The identification of a new Giardia duodenalis assemblage in marine vertebrates and a preliminary analysis of G. duodenalis population biology in marine systems

Abstract

Giardia duodenalis is an intestinal parasite of many vertebrates. The presence of G. duodenalis in the marine environment due to anthropogenic and wildlife activity is well documented, including the contributions from untreated sewage and storm water, agricultural run-off and droppings from terrestrial animals. Recently, studies have detected this protistan parasite in the faeces of marine vertebrates such as whales, dolphins, seals and shore birds. To explore the population biology of G. duodenalis in marine life, we determined the prevalence of G. duodenalis in two species of seal (Halichoerus grypus, Phoca vitulina vitulina and Phoca vitulina richardsi) from the east and west coasts of the USA, sequenced two loci from G. duodenalis-positive samples to assess molecular diversity and examined G. duodenalis distribution amongst these seals and other marine vertebrates along the east coast. We found a significant difference in the presence of G. duodenalis between east and west coast seal species. Only the zoonotic lineages of G. duodenalis, Assemblages A and B and a novel lineage, which we designated as Assemblage H, were identified in marine vertebrates. Assemblages A and B are broadly distributed geographically and show a lack of host specificity. Only grey seal (Halichoerus grypus) samples and one gull sample (Larus argentatus) from a northern location of Cape Cod, Massachusetts, USA, showed the presence of Assemblage H haplotypes; only one other study of harbour seals from the Puget Sound region of Washington, USA previously recorded the presence of an Assemblage H haplotype. Assemblage H sequences form a monophyletic clade that appears as divergent from the other seven Assemblages of G. duodenalis as these assemblages are from each other. The discovery of a previously uncharacterised lineage of G. duodenalis suggests that this parasite has more genetic diversity and perhaps a larger host range than previously believed.

Fig. 1

Sampling sites along the east and west coasts of the United States of America (USA). A) East coast sampling sites. East coast samples originated primarily from locations surrounding Cape Cod, Massachusetts (MA), USA but also include areas south of Cape Cod---Marion and Fall River, MA---and north of Cape Cod---Appledore Island, Maine (ME) and Kent Island, New Brunswick (NB), Canada. Pie graphs represent the proportion of times an assemblage was found at each sampling site, with red representing Assemblage A, blue representing Assemblage B and green representing Assemblage H. The white number centered in the pie chart represents the total number of times Assemblages were detected. The number of individual samples taken from each site is indicated after the sampling site. Both sites from this study and that of Lasek-Nesselquist et al. (2008) are indicated in the figure. * Indicates sampling sites from this study only. ** Indicates seal (Atlantic harbor seal and grey seal) sampling sites from this study with the proportion of seals that tested positive listed. N and S indicate the North/South delineation used for dividing sampling sites into regions for analysis of molecular variance (AMOVA). B) West coast Pacific harbor seal sampling sites, which are all located in California, USA. Sites are represented by circles. Black circles indicate sites negative for Giardia duodenalis. Blue circles indicate sampling sites positive for G. duodenalis Assemblage B. The proportion of positive seals at each subdivision is indicated. The San Francisco (SF) Bay Area encompasses Mid, North, and South SF Bay sites. Specifically, the one SF Bay Area positive seal sample derived from Mid SF Bay. The horizontal line indicates the north - south regional division used for AMOVA. Maps were generated by the National Atlas of the USA at www.nationalatlas.gov with the mapmaker application.

Fig. 1

Sampling sites along the east and west coasts of the United States of America (USA). A) East coast sampling sites. East coast samples originated primarily from locations surrounding Cape Cod, Massachusetts (MA), USA but also include areas south of Cape Cod---Marion and Fall River, MA---and north of Cape Cod---Appledore Island, Maine (ME) and Kent Island, New Brunswick (NB), Canada. Pie graphs represent the proportion of times an assemblage was found at each sampling site, with red representing Assemblage A, blue representing Assemblage B and green representing Assemblage H. The white number centered in the pie chart represents the total number of times Assemblages were detected. The number of individual samples taken from each site is indicated after the sampling site. Both sites from this study and that of Lasek-Nesselquist et al. (2008) are indicated in the figure. * Indicates sampling sites from this study only. ** Indicates seal (Atlantic harbor seal and grey seal) sampling sites from this study with the proportion of seals that tested positive listed. N and S indicate the North/South delineation used for dividing sampling sites into regions for analysis of molecular variance (AMOVA). B) West coast Pacific harbor seal sampling sites, which are all located in California, USA. Sites are represented by circles. Black circles indicate sites negative for Giardia duodenalis. Blue circles indicate sampling sites positive for G. duodenalis Assemblage B. The proportion of positive seals at each subdivision is indicated. The San Francisco (SF) Bay Area encompasses Mid, North, and South SF Bay sites. Specifically, the one SF Bay Area positive seal sample derived from Mid SF Bay. The horizontal line indicates the north - south regional division used for AMOVA. Maps were generated by the National Atlas of the USA at www.nationalatlas.gov with the mapmaker application.

