Alexander Primus

Evolution of a malaria resistance gene in wild primates

The ecology, behaviour and genetics of our closest living relatives, the nonhuman primates, should help us to understand the evolution of our own lineage. Although a large amount of data has been amassed on primate ecology and behaviour, much less is known about the functional and evolutionary genetic aspects of primate biology, especially in wild primates. As a result, even in well-studied populations in which nongenetic factors that influence adaptively important characteristics have been identified, we have almost no understanding of the underlying genetic basis for such traits. Here, we report on the functional consequences of genetic variation at the malaria-related FY (DARC) gene in a well-studied population of yellow baboons (Papio cynocephalus) living in Amboseli National Park in Kenya. FY codes for a chemokine receptor normally expressed on the erythrocyte surface that is the known entry point for the malarial parasite Plasmodium vivax. We identified variation in the cis-regulatory region of the baboon FY gene that was associated with phenotypic variation in susceptibility to Hepatocystis, a malaria-like pathogen that is common in baboons. Genetic variation in this region also influenced gene expression in vivo in wild individuals, a result we confirmed using in vitro reporter gene assays. The patterns of genetic variation in and around this locus were also suggestive of non-neutral evolution, raising the possibility that the evolution of the FY cis-regulatory region in baboons has exhibited both mechanistic and selective parallels with the homologous region in humans. Together, our results represent the first reported association and functional characterization linking genetic variation and a complex trait in a natural population of nonhuman primates.

Phenotypic and molecular characterization of a novel strongly hemolytic Brachyspira species, provisionally designated "Brachyspira hampsonii".

Since 2007, outbreaks of severe bloody diarrhea and hemorrhagic colitis have been reported in the United States and Canada. Though the primary causative agent of swine dysentery is Brachyspira hyodysenteriae, which is strongly hemolytic, the current report describes the isolation of a novel strongly hemolytic Brachyspira sp. This novel Brachyspira sp. was identified from clinical submissions at the Minnesota Veterinary Diagnostic Laboratory, and 40 of such isolates were obtained from 22 clinical submissions representing 5 states. Isolates were confirmed to be different from any known Brachyspira sp. on the basis of phylogenetic analysis of nucleotide sequences of nox and 16S ribosomal RNA (rRNA) genes. Phylogenetic analyses grouped all isolates into 2 clades (clades I and II), and grouping patterns were similar for both nox and 16S rRNA gene sequence analyses. Phenotypically, all isolates were indole and hippurate negative, and enzymatic profiling indicated 2 types of profiles, irrespective of the phylogenetic grouping, differing only in the production of β-glucosidase. The results suggest that a potentially virulent new species of Brachyspira sp., provisionally named “Brachyspira hampsonii ”, is circulating among swine herds in the United States.

Comparison of Cloacal and Oropharyngeal Samples for the Detection of Avian Influenza Virus in Wild Birds

Abstract This study was conducted to compare oropharyngeal (OP) and cloacal samples of wild birds (n  =  137) for the detection and isolation of avian influenza virus (AIV). A total of 39 (28.5%) cloacal and 85 (62.0%) OP samples were positive for AIV by real-time reverse transcription–PCR (RRT-PCR). The AIV nucleic acid was detected in both cloacal and OP samples from 27 (19.7%) birds, in cloacal samples only from 12 (8.8%) birds, and in OP samples only from 58 (42.3%) birds. Thus, a total of 97 (70.8%) birds were AIV positive by RRT-PCR. The cycle threshold values for the cloacal samples ranged from 16.6 to 36.9 (mean 31.5), and those for OP samples ranged from 18 to 38.9 (mean 34.9). Of the cloacal samples, 12 were positive for H5 subtype influenza virus by RRT-PCR, with one being low pathogenic H5N1. In contrast, five of the OP samples were H5 positive, but none was H5N1. None of the cloacal or OP samples was H7 positive. Eight cloacal samples yielded AIV on inoculation in embryonated chicken eggs, while only one isolate was obtained from OP samples. Thus, from testing of 137 birds, only nine (6.6%) AIV isolates were obtained. The isolates from cloacal samples were subtyped as H6N1 (n  =  5), H3N8 (n  =  2), and H4N8 (n  =  1), and the isolate from OP sample was subtyped as H6N1. No virus was isolated from the corresponding cloacal sample of the bird whose OP sample yielded AIV on virus isolation. These results suggest that surveillance programs for detection of AIV by RRT-PCR may include both sample types (cloacal and OP) to obtain a better picture of AIV prevalence, and OP samples may yield additional isolates of AIV when tested in conjunction with cloacal samples.

