Parasites lost: using natural history collections to track disease change across deep time

Museums and other natural history collections (NHC) worldwide house millions of specimens. With the advent of molecular genetic approaches these collections have become the source of many fascinating population studies in conservation genetics that contrast historical with present-day genetic diversity. Recent developments in molecular genetics and genomics and the associated statistical tools have opened up the further possibility of studying evolutionary change directly. As we discuss here, we believe that NHC specimens provide a largely underutilized resource for such investigations. However, because DNA extracted from NHC samples is degraded, analyses of such samples are technically demanding and many potential pitfalls exist. Thus, we propose a set of guidelines that outline the steps necessary to begin genetic investigations using specimens from NHC.

This method is designed to maximize recovery of PCR-amplifiable DNA from ancient bone and teeth specimens and at the same time to minimize co-extraction of substances that inhibit PCR. This is achieved by a combination of DNA extraction from bone powder using a buffer consisting solely of EDTA and proteinase K, and purification of the DNA by binding to silica in the presence of high concentrations of guanidinium thiocyanate. All steps are performed at room temperature (20-23 degrees C), thereby reducing further degradation of the already damaged and fragile ancient DNA and providing an optimal trade-off between DNA release and degradation. Furthermore, the purification step removes most of the various types of PCR inhibitors present in ancient bone samples, thereby optimizing the amount of ancient DNA available for subsequent enzymatic manipulation, such as PCR amplification. The protocol presented here allows DNA extraction from ancient bone and teeth with a minimum of working steps and equipment and yields DNA extracts within 2 working days.

One of the biggest threats facing amphibian species and population survival worldwide is the disease chytridiomycosis, caused by the chytrid fungus, Batrachochytrium dendrobatidis ( 1 , 2 ). Chytridiomycosis was proposed as the cause of death in frog populations in the rain forests of Australia and Panama and was associated with the decline of frog populations in Ecuador, Venezuela, New Zealand, and Spain ( 3 – 6 ). Evidence for a countrywide decline in frog populations in South Africa is lacking ( 7 ), and local declines of several species have been ascribed to two main threats, habitat destruction and pollution ( 8 ). Chytridiomycosis is known in South Africa from infections in Xenopus laevis, Afrana fuscigula, and Strongylopus grayii ( 9 – 11 ). Through surveys of extant and archived specimens, Batrachochytrium has been found in every continent that has amphibians, except Asia ( 6 , 9 , 12 , 13 ). Since B. dendrobatidis has been recognized as an emerging pathogen, whose spread is facilitated by the international and intranational movement of amphibians ( 1 ), identifying its origin will be useful. Some emerging infectious diseases arise when pathogens that have been localized to a single host or small geographic region go beyond previous boundaries ( 14 ). If B. dendrobatidis emerged in this fashion, we hypothesize that the source would meet the following criteria: 1) the hosts would show minimal or no apparent clinical effects, 2) the site would be the place of the earliest known global occurrence, 3) the date of this occurrence would precede any amphibian declines in pristine areas (i.e., late 1970s), 4) the prevalence in the source host or hosts would be stable over time, 5) no geographic spreading pattern would be observed over time in the region, 6) a feasible means of global dissemination of Batrachochytrium from the region of origin would be identified, and 7) B. dendrobatidis would show a greater genetic variation in the host region than in more recently invaded regions. B. dendrobatidis is common in African frogs from Ghana, Kenya, South Africa, and Western Africa ( 12 , 15 ) and declines in frog populations are poorly documented in Africa ( 7 , 16 ). These factors, combined with the global trade in X. laevis and X. tropicalis, prompted us to investigate the likelihood that Africa was the origin of Batrachochytrium and that the trade in Xenopus spp. played a key role in its global dissemination. Within the Xenopus genus, X. laevis is distributed over the greatest area in sub-Saharan Africa. X. laevis occupies most bodies of water in savannah habitats from the Cape of Good Hope to Nigeria and Sudan ( 17 , 18 ). We report the earliest case of the amphibian chytrid found in any amphibian and present epidemiologic evidence to support the hypothesis that B. dendrobatidis originated in Africa. In this article, chytridiomycosis refers to infection of amphibians by B. dendrobatidis. Materials and Methods A retrospective survey was conducted on archived specimens of the genus Xenopus housed in five southern Africa institutions, Bayworld (Port