High chromosomal mobility of rDNA clusters in holocentric chromosomes of Triatominae, vectors of Chagas disease (Hemiptera-Reduviidae).

The subfamily Triatominae (Hemiptera-Reduviidae) includes more than 150 blood-sucking species, potential vectors of the protozoan Trypanosoma cruzi, causative agent of Chagas disease. A distinctive cytogenetic characteristic of this group is the presence of extremely stable chromosome numbers. Unexpectedly, the analyses of the chromosomal location of ribosomal gene clusters and other repetitive sequences place Triatominae as a significantly diverse hemipteran subfamily. Here, we advance the understanding of Triatominae chromosomal evolution through the analysis of the 45S rDNA cluster chromosomal location in 92 Triatominae species. We found the 45S rDNA clusters in one to four loci per haploid genome with different chromosomal patterns: On one or two autosomes, on one, two or three sex chromosomes, on the X chromosome plus one to three autosomes. The movement of 45S rDNA clusters is discussed in an evolutionary context. Our results illustrate that rDNA mobility has been relatively common in the past and in recent evolutionary history of the group. The high frequency of rDNA patterns involving autosomes and sex chromosomes among closely related species could affect genetic recombination and the viability of hybrid populations, which suggests that the mobility of rDNA clusters could be a driver of species diversification.

High Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) prevalence in Triatoma sanguisuga (Hemiptera: Redviidae) in southeastern Louisiana.

From May through November 2007, intensive weekly surveys at the site of a previously reported autochthonous human case of Chagas parasite infection resulted in the collection of 298 Triatoma sanguisuga (Leconte) specimens, of which 60.4% (180) were polymerase chain reaction positive for Trypanosoma cruzi Chagas. All were adults, in a ratio of approximately 1:1 female to male, indicating that the domicile was not colonized, but was a destination for these host-seeking adults. We report on seasonal activity pattern, T. cruzi prevalence in T. sanguisuga, and attempts at insect exclusion and control at the case residence.

High genetic diversity in a single population of Triatoma sanguisuga (LeConte, 1855) inferred from two mitochondrial markers: Cytochrome b and 16S ribosomal DNA.

Chagas disease remains endemic across much of Latin America, but is largely enzootic--restricted to wild mammals and triatomine vectors in the United States. Within the United States, there are ten recognized species of triatomines and 18 mammals reported naturally infected with Trypanosoma cruzi, the causative agent of Chagas disease. However, only six cases of autochthonous vector-borne transmission of T. cruzi to humans have been reported in the United States. As a follow-up to the sixth reported case, triatomine insects were collected from the index case site, in a rural area of New Orleans, LA, USA. During the summer months of 2006 and 2007, 344 Triatoma sanguisuga were collected and showed a T. cruzi infection prevalence of 56%. A subset of these insects was analyzed to infer intraspecific genetic variation from a 606 bp fragment of cytochrome b (n=54) and a 340 bp fragment of 16S ribosomal DNA (n=17). From the 54 samples, 37 cytb haplotypes (H(d)=0.978) were observed and 14.7% of positions were polymorphic. Phylogenetic analysis divides the population into two distinct groups with an average pairwise genetic distance of ~5%. The 16S rDNA sequences revealed 6 haplotypes among 17 specimens (H(d)=0.713) with 1.2% of the positions exhibiting polymorphisms. 16S polymorphism data support the concept of two groups within this single population. The genetic distance of Group 1 from Group 2 suggests that Group 1 could represent a sub-species as this level of divergence is similar to that observed among other triatomine subspecies.

Comparison of polymerase chain reaction on fresh tissue samples and fecal drops on filter paper for detection of Trypanosoma cruzi in Rhodnius prolixus.

PCR detection of Trypanosoma cruzi in Rhodnius prolixus using fresh tissue or fecal drops on filter paper showed comparable results: 38.7% infection rate using the fresh tissue sample and 37.9% by dried fecal drop.

Comparison of polymerase chain reaction on fresh tissue samples and fecal drops on filter paper for detection of Trypanosoma cruzi in Rhodnius prolixus

PCR detection of Trypanosoma cruzi in Rhodnius prolixus using fresh tissue or fecal drops on filter paper showed comparable results: 38.7 percent infection rate using the fresh tissue sample and 37.9 percent by dried fecal drop

Utility of the polymerase chain reaction in detection of Trypanosoma cruzi in Guatemalan Chagas' disease vectors.

