Development of a natural molluscicide prototype kit (MoluSchall) for the control of schistosomiasis mansoni transmission.

INTRODUCTION: In Brazil, Biomphalaria glabrata, B. tenagophila, and B. straminea are intermediate hosts of Schistosoma mansoni, the etiological agent of schistosomiasis mansoni. Molluscicide use is recommended by the WHO for controlling the transmission of this parasite. Euphorbia milii latex has shown promising results as an alternative molluscicide. Thus, a natural molluscicide prototype kit based on freeze-dried E. milii latex was developed and evaluated against Biomphalaria spp. METHODS: E. milii latex was collected, processed, and lyophilized. Two diluents were defined for freeze-dried latex rehydration, and a prototype kit, called MoluSchall, was produced. A stability test was conducted using prototype kits stored at different temperatures, and a toxicity assay was performed using Danio rerio. Additionally, MoluSchall was tested against B. glabrata under semi-natural conditions according to defined conditions in the laboratory. RESULTS: MoluSchall was lethal to three Brazilian snail species while exhibiting low toxicity to D. rerio. Regardless of storage temperature, MoluSchall was stable for 24 months and was effective against B. glabrata under semi-natural conditions, with the same LD100 as observed under laboratory conditions. CONCLUSIONS: MoluSchall is a natural, effective, and inexpensive molluscicide with lower environmental toxicity than existing molluscicides. Its production offers a possible alternative strategy for controlling S. mansoni transmission.

Evaluation of post-mortem diagnostic tests' sensitivity and specificity for bovine tuberculosis using Bayesian latent class analysis.

This study aimed to evaluate the performance of real-time PCR (qPCR), ELISA IDEXX™, and bacterial isolation as post-mortem diagnostic tests in animals with lesions compatible with bovine tuberculosis detected by Brazilian Federal Inspection Service as part of the bovine tuberculosis active surveillance. Bayesian latent class models were used to estimate diagnostic tests' sensitivity, specificity, correlations, predictive values and frequency of infected animals. Samples of tuberculosis-suggestive lesions collected by FIS sanitary inspection routine in slaughterhouses from 11 Brazilian states were analyzed. Isolation was the most sensitive technique, 94.54% (95% Credible Interval (CrI) 90.09%-97.65%), qPCR was 64.69% (95% CrI 54.41%-74.15%) sensitive and ELISA IDEXX™ 26.74% (95% CrI 22.82%-30.97%). Tests' specificities were 98.19% (95% CrI 95.75%-99.45%), 93.49% (95% CrI 79.28%-99.66%), 95.53% (95% CrI 91.71%-98.02%) respectively. Despite its low sensitivity, ELISA IDEXX™ was able to identify positive samples that were not detected by the other techniques. These samples had high probability to be true positives given ELISA's positive predictive value. The correlations between qPCR and isolation were neither biologically nor statistically significant. The low sensitivity of the qPCR is a limiting factor to its use as a post-mortem diagnosis in bovine tuberculosis suggestive lesions. Its use could be recommended in situations of high prevalence, or in parallel association with other tests, such as ELISA IDEXX™. ELISA IDDEX™ should not be used as a unique test, or in substitution of the other tests, for the post-mortem diagnosis of bovine tuberculosis due to its sensitivity.

Development of a natural molluscicide prototype kit (MoluSchall) for the control of schistosomiasis mansoni transmission

Abstract INTRODUCTION In Brazil, Biomphalaria glabrata, B. tenagophila, and B. straminea are intermediate hosts of Schistosoma mansoni, the etiological agent of schistosomiasis mansoni. Molluscicide use is recommended by the WHO for controlling the transmission of this parasite. Euphorbia milii latex has shown promising results as an alternative molluscicide. Thus, a natural molluscicide prototype kit based on freeze-dried E. milii latex was developed and evaluated against Biomphalaria spp. METHODS E. milii latex was collected, processed, and lyophilized. Two diluents were defined for freeze-dried latex rehydration, and a prototype kit, called MoluSchall, was produced. A stability test was conducted using prototype kits stored at different temperatures, and a toxicity assay was performed using Danio rerio. Additionally, MoluSchall was tested against B. glabrata under semi-natural conditions according to defined conditions in the laboratory. RESULTS MoluSchall was lethal to three Brazilian snail species while exhibiting low toxicity to D. rerio. Regardless of storage temperature, MoluSchall was stable for 24 months and was effective against B. glabrata under semi-natural conditions, with the same LD100 as observed under laboratory conditions. CONCLUSIONS MoluSchall is a natural, effective, and inexpensive molluscicide with lower environmental toxicity than existing molluscicides. Its production offers a possible alternative strategy for controlling S. mansoni transmission.

Molecular Typing of Mycobacterium bovis from Cattle Reared in Midwest Brazil.

Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), the pathogen responsible for serious economic impact on the livestock sector. In order to obtain data on isolated M. bovis strains and assist in the control and eradication program for BTB, a cross sectional descriptive molecular epidemiology study in the Brazilian Midwest was conducted. Through spoligotyping and 24-loci MIRU-VNTR methods, 37 clinical isolates of M. bovis circulating in the region were analyzed, 10 isolated from the state of Mato Grosso, 12 from the state of Mato Grosso do Sul and 15 from the state of Goiás. The spoligotyping analysis identified 10 distinct M. bovis profiles (SB0121 n = 14, SB0295 n = 6, SB0140 n = 6, SB0881 n = 3, SB1144 n = 2, SB1145 n = 2, SB0134 n = 1, SB1050 n = 1, SB1055 n = 1, SB1136 n = 1) grouped in six clusters and four orphan patterns. The MIRU-VNTR 24-loci grouped the same isolates in six clusters and 22 unique orphan patterns, showing higher discriminatory power than spoligotyping. When associating the results of both techniques, the isolates were grouped in five clusters and 24 unique M. bovis profiles. Among the 24-loci MIRU-VNTR evaluated, two, ETR-A and QUB 11b loci, showed high discriminatory ability (h = ≥ 0.50), while MIRU 16, MIRU 27, ETR-B, ETR-C, Mtub21 and QUB 26 loci showed moderate ability (h = 0.33 or h = 0.49) and were the most effective in evaluating the genotypic similarities among the clinical M. bovis isolate samples. Herein, the 29 patterns found amongst the 37 isolates of M. bovis circulating in the Brazilian Midwest can be due to the animal movement between regions, municipalities and farms, thus causing the spread of various M. bovis strains in herds from Midwest Brazil.

Draft Genome Sequence of Mycobacterium bovis Strain AN5, Used for Production of Purified Protein Derivative.

Mycobacterium bovis strain AN5 has been used to produce purified protein derivative (PPD) for the intradermal test for bovine tuberculosis since it was introduced in 1948. This work reports the draft genome sequence of M. bovis AN5, which is used for the production of bovine PPD in Brazil, as well as comparisons to other strains of M. bovis and Mycobacterium tuberculosis.

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil.

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.

Spoligotyping and variable number tandem repeat analysis of Mycobacterium bovis isolates from cattle in Brazil.

We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.

Spoligotyping and variable number tandem repeat analysis of Mycobacterium bovis isolates from cattle in Brazil

We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.

Evaluation of adenosine deaminase seric activity in the diagnosis of bovine tuberculosis.

Determination of seric levels of adenosine deaminase (ADA), an enzyme produced by monocytes/macrophages and lymphocytes, has been used in the diagnosis of human tuberculosis (TB). In the present study, ADA seric activity was evaluated comparatively to the comparative tuberculin test in the diagnosis of bovine tuberculosis. Two hundred fifty-six cattle were classified by origin and by the comparative tuberculin test as TB-positive animals (n = 52, from herds where the Mycobacterium bovis had previously been isolated), and TB-negative animals (n = 204, TB-free herds). The mean ADA seric value from the TB-positive group (4.45 +/- 2.33 U/L) was significantly lower (p = 0.008) than that observed in sera from the TB-negative group (6.12 +/- 4.47 U/L). When animals from a herd with clinical cases of enzootic bovine leukosis of TB-negative group were withdrawn from analysis, the mean ADA seric values of TB-negative group (5.12 +/- 3.75 U/L) was not significantly different anymore from that of the TB-positive group (p = 0.28). There was no agreement in the diagnosis of bovine TB between comparative tuberculin test and determination of ADA seric values, using two different cutoff points, being 6.12 U/L and 15.0 U/L, (kappa = -0.086 and kappa = -0.082, respectively). In conclusion, the determination of ADA seric activity was not a good auxiliary test for bovine TB, because it was not able to distinguish between TB-positive and TB-negative animals.

Evaluation of adenosine deaminase seric activity in the diagnosis of bovine tuberculosis

Determination of seric levels of adenosine deaminase (ADA), an enzyme produced by monocytes/macrophages and lymphocytes, has been used in the diagnosis of human tuberculosis (TB). In the present study, ADA seric activity was evaluated comparatively to the comparative tuberculin test in the diagnosis of bovine tuberculosis. Two hundred fifty-six cattle were classified by origin and by the comparative tuberculin test as TB-positive animals (n = 52, from herds where the Mycobacterium bovis had previously been isolated), and TB-negative animals (n = 204, TB-free herds). The mean ADA seric value from the TB-positive group (4.45 ± 2.33 U/L) was significantly lower (p = 0.008) than that observed in sera from the TB-negative group (6.12 ± 4.47 U/L). When animals from a herd with clinical cases of enzootic bovine leukosis of TB-negative group were withdrawn from analysis, the mean ADA seric values of TB-negative group (5.12 ± 3.75 U/L) was not significantly different anymore from that of the TB-positive group (p = 0.28). There was no agreement in the diagnosis of bovine TB between comparative tuberculin test and determination of ADA seric values, using two different cutoff points, being 6.12 U/L and 15.0 U/L, (kappa = -0.086 and kappa = -0.082, respectively). In conclusion, the determination of ADA seric activity was not a good auxiliary test for bovine TB, because it was not able to distinguish between TB-positive and TB-negative animals.