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In this study, we describe the genomes of two novel candidate species of non-nitrogen fixing Frankia that were isolated from the root nodules of Coriaria nepalensis and Alnus glutinosa, genospecies CN and Ag, respectively. Comparative genomic analyses revealed that both genospecies lack genes essential for nitrogen-fixation and possess genes involved in the degradation of plant cell walls. Additionally, we found distinct biosynthetic gene clusters in each genospecies. The availability of these genomes will contribute to the study of the taxonomy and evolution of actinorhizal symbioses.

Head-starting of the federally endangered Houston toad (Anaxyrus houstonensis), that is, the release of egg strands, tadpoles, and metamorphic juveniles produced in captivity into the original breeding ponds, requires assessment of potential threats for the transmission of pathogens from captive to free-ranging toads. We used Illumina-based 16S rRNA V3 amplicon sequencing to investigate the community structure of bacteria from skin lesions of captive Houston toad and habitat (pond) samples. Proteobacteria, alone or together with Actinobacteria and, in some samples, Cyanobacteria represented virtually all reads in tissue lesion samples, whereas pond samples were much more diverse, with Acidobacteria, Actinobacteria, Bacteriodetes, Chloroflexi, Cyanobacteria, Firmicutes, Planctomycetes, Proteobacteria, and Verrucomicrobia present with little variation between samples. If present in lesions, Actinobacteria were largely represented by Mycobacteriaceae, and here mainly by one sequence identical to sequences of members of the Mycobacterium chelonae-abscessus complex. In pond samples, mycobacteria represented only a small portion of the actinobacteria, although at higher diversity with six distinct reads. Sequences for reads obtained from pond samples were identical to those representing the M. chelonae-abscessus complex, a group with Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium avium, a group with Mycobacterium vaccae, Mycobacterium fortuitum, Mycobacterium poriferae, and a group with Mycobacterium elephantis and Mycobacterium celeriflavum, whereas sequences of high similarity were detected for reads related to those of Mycobacterium holsaticum, Mycobacterium pallens, and Mycobacterium obuense, and Mycobacterium goodii. Our results indicated that lesions observed on the Houston toad in captivity are not the result of mycobacteria in every case, and that the presence of mycobacteria in the captive colony does not represent a novel pathogen threat to the wild populations because such bacteria are also seen in the natural pond habitats for the Houston toad.

Members of the nitrogen-fixing actinobacterial genus Frankia are typically isolated from root nodules and generally infective on the same plant species. Several Frankia strains originally isolated from Casuarina species, however, have been found to be non-infective on Casuarina species. The goal of this study was to investigate the potential role of infective isolates from Casuarina species on the potential establishment of these non-infective Frankia strains in root nodule formation on Casuarina equisetifolia. Soil microcosms were established with plants of C. equisetifolia and inoculated with Frankia casuarinae strain CcI3 or cluster 3 strain R43, or combinations of both at different densities. Basic plant growth characteristics, root nodule formation and localization of both Frankia strains in nodule periderm and cortex, as well as population development in soils were monitored. The presence of strain R43 did not affect plant growth performance nor root nodule formation, while inoculation with strain CcI3 enhanced plant growth and resulted in root nodule formation. qPCR analyses on selected nodule lobes revealed the presence of strain CcI3 in cortex samples in all treatments, while strain R43 was not detected in any cortex samples but in 40% of the periderm samples from lobes from treatments with highest inoculation values. In situ hybridization detected cells of strain R43 on the outside of the nodules, i.e. on the periderm only. These results demonstrate that the cluster 3 Frankia strain R43 is not co-infecting root nodules formed by the F. casuarinae strain CcI3 on C. equisetifolia, but has likely been isolated as a surface contaminant from Casuarina nodules.

