Micro-Addition of Silver to Copper: One Small Step in Composition, a Change for a Giant Leap in Biocidal Activity.

The use of copper as an antimicrobial agent has a long history and has gained renewed interest in the context of the COVID-19 pandemic. In this study, the authors investigated the antimicrobial properties of an alloy composed of copper with a small percentage of silver (Cu-0.03% wt.Ag). The alloy was tested against various pathogens, including Escherichia coli, Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, and the H1N1 virus, using contact exposure tests. Results showed that the alloy was capable of inactivating these pathogens in two hours or less, indicating its strong antimicrobial activity. Electrochemical measurements were also performed, revealing that the small addition of silver to copper promoted a higher resistance to corrosion and shifted the formation of copper ions to higher potentials. This shift led to a slow but continuous release of Cu2+ ions, which have high biocidal activity. These findings show that the addition of small amounts of silver to copper can enhance its biocidal properties and improve its effectiveness as an antimicrobial material.

Genetic parameters for various semen production and quality traits and indicators of male and female reproductive performance in Nellore cattle.

BACKGROUND: Given the economic relevance of fertility and reproductive traits for the beef cattle industry, investigating their genetic background and developing effective breeding strategies are paramount. Considering their late and sex-dependent phenotypic expression, genomic information can contribute to speed up the rates of genetic progress per year. In this context, the main objectives of this study were to estimate variance components and genetic parameters, including heritability and genetic correlations, for fertility, female precocity, and semen production and quality (andrological attributes) traits in Nellore cattle incorporating genomic information. RESULTS: The heritability estimates of semen quality traits were low-to-moderate, while moderate-to-high estimates were observed for semen morphological traits. The heritability of semen defects ranged from low (0.04 for minor semen defects) to moderate (0.30 for total semen defects). For seminal aspect (SMN_ASPC) and bull reproductive fitness (BULL_FIT), low (0.19) and high (0.69) heritabilities were observed, respectively. The heritability estimates for female reproductive traits ranged from 0.16 to 0.39 for rebreeding of precocious females (REBA) and probability of pregnancy at 14 months (PP14), respectively. Semen quality traits were highly genetically correlated among themselves. Moderate-to-high genetic correlations were observed between the ability to remain productive in the herd until four years of age (stayability; STAY) and the other reproductive traits, indicating that selection for female reproductive performance will indirectly contribute to increasing fertility rates. High genetic correlations between BULL_FIT and female reproductive traits related to precocity (REBA and PP14) and STAY were observed. The genetic correlations between semen quality and spermatic morphology with female reproductive traits ranged from -0.22 (REBA and scrotal circumference) to 0.48 (REBA and sperm vigor). In addition, the genetic correlations between REBA with semen quality traits ranged from -0.23 to 0.48, and with the spermatic morphology traits it ranged from -0.22 to 0.19. CONCLUSIONS: All male and female fertility and reproduction traits evaluated are heritable and can be improved through direct genetic or genomic selection. Selection for better sperm quality will positively influence the fertility and precocity of Nellore females. The findings of this study will serve as background information for designing breeding programs for genetically improving semen production and quality and reproductive performance in Nellore cattle.

Genome-Wide Association Analysis Reveals Novel Loci Related with Visual Score Traits in Nellore Cattle Raised in Pasture-Based Systems.

Body conformation traits assessed based on visual scores are widely used in Zebu cattle breeding programs. The aim of this study was to identify genomic regions and biological pathways associated with body conformation (CONF), finishing precocity (PREC), and muscling (MUSC) in Nellore cattle. The measurements based on visual scores were collected in 20,807 animals raised in pasture-based systems in Brazil. In addition, 2775 animals were genotyped using a 35 K SNP chip, which contained 31,737 single nucleotide polymorphisms after quality control. Single-step GWAS was performed using the BLUPF90 software while candidate genes were identified based on the Ensembl Genes 69. PANTHER and REVIGO platforms were used to identify key biological pathways and STRING to create gene networks. Novel candidate genes were revealed associated with CONF, including ALDH9A1, RXRG, RAB2A, and CYP7A1, involved in lipid metabolism. The genes associated with PREC were ELOVL5, PID1, DNER, TRIP12, and PLCB4, which are related to the synthesis of long-chain fatty acids, lipid metabolism, and muscle differentiation. For MUSC, the most important genes associated with muscle development were SEMA6A, TIAM2, UNC5A, and UIMC1. The polymorphisms identified in this study can be incorporated in commercial genotyping panels to improve the accuracy of genomic evaluations for visual scores in beef cattle.

Microencapsulation of DEET in Solid Lipid Microparticles: production, characterization and safety evaluation.