Functional consequences of genetic variation in primates on tyrosine hydroxylase (TH) expression in vitro

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, is known to contain naturally occurring genetic variation in its promoter region that associates with a number of neuropsychological disorders. As such, examining non-coding regions is important for understanding tyrosine hydroxylase function in human health and disease. We examined approximately 2 kb upstream of the translation start site within humans and non-human primates to obtain a fine resolution map of evolutionarily and functionally relevant cis-regulatory differences. Our study investigated Macaca mulatta, Pan troglodytes, Gorilla gorilla, and Homo sapiens haplotypes using transient dual-luciferase transfection in three neuroblastoma cell lines to assay the impact of naturally occurring sequence variation on expression level. In addition to trans effects between cell lines, there are several significant expression differences between primate species, but the most striking difference was seen between human haplotypes in one cell line. Underlying this variation are numerous sequence polymorphisms, two of which influence expression within humans in a non-additive and cell line-specific manner. This study highlights functional consequences of tyrosine hydroxylase genetic variation in primates. Additionally, the results emphasize the importance of examining more than one cell line, the existence of multiple functional variants in a given promoter region and the presence of non-additive cis-interactions.Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, is known to contain naturally occurring genetic variation in its promoter region that associates with a number of neuropsychological disorders. As such, examining non-coding regions is important for understanding tyrosine hydroxylase function in human health and disease. We examined approximately 2 kb upstream of the translation start site within humans and non-human primates to obtain a fine resolution map of evolutionarily and functionally relevant cis-regulatory differences. Our study investigated Macaca mulatta, Pan troglodytes, Gorilla gorilla, and Homo sapiens haplotypes using transient dual-luciferase transfection in three neuroblastoma cell lines to assay the impact of naturally occurring sequence variation on expression level. In addition to trans effects between cell lines, there are several significant expression differences between primate species, but the most striking difference was seen between human haplotypes in one cell line. Underlying this variation are numerous sequence polymorphisms, two of which influence expression within humans in a non-additive and cell line-specific manner. This study highlights functional consequences of tyrosine hydroxylase genetic variation in primates. Additionally, the results emphasize the importance of examining more than one cell line, the existence of multiple functional variants in a given promoter region and the presence of non-additive cis-interactions.

IDENTIFICATION OF FRANCISELLA NOATUNENSIS IN NOVEL HOST SPECIES FRENCH GRUNT (HAEMULON FLAVOLINEATUM) AND CAESAR GRUNT (HAEMULON CARBONARIUM)

Abstract:  Francisella noatunensis is an emerging pathogen of fish that has been isolated from several cultured species worldwide. Here presented is a case involving several hundred marine grunts that were caught near the Florida Keys for display in public aquaria. These fish were maintained in a recirculating system where they began to experience mortalities approximately two weeks post-stocking. Postmortem examination revealed disseminated systemic granulomatous disease most severely affecting spleen and kidney. Splenic and renal tissue homogenates inoculated in modified Thayer Martin agar media yielded colonies consistent with F. noatunensis 4 days post inoculation. Bacterial colonies and tissues were confirmed positive after real-time PCR amplification of the intracellular growth loci gene (iglC) specific for F. noatunensis subspecies orientalis. Consequently, multiple novel host species for this pathogen were identified, including the French grunt (Haemulon flavolineatum) and the Caesar grunt (Haemulon carbonarium).

Evolution and development: Wnts in deep time.

