Robert J. Bildfell

Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the northwest United States.

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.

Molecular Evidence That the Range of the Vancouver Island Outbreak of Cryptococcus gattii Infection Has Expanded into the Pacific Northwest in the United States

Cryptococcus neoformans frequently causes fungal meningitis in immunocompromised patients, whereas the related species C. gattii is restricted to tropical and subtropical regions,where it usually infects immunocompetent individuals.An outbreak of C. gattii infection that began in 1999 on Vancouver Island has resulted in endemic C. gattii infection and caused numerous human and veterinary infections; the outbreaks range has spread to mainland British Columbia. The outbreak-related isolates have been molecular type VGIIa, the major genotype, or VGIIb, the minor genotype. Since 2006, human and veterinary cases of C. gattii infection have emerged in Washington and Oregon. Multilocus sequence typing demonstrates the spread of C. gattii VGIIa and VGIIb from Vancouver Island to the Pacific Northwest. Clinical strains recovered in Oregon represent a unique VGIIc genotype.

Coxiella Burnetii Infection is Associated with Placentitis in Cases of Bovine Abortion

A positive score on a modified acid-fast (MAF)-stained smear test of fresh placenta was used to identify a group of bovine abortion submissions believed to be infected with Coxiella burnetii. Immunohistochemical (IHC) testing for Coxiella and Chlamydia antigens was performed on 14 MAF smear-positive cases as well as 29 MAF smear-negative cases Received during the study period. Nine MAF smear-positive cases as well as 1 MAF smear-negative case were Coxiella-positive via the IHC test. No placentas were positive for Chlamydia antigen. Various histopathologic features were categorized for all placentas and the presence or absence of selected risk categories was also graded for each case. The results between Coxiella IHC-positive cases and Coxiella IHC-negative/MAF-negative cases were compared using Fishers exact test (P value at 95% confidence). Significant associations were found between Coxiella IHC-positive cases and the presence of placental inflammation (P = 0.0027), placental necrosis (P = 0.012), fetal pneumonia (P = 0.0152), and the visibility of Coxiella-like organisms within trophoblasts on hematoxylin and eosin–stained sections (P < 0.0001). Histopathologic features of Coxiella IHC-positive placentas included infiltration of the chorionic stroma by mononuclear cells, necrosis of chorionic trophoblasts, and focal exudation of fibrin and neutrophils. The results indicate that MAF smears are a good screening tool for the presence of Coxiella in placentas from bovine abortion cases and that the detection of this pathogen in aborted placentas via traditional staining or IHC methods is usually associated with placentitis.

Investigation of Koi Herpesvirus Latency in Koi

ABSTRACT Koi herpesvirus (KHV) has recently been classified as a member of the family of Alloherpesviridae within the order of Herpesvirales. One of the unique features of Herpesviridae is latent infection following a primary infection. However, KHV latency has not been recognized. To determine if latency occurs in clinically normal fish from facilities with a history of KHV infection or exposure, the presence of the KHV genome was investigated in healthy koi by PCR and Southern blotting. KHV DNA, but not infectious virus or mRNAs from lytic infection, was detected in white blood cells from investigated koi. Virus shedding was examined via tissue culture and reverse transcription-PCR (RT-PCR) testing of gill mucus and feces from six koi every other day for 1 month. No infectious virus or KHV DNA was detected in fecal secretion or gill swabs, suggesting that neither acute nor persistent infection was present. To determine if KHV latent infections can be reactivated, six koi were subjected to a temperature stress regime. KHV DNA and infectious virus were detected in both gill and fecal swabs by day 8 following temperature stress. KHV DNA was also detectable in brain, spleen, gills, heart, eye, intestine, kidney, liver, and pancreas in euthanized koi 1 month post-temperature stress. Our study suggests that KHV may become latent in leukocytes and other tissues, that it can be reactivated from latency by temperature stress, and that it may be more widespread in the koi population than previously suspected.