DEET is considered the gold standard for insect repellent products. However, it behaves as a strong skin permeant. DEET was encapsulated in Solid Lipid Microparticles (SLM) and characterized in terms of morphology, particle size, cytotoxicity and ex vivo permeation. The particles exhibited micrometric size with a spherical shape. In addition, we developed and validated an analytical method for DEET quantification by high performance liquid chromatography (HPLC), which was selective, linear, precise, accurate and robust. The toxicity test in cell culture of keratinocytes, fibroblasts and macrophages showed that the formulation did not present cytotoxicity. The SLM were able to decrease the skin permeation of DEET in relation to the free active in ethanol with gain in the safe. Microparticles were able to increase the skin retention of DEET, which can contribute to extend the time of repellent action. The results showed that Solid Lipid Microparticles are safe and promising topical formulation to insect bite prevention.

Genome-wide association study and pathway analysis for fat deposition traits in nellore cattle raised in pasture-based systems.

Genome-wide association study (GWAS) is a powerful tool to identify candidate genes and genomic regions underlying key biological mechanisms associated with economically important traits. In this context, the aim of this study was to identify genomic regions and metabolic pathways associated with backfat thickness (BFT) and rump fat thickness (RFT) in Nellore cattle, raised in pasture-based systems. Ultrasound-based measurements of BFT and RFT (adjusted to 18 months of age) were collected in 11,750 animals, with 39,903 animals in the pedigree file. Additionally, 1,440 animals were genotyped using the GGP-indicus 35K SNP chip, containing 33,623 SNPs after the quality control. The single-step GWAS analyses were performed using the BLUPF90 family programs. Candidate genes were identified through the Ensembl database incorporated in the BioMart tool, while PANTHER and REVIGO were used to identify the key metabolic pathways and gene networks. A total of 18 genomic regions located on 10 different chromosomes and harbouring 23 candidate genes were identified for BFT. For RFT, 22 genomic regions were found on 14 chromosomes, with a total of 29 candidate genes identified. The results of the pathway analyses showed important genes for BFT, including TBL1XR1, AHCYL2, SLC4A7, AADAT, VPS53, IDH2 and ETS1, which are involved in lipid metabolism, synthesis of cellular amino acids, transport of solutes, transport between Golgi Complex membranes, cell differentiation and cellular development. The main genes identified for RFT were GSK3ß, LRP1B, EXT1, GRB2, SORCS1 and SLMAP, which are involved in metabolic pathways such as glycogen synthesis, lipid transport and homeostasis, polysaccharide and carbohydrate metabolism. Polymorphisms located in these candidate genes can be incorporated in commercial genotyping platforms to improve the accuracy of imputation and genomic evaluations for carcass fatness. In addition to uncovering biological mechanisms associated with carcass quality, the key gene pathways identified can also be incorporated in biology-driven genomic prediction methods.

Matrix Assisted Laser Desorption Ionization-Time-of-Flight mass spectrometry identification of Mycobacterium bovis in Bovinae.

In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M. bovis from other members of the Mycobacterium tuberculosis complex with 100% specificity, 90.91% sensitivity and 91.25% reliability. MALDI-TOF MS methodology described herein provides successful identification of M. bovis within bovine/bubaline clinical samples, demonstrating its usefulness for bovine tuberculosis diagnosis in the future.

Survey of Salmonella spp. in beef meat for export at slaughterhouses in Brazil / Pesquisa de Salmonella spp. em carcaças bovinas durante o processamento em abatedouros-frigoríficos exportadores

The aim of the present study was to investigate the presence of Salmonella spp. in samples collected from beef meat at three points of the slaughter line (after skinning, washing and cooling) at three slaughterhouses in Brazil that export meat. Detection was based on ISO 6579:2002 and confirmed by PCR and qPCR. The isolates were typified using slide agglutination tests and PFGE. The antibiotic sensitivity profile was determined using the disk diffusion method. Contamination was detected in only one slaughterhouse. The overall frequency of contamination by Salmonella spp. was 6.7% of carcasses (6/90) and 2.6% of carcass surface samples (7/270). All isolates were confirmed by PCR and qPCR. The serological analysis and the PFGE showed a single profile: Typhimurium. The strains demonstrated 100% susceptibility to ampicillin, cefotaxime, ciprofloxacin, chloramphenicol, gentamicin and tetracycline. Positive carcasses after cooling pose a direct risk to consumers, since the meat is considered ready to be marketed after this process.(AU)

O objetivo deste trabalho foi investigar a presença de Salmonella spp. em amostras coletadas de carcaças de bovinos, em três pontos da linha de abate (após a esfola, lavagem e refrigeração) de três frigoríficos exportadores no Brasil. A detecção foi realizada pela ISO 6579:2002, e confirmada por PCR e qPCR. Os isolados foram tipificados por testes de soroaglutinação e PFGE e avaliado o perfil de sensibilidade aos antibióticos pelo método de difusão em disco. A contaminação foi detectada em apenas um abatedouro-frigorífico. As contaminações das carcaças (n=90) e amostras de carne (n=270) por Salmonella spp. foram 6 (6,7%) e 7 (2,6%), respectivamente. