Migratory Shorebird Gut Microbes are not Associated with Bivalve Prey in Monsoon Tropical Australia.

Migratory animals can carry symbionts over long distances. While well-studied for parasite and pathogen transmission, less is known about use of this route by other symbiotic taxa, particularly those non-pathogenic. Here we ask the question of whether gut bacteria can be spread between continents by long-distance bird migration, although gut microbiomes in birds may not be as stable or persistent as those of non-volant animals. We used amplicon sequencing of both bacterial 16S rRNA gene and Vibrio-centric hsp60 gene to determine whether the faecal bacteria of migratory great knots (Calidris tenuirostris) also occur in their main food source in Northern Australia or in nearby sand, comparing samples before and after the birds' long-distance migration. Our data suggest that there is little connectivity among the bacterial microbiomes, except in the bivalve prey. Our results are consistent with previous studies finding that bird faecal microbiomes were not host-specific and contrast with those showing an influence of diet on bird faecal bacteria. We also found little connectivity among Vibrio spp. However, although faecal sample sizes were small, the dominance of different individual Vibrio spp. suggests that they may have been well-established in knot guts and thus capable of moving with them on migration. We suggest that the physiological impacts of a long-distance migration may have caused shifts in the phyla comprising great knot faecal communities.

Assessing spatial distribution, genetic variants, and virulence of pathogen Mycoplasma agassizii in threatened Mojave desert tortoises.

Mojave desert tortoises (Gopherus agassizii), a threatened species under the US Endangered Species Act, are long-lived reptiles that experience a chronic respiratory disease. The virulence of primary etiologic agent, Mycoplasma agassizii, remains poorly understood, but it exhibits temporal and geographic variability in causing disease outbreaks in host tortoises. Multiple attempts to culture and characterize the diversity of M. agassizii have had minimal success, even though this opportunistic pathogen chronically persists in nearly every population of Mojave desert tortoises. The current geographic range and the molecular mechanisms of virulence of the type-strain, PS6T, are unknown, and the bacterium is thought to have low-to-moderate virulence. We designed a quantitative polymerase chain reaction (qPCR) targeting three putative virulence genes annotated on the PS6T genome as exo-α-sialidases, enzymes which facilitate growth in many bacterial pathogens. We tested 140 M. agassizii-positive DNA samples collected from 2010 to 2012 across the range of Mojave desert tortoises. We found evidence of multiple-strain infections within hosts. We also found the prevalence of these sialidase-encoding genes to be highest in tortoise populations surrounding southern Nevada, the area from which PS6T was originally isolated. We found a general pattern of loss or reduced presence of sialidase among strains, even within a single host. However, in samples that were positive for any of the putative sialidase genes, one particular gene (528), was positively associated with bacterial loads of M. agassizii and may act as a growth factor for the bacterium. Our results suggest three evolutionary patterns: (1) high levels of variation, possibly due to neutral changes and chronic persistence, (2) a trade-off between moderate virulence and transmission, and (3) selection against virulence in environmental conditions known to be physiologically stressful to the host. Our approach of quantifying genetic variation via qPCR represents a useful model of studying host-pathogen dynamics.

EFFECTS OF MYCOPLASMA GALLISEPTICUM INFECTION ON PREENING BEHAVIORS AND FEATHER QUALITY IN HOUSE FINCHES (HAEMORHOUS MEXICANUS).

Infections can have far-reaching sublethal effects on wildlife, including reduced maintenance of external structures. For many wildlife taxa, daily maintenance of external structures (termed preening in birds) is critical to fitness, but few studies have examined how infections alter such maintenance. Mycoplasma gallisepticum is a common pathogen in free-living House Finches (Haemorhous mexicanus), where it causes mycoplasmal conjunctivitis. Despite documented behavioral changes associated with M. gallisepticum infections in finches, no studies have examined how preening behavior may change with infection and how potential differences in preening may affect feather quality. To test this, we experimentally inoculated captive House Finches with M. gallisepticum or a control treatment, and we collected behavioral and feather quality data to detect potential changes in feather maintenance due to infection. We found that finches infected with M. gallisepticum preened significantly less often, and within the infected treatment, birds with the highest conjunctivitis severity preened the least often. However, there was no difference in the quality scores for secondary flight feathers collected from control versus infected birds. We also assayed feather water retention and found that the degree of water retention correlated with our feather quality scores, such that feathers with poor scores retained more water. However, as with quality scores, feather water retention did not differ with infection; this may be due to the controlled environment that the birds experienced while in captivity. Our data suggest that, in addition to sickness behaviors previously observed in finches, M. gallisepticum infection decreases other behaviors critical to survival, such as preening. While the consequences of reduced preening on feather maintenance were not apparent in captive conditions, further work is needed to determine whether House Finches in the wild that are infected with M. gallisepticum experience a fitness cost, such as increases in ectoparasite loads, due to this reduced feather maintenance.

