Genetic divergence of Toxoplasma gondii strains associated with ocular toxoplasmosis, Brazil.Previous studies have shown a high prevalence of toxoplasmosis Toxoplasmosis Definition
Toxoplasmosis is an infectious disease caused by the one-celled protozoan parasite Toxoplasma gondii. Although most individuals do not experience any symptoms, the disease can be very serious, and even fatal, in and the frequent occurrence of ocular disease in Brazil. To identify the genotypes of parasite strains associated with ocular disease, we compared 25 clinical and animal isolates of Toxoplasma gondii Tox·o·plas·ma gon·di·i
A sporozoan species that is an intracellular parasite in a variety of vertebrates and is the causative agent of toxoplasmosis. from Brazil to previously characterized clonal lineages from North America and Europe. Multilocus nested polymerase chain reaction Nested polymerase chain reaction is a modification of polymerase chain reaction intended to reduce the contaminations in products due to the amplification of unexpected primer binding sites. analysis was combined with direct sequencing of a polymorphic polymorphic - polymorphism intron Intron
In split genes, a portion that is included in ribonucleic acid (RNA) transcripts but is removed from within a transcript during RNA processing and is rapidly degraded. to classify strains by phylogenetic phy·lo·ge·net·ic
1. Of or relating to phylogeny or phylogenetics.
2. Relating to or based on evolutionary development or history. methods. The genotypes of T. gondii strains isolated from Brazil were highly divergent when compared to the previously described clonal lineages. Several new predominant genotypes were identified from different regions of Brazil Brazil is currently divided in five regions, by the Instituto Brasileiro de Geografia e Estatistica (IBGE). These divisions are composed by states with similar cultural, economical, historical and social aspects, and although through the scientific point of view information given by this , including 2 small outbreaks attributable to foodborne or waterborne infection. These findings show that the genetic makeup of T. gondii is more complex than previously recognized and suggest that unique or divergent genotypes may contribute to different clinical outcomes of toxoplasmosis in different localities.
Toxoplasma gondii is an obligate intracellular parasite Obligate intracellular parasites are parasitic microorganisms that cannot reproduce outside their host cell, forcing the host to assist in the parasite's reproduction.
Obligate intracellular parasites of humans include:
1. Having no evident sex or sex organs; sexless.
2. cycle and sexual cycle; the asexual cycle occurs in a wide range of intermediate hosts, and the sexual cycle occurs exclusively in feline hosts, which shed infectious oocysts in their feces (1). T. gondii is mainly transmitted by ingesting cysts contained within tissues of a chronically infected host or by ingesting sporulated oocysts from fecally contaminated contaminated,
v 1. made radioactive by the addition of small quantities of radioactive material.
2. made contaminated by adding infective or radiographic materials.
3. an infective surface or object. food or water (2). T. gondii is an influential foodborne pathogen foodborne pathogen Public health A pathogen–especially bacteria, for which the 'vector' is itself a food. See Airline food. in the United States (3) and a frequent cause of waterborne infection in parts of Brazil (4,5).
Despite having a sexual phase in its life cycle, the population structure of T. gondii is markedly clonal (6). Most strains analyzed from North America and Europe belong to 1 of 3 clonal lineages known as types I, II, and III (7-9). A small number (<5%) of isolates contain different combinations of the same alleles seen in the clonal types, which indicates that recombination recombination, process of "shuffling" of genes by which new combinations can be generated. In recombination through sexual reproduction, the offspring's complete set of genes differs from that of either parent, being rather a combination of genes from both parents. occurs infrequently in the wild (7). Additionally, strains with more divergent genotypes have been isolated from locations such as French Guiana (10).
The serologic se·rol·o·gy
n. pl. se·rol·o·gies
1. The science that deals with the properties and reactions of serums, especially blood serum.
2. prevalence of T. gondii infection in Brazil includes 50%-80% of the adult population, with the highest values found in northern and southern states (5). Waterborne transmission has been implicated im·pli·cate
tr.v. im·pli·cat·ed, im·pli·cat·ing, im·pli·cates
1. To involve or connect intimately or incriminatingly: evidence that implicates others in the plot.
2. in high rates of T. gondii seropositivity Seropositivity is the presence of a certain antibody in a blood sample. A patient with seropositivity for a particular antigen or agent is termed seropositive. in northern Rio de Janeiro Rio de Janeiro, city, Brazil
Rio de Janeiro (rē`ō də zhänā`rō, Port. rē` thĭ zhənĕē`r State (5) and in a toxoplasmosis outbreak in Santa Isabel do Ivai in southern Paranfi (4). High levels of ocular disease are associated with toxoplasmosis in Brazil (11). In the southern city of Erechim, Brazil, 184 (17.7%) of 1,042 adults were found to have retinal scars, thought to be caused by toxoplasmosis (12). Epidemiologic data indicate that many cases of ocular disease are acquired after birth rather than congenitally (13-15). Whether the increased prevalence and severity of ocular toxoplasmosis in Brazil are attributable to host or parasite genetic factors or differences in exposure rate is uncertain.
Polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is (PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) )-based typing at the SAG2 locus has been used previously to suggest that type I strains predominate in Brazilian patients with ocular toxoplasmosis (16). While the SAG2 marker provides accurate genotyping for most strains within the clonal lineages, it cannot detect recombinant strains or those with unusual genotypes (17). In fact, the exclusive use of any single locus may misrepresent mis·rep·re·sent
tr.v. mis·rep·re·sent·ed, mis·rep·re·sent·ing, mis·rep·re·sents
1. To give an incorrect or misleading representation of.
2. the genotype of recombinant or unusual genotypes as having a simple genotype. This problem is partially alleviated by multilocus analysis, and random amplified polymorphic DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. (RAPD RAPD Randomly Amplified Polymorphic DNA
RAPD relative afferent pupillary defect (ophthalmology; aka Marcus-Gunn Pupil) )-PCR analysis of Brazilian T. gondii strains with multiple markers showed that most strains contain both type I and III alleles at different loci loci
[L.] plural of locus.
loci Plural of locus, see there (18). However, PCR-based markers, such as restriction fragment length polymorphism restriction fragment length polymorphism
n. Abbr. RFLP
Intraspecies variations in the length of DNA fragments generated by the action of restriction enzymes and caused by mutations that alter the sites at which these enzymes act, changing (RFLP RFLP
restriction fragment length polymorphism
restriction fragment length polymorphism.
RFLP ) or RAPD, underestimate the true rate of nucleotide divergence and thus may not accurately classify T. gondii strains from new regions. For example, a high degree of polymorphism polymorphism, of minerals, property of crystallizing in two or more distinct forms. Calcium carbonate is dimorphous (two forms), crystallizing as calcite or aragonite. Titanium dioxide is trimorphous; its three forms are brookite, anatase (or octahedrite), and rutile. is detected at the GRA GRA Graphic Arts
GRA Grande Raccordo Anulare (circular highway surrounding Rome, Italy)
GRA Graduate Research Assistant
GRA Georgia Research Alliance
GRA Graduate Research Assistantship
GRA Guyana Revenue Authority 6 locus by sequence analysis (9 allelic al·lele
One member of a pair or series of genes that occupy a specific position on a specific chromosome.
[German Allel, short for Allelomorph, allelomorph, from English sequences among 30 strains), whereas PCR-RFLP PCR-RFLP Polymerase Chain Reaction–Restriction Fragment Length Polymorphism analysis differentiates only 3 groups among these same strains (19).
We have recently described a sensitive method for multilocus genotyping consisting of nested PCR (nPCR) amplification of 4 different RFLP markers (SAG2, GRA6, SAG3, and BTUB). When combined with sequencing of the UPRT-1 intron, multilocus nPCR typing provides a robust means to classify strains as having clonal, recombinant, or novel genotypes (17). Multilocus nPCR analysis also can detect as few as 5 parasite genomes and thus is applicable to low-volume samples containing few parasites, as is typical of clinical specimens (1 7). In this study, we examined a group of Brazilian T. gondii strains from animal and human sources, including several outbreaks, to examine the population structure of T. gondii in Brazil.
Patients were examined at Clinica Silveira, Erechim, Rio Grande do Sul Rio Grande do Sul (rē` grän`dĭ th s State, by indirect ophthalmoscopy ophthalmoscopy /oph·thal·mos·co·py/ (of?thal-mos´kah-pe) examination of the eye by means of the ophthalmoscope.
medical ophthalmoscopy that performed for diagnostic purposes. or biomicroscopy by using a slit-lamp microscope slit-lamp microscope Ophthalmology A low-power microscope fitted with a specialized illuminating system for generating collimated light, used to examine the anterior eye–conjunctiva, sclera, cornea, iris, lens, anterior chamber, and to visualize ocular fluids . Ocular disease was evaluated on the basis of parameters described previously (11). Patient consent was obtained at the time of sample acquisition. Recently acquired toxoplasmosis was confirmed by serologic tests that monitor immunoglobulin G immunoglobulin G
n. Abbr. IgG
The most abundant class of antibodies found in blood serum and lymph and active against bacteria, fungi, viruses, and foreign particles. Immunoglobulin G antibodies trigger action of the complement system. (IgG) and IgM by enzyme-linked immunosorbent assay enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay (ELISA)
A diagnostic blood test used to screen patients for AIDS or other viruses. (Abbott Laboratories, Abbott Park, IE, USA) (11). Ocular toxoplasmosis was diagnosed on the basis of recurrent episodes of necrotizing necrotizing /nec·ro·tiz·ing/ (nek´ro-tiz?ing) causing necrosis.
