Effects of residential acaricide treatments on patterns of pathogen coinfection in blacklegged ticks.

Medically important ixodid ticks often carry multiple pathogens, with individual ticks frequently coinfected and capable of transmitting multiple infections to hosts, including humans. Acquisition of multiple zoonotic pathogens by immature blacklegged ticks (Ixodes scapularis) is facilitated when they feed on small mammals, which are the most competent reservoir hosts for Anaplasma phagocytophilum (which causes anaplasmosis in humans), Babesia microti (babesiosis) and Borrelia burgdorferi (Lyme disease). Here, we used data from a large-scale, long-term experiment to ask whether patterns of single and multiple infections in questing nymphal I. scapularis ticks from residential neighbourhoods differed from those predicted by independent assortment of pathogens, and whether patterns of coinfection were affected by residential application of commercial acaricidal products. Quantitative polymerase chain reaction was used for pathogen detection in multiplex reactions. In control neighbourhoods and those treated with a fungus-based biopesticide deployed against host-seeking ticks (Met52), ticks having only single infections of either B. microti or B. burgdorferi were significantly less common than expected, whereas coinfections with these 2 pathogens were significantly more common. However, use of tick control system bait boxes, which kill ticks attempting to feed on small mammals, eliminated the bias towards coinfection. Although aimed at reducing the abundance of host-seeking ticks, control methods directed at ticks attached to small mammals may influence human exposure to coinfected ticks and the probability of exposure to multiple tick-borne infections.

Effects of Tick-Control Interventions on Tick Abundance, Human Encounters with Ticks, and Incidence of Tickborne Diseases in Residential Neighborhoods, New York, USA.

Tickborne diseases (TBDs) such as Lyme disease result in ≈500,000 diagnoses annually in the United States. Various methods can reduce the abundance of ticks at small spatial scales, but whether these methods lower incidence of TBDs is poorly understood. We conducted a randomized, replicated, fully crossed, placebo-controlled, masked experiment to test whether 2 environmentally safe interventions, the Tick Control System (TCS) and Met52 fungal spray, used separately or together, affected risk for and incidence of TBDs in humans and pets in 24 residential neighborhoods. All participating properties in a neighborhood received the same treatment. TCS was associated with fewer questing ticks and fewer ticks feeding on rodents. The interventions did not result in a significant difference in incidence of human TBDs but did significantly reduce incidence in pets. Our study is consistent with previous evidence suggesting that reducing tick abundance in residential areas might not reduce incidence of TBDs in humans.

Resolution of hepatitis E virus infection in CD8+ T cell-depleted rhesus macaques.

BACKGROUND & AIMS: HEV is a significant cause of acute hepatitis globally. Some genotypes establish persistent infection when immunity is impaired. Adaptive immune mechanisms that mediate resolution of infection have not been identified. Herein, the requirement for CD8+ T cells to control HEV infection was assessed in rhesus macaques, a model of acute and persistent HEV infection in humans. METHODS: Rhesus macaques were untreated or treated with depleting anti-CD8α monoclonal antibodies before challenge with an HEV genotype (gt)3 isolate derived from a chronically infected human patient. HEV replication, alanine aminotransferase, anti-capsid antibody and HEV-specific CD4+ and CD8+ T cell responses were assessed after infection. RESULTS: HEV control in untreated macaques coincided with the onset of a neutralizing IgG response against the ORF2 capsid and liver infiltration of functional HEV-specific CD4+ and CD8+ T cells. Virus control was delayed by 1 week in CD8+ T cell-depleted macaques. Infection resolved with onset of a neutralizing IgG antibody response and a much more robust expansion of CD4+ T cells with antiviral effector function. CONCLUSIONS: Liver infiltration of functional CD8+ T cells coincident with HEV clearance in untreated rhesus macaques, and a 1-week delay in HEV clearance in CD8+ T cell-depleted rhesus macaques, support a role for this subset in timely control of virus replication. Resolution of infection in the absence of CD8+ T cells nonetheless indicates that neutralizing antibodies and/or CD4+ T cells may act autonomously to inhibit HEV replication. HEV susceptibility to multiple adaptive effector mechanisms may explain why persistence occurs only with generalized immune suppression. The findings also suggest that neutralizing antibodies and/or CD4+ T cells should be considered as a component of immunotherapy for chronic infection. LAY SUMMARY: The hepatitis E virus (HEV) is a major cause of liver disease globally. Some genetic types (genotypes) of HEV persist in the body if immunity is impaired. Our objective was to identify immune responses that promote clearance of HEV. Findings indicate that HEV may be susceptible to multiple arms of the immune response that can act independently to terminate infection. They also provide a pathway to assess immune therapies for chronic HEV infection.