Geographic variation in bacterial assemblages on cane toad skin is influenced more by local environments than by evolved changes in host traits.

Bacterial assemblages on amphibian skin may play an important role in protecting hosts against infection. In hosts that occur over a range of environments, geographic variation in composition of bacterial assemblages might be due to direct effects of local factors and/or to evolved characteristics of the host. Invasive cane toads (Rhinella marina) are an ideal candidate to evaluate environmental and genetic mechanisms, because toads have evolved major shifts in physiology, morphology, and behavior during their brief history in Australia. We used samples from free-ranging toads to quantify site-level differences in bacterial assemblages and a common-garden experiment to see if those differences disappeared when toads were raised under standardised conditions at one site. The large differences in bacterial communities on toads from different regions were not seen in offspring raised in a common environment. Relaxing bacterial clustering to operational taxonomic units in place of amplicon sequence variants likewise revealed high similarity among bacterial assemblages on toads in the common-garden study, and with free-ranging toads captured nearby. Thus, the marked geographic divergence in bacterial assemblages on wild-caught cane toads across their Australian invasion appears to result primarily from local environmental effects rather than evolved shifts in the host.

Potential Facilitation Between a Commensal and a Pathogenic Microbe in a Wildlife Disease.

We assessed the potential for microbial interactions influencing a well-documented host-pathogen system. Mycoplasma agassizii is the known etiological agent of upper respiratory tract disease in Mojave desert tortoises (Gopherus agassizii), but disease in wild animals is extremely heterogeneous. For example, a much larger proportion of animals harbor M. agassizii than those that develop disease. With the availability of a new quantitative PCR assay for a microbe that had previously been implicated in disease, Pasteurella testudinis, we tested 389 previously collected samples of nasal microbes from tortoise populations across the Mojave desert. We showed that P. testudinis is a common commensal microbe. However, we did find that its presence was associated with higher levels of M. agassizii among the tortoises positive for this pathogen. The best predictor of P. testudinis prevalence in tortoise populations was average size of tortoises, suggesting that older populations have higher levels of P. testudinis. The prevalence of co-infection in populations was associated with the prevalence of URTD, providing additional evidence for an indirect interaction between the two microbes and inflammatory disease. We showed that URTD, like many chronic, polymicrobial diseases involving mucosal surfaces, shows patterns of a polymicrobial etiology.

Antibiotic perturbation of gut bacteria does not significantly alter host responses to ocular disease in a songbird species.

Bacterial communities in and on wild hosts are increasingly appreciated for their importance in host health. Through both direct and indirect interactions, bacteria lining vertebrate gut mucosa provide hosts protection against infectious pathogens, sometimes even in distal body regions through immune regulation. In house finches (Haemorhous mexicanus), the bacterial pathogen Mycoplasma gallisepticum (MG) causes conjunctivitis, with ocular inflammation mediated by pro- and anti-inflammatory cytokines and infection triggering MG-specific antibodies. Here, we tested the role of gut bacteria in host responses to MG by using oral antibiotics to perturb bacteria in the gut of captive house finches prior to experimental inoculation with MG. We found no clear support for an impact of gut bacterial disruption on conjunctival pathology, MG load, or plasma antibody levels. However, there was a non-significant trend for birds with intact gut communities to have greater conjunctival pathology, suggesting a possible impact of gut bacteria on pro-inflammatory cytokine stimulation. Using 16S bacterial rRNA amplicon sequencing, we found dramatic differences in cloacal bacterial community composition between captive, wild-caught house finches in our experiment and free-living finches from the same population, with lower bacterial richness and core communities composed of fewer genera in captive finches. We hypothesize that captivity may have affected the strength of results in this experiment, necessitating further study with this consideration. The abundance of anthropogenic impacts on wildlife and their bacterial communities, alongside the emergence and spread of infectious diseases, highlights the importance of studies addressing the role of commensal bacteria in health and disease, and the consequences of gut bacterial shifts on wild hosts.

Protection Generated by Prior Exposure to Pathogens Depends on both Priming and Challenge Dose.

