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Plasmodium falciparum is the parasite responsible for most malaria cases globally. The risk of transfusion-transmitted malaria (TTM) is mitigated by donor deferrals and blood screening strategies, which adversely impact blood availability. Previous studies showed robust inactivation of P. falciparum using nucleic acid-targeting pathogen reduction technologies (PRT) for the treatment of plasma and platelet components or whole blood (WB). The efficacy of the amustaline-glutathione (GSH) PRT to inactivate P. falciparum is here evaluated in red blood cells (RBC), as well the impact of PRT on parasite loads, stages, and strains.RBC units resuspended in AS-1 or AS-5 additive solutions were spiked with ring stage-infected RBC and treated with the amustaline-GSH PRT. Parasite loads and viability were measured in samples at the time of contamination, and after treatment, using serial 10-fold dilutions of the samples in RBC cultures maintained for up to 4 weeks.P. falciparum viability assays allow for the detection of very low levels of parasite. Initial parasite titer was5.2 logAmustaline-GSH PRT demonstrated robust efficacy to inactivate malaria parasites in RBC concentrates. This study completes the portfolio of studies demonstrating the efficacy of nucleic acid-targeting PRTs to mitigate TTM risks as previously reported for platelet concentrates, plasma, and WB.

Background The reemergence of yellow fever virus (YFV) in Africa and Brazil, and massive vaccine campaigns triggered to contain the outbreaks, have raised concerns over blood transfusion safety and availability with increased risk of YFV transfusion-transmitted infections (TTIs) by native and vaccine-acquired YFV. Blood donor deferral for 2 to 4 weeks following live attenuated YFV vaccination, and deferral for travel to endemic/epidemic areas, may result in blood donor loss and impact platelet component (PC) stocks. This study investigated the efficacy of INTERCEPT Blood System pathogen reduction (PR) with use of amotosalen and ultraviolet A (UVA) light to inactivate high levels of YFV in PCs. Materials Four units of apheresis platelets prepared in 35% plasma/65% platelet additive solution (PC-PAS) and 4 units of PC in 100% human plasma (PC-Plasma) were spiked with high infectious titers of YFV (YFV-17D vaccine strain). YFV-17D infectious titers were measured by plaque assay and expressed as plaque-forming units (PFU) before and after amotosalen/UVA treatment to determine log reduction. Results The mean YFV-17D infectious titers in PC before inactivation were 5.5 ± 0.1 log PFU/mL in PC-PAS and 5.3 ± 0.1 log PFU/mL in PC-Plasma. No infectivity was detected immediately after amotosalen/UVA treatment. Conclusion The amotosalen/UVA PR system inactivated high titers of infectious YFV-17D in PC. This PR technology could reduce the risk of YFV TTI and help secure PC supplies in areas experiencing YFV outbreaks where massive vaccination campaigns are required.

Background The INTERCEPT Blood System pathogen reduction technology (PRT), which uses amotosalen and ultraviolet A light treatment (amotosalen/UV-PRT), inactivates pathogens in plasma and platelet components (PCs). This review summarizes data describing the inactivation efficacy of amotosalen/UVA-PRT for a broad spectrum of viruses and parasites. Methods Twenty-five enveloped viruses, six nonenveloped viruses (NEVs), and four parasites species were evaluated for sensitivity to amotosalen/UVA-PRT. Pathogens were spiked into plasma and PC at high titers. Samples were collected before and after PRT and assessed for infectivity with cell cultures or animal models. Log reduction factors (LRFs) were defined as the difference in infectious titers before and after amotosalen/UV-PRT. Results LRFs of ≥4.0 log were reported for 19 pathogens in plasma (range, ≥4.0 to ≥7.6), 28 pathogens in PC in platelet additive solution (PC-PAS; ≥4.1-≥7.8), and 14 pathogens in PC in 100% plasma (PC-100%; (≥4.3->8.4). Twenty-five enveloped viruses and two NEVs were sensitive to amotosalen/UV-PRT; LRF ranged from >2.9 to ≥7.6 in plasma, 2.4 or greater to greater than 6.9 in PC-PAS and >3.5 to >6.5 in PC-100%. Infectious titers for four parasites were reduced by >4.0 log in all PC and plasma (≥4.9 to >8.4). Conclusion Amotosalen/UVA-PRT demonstrated effective infectious titer reduction for a broad spectrum of viruses and parasites. This confirms the capacity of this system to reduce the risk of viral and parasitic transfusion-transmitted infections by plasma and PCs in various geographies.