Vascular delivery of rAAVrh74.MCK.GALGT2 to the gastrocnemius muscle of the rhesus macaque stimulates the expression of dystrophin and laminin α2 surrogates.

Overexpression of GALGT2 in skeletal muscle can stimulate the glycosylation of α dystroglycan and the upregulation of normally synaptic dystroglycan-binding proteins, some of which are dystrophin and laminin α2 surrogates known to be therapeutic for several forms of muscular dystrophy. This article describes the vascular delivery of GALGT2 gene therapy in a large animal model, the rhesus macaque. Recombinant adeno-associated virus, rhesus serotype 74 (rAAVrh74), was used to deliver GALGT2 via the femoral artery to the gastrocnemius muscle using an isolated focal limb perfusion method. GALGT2 expression averaged 44 ± 4% of myofibers after treatment in macaques with low preexisting anti-rAAVrh74 serum antibodies, and expression was reduced to 9 ± 4% of myofibers in macaques with high preexisting rAAVrh74 immunity (P < 0.001; n = 12 per group). This was the case regardless of the addition of immunosuppressants, including prednisolone, tacrolimus, and mycophenolate mofetil. GALGT2-treated macaque muscles showed increased glycosylation of α dystroglycan and increased expression of dystrophin and laminin α2 surrogate proteins, including utrophin, plectin1, agrin, and laminin α5. These experiments demonstrate successful transduction of rhesus macaque muscle with rAAVrh74.MCK.GALGT2 after vascular delivery and induction of molecular changes thought to be therapeutic in several forms of muscular dystrophy.

Impacts Over Time of Neighborhood-Scale Interventions to Control Ticks and Tick-Borne Disease Incidence.

Background: Controlling populations of ticks with biological or chemical acaricides is often advocated as a means of reducing human exposure to tick-borne diseases. Reducing tick abundance is expected to decrease immediate risk of tick encounters and disrupt pathogen transmission cycles, potentially reducing future exposure risk. Materials and Methods: We designed a placebo-controlled, randomized multiyear study to assess whether two methods of controlling ticks-tick control system (TCS) bait boxes and Met52 spray-reduced tick abundance, tick encounters with people and outdoor pets, and reported cases of tick-borne diseases. The study was conducted in 24 residential neighborhoods in a Lyme disease endemic zone in New York State. We tested the hypotheses that TCS bait boxes and Met52, alone or together, would be associated with increasing reductions in tick abundance, tick encounters, and cases of tick-borne disease over the 4-5 years of the study. Results: In neighborhoods with active TCS bait boxes, populations of blacklegged ticks (Ixodes scapularis) were not reduced over time in any of the three habitat types tested (forest, lawn, shrub/garden). There was no significant effect of Met52 on tick abundance overall, and there was no evidence for a compounding effect over time. Similarly, we observed no significant effect of either of the two tick control methods, used singly or together, on tick encounters or on reported cases of tick-borne diseases in humans overall, and there was no compounding effect over time. Thus, our hypothesis that effects of interventions would accumulate through time was not supported. Conclusions: The apparent inability of the selected tick control methods to reduce risk and incidence of tick-borne diseases after years of use requires further consideration.

Effects of Neighborhood-Scale Acaricidal Treatments on Infection Prevalence of Blacklegged Ticks (Ixodes scapularis) with Three Zoonotic Pathogens.

Acaricides are hypothesized to reduce human risk of exposure to tick-borne pathogens by decreasing the abundance and/or infection prevalence of the ticks that serve as vectors for the pathogens. Acaricides targeted at reservoir hosts such as small mammals are expected to reduce infection prevalence in ticks by preventing their acquisition of zoonotic pathogens. By reducing tick abundance, reservoir-targeted or broadcast acaricides could reduce tick infection prevalence by interrupting transmission cycles between ticks and their hosts. Using an acaricide targeted at small-mammal hosts (TCS bait boxes) and one sprayed on low vegetation (Met52 fungal biocide), we tested the hypotheses that infection prevalence of blacklegged ticks with zoonotic pathogens would be more strongly diminished by TCS bait boxes, and that any effects of both acaricidal treatments would increase during the four years of deployment. We used a masked, placebo-controlled design in 24 residential neighborhoods in Dutchess County, New York. Analyzing prevalence of infection with Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti in 5380 nymphal Ixodes scapularis ticks, we found little support for either hypothesis. TCS bait boxes did not reduce infection prevalence with any of the three pathogens compared to placebo controls. Met52 was associated with lower infection prevalence with B. burgdorferi compared to placebo controls but had no effect on prevalence of infection with the other two pathogens. Although significant effects of year on infection prevalence of all three pathogens were detected, hypothesized cumulative reductions in prevalence were observed only for B. burgdorferi. We conclude that reservoir-targeted and broadcast acaricides might not generally disrupt pathogen transmission between reservoir hosts and tick vectors or reduce human risk of exposure to tick-borne pathogens.

