Robust inactivation of Plasmodium falciparum in red blood cell concentrates using amustaline and glutathione pathogen reduction.

BACKGROUND: 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. STUDY DESIGN AND METHODS: 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. RESULTS: P. falciparum viability assays allow for the detection of very low levels of parasite. Initial parasite titer was >5.2 log10 /ml in AS-1/5 RBC. No infectious parasites were detected in amustaline-GSH-treated samples after 4 weeks of culture. Amustaline-GSH inactivated high parasite loads regardless of parasite stages and strains. Amustaline readily penetrates the parasite, irreversibly blocks development, and leads to parasite death and expulsion from RBC. DISCUSSION: Amustaline-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.

Inactivation of yellow fever virus with amotosalen and ultraviolet A light pathogen-reduction technology.

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.

Inactivation of Plasmodium falciparum in whole blood using the amustaline and glutathione pathogen reduction technology.

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.

Inactivation of a broad spectrum of viruses and parasites by photochemical treatment of plasma and platelets using amotosalen and ultraviolet A light.

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.

Two boundaries separate Borrelia burgdorferi populations in North America.

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.

Genetic structure of Pacific Flyway avian influenza viruses is shaped by geographic location, host species, and sampling period.

The eight gene segments of avian influenza virus (AIV) reassort frequently and rapidly to generate novel genotypes and subtypes that are transmissible to a broad range of hosts. There is evidence that AIV can have a restricted host range and can segregate in space and time. Host-virus relationships at the species, geographic, and spatial scales have not been fully defined for AIV populations of the Pacific Flyway, particularly among the diverse waterfowl that occupy the Flyway in Alaska and California. Using the sequence analysis program Bayesian Tip-association Significance testing (BaTS) created for analysis of phylogeny-trait associations, we determined whether the genetic structure of Pacific Flyway AIVs sampled between 2006 and 2008 was influenced by the host species, geographic location of virus collection, and time of sampling. In posterior sets of trees, genetically similar viruses clustered by host species for thick-billed murres and glaucous gulls (order Charadriiformes), and for northern shovelers, northern pintails, and mallards (order Anseriformes). AIVs from Alaska and California were strongly spatially structured, clustering separately by region across all segments. The timing of sampling influenced the genetic structure of California AIV gene segments, possibly reflecting waves of host species movement into wintering areas. The strength of phylogeny-trait association varied by virus segment and by trait of interest, which we hypothesize is related to the frequent genetic reassortment and interspecies transmission in waterfowl.

Genetic diversity of Borrelia burgdorferi and detection of B. bissettii-like DNA in serum of north-coastal California residents.

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.

Transcriptome changes in Culex quinquefasciatus (Diptera: Culicidae) salivary glands during West Nile virus infection.

Persistent West Nile virus (WNV) infection in the mosquito Culex quinquefasciatus Say (Diptera: Culicidae) is associated with pathological changes in the salivary glands, including apoptotic cell death and a corresponding reduction in virus transmission over time. The vector host response to WNV infection and the molecular basis of WNV pathogenesis in Cx. quinquefasciatus was investigated using oligonucleotide microarrays designed to detect differences in the salivary gland transcriptome between WNV-infected mosquitoes and uninfected controls. Transcripts with increased abundance in infected salivary glands included those related to immunity, transcription, protein transport and degradation, amino acid and nucleotide metabolism, signal transduction, and cellular detoxification. Microarray-based analysis detected a decrease in transcript levels of a Culex inhibitor of apoptosis gene (IAP-1) and a decrease in abundance of 11 transcripts encoding salivary gland proteins. Transcript levels for an endonuclease, a proline-rich mucin, and several D7 protein family members also decreased. Transcripts with the greatest change in abundance during infection had either no similarity to sequences found in GenBank, VectorBase, and FlyBase, or were similar to sequences with uncharacterized protein products. These transcripts represent exciting targets for future analysis. Results from this study suggest that WNV infection influences transcriptional changes in an invertebrate host target tissue that may confer an advantage to the replicating virus, induce a host defense response, and alter the composition of vector saliva. The ramifications of these changes are discussed in terms of mosquito vector competence and WNV pathogenesis.

Population structure of the lyme borreliosis spirochete Borrelia burgdorferi in the western black-legged tick (Ixodes pacificus) in Northern California.

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.

Avian trichomonosis mortality events in band-tailed pigeons (Patagioenas fasciata) in California during winter 2014-2015.

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.

Spatial clustering of Borrelia burgdorferi sensu lato within populations of Allen's chipmunks and dusky-footed woodrats in northwestern California.