Causing the death of a specific area of tissue. Human bites frequently cause necrotizing infections. retinochoroiditis. Venous blood venous blood
n. Abbr. v
Blood that has passed through the capillaries of various tissues other than the lungs, is found in the veins, in the right chambers of the heart, and in pulmonary arteries, and is usually dark red as a result of a was collected before treatment in Vacutainer tubes containing heparin and cells, and serum was separated by centrifugation Centrifugation
A mechanical method of separating immiscible liquids or solids from liquids by the application of centrifugal force. This force can be very great, and separations which proceed slowly by gravity can be speeded up enormously in centrifugal . The buffy-coat layer was removed, frozen at -20[degrees]C, and shipped to Washington University, Saint Louis, for analysis. Blood was also obtained from patients infected during 2 small outbreaks of toxoplasmosis in Santa Vitoria do Palmar, Rio Grande do Sul State, and Agronomica, Santa Catarina State, and processed in a similar manner. Buffy-coat samples were processed by using the DNAeasy tissue extraction kit (Qiagen Inc., Valencia, CA, USA) before PCR analysis.
Parasite Strains and Tissue Samples
Reference strains consisted of representative members of the 3 clonal lineages originally isolated from human or animal infections in North America and Europe. Reference strains for the clonal types included: 4 type I strains: ENT ENT ears, nose, and throat (otorhinolaryngology).
ear, nose, and throat
ear, nose and throat.
ENT Ears, nose & throat; formally, otorhinolaryngology (ATCC ATCC American Type Culture Collection, see there 50850), RH (ATCC 50838), GT1, and VEL VEL - LISP70 (ATCC 50852); 3 type II strains: Me49 (ATCC 50840), DEG (ATCC 50855), and PIH PIH
prolactin-inhibitory hormone. (ATCC 50857); and 3 type III strains: CTG CTG Cartridge
CTG Center for Technology in Government (SUNY, Albany, New York)
CTG Center for Technology in Government
CTG Computer Task Group (IT consulting company; Buffalo, NY, USA) (ATCC 50842), STRL STRL Sea Trial , and VEG (ATCC 50861). In addition, 3 previously reported strains with more divergent genotypes were included: CAST (ATCC 50868), COUG COUG Chicago Oracle Users Group , and MAS (ATCC 50870) (20). Parasite strains were grown in human fibroblast fibroblast /fi·bro·blast/ (fi´bro-blast)
1. an immature fiber-producing cell of connective tissue capable of differentiating into chondroblast, collagenoblast, or osteoblast.
2. cells, harvested after natural egress See ingress. from host cells and purified as above; cell lysates were prepared for PCR as described previously (17). Five Brazilian T. gondii strains isolated from Belo Horizonte, Minas Gerais, and one from Sao Paulo, as described previously (18), were also included.
Additional samples of porcine porcine /por·cine/ (por´sin) pertaining to swine.
pertaining to pig. See also hog (1), swine.
porcine circovirus 1
a nonpathogenic virus. tissue collected from abattoirs in the Erechim region were included (R.N. Belfort, unpub, data). Six samples were analyzed, 2 negative controls and 4 samples that were positive by PCR (data not shown). Tissue samples were extracted with DNAzol, followed by an equal volume of chloroform chloroform (klôr`əfôrm) or trichloromethane (trī'klôrōmĕth`ān), CHCl3 . Polyacryl carrier (Molecular Research Center, Inc., Cincinnati, OH, USA) was added (5 [micro]L) to the aqueous phase aqueous phase
The water portion of a system consisting of two liquid phases, one that is primarily water and a second that is a liquid immiscible with water. , and DNA was precipitated by adding an equal volume of ethanol and centrifugation at 5,000 x g for 10 min.
Genotyping Isolates by PCR-RFLP
Multilocus nPCR analysis of 4 different loci was based on the markers 5'-SAG2, 3'-SAG2, BTUB, GRA6, and SA G3 (17). Amplification was performed as described previously (20), and negative controls consisted of sterile, distilled water or proteinase proteinase /pro·tein·ase/ (pro´ten-as?) endopeptidase.
A protease that begins the hydrolytic breakdown of proteins usually by splitting them into polypeptide chains. K-treated cell lysate ly·sate
The cellular debris and fluid produced by lysis. of noninfected host cells. The amplified products were digested with appropriate restriction enzymes for different loci, and the resulting fragments were analyzed by 3% agarose gel electrophoresis Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA, RNA, or protein molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). , stained with ethidium bromide, and imaged by an Alpha Imager version 5.5 camera (Alpha Innotech Corp., San Leandro, CA, USA).
