TRYPANOSOMA CRUZI IN FREE-RANGING MAMMALIAN POPULATIONS IN SOUTH TEXAS, USA.

Chagas disease, also known as American trypanosomiasis, is caused by the flagellate protozoan Trypanosoma cruzi. It is a significant health concern in South and Central America, where millions of people are infected or at risk of infection, and is an emerging health concern in the US. The occurrence of Chagas disease in natural environments is supported by mammal host species, but those primary species may vary based on geographic location. In South Texas, the primary host species for the disease is poorly understood, and required a field study to determine the spatial distribution of T. cruzi prevalence in free-ranging mammals. Our study objectives were to determine the spatial distribution and prevalence of T. cruzi parasites in free-ranging mammals. We compared T. cruzi prevalence among species, among vegetative communities, and among different topographies (i.e., floodplain versus upland). From December 2011 through December 2013, 450 blood and tissue samples from geolocated free-ranging wildlife mammal species were analyzed with the use of polymerase chain reaction to detect protozoan T. cruzi DNA. We also calculated mammal abundance with the use of mark-recapture methodology and recorded capture-site characteristics such as vegetation structure. We found that animals in grasslands had a significantly lower infection rate when summed across all species compared with animals in dense hardwoods and semi-improved woodlands (P=0.001). A higher percentage of infections were found in the lower-elevation floodplain-65% (28/43) of animals sampled, compared to upland areas-25% (9/36) of animals sampled. Our study suggested that common free-ranging meso-mammals supported T. cruzi in natural environments and are of public health concern in South Texas. Mitigation strategies should consider a range of management activities to include vegetation management, selective application of insecticides, and changes in human behavior in high-risk areas.

Genome-wide polymorphism and comparative analyses in the white-tailed deer (Odocoileus virginianus): a model for conservation genomics.

The white-tailed deer (Odocoileus virginianus) represents one of the most successful and widely distributed large mammal species within North America, yet very little nucleotide sequence information is available. We utilized massively parallel pyrosequencing of a reduced representation library (RRL) and a random shotgun library (RSL) to generate a complete mitochondrial genome sequence and identify a large number of putative single nucleotide polymorphisms (SNPs) distributed throughout the white-tailed deer nuclear and mitochondrial genomes. A SNP validation study designed to test specific classes of putative SNPs provides evidence for as many as 10,476 genome-wide SNPs in the current dataset. Based on cytogenetic evidence for homology between cow (Bos taurus) and white-tailed deer chromosomes, we demonstrate that a divergent genome may be used for estimating the relative distribution and density of de novo sequence contigs as well as putative SNPs for species without draft genome assemblies. Our approach demonstrates that bioinformatic tools developed for model or agriculturally important species may be leveraged to support next-generation research programs for species of biological, ecological and evolutionary importance. We also provide a functional annotation analysis for the de novo sequence contigs assembled from white-tailed deer pyrosequencing reads, a mitochondrial phylogeny involving 13,722 nucleotide positions for 10 unique species of Cervidae, and a median joining haplotype network as a putative representation of mitochondrial evolution in O. virginianus. The results of this study are expected to provide a detailed template enabling genome-wide sequence-based studies of threatened, endangered or conservationally important non-model organisms.

Molecular detection of Babesia bovis and Babesia bigemina in white-tailed deer (Odocoileus virginianus) from Tom Green County in central Texas.

Serologic and molecular evidence suggest that white-tailed deer in South Texas and North Mexico carry the agents of bovine babesiosis, Babesia bovis and Babesia bigemina. To determine if white-tailed deer in central Texas, which is outside the known occurrence of the vector tick at this time, harbor these parasites, blood samples from free-ranging and captive white-tailed deer (Odocoileus virginianus) in Tom Green County were tested by polymerase chain reaction (PCR) assays for B. bovis and B. bigemina 18S rDNA. Of the 25 samples tested, three (12%) were positive by nested PCR for B. bovis. This identity was confirmed by sequence analysis of the cloned 18S rDNA PCR product. Further confirmation was made by sequence analysis of the rRNA internal transcribed spacer (ITS) 1, 5.8S rRNA gene, and ITS 2 genomic region in two (representing samples from two different ranches) of the B. bovis positive samples. Three samples were positive by B. bigemina nested PCR, but sequencing of the cloned products confirmed only one animal positive for B. bigemina; Theileria spp. DNA was amplified from the other two animal samples. In addition to Theileria spp., two genotypically unique Babesia species sequences were identified among the cloned sequences produced by the B. bigemina primers in one sample. Phylogenetic analysis showed no separation of the deer B. bovis or B. bigemina 18S rDNA, or deer B. bovis ITS region sequences from those of bovine origin. Clarification of the possible role of white-tailed deer as reservoir hosts in maintaining these important pathogens of cattle is critical to understanding whether or not deer contribute to the epidemiology of bovine babesiosis.

