigmodon hispidus), 1 cotton mouse (Peromyscus gossypinus), 1 eastern grey squirrel (Sciurus carolinensis), and 1 woodchuck (Marmota monax). Phylogenetic analyses and comparison with sequences in the existing database revealed distinct groups of Hepatozoon spp., with clusters formed by sequences obtained from scavengers and carnivores (opossum, raccoons, canids, and felids) and those obtained from rodents. Surprisingly, Hepatozoon spp. sequences from wild rabbits were most closely related to sequences obtained from carnivores (97.2% identical), and the sequence from the boa constrictor was most closely related to the rodent cluster (97.4% identical). These data are consistent with recent work identifying prey-predator transmission cycles in Hepatozoon spp. and suggest this pattern may be more common than previously recognized.

Doxycycline is the treatment of choice for canine monocytic ehrlichiosis (CME), a well-characterized disease and valuable model for tick-borne zoonoses. Conflicting reports of clearance of Ehrlichia canis after treatment with doxycycline suggested that the disease phase during which treatment is initiated influences outcomes of these treatments. The purpose of this study was to evaluate the efficacy of a 28-day doxycycline regimen for clearance of experimental E. canis infections from dogs treated during three phases of the disease. Ten dogs were inoculated with blood from E. canis carriers and treated with doxycycline during acute, subclinical, or chronic phases of CME. Daily rectal temperatures and semiweekly blood samples were monitored from each dog, and Rhipicephalus sanguineus ticks were acquisition fed on each dog for xenodiagnosis. Blood collected from dogs treated during acute or subclinical CME became PCR negative for E. canis as clinical parameters improved, but blood samples collected from dogs treated during chronic CME remained intermittently PCR positive. R. sanguineus ticks fed on dogs after doxycycline treatments became PCR positive for E. canis, regardless of when treatment was initiated. However, fewer ticks became PCR positive after feeding on two persistently infected dogs treated with doxycycline followed by rifampin, suggesting that antibiotic therapy can reduce tick acquisition of E. canis.

During the last two decades, expanding industrial activity in east Asia has led to increased production of airborne pollutants that can be transported to North America. Previous efforts to detect this trans-Pacific pollution have relied upon remote sensing and remote sample locations. We tested whether Pb isotope ratios in airborne particles can be used to directly evaluate the Asian contribution to airborne particles of anthropogenic origin in western North America, using a time series of samples from a pair of sites upwind and downwind of the San Francisco Bay Area. Our results for airborne Pb at these sites indicate a median value of 29% Asian origin, based on mixing relations between distinct regional sample groups. This trans-Pacific Pb is present in small quantities but serves as a tracer for airborne particles within the growing Asian industrial plume. We then applied this analysis to archived samples from urban sites in central California. Taken together, our results suggest that the analysis of Pb isotopes can reveal the distribution of airborne particles affected by Asian industrial pollution at urban sites in northern California. Under suitable circumstances, this analysis can improve understanding of the global transport of pollution, independent of transport models.

There is no labeled treatment for dogs with American canine hepatozoonosis (ACH), but the drug therapies discussed in this article, although not rapidly curative, may be successful in alleviating acute clinical signs, prolonging life, reducing the number of clinical relapses, and enhancing quality of life. This article also describes a pilot trial conducted to assess the efficacy of a novel treatment approach with ponazuril as a stand-alone parasiticide administered for 4 weeks without follow-up decoquinate treatment. Although extended ponazuril treatment in combination with NSAID administration did ameliorate acute clinical signs associated with ACH, the parasite was not completely cleared with this treatment protocol alone. Long-term decoquinate therapy remains a critical component of successful treatment of ACH.

Background: In a murine model of inflammatory bowel disease (IBD), treatment of colitis in IL-10 gene-deficient mice with the parasitic helminth Heligmosomoides polygyrus ameliorates colonic inflammation. The cellular and molecular mechanisms driving this therapeutic host response are being studied vigorously. One proposed mechanism is that H. polygyrus infection favors the outgrowth or suppression of certain bacteria, which in turn help modulate host immunity., Methods: To quantify the effect of H. polygyrus infection on the composition of the gastrointestinal (GI) tract microbiota, we conducted two independent microbial ecology analyses of C57BL/6 mice. We obtained and analyzed 3,353 bacterial 16S rRNA encoding gene sequences from the ileum and cecum of infected and uninfected mice as well as incective H. polygyrus larvae at the outset of the second experiment and adult worms taken directly from the mouse duodenum at the end of the second experiment., Results: We found that a significant shift in the abundance and relative distribution of bacterial species in the ileum of mice is associated with H. polygyrus infection. Members of the bacterial family Lactobacillaceae significantly increased in abundance in the ileum of infected mice reproducibly in two independent experiments despite having different microbiotas present at the outset of each experiment., Conclusions: These data support the concept that helminth infection shifts the composition of intestinal bacteria. The clinical consequences of these shifts in intestinal flora are yet to be explored.