Background Risk of transfusion-transmitted (TT) malaria is mainly associated with whole blood (WB) or red blood cell (RBC) transfusion. Risk mitigation relies mostly on donor deferral while a limited number of countries perform blood testing, both negatively impacting blood availability. This study investigated the efficacy of the pathogen reduction system using amustaline and glutathione (GSH) to inactivate Plasmodium falciparum in WB. Study design and methods WB units were spiked with ring stage P. falciparum infected RBCs. Parasite loads were measured in samples at time of infection, after 24 hours at room temperature (RT), and after a 24-hour incubation at RT post-treatment with 0.2 mM amustaline and 2 mM GSH. Serial 10-fold dilutions of the samples were inoculated to RBC cultures and maintained up to 4 weeks. Parasitemia was quantified by cytometry. Results The P. falciparum viability assay has a limit of detection of a single live parasite per sample. Input parasite titer was >5.7 log10 TCID50 per mL. A 24-hour incubation at RT paused parasite development in controls, but they retained viability and infectivity when tested in culture. In contrast, no infectious parasites were detected in the amustaline/GSH-treated sample after 4 weeks of culture. Conclusion A robust level of P. falciparum inactivation was achieved in WB using amustaline/GSH treatment. Parasite log reduction was >5.7 log10 TCID50 per mL. Development of such a pathogen reduction system may provide an opportunity to reduce the risk of TT malaria and improve blood availability.

Avian trichomonosis, caused by the flagellated protozoan parasite Trichomonas gallinae, has variable pathogenicity among bird species ranging from asymptomatic infections to severe disease periodically manifesting in epidemic mortality. Traditionally, columbids are identified as highly susceptible to infection with occasional spillover into raptors that prey on infected birds. We identified avian trichomonosis in two dead California spotted owls (Strix occidentalis occidentalis) and three dead northern spotted owls (S. o. caurina) in California during 2011–2015; infection was confirmed in four owls by PCR. Pathologic lesions associated with trichomonosis in the owls included caseonecrotic lesions of the upper palate accompanied by oropharyngitis, cellulitis, myositis, and/or sinusitis. Spotted owls are known to mainly feed on small mammals; therefore, the source of infection as well as the significance of the disease in spotted owls is unclear. These owl trichomonosis cases coincided temporally and spatially with three trichomonosis epidemics in band-tailed pigeons (Patagioenas fasciata monilis). The same parasite, T. gallinae subtype A2, was isolated from the spotted owls and band-tailed pigeons, suggesting the owls became infected when opportunistically feeding on pigeons during mortality events. Avian trichomonosis is an important factor in the decline of the Pacific Coast band-tailed pigeon population with near-annual mortality events during the last 10 years and could have conservation implications for raptor species at risk, particularly those that are facing multiple threats., Graphical abstract Image 1, Highlights • Band-tailed pigeons evaluated for trichomonad infections during mortality events. • Spotted owls evaluated for trichomonad infections coinciding with mortality events. • Trichomonads characterized using ITS1/5.8S rRNA/ITS2 and Fe-hydrogenase loci. • Trichomonas gallinae subtype A2 was predominant parasite in both pigeons and owls.