Chitosan Dressing Provides Hemostasis in Swine Femoral Arterial Injury Model

Objective. Chitosan dressings have been shown to be effective in improving survival of severe parenchymal injuries in an animal model andin treating prehospital combat casualties. Our goal was to test the efficacy of chitosan acetate dressings in providing durable hemostasis in a high-flow arterial wound model. Methods. A proximal arterial injury was created with 2.7-mm vascular punches in both femoral arteries of fourteen anesthetized swine. By using a crossover design, 48-ply gauze (48PG) or a chitosan dressing (HC) was applied with pressure to the injury for 3 minutes andthen released. If hemostasis was not maintained for 30 minutes, a second identical attempt was made by using the same dressing type. If hemostasis was still not achieved, the dressing was considered an acute failure andthe alternate dressing type was applied. If failure of hemostasis occurred between 30 and240 minutes after application, the dressing was considered a chronic failure andthe artery was ligated. Results. All 25/25 (100%) of the HC tests and3/14 (21%) of the 48PG maintained hemostasis for 30 minutes. At 240 minutes, 21/25 (84%) of the HC tests and1/14 (7%) of the 48PG maintained hemostasis. Statistical analysis by Fischers exact test shows a significant (p < 0.001) difference in hemostatic efficacy between the 48PG andHC groups in this model, both at 30 minutes andat 240 minutes. Conclusion. Chitosan acetate hemorrhage control dressings provided superior hemostasis to 48 ply gauze in high inguinal femoral arterial injuries. Chitosan-based dressings may provide prehospital treatment options for hemostasis in patients with severe hemorrhagic arterial injuries.

Identification of Mycobacterium avium pathogenicity island important for macrophage and amoeba infection

The ability to infect macrophages is a common characteristic shared among many mycobacterial species. Mycobacterium avium, Mycobacterium tuberculosis, and Mycobacterium kansasii enter macrophages, using the complement receptors CR1, CR3, CR4, and the mannose receptor. To identify M. avium genes and host cell pathways involved in the bacterial uptake by macrophages, we screened a M. avium transposon mutant library for the inability to enter macrophages. Uptake-impaired clones were selected. Sequence of six M. avium clones identified one gene involved in glycopeptidolipid biosynthesis, one gene encoding the conserved membrane protein homologue to the M. avium subsp. paratuberculosis MAP2446c gene and four others belonging to the same region of the chromosome. Analysis of the chromosome region revealed a pathogenicity island inserted between two tRNA sequences with 58% of G+C content versus 69% in the M. avium genome. The region is unique for M. avium and is not present in M. tuberculosis or M. paratuberculosis. Although the mutants did not differ from the WT bacterium regarding the binding to macrophage cell membrane, analysis of macrophage proteins after 1 h infection revealed a deficiency in the mutant to phosphorylate certain proteins on uptake. To understand M. avium interaction with two evolutionarily distinct hosts, the mutants were evaluated for Acanthamoeba castellanii invasion. The defect in the ability of the mutants to invade both cells was highly similar, suggesting that M. avium might have evolved mechanisms that are used to enter amoebas and human macrophages.

Dietary CLA alters yolk and tissue FA composition and hepatic histopathology of laying hens.

The effect of dietary CLA along with n-3 PUFA on yolk FA profile and hepatic lipid accumulation was investigated. Laying hens (n=40) were randomly assigned to four experimental diets containing 0, 0.5, 1.0, or 2.0% CLA. Menhaden oil was used as the source of n-3 PUFA. Dietary CLA did not affect the total lipid content of egg yolk (P>0.05). The amounts of CLA isomers (cis-9 trans-11, trans-10 cis-12) in the egg yolk were proportional to the levels of CLA in the diet (P<0.05). The total CLA content in the egg yolk was 0, 0.97, 2.4, and 5.3 wt%, respectively (P<0.05). Addition of CLA resulted in an increase in saturated FA (P<0.05) with a concomitant reduction in monounsaturated FA (P<0.05) in the yolk, liver, abdominal fat, breast, and thigh muscle. No difference in saturated and monounsaturated FA content in heart and spleen tissue was noted. Dietary CLA at all concentrations resulted in an increase (P<0.05) in the total number of fat vacuoles and lipid infiltration in hepatocytes. The number of cells with 75% or higher lipid vacuolation in the cytoplasm was also increased (P<0.05) by 2.0% CLA. Dietary CLA at 0.5% levels resulted in an increase (P<0.05) in the total lipid content of hepatic tissue. The total lipid content in leg muscle was lower (P<0.05) in CLA-fed birds. However, no effect of CLA on lipid content of breast muscle, heart, spleen and adipose tissue was observed (P>0.05). The current study used CLA in a FFA form. The effects of using CLA in other form such as TG on avian hepatic tissue need to be investigated.

Incidence of Polysaccharide Storage Myopathy in Draft Horse-Related Breeds: A Necropsy Study of 37 Horses and a Mule

Skeletal muscle samples from 38 draft horse–related animals 1–23 years of age were evaluated for evidence of aggregates of glycogen and complex polysaccharide characteristic of equine polysaccharide storage myopathy (EPSSM). Cardiac muscle from 12 of these horses was also examined. Antemortem serum levels of creatine kinase (CK) and aspartate aminotransferase (AST) from 9 horses with EPSSM and 5 horses without EPSSM were compared. Skeletal muscle from 17 horses contained inclusions of periodic acid–Schiff (PAS)-positive, amylase-resistant complex polysaccharide. Similar inclusions were also present in the cardiac muscle of 1 horse. A vacuolar myopathy with aggregates of PAS-positive, amylase-sensitive glycogen was seen in 8 other horses, and these findings are also considered diagnostic for EPSSM. Antemortem serum activities of CK and AST were often higher in EPSSM horses than in horses without EPSSM. Using the presence of amylase-resistant complex polysaccharide as the criterion for diagnosis of EPSSM, the incidence in this population was 45%. Inclusion of horses with aggregates of glycogen but no amylase-resistant complex polysaccharide as representative of the range of pathologic findings in horses with EPSSM resulted in a 66% incidence in this population.

The influence of diesel exhaust on polycyclic aromatic hydrocarbon-induced DNA damage, gene expression and tumor initiation in Sencar mice in vivo

The carcinogenic effects of individual polycyclic aromatic hydrocarbons (PAH) are well established. However, their potency within an environmental complex mixture is uncertain. We evaluated the influence of diesel exhaust particulate matter on PAH-induced cytochrome P450 (CYP) activity, PAH-DNA adduct formation, expression of certain candidate genes and the frequency of tumor initiation in the two-stage Sencar mouse model. To this end, we monitored the effects of treatment of mice with diesel exhaust, benzo[a]pyrene (BP), dibenzo[a,l]pyrene (DBP), or a combination of diesel exhaust with either carcinogenic PAH. The applied diesel particulate matter (SRM(1975)) altered the tumor initiating potency of DBP: a statistically significant decrease in overall tumor and carcinoma burden was observed following 25 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA), compared with DBP exposure alone. From those mice that were treated at the beginning of the observation period with 2 nmol DBP all survivors developed tumors (9 out of 9 animals, 100%). Among all tumors counted at the end, nine carcinomas were detected and an overall tumor incidence of 2.6 tumors per tumor-bearing animal (TBA) was determined. By contrast, co-treatment of DBP with 50mg SRM(1975) led to a tumor rate of only 66% (19 out of 29 animals), occurrence of only three carcinomas in 29 animals and an overall rate of 2.1 tumors per TBA (P=0.04). In contrast to the results with DBP, the tumor incidence induced by 200 nmol BP was found slightly increased when co-treatment with SRM(1975) occurred (71% vs. 85% after 25 weeks). Despite this difference in tumor incidence, the numbers of carcinomas and tumors per TBA did not differ statistically significant between both treatment groups possibly due to the small size of the BP treatment group. Since bioactivation of DBP, but not BP, predominantly depends on CYP1B1 enzyme activity, SRM(1975) affected PAH-induced carcinogenesis in an antagonistic manner when CYP1B1-mediated bioactivation was required. The explanation most likely lies in the much stronger inhibitory effects of certain PAHs present in diesel exhaust on CYP1B1 compared to CYP1A1. In the present study we also found molecular markers such as highly elevated AKR1C21 and TNFRSF21 gene expression levels in tumor tissue derived from animals co-treated with SRM(1975) plus DBP. Therefore we validate microarray data as a source to uncover transcriptional signatures that may provide insights into molecular pathways affected following exposure to environmental complex mixtures such as diesel exhaust particulates.

Cryptococcus gattii with bimorphic colony types in a dog in Western Oregon: additional evidence for expansion of the Vancouver Island outbreak

Cryptococcus gattii was isolated from a 1.5-year-old dog with systemic cryptococcosis in Oregon. The dog had no link to Vancouver Island or British Columbia, Canada. Samples from a nasal swab and from a granulomatous mass within the cranial cavity were pooled for culture. Colonies on Sabouraud dextrose agar were mucoid and exhibited bimorphic morphology, melanin-pigmented and unpigmented. Pigmented colonies were encapsulated budding spherical yeast, whereas unpigmented colonies were of unencapsulated ovoid budding yeast. In addition to defective melanin production, the unpigmented colony type exhibited defective mating. Genetic analysis by high-resolution multilocus sequence typing revealed that the 2 isolates are genetically identical at 8 unlinked loci tested and that the 2 isolates are both the VGIIa Vancouver Island major genotype. Findings are consistent with expansion of the Vancouver Island outbreak onto the mainland Pacific Northwest region of the United States.