Fig. 2

Glutamate dehydrogenase (gdh) gene tree depicting the relationships among all known Giardia duodenalis assemblages (A–G) and the newly characterized Assemblage H generated using Bayesian analysis. Sequences were 715 bp in length and chosen to maximize phylogenetic resolution while still representing the majority of the Assemblage H variants described. The gene tree is a consensus of 75,000 trees and rooted with Giardia ardeae and Spironucleus vortens. Posterior probabilities > 0.70 are shown below each branch. Bootstrap support > 50 from maximum likelihood (ML) analysis are shown above branches. The area of the triangle corresponds to the amount of variation within a clade. Inset highlights the variation and sub-structuring within Assemblage H and includes all variants (292–715 bp). Grey refers to grey seal.

Fig. 3

Seal mtDNA maximum likelihood tree. H2, H8, H9, H12 and H13 refer to grey seal (Halichoerus grypus) mtDNA haplotypes. P1, P3-P7, P10, P11, P14 and P15 refer to Atlantic and Pacific harbor seal (Phoca vitulina vitulina and Phoca vitulina richardsi) mtDNA haplotypes. All grey seal and Atlantic harbor seal samples derive from Massachusetts (east coast USA). All Pacific harbor seal samples derive from California (west coast, USA). Mid, North and South SF Bay refer to Mid, North, and South San Francisco Bay, which comprise the San Francisco Bay Area. The tree shows the number of times each haplotype was detected at a sampling site. Boldface indicates Giardia duodenalis positive seal samples. Bootstrap values > 50 are shown above branches and posterior probabilities > 0.70 from Bayesian analysis are shown below branches.

Fig. 4

Giardia duodenalis glutamate dehydrogenase (gdh) Bayesian tree derived from east coast marine animal fecal samples. The gene tree is a consensus of 75,000 sampled trees. Boldface indicates sequences generated from this study. Grey, Ahs, Phs and harp refer to grey seal, Atlantic harbor seal, Pacific harbor seal and harp seal, respectively. Boostrap values > 50 are shown above branches and posterior probabilities > 0.70 are shown below branches unless a boostrap value is not present. The shaded box highlights Assemblage H sequences derived from 10 grey seals from Jeremy Point, Wellfleet, Massachusetts (MA, USA) and one gull from Dennis, MA. Sampling site abbreviations are included with each sample: KI, Kent Island, Canada; We, Wellfleet, MA; De, Dennis, MA; Sa, Sandwich, MA; Or, Orleans, MA; Ch, Chatham, MA; Mo, Monomoy, MA; Mu, Muskeget, MA; MV, Martha’s Vineyard Island, MA; NI, Nantucket Island, MA; AI, Appledore Island, Maine; Ma, Marion, MA. A1 and A2 denote subgroups of Assemblage A.

Fig. 5

Giardia duodenalis triose phosphate isomerase (tpi) tree representing a consensus of 75,000 sampled trees from Bayesian analysis. Sequences derive from east and west coast marine animal fecal samples. Boldface indicates sequences generated from this study. The shaded box highlights the cluster of G. duodenalis sequences from California (CA) Pacific harbor seals. Sampling site abbreviations are included with each sample: KI, Kent Island, Canada; We, Wellfleet, Massachusetts (MA); De, Dennis, MA; Sa, Sandwich, MA; Or, Orleans, MA; Ch, Chatham, MA; Mo, Monomoy, MA, Mu, Muskeget, MA; MV, Martha’s Vineyard Island, MA; NI, Nantucket Island, MA; AI, Appledore Island, Maine; Ma, Marion, MA; Mn, Marin, CA; SF, (Mid) San Francisco Bay Area, CA. Posterior probabilities > 0.70 are shown below branches and bootstrap support > 50 from a maximum likelihood analysis are shown above branches. A1 and A2 denote sub-groups of Assemblage A