Evidence for reductive genome evolution and lateral acquisition of virulence functions in two Corynebacterium pseudotuberculosis strains

Background

Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity.

Methodology and Findings

We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer.

Conclusions

These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers {"type":"entrez-nucleotide","attrs":{"text":"CP001809","term_id":"340539261","term_text":"CP001809"}}CP001809 and {"type":"entrez-nucleotide","attrs":{"text":"CP001829","term_id":"302205157","term_text":"CP001829"}}CP001829.     

Epidemiological surveillance of birth defects compatible with thalidomide embryopathy in Brazil

The thalidomide tragedy of the 1960s resulted in thousands of children being born with severe limb reduction defects (LRD), among other malformations. In Brazil, there are still babies born with thalidomide embryopathy (TE) because of leprosy prevalence, availability of thalidomide, and deficiencies in the control of drug dispensation. Our objective was to implement a system of proactive surveillance to identify birth defects compatible with TE. Along one year, newborns with LRD were assessed in the Brazilian hospitals participating in the Latin-American Collaborative Study of Congenital Malformations (ECLAMC). A phenotype of LRD called thalidomide embryopathy phenotype (TEP) was established for surveillance. Children with TEP born between the years 2000-2008 were monitored, and during the 2007-2008 period we clinically investigated in greater detail all cases with TEP (proactive period). The period from 1982 to 1999 was defined as the baseline period for the cumulative sum statistics. The frequency of TEP during the surveillance period, at 3.10/10,000 births (CI 95%: 2.50-3.70), was significantly higher than that observed in the baseline period (1.92/10,000 births; CI 95%: 1.60-2.20), and not uniformly distributed across different Brazilian regions. During the proactive surveillance (2007-2008), two cases of suspected TE were identified, although the two mothers had denied the use of the drug during pregnancy. Our results suggest that TEP has probably increased in recent years, which coincides with the period of greater thalidomide availability. Our proactive surveillance identified two newborns with suspected TE, proving to be a sensitive tool to detect TE. The high frequency of leprosy and the large use of thalidomide reinforce the need for a continuous monitoring of TEP across Brazil.     

Development and evaluation of a new lateral flow immunoassay for serodiagnosis of human fasciolosis

Background

Human fasciolosis is a re-emerging disease worldwide and is caused by species of the genus Fasciola (F. hepatica and F. gigantica). Human fasciolosis can be diagnosed by classical coprological techniques, such as the Kato-Katz test, to reveal parasite eggs in faeces. However, although 100% specific, these methods are generally not adequate for detection of acute infections, ectopic infections, or infections with low number of parasites. In such cases immunological methods may be a good alternative and are recommended for use in major hospitals where trained personnel are available, although they are not usually implemented for individual testing.

Methodology/Principal Findings

We have developed a new lateral flow test (SeroFluke) for the serodiagnosis of human fasciolosis. The new test was constructed with a recombinant cathepsin L1 from F. hepatica, and uses protein A and mAb MM3 as detector reagents in the test and control lines, respectively. In comparison with an ELISA test (MM3-SERO) the SeroFluke test showed maximal specificity and sensitivity and can be used with serum or whole blood samples.

Conclusions/Significance

The new test can be used in major hospitals in hypoendemic countries as well as in endemic/hyperendemic regions where point-of-care testing is required.     

Relationship between plant development, tannin concentration and insects associated with Copaifera langsdorffii (Fabaceae)

Plant development is the main factor that determines the insect-ontogeny interaction, since it leads to variations in resource quality and availability. The aim of this study was to test the hypothesis that plant development and varying tannin concentration leads to changes in species richness, abundance and composition of ants, free-feeding herbivores and galling insects associated with Copaifera langsdorffii (Fabaceae). The plant ontogeny and tannin concentration effects on insects were tested on 60 individuals with height varying from 0.9 to 11.0 m. A positive correlation was observed for tree height and species richness and abundance of ants, free-feeding and galling insects. In contrast, we did not find a significant relation between leaf tannin concentration and plant height, or richness and abundance of the different insect guilds. The assemblage of ants (composition of species) did not change between saplings and adults of C. langsdorffii. However, the assemblage of free-feeding herbivores and galling insects varied between the two development stages studied. The present study reveals an ontogenetic succession pattern for herbivore insects along the C. langsdorffii growth, probably due to both indirect and direct benefits from the host plant architecture and quality. Those plants with more complex architectures should support a wider diversity of insects, since they present higher number of sites for egg laying, housing, feeding and better environmental conditions. This is the first work to investigate the host plant ontogeny effect on insects in Cerrado “Savanna” vegetation. The pattern described, along with other previous studies, suggests a vast occurrence of ontogenetic succession in tropical areas.     