Avian trichomonosis is an upper digestive tract disease of birds typically caused by the protozoan parasite Trichomonas gallinae. In California (U.S.A), trichomonosis is known to cause periodic epidemics in the Pacific Coast band-tailed pigeon (Patagioenas fasciata monolis), a migratory upland game bird. We summarize the mortality events that occurred during winter 2014–2015 including the duration, estimated mortality, pathology, and genetic identity of infecting parasites. Increased mortality was reported from locations in 25 counties between November 2014 and June 2015. Based on reports, carcasses received, wildlife rehabilitation center admissions, site visits, and regular monitoring by local personnel, total mortality was estimated at 18,440. At necropsy, birds had multiple coalescing lesions in the oral cavity involving the upper palate and/or around the tongue and glottis, esophagus, crop, and/or proventriculus. Birds collected from Contra Costa (63.9%; 30/47); Marin (75.0%; 6/8), San Mateo (46.7%; 14/30), and Santa Clara (35.0%; 37/106) counties were more likely to have lesions extending into their head involving muscle, sinuses, ear canals, eye sockets, and bone (χ2 = 62.9; df = 11; P < 0.001). Histopathologic findings included pharyngitis, esophagitis, myositis, and air sacculitis of the pneumatic bone of the skull. Mixed bacterial colonies were found multifocally at the fronts of the necrosis in six of the eleven birds examined histologically. Infecting trichomonads included T. gallinae subtype A2 (n = 5), un-typed T. gallinae (n = 4), mixed infection with T. gallinae subtype A2 and T. stableri (n = 1), and mixed infection with un-typed T. gallinae and T. stableri (n = 1). The winter 2014–2015 epidemic was the largest on record in terms of duration, locations, and birds affected. Infection dynamics may have been exacerbated by the drought in California. Increased monitoring of band-tailed pigeons is needed to understand the long-term impacts of large-scale mortality events on their population. Keywords: Avian trichomonosis, Band-tailed pigeon, Epidemic, Trichomonas gallinae, ITS1/5.8S rRNA/ITS2, Fe-hydrogenase

Graphical abstract, Highlights • Trichomonad protozoa infecting free-ranging band-tailed pigeons were characterized. • Trichomonas gallinae and novel species T. stableri were isolated in sick birds. • T. stableri is significantly smaller in length and width than T. gallinae. • T. stableri is genetically more similar to T. vaginalis than to T. gallinae. • T. stableri is a secondary agent of trichomonosis in band-tailed pigeons., Trichomonas gallinae is a ubiquitous flagellated protozoan parasite, and the most common etiologic agent of epidemic trichomonosis in columbid and passerine species. In this study, free-ranging Pacific Coast band-tailed pigeons (Patagioenas fasciata monilis) in California (USA) were found to be infected with trichomonad protozoa that were genetically and morphologically distinct from T. gallinae. In microscopic analysis, protozoa were significantly smaller in length and width than T. gallinae and were bimodal in morphology. Phylogenetic analysis of the ITS1/5.8S/ITS2, rpb1, and hydrogenosomal Fe-hydrogenase regions revealed that the protozoan shares an ancestor with Trichomonas vaginalis, the sexually-transmitted agent of trichomoniasis in humans. Clinical and pathologic features of infected birds were similar to infections with T. gallinae. Evidence presented here strongly support taxonomical distinction of this parasite, which we hereby name Trichomonas stableri n. sp. This work contributes to a growing body of evidence that T. gallinae is not the sole etiologic agent of avian trichomonosis, and that the incorporation of molecular tools is critical in the investigation of infectious causes of mortality in birds.

The primary challenge to mountain lion population viability in California is habitat loss and fragmentation. These habitat impacts could enhance disease risk by increasing contact with domestic animals and by altering patterns of exposure to other wild felids. We performed a serologic survey for feline pathogens in California mountain lions (Puma concolor) using 490 samples from 45 counties collected from 1990 to 2008. Most mountain lions sampled were killed because of depredation or public safety concerns and 75% were adults. Pathogens detected by serosurvey in sampled mountain lions included feline panleukopenia virus (39.0%), feline calicivirus (33.0%), feline coronavirus (FCoV, 15.1%), feline herpesvirus (13.0%), heartworm (12.4%), feline leukemia virus (5.4%), and canine distemper virus (3%). An outbreak of heartworm exposure occurred from 1995 to 2003 and higher than expected levels of FCoV-antibody-positive mountain lions were observed from 2005 to 2008, with foci in southern Mendocino and eastern Lake counties. We show that the majority of mountain lions were exposed to feline pathogens and may be at risk of illness or fatality, particularly kittens. Combined with other stressors, such as ongoing habitat loss, infectious disease deserves recognition for potential negative impact on mountain lion health and population viability.