For effective control programs, accurate assessment of Trypanosoma cruzi infection in vectors is essential and has traditionally been performed by microscopic examination. For particular vectors and not others, polymerase chain reaction (PCR) analysis of fecal samples recently has been shown to be an effective means of detection. The sensitivities of the PCR and microscopy for detection of T. cruzi in different anatomic sites were compared in the two major vectors of Guatemala, Triatoma dimidiata and Rhodnius prolixus. Preliminary studies established that T. cruzi can be detected by the PCR in the presence of 90% T. rangeli. One hundred thirty-five vectors were collected, and samples were obtained from the rectum, intestines, and stomach and analyzed by microscopy and the PCR. For Triatoma dimidiata rectal samples, the PCR sensitivity (39.1% T. cruzi positive) and the microscopic sensitivity (24.6% positive) was not significantly different. However, in R. prolixus, the PCR proved significantly more sensitive than microscopy: 57.6% positive by PCR compared with 22.7% by microscopy. Rectal samples showed the highest rates of infection followed by intestine and stomach samples. However, 10.5% of the Rhodnius infections would have been missed if only the rectal sample had been analyzed. Thus, the PCR is significantly more sensitive than microscopy for detection of T. cruzi in R. prolixus. Analysis of anatomic sites in addition to the rectal sample may be necessary for accurate assessment of infection in particular vectors.

Inhibition of protein synthesis results in super-induction of procyclin (PARP) RNA levels.

Procyclin is an abundant surface antigen found exclusively on the procyclic forms of African trypanosomes. We are interested in the induction of procyclin gene expression during differentiation from bloodstream forms. We find that increased levels of procyclin RNA are evident as early as 15 min after triggering differentiation. The increase in procyclin RNA levels requires the temperature shift from 37 degrees C to 27 degrees C and is aided by addition of the tricarboxylic acid cycle intermediate cis-aconitate. Maximal induction is observed with a combination of three triggers of differentiation: citrate, cis-aconitate and the temperature shift. Protein synthesis does not appear to be required for induction of procyclin RNA during differentiation. In fact, addition of protein synthesis inhibitors results in super-induction of procyclin RNA levels, even under conditions where no induction is normally observed (i.e., at 37 degrees C in the absence of citrate and cis-aconitate). This super-induction was observed with four different protein synthesis inhibitors that affect different stages of translation. Thus, the accumulation of procyclin transcripts may be under the control of a negative regulator whose effective levels are reduced during differentiation from bloodstream to procyclic forms.

cis- and trans-acting regulation of gene expression of equine infectious anemia virus.

Deletion analysis of the equine infectious anemia virus long terminal repeat revealed that sequences responsive to virus-specific transactivation are located within the region spanning the transcriptional start site (-31 to +22). In addition, an active exon of a trans-acting factor (tat) was identified downstream of pol and overlapping env (nucleotides 5264 to 5461). Activation by tat is accompanied by an increase in the steady-state levels of mRNA directed by the equine infectious anemia virus long terminal repeat.

Characterization of equine infectious anemia virus long terminal repeat.

The long terminal repeats (LTRs) of equine infectious anemia virus (EIAV) were examined with respect to their ability to function as transcriptional promoters in various cellular environments. Nucleotide sequence analyses of the LTRs derived from two unique proviral clones revealed the requisite consensus transcription and processing signals. One of the proviruses possessed a duplication of a 16-base-pair sequence in the CCAAT box region of the LTR which was absent in the other provirus. To assess its functional activity, each LTR was coupled to the bacterial chloramphenicol acetyltransferase gene and transfected onto various cell lines, including matched cultures of EIAV-infected and uninfected cells. The levels of chloramphenicol acetyltransferase activity directed by the EIAV LTRs were between 250 and 900 times greater in EIAV-infected cells compared with their uninfected counterparts. Thus, EIAV expression appears to be activated by a virus-induced trans-activation phenomenon analogous to that recently shown to amplify expression of certain other lentiviruses.

Prognosis of asymptomatic ulcerating carotid lesions.

To determine the proper approach to asymptomatic carotid bifurcation ulcerated plaque (UP), 79 patients with 91 asymptomatic UPs were identified angiographically, and a 96% follow-up was obtained with a mean duration of three years. The cumulative stroke rate by life-table analysis was 1% at seven years. Sixty-three UPs in 55 patients were classified as small, and of these patients, transient ischemic attacks (TIAs) that were appropriate to the lesion developed in three and stroke in one (7% cumulative symptom rate). Twenty-four UPs in 21 patients were classified as large, and a TIA developed in one patient (9%), but no strokes were observed in this group. The cumulative mortality was 17% at three years and 52% at seven years. Life-table curves of several subgroups were compared and showed no significant differences in either stroke rate or mortality between any of these groups. On the basis of these data, and particularly the seven-year stroke rate of 1%, prophylactic carotid endarterectomy is not justified for asymptomatic carotid bifurcation ulcerations.