Restriction fragments were scored visually as present or absent, and a genetic distance matrix was calculated from the proportion of shared restriction sites by using the equation of Nei and Li (21). The neighbor-joining method was used to analyze the distance matrix, and dendrograms were generated by using the phylogenetic analysis program PAUP PAUP Phylogenetic Analysis Using Parsimony * version 4.0b (22). Bootstrap See boot.
(operating system, compiler) bootstrap - To load and initialise the operating system on a computer. Normally abbreviated to "boot". From the curious expression "to pull oneself up by one's bootstraps", one of the legendary feats of Baron von Munchhausen. analysis was conducted for 1,000 replicates to obtain confidence estimates for the taxonomic groupings. The conditions were set to distance, neighbor-joining, with mean character differences, and dendrograms were constructed by using the 50% majority rule.
UPRT-1 Intron Sequences Analysis
Sequence divergence among strains of T. gondii was determined at the uracil uracil (yr`əsĭl), organic base of the pyrimidine family. It was isolated from herring sperm and also produced in a laboratory in 1900–1901. phosphoribosyl transferase transferase /trans·fer·ase/ (trans´fer-as) a class of enzymes that transfer a chemical group from one compound to another.
n. (UPRT UPRT Uracil Phosphoribosyltransferase
UPRT University of Pécs Roundtable (Pecs, Hungary conference) ) intron 1 sequence (GenBank accession no. AY143141), as described previously (17). Following the previously described nPCR amplification of the UPRT-1 intron, a third set of internal primers was used for sequencing: UPRT-1 seqF 5'-CTCGTCCTCGTTTTCCTT-3' and UPRT-1 seqR 5'-TGAAAGGAAGCACGTAAAGT-3'. Sequencing was conducted on 3 independent PCR-amplified templates by using BigDye cycle sequencing (Applied Biosystems, Foster City, CA, USA) (conducted by SeqWright DNA Technology Services, Houston, TX, USA). ClustalX/W (23) was used to align the sequences for comparison with default settings. After removal of primer sequences, the UPRT-1 intron sequence used for comparison was 467 bp in length. Unrooted phylogenetic comparisons were conducted with distance and parsimony par·si·mo·ny
1. Unusual or excessive frugality; extreme economy or stinginess.
2. Adoption of the simplest assumption in the formulation of a theory or in the interpretation of data, especially in accordance with the rule of methods by using PAUP* 4.0b (22). The conditions were set to distance (mean character difference, minimal evolution, negative branches = 0), and 1,000 bootstrap replicates were performed by using the BioNeighbor-Joining algorithm. Alternatively, parsimony analysis was conducted by heuristic stepwise stepwise
incremental; additional information is added at each step.
stepwise multiple regression
used when a large number of possible explanatory variables are available and there is difficulty interpreting the partial regression searching, with bootstrapping Bootstrapping
A procedure used to calculate the zero coupon yield curve from market figures.
Since the T-bills offered by the government are not available for every time period, the bootstrapping method is used to fill in the missing figures in order to derive the for >1,000 replicates. Consensus trees were drawn with an arbitrary root according to the bootstrap 50% majority rule.
Brazil has a high prevalence of ocular toxoplasmosis, and many of these cases are recurrent and serious in nature (11,12). This situation prompted us to consider whether sampling patient blood might allow diagnosis of recent (acute) or recurrent infection by direct PCR amplification. Blood was collected from 77 patients seen at the Clinica Silveira, Erechim, Brazil, from 2003 to 2005, and the buffy-coat that contained leukocytes was separated by centrifugation and used for analysis. Nested PCR analysis of these samples by using the SAG3 gene showed that 11 of 77 were positive, including 6 patients with acute disease and 5 patients with recurrent disease (Table) (locations of the patients with positive samples are shown in Figure 1). We also analyzed several sets of samples from 2 small outbreaks of acute toxoplasmosis. The first in Santa Vitoria do Palmar consisted of 10 persons from a single family that shared a meal of home-cured sausage that contained pork. Symptoms in infected persons included lymphadenitis Lymphadenitis Definition
Lymphadenitis is the inflammation of a lymph node. It is often a complication of a bacterial infection of a wound, although it can also be caused by viruses or other disease agents. , myalgia myalgia /my·al·gia/ (mi-al´jah) muscular pain.myal´gic
epidemic myalgia see under pleurodynia.
n. , fever, headache, and sweating. One of these patients, a 53-year-old woman, had severe retinochoroiditis. Only a single sample from these 10 persons was positive by SAG3 nPCR analysis of buffy-coat cells. A second outbreak consisted of 8 infected persons from Agronomica, a town of [approximately equal to] 4,000 residents located 200 km from Florianopolis. These 8 persons shared the same source of nontreated water in a common neighborhood, and their illness was likely caused by waterborne infection. Three of these persons had positive results by SA G3 nPCR.