Spatial variation in risk for tick-borne diseases in residential areas of Dutchess County, New York.

Although human exposure to the ticks that transmit Lyme-disease bacteria is widely considered to occur around people's homes, most studies of variation in tick abundance and infection are undertaken outside residential areas. Consequently, the patterns of variation in risk of human exposure to tick-borne infections in these human-dominated landscapes are poorly understood. Here, we report the results of four years of sampling for tick abundance, tick infection, tick encounters, and tick-borne disease reports on residential properties nested within six neighborhoods in Dutchess County, New York, USA, an area of high incidence for Lyme and other tick-borne diseases. All properties were within neighborhoods that had been randomly assigned as placebo controls in The Tick Project; hence, none were treated to reduce tick abundance during the period of investigation, providing a unique dataset of natural variation within and between neighborhoods. We estimated the abundance of host-seeking blacklegged ticks (Ixodes scapularis) in three types of habitats on residential properties-forests, lawns, and gardens. In forest and lawn habitats, some neighborhoods had consistently higher tick abundance. Properties within neighborhoods also varied consistently between years, suggesting hot spots and cold spots occurring at a small (~ 1-hectare) spatial scale. Across neighborhoods, the abundance of nymphal ticks was explained by neither the amount of forest in that neighborhood, nor by the degree of forest fragmentation. The proportion of ticks infected with three common tick-borne pathogens did not differ significantly between neighborhoods. We observed no effect of tick abundance on human encounters with ticks, nor on either human or pet cases of tick-borne diseases. However, the number of encounters between ticks and outdoor pets in a neighborhood was negatively correlated with the abundance of questing ticks in that neighborhood. Our results reinforce the need to understand how human behavior and neglected ecological factors affect variation in human encounters with ticks and cases of tick-borne disease in residential settings.

Sustained alpha-sarcoglycan gene expression after gene transfer in limb-girdle muscular dystrophy, type 2D.

OBJECTIVE: The aim of this study was to attain long-lasting alpha-sarcoglycan gene expression in limb-girdle muscular dystrophy, type 2D (LGMD2D) subjects mediated by adeno-associated virus (AAV) gene transfer under control of a muscle specific promoter (tMCK). METHODS: rAAV1.tMCK.hSGCA (3.25 × 10¹¹ vector genomes) was delivered to the extensor digitorum brevis muscle of 3 subjects with documented SGCA mutations via a double-blind, randomized, placebo controlled trial. Control sides received saline. The blind was not broken until the study was completed at 6 months and all results were reported to the oversight committee. RESULTS: Persistent alpha-sarcoglycan gene expression was achieved for 6 months in 2 of 3 LGMD2D subjects. Markers for muscle fiber transduction other than alpha-sarcoglycan included expression of major histocompatibility complex I, increase in muscle fiber size, and restoration of the full sarcoglycan complex. Mononuclear inflammatory cells recruited to the site of gene transfer appeared to undergo programmed cell death, demonstrated by terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling and caspase-3 staining. A patient failing gene transfer demonstrated an early rise in neutralizing antibody titers and T-cell immunity to AAV, validated by enzyme-linked immunospot on the second day after gene injection. This was in clear distinction to other participants with satisfactory gene expression. INTERPRETATION: The findings of this gene replacement study in LGMD2D subjects have important implications not previously demonstrated in muscular dystrophy. Long-term, sustainable gene expression of alpha-sarcoglycan was observed following gene transfer mediated by AAV. The merit of a muscle-specific tMCK promoter, not previously used in a clinical trial, was evident, and the potential for reversal of disease was displayed.

Persistent expression of FLAG-tagged micro dystrophin in nonhuman primates following intramuscular and vascular delivery.

Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Nonhuman primates (NHPs) provide the ideal model to optimize vector delivery across a vascular barrier and provide accurate dose estimates for widespread transduction. To address vascular delivery and dosing in rhesus macaques, we have generated a fusion construct that encodes an eight amino-acid FLAG epitope at the C-terminus of micro-dystrophin to facilitate translational studies targeting DMD. Intramuscular (IM) injection of AAV8.MCK.micro-dys.FLAG in the tibialis anterior (TA) of macaques demonstrated robust gene expression, with muscle transduction (50-79%) persisting for up to 5 months. Success by IM injection was followed by targeted vascular delivery studies using a fluoroscopy-guided catheter threaded through the femoral artery. Three months after gene transfer, >80% of muscle fibers showed gene expression in the targeted muscle. No cellular immune response to AAV8 capsid, micro-dystrophin, or the FLAG tag was detected by interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot (ELISpot) at any time point with either route. In summary, an epitope-tagged micro-dystrophin cassette enhances the ability to evaluate site-specific localization and distribution of gene expression in the NHP in preparation for vascular delivery clinical trials.

Limb-girdle muscular dystrophy type 2D gene therapy restores alpha-sarcoglycan and associated proteins.

OBJECTIVE: alpha-Sarcoglycan deficiency results in a severe form of muscular dystrophy (limb-girdle muscular dystrophy type 2D [LGMD2D]) without treatment. Gene replacement represents a strategy for correcting the underlying defect. Questions related to this approach were addressed in this clinical trial, particularly the need for immunotherapy and persistence of gene expression. METHODS: A double-blind, randomized controlled trial using rAAV1.tMCK.hSGCA injected into the extensor digitorum brevis muscle was conducted. Control sides received saline. A 3-day course of methylprednisolone accompanied gene transfer without further immune suppression. RESULTS: No adverse events were encountered. SGCA gene expression increased 4-5-fold over control sides when examined at 6 weeks (2 subjects) and 3 months (1 subject). The full sarcoglycan complex was restored in all subjects, and muscle fiber size was increased in the 3-month subject. Adeno-associated virus serotype 1 (AAV1)-neutralizing antibodies were seen as early as 2 weeks. Neither CD4+ nor CD8+ cells were increased over contralateral sides. Scattered foci of inflammation could be found, but showed features of programmed cell death. Enzyme-linked immunospot (ELISpot) showed no interferon-gamma response to alpha-SG or AAV1 capsid peptide pools, with the exception of a minimal capsid response in 1 subject. Restimulation to detect low-frequency capsid-specific T cells by ELISpot assays was negative. Results of the first 3 subjects successfully achieved study aims, precluding the need for additional enrollment. INTERPRETATION: The finding of this gene replacement study in LGMD2D has important implications for muscular dystrophy. Sustained gene expression was seen, but studies over longer time periods without immunotherapy will be required for design of vascular delivery gene therapy trials.

Erratum: Role of Interventional Radiologist in the Management of Acute Pulmonary Embolism.

[This corrects the article DOI: 10.1055/s-0039-3401841.].

Role of Interventional Radiologist in the Management of Acute Pulmonary Embolism.

Pulmonary embolism is a common cause of morbidity and mortality which continues to increase in overall incidence. Because it can occur with a wide range of clinical presentations, different guidelines have been developed for appropriate risk stratification of patients; interventional radiology plays a vital role in the management of both massive and submassive pulmonary embolism. Catheter-directed therapy, including mechanical and aspiration thrombectomy, standard catheter-directed thrombolysis, and ultrasound-accelerated thrombolysis, has many benefits, including lower thrombolytic doses and intraclot administration of thrombolytic therapy. While the role of catheter-directed therapy is still being developed, four important prospective studies have demonstrated its safety and efficacy. Additional studies comparing short- and long-term clinical outcomes in patients treated with catheter-directed therapy versus anticoagulation are the next step in understanding its role within the management of submassive pulmonary embolism. Furthermore, multidisciplinary pulmonary embolism response teams, in which interventional radiology plays a crucial role, are becoming essential to appropriately managing pulmonary embolism patients, including selection of those who may benefit from catheter-directed therapy.

Arterial-portal fistula treated with hepatic arterial embolization and portal venous aneurysm stent-graft exclusion complicated by type 2 endoleak.

Intrahepatic arterioportal fistulas may be complicated by portal hypertension. An associated portal venous aneurysm (PVA) may impinge upon adjacent structures or rupture. We present a 65-year-old man with an intrahepatic Intrahepatic arterioportal fistula and 6.4 × 5.8 cm right portal vein aneurysm extending within 0.4 cm of the hepatic margin, associated with pain concerning for impending rupture. The PVA was refractory to transarterial embolization due to recruitment of arterial collaterals. Therefore, it was additionally excluded from the portal vein with a 12 mm × 9.5 cm venous stent graft. Although endovascular therapy thrombosed the aneurysm and improved symptoms, it was complicated by a type 2 endoleak into the PVA.