Oral vaccination with microencapsuled strain 19 vaccine confers enhanced protection against Brucella abortus strain 2308 challenge in red deer (Cervus elaphus elaphus).

Bison (Bison bison) and elk (Cervus elaphus nelsoni) in the Greater Yellowstone Area (GYA), USA, are infected with Brucella abortus, the causative agent of bovine brucellosis, and they serve as a wildlife reservoir for the disease. Bovine brucellosis recently has been transmitted from infected elk to cattle in Montana, Wyoming, and Idaho and has resulted in their loss of brucellosis-free status. An efficacious Brucella vaccine with a delivery system suitable for wildlife would be a valuable tool in a disease prevention and control program. We evaluated Strain 19 (S19) in a sustained release vehicle consisting of alginate microspheres containing live vaccine. In a challenge study using red deer (Cervus elaphus elaphus) as a model for elk, alginate, a naturally occurring polymer combined with a protein of Fasciola hepatica vitelline protein B was used to microencapsulate S19. Red deer were orally or subcutaneously immunized with 1.5 x 10(10) colony-forming units (CFUs) using microencapsulated S19. Humoral and cellular profiles were analyzed bimonthly throughout the study. The vaccinated red deer and nonvaccinated controls were challenged 1 yr postimmunization conjunctivally with 1 x 10(9) CFUs of B. abortus strain 2308. Red deer vaccinated with oral microencapsulated S19 had a statistically significant lower bacterial tissue load compared with controls. These data indicate for the first time that protection against Brucella-challenge can be achieved by combining a commonly used vaccine with a novel oral delivery system such as alginate-vitelline protein B microencapsulation. This system is a potential improvement for efficacious Brucella-vaccine delivery to wildlife in the GYA.

Enhanced immune response of red deer (Cervus elaphus) to live rb51 vaccine strain using composite microspheres.

Brucellosis is an important zoonotic disease of nearly worldwide distribution. The occurrence of the infection in humans is largely dependent on the prevalence of brucellosis in animal reservoirs, including wildlife. The current vaccine used for cattle Brucella abortus strain RB51, has proven ineffective in protecting bison (Bison bison) and elk (Cervus nelsoni) from infection and abortion. To test possible improvements in vaccine efficacy, a novel approach of immunization was examined from April 2004 to November 2006 using alginate composite microspheres containing a nonimmunogenic, eggshell-precursor protein of the parasite Fasciola hepatica (Vitelline protein B, VpB) to deliver live vaccine strain RB51. Red deer (Cervus elaphus), used as a model for elk, were vaccinated orally (PO) or subcutaneously (SC) with 1.5x10(10) viable organisms per animal. Humoral responses postvaccination (immunoglobulin G [IgG] levels), assessed at different time points, indicated that capsules containing live RB51 elicited an anti-Brucella specific IgG response. Furthermore, the encapsulated vaccine elicited a cell-mediated response that the nonencapsulated vaccinates failed to produce. Finally, red deer were challenged with B. abortus strain 19 by conjunctival exposure. Only animals that received encapsulated RB51 vaccine by either route exhibited a significant reduction in bacterial counts in their spleens. These data suggest that alginate-VpB microspheres provide a method to enhance the RB51 vaccine performance in elk.