© Wildlife Disease Association 2016. Avian trichomonosis, a disease typically caused by the protozoan parasite Trichomonas gallinae, is a well recognized cause of death in many avian species. In California, US, trichomonosis has caused periodic epidemics in Pacific Coast Band-tailed Pigeons (Patagioenas fasciata monilis). We summarize reported mortality events and investigate ecologic drivers and population impacts associated with epidemic mortality due to trichomonosis in Band-tailed Pigeons. Between 1945 and 2014, 59 mortality events involving Band-tailed Pigeons were reported in California with the number of reported events increasing over time. Estimated mortality for these events was variable, ranging between 10 and 10,000 pigeons. Events were most-frequently reported in Monterey (19%; 11/59) and San Luis Obispo (8%; 5/59) counties. Events often started in January (32%; 9/28) and February (50%; 14/28) and lasted 5–68 d. Impacts of mortality events on pigeon populations were indicated by Breeding Bird Survey and Christmas Bird Count abundance indices, which showed a decline in outbreak years compared to nonoutbreak years. Environmental conditions most associated with outbreak years included higher average temperatures between January and March, the period most associated with mortality events, and lower average precipitation in December just prior to mortality events. In Monterey County, events tended to occur in winters following higher acorn production of coast live oaks (Quercus agrifolia) in the fall. Weather and food abundance could be related to increased transmission or enhanced viability of Trichomonas spp. Although estimated mortality due to avian trichomonosis was highly variable across years, cumulative losses were substantial and likely to have a negative impact on population size.

Tissue-cyst forming coccidia in the family Sarcocystidae are etiologic agents of protozoal encephalitis in marine mammals including the federally listed Southern sea otter (Enhydra lutris). California sea lions (Zalophus californianus), whose coastal habitat overlaps with sea otters, are definitive hosts for coccidian protozoa provisionally named Coccidia A, B and C. While Coccidia A and B have unknown clinical effects on aquatic wildlife hosts, Coccidia C is associated with severe protozoal disease in harbor seals (Phoca vitulina). In this study, we conducted surveillance for protozoal infection and fecal shedding in hospitalized and free-ranging California sea lions on the Pacific Coast and examined oocyst morphology and phenotypic characteristics of isolates via mouse bioassay and cell culture. Coccidia A and B were shed in similar frequency, particularly by yearlings. Oocysts shed by one free-ranging sea lion sampled at Año Nuevo State Park in California were previously unidentified in sea lions and were most similar to coccidia infecting Guadalupe fur seals (Arctocephalus townsendi) diagnosed with protozoal disease in Oregon (USA). Sporulated Coccidia A and B oocysts did not replicate in three strains of mice or in African green monkey kidney cells. However, cultivation experiments revealed that the inoculum of fecally-derived Coccidia A and B oocysts additionally contained organisms with genetic and antigenic similarity to Sarcocystis neurona; despite the absence of detectable free sporocysts in fecal samples by microscopic examination. In addition to the further characterization of Coccidia A and B in free-ranging and hospitalized sea lions, these results provide evidence of a new role for sea lions as putative mechanical vectors of S. neurona, or S. neurona-like species. Future work is needed to clarify the distribution, taxonomical status, and pathogenesis of these parasites in sea lions and other marine mammals that share their the near-shore marine environment., Graphical abstract, Highlights • Diverse coccidian protozoa shed by California sea lions (CSL) were characterized. • Oocyst shedding patterns, taxonomy, morphology and pathogenicity were examined. • Mice and cell cultures were not susceptible to Coccidia A or B of CSL origin. • Sarcocystis neurona-like zoites grew in cells inoculated with CSL fecal samples. • California sea lions may serve as mechanical vectors of an S. neurona-like organism.

In the far-western United States, the nymphal stage of the western black-legged tick, Ixodes pacificus, has been implicated as the primary vector to humans of Borrelia burgdorferi sensu stricto (hereinafter referred to as B. burgdorferi), the causative agent of Lyme borreliosis in North America. In the present study, we sought to determine if infection prevalence with B. burgdorferi in I. pacificus nymphs and the density of infected nymphs differ between dense-woodland types within Mendocino County, California, and to develop and evaluate a spatially-explicit model for density of infected nymphs in dense woodlands within this high-incidence area for Lyme borreliosis. In total, 4.9% (264) of 5431 I. pacificus nymphs tested for the presence of B. burgdorferi were infected. Among the 78 sampling sites, infection prevalence ranged from 0 to 22% and density of infected nymphs from 0 to 2.04 per 100 m(2). Infection prevalence was highest in woodlands dominated by hardwoods (6.2%) and lowest for redwood (1.9%) and coastal pine (0%). Density of infected nymphs also was higher in hardwood-dominated woodlands than in conifer-dominated ones that included redwood or pine. Our spatial risk model, which yielded an overall accuracy of 85%, indicated that warmer areas with less variation between maximum and minimum monthly water vapor in the air were more likely to include woodlands with elevated acarological risk of exposure to infected nymphs. We found that 37% of dense woodlands in the county were predicted to pose an elevated risk of exposure to infected nymphs, and that 94% of the dense-woodland areas that were predicted to harbor elevated densities of infected nymphs were located on privately-owned land.

