Abiotic and Biotic Contributors to Support Inter-Epidemic Francisella tularensis in an Agricultural Peri-Urban Environment.

To characterize the inter-epidemic ecology of Francisella tularensis, we surveyed vertebrates and invertebrates for the abundance, spatial distribution, and status of infection at a site in northern California that had evidence of endemic type B tularemia. We collected 2910 mosquitoes, 77 biting flies, 704 ticks, 115 mammals, and 1911 aquatic invertebrates in 2013-2014. Real-time PCR on all mosquitoes, 40 biting flies, 113 aquatic invertebrates, and 650 ticks did not detect F. tularensis DNA. Indirect enzyme linked immunosorbent assay (ELISA) on 109 mammals revealed 2 (of 2, 100%) seropositive feral cats, 1 (of 24, 4.5%) seropositive black rat, and 5 (of 10, 50%) seropositive Virginia opossums. A riparian reserve, ∼1 km from the primate research center, had the highest seroprevalence in mammals and the highest capture success for invertebrate vectors whereas opossums, cats, and ground squirrels in close proximity to the primate center had high seroprevalence and abundant fleas. Well-vegetated regions with standing water appeared to be ideal habitats for biotic components of tularemia enzootic persistence. Mesocarnivores may facilitate the spread of F. tularensis, and high densities of rodents and their fleas may be a mechanism for amplification and spillover.

A method to preserve low parasitaemia Plasmodium-infected avian blood for host and vector infectivity assays.

BACKGROUND: Avian malaria vector competence studies are needed to understand more succinctly complex avian parasite-vector-relations. The lack of vector competence trials may be attributed to the difficulty of obtaining gametocytes for the majority of Plasmodium species and lineages. To conduct avian malaria infectivity assays for those Plasmodium spp. and lineages that are refractory to in vitro cultivation, it is necessary to obtain and preserve for short periods sufficient viable merozoites to infect naïve donor birds to be used as gametocyte donors to infect mosquitoes. Currently, there is only one described method for long-term storage of Plasmodium spp.-infected wild avian blood and it is reliable at a parasitaemia of at least 1%. However, most naturally infected wild-caught birds have a parasitaemia of much less that 1%. To address this problem, a method for short-term storage of infected wild avian blood with low parasitaemia (even ≤ 0.0005%) has been explored and validated. METHODS: To obtain viable infective merozoites, blood was collected from wild birds using a syringe containing the anticoagulant and the red blood cell preservative citrate phosphate dextrose adenine solution (CPDA). Each blood sample was stored at 4 °C for up to 48 h providing sufficient time to determine the species and parasitaemia of Plasmodium spp. in the blood by morphological examination before injecting into donor canaries. Plasmodium spp.--infected blood was inoculated intravenously into canaries and once infection was established, Culex stigmatosoma, Cx. pipiens and Cx. quinquefasciatus mosquitoes were then allowed to feed on the infected canaries to validate the efficacy of this method for mosquito vector competence assays. RESULTS: Storage of Plasmodium spp.--infected donor blood at 4 °C yielded viable parasites for 48 h. All five experimentally-infected canaries developed clinical signs and were infectious. Pathologic examination of three canaries that later died revealed splenic lesions typical of avian malaria infection. Mosquito infectivity assays demonstrated that Cx. stigmatosoma and Cx. pipiens were competent vectors for Plasmodium cathemerium. CONCLUSIONS: A simple method of collecting and preserving avian whole blood with malaria parasites of low parasitaemia (≤ 0.0005%) was developed that remained viable for further experimental bird and mosquito infectivity assays. This method allows researchers interested in conducting infectivity assays on target Plasmodium spp. to collect these parasites directly from nature with minimal impact on wild birds.

Surveillance for Ixodes pacificus and the tick-borne pathogens Anaplasma phagocytophilum and Borrelia burgdorferi in birds from California's Inner Coast Range.