Elizabeth), Natal Museum (Pietermaritzburg), National Museum (Bloemfontein), South African Museum (Cape Town), and Transvaal Museum (Pretoria). Specimens in these museums had been collected for archiving by a large number of persons for various purposes and had not been selected for a systematic survey of amphibian disease. Specimens were collected mainly from South Africa, Lesotho, and Swaziland. A piece (3 x 3 mm) of the interdigital webbing was removed from one hind foot of each specimen of X. gilli, X. muelleri, and X. laevis. Tissue was prepared for histologic examination with routine techniques ( 19 ). Sections were cut at 6 μm and stained with hematoxylin and eosin. Chytridiomycosis was diagnosed by using described criteria ( 20 ). Sections from the two specimens diagnosed as having chytridiomycosis with hematoxylin and eosin before 1971 (one collected in 1938, the other in 1943) were confirmed with the more specific immunoperoxidase test ( 21 ) to increase the confidence of the diagnosis. Measurements of sporangia were performed with a calibrated eyepiece and expressed as mean ± standard deviation (SD). Histologic slides were examined "blind," without reference to dates that the frogs were collected, to decrease any opportunity for bias in diagnosis. Exact versions of chi-square tests were used to analyze bivariate associations between chytridiomycosis prevalence and host species, region in South Africa (southwestern, eastern, and central), and season. Bivariate time trends of prevalences were analyzed by exact chi-square tests for trend. Multivariate logistic regression models were applied to assess potential confounding effect of species, region, and season on the time trend of chytridiomycosis prevalence. Confidence intervals (CI) were calculated by using exact binomial probabilities. Longitudinal and latitudinal historical patterns of spread were analyzed with linear regression models. Results Zoosporangia with a diameter (mean ± SD) of 5.2 ± 0.72 μm (maximum 6 μm) were seen in the stratum corneum of the digital webbing of infected frogs (Figure 1). Most sporangia were empty spherical structures, but occasional sporangia were observed with developing stages, septa, or discharge papillae. The structures stained brown (indicating positivity) in the immunoperoxidase test with the specific anti-Batrachochytrium antibody (Figure 1). Lesions usually associated with chytridiomycosis, including hyperplasia of the epidermis and hyperkeratosis of the stratum corneum, were mild and localized to areas of infection. Figure 1 Micrographs of immunoperoxidase stained sections through the interdigital webbing of Xenopus gilli, showing the morphologic features and size of zoosporangia consistent with Batrachochytrium dendrobatidis. A) Arrow a indicates localized hyperplastic epidermal response; arrow b indicates an uninfected region of the epidermis. B) Arrows indicate two zoosporangia with internal septa. Circle indicates location of the infection in the stratum corneum. Bar, 10 μm. Overall, chytridiomycosis prevalence from the survey was 2.7% (19 positives out of 697 specimens) and did not differ significantly across species (p = 0.7; Table 1). The earliest date for a chytridiomycosis-positive specimen was 1938 in an X. laevis collected from the Western Cape coastal lowland. This specimen is housed in the South African Museum, Cape Town (SAMZR 18927). The next earliest positive specimen detected was an X. gilli from 1943 (specimen number NMB 112, National Museum, Bloemfontein). The distribution of dates specimens were collected was greatly skewed to the latter half of the 20th century (Table 2). The breakdown for the time interval 1871–1940 is presented in order (decade, number of frogs infected/number of frogs examined) as follows: 1871–1880, 0/1; 1881–1890, 0/0; 1891–1900, 0/6; 1901–1910, 0/6; 1911–1920, 0/4; 1921–1930, 0/2; 1931–1940, 1/37. No statistically significant change of chytridiomycosis prevalence occurred over the decades since the 1940s (p = 0.36), or when the broader interval of pre-1971 is used as the baseline for the calculations (p = 0.22; Figure 2). No evidence for any trend in prevalence over time could be found using multivariate modeling where the odds ratios for the time intervals were adjusted for the potential confounders of species, season, and region. The multivariate odds ratios in these models were not significant and very similar to the bivariate findings, which indicate no confounding effects. The prevalence of chytridiomycosis in South Africa showed no significant change over time after 1940. No significant change of the geographic distribution of chytridiomycosis was detected after 1973. By 1973 the distribution of chytridiomycosis, as proved by positive specimens, covered already the area from 27° to 34° latitude and 18.25° to 32.5° longitude. This finding implies that positive specimens were detected from all regions of southern Africa by 1973. Infected frogs were found in 5 of the 9 provinces in South Africa, including the Western Cape (5 of 171), Northern Cape (2 of 22), Free State (6 of 141), Kwazulu-Natal (3 of 152), and Eastern Cape (1 of 137), as well as in Swaziland (2 of 42). Prevalence of B. dendrobatidis did not differ (p = 0.24) between the designated three broader regions with prevalences of 3.0% in the southwest, 3.8% in the central region, and 1.5% in the eastern region. Overall, the seasons (wet versus dry) when the specimens were collected were not significantly associated with prevalence (p = 0.22). Only in the eastern region, was a significantly higher prevalence found in the wet season than the dry season. Table 1 Prevalence of chytridiomycosis in archived Xenopus spp. from southern Africaa Species No. examined % positive (95% CI) Earliest positive detected Country Xenopus laevis 583 2.6 (1.5–4.2) 1938 South Africa X. meulleri 53 3.8 (0.5–13.0) 1991 Swaziland X. gilli 61 3.3 (0.4–11.4) 1943 South Africa Total 697 2.7 ap = 0.7; CI, confidence interval. Table 2 Prevalence of chytridiomycosis in archived Xenopus, by time intervalsa Time interval No. examined No. positives % positive (95% CI) 1871–1940 56 1 1.8 (0.0–9.6) 1941–1950 16 1 6.3 (0.2–30.2) 1951–1960 63 0 0.0 (0.0–5.7) 1961–1970 17 0 0.0 (0.0–19.5) 1971–1980 230 6 2.6 (1.0–5.6) 1981–1990 145 3 2.1 (0.4–5.9) 1991–2001 170 8 4.7 (2.0–9.0) Total 697 19 2.7 (1.7–4.2) ap = 0.36; CI, confidence interval. Figure 2 Historical time-trend of chytridiomycosis prevalence in southern Africa. No significant change was shown in the prevalence over time (p = 0.22, 95% confidence interval). Discussion Our study has extended the date for the earliest case of chytridiomycosis in wild amphibians by 23 years. The next earliest case outside South Africa was found in Rana clamitans from Saint-Pierre-de-Wakefield, Québec, Canada, in 1961 ( 22 ). After the case in Canada, the earliest cases from other countries follow sequentially over a period of 38 years from 1961 to 1999 (Figure 3). Figure 3 Time bar indicating when chytridiomycosis first appeared in the major centers of occurrence in relation to each other. Following a 23-year interruption in occurrences after the Xenopus laevis infection in 1938, records outside Africa appear with increasing frequency up until the present; North America ( 22 ), Australia ( 2 , 23 ), South America ( 5 ), Central America ( 24 ), Europe ( 6 ), Oceania (New Zealand) ( 25 ). X. laevis in the wild does not show clinical signs, nor has it experienced any sudden die-offs. Moreover, only subclinical chytrid infections have been observed among captive colonies of X. laevis ( 26 , 27 ). A frog of a related species, X. tropicalis, died in captivity from chytridiomycosis, it was suspected of having contracted the fungus from X. laevis ( 27 ). An ideal host for transmission of chytridiomycosis through international translocation would be a species of amphibian that does not become diseased or die from the infection; hence, X. laevis could take on the role of a natural carrier. The sudden appearance of chytridiomycosis can best be explained by the hypothesis that B. dendrobatidis was recently introduced into new regions and subsequently infected novel host species ( 1 ). Dispersal of B. dendrobatidis between countries is most likely by the global transportation of amphibians ( 1 , 2 , 23 , 28 , 29 ). The World Organization for Animal Health has recently placed amphibian chytridiomycosis on the Wildlife Diseases List in recognition of this risk. If Africa is the source of B. dendrobatidis, a feasible route of dissemination by infected amphibians needs to be identified. Some members of the family Pipidae have been exported, in particular Hymenochirus curtipes and X. laevis, to North America and Europe ( 30 ). In terms of a most likely candidate for spread from Africa, the number of frogs and geographic dissemination favor X. laevis. Soon after discovery of the pregnancy assay for humans in 1934 ( 30 ), enormous quantities of the species were caught in the wild in southern Africa and exported around the world. The pregnancy assay is based on the principle that ovulation in X. laevis is induced by injection with urine from pregnant women because of high levels of gonadotropic hormones in the urine ( 31 , 32 ). X. laevis was selected as the most suitable amphibian for investigating the mechanism of the mating reflex because of the relative ease with which the animal can be maintained in captivity ( 33 ). For 34 years, the trade in X. laevis in South Africa was controlled by the then Cape of Good Hope Inland Fisheries Department (Western Cape Nature Conservation Board) at the Jonkershoek Fish Hatchery. As an indication of the numbers involved in this trade, 10,866 frogs were distributed in 1949, of which 3,803 (35%) were exported, and of the 20,942 frogs distributed in 1970, a total of 4,950 (24%) were shipped abroad ( 34 , 35 ). After the introduction of nonbiologic pregnancy tests, X. laevis became important as a model for the scientific study of immunity and later embryology and molecular biology. X. laevis could have carried the disease globally, particularly if the prevalence was similar to that seen in wild-caught X. laevis today. In the importing country, escaped