We investigated the role of the western gray squirrel (Sciurus griseus) as a reservoir host of the Lyme disease spirochete Borrelia burgdorferi. A survey of 222 western gray squirrels in California showed an overall prevalence of B. burgdorferi infection of 30%, although at a county level, prevalence of infection ranged from 0% to 50% by polymerase chain reaction. Laboratory trials with wild-caught western gray squirrels indicated that squirrels were competent reservoir hosts of the Lyme disease bacterium and infected up to 86% of feeding Ixodes pacificus larvae. Infections were long-lasting (up to 14 months), which demonstrated that western gray squirrels can maintain B. burgdorferi trans-seasonally. Non-native eastern gray squirrels (Sciurus carolinensis) and fox squirrels (Sciurus niger) were infrequently infected with B. burgdorferi.

To evaluate the potential for nonviremic transmission (NVT) of West Nile virus (WNV) to occur in nature, we examined the effect of increasing spatial and temporal separation between co-feeding mosquitoes on the efficiency of nonviremic transmission and the potential of a West Nile virus bridge vector species, Aedes albopictus, to be infected via nonviremic transmission. West Nile virus-infected (donor) Culex pipiens quinquefasciatus were allowed to feed on a mouse for 5 minutes followed by non-infected (recipient) mosquitoes with increasing spatial (0, 10, 20, 30, 40, or 50 mm) or temporal (0, 15, 30, 45, or 60 min) separation from the site or time of donor feeding, respectively. Recipients became infected when feeding up to 40 mm from the donor and up to 45 minutes after donor feeding. Additionally, nonviremic transmission of West Nile virus from Cx. p. quinquefasciatus to Ae. albopictus was observed.

The effect of long-term West Nile virus (WNV) infection on Culex salivary gland morphology and viability was evaluated by transmission electron microscopy during a four week period post-blood feeding. These studies showed that apoptosis and other cytopathologic changes occurred more frequently in WNV-infected mosquitoes compared with uninfected controls. The effect of long-term infection on WNV transmission was evaluated by titering virus in saliva over the same time period. Although the mean titer of WNV in mosquito saliva did not change significantly over time, the percentage of saliva samples containing WNV decreased. Because of the importance of saliva in blood meal acquisition and virus delivery, salivary gland pathology has the potential to affect mosquito feeding behavior and virus transmission. Results from this study add to a growing body of evidence that arbovirus infections in mosquito vectors can be cytopathic, and offer a potential mechanism for virus-induced cell death in mosquitoes.

Point mutations and inserts in the β3-β4 region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are associated with resistance to nucleoside analog inhibitors. This report describes HIV-1 strains from seven patients that were found to have a 3-bp deletion in the β3-β4 region of the RT gene. These patient strains also had a mean of 6.2 drug resistance-associated mutations in their RT genes (range, 3 to 10 mutations). The deletion was most frequently found in strains with the Q151M mutation. Nonnucleoside RT inhibitor mutations were found in six of seven strains. Culture-based drug sensitivity assays showed that deletion-containing isolates had reduced susceptibility to four to eight RT inhibitors. Site-directed mutagenesis experiments showed that the deletion alone conferred reduced susceptibility to nucleoside analogs. Changes in the three-dimensional models of the RT deletion mutants were consistently observed at the β3-β4 loop and at helices C and E in both the presence and the absence of dTTP. Loss of hydrogen bonds between the RT and dTTP were also observed in the RT deletion mutant. These results suggest that the deletion in the RT gene contributes to resistance to several nucleoside analogs through a complex interaction with other mutations in the RT gene.