We used PCR and a novel serologic assay to determine infection and exposure rates to Ehrlichia ewingii in dogs from an area of northeast Oklahoma and northwest Arkansas where Amblyomma americanum ticks are abundant. Of 143 dogs assayed, 13 (9.1%) harbored E. ewingii by PCR and 64 (44.8%) had antibodies to E. ewingii detected using a peptide-based microtiter plate ELISA. Dogs were more likely (P=0.001) to be positive by PCR if sampled in August (30.8%) but no association was found between seropositive status and month of collection of sample (P>0.05). Additional testing revealed PCR evidence of Ehrlichia chaffeensis (4/143; 2.8%) and Anaplasma platys (5/143; 3.5%) as well as antibodies reactive to E. chaffeensis (25/143; 17.5%), Ehrlichia canis (2/143; 1.4%), and Anaplasma spp. (8/143; 5.6%). Testing of another 200 dogs from the area revealed additional PCR and/or serologic evidence of E. ewingii, E. canis, E. chaffeensis, and A. platys. None of the 343 dogs evaluated had evidence of Borrelia burgdorferi exposure. These data support the interpretation that E. ewingii may be the primary agent of canine ehrlichiosis in this region, and suggest that diagnostic evaluation of dogs suspected to have a tick-borne disease should include assays targeting this organism.

The intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), described by Sir Arnold Theiler in 1910, is endemic worldwide in tropical and subtropical areas. Infection of cattle with A. marginale causes bovine anaplasmosis, a mild to severe hemolytic disease that results in considerable economic loss to both dairy and beef industries. Transmission of A. marginale to cattle occurs biologically by ticks and mechanically by biting flies and by blood-contaminated fomites. Both male ticks and cattle hosts become persistently infected with A. marginale and serve as reservoirs of infection. While erythrocytes are the major site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks that begins by infection of gut cells, and transmission to susceptible hosts occurs from salivary glands during feeding. Major surface proteins (MSPs) play a crucial role in the interaction of A. marginale with host cells, and include adhesion proteins and MSPs from multigene families that undergo antigenic change and selection in cattle, thus contributing to maintenance of persistent infections. Many geographic strains of A. marginale have been identified worldwide, which vary in genotype, antigenic composition, morphology and infectivity for ticks. Isolates of A. marginale may be maintained by independent transmission events and a mechanism of infection/exclusion in cattle and ticks. The increasing numbers of A. marginale genotypes identified in some geographic regions most likely resulted from intensive cattle movement. However, concurrent A. marginale strain infections in cattle was reported, but these strains were more distantly related. Phylogenetic studies of selected geographic isolates of A. marginale, using msp4 and msp1alpha, provided information about the biogeography and evolution of A. marginale, and msp1alpha genotypes appear to have evolved under positive selection pressure. Live and killed vaccines have been used for control of anaplasmosis and both types of vaccines have advantages and disadvantages. Vaccines have effectively prevented clinical anaplasmosis in cattle but have failed to block A. marginale infection. Vaccines are needed that can prevent clinical disease and, simultaneously, prevent infection in cattle and ticks, thus eliminating these hosts as reservoirs of infection. Advances in genomics, proteomics, immunology and biochemical and molecular technologies during the last decade have been applied to research on A. marginale and related organisms, and the recent development of a cell culture system for A. marginale has provided a format for studying the pathogen/tick interface. Recent advancements and new research methodologies should provide additional opportunities for development of new strategies for control and prevention of bovine anaplasmosis.