We investigated the involvement of birds in the ecology of the western black-legged tick, Ixodes pacificus, and its associated zoonotic bacteria, Borrelia burgdorferi and Anaplasma phagocytophilum, at two interior coast-range study sites in northern California. Anaplasma phagocytophilum, the agent of granulocytic anaplasmosis (GA), and B. burgdorferi s.s., the agent of Lyme disease (LD), are tick-borne pathogens that are well established in California. We screened blood and ticks from 349 individual birds in 48 species collected in 2011 and 2012 using pathogen-specific PCR. A total of 617 immature I. pacificus was collected with almost three times as many larvae than nymphs. There were 7.5 times more I. pacificus at the Napa County site compared to the Yolo County site. Two of 74 (3%) nymphal pools from an Oregon junco (Junco hyemalis) and a hermit thrush (Catharus guttatus) and 4 individual larvae (all from Oregon juncos) were PCR-positive for B. burgdorferi. Blood samples from a golden-crowned sparrow (Zonotrichia atricapilla) and a European starling (Sturnus vulgaris) were positive for A. phagocytophilum DNA at very low levels. Birds that forage on ground or bark and nest on the ground, as well as some migratory species, are at an increased risk for acquiring I. pacificus. Our findings show that birds contribute to the ecologies of LD and GA in California by serving as a blood-meal source, feeding and transporting immature I. pacificus, and sometimes as a source of Borrelia infection.

Stone Lakes virus (family Togaviridae, genus Alphavirus), a variant of Fort Morgan virus isolated from swallow bugs (Hemiptera: Cimicidae) west of the Continental Divide.

Multiple isolates of an alphaviruses within the western equine encephalomyelitis-serocomplex that were related closely to Ft. Morgan and its variant Buggy Creek virus were made from swallow bugs, Oeciacus vicarius Horvath (Hemiptera: Cimicidae), collected from cliff swallow (Petrochelidon pyrrhonota) nests at the Stone Lakes National Wildlife Refuge, Sacramento County, CA, during the summers of 2005 and 2006. This virus (hereafter Stone Lakes virus, family Togaviridae, genus Alphavirus, STLV) was the first record of this viral group west of the Continental Divide. STLV replicated well in Vero and other vertebrate cell cultures but failed to replicate in C6/36 cells or infect Culex tarsalis Coquillett mosquitoes. STLV failed to produce elevated viremias in adult chickens or house sparrows and was weakly immunogenic. In addition, STLV was not isolated from cliff swallow nestlings nor was antibody detected in adults collected at mist nets. We suggest that STL and related swallow bug viruses may be primarily infections of cimicids that are maintained and amplified either by vertical or nonviremic transmission and that cliff swallows may primarily be important as a bloodmeal source for the bugs rather than as an amplification host for the viruses.

Role of communally nesting ardeid birds in the epidemiology of West Nile virus revisited.

Although herons and egrets in the family Ardeidae frequently have been associated with viruses in the Japanese encephalitis virus serocomplex, communal nesting colonies do not appear to be a focus of early season and rapid amplification of West Nile virus (WNV) in California. Evidence for repeated WNV infection was found by testing living and dead nestlings collected under trees with mixed species ardeid colonies nesting above in an oak grove near the University of California arboretum in Davis and in a Eucalyptus grove at a rural farmstead. However, mosquito infection rates at both nesting sites were low and positive pools did not occur earlier than at comparison sites within the City of Davis or at the Yolo Bypass wetlands managed for rice production and waterfowl habitat. Black-crowned night herons (Nycticorax nycticorax) were the most abundant and frequently infected ardeid species, indicating that WNV may be an important cause of mortality among nestlings of this species.

Impact of aerial spraying of pyrethrin insecticide on Culex pipiens and Culex tarsalis (Diptera: Culicidae) abundance and West Nile virus infection rates in an urban/suburban area of Sacramento County, California.

In response to an epidemic amplification of West Nile virus (family Flaviviridae, genus Flavivirus, WNV), the Sacramento and Yolo Mosquito and Vector Control District (SYMVCD) sprayed ultralow-volume (ULV) formulations of pyrethrin insecticide (Evergreen EC 60-6: 6% pyrethrin insecticide, 60% piperonylbutoxide; MGK, Minneapolis, MN, applied as 0.003 kg/ha [0.0025 lb/acre] ) over 218 km2 in north Sacramento and 243.5 km2 in south Sacramento on three consecutive evenings in August 2005. We evaluated the impact of this intervention in north Sacramento on the abundance and WNV infection rates of Culex pipiens L. and Culex tarsalis Coquillett. Mortality rates of caged Cx. tarsalis sentinels ranged from 0% under dense canopy to 100% in open fields. A comparison of weekly geometric mean mosquito abundance in CO2-baited traps in sprayed and unsprayed areas before and after treatment indicated a 75.0 and 48.7% reduction in the abundance of Cx. pipiens and Cx. tarsalis, respectively. This reduction was statistically significant for Cx. pipiens, the primary vector of WNV, with highest abundance in this urban area, but not for Cx. tarsalis, which is more associated with rural areas. The infection rates of WNV in Cx. pipiens and Cx. tarsalis collected from the spray zone were 8.2 and 4.3 per 1,000 female mosquitoes in the 2 wk before and the 2 wk after applications of insecticide, respectively. In comparison, WNV infection rates in Cx. pipiens and Cx. tarsalis collected at same time interval in the unsprayed zone were 2.0 and 8.7 per 1,000, respectively. Based on the reduction in vector abundance and its effects on number of infective bites received by human population, we concluded that the aerial application ofpyrethrin insecticide reduced the transmission intensity of WNV and decreased the risk of human infection.

