J. Delton Hanson

Using museum collections to detect pathogens.

To the Editor: Natural history museum collections have evolved in recent years to meet the challenges of current and future interdisciplinary scientific studies. Many natural history museums have built tissue collections and made digital information (e.g., photographs, publications, geographic coordinates) freely available on the Internet. These collections provide endless opportunities to conduct studies, including temporal and spatial surveys of emerging and reemerging pathogens (1). We report an example of a museum collection being useful in detecting Trypanosoma cruzi, the etiologic agent of Chagas disease, in the southern plains woodrat (Neotoma micropus) in southern Texas. This finding is of interest in the epidemiology of Chagas disease because the climatic characteristics and demographics of the region are similar to areas in Latin America where Chagas disease is an important zoonotic agent that infects ≈20 million persons (2). Tissue samples from N. micropus woodrats archived in the Natural Science Research Laboratory at the Museum of Texas Tech University were evaluated for T. cruzi DNA by PCR methods. All samples were originally collected during March 2001–June 2003 from the Chaparral Wildlife Management Area in southern Texas (28o18′N, 99o24′W), 86 km west of the Mexico–US border; some samples had been used previously in other research projects (3). Individual rodents were captured with live traps (n = 13) or by excavating middens in which all the nest occupants were collected by hand (n = 146). Animals were later euthanized and tissue samples (heart, kidney, liver, lung, muscle, spleen) were obtained. Tissues were immediately frozen in liquid nitrogen and permanently stored in ultralow-temperature freezers. We extracted 1 DNA sample from each animal’s liver for use in this survey. DNA amplification was performed by using primers specific to T. cruzi (TCZ1 and TCZ2) (4) under previously standardized conditions and positive controls (5). T. cruzi DNA was detected in 42 (26.4%) of 159 woodrat samples tested. Males were infected significantly more often (31/82) than females (11/73); sex was not determined for 4 individuals (Score test for a binomial proportion, z = –4.0, p<0.01). Adults had a nonsignificant higher prevalence (24/92) than all other individuals in the remaining age categories combined (14/54) (age was not determined for 13 individuals) (Score test for a binomial proportion, z = –0.02, p = 0.98). Middens that harbored infected individuals (n = 28, mean = 1.8) were not significantly (t = 0.79, df = 84, p = 0.43) more populated than middens that harbored uninfected individuals (n = 58, mean = 1.6). Woodrats had been shown by using microscopy to be infected by T. cruzi and T. cruzi–like organisms (6); however, no definitive DNA-based confirmation had been performed (6,7). The results of this research confirm the infection of N. micropus woodrats with T. cruzi and show a higher prevalence than that reported in previous studies that used other diagnostic methods. These results also point to woodrats as a potentially important reservoir of T. cruzi in North America. We hypothesize that the high prevalence is a consequence of the nest-building habits of these rodents. These nests are complexes of dry branches, grasses, and leaves, with a mean diameter of 84 cm, and offer easy access and permanent refuge to triatomine bugs. Woodrats have been found in association with at least 5 triatomine species: Triatoma gerstaeckeri, T. lecticularia, T. neotomae, T. protracta, and T. sanguisuga (8). Another factor for consideration is woodrats’ multigenerational midden use, which may enable the permanent occurrence of triatomine colonies and therefore maintain long-term circulation of T. cruzi. Whereas recent characterizations of North American strains have included isolates from other mammalian reservoir hosts (9), the genotyping of parasites from N. micropus woodrats and other woodrats is still to be done. Despite successful results from tracking pathogens by using material deposited in natural history museum collections (10), this practice is not common. We suggest that natural history museum collections be used more frequently, especially for surveying and genotyping T. cruzi in mammals, because of the importance of such information in clarifying the epidemiology and the evolutionary history of this pathogen.

