LEPTOSPIRA PREVALENCE AND ITS ASSOCIATION WITH RENAL PATHOLOGY IN MOUNTAIN LIONS (PUMA CONCOLOR) AND BOBCATS (LYNX RUFUS) IN CALIFORNIA, USA.

Leptospirosis is reported infrequently in wild and domestic felids. We estimated the prevalence of Leptospira spp. infection and exposure using real-time PCR and serology, respectively, in 136 mountain lions (Puma concolor) and 39 bobcats (Lynx rufus) that died or were euthanized between 2009 and 2017 from several regions of California, US. Felids were classified as Leptospira-positive if they were test-positive using real-time PCR targeting the LipL32 gene of pathogenic Leptospira spp. or microscopic agglutination test for six serovars of Leptospira spp. The overall Leptospira spp. prevalence was 46% (63/136) for mountain lions and 28% (11/39) for bobcats. The most common serovar detected in both felid species was Leptospira interrogans serovar Pomona. Age class and geographic location were significantly associated with Leptospira spp. in mountain lions, but not in bobcats. Interstitial nephritis, predominately lymphocytic, was diagnosed in 39% (41/106) of mountain lions and 16% (4/25) of bobcats evaluated histologically and was significantly associated with being Leptospira spp.-positive in both species. Our findings suggest that Leptospira spp. infection is common and widespread in California's wild felids and may have clinical impacts on renal and overall health of individuals. Key words: Bobcat, Leptospira spp., leptospirosis, Lynx rufus, mountain lion, nephritis, pathology, Puma concolor.

Cross-sectional evaluation of multiple epidemiological cycles of Leptospira species in peri-urban wildlife in California.

OBJECTIVE: To perform a cross-sectional survey to estimate prevalence of and potential risk factors for Leptospira spp infection and exposure in peri-urban wildlife throughout California. ANIMALS: 723 animals representing 12 wildlife species. PROCEDURES: Blood and urine samples were obtained from wildlife in California from 2007 to 2017. Live animals were captured in humane traps, anesthetized, and released. Carcasses were donated by wildlife services and necropsied for urine, blood, and kidney tissue samples. Samples were tested for antibodies against 6 serovars of Leptospira spp with a microscopic agglutination test and for pathogenic Leptospira spp DNA with a real-time PCR assay targeting the LipL32 gene. Potential risk factors for Leptospira spp exposure were assessed by logistic regression. Genetic relatedness of Leptospira spp were assessed with DNA sequencing of the rrs2 gene and multiple locus sequence analysis. RESULTS: Statewide Leptospira spp seroprevalence was 39.1%, and prevalence of positive PCR assay results for Leptospira spp DNA was 23.0%. Risk factors for Leptospira spp exposure included being an adult, being from northern California, and being a western gray squirrel, coyote, striped skunk, raccoon, gray fox, or mountain lion. Antibodies against serovar Pomona predominated in most species, followed by serovar Copenhageni. Complete rrs2 sequences were identified as Leptospira interrogans and multiple locus sequence type analysis revealed sequence type 140. CONCLUSIONS AND CLINICAL RELEVANCE: Pathogenic Leptospira spp appeared to be common and widespread among peri-urban wildlife in California. Our data highlight the potential for exposure to infectious disease for both humans and domestic animals at the urban-wildland interface.

Borrelia burgdorferi and Anaplasma phagocytophilum Genospecies in Northern California.

