Daniel Rejmanek

Detection of Lawsonia intracellularis by Real-time PCR in the Feces of Free-living Animals from Equine Farms with Documented Occurrence of Equine Proliferative Enteropathy

The objective of this study was to determine whether Lawsonia intracellularis was present in the feces of free-living animals collected on two equine premises with documented occurrence of equine proliferative enteropathy (EPE). Fresh feces from black-tailed jackrabbits (Lepus californicus, n=100), striped skunks (Mephitis mephitis, n=22), feral cats (Felis catus, n=14), Brewers Blackbirds (Euphagus cyanocephalus, n=10), Virginian opossums (Didelphis virginiana, n=9), raccoons (Procyon lotor, n=4), California ground squirrels (Spermophilus beecheyi, n=3), and coyotes (Canis latrans, n=2) were collected from August 2006 to January 2007 either from the ground while walking the premises or after trapping the animals using live traps. Nucleic acid purified from feces was directly processed for polymerase chain reaction (PCR) analysis using a real-time PCR assay targeting the aspartate ammonia lyase gene of L. intracellularis. Purified DNA samples were also precipitated, preamplified for L. intracellularis, and analyzed using the same real-time PCR assay, to increase the detection limit to one L. intracellularis organism per extracted sample. Feces from jackrabbits, striped skunks, Virginian opossums, and coyotes tested PCR positive for L. intracellularis, whereas all feces from feral cats, Brewers Blackbirds, raccoons, and ground squirrels tested PCR negative for L. intracellularis. PCR testing on DNA extracted directly from feces was positive for L. intracellularis in six of 164 fecal samples. When DNA purification from feces was followed by a precipitation and preamplification step, five additional fecal samples tested PCR positive for L. intracellularis (11/164). The largest number of PCR positive L. intracellularis fecal samples was observed in striped skunks, followed by Virginian opossums, jackrabbits, and coyotes. This is the first description of L. intracellularis in these four species. Because the fecal samples were collected at equine farms with confirmed cases of EPE, striped skunks, Virginian opossums, jackrabbits, and coyotes may act as potential sources of infection to susceptible weanlings.

Molecular characterization reveals distinct genospecies of Anaplasma phagocytophilum from diverse North American hosts

Anaplasma phagocytophilum is an emerging tick-borne pathogen that infects humans, domestic animals and wildlife throughout the Holarctic. In the far-western United States, multiple rodent species have been implicated as natural reservoirs for A. phagocytophilum. However, the presence of multiple A. phagocytophilum strains has made it difficult to determine which reservoir hosts pose the greatest risk to humans and domestic animals. Here we characterized three genetic markers (23S-5S rRNA intergenic spacer, ank and groESL) from 73 real-time TaqMan PCR-positive A. phagocytophilum strains infecting multiple rodent and reptile species, as well as a dog and a horse, from California. Bayesian and maximum-likelihood phylogenetic analyses of all three genetic markers consistently identified two major clades, one of which consisted of A. phagocytophilum strains infecting woodrats and the other consisting of strains infecting sciurids (chipmunks and squirrels) as well as the dog and horse strains. In addition, analysis of the 23S-5S rRNA spacer region identified two unique and highly dissimilar clades of A. phagocytophilum strains infecting several lizard species. Our findings indicate that multiple unique strains of A. phagocytophilum with distinct host tropisms exist in California. Future epidemiological studies evaluating human and domestic animal risk should incorporate these distinctions.