Rodent species were assessed as potential hosts of Trypanosoma cruzi, the etiologic agent of Chagas disease, from five sites throughout Texas in sylvan and disturbed habitats. A total of 592 rodents were captured, resulting in a wide taxonomic representation of 11 genera and 15 species. Heart samples of 543 individuals were successfully analyzed by SybrGreen-based quantitative PCR (qPCR) targeting a 166 bp fragment of satellite DNA of T. cruzi. Eight rodents representing six species from six genera and two families were infected with T. cruzi. This is the first report of T. cruzi in the pygmy mouse (Baiomys taylori) and the white-footed mouse (Peromyscus leucopus) for the USA. All infected rodents were from the southernmost site (Las Palomas Wildlife Management Area). No differences in pathogen prevalence existed between disturbed habitats (5 of 131 tested; 3.8%) and sylvan habitats (3 of 40 tested; 7.5%). Most positives (n = 6, 16% prevalence) were detected in late winter with single positives in both spring (3% prevalence) and fall (1% prevalence). Additionally, 30 Triatoma insects were collected opportunistically from sites in central Texas. Fifty percent of these insects, i.e., 13 T. gerstaeckeri (68%), and two T. lecticularia (100%) were positive for T. cruzi. Comparative sequence analyses of 18S rRNA of samples provided identical results with respect to detection of the presence or absence of T. cruzi and assigned T. cruzi from rodents collected in late winter to lineage TcI. T. cruzi from Triatoma sp. and rodents from subsequent collections in spring and fall were different, however, and could not be assigned to other lineages with certainty.

Targeted Illumina sequencing of nitrogenase reductase (nifH) gene fragments and analyses of pair-end reads through a modified QIIME pipeline were used to assess the diversity of the actinomyceteous genus Frankia in three soils. Soils were vegetated with host or non-host plants, and included locations in Illinois (ABA, host), Colorado (CoMt, non-host), and Wisconsin (FMWI, non-host). After filtering, seven unique sequences were recovered for soil ABA, six for CoMt, and four sequences for FMWI. These sequences were included in a Bayesian topology anchored by published sequence data from pure cultures of Frankia. Sequences from all three soils showed affinities to Frankia strains from both the Alnus and Elaeagnus host infection groups. Reads representing Casuarina-infective strains were not detected. Four sequences from soil CoMt and five sequences from soil ABA did not cluster, at 97% similarity, into a shared OTU that contained a cultured relative. These results demonstrate that targeted Illumina sequencing provides an efficient and economical method for assessing haplotype diversity of ecofunctional genes (e.g. nifH) at the genus level in microorganisms that perform important ecosystem functions.

Using 8 polymorphic microsatellite loci, we explored genetic variability in Texas spiny softshell turtles (Apalone spinifera emoryi) in the region of Big Bend National Park (BBNP) and the Lower Rio Grande Valley (LRGV), which are located in western and southern regions, respectively, of the distribution of the subspecies in Texas. The presence of multiple anthropogenic stressors, such as river flow alterations, human population expansion, and direct harvest, motivated us to evaluate whether genetic consequences of these stressors have become apparent in this species. A low but significant level of genetic differentiation was detectable between these 2 regions. There was also detectable isolation by distance among the turtles in LRGV but not among turtles in BBNP, possibly because the LRGV localities were discontinuous ponds, whereas the BBNP localities were continuously joined stretches of the Rio Grande. We detected no evidence of a recent population bottleneck in BBNP or the LRGV. However, turt...

Freshwater turtles from 28 species representing 6 families, categorized as “wild,” “captive,” or “wild-caught, captive-raised,” were analyzed for the presence of intraerythrocytic, parasitic protozoa. Microscopic analyses of blood smears from 327 individual turtles revealed parasites in the blood of 29% of these individuals (n = 96), with levels of parasitemia ranging from 0.003% to 2.1%. SYBR ® Green–based quantitative polymerase chain reaction confirmed these results with 29% overall prevalence (94 of 324), with 66% prevalence in the Geoemydidae, 26% prevalence in the Emydidae, and 23% prevalence in the Kinosternidae. No infections were detected in members of the Chelidae, Pelomedusidae, and Trionychidae. Prevalence was highest in wild turtles with 67%–100% from 7 locations, followed by wild-caught, captive-raised turtles with 4%–26% from 2 locations, while detections in captive turtles were zero in 2 locations and 3% in the remaining third location. Comparative sequence analyses of 583-bp amplicons of 18S rRNA gene fragments allowed us to identify 80 Haemogregarina infections and one Hemolivia infection. Parasites representing the genus Hepatozoon were not detected. Because parasites were generally detected in wild turtles and only rarely in captive turtles, captive breeding with subsequent release of turtles would seem to pose little risk to free-ranging turtles with regard to the spread of these parasites. On the contrary, wild turtles would be more likely sources of parasite transmission to captive populations if used in breeding programs.