The sensu lato (s.l.) complexes of Borrelia burgdorferi and Anaplasma phagocytophilum include pathogenic genospecies each with distinct ecologies in northern California, yet, most work conflates the genospecies of each pathogen into one sensu lato species. Detailed understanding of the differences in geographic distributions and ecology among genospecies is lacking. We aimed to evaluate whether two B. burgdorferi and two A. phagocytophilum genospecies in high-risk locations in coastal northern California were spatially clustered and if presence of a particular genospecies was associated with geographical site, host species, or other demographic or ecological variables. DNA sequencing was performed to differentiate genospecies of Borreliae and Anaplasma from PCR-positive dusky-footed woodrats (Neotoma fuscipes) and sciurids (chipmunks, Tamias spp., and Douglas squirrels, Tamiasciurus douglasii) at four sites in northwestern California. Logistic regression was performed to assess associations of genospecies with the predictor variables host species, host sex, site, season, and year. Spatial clustering was assessed using a Poisson spatial scan statistic in SaTScan. Host species was a significant predictor for Borrelia bissettiae, B. burgdorferi sensu stricto (s.s.), A. phagocytophilum s.s., and the DU1 Anaplasma genospecies. Woodrats were significantly more likely to be PCR-positive for B. bissettiae and A. phagocytophilum DU1 genospecies, while A. phagocytophilum s.s. and B. burgdorferi s.s. were significantly associated with sciurids. We report a single Borrelia lanei in an Allen's chipmunk (Tamias senex) from the Hoopa Valley Tribal Reservation. A significant spatial cluster of A. phagocytophilum s.s. was detected at Hendy Woods State Park in Mendocino County. These results highlight the need to better understand genospecies partitioning according to host species to further assess human risks, aid in future surveillance, and inform targeted research.

Raccoons (Procyon lotor) and Striped Skunks (Mephitis mephitis) as Potential Reservoirs of Leptospira spp. in California.

Leptospirosis is a globally important, fatal disease of humans, and over 160 species of animals are associated with more than 250 bacterial serovars in 64 species, but its ecology varies regionally and has changed over time with expansion of human development on previously agricultural and wild land. Sporadic human cases and clusters of canine leptospirosis, primarily attributable to Leptospira interrogans serogroup Pomona, have been detected in northern California. Small mesocarnivores such as raccoons and skunks frequent peridomestic space across much of the western United States and could serve as reservoirs for human and canine leptospirosis. We aimed to summarize the prevalence of infection with pathogenic leptospires in skunk and raccoon renal and urinary samples across broad geographic zones in California, and to determine whether prevalence changed during wet and dry seasons, and as functions of host species and demographic characters. Overall, 25.6% (22/86 tested) of raccoons and 28.5% (39/137 tested) of skunks were PCR-positive for Leptospira spp. in either renal tissue or urine, with leptospiral DNA in 22.0% of kidney samples and 18.8% of urine samples from raccoons and 27.8% and 14.5% of kidney and urine samples from skunks, respectively. Raccoons from the Central California and skunks from the San Francisco Bay Area had the highest overall PCR-prevalence (35.7% and 44.4%), respectively, and adults were more likely to be PCR-positive for Leptospira spp. than juveniles. There was moderate agreement between urine and renal tissue Leptospira spp. PCR with sensitivity for both host species in renal tissue of 0.86-0.97 and 0.42-0.64 in urine. Cases of human leptospirosis are thought to be underrecognized in the continental United States and possibly increasing in some states, including California. Our data document regionally high rates of infection in common mesocarnivores, which can pose a threat to humans and dogs, revealing an important periurban epidemiological cycle.

Evaluation of Proctophyllodes huitzilopochtlii on feathers from Anna's (Calypte anna) and Black-chinned (Archilochus alexandri) Hummingbirds: Prevalence assessment and imaging analysis using light and tabletop scanning electron microscopy.

Proctophyllodes huitzilopochtlii Atyeo & Braasch 1966 (Acariformes: Astigmata: Proctophyllodidae), a feather mite, was found on feathers collected from five hummingbird species in California. This mite has not been previously documented on feathers from Anna's (Calypte anna [Lesson 1829]) or Black-chinned (Archilochus alexandri [Bourcier & Mulsant 1846]) Hummingbirds. A total of 753 hummingbirds were evaluated for the presence of mites by species (Allen's n = 112; Anna's n = 500; Black-chinned n = 122; Rufous n = 18; Calliope n = 1), sex (males n = 421; females n = 329; 3 unidentified), and age (juvenile n = 199; after-hatch-year n = 549; 5 unidentified). Of these 753 hummingbirds evaluated, mites were present on the rectrices of 40.9% of the birds. Significantly more Anna's Hummingbirds were positive for rectricial mites (59.2%) compared with 8.2% of Black-chinned, 0.9% of Allen's, 5.6% of Rufous Hummingbirds, and 0% for Calliope (p-value < 0.0001). Across all hummingbird species, male hummingbirds (44.9%) had a higher prevalence of rectricial mites compared to female hummingbirds (36.2%; p-value = 0.004), while juvenile hummingbirds (46.2%) had a non-significantly higher prevalence compared to after-hatch-year hummingbirds (39.0%; p-value = 0.089). On average, the percentage of the long axis of the rachis occupied by mites for the outer rectrices (R4 and R5) was 19%, compared to 11% for inner rectrices (R1 and R2), a significant difference (p-value = <0.0001). There was a marginal lack of significance for symmetrical distribution of tail mites with the mean left side percentage of long axis of the rachis occupied by mites being 16% and very close to the mean right side score of 18% (p-value = 0.003). The identification of the feather mite species was based on light microscopic morphometry, and mite distribution on feathers was further evaluated using tabletop scanning electron microscopy (TSEM). The hummingbird-feather mite relationship is not well understood, but the specialized TSEM technique may be especially useful in examining natural positioning and developmental aspects of the mites since it allows in situ feather examination of live mites.