Free-living hosts encounter pathogens at a wide range of frequencies and concentrations, including low doses that are largely aclinical, creating a varied landscape of exposure history and reinfection likelihood. While several studies show that higher priming doses result in stronger immunological protection against reinfection, it remains unknown how the reinfection challenge dose and priming dose interact to determine the likelihood and severity of reinfection. We manipulated both priming and challenge doses of Mycoplasma gallisepticum, which causes mycoplasmal conjunctivitis, in captive house finches (Haemorhous mexicanus), to assess reinfection probability and severity. We found a significant interaction between priming and challenge doses on reinfection probability, with the likelihood of reinfection by a high but not a low challenge dose decreasing exponentially at higher priming doses. While this interaction was likely driven by lower average infection probabilities for low-dose versus high-dose challenges, even the highest priming dose provided only negligible protection against reinfection from low-dose challenges. Similarly, pathogen loads during reinfection were significantly reduced with increasing priming doses only for birds reinfected at high but not low doses. We hypothesize that these interactions arise to some degree from fundamental differences in host immune responses across doses, with single low doses only weakly triggering host immune responses. Importantly, our results also demonstrate that reinfections can occur from a variety of exposure doses and across diverse degrees of standing immunity in this system. Overall, our study highlights the importance of considering both initial and subsequent exposure doses where repeated exposure to a pathogen is common in nature.

Experimental test of microbiome protection across pathogen doses reveals importance of resident microbiome composition.

The commensal microbes inhabiting a host tissue can interact with invading pathogens and host physiology in ways that alter pathogen growth and disease manifestation. Prior work in house finches (Haemorhous mexicanus) found that resident ocular microbiomes were protective against conjunctival infection and disease caused by a relatively high dose of Mycoplasma gallisepticum. Here, we used wild-caught house finches to experimentally examine whether protective effects of the resident ocular microbiome vary with the dose of invading pathogen. We hypothesized that commensal protection would be strongest at low M. gallisepticum inoculation doses because the resident microbiome would be less disrupted by invading pathogen. Our five M. gallisepticum dose treatments were fully factorial with an antibiotic treatment to perturb resident microbes just prior to M. gallisepticum inoculation. Unexpectedly, we found no indication of protective effects of the resident microbiome at any pathogen inoculation dose, which was inconsistent with the prior work. The ocular bacterial communities at the beginning of our experiment differed significantly from those previously reported in local wild-caught house finches, likely causing this discrepancy. These variable results underscore that microbiome-based protection in natural systems can be context dependent, and natural variation in community composition may alter the function of resident microbiomes in free-living animals.

Mycoplasma agassizii, an opportunistic pathogen of tortoises, shows very little genetic variation across the Mojave and Sonoran Deserts.

Mycoplasma agassizii is a common cause of upper respiratory tract disease in Mojave desert tortoises (Gopherus agassizii). So far, only two strains of this bacterium have been sequenced, and very little is known about its patterns of genetic diversity. Understanding genetic variability of this pathogen is essential to implement conservation programs for their threatened, long-lived hosts. We used next generation sequencing to explore the genomic diversity of 86 cultured samples of M. agassizii collected from mostly healthy Mojave and Sonoran desert tortoises in 2011 and 2012. All samples with enough sequencing coverage exhibited a higher similarity to M. agassizii strain PS6T (collected in Las Vegas Valley, Nevada) than to strain 723 (collected in Sanibel Island, Florida). All eight genomes with a sequencing coverage over 2x were subjected to multiple analyses to detect single-nucleotide polymorphisms (SNPs). Strikingly, even though we detected 1373 SNPs between strains PS6T and 723, we did not detect any SNP between PS6T and our eight samples. Our whole genome analyses reveal that M. agassizii strain PS6T may be present across a wide geographic extent in healthy Mojave and Sonoran desert tortoises.

House finches with high coccidia burdens experience more severe experimental Mycoplasma gallisepticum infections.

Parasites co-infecting hosts can interact directly and indirectly to affect parasite growth and disease manifestation. We examined potential interactions between two common parasites of house finches: the bacterium Mycoplasma gallisepticum that causes conjunctivitis and the intestinal coccidian parasite Isospora sp. We quantified coccidia burdens prior to and following experimental infection with M. gallisepticum, exploiting the birds' range of natural coccidia burdens. Birds with greater baseline coccidia burdens developed higher M. gallisepticum loads and longer lasting conjunctivitis following inoculation. However, experimental inoculation with M. gallisepticum did not appear to alter coccidia shedding. Our study suggests that differences in immunocompetence or condition may predispose some finches to more severe infections with both pathogens.

