Genetic Assemblages Research Articles

Occurrence and Diversity ofGiardia duodenalisAssemblages in Livestock in theUK

SummaryGiardia duodenalis is a common intestinal parasite in humans and a wide range of livestock species. It is a genetically heterogeneous parasite that has been characterized in seven distinct genetic assemblages or cryptic species, and molecular markers can be used to differentiate both animal-specific and potentially zoonotic genotypes. Little is known about G. duodenalis and the range of assemblages occurring in domestic livestock species in the UK. Here, we present data on the occurrence and molecular diversity of G. duodenalis detected in the faeces or large intestinal contents of cattle, sheep, pigs, goats and camelids from farms in the north-west of England. Both healthy and clinically diseased animals were included in the survey. The presence of Giardia spp. and assemblages was determined by sequencing of the small-subunit ribosomal RNA gene. The potential association of infection with various clinical and epidemiological parameters was studied in cattle using both univariate and multivariate analyses. Giardia spp. were detected in 127 (34.3%) of the 370 animals tested. G. duodenalis assemblage E was found to be predominant in cattle and sheep, followed by assemblage A. Mixed infections with assemblages A and E were also detected. Interestingly, some cattle, sheep and pigs were found to be infected with more unexpected assemblages (C, D, F). Pre-weaned calves were more likely to test positive than adult animals, but no association between the occurrence of overt intestinal disease and G. duodenalis infection was detected. The common occurrence of assemblage A and the finding of unusual assemblages in atypical hosts suggest that in future, a multilocus analysis should be used to confirm the actual diversity of G. duodenalis in livestock and the presence of potentially zoonotic genotypes. These data also suggest that there is a need to re-evaluate the clinical significance of G. duodenalis infection in livestock.

Rapid identification of Giardia duodenalis assemblages in NSW using terminal-restriction fragment length polymorphism

Humans are infected by 2 genetic assemblages (A and B) of Giardia duodenalis, a protozoan parasite that causes gastro-intestinal disease. Sub-assemblages AI, AII, BIII and BIV are commonly identified in human cases. Detection requires amplification of G. duodenalis loci. Subsequent DNA sequencing or restriction fragment length polymorphism (RFLP) identifies sub-assemblages but is expensive (DNA sequencing) or insensitive (RFLP). This study investigated a fluorescence-based detection method, using terminal-restriction fragment length polymorphism (T-RFLP) of the glutamate dehydrogenase gene to characterize human infections. Clinical samples (n=73), positive for Giardia were collected in New South Wales, Australia, and were used to evaluate T-RFLP detection. The accuracy and sensitivity of T-RFLP detection was established by comparison to DNA sequencing and RFLP. Sub-assemblage assignment by T-RFLP identified BIV as the common subtype in N.S.W cases, whilst AI, AII and BIII were also detected. When compared to DNA sequencing and RFLP, analysis by T-RFLP was a reliable and reproducible method. Automated fluorescent detection enabled accurate sizing of restriction fragments and provided a sensitive alternative to RFLP. Discrimination of sub-assemblages by T-RFLP was comparable to DNA sequencing, but was efficient and inexpensive. The protocol described here provides a rapid and sensitive diagnostic tool for routine sample screenings in epidemiological research.

Resurrecting an extinct salmon evolutionarily significant unit: archived scales, historical DNA and implications for restoration

Archival scales from 603 sockeye salmon (Oncorhynchus nerka), sampled from May to July 1924 in the lower Columbia River, were analysed for genetic variability at 12 microsatellite loci and compared to 17 present-day O.nerka populations-exhibiting either anadromous (sockeye salmon) or nonanadromous (kokanee) life histories-from throughout the Columbia River Basin, including areas upstream of impassable dams built subsequent to 1924. Statistical analyses identified four major genetic assemblages of sockeye salmon in the 1924 samples. Two of these putative historical groupings were found to be genetically similar to extant evolutionarily significant units (ESUs) in the Okanogan and Wenatchee Rivers (pairwise F(ST) = 0.004 and 0.002, respectively), and assignment tests were able to allocate 77% of the fish in these two historical groupings to the contemporary Okanogan River and Lake Wenatchee ESUs. A third historical genetic grouping was most closely aligned with contemporary sockeye salmon in Redfish Lake, Idaho, although the association was less robust (pairwise F(ST) = 0.060). However, a fourth genetic grouping did not appear to be related to any contemporary sockeye salmon or kokanee population, assigned poorly to the O.nerka baseline, and had distinctive early return migration timing, suggesting that this group represents a historical ESU originating in headwater lakes in British Columbia that was probably extirpated sometime after 1924. The lack of a contemporary O.nerka population possessing the genetic legacy of this extinct ESU indicates that efforts to reestablish early-migrating sockeye salmon to the headwater lakes region of the Columbia River will be difficult.