The prevalence of salmonellae in the intestines of the invasive suckermouth catfish Hypostomus plecostomus was assessed in the San Marcos River, just down-stream of its spring-fed headwaters. In 2014, H. plecostomus, sediment, and water samples were collected during 15 sampling events. A combination of semi-selective enrichment and quantitative polymerase chain reaction (qPCR) revealed the presence of salmonellae in 45% of the fish intestines across the entire year, with a prevalence range of 13–100% per sampling event. Repetitive element sequence-based PCR (rep-PCR) and multi-locus sequence typing (MLST) revealed a high diversity of salmonellae from fish intestine samples at individual sampling times, single or multiple presence of rep-PCR patterns and serotypes within individual fish, and identical rep-PCR patterns and serotypes for different fish within and across sampling events. Overall, 15 serotypes were identified by MLST, with a diversity range between one and seven serotypes per sampling event. Some serotypes were retrieved only once, while others were detected more frequently. A few serotypes were retrieved at several sampling times, nearly evenly distributed over the entire sampling period. Prevalence and diversity were independent of precipitation events, indicating the potential presence of environmental strains that are capable of long-term persistence in the environment.

Actinorhizal plants form nitrogen-fixing root nodules in symbiosis with soil-dwelling actinobacteria within the genus Frankia , and specific Frankia taxonomic clusters nodulate plants in corresponding host infection groups. In same-soil microcosms, we observed that some host species were nodulated ( Alnus glutinosa , Alnus cordata , Shepherdia argentea , Casuarina equisetifolia ) while others were not ( Alnus viridis , Hippophaë rhamnoides ). Nodule populations were represented by eight different sequences of nifH gene fragments. Two of these sequences characterized frankiae in S. argentea nodules, and three others characterized frankiae in A. glutinosa nodules. Frankiae in A. cordata nodules were represented by five sequences, one of which was also found in nodules from A. glutinosa and C. equisetifolia , while another was detected in nodules from A. glutinosa . Quantitative PCR assays showed that vegetation generally increased the abundance of frankiae in soil, independently of the target gene (i.e., nifH or the 23S rRNA gene). Targeted Illumina sequencing of Frankia -specific nifH gene fragments detected 24 unique sequences from rhizosphere soils, 4 of which were also found in nodules, while the remaining 4 sequences in nodules were not found in soils. Seven of the 24 sequences from soils represented >90% of the reads obtained in most samples; the 2 most abundant sequences from soils were not found in root nodules, and only 2 of the sequences from soils were detected in nodules. These results demonstrate large differences between detectable Frankia populations in soil and those in root nodules, suggesting that root nodule formation is not a function of the abundance or relative diversity of specific Frankia populations in soils. IMPORTANCE The nitrogen-fixing actinobacterium Frankia forms root nodules on actinorhizal plants, with members of specific Frankia taxonomic clusters nodulating plants in corresponding host infection groups. We assessed Frankia diversity in root nodules of different host plant species, and we related specific populations to the abundance and relative distribution of indigenous frankiae in rhizosphere soils. Large differences were observed between detectable Frankia populations in soil and those in root nodules, suggesting that root nodule formation is not a function of the abundance or relative diversity of specific Frankia populations in soils but rather results from plants potentially selecting frankiae from the soil for root nodule formation. These data also highlight the necessity of using a combination of different assessment tools so as to adequately address methodological constraints that could produce contradictory data sets.

The nodule-forming actinobacterial genus Frankia can generally be divided into 4 taxonomic clusters, with clusters 1, 2, and 3 representing nitrogen-fixing strains of different host infection groups and cluster 4 representing atypical, generally non-nitrogen-fixing strains. Recently, quantitative PCR (qPCR)-based quantification methods have been developed for frankiae of clusters 1 and 3; however, similar approaches for clusters 2 and 4 were missing. We amended a database of partial 23S rRNA gene sequences of Frankia strains belonging to clusters 1 and 3 with sequences of frankiae representing clusters 2 and 4. The alignment allowed us to design primers and probes for the specific detection and quantification of these Frankia clusters by either Sybr Green- or TaqMan-based qPCR. Analyses of frankiae in different soils, all obtained from the same region in Illinois, USA, provided similar results, independent of the qPCR method applied, with abundance estimates of 10 × 10 5 to 15 × 10 5 cells (g soil) −1 depending on the soil. Diversity was higher in prairie soils (native, restored, and cultivated), with frankiae of all 4 clusters detected and those of cluster 4 dominating, while diversity in soils under Alnus glutinosa , a host plant for cluster 1 frankiae, or Betula nigra , a related nonhost plant, was restricted to cluster 1 and 3 frankiae and generally members of subgroup 1b were dominating. These results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees. IMPORTANCE Root nodule formation by the actinobacterium Frankia is host plant specific and largely, but not exclusively, correlates with assignments of strains to specific clusters within the genus. Due to the lack of adequate detection and quantification tools, studies on Frankia have been limited to clusters 1 and 3 and generally excluded clusters 2 and 4. We have developed tools for the detection and quantification of clusters 2 and 4, which can now be used in combination with those developed for clusters 1 and 3 to retrieve information on the ecology of all clusters delineated within the genus Frankia . Our initial results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees.