Disease Exposure and Antifungal Bacteria on Skin of Invasive Cane Toads, Australia.

Cane toads, an invasive species in Australia, are resistant to fungal pathogens affecting frogs worldwide (Batrachochytrium dendrobatidis). From toad skin swabs, we detected higher proportions of bacteria with antifungal properties in Queensland, where toad and pathogen distributions overlap, than in other sites. This finding suggests that site-specific pathogen pressures help shape skin microbial communities.

Host species, pathogens and disease associated with divergent nasal microbial communities in tortoises.

Diverse bacterial communities are found on every surface of macro-organisms, and they play important roles in maintaining normal physiological functions in their hosts. While the study of microbiomes has expanded with the influx of data enabled by recent technological advances, microbiome research in reptiles lags behind other organisms. We sequenced the nasal microbiomes in a sample of four North American tortoise species, and we found differing community compositions among tortoise species and sampling sites, with higher richness and diversity in Texas and Sonoran desert tortoises. Using these data, we investigated the prevalence and operational taxonomic unit (OTU) diversity of the potential pathogen Pasteurella testudinis and found it to be common, abundant and highly diverse. However, the presence of this bacterium was not associated with differences in bacterial community composition within host species. We also found that the presence of nasal discharge from tortoises at the time of sampling was associated with a decline in diversity and a change in microbiome composition, which we posit is due to the harsh epithelial environment associated with immune responses. Repeated sampling across seasons, and at different points of pathogen colonization, should contribute to our understanding of the causes and consequences of different bacterial communities in these long-lived hosts.

Skin bacterial diversity is higher on lizards than sympatric frogs in tropical Australia.

Animal skin acts as a barrier between the organism and its environment and provides the first line of defense against invading pathogens. Thus, skin surfaces harbor communities of microbes that are interacting with both the host and its environment. Amphibian skin bacteria form distinct communities closely tied to their host species, but few studies have compared bacterial communities between amphibians and other, non-amphibian sympatric animals. Notably, skin microbes on reptiles have gained little attention. We used next-generation sequencing technology to describe bacterial communities on the skin of three lizard species and compared them to bacteria on six cohabiting frog species in the Northern Territory of Australia. We found bacterial communities had higher richness and diversity on lizards than frogs, with different community composition between reptiles and amphibians and among species. Core bacteria on the three lizard species overlapped by over 100 operational taxonomic units. The bacterial communities were similar within species of frogs and lizards, but the communities tended to be more similar between lizard species than between frog species and when comparing lizards with frogs. The diverse bacteria found on lizards invites further questions on how and how well reptiles interact with microorganisms through their scaly skin.

High quality draft genome sequences of Mycoplasma agassizii strains PS6T and 723 isolated from Gopherus tortoises with upper respiratory tract disease.

Mycoplasma agassizii is one of the known causative agents of upper respiratory tract disease (URTD) in Mojave desert tortoises (Gopherus agassizii) and in gopher tortoises (Gopherus polyphemus). We sequenced the genomes of M. agassizii strains PS6T (ATCC 700616) and 723 (ATCC 700617) isolated from the upper respiratory tract of a Mojave desert tortoise and a gopher tortoise, respectively, both with signs of URTD. The PS6T genome assembly was organized in eight scaffolds, had a total length of 1,274,972 bp, a G + C content of 28.43%, and contained 979 protein-coding genes, 13 pseudogenes and 35 RNA genes. The 723 genome assembly was organized in 40 scaffolds, had a total length of 1,211,209 bp, a G + C content of 28.34%, and contained 955 protein-coding genes, seven pseudogenes, and 35 RNA genes. Both genomes exhibit a very similar organization and very similar numbers of genes in each functional category. Pairs of orthologous genes encode proteins that are 93.57% identical on average. Homology searches identified a putative cytadhesin. These genomes will enable studies that will help understand the molecular bases of pathogenicity of this and other Mycoplasma species.

High quality draft genome sequence of Mycoplasma testudineum strain BH29T, isolated from the respiratory tract of a desert tortoise.

Mycoplasma testudineum is one of the pathogens that can cause upper respiratory tract disease in desert tortoises, Gopherus agassizii. We sequenced the genome of M. testudineum BH29T (ATCC 700618T = MCCM 03231T), isolated from the upper respiratory tract of a Mojave desert tortoise with upper respiratory tract disease. The sequenced draft genome, organized in 25 scaffolds, has a length of 960,895 bp and a G + C content of 27.54%. A total of 788 protein-coding sequences, six pseudogenes and 35 RNA genes were identified. The potential presence of cytadhesin-encoding genes is investigated. This genome will enable comparative genomic studies to help understand the molecular bases of the pathogenicity of this and other Mycoplasma species.