Evaluation of a PCR protocol for sensitive detection of Giardia intestinalis in human faeces

Giardia intestinalis is a protozoan parasite and a human pathogen. It is a leading cause of human diarrheal disease and a significant cause of morbidity worldwide. At the molecular level, G. intestinalis is a species complex, consisting of genetic assemblages (A to G) and sub-assemblage strains. The genotypes that cause human disease have been characterised to assemblages A and B, and include strains AI, AII, BIII and BIV. PCR amplification of diagnostic loci is used to genotype samples and is required to understand different transmission cycles within communities. A multi-locus approach is required for validation of Giardia genotyping and molecular diagnostic techniques that are efficient across numerous loci have not been established. This study evaluated several published protocols for the 18S small subunit ribosomal RNA (18S rRNA) and glutamate dehydrogenase genes (gdh) genes. Assays were compared using spiked faecal samples and by measuring the concentration of DNA generated following DNA extraction and PCR amplification. An optimal molecular method for G. intestinalis identification was established from direct DNA extraction of faecal material and GC-rich PCR chemistry. The protocol was applied to 50 clinical samples and produced PCR success rates of 90% and 94% at the 18S rRNA and gdh loci. Cyst concentration prior to DNA extraction was not necessary, and the optimal protocol was highly sensitive and an efficient method for testing clinical samples.

Evolutionary history of the Karoo bush rat, Myotomys unisulcatus (Rodentia: Muridae): disconcordance between morphology and genetics

Morphological characters have historically been used as the basis for mammalian taxonomic designations and, in a geographic context, subspecies descriptions. Geographic genetic structuring of a species, however, often reflects a contrasting classification for sampled populations. To investigate morphological and genetic congruence, geometric morphometrics and phylogeographic mitochondrial DNA sequence analyses of a South African plains-dwelling species, Myotomys unisulcatus, the Karoo bush rat, was performed across its range. A Bayesian population structure analysis identified two closely-related distinct genetic assemblages: the first contains populations from both the eastern, southern, and western parts of the species range (coastal lowland group), and the second comprises individuals from the Little Karoo (central interior group). Areas of sharp elevation (the Great Escarpment), coupled to vegetational differences, appeared to be the main factor limiting gene flow between these two groups. Geometric morphometric analyses on the ventral and dorsal views of the crania of M. unisulcatus failed to support the genetic groupings. Instead environmental factors in the respective biomes appeared to play a more important role in shaping the crania of both genders. The contrasting patterns obtained between morphology and genetics in M. unisulcatus is probably indicative of phenotypic plasticity throughout the range of the species, and it is hypothesized that regional environmental factors play a prominent role in explaining geographic morphological variation within the species. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102, 510–526.

Giardiasis in dogs and cats: update on epidemiology and public health significance

Molecular data have defined seven genetic Assemblages of Giardia duodenalis, named A-G. Humans are infected with Assemblages A and B, dogs primarily with C and D, and cats with F. Assemblage A has been subclassified into subtypes A-I to A-IV: A-I has been reported in humans and animals, A-II in humans, and A-III and IV exclusively in animals. Assemblage B has broad host specificity infecting humans and animals. Recently, small numbers of dogs and cats have been reported to also carry Assemblages A-I or B. Because these genotypes are found primarily in humans, and no comprehensive studies to address zoonotic transmission of G. duodenalis are yet available, the potential role of dogs and cats cannot be conclusively excluded.