Vertebrate hosts of Ixodes pacificus (Acari: Ixodidae) in California.

The western black-legged tick, Ixodes pacificus Cooley & Kohls, is an important parasite and vector of disease agents that affect human and animal health in the western United States. This paper presents a review of all published California host records for I. pacificus. Unpublished data from public health, academic, and vector control agencies and researchers were reviewed as well. Host species were identified for each active life stage (larvae, nymph and adult). A total of 108 vertebrate species in three classes (Mammalia, Aves, and Reptilia) were identified as hosts for at least one life stage of I. pacificus. Adult I. pacificus were recorded from 29 species of mammals, 2 species of birds, and 1 reptile species. Nymphal I. pacificus were recorded from 30 species of mammals, 38 species of birds, and 8 reptile species. Larval I. pacificus were recorded from 29 species of mammals, 43 species of birds, and 8 species of reptiles. A table depicting the taxonomic classification of host species is provided. This review adds eight new host records to the California list of recognized vertebrate host species for I. pacificus.

An avian contribution to the presence of Ixodes pacificus (Acari: Ixodidae) and Borrelia burgdorferi on the Sutter Buttes of California.

Birds from 45 species were sampled during three spring seasons from an isolated canyon on the Sutter Buttes in California for the presence of subadult stages of Ixodes pacificus Cooley & Kohls, and for infection with Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner. These birds were found to have an infestation prevalence of 45%, a density of 1.7 ticks per bird, and an intensity of 3.8 ticks per infested bird. There was a significant difference in the I. pacificus infestations between canopy and ground-dwelling birds. Birds also demonstrated an overall infection with B. burgdorferi of 6.4% with significant difference between bird species. Amplification and subsequent sequencing of the 23s-5s rRNA intergenic spacer region of the Borrelia genome from one bird, a hermit thrush, Catharus guttatus (Nuttall), showed that the infection in this bird was caused by B. burgdorferi sensu stricto; the first such finding in a bird from the far west. Our results suggest that birds play a role in the distribution and maintenance of I. pacificus, and possibly of B. burgdoferi, at the Sutter Buttes, CA.

The western black-legged tick, Ixodes pacificus, in the Sutter Buttes.

The western black-legged tick, Ixodes pacificus, was collected from Huff Canyon in the Sutter Buttes in north central California, Sutter County. The Sutter Buttes are within the Sacramento Valley and outside the normal distribution of I. pacificus in California. Adult I. pacificus were collected via flagging as they quested along deer trails; subadult life stages were collected from three species of lizard, Elgaria multicarinata, Sceloporus occidentalis, and S. graciosus. Adult ticks were tested for the presence of spirochetes using the standard IFA with a monoclonal antibody specific for OSPA. Of the ticks tested, 3.3% were identified as positive for the presence of the spirochete Borrelia burgdorferi. These results present the first published account of Ixodes pacificus as well as the first detection of B. burgdorferi in the Sutter Buttes, Sutter County, California.

Hematology and serum biochemistry values of dusky-footed wood rat (Neotoma fuscipes).

Serum chemistry values and complete blood counts were determined for 36 wild dusky-footed wood rats (Neotoma fuscipes) from Sonoma and western Yolo County, California (USA) in summer 1999 and spring 2001. All wood rats had adequate body condition and were hydrated. Many hematologic and biochemical values were comparable to those for house rat (Rattus rattus). There were differences between wood rats tested immediately after capture (those from Yolo County) and after a week of habituation in the laboratory (Sonoma County). Significant differences were noted in red blood cell counts, hemoglobin, hematocrit, neutrophil:lymphocyte ratio, glucose, alanine transaminase, aspartate aminotransferase, and alkaline phosphatase values. The neutrophil:lymphocyte ratio may have been iatrogenically modified in the wood rats tested immediately after capture by stress-induced neutrophilia and lymphopenia. Eosinophilia may have been associated with parasites such as botflies in four individuals, and hyperglycemia in three individuals could have been associated with stress. The cause of elevated enzymes in the animals tested after laboratory habituation is unclear. The hematologic and biochemical values of these apparently healthy wood rats provide valuable baseline information for use in further medical studies performed with this species.