Host surveys, ixodid tick biology and transmission scenarios as related to the tick-borne pathogen, Ehrlichia canis.

The ehrlichioses have been subject to increasing interest from veterinary and public health perspectives, but experimental studies of these diseases and their etiologic agents can be challenging. Ehrlichia canis, the primary etiologic agent of canine monocytic ehrlichiosis, is relatively well characterized and offers unique advantages and opportunities to study interactions between a monocytotropic pathogen and both its vertebrate and invertebrate hosts. Historically, advances in tick-borne disease control strategies have typically followed explication of tick-pathogen-vertebrate interactions, thus it is reasonable to expect novel, more sustainable approaches to control of these diseases as the transmission of their associated infections are investigated at the molecular through ecological levels. Better understanding of the interactions between E. canis and its canine and tick hosts would also elucidate similar interactions for other Ehrlichia species as well as the potential roles of canine sentinels, reservoirs and models of tick-borne zoonoses. This article summarizes natural exposure studies and experimental investigations of E. canis in the context of what is understood about biological vectors of tick-borne Anaplasmataceae.

Serial FDG-PET/CT Imaging in the Management of Cardiac Sarcoidosis.

A 60-year-old woman with biopsy-proven cardiac sarcoidosis status post implantable cardioverter defibrillator placement presented with periodic dizziness and dyspnea on exertion. Myocardial perfusion scan demonstrated a moderate sized, fixed perfusion defect along the mid to proximal anteroseptal wall of the left ventricle, with better perfusion on stress images. FDG-PET/CT demonstrated corresponding focal FDG avidity of the mid to proximal anteroseptal wall, suggestive of active cardiac sarcoidosis. Because of severe side effects, mycophenolate mofetil was discontinued, and the patient received low-dose steroid therapy. At 7-month follow-up FDG PET/CT, there was increased FDG avidity in the same regions, indicating worsening cardiac sarcoidosis.

Experimental infection of dairy calves with Ehrlichia chaffeensis.

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.

Delayed Tumor Seeding Along Axillary Lymph Node Biopsy Tract Visualized on FDG PET/CT.

A 59-year-old woman with right breast cancer underwent an ultrasound-guided core needle biopsy of a right axilla lymph node. Pathology results revealed metastases from breast cancer. The patient had serial FDG PET/CT scans for further workup and therapy assessment during the disease course. Hypermetabolic tumor seeding along the needle biopsy tract was not evident at FDG PET/CT scan performed 5 weeks after biopsy but became apparent at 10 weeks after biopsy and progressed subsequently.

Transstadial and intrastadial experimental transmission of Ehrlichia canis by male Rhipicephalus sanguineus.

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.

A GLP-Compliant Toxicology and Biodistribution Study: Systemic Delivery of an rAAV9 Vector for the Treatment of Mucopolysaccharidosis IIIB.

No treatment is currently available for mucopolysaccharidosis (MPS) IIIB, a neuropathic lysosomal storage disease due to defect in α-N-acetylglucosaminidase (NAGLU). In preparation for a clinical trial, we performed an IND-enabling GLP-toxicology study to assess systemic rAAV9-CMV-hNAGLU gene delivery in WT C57BL/6 mice at 1 × 10(14) vg/kg and 2 × 10(14) vg/kg (n = 30/group, M:F = 1:1), and non-GLP testing in MPS IIIB mice at 2 × 10(14) vg/kg. Importantly, no adverse clinical signs or chronic toxicity were observed through the 6 month study duration. The rAAV9-mediated rNAGLU expression was rapid and persistent in virtually all tested CNS and somatic tissues. However, acute liver toxicity occurred in 33% (5/15) WT males in the 2 × 10(14) vg/kg cohort, which was dose-dependent, sex-associated, and genotype-specific, likely due to hepatic rNAGLU overexpression. Interestingly, a significant dose response was observed only in the brain and spinal cord, whereas in the liver at 24 weeks postinfection (pi), NAGLU activity was reduced to endogenous levels in the high dose cohort but remained at supranormal levels in the low dose group. The possibility of rAAV9 germline transmission appears to be minimal. The vector delivery resulted in transient T-cell responses and characteristic acute antibody responses to both AAV9 and rNAGLU in all rAAV9-treated animals, with no detectable impacts on tissue transgene expression. This study demonstrates a generally safe and effective profile, and may have identified the upper dosing limit of rAAV9-CMV-hNAGLU via systemic delivery for the treatment of MPS IIIB.