Reactive oxygen species production and mitochondrial dysfunction contribute to quercetin induced death in Leishmania amazonensis

Background

Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects more than 12 million people worldwide. Quercetin has generated considerable interest as a pharmaceutical compound with a wide range of therapeutic activities. One such activity is exhibited against the bloodstream parasite Trypanosoma brucei and amastigotes of Leishmania donovani. However, the mechanism of protozoan action of quercetin has not been studied.

Methodology/Principal Findings

In the present study, we report here the mechanism for the antileishmanial activity of quercetin against Leishmania amazonensis promastigotes. Quercetin inhibited L. amazonensis promastigote growth in a dose- and time- dependent manner beginning at 48 hours of treatment and with maximum growth inhibition observed at 96 hours. The IC₅₀ for quercetin at 48 hours was 31.4 µM. Quercetin increased ROS generation in a dose-dependent manner after 48 hours of treatment. The antioxidant GSH and NAC each significantly reduced quercetin-induced cell death. In addition, quercetin caused mitochondrial dysfunction due to collapse of mitochondrial membrane potential.

Conclusions/Significance

The effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. Quercetin has been described as a pro-oxidant, generating ROS which are responsible for cell death in some cancer cells. Mitochondrial membrane potential loss can be brought about by ROS added directly in vitro or induced by chemical agents. Taken together, our results demonstrate that quercetin eventually exerts its antileishmanial effect on L. amazonensis promastigotes due to the generation of ROS and disrupted parasite mitochondrial function.     

Nociceptive Response and Adenine Nucleotide Hydrolysis in Synaptosomes Isolated from Spinal Cord of Hypothyroid Rats

Purinergic system exerts a significant influence on the modulation of pain pathways at the spinal site. Adenosine has antinociceptive properties in experimental and clinical situations, while ATP exerts pronociceptive actions in different pain models. In this study we investigated the hydrolysis of ATP to adenosine in synaptosomes from spinal cord in parallel with the nociceptive response of rats at different ages after hypothyroidism induction. Hypothyroidism elicited a significant increase in AMP hydrolysis to adenosine in synaptosomes from spinal cord of rats subjected to neonatal hypothyroidism and in 420-day-old rats submitted to thyroidectomy. Accordingly, these rats presented an analgesic response as a consequence of hypothyroidism. In contrast, the ATP hydrolysis was decreased in the spinal cord of 60-day-old hypothyroid rats in parallel with a significant increase in nociceptive response. These results indicate the involvement of adenine nucleotides in the control of the hypothyroidism-induced nociceptive response during development.     

A method for testing the host specificity of ectoparasites: give them the opportunity to choose

Host-choice experiments were carried out with rodent and bat ectoparasites on Ilha Grande, state of Rio de Janeiro, Brazil. We constructed experimental chambers that enclosed three different rodent or bat host species, and then introduced a selected set of ectoparasitic arthropods. When given the opportunity to choose among host species, the ectoparasites showed a strong tendency to select their primary hosts, and reject novel host species. These kinds of simple experiments can be valuable tools for assessing the ability of ectoparasites to locate and discern differences between host species, and make choices about which hosts to infest, and which hosts to avoid.     