Congenital Transmission of Toxoplasma gondii in Deer Mice (Peromyscus maniculatus) After Oral Oocyst Infection

Abstract To investigate how different routes of Toxoplasma gondii transmission influence the antibody response and infection status of deer mice (Peromyscus maniculatus), 80 mice were orally infected with 1, 5, 10, or 100 T. gondii oocysts. Ten weeks postinfection, 15 T. gondii–seropositive female mice were bred and allowed to produce 2 litters. Evidence of persistent T .gondii infection in orally infected mice was detected by serology and DNA amplification in mice from all 4 oocyst treatment groups, including those that received only a single T. gondii oocyst. Congenital transmission of T. gondii was detected by polymerase chain reaction (PCR) in 7/8 first and 4/7 second litters. Toxoplasma gondii was also detected by PCR in 9/30 congenitally infected offspring 16 wk after birth, despite the fact that detectable serological titers had waned. These findings raise questions about the applicability of serological testing to assess the prevalence of T. gondii infection in deer mice and other rodents in the wild. Additionally, the detection of frequent congenital transmission suggests that deer mice could help maintain T. gondii in the environment even in the absence of definitive feline hosts.

Using molecular epidemiology to track Toxoplasma gondii from terrestrial carnivores to marine hosts: implications for public health and conservation.

Background Environmental transmission of the zoonotic parasite Toxoplasma gondii, which is shed only by felids, poses risks to human and animal health in temperate and tropical ecosystems. Atypical T. gondii genotypes have been linked to severe disease in people and the threatened population of California sea otters. To investigate land-to-sea parasite transmission, we screened 373 carnivores (feral domestic cats, mountain lions, bobcats, foxes, and coyotes) for T. gondii infection and examined the distribution of genotypes in 85 infected animals sampled near the sea otter range. Methodology/Principal Findings Nested PCR-RFLP analyses and direct DNA sequencing at six independent polymorphic genetic loci (B1, SAG1, SAG3, GRA6, L358, and Apico) were used to characterize T. gondii strains in infected animals. Strains consistent with Type X, a novel genotype previously identified in over 70% of infected sea otters and four terrestrial wild carnivores along the California coast, were detected in all sampled species, including domestic cats. However, odds of Type X infection were 14 times higher (95% CI: 1.3–148.6) for wild felids than feral domestic cats. Type X infection was also linked to undeveloped lands (OR = 22, 95% CI: 2.3–250.7). A spatial cluster of terrestrial Type II infection (P = 0.04) was identified in developed lands bordering an area of increased risk for sea otter Type II infection. Two spatial clusters of animals infected with strains consistent with Type X (P≤0.01) were detected in less developed landscapes. Conclusions Differences in T. gondii genotype prevalence among domestic and wild felids, as well as the spatial distribution of genotypes, suggest co-existing domestic and wild T. gondii transmission cycles that likely overlap at the interface of developed and undeveloped lands. Anthropogenic development driving contact between these cycles may increase atypical T. gondii genotypes in domestic cats and facilitate transmission of potentially more pathogenic genotypes to humans, domestic animals, and wildlife.

Molecular characterization of Sarcocystis neurona strains from opossums (Didelphis virginiana) and intermediate hosts from Central California.

Sarcocystis neurona is a significant cause of neurological disease in horses and other animals, including the threatened Southern sea otter (Enhydra lutris nereis). Opossums (Didelphis virginiana), the only known definitive hosts for S. neurona in North America, are an introduced species in California. S. neurona DNA isolated from sporocysts and/or infected tissues of 10 opossums, 6 horses, 1 cat, 23 Southern sea otters, and 1 harbor porpoise (Phocoena phocoena) with natural infections was analyzed based on 15 genetic markers, including the first internal transcribed spacer (ITS-1) region; the 25/396 marker; S. neurona surface antigen genes (snSAGs) 2, 3, and 4; and 10 different microsatellites. Based on phylogenetic analysis, most of the S. neurona strains segregated into three genetically distinct groups. Additionally, fifteen S. neurona samples from opossums and several intermediate hosts, including sea otters and horses, were found to be genetically identical across all 15 genetic markers, indicating that fatal encephalitis in Southern sea otters and equine protozoal myeloencephalitis (EPM) in horses is strongly linked to S. neurona sporocysts shed by opossums.

Temporal patterns of tick-borne granulocytic anaplasmosis in California

Granulocytic anaplasmosis (GA) is a tick-borne emerging infectious disease caused by the bacterium Anaplasma phagocytophilum. From fall 2005 to spring 2007, A. phagocytophilum infection prevalence in small mammals and tick abundance were monitored at 4 study sites in coastal California. The abundance of different life stages of questing Ixodes pacificus ticks fluctuated seasonally with the number of adults peaking December to February, nymphs peaking May to July, and larvae peaking April to June. Numerous Ixodes tick species were found attached to dusky-footed woodrats (Neotoma fuscipes), chimunks (Tamias spp.), and deer mice (Peromyscus maniculatus); however, attached tick larvae on all 3 rodent species were primarily I. pacificus, attached nymphs were primarily I. angustus, and adults were either I. ochotonae, I. spinipalpis, or I. woodi. A. phagocytophilum DNA was detected by PCR in 2.2% (n=275, 95% C.I.=0.09-4.9) of sampled ticks. The overall A. phagocytophilum seroprevalence among small mammals was 7.4% (n=654, 95% C.I.=5.5-9.7) while 7.2% (n=125, 95% C.I.=3.5-13.4) of the animals were found to be PCR-positive. Seropositive animals included woodrats, chipmunks, and deer mice, although only woodrats and chipmunks had PCR-detectable infections. Seroprevalence varied temporally among species with the majority of exposed deer mice detected in fall 2006 and the majority of exposed woodrats and chipmunks identified in spring 2007. This study highlights the importance of multiple-year monitoring of both vectors and wildlife hosts in order to better understand the complex ecology of A. phagocytophilum and other related tick-borne disease agents.

Prevalence and risk factors associated with Sarcocystis neurona infections in opossums (Didelphis virginiana) from central California.

Sarcocystis neurona, a protozoal parasite shed by opossums (Didelphis virginiana), has been shown to cause significant morbidity and mortality in horses, sea otters, and other marine mammals. Over the course of 3 years (fall 2005-summer 2008), opossums from central California were tested for infection with S. neurona. Of 288 opossums sampled, 17 (5.9%) were infected with S. neurona based on the molecular characterization of sporocysts from intestinal scrapings or feces. Risk factors evaluated for association with S. neurona infection in opossums included: age, sex, location, season, presence of pouch young in females, concomitant infection, and sampling method (live-trapped or traffic-killed). Multivariate logistic regression analysis identified that opossums in the Central Valley were 9 times more likely to be infected than those near the coast (p=0.009). Similarly, opossum infection was 5 times more likely to be detected during the reproductive season (March-July; p=0.013). This first investigation of S. neurona infection prevalence and associated risk factors in opossums in the western United States can be used to develop management strategies aimed at reducing the incidence of S. neurona infections in susceptible hosts, including horses and threatened California sea otters (Enhydra lutris neries).

Evolutionary Dynamics and Global Diversity of Influenza A Virus

ABSTRACT The increasing number of zoonotic infections caused by influenza A virus (IAV) subtypes of avian origin (e.g., H5N1 and H7N9) in recent years underscores the need to better understand the factors driving IAV evolution and diversity. To evaluate the current feasibility of global analyses to contribute to this aim, we evaluated information in the public domain to explore IAV evolutionary dynamics, including nucleotide substitution rates and selection pressures, using 14 IAV subtypes in 32 different countries over a 12-year period (2000 to 2011). Using geospatial information from 39,785 IAV strains, we examined associations between subtype diversity and socioeconomic, biodiversity, and agricultural indices. Our analyses showed that nucleotide substitution rates for 11 of the 14 evaluated subtypes tended to be higher in Asian countries, particularly in East Asia, than in Canada and the United States. Similarly, at a regional level, subtypes H5N1, H5N2, and H6N2 exhibited significantly higher substitution rates in East Asia than in North America. In contrast, the selection pressures (measured as ratios of nonsynonymous to synonymous evolutionary changes [dN/dS ratios]) acting on individual subtypes showed little geographic variation. We found that the strongest predictors for the detected subtype diversity at the country level were reporting effort (i.e., total number of strains reported) and health care spending (an indicator of economic development). Our analyses also identified major global gaps in IAV reporting (including a lack of sequences submitted from large portions of Africa and South America and a lack of geolocation information) and in broad subtype testing which, until addressed, will continue to hinder efforts to track the evolution and diversity of IAV around the world. IMPORTANCE In recent years, an increasing number of influenza A virus (IAV) subtypes, including H5N1, H7N9, and H10N8, have been detected in humans. High fatality rates have led to an increased urgency to better understand where and how novel pathogenic influenza virus strains emerge. Our findings showed that mutational rates of 11 commonly encountered subtypes were higher in East Asian countries than in North America, suggesting that there may be a greater risk for the emergence of novel pathogenic strains in East Asia. In assessing the potential drivers of IAV subtype diversity, our analyses confirmed that reporting effort and health care spending were the best predictors of the observed subtype diversity at the country level. These findings underscore the need to increase sampling and reporting efforts for all subtypes in many undersampled countries throughout the world.