The Pacific Coast band-tailed pigeon (Patagioenas fasciata monilis) is a migratory game bird of North America that is at risk for population decline. Epidemics of avian trichomonosis caused by upper digestive tract infection with Trichomonas spp. protozoa in these and other doves and pigeons of the United States are sporadic, but can involve tens of thousands of birds in a single event. Herein, we analyze the role of trichomonosis in band-tailed pigeon mortality and relate spatial, temporal and demographic patterns of parasite transmission to the genetic background of the infecting organism. Infections were most common in adult birds and prevalence was high in band-tailed pigeons sampled at mortality events (96%) and rehabilitation centers (36%) compared to those that were hunter-killed (11%) or live-caught (4%). During non-epidemic periods, animals were primarily infected with T. gallinae Fe-hydrogenase subtype A2, and were less often infected with either T. gallinae subtype A1 (the British finch epidemic strain), T. stableri n. sp. (a T. vaginalis-like species), or Tritrichomonas blagburni n. sp.-like organisms. Birds sampled during multiple epidemics in California were only infected with T. gallinae subtype A2 and T. stableri. The non-clonal etiology of avian trichomonosis outbreaks in band-tailed pigeons and the risk of spill-over to raptor and passerine species highlights the need for additional studies that clarify the host range and evolutionary relationships between strains of Trichomonas spp. in regions of trichomonosis endemicity. © 2014 Elsevier B.V.

Understanding the spread of infectious diseases is crucial for implementing effective control measures. For this, it is important to obtain information on the contemporary population structure of a disease agent and to infer the evolutionary processes that may have shaped it. Here, we investigate on a continental scale the population structure of Borrelia burgdorferi , the causative agent of Lyme borreliosis (LB), a tick-borne disease, in North America. We test the hypothesis that the observed population structure is congruent with recent population expansions and that these were preceded by bottlenecks mostly likely caused by the near extirpation in the 1900s of hosts required for sustaining tick populations. Multilocus sequence typing and complementary population analytical tools were used to evaluate B. burgdorferi samples collected in the Northeastern, Upper Midwestern, and Far-Western United States and Canada. The spatial distribution of sequence types (STs) and inferred population boundaries suggest that the current populations are geographically separated. One major population boundary separated western B. burgdorferi populations transmitted by Ixodes pacificus in California from Eastern populations transmitted by I. scapularis ; the other divided Midwestern and Northeastern populations. However, populations from all three regions were genetically closely related. Together, our findings suggest that although the contemporary populations of North American B. burgdorferi now comprise three geographically separated subpopulations with no or limited gene flow among them, they arose from a common ancestral population. A comparative analysis of the B. burgdorferi outer surface protein C ( ospC ) gene revealed novel linkages and provides additional insights into the genetic characteristics of strains.

Urban and agricultural land use may increase the risk of disease transmission among wildlife, domestic animals, and humans as we share ever-shrinking and fragmented habitat. American badgers (Taxidae taxus), a species of special concern in California, USA, live in proximity to urban development and often share habitat with livestock and small peridomestic mammals. As such, they may be susceptible to pathogens commonly transmitted at this interface and to anticoagulant rodenticides used to control nuisance wildlife on agricultural lands. We evaluated free-ranging badgers in California for exposure to pathogens and anticoagulant rodenticides that pose a risk to wildlife, domestic animals, or public health. We found serologic evidence of badger exposure to Francisella tularensis, Toxoplasma gondii, Anaplasma phagocytophilum, canine distemper virus, and three Bartonella species: B. henselae, B. clarridgeiae, and B. vinsonii subsp. berkhoffii. Badger tissues contained anticoagulant rodenticides brodifacoum and bromadiolone, commonly used to control periurban rodent pests. These data provide a preliminary investigation of pathogen and toxicant exposure in the wild badger population.

In North America, Lyme borreliosis (LB) is a tick-borne disease caused by infection with the spirochete Borrelia burgdorferi . We studied the genetic diversity of LB spirochetes in north-coastal California residents. Spirochete DNA was detected in 23.7% (27/114) of the study subjects using a PCR protocol optimized for increased sensitivity in human sera. Californians were most commonly infected with B. burgdorferi ospC genotype A, a globally widespread spirochete associated with high virulence in LB patients. Sequence analysis of rrf-rrl and p66 loci in 11% (3/27) of the PCR-positive study subjects revealed evidence of infection with an organism closely related to B. bissettii . This spirochete, heretofore associated with LB only in Europe, is widely distributed among ticks and wildlife in North America. Further molecular testing of sera from residents in areas where LB is endemic is warranted to enhance our understanding of the geographic distribution and frequency of occurrence of B. bissettii -like infections.