Background: The range of American canine hepatozoonosis (ACH) is expanding from the southern USA northward. Transmission of Hepatozoon americanum occurs by ingestion of infected Gulf Coast ticks, Amblyomma maculatum. The source of the protozoan for the tick remains undetermined; infected dogs are unusual hosts for the tick., Objective: Compare possible sources of infection by field investigations of 2 multiple-dog outbreaks of ACH., Animals: Twenty-eight privately owned dogs (Canis familiaris), 1 coyote (Canis latrans), 31 wild-trapped cotton rats (Sigmodon hispidus), 24 wild-trapped field mice (Peromyscus leucopus), and 9 wild-caught rabbits (Sylvilagus spp.) from sites in eastern Oklahoma were monitored for hepatozoonosis. Six laboratory-raised cotton rats (S. hispidus), 6 Sprague-Dawley rats (Rattus norvegicus), 6 C57BL/6J-Lystbg-J/J mice (Mus musculus), 6 outbred white mice (M. musculus), 6 New Zealand white rabbits (Oryctolagus cuniculus), and 2 dogs were acquired through commercial vendors for experimental transmission trials of H. americanum., Methods: Four of 15 dogs in a rural neighborhood and 5/12 hunting Beagles were confirmed to be infected by blood smear examination, muscle biopsy, and polymerase chain reaction assay of the 18S rRNA gene of Hepatozoon species. Histories and tick host preferences led to field collections of common prey of canids and experimental transmission trials of H. americanum to selected prey (M. musculus, S. hispidus, R. norvegicus, and O. cuniculus)., Results: Dogs with ready access to prey (4/15 dogs) or that were fed prey retrieved from hunts (5/12 hunting Beagles) became infected, providing evidence that predation is an important epidemiologic component of ACH infection. Experimental transmission studies identified a quiescent, infectious stage (cystozoite) of the parasite that provides an alternate mode of transmission to canids through predation, demonstrating that cotton rats, mice, and rabbits but not brown rats may act as paratenic hosts of H. americanum., Conclusions and Clinical Importance: Predation of prey harboring infected A. maculatum or containing cystozoites of H. americanum in their tissues provide 2 modes of transmission of ACH to dogs, putting unconfined dogs at increased risk of infection in endemic areas.

Inflammatory lesions containing parasitic cystozoites developed in multiple organs and tissues of laboratory-raised Oryctolagus cuniculus that were administered approximately 100 sporulated oocysts of Hepatozoon americanum (Oklahoma isolate, GenBank accession AF176836) orally. The predominantly granulomatous inflammatory lesions were detected histologically 8 weeks after exposure to oocysts. Cystozoites, recognized by cresent-shaped, uninucleated bodies surrounded by an accumulation of globular, PAS-positive polysaccharide material, were evident within macrophages as monozoic and dizoic cysts. Neither meronts nor gamonts were detected in any of the laboratory-raised lagomorphs during the 24-week observation period. Nested PCR assay of rabbit tissues for a 488 bp fragment of the 18S rRNA Hepatozoon spp. gene was positive at 8 and 24 weeks post-exposure. The sequence was 97.1% similar with sequence from the H. americanum carrier used to infect ticks. A Hepatozoon-free dog fed tissues from the 24-week post-exposure rabbit principal developed American canine hepatozoonosis. Gamonts were first detected 5 weeks after the dog ingested the rabbit tissues containing cystozoites. PCR assay of blood from the dog was positive for the Hepatozoon spp. gene fragment. Sequencing confirmed that the parasite in the dog was H. americanum.

Canine hepatozoonosis is caused by Hepatozoon canis and Hepatozoon americanum, apicomplexan parasites transmitted to dogs by ingestion of infectious stages. Although the two agents are phylogenetically related, specific aspects, including characteristics of clinical disease and the natural history of the parasites themselves, differ between the two species. Until recently, H. canis infections had not been clearly documented in North America, and autochthonous infection with H. americanum has yet to be reported outside of the southern United States. However, recent reports demonstrate H. canis is present in areas of North America where its vector tick, Rhipicephalus sanguineus, has long been endemic, and that the range of H. americanum is likely expanding along with that of its vector tick, Amblyomma maculatum; co-infections with the two organisms have also been identified. Significant intraspecific variation has been reported in the 18S rRNA gene sequence of both Hepatozoon spp.-infecting dogs, suggesting that each species may represent a complex of related genogroups rather than well-defined species. Transmission of H. americanum to dogs via ingestion of cystozoites in muscle of infected vertebrates was recently demonstrated, supporting the concept of predation as a means of natural transmission. Although several exciting advances have occurred in recent years, much remains to be learned about patterns of infection and the nature of clinical disease caused by the agents of canine hepatozoonosis in North America.

Ehrlichia canis is the etiologic agent of canine monocytic ehrlichiosis (CME) and is a useful model for zoonotic tick-borne pathogens, many of which infect dogs. The purpose of this study was to evaluate rifampin and doxycycline regimens for clearance of E. canis infections in addition to alleviation of CME. Beagles were infected with E. canis by intravenous inoculation with carrier blood and treated with either rifampin or doxycycline after the acute phase of CME. Improved hematological values demonstrated that both treatments effectively relieved signs of the disease. Peripheral blood from all dogs became PCR negative after antibiotic treatment, suggesting that these infections were eliminated and that rifampin is an effective alternative chemotherapeutic agent for treatment of CME.

Hepatozoon americanum cystozoites from experimentally infected, laboratory-raised rodents were fed to a Hepatozoon-free dog. Gamonts were detected by examination of blood smear 42 and 56 days post-exposure. PCR analysis of blood was positive for the 18S rRNA Hepatozoon gene on days gamonts were demonstrated. Meronts were detected histologically in a skeletal muscle biopsy 90 days after ingestion of cystozoites. Sequencing confirmed that the parasite in the dog was H. americanum. Xenodiagnosis was conducted by replete feeding of Ambylomma maculatum larvae on the dog; 40 days after detachment, sporulated oocysts were recovered from recently molted nymphs.

Laboratory-raised cotton rats (Sigmodon hispidus), outbred white mice (Mus musculus), and C57BL/6J-Lystbg-J/J mice (M. musculus) that were administered approximately 50 sporulated oocysts of Hepatozoon americanum (AF176836) by gavage developed inflammatory lesions containing parasitic cystozoites in cardiac and skeletal muscle, kidney, and lung. Sprague-Dawley rats (Rattus norvegicus) similarly exposed showed no evidence of infection. Cystozoites were first detected by histopathologic examination four weeks after exposure to oocysts. Globular, PAS-positive material accumulated around the cystozoites as the duration of infection lengthened. Nested PCR analysis of tissues collected 16 weeks post-exposure was positive for the 18S rRNA Hepatozoon sp. gene and the DNA sequence of the fragment amplified was 99.6% and 99.8% identical to H. americanum sequences previously reported from naturally-infected dogs (AF176836 and AY864676, respectively). Merogonous and gamontogonous stages of the parasite were not detected in any of the cystozoite-infected rodents.

Human monocytic ehrlichiosis (HME) is a zoonotic emerging tick-borne disease with clinical signs that range from mild symptoms to multiple organ failure and death. Ehrlichia chaffeensis, the aetiologic agent of HME, is reported to infect a divergent range of mammals. Although cattle are common hosts of the primary vector of this pathogen, the susceptibility of this host to E. chaffeensis has not been reported to date. This study was undertaken to determine if cattle could provide a useful infection model of E. chaffeensis. Dairy calves were injected with DH82 cells infected with the Arkansas, St Vincent or 91HE17 strain of E. chaffeensis, and monitored for signs of clinical ehrlichiosis and for infection of peripheral blood and ticks by PCR assay. Splenectomized and spleen-intact calves were injected with cryopreserved stabilates of E. chaffeensis-infected DH82 cells for the first experiment. Mild clinical signs were occasionally observed among these calves, and only two blood samples were PCR-positive, while several ticks fed on each calf tested PCR-positive. The second experiment involved injection of normal calves with active cultures of the same E. chaffeensis strains. Interestingly, three of six calves inoculated with active cultures became recumbent and died or had to be euthanized. All of the surviving calves in this experiment tested PCR-positive on multiple dates, but fewer ticks fed on these calves were PCR-positive. These results suggest that a bovine disease model could facilitate the understanding of factors that affect the severity of HME.

Eighteen of 31 (58%) cotton rats (Sigmodon hispidus) and 8 of 24 (33.3%) white-footed mice (Peromyscus leucopus) that were wild-trapped from 4 American canine hepatozoonosis endemic sites in Oklahoma were infected with Hepatozoon species. The predilection organ for merogony of the Hepatozoon species in cotton rats was the liver. Meronts were not detected in any of the white-footed mice. A 488 bp DNA fragment that includes a variable region of the 18S rRNA Hepatozoon gene amplified from blood or tissue of these infected animals. Sequences from eight cotton rats were 100% identical to each other as were sequences from three white-footed mice 100% identical to each other. The cotton rat sequence and the white-footed mouse sequence were 98.8% identical, differing in 6 bp of the 488 bp fragment. The DNA sequence from cotton rats was 97.7% identical to a Hepatozoon sp. described in a large bandicoot rat from Thailand and 97.5% identical to a Hepatozoon sp. in a bank vole from Brazil. The sequence from white-footed mice was 98.6% identical to the bandicoot rat sequence and 98.4% identical to the bank vole sequence. However, the sequences were only 90.6% (cotton rat) and 91.4% (white-footed mouse) identical to H. americanum. These findings suggest that the rodents are obligate intermediate hosts for distinct Hepatozoon spp., but not H. americanum.

Doxycycline generally alleviates clinical monocytic ehrlichiosis, but its efficacy in the control of monocytotropic ehrlichial pathogens requires further investigation. In this study, Ehrlichia canis was detected in dogs treated with doxycycline for 14 days and in ticks fed on these dogs, suggesting that treated dogs can remain reservoirs for E. canis.

To characterize phylogenetically the species which causes canine hepatozoonosis at two rural areas of Rio de Janeiro State, Brazil, we used universal or Hepatozoon spp. primer sets for the 18S SSU rRNA coding region. DNA extracts were obtained from blood samples of thirteen dogs naturally infected, from four experimentally infected, and from five puppies infected by vertical transmission from a dam, that was experimentally infected. DNA of sporozoites of Hepatozoon americanum was used as positive control. The amplification of DNA extracts from blood of dogs infected with sporozoites of Hepatozoon spp. was observed in the presence of primers to 18S SSU rRNA gene of Hepatozoon spp., whereas DNA of H. americanum sporozoites was amplified in the presence of either universal or Hepatozoon spp.-specific primer sets; the amplified products were approximately 600bp in size. Cloned PCR products obtained from DNA extracts of blood from two dogs experimentally infected with Hepatozoon sp. were sequenced. The consensus sequence, derived from six sequence data sets, were blasted against sequences of 18S SSU rRNA of Hepatozoon spp. available at GenBank and aligned to homologous sequences to perform the phylogenetic analysis. This analysis clearly showed that our sequence clustered, independently of H. americanum sequences, within a group comprising other Hepatozoon canis sequences. Our results confirmed the hypothesis that the agent causing hepatozoonosis in the areas studied in Brazil is H. canis, supporting previous reports that were based on morphological and morphometric analyses.

The occurrence of hypolithic cyanobacteria colonizing translucent stones was quantified along the aridity gradient in the Atacama Desert in Chile, from less arid areas to the hyperarid core where photosynthetic life and thus primary production reach their limits. As mean rainfall declines from 21 to

Transmission of Hepatozoon spp. to dogs was investigated using four species of ixodid ticks: Rhipicephalus sanguineus, Amblyomma aureolatum, Amblyomma ovale and Amblyomma cajennense. We collected completely or partially engorged adult ticks of these species from dogs that were naturally infested and positive for Hepatozoon spp. We selected some of these ixodids and inoculated them orally in four negative dogs. The other ticks were dissected and examined for oocysts. Of all dogs inoculated orally with R. sanguineus, A. aureolatum, A. cajennense and A. ovale, only the animal that received the macerate of A. ovale was positive; evidence (gametocytes in peripheral blood) of infection was found 63 days after inoculation. Among all dissected ticks, we found only two oocysts; these were similar to those of Hepatozoon canis, and both were recovered from a single A. ovale specimen. We inoculated sporozoites recovered from the oocysts intraperitoneally into a Hepatozoon spp. negative dog, and circulating gametocytes were detected 84 days later. Our study demonstrated that A. ovale can be a vector of Hepatozoon spp. in Brazil.

American canine hepatozoonosis is caused by Hepatozoon americanum, a protozoan parasite, the definitive host of which is the tick, Amblyomma maculatum. Infection of the dog follows ingestion of ticks that harbor sporulated H. americanum oocysts. Following penetration of the intestinal mucosa, sporozoites are disseminated systemically and give rise to extensive asexual multiplication in cells located predominantly in striated muscle. The parasitized canine cells in "onion skin" cysts and in granulomas situated within skeletal muscle, as well as those in peripheral blood leukocytes (PBL), were identified as macrophages by use of fine structure morphology and/or immunohistochemical reactivity with macrophage markers. Additionally, two basic morphologic forms of the parasite were observed in macrophages of granulomas and PBLs. The forms were presumptively identified as merozoites and gamonts. The presence of a "tail" in some gamonts in PBLs indicated differentiation toward microgametes. Recognition of merozoites in PBLs supports the contention that hematogenously redistributed merozoites initiate repeated asexual cycles and could explain persistence of infection for long periods in the vertebrate host. Failure to clearly demonstrate a host cell membrane defining a parasitophorous vacuole may indicate that the parasite actively penetrates the host cell membrane rather than being engulfed by the host cell, as is characteristic of some protozoans.

The acquisition and transmission of rickettsial pathogens by different tick developmental stages has important epidemiological implications. The purpose of this study was to determine if male Rhipicephalus sanguineus can experimentally acquire and transmit Ehrlichia canis in the absence of female ticks. Two trials were performed where nymphal and male R. sanguineus were simultaneously acquisition fed on the same infected donor hosts, and transstadially or intrastadially exposed male ticks were fed on separate pathogen-free dogs as a test for transmission. A single-step p30-based PCR assay was used to test canine and tick hosts for E. canis infections before and after tick feeding. E. canis was detected after either intrastadial or transstadial passage in male ticks, the organism remained detectable in both tick groups after transmission feeding, and both tick groups transmitted the rickettsia to susceptible dogs. Infection of dogs via tick feeding resulted in milder clinical signs and lower antibody titers than intravenous inoculation of carrier blood, but further investigation is needed to understand the mechanisms responsible for this observation. These results demonstrate that male R. sanguineus can take multiple feedings, and that they can both acquire and transmit E. canis in the absence of female ticks. This tick development stage could be important in transmission of E. canis, and perhaps related pathogens, between vertebrate hosts under natural and experimental conditions.

Each of five adult and four juvenile coyotes (Canis latrans) was exposed to an oral dose of 50 Hepatozoon americanum oocysts recovered from Amblyomma maculatum ticks that previously fed on either naturally infected domestic dogs (Canis familiaris) or naturally infected wild coyotes. All coyotes exposed to H. americanum became infected, regardless of isolate source, and all exhibited mild to moderate clinical disease that simulated American canine hepatozoonosis in naturally infected dogs. At 100 days postexposure, parasitemia was greater in juvenile than adult coyotes (0.9% and 0.3%, respectively); radiographic imaging of femurs revealed moderate exostosis in all juveniles and mild to moderate new bone growth in four of five (80%) adult coyotes. Gross postmortem analysis of bone lesions demonstrated variation between age groups of coyotes but not between isolates of H. americanum. Microscopic evaluation of skeletal muscle revealed that parasite-induced lesions were significantly more numerous (t = 5.0, df = 7, P = 0.001) in juvenile than adult coyotes. Results of this study indicate that juvenile and adult coyotes are equally susceptible to experimental infection with H. americanum isolated from domestic dog and wild coyote sources. The age of coyotes at the time of exposure, and possibly the number of H. americanum oocysts ingested, might influence morbidity and mortality, but it appears that both adult and juvenile coyotes could be reservoirs of H. americanum.

Sequence analysis of the ribosomal DNA second internal transcribed spacer (ITS 2) region in 2 spatially distinct populations of Amblyomma americanum (L.) revealed intraspecific variation. Nucleotide sequences from multiple DNA extractions and several polymerase chain reaction amplifications of eggs from mixed-parentage samples from both populations of ticks revealed that 12 of 1,145 (1.0%) sites varied. Three of the 12 sites of variation were distinct between the 2 A. americanum populations, which corresponded to a rate of 0.26%. Phylogenetic analysis based on ITS 2 sequences provided strong support (i.e., bootstrap value of 80%) that wild A. americanum clustered into a distinguishable group separate from those derived from colony ticks.

Detection of Ehrlichia chaffeensis is necessary to study interactions between the parasite and its vertebrate and invertebrate hosts. The purpose of this study was to develop a sensitive, specific PCR assay for E. chaffeensis based on the outer membrane protein gene, p28. Candidate primer sets were identified and ranked based on annealing scores, similarities to three major p28 sequence clusters, dissimilarity to E. canis p30, an ortholog of p28, and the proximities of flanking primer sequences for nested PCR. The relative sensitivities of five optimized single-step and two nested PCR assays were determined, and the most sensitive assay was found to be a single-step PCR that was as much as 1000-fold more sensitive than a standard 16S rDNA-based nested PCR assay. This p28-based PCR assay amplified the target amplicon from isolates representative of all three major clusters of known p28 sequences, and this assay did not amplify template prepared from either of the two species most closely related to E. chaffeensis, E. canis and E. muris. These results indicate that this sensitive, specific and isolate-universal single-step PCR assay will be a useful tool in characterizing the transmission of this important zoonotic pathogen.

Parasitic life stages of Amblyomma maculatum Koch were collected from domestic cattle and several species of wild mammals during a 3.5-yr study (May 1998-October 2001) in north-central Oklahoma. Adult ticks were the predominant life stage collected from cattle, white-tailed deer, coyotes, and raccoons, whereas only immature ticks were collected from cotton rats and white-footed mice. The prevalence of adult A. maculatum on white-tailed deer (n = 15) examined in June, July, and August 1998 was 80, 100, 100%, respectively. The prevalence of adult A. maculatum on cattle (n = 84) ranged from 52% in February 1999 to 100% in May 1999. The prevalence of adult A. maculatum on coyotes (n = 16) was 100% in April 1998 and 43% on coyotes (n = 7) examined in January 2001. The prevalence of adult A. maculatum on raccoons (n = 23) examined during May, June, and July 1999 was 13%. No A. maculatum of any life stage were recovered from opossums (n = 7). Nine hundred forty-five rodents were trapped over 294 trap-nights; prevalence of A. maculatum larvae and nymphs on cotton rats (n = 395) was 34 and 15%, respectively, whereas on white-footed mice (n = 517), prevalence was 1.5 and 1.4%, respectively. No A. maculatum were recovered from pack rats (n = 33). There were significant differences (P = 0.0001) in larval infestation prevalence between cotton rats and white-footed mice in the spring, summer, and fall and for nymphs in the spring and summer. Results of A. maculatum parasitism and seasonal occurrence on hosts in this study are compared with previous research conducted in Oklahoma and with collection records of A. maculatum in the Entomology Museum at Oklahoma State University.

American canine hepatozoonosis (ACH) is a tick-borne disease that is spreading in the southeastern and south-central United States. Characterized by marked leukocytosis and periosteal bone proliferation, ACH is very debilitating and often fatal. Dogs acquire infection by ingesting nymphal or adult Gulf Coast ticks (Amblyomma maculatum) that, in a previous life stage, ingested the parasite in a blood meal taken from some vertebrate intermediate host. ACH is caused by the apicomplexan Hepatozoon americanum and has been differentiated from Old World canine hepatozoonosis caused by H. canis. Unlike H. canis, which is transmitted by the ubiquitous brown dog tick (Rhipicephalus sanguineus), H. americanum is essentially an accidental parasite of dogs, for which Gulf Coast ticks are not favored hosts. The geographic portrait of the disease parallels the known distribution of the Gulf Coast tick, which has expanded in recent years. Thus, the endemic cycle of H. americanum involves A. maculatum as definitive host and some vertebrate intermediate host(s) yet to be identified. Although coyotes (Canis latrans) are known to be infected, it is not known how important this host is in maintaining the endemic cycle. This review covers the biology of the parasite and of the tick that transmits it and contrasts ACH with classical canine hepatozoonosis. Clinical aspects of the disease are discussed, including diagnosis and treatment, and puzzling epidemiologic issues are examined. Brief consideration is given to the potential for ACH to be used as a model for study of angiogenesis and of hypertrophic osteoarthropathy.

To determine the persistence of Hepatozoon americanum in a naturally infected dog, skeletal muscle biopsies were performed at approximately 6-mo intervals over a period of 5.5 yr, and the samples were examined for presence of lesions of American canine hepatozoonosis (ACH). Nymphal Amblyomma maculatum (Gulf Coast tick) were allowed to feed to repletion on the dog periodically over the 5.5-yr period, and adult ticks were dissected and examined for presence of H. americanum oocysts. With 3 exceptions, the biopsied muscle contained lesions characteristic of ACH; no evidence of infection was found at 36, 54, and 67 mo after the original diagnosis. In every instance, nymphal Gulf Coast ticks became infected, indicating that dogs naturally infected with H. americanum can remain infectious for Gulf Coast ticks for at least 5.5 yr. Skeletal muscle biopsy is a reasonably reliable method of determining whether dogs are infected with the parasite. Xenodiagnosis using nymphal Gulf Coast ticks is an even more sensitive method, but the procedure is practicable only experimentally. Design of prevention and control measures for ACH must take into account knowledge that the parasite can survive in dogs, and presumably other vertebrate host(s), for long periods. Preventing ingestion of Gulf Coast ticks is an effective control measure.