Distribution and prevalence of vector-borne diseases in California chipmunks (Tamias spp.).

California, with 13 chipmunk (Tamias) species, has more than any other state or country, occupying habitats ranging from chaparral to the high peaks of the Sierra Nevada. Chipmunks host zoonotic pathogens including Yersinia pestis, Anaplasma phagocytophilum, relapsing fever (RF) Borrelia spp., Borrelia burgdorferi, and spotted fever group (SFG) Rickettsia species. Chipmunk species are often not differentiated by public health workers, yet different species utilize different ecological niches and may have intrinsically different capacities for maintaining vector-borne pathogens and infecting vectors. We surveyed over 700 individuals from nine species of chipmunks throughout California for exposure to and infection by Y. pestis, A. phagocytophilum, RF Borrelia spp., Borrelia burgdorferi, and SFG Rickettsia species. DNA of all five pathogens was found and all chipmunks except Merriam's chipmunk (T. merriami) were PCR-positive for at least one of the pathogens. Anaplasma phagocytophilum was most common (40.0%, 2/5) in Sonoma chipmunks (T. sonomae) from Marin county and B. burgdorferi most common (37.5%, 27/72) in redwood chipmunks (T. ochrogenys) from Mendocino county. RF Borrelia spp. was detected in 2% (6/297) of redwood chipmunks in Mendocino county and 10% (1/10) of both least (T. minimus) and lodgepole (T. speciosus) chipmunks in the western Sierra. Exposure to SFG Rickettsia spp. was found in the Northern Coastal region (Del Norte, Humboldt and Mendocino counties) and in the northern and western Sierra in several species of chipmunks. Y. pestis infection was found only in the western Sierra-in a yellow-pine (T. amoenus) and a long-eared (T. quadrimaculatus) chipmunk. Though more data are needed to thoroughly understand the roles that different chipmunk species play in disease transmission, our findings suggest that some chipmunk species may be more important to the maintenance of vector-borne diseases than others within each geographic area.

Distribution and Diversity of Borrelia burgdorferi Sensu Lato Group Bacteria in Sciurids of California.

California has a remarkable diversity of squirrel and chipmunk species (sciurids), and five named and several unnamed genospecies in the Borrelia burgdorferi sensu lato group (BBSL) of bacteria as well, many of which utilize sciurids as reservoirs. We investigated the prevalence, spatial distribution, and diversity of BBSL in sciurids of California by literature search, PCR of 585 ear tissue samples from 15 sciurid species prospectively collected across 19 California counties, and DNA sequencing when possible. Seven publications documented BBSL infections in western gray squirrels (Sciurus griseus), fox squirrels (Sciurus niger), eastern gray squirrels (Sciurus carolinensis), Douglas squirrels (Tamiasciurus douglasii), and redwood chipmunks (Tamias ochrogenys) in northern California. Prospective sampling added new BBSL infection records for long-eared chipmunks (Tamias quadrimaculatus), Allen's chipmunks (Tamias senex), and Siskiyou chipmunks (Tamias siskiyou). Infection was detected in the Mendocino, North Coast, West Sierra, and Central Valley regions of California. The overall PCR prevalence was 9.4% (n = 585), and exceeded 40% (n = 84) in Mendocino and farther north along the Pacific coast. Redwood (40.7%, n = 81) and Siskiyou (22.2%, n = 18) chipmunks had the highest prevalence of BBSL infection. BBSL infections were associated with arboreal and semiarboreal sciurid species and species occurring in conifer forests. Western gray squirrels and Allen's chipmunks in Humboldt County and redwood chipmunks in Mendocino County were infected with B. burgdorferi sensu stricto, while we identified Borrelia bissettiae in Douglas squirrels and Siskiyou chipmunks in Humboldt and Del Norte Counties. This indicates that further study of sciurids can aid in describing the ecology of BBSL in California.