[FIGURE 1 OMITTED]
To determine the genotype of T. gondii strains present in clinical samples from Brazil, multilocus nPCR was conducted by using 4 independent markers, SAG2, BTUB, GRA6, and SAG3, as described previously (17). The 15 clinical samples found to be positive for SAG3 were genotyped for most of these markers, although in some cases, insufficient material was available to type all markers (Table). We compared these isolates to strains previously characterized from Brazil and to the clonal lineages common in North America and Europe. In total, 38 strains were subjected to multilocus nPCR analysis, and after restriction digestion and gel electrophoresis of the products, the strains were classified on the basis of the alleles present relative to the reference strains (Table, Figure 2) (17). Three of the ocular toxoplasmosis samples carried alleles characteristic of type I strains at 3 or more independent markers, and 2 Brazilian chicken strains possessed alleles typical of type III strains at all loci. All of the remaining Brazilian samples had genotypes consisting of different combinations of alleles seen in the clonal types. Nine clinical samples, including samples from Agronomica from 2 outbreaks, possessed the same profile that consisted of alleles typical of type I and type II lineages. The nPCR assay used here can detect both alleles equally well for all the makers studied, yet in no case were 2 alleles detected at a single locus within a single strain (data not shown). Consequently, the genotypes observed in Brazilian isolates cannot be explained by "mixtures" of >1 strain in a given patient or sample.
[FIGURE 2 OMITTED]
The percentage of nucleotide divergence between strains was estimated from the proportion of shared restriction sites at each locus, and a distance matrix was used to construct a dendrogram A dendrogram is a tree diagram frequently used to illustrate the arrangement of the clusters produced by a clustering algorithm (see cluster analysis). Dendrograms are often used in computational biology to illustrate the clustering of genes. by using neighbor-joining analysis (Figure 3). All the strains belonging to type II were clustered together with a high degree of confidence. All type III strains and 2 chicken strains from Brazil were grouped together with a similarly high confidence. The 4 type I reference strains and 3 human Brazilian clinical isolates (ER8, ER11, and AG2) were clustered together. However, most Brazilian T. gondii strains were clustered into 2 new groups that were intermediate between types I and III. These results suggest the presence of at least 2 additional haplotypes that are prevalent in Brazil and which differ from North America and European lineages. MAS, which was isolated from a patient with a congenital case of toxoplasmosis in France, clustered with 1 of these Brazilian haplotypes.
[FIGURE 3 OMITTED]
To more accurately assess genetic divergence, we characterized the strains by UPRT-1 intron sequencing, a method that is highly sensitive for detecting divergent strains (17). UPRT-1 intron sequences from 35 strains (1 clinical sample was not available in sufficient quantity for analysis, and 2 samples gave unsatisfactory sequence quality) were aligned by using Clustal X (online Appendix Figure, available at http://www.cdc.gov/ncidod/EID/ vol12no06/06-0025-appG.htm), and the relative divergence of different Brazilian strains was determined by phylogenetic comparison. The results of parsimony and distance analysis were similar and the neighbor-joining distance analysis is shown in Figure 4. Because of the strongly biallelic pattern of T. gondii, types II and III are identical at the UPRT locus, while type I possess a unique haplotype haplotype /hap·lo·type/ (-tip) the group of alleles of linked genes, e.g., the HLA complex, contributed by either parent; the haploid genetic constitution contributed by either parent.
n. distinguished by 6 single nucleotide polymorphisms (17,20). Most Brazilian T. gondii strains (13 of 22) shared a new allele allele (əlēl`): see genetics.
Any one of two or more alternative forms of a gene that may occur alternatively at a given site on a chromosome. that was distinguished by 6 additional polymorphisms not seen in the clonal lineages (online Appendix Figure). This new Brazilian allele was also shared by the previously characterized divergent strain MAS (Figure 4). Additionally, 3 outbreak strains (AG 1, AG2, AG3) from Agronomica and 1 strain each from chickens (MG4) and pigs (P3) were found in this group that otherwise contained a majority of ocular toxoplasmosis isolates from the Erechim region. Other strains from Brazil contained equally divergent but unique alleles that in some cases formed smaller groups (i.e., Pl, P2, P4 and MG1, ER4) (Figure 4). Only a single Brazilian strain (ER8) contained a haplotype characteristic of 1 of the clonal lineages, and this strain was identical to the type I lineage in both the PCR-RFLP and UPRT-1 intron trees.