Factors potentially contributing to the lower incidence of Lyme borreliosis (LB) in the far-western than in the northeastern United States include tick host-seeking behavior resulting in fewer human tick encounters, lower densities of Borrelia burgdorferi -infected vector ticks in peridomestic environments, and genetic variation among B. burgdorferi spirochetes to which humans are exposed. We determined the population structure of B. burgdorferi in over 200 infected nymphs of the primary bridging vector to humans, Ixodes pacificus , collected in Mendocino County, CA. This was accomplished by sequence typing the spirochete lipoprotein ospC and the 16S-23S rRNA intergenic spacer (IGS). Thirteen ospC alleles belonging to 12 genotypes were found in California, and the two most abundant, ospC genotypes H3 and E3, have not been detected in ticks in the Northeast. The most prevalent ospC and IGS biallelic profile in the population, found in about 22% of ticks, was a new B. burgdorferi strain defined by ospC genotype H3. Eight of the most common ospC genotypes in the northeastern United States, including genotypes I and K that are associated with disseminated human infections, were absent in Mendocino County nymphs. ospC H3 was associated with hardwood-dominated habitats where western gray squirrels, the reservoir host, are commonly infected with LB spirochetes. The differences in B. burgdorferi population structure in California ticks compared to the Northeast emphasize the need for a greater understanding of the genetic diversity of spirochetes infecting California LB patients.

To evaluate the potential for nonviremic transmission (NVT) of West Nile virus (WNV) to occur in nature, we examined the effect of increasing spatial and temporal separation between co-feeding mosquitoes on the efficiency of nonviremic transmission and the potential of a West Nile virus bridge vector species, Aedes albopictus, to be infected via nonviremic transmission. West Nile virus-infected (donor) Culex pipiens quinquefasciatus were allowed to feed on a mouse for 5 minutes followed by non-infected (recipient) mosquitoes with increasing spatial (0, 10, 20, 30, 40, or 50 mm) or temporal (0, 15, 30, 45, or 60 min) separation from the site or time of donor feeding, respectively. Recipients became infected when feeding up to 40 mm from the donor and up to 45 minutes after donor feeding. Additionally, nonviremic transmission of West Nile virus from Cx. p. quinquefasciatus to Ae. albopictus was observed.

The effect of long-term West Nile virus (WNV) infection on Culex salivary gland morphology and viability was evaluated by transmission electron microscopy during a four week period post-blood feeding. These studies showed that apoptosis and other cytopathologic changes occurred more frequently in WNV-infected mosquitoes compared with uninfected controls. The effect of long-term infection on WNV transmission was evaluated by titering virus in saliva over the same time period. Although the mean titer of WNV in mosquito saliva did not change significantly over time, the percentage of saliva samples containing WNV decreased. Because of the importance of saliva in blood meal acquisition and virus delivery, salivary gland pathology has the potential to affect mosquito feeding behavior and virus transmission. Results from this study add to a growing body of evidence that arbovirus infections in mosquito vectors can be cytopathic, and offer a potential mechanism for virus-induced cell death in mosquitoes.

A trans -packaging system for West Nile virus (WNV) subgenomic replicon RNAs (repRNAs), deleted for the structural coding region, was developed. WNV repRNAs were efficiently encapsidated by the WNV C/prM/E structural proteins expressed in trans from replication-competent, noncytopathic Sindbis virus-derived RNAs. Infectious virus-like particles (VLPs) were produced in titers of up to 10 9 infectious units/ml. WNV VLPs established a single round of infection in a variety of different cell lines without production of progeny virions. The infectious properties of WNV and VLPs were indistinguishable when efficiencies of infection of a number of different cell lines and inhibition of infection by neutralizing antibodies were determined. To investigate the usefulness of VLPs to address biological questions in vivo, Culex pipiens quinquefasciatus mosquitoes were orally and parenterally infected with VLPs, and dissected tissues were analyzed for WNV antigen expression. Antigen-positive cells in midguts of orally infected mosquitoes were detected as early as 2 days postinfection and as late as 8 days. Intrathoracic inoculation of VLPs into mosquitoes demonstrated a dose-dependent pattern of infection of secondary tissues and identified fat body, salivary glands, tracheal cells, and midgut muscle as susceptible WNV VLP infection targets. These results demonstrate that VLPs can serve as a valuable tool for the investigation of tissue tropism during the early stages of infection, where virus spread and the need for biosafety level 3 containment complicate the use of wild-type virus.