Leptospirosis, an emerging infectious disease caused by bacteria of the genus Leptospira, is thought to be the most widespread zoonotic disease in the world. A first step in preventing the spread of Leptospira is delineating the animal reservoirs that maintain and disperse the bacteria. Quantitative PCR (qPCR) methods targeting the LipL32 gene were used to analyze kidney samples from 124 House mice (Mus musculus), 94 Black rats (Rattus rattus), 5 Norway rats (R. norvegicus), and 89 small Indian mongooses (Herpestes auropunctatus) from five cattle farms in Puerto Rico. Renal carriage of Leptospira was found in 38% of the sampled individuals, with 59% of the sampled mice, 34% of Black rats, 20% of Norway rats, and 13% of the mongooses. A heterogeneous distribution of prevalence was also found among sites, with the highest prevalence of Leptospira-positive samples at 52% and the lowest at 30%. Comparative sequence analysis of the LipL32 gene from positive samples revealed the presence of two species of Leptospira, L. borgpetersenii and L. interrogans in mice, detected in similar percentages in samples from four farms, while samples from the fifth farm almost exclusively harbored L. interrogans. In rats, both Leptospira species were found, while mongooses only harbored L. interrogans. Numbers tested for both animals, however, were too small (n = 7 each) to relate prevalence of Leptospira species to location. Significant associations of Leptospira prevalence with anthropogenic landscape features were observed at farms in Naguabo and Sabana Grande, where infected individuals were closer to human dwellings, milking barns, and ponds than were uninfected individuals. These results show that rural areas of Puerto Rico are in need of management and longitudinal surveillance of Leptospira in order to prevent continued infection of focal susceptible species (i.e. humans and cattle)., Author summary Leptospirosis is a global zoonotic disease that recently has shown an increase of human cases in many regions, Puerto Rico being one of them. To decrease human Leptospira infections it is necessary to ascertain the role of animal reservoirs to maintain the pathogen in the environment. However, no studies of wild mammal reservoirs in Puerto Rico have been done since 1963. We addressed the prevalence of Leptospira in four species of introduced wild mammals, which included three commensal rodent species (Mus and Rattus spp.) and the small Indian mongoose (Herpestes auropunctatus) on dairy farms in Puerto Rico. Pathogen prevalence ranged from high to moderate in mice and mongooses, respectively. We also found that the pathogen is not distributed homogeneously among the sites we sampled and that landscape features, both natural and manmade, can play a role in the distribution of Leptospira. Our study provides an initial exploration of wild mammal reservoirs for Leptospira in Puerto Rico, and our data highlight the need for management of these species to potentially decrease pathogen transmission rates in both humans and cattle.

Six polymorphic microsatellite loci were isolated and characterized in the threatened Mexican beaded lizard, Heloderma horridum. The number of alleles per locus ranged from three to 12, with observed heterozygosity estimates ranging from 0.00 to 0.77, and expected heterozygosity estimates ranging from 0.00 to 0.73. These microsatellites will provide a valuable tool for the investigation of the genetic variation and structure of both wild and captive H. horridum populations.

We characterize eight polymorphic microsatellite loci for the San Esteban chuckwalla, Sauromalus varius, which are being used as markers in a study of the genetic results from a decade of captive breeding in this endangered species. Observed heterozygosity estimates for S. varius ranged from 0.00 to 0.30, whereas expected heterozygosity estimates ranged from 0.06 to 0.58.