Influence of high-intensity exercise training and anabolic androgenic steroid treatment on rat tissue glycogen content

To increase tissue glycogen content many athletes use anabolic androgenic steroids (AAS). However, the literature concerning the effects of androgens on glycogen metabolism is conflicting. This study aimed to determine the influence of training and AAS on body weight (bw), triglycerides, glucose, tissue glycogen and transaminases levels. Male Wistar rats, randomized into four groups (sedentary vehicle (SV), sedentary AAS (SA), trained vehicle (TV) and trained AAS (TA)), were treated with nadrolone (5 mg/Kg, 2x/week, i.m.) or vehicle. Trained rats performed jumps into water (4 sets, 10 repetitions, 30 sec rest) carrying a 50-70% body wt-load strapped to the chest (5 days/week,6 weeks). Two days after the last session, the animals were killed (bifatorial ANOVA+Tukey test; P < 0.05). Trained animals presented lower bw (TV:345+/-7 vs. SV:380+/-7 and TA:328+/-4 vs SA:370+/-11 g) and triglycerides levels (TV:77+/-3 vs. SV:98+/-4 and TA:79+/-3 vs. SA:98+/-8 mg/dL) and higher glycogen content in liver (TV:5.3+/-0.2 vs. SV:3.9+/-0.1 and TA:5.3+/-0.3 vs. SA:4.6+/-0,2 mg/100 mg) and in gastrocnemious (TV:0.70+/-0.02 vs. SV:0.49+/-0.01 and TA:0.73+/-0.03 vs. SA:0.57+/-0.02 mg/100 mg) than sedentary ones. In the cardiac muscle, the association between training and AAS increased glycogen content (TA:0.19+/-0.01 > SV:0.13+/-0.01=TV:0.13+/-0.01=SA:0.14+/-0.01 mg/100 mg). In the soleus AAS increased glycogen (SA:0.53+/-0.03 vs. SV:0.43+/-0.01 and TA:0.58+/-0.02 vs. TV:0.48+/-0.01 mg/100 mg). Exercise training and AAS had no effect on blood glucose and transaminases levels. Training and AAS effects on glycogen supercompensation are tissue-dependent and the effects of association between them were only observed in the cardiac muscle. These data emphasize the necessity of more studies to confirm greater effects of AAS than those promoted by physical exercise.     

BCG modulation of anaphylactic antibody response, airway inflammation and lung hyperreactivity in genetically selected mouse strains (Selection IV-A)

The effect of Bacillus Calmette-Guérin (BCG) treatment in allergic pulmonary reaction was studied in mice genetically selected accordingly to a High (H-IVA) or Low (L-IVA) antibody responsiveness. Mice were immunized with ovalbumin (OVA) or OVA plus BCG. Two days after nasal antigenic challenge, seric IgE and IgG1 anti-OVA, eosinophils in pulmonary tissue, inflammatory cells in bronchoalveolar lavage and the compliance and conductance of respiratory system were evaluated. H-IVA mice were found more susceptible than L-IVA, and BCG was able to inhibit simultaneously the production of IgE, the bronchopulmonary inflammation and bronchial hyperresponsiveness in these genetically selected mice.     

Adoption of shelter dogs in a Brazilian community: assessing the caretaker profile

A survey in Ibiuna, Sao Paulo, Brazil, of caregivers (owners) who adopted shelter dogs assessed length of ownership, proportion of male and female dogs adopted, and owners' characteristics. It addressed breeding, neutering, vaccination, and veterinary care. It used no testing to provide a good “match ”between dog and future owner. Of adopted dogs, 58% were male. Only 36% of owners were located. Mean ownership length was 14.8 months (95% confidence interval = 12.4 to 17.2 months), estimated through a survival analysis method. Of adopted dogs, 40.9% lived with their owners; 34.9% had died (some had lived on the streets); 15.0% were donated; 4.3% ran away; 3.2% were returned to the city shelter. Of interviewees, 57% reported no difficulties with the adoption; 23.1% cited the animal's illness and death as the main difficulty. For contraception, 87 owners (46.7%) chained dogs to prevent contact with other animals; 56.5% were against neutering. Reasons given were compassion (58.1%), unnecessary procedure (11.4%), cost (9.5%), and behavior change (4.8%). This research motivated a design for Ibiuna shelter dog adoption to improve the proportion of successful adoptions.