Unique strains of Anaplasma phagocytophilum segregate among diverse questing and non-questing Ixodes tick species in the western United States

The emerging tick-borne pathogen Anaplasma phagocytophilum infects humans, domestic animals, and wildlife throughout the Holarctic. In the western US, the ecology of A. phagocytophilum is particularly complex, with multiple pathogen strains, tick vectors, and reservoir hosts. A recent phylogenetic analysis of A. phagocytophilum strains isolated from various small mammal hosts in California documented distinct clustering of woodrat strains separate from sciurid (chipmunk and squirrel) strains. Here, we identified strains of A. phagocytophilum in various Ixodes tick species in California and related these genotypes to those found among reservoir and clinical hosts from the same areas. The sequences from all of the nidicolous (nest-dwelling) Ixodes ticks grouped within a clade that also contained all of the woodrat-origin A. phagocytophilum strains. Two of the I. pacificus sequences were also grouped within this woodrat clade, while the remaining five belonged to a less genetically diverse clade that included several sciurid-origin strains as well as a dog, a horse, and a human strain. By comparing A. phagocytophilum strains from multiple sources concurrently, we were able to gain a clearer picture of how A. phagocytophilum strains in the western US are partitioned, which hosts and vectors are most likely to be infected with a particular strain, and which tick species and reservoir hosts pose the greatest health risk to humans and domestic animals.

Evolution of antigen variation in the tick-borne pathogen Anaplasma phagocytophilum

Anaplasma phagocytophilum is an obligately intracellular tick-transmitted bacterial pathogen of humans and other animals. During the course of infection, A. phagocytophilum utilizes gene conversion to shuffle ∼100 functional pseudogenes into a single expression cassette of the msp2(p44) gene, which codes for the major surface antigen and major surface protein 2 (MSP2). The role and extent of msp2(p44) recombination, particularly in hosts that only experience acute infections, is not clear. In the present study, we explored patterns of recombination and expression of the msp2(p44) gene of A. phagocytophilum in a serially infected mouse model. Even though the bacterium was passed rapidly among mice, minimizing the opportunities for the host to develop adaptive immunity, we detected the emergence of 34 unique msp2(p44) expression cassette variants. The expression of msp2(p44) pseudogenes did not follow a consistent pattern among different groups of mice, although some pseudogenes were expressed more frequently than others. In addition, among 263 expressed pseudogenes, 3 mosaic sequences each consisting of 2 different pseudogenes were identified. Population genetic analysis showed that genetic diversity and subpopulation differentiation tended to increase over time until stationarity was reached but that the variance that was observed in allele (expressed pseudogene) frequency could occur by drift alone only if a high variance in bacterial reproduction could be assumed. These findings suggest that evolutionary forces influencing antigen variation in A. phagocytophilum may comprise random genetic drift as well as some innate but apparently nonpurifying selection prior to the strong frequency-dependent selection that occurs cyclically after hosts develop strong adaptive immunity.