GENETIC DIVERSITY WITHIN THE SOUTHERN PLAINS WOODRAT (NEOTOMA MICROPUS) IN SOUTHERN TEXAS

Abstract Genetic diversity within a population of the southern plains woodrat was examined using DNA sequences (967 base pairs [bp]) obtained from the control or d-loop region of the mitochondrial genome. One hundred fourteen individuals from 10 collection sites were assigned to 42 haplotypes. Haplotype diversity values were moderate to high (0.974 overall and ranged from 0.524 to 0.964 across collecting sites), whereas nucleotide diversity values were low (0.008 overall and ranged from 0.001 to 0.010 across sites), indicating that this population possesses a high number of closely related haplotypes. Seventy-nine percent of the genetic variability was partitioned within groups that corresponded to the collecting sites. In addition, 13 samples from Texas, New Mexico, and Mexico were included as references for evaluating the evolutionary history of haplotypes. Nested clade analysis revealed that restricted gene flow with isolation by distance in conjunction with contiguous range expansion was responsible for the observed pattern of genetic diversity. A test of neutrality supported the diagnosis of restricted gene flow, but failed to support contiguous range expansion due solely to population growth. Examination of the spatial distribution of the haplotypes indicated that most haplotypes were restricted to a single collecting site; however, a small number of haplotypes were found at 2 or more sites. A phylogenetic analysis indicated that some haplotypes (28.6%) were restricted to the study area whereas the remaining haplotypes occupied a broader geographic region.

Molecular divergence within the Oryzomys palustris complex: evidence for multiple species

Abstract Nucleotide sequences from 94 individuals representing the Oryzomys palustris complex (O. palustris and O. couesi) were examined to assess phylogenetic relationships and taxonomic boundaries. Sequence data from the entire mitochondrial cytochrome-b (Cytb; 1,143 base pairs [bp]), a portion of exon 1 of the nuclear interphotoreceptor retinoid-binding protein (1,266 bp), and intron 2 of the alcohol dehydrogenase 1 (580 bp) genes were analyzed using phylogenetic methods (maximum parsimony and Bayesian inference). In the Cytb analysis, individuals recognized as O. palustris and O. couesi formed reciprocally monophyletic clades supporting their recognition as species; however, additional phylogenetically informative groups were present within each of the 2 nominate clades. In addition, levels of genetic divergence within the currently recognized taxa exceeded values normally associated with intraspecies variation. Together, the phylogenetic and genetic divergence data imply that consideration should be given to recognizing 4 additional species in this complex.

RAPID RECOVERY OF RODENT POPULATIONS FOLLOWING SEVERE DROUGHT

Abstract Below normal precipitation during June 2001 through June 2002 was recorded at Chaparral Wildlife Management Area, near Catarina, Texas. This drought was followed by a 13-mo period (July 2002 through July 2003) of above average precipitation, providing an opportunity to examine the response of 5 species of rodents to drought conditions. Comparison of the number of unique individuals captured during the drought to the post-drought revealed a significant increase in the total number of individuals (approximately 500%) and in the number of individuals per species. A 3-mo lag time was observed between the end of the drought and the onset of population recovery. Although an increase in population size was anticipated as precipitation patterns returned to normal, the magnitude and rapidity of the recovery exceeded predictions and documented the resilience of rodents to adverse climatic conditions.

Diversity and phylogenetic relationships among the North American Tacaribe serocomplex viruses (Family Arenaviridae)

The purpose of this study was to extend our knowledge of the genetic diversity and phylogenetic relationships among the North American Tacaribe serocomplex viruses. Analyses of glycoprotein precursor gene sequence data separated the North American arenaviruses into 7 major phylogenetic groups. The results of analyses of Z gene and nucleocapsid protein gene sequence data were not remarkably different from the glycoprotein precursor gene tree. In contrast, the tree generated from RNA-dependent RNA polymerase gene sequences differed from the glycoprotein precursor gene tree with regard to phylogenetic relationships among the viruses associated with woodrats captured in the western United States, Texas, or northern Mexico. Further analyses of the polymerase gene sequence data set suggested that the difference in topology was a consequence of incongruence among the gene tree data sets or chance rather than genetic reassortment or recombination between arenaviruses.