Prevalence and clinical correlations of genetic subtypes ofGiardia lambliain an urban setting

The clinical significance of different genetic subtypes or assemblages of Giardia lamblia is uncertain. Cases of giardiasis in south-west London between 1999 and 2005 were studied, comparing molecular-typing results with clinical and epidemiological findings from routine surveillance. We identified 819 cases, of whom 389 returned surveillance questionnaires. A subset of 267 faecal samples was submitted for typing by sequencing of the triose phosphate isomerase (tpi) and ribosomal RNA genes, and/or a separate duplex PCR of the tpi gene. Typing was successful in 199 (75%) samples by at least one of the molecular methods. Assemblage A accounted for 48 (24%) samples and Assemblage B for 145 (73%); six (3%) were mixed. Both assemblages had similar seasonality, age distribution and association with travel. Clinical features were available for 59 successfully typed cases: both assemblages caused similar illness, but Assemblage A was significantly more frequently associated with fever than Assemblage B.

Alpha 2 giardin is an assemblage A-specific protein of human infectiveGiardia duodenalis

Of the 7 genetic assemblages of the parasite Giardia duodenalis only 2 (A and B) are known to cause infections in humans. These assemblages have been characterized in detail at the genomic level but few studies have examined differences in the proteins expressed. Employing one and two-dimensional PAGE we have identified an assemblage A-specific protein of human infective G. duodenalis; alpha 2 giardin. The protein difference was evident using both electrophoretic techniques. Alpha 2 giardin is known to be a structural protein and associates with the caudal flagella and the plasma membrane; however, its exact function is unknown. Although several proteins unique to assemblage B were also observed, we were unable to identify these proteins due to a lack of genomic data available for assemblage B isolates. Together, these proteins represent distinct phenotypic differences between the human infective assemblages of G. duodenalis and support the need to revise the taxonomy of this parasite.

Phylogeography of the Southwestern Atlantic menhaden genus Brevoortia (Clupeidae, Alosinae)

Among pelagic fish, the Southwestern Atlantic menhaden genus Brevoortia (Clupeidae, Alosinae) constitutes an important species model to investigate the patterns of genetic differentiation. It is abundant in the Rio de la Plata estuary and in the Atlantic coastal lagoons system from Uruguay and Southern Brazil. To access in the taxa discrimination and population structure in Brevoortia we perform a phylogeographic approach based on mitochondrial cytochrome b (cyt-b) sequences including 240 individuals from 16 collecting sites. Among the 720 bp cyt-b sequenced, 199 correspond to variables and 88 to phylogenetically informative sites. High values of haplotype diversity (h = 1.000) and nucleotide diversity (π = 0.061), as well as an average of 0.084 polymorphic segregating sites and 46 different haplotypes were found. Maximum likelihood analysis based on the GTR + I + G model and Bayesian inference strongly support the idea that B. aurea is the only species of the genus inhabiting the Southwestern Atlantic region. Our analyses revealed a complex population pattern characterized by the existence of long-term highly structured genetic assemblages of mixed stocks. Each monophyletic entity included individuals from different collecting sites, different age groups and collected in different years. Our data also suggest that the recruitment of unrelated mtDNA haplotypes carried out by individuals within schools could be occurring in the same nursery areas revealing the existence of many different maternal lineages. A scenario where different simultaneously and successively mixed mtDNA lineages remain historically connected through basal haplotypes among different clades could explains more accurately the complex and ordered metapopulation dynamic found in this pelagic fish.

Genetic characterization of captive Cuban crocodiles (Crocodylus rhombifer) and evidence of hybridization with the American crocodile (Crocodylus acutus)

There is a surprising lack of genetic data for the Cuban crocodile (Crocodylus rhombifer), especially given its status as a critically endangered species. Samples from captive individuals were used to genetically characterize this species in comparison with other New World crocodilians. Partial mitochondrial sequence data were generated from cyt-b (843 bp) and the tRNA(Pro)- tRNA(Phe)-D-loop region (442 bp). Phylogenetic analyses were performed by generating maximum parsimony, maximum likelihood, and Bayesian-based topologies. In addition, in an effort to identify species-specific alleles, ten polymorphic microsatellite loci were genotyped. Distance and model-based clustering analyses were performed on microsatellite data, in addition to a model-based assignment of hybrid types. Both mitochondrial and nuclear markers identified two distinct C. rhombifer genetic sub-clades (alpha and beta); and microsatellite analyses revealed that most admixed individuals were F(2) hybrids between C. rhombifer-alpha and the American crocodile (C. acutus). All individuals in the C. rhombifer-beta group were morphologically identified as C. acutus and formed a distinct genetic assemblage. J. Exp. Zool. 309A:649-660, 2008. (c) 2008 Wiley-Liss, Inc.

Genetic variation among trout in the River Neretva basin, Bosnia and Herzegovina

Mitochondrial DNA haplotypes, characteristic of the Adriatic, Danubian and Atlantic lineages of brown trout Salmo trutta and of Salmo obtusirostris were found in trout inhabiting the River Neretva basin. With the exception of the one associated with softmouth trout, haplotypes were not correlated with operational taxonomic units based on phenotype. Nuclear DNA analysis identified four genetic assemblages corresponding to S. obtusirostris, different geographically confined autochthonous S. trutta populations, introduced S. trutta and a genetically heterogeneous group located between S. obtusirostris and S. trutta in the dendrogram of individuals, indicating the existence of hybrid swarms in the Neretva basin. Genetic assemblages corresponding to Salmo marmoratus and the recently proposed Salmo cf. montenigrinus were not detected. The presence of genetic intermediates indicates that the studied taxa are not completely reproductively isolated and that genetic stability has been either anthropogenically interrupted or not yet achieved among Neretva trout. This finding should be considered in management decisions since such an unstable community must be particularly susceptible to breakdown in genetic population structure as a result of hybridization between native and non‐native introduced trout stocks.

Microsatellite DNA analysis of rainbow trout (Oncorhynchus mykiss) from western Alberta, Canada: native status and evolutionary distinctiveness of “Athabasca” rainbow trout

Molecular genetic assays can contribute to conservation of aquatic taxa by assessing evolutionary and taxonomic distinctiveness, levels of genetic variation within and between populations, and the degree of introgression with introduced taxa. The Athabasca River drainage of␣western Alberta, Canada is one of only three (and the largest) drainages flowing east of the continental divide that contain native populations of rainbow trout (Salmonidae: Oncorhynchus mykiss). The “Athabasca” rainbow trout has been considered a preglacial relict worthy of special conservation measures. In addition, the native range of Athabasca rainbow trout has seen many instances of introductions of non-native populations since the beginning of the 20th century. We assayed rainbow trout from the Athabasca River drainage, from hatchery populations, and from representative populations in adjacent regions (N = 49 localities) for variation at 10 microsatelite loci to assess the level of evolutionary distinctiveness of Athabasca rainbow trout, and to assess the levels of introgression with non-native hatchery fish. We found that native Athabasca rainbow trout did not form a distinctive genetic assemblage and that the greatest amount of allele frequency variation was attributable to contemporary drainage systems (29.3%) rather than by a Athabasca/non-Athabasca distinction (12.6%). We found that 78% of all fish were confidently assigned to a “wild” rather than a “hatchery” genetic grouping and that most of the inferred introgression with hatchery fish was restricted to a few localities (N = 6). Our results suggest that: (i)␣Athabasca River rainbow trout are likely postglacial immigrants from adjacent populations of the Fraser River, and (ii) that there is no evidence of widespread introgression of hatchery alleles into native Athabasca River drainage rainbow trout.

Population and conservation genetics of the gopher tortoise (Gopherus polyphemus)

The gopher tortoise (Gopherus polyphemus) is an important member of the sandhill, longleaf pine, and scrub ecosystems in the southeastern United States. Even though it is currently protected throughout its range, tortoise populations continue to decline. We assessed genetic diversity at nine microsatellite loci in 300 individuals from 21 locations throughout Florida and southern Georgia. Tortoise populations are clearly subdivided into at least eight genetic assemblages with an \(\bar{F}_{\rm ST}=0.24\pm 0.11\). Furthermore, we found indications of anthropogenic effects in the form of population bottlenecks in five populations and putative admixture in four. From these data, we recommend that the populations be managed to maintain existing genetic structure without further isolation of populations and the establishment of a holistic␣database to include genetic and demographic information useful for relocation and management purposes.