Multiple-host pathogens in domestic hunting dogs in Nicaragua's Bosawás Biosphere Reserve.

Nicaragua's Bosawás Biosphere Reserve is a vast forested area inhabited largely by indigenous Mayangna and Miskitu people. Most Bosawás residents rely on subsistence hunting and swidden agriculture, and hunting dogs are important for finding and securing wild game. We investigated the health of hunting dogs in three communities differing in location, size, and economy. Dogs in all communities were nutritionally compromised and experienced a heavy burden of disease. Seroprevalence of canine distemper, canine parvovirus, Rickettsia rickettsii, and Leptospira spp. exceeded 50% of dogs. At least one dog was actively shedding leptospires in urine, and many dogs were anemic and/or dehydrated. These dogs interact with wildlife in the forest and humans and domestic livestock in the communities, and may therefore serve as sources of zoonotic and wildlife diseases. Bosawás represents one of the largest intact tracts of habitat for jaguars (Panthera onca) in Central America, and given that these communities are located within the forest, jaguars may be at risk from disease spillover from hunting dogs. Dog owners reported that four of 49 dogs had been attacked and killed by jaguars in the past year, and that retaliatory killing of jaguars was sometimes practiced. Disease spillover from dogs to wildlife could occur both in the course of dogs' hunting activities as well as during jaguar attacks. A better understanding of dog depredation by jaguars, pathogen exposure in jaguars, and a management strategy for the hunting dog population, are urgently needed to mitigate these dual threats to jaguars, improve the lives of hunting dogs, and safeguard the health of their owners.

Seroepidemiologic Survey of Potential Pathogens in Obligate and Facultative Scavenging Avian Species in California.

Throughout the world, populations of scavenger birds are declining rapidly with some populations already on the brink of extinction. Much of the current research into the factors contributing to these declines has focused on exposure to drug residues, lead, and other toxins. Despite increased monitoring of these declining populations, little is known about infectious diseases affecting scavenger bird species. To assess potential infectious disease risks to both obligate and facultative scavenger bird species, we performed a serosurvey for eleven potential pathogens in three species of scavenging birds in California: the California condor (Gymnogyps californianus), turkey vulture (Cathartes aura) and golden eagle (Aquila chrysaetos). California condors were seropositive for avian adenovirus, infectious bronchitis virus, Mycoplasma gallisepticum, avian paramyxovirus-2, West Nile virus (WNV) and Toxoplasma gondii. Golden eagles were seropositive for avian adenovirus, Chlamydophila psittaci and Toxoplasma gondii, and turkey vultures were seropositive for avian adenovirus, Chlamydophila psittaci, avian paramyxovirus-1, Toxoplasma gondii and WNV. Risk factor analyses indicated that rearing site and original release location were significantly associated with a positive serologic titer to WNV among free-flying condors. This study provides preliminary baseline data on infectious disease exposure in these populations for aiding in early disease detection and provides potentially critical information for conservation of the endangered California condor as it continues to expand its range and encounter new infectious disease threats.

Oral shedding of Bartonella in cats: correlation with bacteremia and seropositivity.