[FIGURE 4 OMITTED]
T. gondii is highly prevalent in Brazil, where human infection is associated with an unusually high occurrence of ocular disease in some locations. We examined the genotype of T. gondii strains collected from a variety of sources in southern Brazil. Included in this study were a group of patients seen at an eye clinic in Erechim, a region known for high levels of ocular toxoplasmosis (11). We also examined strains from several small outbreaks from nearby regions and compared these strains to animal isolates from Erechim and the more central region of Minas Gerais. Multilocus PCR-RFLP and sequenced-based analysis showed that they differ substantially from the previously described clonal lineages and instead define several new haplotypes that appear to be predominant in Brazil. The abundance of genotypes that do not fit the conventional classification shows the global pattern of T. gondii population structure to be more complex than previously thought. These findings have implications for the transmission of T. gondii as a waterborne and foodborne human pathogen and for studies on the role of genetic composition in virulence, pathogenesis, and life cycle dynamics.
Most human infections of T. gondii are not clinically severe and progress rapidly to a chronic state that is characterized by semidormant tissue cysts (2,24). During the chronic infection, parasites are generally not found in circulation, and obtaining parasites without performing invasive procedures such as tissue biopsy is relatively difficult. Previous reports have suggested that parasites may be found circulating in blood during reactivation reactivation
to become active after a period of quiescence or, as in bacterial and viral infections, latency.
cross reactivation of toxoplasmosis in AIDS patients (25,26). Our studies show that by using highly sensitive and specific nPCR, small numbers of parasites may be detected in circulating blood from some patients with either acute-onset or recurrent ocular toxoplasmosis. False-positive PCR amplification did not appear to be a substantial problem, as shown by consistently negative results for water and host-cell-only samples and the fact that the genotypes of clinical strains did not resemble common laboratory strains that would be the likely source of any contamination.
Genotyping T. gondii strains found in clinical and animal samples from Brazil showed that all strains except 1 (ER8) had different genotypes from clonal lineages that predominate in North America and Europe. When analyzed by multilocus PCR-RFLP, these new South American genotypes initially appeared to be composed of different combinations of alleles seen in the clonal types, similar to findings of a previous report from Brazil (18). This pattern could indicate that Brazilian strains of T. gondii undergo more frequent sexual recombination, resulting in mixed genotypes. However, the true extent of sequence divergence is not captured by multilocus RFLP analysis. We have previously shown that direct sequencing of introns from housekeeping genes provides a more accurate picture of sequence divergence (17,20). Introns are also likely to be selectively neutral and therefore well suited for phylogenetic comparisons (27). In the present study, when the UPRT-1 intron sequence was compared, all strains from Brazil except 1 (ER8) had multiple additional polymorphisms not seen in the clonal lineages. This locus indicates a low genetic diversity in T. gondii strains in Brazil, although they include genotype(s) uncommon in North America and Europe. Both the RFLP and intron analysis indicate several predominant haplotypes in Brazil, along with less common unique genotypes. Further studies will be necessary to define the population structure of T. gondii in Brazil and other South American locations.
The high seropositivity to T. gondii (11,28,29), combined with unusually high levels of ocular disease in some regions, shows that toxoplasmosis is a notable health problem in Brazil. Previous studies have shown a high prevalence of T. gondii in food animals such as pigs (30) and chickens (31) and in companion animals such as dogs (32) and cats (33). Although companion animals are not typically a source of human infection, the high prevalence in these species indicates a high level of transmission in Brazil. A recent survey of pig samples obtained from abattoirs in the Erechim region indicated a high prevalence of T. gondii (35%-66% positive by PCR) (R.N. Belfort, unpub, data). Previous studies have shown a high level of recurrent ocular disease from this region, where 17.7% adults were found to have retinal scars, likely due to toxoplasmosis (11,12). In addition, drinking unfiltered Please wikify (format) this article or section as suggested in the Guide to layout and the Manual of Style.
Remove this template after wikifying. This article has been tagged since water has been associated with an increased risk of T. gondii seropositivity in north Rio de Janeiro State, Brazil (5).
Collectively, these epidemiologic features suggest that infection with T. gondii in Brazil is more likely to lead to serious ocular disease, even in otherwise healthy persons. The extent to which host genetics, immune status, and exposure rate contribute to this pattern is unknown. However, an obvious difference is the markedly different genetic makeup of Brazilian strains of T. gondii. Previous studies of recurrent ocular toxoplasmosis in patients in the United States have also shown an elevated frequency of unusual genotypes (34). Although small animal models have been used for evaluating virulence traits of T. gondii strains (6), comparisons have not yet been made between North American North American
named after North America.