A recent sea anemone study reveals that the genes controlling body plan development evolved much earlier than previously thought. Evolutionary and developmental biologists often use comparative data on extant organisms to reconstruct morphological and developmental phenotypes of ancestral forms. It is the nature of the developmental mechanisms that may have been used by the earliest metazoans that is particularly controversial. In a report recently published in Nature, Kusserow and colleagues (2005) have taken this approach to reconstruct the developmental mechanisms that were functioning in the last common ancestor of bilatarians and cnidarians (a Precambrian form which gave rise to nearly all multicellular animals). Based on their investigation of the sea anemone Nematostella vectensis, these authors conclude that a large family of Wnt genes evolved much earlier than previously thought and played a crucial role in the diversification of metazoan body plans. The Wnt genes encode a family of signaling proteins that mediate a variety of critical intercellular signaling events in animal development and adult tissue homeostasis. Secreted Wnt proteins act on target cells by binding cell surface receptors belonging to the Frizzled family. These receptors activate one of at least three different intracellular pathways: the ‘canonical Wnt/b-catenin’ pathway, the ‘Wnt/planar polarity’ pathway and the ‘Wnt/Ca2þ ’ pathway (reviewed by Veeman et al, 2003; Logan and Nusse, 2004). All major animal phyla, with the possible exception of Porifera (sponges), have Wnt genes and most Wnt genes can be placed in one of 12 subfamilies. The number of Wnt subfamilies varies between representative species from different animal phyla: Drosophila has seven, Caenorhabditis has five and humans have 11. In a phylogenetic analysis of Wnt genes from representative species in all three main divisions of bilaterally symmetrical animals – the deuterostomes, ecdysozoans and lophotrochozoans – Prud’homme’s group (2002) concluded that at least nine of these Wnt subfamilies were present in the common ancestor of all bilaterians. They ascribed the smaller number of Wnt subfamilies in representatives with less than nine subfamilies to gene loss during evolution. At the time they did this work, only one Wnt gene was known for a representative of the Phylum Cnidaria; it had been discovered in Hydra where Hobmayer et al (2000) showed that the Wnt signaling pathway played a causal role in bud formation during asexual reproduction. More recently, Kusserow et al (2005) isolated 12 Wnt genes from another cnidarian, Nematostella. A phyogenetic analysis of these genes with those from deuterostome, ecdysozoan and lophotrochozoan representatives indicate that the Wnt genes present in Nematostella belong to 11 different subfamilies. Kusserow and his co-workers argue that an expanded Wnt gene family was a feature of the common ancestor that gave rise to cnidarians and bilaterians, and that an expanded Wnt family was not a bilaterian novelty. To gain insight into the developmental roles that these Wnt genes might have, Kusserow and his co-workers examined the expression profiles of many of these genes. Before describing their results, it is worth nothing that most species in the phylum Cnidaria, including Nematostella, are radially symmetrical animals with only two germ layers: ectoderm and entoderm. The expression of many Wnt genes in Nematostella begins near the onset and site of gastrulation. One subset of Wnt genes is expressed primarily in the future ectoderm while the other is expressed primarily in the entoderm, at different distances from the region that would become the future mouth of the juvenile. As Wnt signaling plays a role in gastrulation and axial differentiation in a number of other animals (reviewed by Miller and Moon, 1996), the expression patterns of these Wnts and a number of other transcriptional regulators in Nematostella (Wikramanayake et al, 2003; Martindale et al, 2004) led these authors to three conclusions. Firstly, that Wnt genes play distinct roles in gastrulation and axial differentiation in Nematostella; secondly, that the ancestor of bilaterians and cnidarians possessed a blastoporal signaling center (mediated by Wnts) that gave rise to gastrulation-based regional specification in bilaterians; and lastly, that specific Wnt genes of that ancestral signaling center played roles in the evolution of mesoderm and neural patterning in bilatarians. While the findings of Kusserow et al (2005) are intriguing, there are several things that one should keep in mind about the conclusions these authors have reached. Although the expression pattern of Wnt genes in Nematostella may be suggestive, a more conclusive analysis of their functions during development requires experimental approaches. Moreover, assessing the developmental functions of genes based solely on expression patterns in animals where very little is known about the development of that animal (as is the case in Nematostella) can be misleading. For example, in vertebrates, nerve cells originate from the ectoderm; in Hydra, nerve cells originate from multipotent stem cells (interstitial cells) that initially form in the entoderm (reviewed by David and Hager, 1994). If neurogenesis proceedes in Nematostella as it does in Hydra, which seems likely based on their phylogenetic proximity, then implications of Wnt function in vertebrate neural patterning and homology of Wnt expression patterns between Nematostella and vertebrates may not be applicable. Lastly, demonstrating true developmental homology – homology at the level of both molecular and developmental function (Wray and Abouheif, 1998) – between signaling molecules in highly disparate taxa can be difficult, even if the developmental function of these molecules has been verified experimentally in your taxa-of-interest. One reason for this is that there are a limited number of signaling pathways that are used during development, and in the course of metazoan evolution, these pathways have been co-opted for new developmental functions many times (Gerhart and Kirschner, 1997). It would be surprising if this is not the case for Wnts. A Primus and G Freeman are at the University of Texas at Austin, Austin, USA.