Cats are the main reservoirs of zoonotic Bartonella henselae, B. clarridgeiae and B. koehlerae, transmitted among cats by cat fleas. No study has investigated the presence of Bartonella in the saliva of bacteremic and non-bacteremic cats to correlate it to the level of bacteremia and the presence or absence of oral lesions. Shelter cats from northern California (n=130) and Michigan (n=50) were tested for Bartonella bacteremia by blood culture, presence of Bartonella antibodies and Bartonella DNA in oral swabs. Bacteremia was detected in 45 (25%) cats, mainly from northern California (n=40), which were highly flea infested and were 4 times more likely to be bacteremic than the non-flea-infested cats from Michigan. Overall, 69 (38.3%) cats had Bartonella PCR positive oral swabs. Bacteremic cats were almost 3 times (P=0.003) more likely to have PCR positive oral swabs (59%, 26/44) than non-bacteremic cats (32.5%, 44/135). However, there was no correlation between cats being bacteremic and having oral lesions. Antibody prevalences for B. henselae and B. clarridgeiae were 30% and 42.8%. B. henselae and B. clarridgeiae seropositive cats were almost 4 times (P=0.0001) and 3 times (P=0.003) more likely to have oral lesions than seronegative cats. Despite a higher prevalence (odds ratio=1.73; 95% confidence interval=0.88-3.38) of oral lesions in cats with oral swabs testing PCR positive, no statistical association could be demonstrated in this cat population.

Diagnostic efficacy of surgeon-performed ultrasound-guided fine needle aspiration: a randomized controlled trial.

OBJECTIVE: To evaluate the clinical efficacy of surgeon-performed, office-based head and neck ultrasound in facilitating diagnostic fine needle aspiration (FNA) of lesions in the head and neck. STUDY DESIGN: A randomized controlled trial of ultrasound-guided FNA versus traditional palpation-guided technique for palpable masses in the head and neck. SETTING: An office-based study performed in a military academic medical center. SUBJECTS AND METHODS: Eighty-one adults older than 18 years of age with a palpable head and neck mass (less than 3 cm in largest diameter) were randomized to ultrasound-guided or traditional palpation-guided FNA of a head and neck mass. Measured variables and outcomes for the study included tissue adequacy rates, tissue type, and operator variability. RESULTS: Following three passes using either palpation or ultrasound guidance, a comparative tissue adequacy rate of 84 percent for ultrasound guidance versus 58 percent for standard palpation was established (P < 0.014). With regard to tissue type, a statistically significant comparative diagnostic advantage for ultrasound guidance was observed in thyroid tissue while remaining statistically insignificant for lymphatic and salivary tissues. No statistical significance was found when comparing the ability of otolaryngology residents versus attending otolaryngologists to obtain ultrasound-guided diagnostic samples. CONCLUSION: Office-based surgeon-performed ultrasound-guided FNA of palpable lesions in the head and neck yields a statistically significant higher diagnostic rate compared to standard palpation technique. Our institutional experience supports the utility of surgeon-performed ultrasound as a core competency in clinical practice.

Structure-based chemical modification strategy for enzyme replacement treatment of phenylketonuria.

Structure-based protein engineering coupled with chemical modifications (e.g., pegylation) is a powerful combination to significantly improve the development of proteins as therapeutic agents. As a test case, phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) was selected for enzyme replacement therapy in phenylketonuria [C.R. Scriver, S. Kaufman, Hyperphenylalaninemia:phenylalanine Hydroxylase Deficiency. The Metabolic and Molecular Bases of Inherited Disease, McGraw-Hill, New York, 2001, Chapter 77], an inherited metabolic disorder (OMIM 261600) causing mental retardation due to deficiency of the enzyme l-phenylalanine hydroxylase (EC 1.14.16.1). Previous in vivo studies of recombinant PAL demonstrated a lowering of blood l-phenylalanine levels; yet, the metabolic effect was not sustained due to protein degradation and immunogenicity [C.N. Sarkissian, Z. Shao, F. Blain, R. Peevers, H. Su, R. Heft, T.M. Chang, C.R. Scriver, A different approach to treatment of phenylketonuria:phenylalanine degradation with recombinant phenylalanine ammonia lyase, Proc. Natl. Acad. Sci. USA 96 (1999) 2339; J.A. Hoskins, G. Jack, H.E. Wade, R.J. Peiris, E.C. Wright, D.J. Starr, J. Stern, Enzymatic control of phenylalanine intake in phenylketonuria, Lancet 1 (1980) 392; C.M. Ambrus, S. Anthone, C. Horvath, K. Kalghatgi, A.S. Lele, G. Eapen, J.L. Ambrus, A.J. Ryan, P. Li, Extracorporeal enzyme reactors for depletion of phenylalanine in phenylketonuria, Ann. Intern. Med. 106 (1987) 531]. Here, we report the 1.6A three-dimensional structure of Rhodosporidium toruloides PAL, structure-based molecular engineering, pegylation of PAL, as well as in vitro and in vivo PKU mouse model studies on pegylated PAL formulations. Our results show that pegylation of R. toruloides PAL leads to promising therapeutic efficacy after subcutaneous injection by enhancing the in vivo activity, lowering plasma phenylalanine, and leading to reduced immunogenicity. The three-dimensional structure of PAL provides a basis for understanding the properties of pegylated forms of PAL and strategies for structure-based re-engineering of PAL for PKU treatment.