American canine hepatozoonosis is an emerging, tick-transmitted infection of domestic dogs caused by a recently recognized species of apicomplexan parasite, Hepatozoon americanum. The known definitive host of the protozoan is the Gulf Coast tick, Amblyomma maculatum. Presently recognized intermediate hosts include the domestic dog and the coyote, Canis latrans. Laboratory-reared larval or nymphal A. maculatum can be infected readily by feeding to repletion on a parasitemic intermediate host; sporogony requires 35-40 days. Transmission of infection to the dog has been produced experimentally by oral administration of mature oocysts or oocyst-containing ticks. Canine disease follows experimental exposure in 4-6 weeks and is characterized by systemic illness, extreme neutrophilic leukocytosis, muscle and bone pain, and proliferation of periosteal bone. Histopathological findings include multifocal skeletal and cardiac myositis associated with escape of mature merozoites from within the host-cell environment. There is also rapid onset of periosteal activation and osteogenesis and, less frequently, glomerulopathy and amyloidosis. Sequential stages of development of H. americanum in both the dog and the tick have been elucidated. Gamonts potentially infectious to ticks have been observed in peripheral blood leukocytes of the dog in as few as 28 days after exposure to oocysts. Young coyotes experimentally exposed to a canine strain of H. americanum acquired disease indistinguishable from that of similarly exposed young dogs.

Williams Syndrome (WS) is marked by a relative strength in verbal cognition coupled with a serious impairment in non-verbal cognition. A strong deficit in numerical cognition has been anecdotally reported in this disorder; however, its nature has not been systematically investigated. Here, we tested 14 children with WS (mean age=7 years 2 months), 14 typically developing controls individually matched on visuo-spatial ability (mean age=3 years 5 months) as well as a larger group of typically developing controls (mean age=3 years 4 months) on two tasks to assess their understanding that counting determines the exact quantity of sets (cardinality principle). The understanding of the cardinality principle in children with WS is extremely delayed and only at the level predicted by their visuo-spatial MA. In this clinical group, only language accounted for a significant amount of the variance in cardinality understanding, whereas in the normal comparison group only visuo-spatial competence predicted the variance. The present findings suggest that visuo-spatial ability plays a greater role than language ability in the actual development of cardinality understanding in typically developing children, whereas the opposite obtains for the clinical group.

N-Arachidonoyldopamine (NADA) was recently identified as an endogenous ligand for the vanilloid type 1 receptor (VR1). Further analysis of the bovine striatal extract from which NADA was isolated indicated the existence of substances corresponding in molecular mass to N-oleoyldopamine (OLDA), N-palmitoyldopamine (PALDA), and N-stearoyldopamine (STEARDA). Quadrupole time-of-flight mass spectrometric analysis of bovine striatal extracts revealed the existence of OLDA, PALDA, and STEARDA as endogenous compounds in the mammalian brain. PALDA and STEARDA failed to affect calcium influx in VR1-transfected human embryonic kidney (HEK) 293 cells or paw withdrawal latencies from a radiant heat source, and there was no evidence of spontaneous pain behavior. By contrast, OLDA induced calcium influx (EC(50) = 36 nm), reduced the latency of paw withdrawal from a radiant heat source in a dose-dependent manner (EC(50) = 0.72 microg), and produced nocifensive behavior. These effects were blocked by co-administration of the VR1 antagonist iodo-resiniferatoxin (10 nm for HEK cells and 1 microg/50 micro;l for pain behavior). These findings demonstrate the existence of an endogenous compound in the brain that is similar to capsaicin and NADA in its chemical structure and activity on VR1. Unlike NADA, OLDA was only a weak ligand for rat CB1 receptors; but like NADA, it was recognized by the anandamide membrane transporter while being a poor substrate for fatty-acid amide hydrolase. Analysis of the activity of six additional synthetic and potentially endogenous N-acyldopamine indicated the requirement of a long unsaturated fatty acid chain for an optimal functional interaction with VR1 receptors.

American canine hepatozoonosis (ACH) caused by Hepatozoon americanum Vincent-Johnson, Macintire, Lindsay, Lenz, Baneth, and Shkap is an emerging, often fatal, tick-borne protozoal disease of dogs in the United States of America. Dogs acquire infection by ingesting ticks that contain oocysts. To understand the invertebrate (definitive) host range of H. americanum, experiments were carried out using four ixodids, Rhipicephalus sanguineus (Latreille), Dermacentor variabilis Say, Amblyomma americanum (L.), and Amblyomma maculatum Koch. Laboratory-reared nymphal ticks were fed on dogs that were either naturally or experimentally infected with H. americanum; when these ticks molted to the adult stage they were either fed to susceptible dogs or were dissected and examined for the presence of oocysts. Mature H. americanum oocysts were found in >90% of A. maculatum (both males and females), whereas oocysts were not found in any of the other three species. These results confirm that A. maculatum is an excellent host and vector for H. americanum and also suggest that this apicomplexan may have a narrow invertebrate host range, at least among ixodid ticks that are likely candidate vectors in the United States.