Molecular characterization of the Rocky Mountain elk (Cervus elaphus nelsoni) PRNP putative promoter.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) affecting deer (Odocoileus spp.), moose (Alces alces), and Rocky Mountain elk (Cervus elaphus nelsoni). Leucine homozygosity at elk PRNP codon 132 has been associated with reduced CWD susceptibility. However, naturally acquired CWD has been detected in elk possessing the 132 Leu/Leu genotype. Recent human and bovine studies indicate that PRNP regulatory polymorphisms may also influence TSE occurrence. Therefore, we generated sequences for the elk PRNP putative promoter (2.2 kb), exon 1 (predicted; 54 bp), intron 1 (predicted; 193 bp), and exon 3 (771 bp). Promoter prediction analysis using CpGProD yielded a single elk PRNP promoter that was homologous to regions of known promoter activity in cow and sheep. Molecular interrogation of the elk PRNP putative promoter revealed 32 diallelic single-nucleotide polymorphisms (SNPs). No variation was detected within the predicted exon 1 or intron 1 sequences. Evaluation of elk PRNP exon 3 revealed 3 SNPs (63Y, 312R, 394W-->Met/Leu). Bayesian haplotype reconstruction resulted in 3 elk PRNP haplotypes, with complete linkage disequilibrium observed between all PRNP putative promoter SNPs and codon 132. The results of this study provide the initial genomic foundation for future comparative and haplotype-based elk PRNP studies.

Effectiveness of Spayvac for reducing white-tailed deer fertility.

Overabundant white-tailed deer (Odocoileus virginianus) populations have been reported in many urban and suburban communities across the United States. Large populations of deer can potentially increase the risk of human-wildlife conflicts, such as deer-vehicle collisions, transmission of disease to humans, and vegetation damage. In 2003, efforts to control white-tailed deer numbers were initiated at the National Aeronautical and Space Agency's (NASA) Lyndon B. Johnson Space Center (JSC) in Houston, Texas, using the long-lasting, single-dose contraceptive SpayVac. Our objectives were to evaluate the effectiveness of SpayVac for reducing white-tailed deer fertility and determine the partial cost for treatment. Between 2003 and 2004, we monitored 45 adult female deer (34 treated with SpayVac, 11 controls treated with a placebo). Fawning rate over 2 yr for deer treated with SpayVac >30 days prior to the rut was 0% (n=31), whereas the fawning rate for control deer was 78% (n=11). Inoculation 1 mo prior to the breeding season was sufficient time to achieve fertility control. We conclude that SpayVac can effectively reduce the fertility of urban white-tailed deer.

Evaluation of novel Brucella melitensis unmarked deletion mutants for safety and efficacy in the goat model of brucellosis.

Pregnant goats were employed to assess unmarked deletion mutant vaccine candidates BMDeltaasp24, BMDeltacydBA, and BMDeltavirB2, as the target host species naturally infected with Brucella melitensis. Goats were assessed for the degree of pathology associated with the vaccine strains as well as the protective immunity afforded by each strain against abortion and infection after challenge with wild-type Brucella melitensis 16M. Both BMDeltaasp24 and BMDeltavirB2 were considered safe vaccine candidates in the pregnant goat model because they did not cause abortion or colonize fetal tissues. BMDeltaasp24 was isolated from the maternal tissues only, indicating a slower rate of clearance of the vaccine strain than for BMDeltavirB2, which was not isolated from any maternal or fetal tissues. Both strains were protective against abortion and against infection in the majority of pregnant goats, although BMDeltaasp24 was more efficacious than BMDeltavirB2 against challenge infection.

Genetic evidence for tula virus in Microtus arvalis and Microtus agrestis populations in Croatia.

To determine the threat of hantavirus infection to U.S. Forces, small mammals were sampled from training areas within Croatia. Of the 152 samples, 20 were positive for Tula virus (TUL), 12 common voles (Microtus arvalis) and eight field voles (Microtus agrestis). Sequences from M. agrestis were found in five and sequences from M. arvalis were found in six of seven sequence groups. The high percentage of the same TUL sequences in M. agrestis and M. arvalis suggests the co-occurrence of this virus in both Microtus species is not an accident. If M. agrestis field voles were accidentally infected with TUL, the percentage of polymerase chain reaction-positive animals should be lower than that of M. arvalis. Because the infection rate in M. arvalis (11.8%) was less than half of that found in M. agrestis (27.6%), it is unlikely that the predominance of positive M. agrestis could be due to accidental exposure. It is much more likely that the Croatian virus is circulating between both rodent species.