Ecology of Catarina Virus (family Arenaviridae) in Southern Texas, 2001–2004

A total of 3941 rodents were captured during a 46-month prospective (mark-recapture) study on the ecology of Catarina virus in southern Texas. Antibody reactive against Catarina virus was found in 73 (11.9%) of 611 southern plains woodrats (Neotoma micropus) and none of 3330 other rodents; strains of Catarina virus were isolated from 6 antibody-negative and 9 antibody-positive southern plains woodrats; and the infections in at least 3 southern plains woodrats were chronic. These results affirm the notion that the southern plains woodrat is the principal host of Catarina virus and suggest that Catarina virus infection is highly specific to N. micropus.

Molecular Systematics of South American Marsh Rats of the Genus Holochilus (Muroidea, Cricetidae, Sigmodontinae)

We present a comprehensive systematic study of Holochilus, a sigmodontine genus of large, herbivorous, and semiaquatic rodents widely distributed in South America. Remarkably, given its complex taxonomic history and large economic as well as epidemiological importance, the alpha taxonomy of Holochilus has not benefited from a molecular-based approach. The study is based on sequences of 1 mitochondrial and 3 nuclear loci that were analyzed by maximum likelihood and Bayesian inference. Analyses include sequences of specimens from localities from Argentina, Bolivia, Brazil, Colombia, Paraguay, Peru, Suriname, and Uruguay, representing all but 2 of the species currently recognized in the genus. Of the 4 data matrices, the mitochondrial data set contains the largest geographic coverage and recovered 6 species-level lineages that form 2 well-supported species groups: the brasiliensis species group formed by H. brasiliensis and H. vulpinus and the sciureus species group composed by H. chacarius, H. sciureus, and 2 currently unnamed forms. Surprisingly, in the cytochrome b gene analyses, the 2 species groups are not sister to each other; i.e., Holochilus is not monophyletic, although these topologies lack significant support. However, the monophyly of Holochilus was supported by the 3 nuclear loci as well as by the combined analysis of all 4 loci. These genealogical results are the basis of taxonomic and biogeographic considerations. Presentamos un estudio sistemático comprensivo sobre Holochilus, un género sigmodontino de grandes roedores herbívoros y semi-acuáticos ampliamente distribuido en América del Sur. Llamativamente, dada su compleja historia taxonómica y el gran impacto económico y epidemiológico, la taxonomía alfa de Holochilus no se ha beneficiado de un abordaje basado en evidencia molecular. El estudio se basa en secuencias de 1 gen mitocondrial y de 3 nucleares que fueron analizadas con máxima verosimilitud e inferencia Bayesiana. Los análisis incluyen secuencias de especímenes colectados en localidades de Argentina, Bolivia, Brasil, Colombia, Paraguay, Perú, Surinam y Uruguay, representando todas, con excepción de 2, las especies actualmente reconocidas en el género. La genealogía mitocondrial, que es la que tiene la mayor cobertura geográfica de Holochilus, recobra 6 linajes de nivel de especie que forman 2 grupos de especies bien apoyados: el grupo de especies brasiliensis integrado por H. brasiliensis y H. vulpinus y el grupo de especies sciureus que está compuesto por H. chacarius, H. sciureus y 2 formas aparentemente sin nominar. Llamativamente, en los análisis basados en el gen mitocondrial los 2 grupos de especies no son hermanos; i.e., Holochilus no es monofilético, aunque esta topología no tiene apoyo significativo. Sin embargo la monofilia de Holochilus es apoyada por los análisis de los 3 genes nucleares y por el análisis combinado de los 4 genes. Estos resultados genealógicos son la base de consideraciones taxonómicas y biogeográficas.