Genome interrelationship in the genusEleusine(Poaceae) as revealed through heteroploid crosses

Genome interrelationship in the genus Eleusine (Poaceae) has always been a matter of considerable in- terest for plant breeders whose major objective was to attain genetic improvement in E. coracana (finger millet), an important crop of Africa and south Asia. E. coracana and E. africana are the two major amphidiploid species of Eleu- sine with AABB genome (2n=4x=36). In attempt to identify the A and B genome donor species, heteroploid crosses were made involving these two tetraploid species and four diploid species viz. E. intermdia, E. indica, E. floccifolia, E. tristachya (all with 2n=2x=18). The resultant F1 hybrids were investigated for cytomorphological details. Male meio- sis studies in pollen mother cells showed regular occurrence of expected 9II+9I at diakinesis/ metaphase I in major- ity of the cells indicating the existence of complete and/or partial homology among crossing parents. The occurrence of occasional quadrivalents had apparently no bearing on the course of meiosis owing to their scant number. Ana- phase I and II were characterized by unequal distribution and micronuclei leading to drastic reduction in pollen stainability (2-8%) and complete seed seterlity. Our observations suggest that E. indica with AA genome is the piv- otal donor species in the evolution of E. africana and E. coracana while E. intermedia (AimAim) and E. tristachya (AtAt) belong to A genomic group of diploid Eleusines. Further these three species form a close genetic assemblage within the genus Eleusine. The assumption that E.floccifolia is a definite B genome donor species is now widely accepted while E. tristachya might also be a candidate for BB genome is completely ruled out there by leaving the question wide open regarding identity of the other B genome donor species, if any.

Genome Analysis of Finger Millet E. coracana by Interspecific Hybridization among Diploid Wild Species of Eleusine (Poaceae)

Four species of Eleusine viz. E. intermedia, E. floccifolia, E. indica and E. tristachya are wild diploid taxa (2n=2x=18) and are presumed to have contributed to the evolution of finger millet, E. coracana, the amphidiploid with AABB genome (2n=2x=36) and an important minor millet grown in Africa and South Asia. E. indica is known to have contributed the A genome to E. coracana, while the information regarding the other genome donor species is not forthcoming. The present investigations were undertaken to clearly understand the genome homology of 3 species viz. E. intermedia, E. floccifolia and E. tristachya vis-a-vis the A genome of E. indica and to analyze the interspecific relationship among these diploid species and their possible contribution to the evolution of E. coracana. Interspecific F1 hybrids of E. intermedia×E. indica, E. intermedia×E. floccifolia and E. tristachya×E. intermedia were produced and studied for morphological and cytological details. The experimental data revealed a high degree of homology at the interspecific level among all 4 species resulting in high frequency of bivalents and a very low frequency of univalents and multivalents. Inspite of good chromosome pairing and bivalent formation, a significant reduction in pollen stainability (8–32%) has been recorded in the F1 hybrids. The data obtained on chromosome associations at diakinesis/ metaphase I of meiosis suggest that these diploid species form a close genetic assemblage within the genus Eleusine. Various factors affecting the pollen stainability and seed fertility are discussed in the light of available information on the subject.

Real-time PCR assay for detection and genotype differentiation of Giardia lamblia in stool specimens.

Real-time PCR, using dual-labeled fluorescent probes targeting the beta-giardin gene, was used to detect Giardia lamblia in human stool specimens and to discriminate between isolates from the two major genetic assemblages of G. lamblia infective to humans, assemblages A and B.

Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP

A PCR-RFLP genotyping tool was developed and used to characterise morphologically identical isolates of Giardia duodenalis from a variety of host species. Primers were designed to amplify a 432 bp region of the glutamate dehydrogenase gene (gdh) from genetic Assemblages AI, AII, BIII, BIV, C, D and E of G. duodenalis. DNA extracted from cultured Giardia trophozoites, Giardia cysts purified from faeces and directly from whole faeces was amplified and sequenced at the gdh and 18SrDNA loci. The gdh sequences were identical with published gdh sequences for each assemblage with a few exceptions. However, in some cases genotyping results obtained using gdh differed from 18SrDNA genotyping results. From gdh sequence information a PCR-RFLP profile was identified for each of the genetic assemblages. PCR-RFLP is a reproducible, reliable and sensitive method for genotyping Giardia. Eight human, 12 cat, 9 dog and 16 cattle faecal isolates were genotyped using PCR-RFLP. This method allows G. duodenalis isolates from human-beings, their companion animals and livestock to be genotyped directly from faeces, leading to valuable information about Giardia genotypes in population without the need for in vitro/in vivo amplification.

Genome Differentiation Patterns in Diploid Species of Elemine (Poaceae)

Three species of Eleusine viz. E. indica, E. tristachya (both annual), and E. floccifolia (perennial)are wild diploid taxa (x = 9) that display nine bivalents at diakinesis/metaphase I of meiosis and high pollen fertility and seed set. Interspecific F1 hybrids, E. indica ×E. floccifolia and E. tristachya ×E. indica, were analysed to investigate species genome affinity and crossing barriers. In E. indica ×E. floccifolia F1, the mean chromosome pairing was 0.38I + 8.78II + 0.008III + 0.004IV, and nearly 81% of the pollen mother cells contained nine bivalents, suggesting that genomes of E.indica and E. floccifolia are homologous/similar. Despite the regular meiosis, F1s were seed sterile. In E. tristachya × E. indica F1, the mean chromosome pairing was 0.191 8.87II % 0.0IIV, and about 89% of the pollen mother cells exhibited nine bivalents, and the F1s were partially fertile, indicating that genomes of E. tristachya and E. indica are also largely homologous. These pieces of cytogenetic information suggest that these three diploid species form a close genetic assemblage within the genus Eleusine. Barriers for gene exchange between these taxa, in nature, are discussed considering the available literature.

Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP

A PCR-RFLP genotyping tool was developed and used to characterise morphologically identical isolates of Giardia duodenalis from a variety of host species. Primers were designed to amplify a 432 bp region of the glutamate dehydrogenase gene ( gdh) from genetic Assemblages AI, AII, BIII, BIV, C, D and E of G. duodenalis. DNA extracted from cultured Giardia trophozoites, Giardia cysts purified from faeces and directly from whole faeces was amplified and sequenced at the gdh and 18SrDNA loci. The gdh sequences were identical with published gdh sequences for each assemblage with a few exceptions. However, in some cases genotyping results obtained using gdh differed from 18SrDNA genotyping results. From gdh sequence information a PCR-RFLP profile was identified for each of the genetic assemblages. PCR-RFLP is a reproducible, reliable and sensitive method for genotyping Giardia. Eight human, 12 cat, 9 dog and 16 cattle faecal isolates were genotyped using PCR-RFLP. This method allows G. duodenalis isolates from human-beings, their companion animals and livestock to be genotyped directly from faeces, leading to valuable information about Giardia genotypes in population without the need for in vitro/in vivo amplification.

Genetic homogeneity of axenic isolates of Giardia intestinalis derived from acute and chronically infected individuals in Mexico.

Twenty-six axenic isolates of Giardia intestinalis, established in Mexico City over an 11-year period from symptomatic and asymptomatic individuals with acute or chronic infections, were typed genetically. A segment of the glutamate dehydrogenase gene was amplified by PCR and examined by restriction analysis using BspH1 and ApaI to determine the major genetic assemblages to which the isolates belonged. This was coupled with the amplification and analysis of segments of variant-specific surface protein genes to determine genetic subgroupings. Despite their heterogeneous clinical backgrounds, the isolates were found to be genetically homogeneous-all belonging to genetic group I of assemblage A. The results show that type A-I G. intestinalis is ubiquitous in Mexico City and that host factors play an important, if not dominant, role in determining the clinical outcome of Giardia infections in humans.