Somatic cell nuclear transfer (SCNT), or cloning, is one of the assisted reproductive technologies currently used in agriculture. Commercial applications of SCNT are presently limited to the production of animals of high genetic merit or the production of the most elite show cattle owing to its relatively low efficiency. In current practice, 20% to 40% of SCNT pregnancies do not result in viable offspring. In an effort to better understand some of the anomalies associated with SCNT pregnancies, we investigated amino acid compositions of first trimester amniotic fluid. In this retrospective study, amniotic fluids were collected from SCNT and control IVF pregnancies at Day 75 of gestation and grouped according to the pregnancy results: control IVF (IVF), viable SCNT pregnancies that resulted in live healthy calves (SCNT-HL), nonviable SCNT pregnancies that were aborted before Day 150 (SCNT-ED), and nonviable SCNT pregnancies that were aborted after Day 150 or produced deceased calves (SCNT-LD). High-performance liquid chromatography (HPLC) was used to analyze the concentrations of 22 amino acids (AAs) in the amniotic fluid samples. There were no differences in average AA concentrations between IVF and SCNT-HL groups, whereas SCNT-LD and SCNT-ED had higher levels of total AA concentrations. Concentrations of asparagine, citruline, arginine, and valine were significantly higher in the SCNT-LD group. Both SCNT-LD and SCNT-ED groups had relatively large intragroup variances in AA concentrations. Urea concentration was also measured in the SCNT amniotic fluid samples. No correlations between urea concentrations and arginine concentrations or pregnancy outcomes were found. The findings in this study not only deepen the understanding on SCNT pregnancy anomalies, but also provide a potentially useful screening tool for assessing viable and nonviable SCNT pregnancies.

Marine turtles are divided into two families, the Dermochelyidae and the Cheloniidae. The majority of species are currently placed within the two tribes of the Cheloniidae, the Chelonini and the Carettini, but debate continues over generic and tribal affinities as well as species boundaries. We used nucleotide sequences (907 bp) from the ND4-LEU tRNA region and the control region (526 bp) of mitochondrial DNA to resolve areas of uncertainty in marine turtle (Chelonioidae) systematics. The ND4-LEU tRNA fragment was more conserved than the fragment from the control region, with sequence divergences ranging from 0.026 to 0.148 and 0.067 to 0.267, respectively. Parsimony analysis based only on the ND4-LEU tRNA data suggests that the hawksbill, Eretmochelys imbricata, lies within the tribe Carettni and is closely related to the genus Caretta, but could not resolve the position of the flatback, Natator depressus. A similar analysis based only on the control region sequence data suggested that N. depressus is affiliated with the Chelonini, but failed to resolve the position of E. imbricata and the loggerhead, Caretta caretta. In contrast to these results, the combination of both data sets with published cytochrome b data produced a phylogeny based on 1924 bp of sequence data which resolves the position of E. imbricata relative to Caretta and Lepidochelys and joins N. depressus as sister to the Carettini. Based on the molecular data, the Chelonini contains the Chelonia species, while the Carettini contains the remaining species of Cheloniidae. The control region sequence divergence between Pacific and Atlantic populations of the leatherback, Dermochelys coriacea, was relatively low (0.0081) when compared with the green turtle, Chelonia mydas (0.071-0.074). Atlantic and Pacific populations of Ch. mydas were found to be paraphyletic with respect to the black turtle, Ch. agassizi, suggesting that the current taxonomic designations within the Pacific Chelonia are questionable. This analysis shows the utility of combining sequence data for different regions of mtDNA that by themselves are insufficient to obtain robust phylogenies.

To expedite linkage studies and positional cloning efforts in the dog, Minimal Screening Set 2 (MSS-2) of 327 canine microsatellite markers has been multiplexed into chromosome-specific panels. MSS-2 provides 9 Mb coverage of the canine genome with no gaps larger than 17.1 Mb and is the most recent and comprehensive set of microsatellites available for whole-genome scans. Markers were labeled with fluorescent dyes based on locations and expected product sizes to facilitate the multiplexing of a maximum number of markers for each chromosome. All markers are amplified using a single thermal cycling program and PCR mix and are optimized for resolution on an ABI 3100 genetic analyzer. Sixty-nine chromosome-specific panels were created by coamplification of a maximum number of markers and subsequent coloading of the remaining markers.