Co-infection does not predict disease signs in Gopherus tortoises.

In disease ecology, the host immune system interacts with environmental conditions and pathogen properties to affect the impact of disease on the host. Within the host, pathogens may interact to facilitate or inhibit each other's growth, and pathogens interact with different hosts differently. We investigated co-infection of two Mycoplasma and the association of infection with clinical signs of upper respiratory tract disease in four congeneric tortoise host species (Gopherus) in the United States to detect differences in infection risk and disease dynamics in these hosts. Mojave Desert tortoises had greater prevalence of Mycoplasma agassizii than Texas tortoises and gopher tortoises, while there were no differences in Mycoplasma testudineum prevalence among host species. In some host species, the presence of each pathogen influenced the infection intensity of the other; hence, these two mycoplasmas interact differently within different hosts, and our results may indicate facilitation of these bacteria. Neither infection nor co-infection was associated with clinical signs of disease, which tend to fluctuate across time. From M. agassizii DNA sequences, we detected no meaningful differentiation of haplotypes among hosts. Experimental inoculation studies and recurrent resampling of wild individuals could help to decipher the underlying mechanisms of disease dynamics in this system.

COMPARISON OF CURRENT METHODS FOR THE DETECTION OF CHRONIC MYCOPLASMAL URTD IN WILD POPULATIONS OF THE MOJAVE DESERT TORTOISE (GOPHERUS AGASSIZII).

Pathogens that cause subclinical diseases or exhibit low infection intensities are difficult to quantify in wild populations. Mojave desert tortoises ( Gopherus agassizii ) have been the focus of much research aimed at measuring the presence of upper respiratory disease (URTD) and URTD-associated pathogens, and techniques used to quantify disease in Gopherus species have also been used for disease surveillance in other species of turtles and tortoises of conservation concern. Published surveys of G. agassizii populations have found a relatively low prevalence of URTD, with most URTD-positive animals exhibiting moderate, intermittent signs of morbidity. Therefore, multiple tests have been developed to quantify URTD including genetic detection of the pathogens Mycoplasma agassizii and Mycoplasma testudineum , detection of M. agassizii -specific antibodies, and standardized quantification of clinical signs of URTD and body condition. These diagnostic tests have only been compared in diseased or moribund, semicaptive animals. We compared diagnostic techniques (TaqMan® and SYBR™ Green qPCR, serology, and visible examination) to detect M. agassizii -associated URTD in 126 wild desert tortoises sampled in Nevada and California, US in 2010. All had healthy body condition indices and none exhibited more than mild-to-moderate visual signs of URTD. Pairwise comparisons of diagnostic techniques indicated poor performance in diagnosing disease in individual animals. We found stronger, but inconsistent, statistical associations among diagnostic techniques at the population level. Our findings have implications for quantifying subclinical respiratory disease in tortoises.

Fine-scale genetic structure among greater sage-grouse leks in central Nevada.

BACKGROUND: Mating systems that reduce dispersal and lead to non-random mating might increase the potential for genetic structure to arise at fine geographic scales. Greater sage-grouse (Centrocercus urophasianus) have a lek-based mating system and exhibit high site fidelity and skewed mating ratios. We quantified population structure by analyzing variation at 27,866 single-nucleotide polymorphisms in 140 males from ten leks (within five lek complexes) occurring in a small geographic region in central Nevada. RESULTS: Lek complexes, and to a lesser extent individual leks, formed statistically identifiable clusters in ordination analyses, providing evidence for fine-scale geographic genetic differentiation. Lek geography predicted genetic differentiation even at a small geographic scale, which could be sharpened by strong site fidelity. Relatedness was also higher among individuals within lek complexes (and leks), suggesting that reproductive skew, where few males participate in most of the successful matings, could also potentially contribute to genetic differentiation. Models incorporating a habitat resistance surface as a proxy for potentially reduced movement due to landscape features indicated that both geographic distance and habitat suitability (i.e. preferred habitat) predicted genetic structure, with no significant effect of man-made barriers to movement (i.e. power lines and roads). Finally, we illustrate how data sets containing fewer loci (<4000) had less statistical precision and failed to detect the full degree of genetic structure. CONCLUSION: Our results suggest that habitat features and lek site geography of sage-grouse shape fine scale genetic structure, and highlight how larger data sets can have increased precision and accuracy for quantifying ecologically relevant genetic structure over small geographic scales.