North American blastomycosis
see North American blastomycosis.
North American cattle tick
see boophilusannulatus. and South American strains in terms of their potential to cause ocular disease.
We have previously advocated using SAG2 for genotyping T. gondii strains, since it is capable of distinguishing all 3 clonal genotypes at a single locus (35). This approach works well in North America and Europe, where the 3 major lineages predominate because of extreme linkage disequilibrium linkage disequilibrium
The nonrandom association between two or more alleles such that certain combinations of alleles are more likely to occur together on a chromosome than other combinations of alleles. (7). Our current findings indicate that most strains from Brazil do not fit the clonal pattern seen in North America. Additionally, T. gondii strains isolated from French Guiana are also genetically distinct from the clonal lineages seen in North America (10). Consequently, studies that rely solely on SAG2 typing will necessarily underrepresent un·der·rep·re·sent
tr.v. un·der·rep·re·sent·ed, un·der·rep·re·sent·ing, un·der·rep·re·sents
To imply or suggest a lower amount, quantity, quality, or degree of than is actually present: the true genetic divergence in many regions. SAG2 typing has been used for genotyping T. gondii isolates from various animals in Brazil (30-33,36), other parts of South America (37,38), and Africa (39,40). Researchers also recently suggested that strains associated with an outbreak of waterborne toxoplasmosis in Parana, Brazil, were type I strains, based solely on genotyping with the SAG2 marker (4). However, analyses based solely on SAG2 almost certainly underestimate the genetic diversity of T. gondii in these regions. Further strain comparisons based on a wider set of sequence-based markers will be necessary to define the global population structure of T. gondii and to resolve the relationships between major strain types seen in different regions. Establishing the population structure of T. gondii is highly relevant to transmission dynamics because the suggestion has been made that recently derived clonal lineages arose through a process of recombination that led to enhanced asexual oral transmission (20). Whether other, more divergent strains also express this trait and to what extent their genetic makeup contributes to transmission are highly relevant to understanding the pathogenesis of toxoplasmosis.
We thank J.P. Dubey, Tovi Lehmann, Chunlei Su, Ben Rosenthal, Michael Grigg, Jim Ajioka, and Blima Fux for helpful comments and discussions and Julie Suetterlin for expert technical assistance.
This study was supported by a grant from the National Institutes of Health (AI059176).
Dr Khan is a postdoctoral research associate at Washington University. His research interests include molecular epidemiology molecular epidemiology Molecular medicine An evolving field that combines the tools of standard epidemiology–case studies, questionnaires and monitoring of exposure to external factors with the tools of molecular biology–eg, restriction endonucleases, and microbial microbial
pertaining to or emanating from a microbe.
the breakdown of organic material, especially feedstuffs, by microbial organisms. pathogenesis.
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(4.) Moura L, Bahia-Oliveira LMG LMG Light Machine Gun
LMG Laurence M. Gould (Antarctic Research Support Vessel, USAP)
LMG Local Marketing Group
LMG Loaf's Merry Guild
LMG Laboratory Molecular Genetics
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Address for correspondence: L. David Sibley, Washington University Medical School, 660 S Euclid Ave, St. Louis, MO 63130, USA; email: email@example.com
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Asis Khan, * Catherine Jordan, * Cristina Muccioli, ([dagger]) Adriana L. Vallochi, ([double dagger]) Luiz V. Rizzo, ([double dagger]) Rubens Belfort Jr, ([dagger]) Ricardo W.A. Vitor, ([section]) Claudio Silveira, ([paragraph]) and L. David Sibley *