Development of pegylated forms of recombinant Rhodosporidium toruloides phenylalanine ammonia-lyase for the treatment of classical phenylketonuria.

Phenylketonuria (PKU) is a metabolic disorder due primarily to mutations in the PAH gene that impair both phenylalanine hydroxylase activity and disposal of l-phenylalanine from the normal diet. Excess phenylalanine is toxic to cognitive development and a low-phenylalanine diet prevents mental retardation, but it is a difficult therapeutic option. Previous studies with recombinant phenylalanine ammonia-lyase, PAL, demonstrated pharmacologic and physiologic proofs of principle for PAL as an alternative therapy for PKU but its immunogenicity was problematic. From a series of formulations of linear and branched polyethylene glycols chemically conjugated to PAL, we have created a parenteral therapeutic agent for PKU treatment. All the pegylated molecules were fully characterized in vitro and the most promising formulations were then tested in vivo in the PKU mouse model. The linear 20-kDa PEG-PAL combination abolished in vivo immunogenicity after repeated challenge while retaining full catabolic activity against phenylalanine, suggesting potential as a novel PKU therapeutic.

Toward PKU enzyme replacement therapy: PEGylation with activity retention for three forms of recombinant phenylalanine hydroxylase.

Phenylketonuria (PKU) is a disease in which phenylalanine and phenylalanine-derived metabolites build up to neurotoxic levels due to mutations in the phenylalanine hydroxylase gene (PAH). Enzyme replacement therapy is a viable option to supply active PAH. However, the inherent protease sensitivity and potential immunogenicity of PAH have precluded adoption of this approach. In this report, we have used polyethylene glycol derivatization (PEGylation) to produce protected forms of PAH for potential therapeutic use. Three recombinantly produced PAH enzymes were reacted with activated PEG species, with the aim of developing a stable and active PKU enzyme replacement. Tetrameric full-length human PAH, dimeric double-truncated (DeltaN102-DeltaC428) human PAH, and monomeric Chromobacterium violaceum PAH were PEGylated with succinimidyl succinate polyethylene glycol of molecular weight 5000 or 20,000 Da. Characterization of the PEGylated species was accomplished with MALDI-TOF mass spectrometry, SDS-PAGE, and specific activity measurements using ESI mass spectrometry. All PEG-derivatized PAH species retained catalytic activity, and, at low numbers of PEG molecules attached, these PEGylated PAH proteins were found to be more active and more stable than their non-derivatized PAH counterparts.

Structural studies on phenylalanine hydroxylase and implications toward understanding and treating phenylketonuria.

Mutations in the gene encoding for phenylalanine hydroxylase (PAH) result in phenylketonuria (PKU) or hyperphenylalaninemia (HPA). Several 3-dimensional structures of truncated forms of PAH have been determined in our laboratory and by others, using x-ray crystallographic techniques. These structures have allowed for a detailed mapping of the >250 missense mutations known to cause PKU or HPA found throughout the 3 domains of PAH. This structural information has helped formulate rules that might aid in predicting the likely effects of unclassified or newly discovered PAH mutations. Also, with the aid of recent crystal structure determinations of co-factor and substrate analogs bound at the PAH active site, the recently discovered tetrahydrobiopterin-responsive PKU/HPA genotypes can be mapped onto the PAH structure, providing a molecular basis for this tetrahydrobiopterin response.