The ecology of Lyme borreliosis is complex in northwestern California, with several potential reservoir hosts, tick vectors, and genospecies of Borrelia burgdorferi sensu lato. The primary objective of this study was to determine the fine-scale spatial distribution of different genospecies in four rodent species, the California ground squirrel (Otospermophilus beecheyi), northern flying squirrel (Glaucomys sabrinus), dusky-footed woodrat (Neotoma fuscipes), and Allen's chipmunk (Neotamias senex). Rodents were live-trapped between June 2004 and May 2005 at the Hoopa Valley Tribal Reservation (HVTR) in Humboldt County, California. Ear-punch biopsies obtained from each rodent were tested by polymerase chain reaction (PCR) and sequencing analysis. The programs ArcGIS and SaTScan were used to examine the spatial distribution of genospecies. Multinomial log-linear models were used to model habitat and host-specific characteristics and their effect on the presence of each borrelial genospecies. The Akaike information criterion (AICc) was used to compare models and determine model fit. Borrelia burgdorferi sensu stricto was primarily associated with chipmunks and B. bissettiae largely with woodrats. The top model included the variables "host species", "month", and "elevation" (weight = 0.84). Spatial clustering of B. bissettiae was detected in the northwestern section of the HVTR, whereas B. burgdorferi sensu stricto was clustered in the southeastern section. We conclude that the spatial distribution of these borreliae are driven at least in part by host species, time-of-year, and elevation.

Nonviremic transmission of West Nile virus: evaluation of the effects of space, time, and mosquito species.

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.

Salivary gland morphology and virus transmission during long-term cytopathologic West Nile virus infection in Culex mosquitoes.

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.

Potentiation of West Nile encephalitis by mosquito feeding.

Mosquitoes infect human beings with arboviruses while taking a blood meal, inoculating virus with their saliva. Mosquito saliva contains compounds that counter host hemostatic, inflammatory, and immune responses. Modulation of these crucial defensive responses may facilitate virus infection. Using a murine model we explored the potential for mosquitoes to impact the course of West Nile virus (WNV) disease by determining whether differences in pathogenesis occurred in the presence or absence of mosquito saliva. Mice inoculated intradermally with 10(4) pfu of WNV subsequent to the feeding of mosquitoes developed more progressive infection, higher viremia, and accelerated neuroinvasion than the mice inoculated with WNV alone. At a lower dose of WNV (10(2) pfu), mice fed upon by mosquitoes had a lower survival rate. This study suggests that mosquito feeding and factors in mosquito saliva can potentiate WNV infection, and offers a possible mechanism for this effect via accelerated infection of the brain.

Avian trichomonosis in spotted owls (Strix occidentalis): Indication of opportunistic spillover from prey.

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.

ECOLOGIC DRIVERS AND POPULATION IMPACTS OF AVIAN TRICHOMONOSIS MORTALITY EVENTS IN BAND-TAILED PIGEONS (PATAGIOENAS FASCIATA) IN CALIFORNIA, USA.

: 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.

Detection and characterization of diverse coccidian protozoa shed by California sea lions.

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.

Ultrastructural study of West Nile virus pathogenesis in Culex pipiens quinquefasciatus (Diptera: Culicidae).

The ultrastructural features of West Nile virus (WNV) replication and dissemination in orally infected Culex pipiens quinquefasciatus Say were analyzed over a 25-d infection period. To investigate the effects of virus replication on membrane induction, cellular organization, and cell viability in midgut and salivary gland tissues, midguts were dissected on days 3, 7, 14, and 21, and salivary glands were collected on days 7, 14, 21, and 25 postinfection (d.p.i.) for examination by transmission electron microscopy (TEM). Whole mosquito heads were embedded for TEM analysis 14 d.p.i. to localize WNV particles and to investigate the effects of replication on nervous tissues of the brain. Membrane proliferation was induced by WNV in the midgut epithelium, midgut muscles, and salivary glands, although extensive endoplasmic reticulum swelling was a unique feature of salivary gland infection. TEM revealed WNV-induced pathology in salivary glands at 14, 21, and 25 d.p.i., and we hypothesize that long-term virus infection of this tissue results in severe cellular degeneration and apoptotic-like cell death. This finding indicates that the efficiency of WNV transmission may decrease with mosquito age postinfection.

West Nile virus dissemination and tissue tropisms in orally infected Culex pipiens quinquefasciatus.

We investigated the spatial and temporal distribution of West Nile virus (WNV) in organs and tissues of Culex pipiens quinquefasciatus mosquitoes for up to 27 days following oral infection. WNV antigen was detected in paraffin-embedded mosquitoes by immunohistochemistry. Immunofluorescence assays were performed on dissected salivary glands and midguts and analyzed by confocal microscopy. We evaluated the route of virus dissemination following midgut escape and the relative importance of amplifying tissues in mosquito susceptibility to infection. WNV infection was persistent in all tissues analyzed including the midgut, salivary glands, nervous system, and fat body and only declined in the cytoplasm of posterior midgut epithelial cells over time. The phenomenon of cell-to-cell spread was observed in the midgut epithelium and WNV intensely infected both circular and longitudinal muscles of the same organ. It is possible that muscle tissue serves as a conduit for virus dissemination and contributes to WNV amplification, particularly late in infection. These findings provide insight into WNV infection dynamics in a highly susceptible, epidemiologically important, North American vector. Our results pave the way for future studies to analyze physical and biological barriers to WNV dissemination in less competent mosquitoes.

Dual-pathogen etiology of avian trichomonosis in a declining band-tailed pigeon population.

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.