frogs, the water they lived in ( 36 ), or both, could have come into contact with local amphibian species, and subsequent transmission of the disease could have followed. The establishment of feral populations of X. laevis in Ascension Island, the United Kingdom, the United States, and Chile in 1944, 1962, the 1960s, and 1985 ( 37 ), respectively, show that transmission could have become ongoing if these feral populations were infected. Although we have demonstrated that B. dendrobatidis was in southern Africa since 1938, our studies provide no indication regarding whether this region was the original source within Africa. B. dendrobatidis has been found in wild frogs in Kenya and in frogs (X. tropicalis and X. laevis) wild-caught in Western Africa and detected after importation into the United States ( 12 , 26 , 27 , 38 ), which indicates that B. dendrobatidis is widely disseminated in Africa. Xenopus consists of 17 species that are found in sub-Saharan Africa, with a varying degree of sympatry between species ( 17 ). The overlap in the distribution and, in some cases, the sharing of habitats could facilitate transmission of B. dendrobatidis between these species. This finding would imply that chytridiomycosis could have originated elsewhere in Africa and spread within multiple host-region combinations. More detailed historical studies of archived African amphibians may indicate whether B. dendrobatidis was originally present in a small area of Africa from which it emerged to occupy large areas of the continent. Until the deficit in distribution data and comparative genetic studies is remedied, locating the source of the origin of B. dendrobatidis within Africa remains speculative. The relationship appears to have coevolved within an anuran host, and the opportunity to disseminate across the globe existed for B. dendrobatidis in southern Africa. If X. laevis did carry B. dendrobatidis out of Africa as we propose, other amphibian species subsequently could have distributed it between and within countries. The American bullfrog, Rana catesbeiana, has been proposed as an important vector, mainly through international trade as a food item, but also within countries as populations established for the food trade escape and spread ( 29 ). The earliest current record for the occurrence of chytridiomycosis in R. catesbeiana is 1978 in South Carolina ( 38 ), 40 years after the first record in southern Africa, but details on the intensity of the search for chytridiomycosis in archived bullfrogs are not available. The transmission of chytridiomycosis globally may involve a series of key steps: 1) occurrence of B. dendrobatidis in an amphibian vector in southern Africa that is relatively resistant to disease (X. laevis), 2) sudden rise in 1935 of export trade in this vector because of technologic advances (Xenopus pregnancy test), 3) escape of the pathogen from the exported Xenopus to establish new foci in other countries (possibly expedited in some countries by establishment of feral populations of X. laevis), 4) transmission into other vector amphibians (food and pet trade), and 5) further transmission to other countries along different trade routes in key amphibian vectors that move in high numbers and become established in commercial populations and closely interact with wild frogs, which likely leads to feral populations (food frogs R. catesbeiana). Spread through native amphibian populations with epidemic disease in some species could have occurred at any point after B. dendrobatidis entered a naïve native species. We have provided epidemiologic evidence that Africa is the origin of the amphibian chytrid fungus. Support for six of the seven criteria proposed for the source of B. dendrobatidis has been demonstrated: 1) the major host (X. laevis) shows minimal or no apparent clinical effects, 2) site of the earliest global occurrence (1938), 3) this date precedes any amphibian declines in pristine areas, 4) the prevalence in the source host or hosts (Xenopus spp.) has been stable over time, 5) no geographic spreading pattern could be observed over time, and 6) a feasible means of global dissemination exists via the international trade in wild-caught X. laevis, which commenced in 1935 and continues today. Criterion 7, greater genetic diversity of B. dendrobatidis at the source, has not been investigated. A low level of genetic variation was shown for 35 strains of B. dendrobatidis and suggested that B. dendrobatidis was a recently emerged clone ( 39 ). The strains had been collected in North America, Australia, Panama, and Africa from wild and captive amphibians. Three strains isolated from captive X. tropicalis in United States had been imported from Ghana. Although these showed no significant differences from the U.S. strains ( 39 ), their assignment to Africa assumes no cross-infection had occurred within the importing facility. Future work on the genetic diversity of B. dendrobatidis in Africa compared with strains from regions outside Africa will add weight to the hypothesis if greater genetic diversity is found in African strains.