* Washington University School of Medicine Washington University School of Medicine, located in St. Louis, Missouri, is one of the most competitive and highly regarded medical schools and biomedical research institutes in the United States. , St. Louis, Missouri, USA; ([dagger]) Paulista School of Medicine, Sao Paulo, Brazil; ([double dagger]) University of Sao Paulo, Sao Paulo, Brazil; ([section]) Federal University of Minas Gerais, Belo Horizonte, Brazil; and ([paragraph]) Clinica Silveira, Erechim, Brazil
Table. Genotypes of Toxoplasma gondii in human ocular toxoplasmosis samples from Brazil * Strain Type of name sample Source ENT C (#) Human RH C Human GT1 C Goat VEL C Human Me49 C Sheep DEG C Human PIH C Human CTG C Cat STRL C Human VEG C Human CAST C Human COUG C Cougar MAS C Human PBR C Dog D3 C Dog CH1 C Chicken CH2 C Chicken EFP C Human SAF C Human 6T A ([dagger][dagger]) Porcine 7T A Porcine 8T A Porcine 9T A Porcine 2147 Cl ([double dagger][double dagger]) Recurrent ocular 2324 Cl Acute ocular 2296 Cl Acute ocular 2323 Cl Acute ocular 2434 Cl Recurrent ocular 2325 Cl Acute ocular 2566 Cl Recurrent ocular 2583 Cl Acute ocular 2612 Cl Recurrent ocular 2670 Cl Recurrent ocular 2728 Cl Acute ocular 2694 Cl Outbreak 2712 Cl Outbreak 2717 Cl Outbreak 2719 Cl Outbreak Locus Strain name 5'-SAG2 ([dagger]) 3'-SAG2 ([double dagger]) ENT 1 ** 1 RH 1 1 GT1 1 1 VEL 1 1 Me49 1 2 DEG 1 2 PIH 1 2 CTG 2 1 STRL 2 1 VEG 2 1 CAST 1 1 COUG 1 2 MAS 1 1 PBR 1 1 D3 1 1 CH1 2 1 CH2 2 1 EFP 1 1 SAF 1 1 6T 1 1 7T 1 1 8T 1 1 9T 1 1 2147 1 1 2324 1 1 2296 1 1 2323 1 1 2434 1 1 2325 1 1 2566 1 2 2583 1 1 2612 1 -- 2670 1 -- 2728 1 1 2694 1 2 2712 1 -- 2717 1 -- 2719 1 1 Locus Strain name BTUB ([section]) SAG3 GRA6 ENT 1,1 1 1 RH 1,1 1 1 GT1 1,1 1 1 VEL 1,1 1 1 Me49 2,2 2 2 DEG 2,2 2 2 PIH 2,2 2 2 CTG 2,1 3 3 STRL 2,1 3 3 VEG 2,1 3 3 CAST 1,1 1 1 COUG 2,2 3 2 MAS 1,1 3 3 PBR 1,1 3 3 D3 1,1 3 3 CH1 1,1 3 3 CH2 1,1 3 3 EFP 1,1 3 3 SAF 1,1 3 3 6T 1,1 3 3 7T 1,1 3 3 8T 1,1 3 2 9T 1,1 3 3 2147 1,1 2 1 2324 1,1 2 1 2296 1,1 2 1 2323 1,1 2 1 2434 1,1 2 1 2325 1,1 3 1 2566 1,1 3 -- ([section][section]) 2583 1,1 1 -- 2612 1,1 2 -- 2670 1,1 2 -- 2728 1,1 1 -- 2694 1,1 3 -- 2712 1,1 2 -- 2717 1,1 1 -- 2719 1,1 2 1 Locus Strain name Genotype Reference ([paragraph]) Location ENT I ENT F RH I RH USA-OH GT1 I GT1 USA-MD VEL I VEL USA-CA Me49 II Me49 USA-CA DEG II DEG F PIH II PIH USA-CA CTG III CTG USA-NIH STRL III STRL USA-CA VEG III VEG USA-CA CAST I CAST USA-CA COUG I/II/III COUG CAN-BC MAS I/III MAS F PBR I/III MG1 SP D3 I/III MG2 MG CH1 III MG3 MG CH2 III MG4 MG EFP I/III MG5 MG SAF I/III MG6 MG 6T I/III P1 EC 7T I/III P2 EC 8T I/II/III P3 EC 9T I/III P4 EC 2147 I/II ER1 ER 2324 I/II ER2 ER 2296 I/II ER3 ER 2323 I/II ER4 EC 2434 I/II ER5 ER 2325 I/III ER6 ER 2566 I/II/III ER7 ER 2583 I ER8 ER 2612 I/II ER9 ER 2670 I/II ER10 EC 2728 I ER11 EC 2694 I/II/III SV SV 2712 I/II AG1 AG 2717 I AG2 AG 2719 I/II AG3 AG * C, culture; A, animal; Cl, clinical; F, France; CA, California; NIH, National Institutes of Health; CAN-BC, British Columbia, Canada; SP, Sao Paulo; MG, Belo Horizonte, Minas Gerais; EC, Erechim City; ER, Erechim region; SV, Santa Vitttoria do Palmar; AG, Agronomica. ([dagger]) Genotypes I and II are the same. ([double dagger]) Genotypes I and III are the same. ([section]) Alleles represent BsEl and Taql, respectively. ([paragraph]) As referred in figures. (#) Culture strains used as reference. ** Alleles defined by pattern in type I strain = 1, second allele = 2; allele 3 is defined by the presence of a second biallelic polymorphism. ([dagger][dagger]) Meat tissue samples, primary source. ([double dagger][double dagger]) Human ocular toxoplasmosis. ([section][section]) Refers to negative amplification product.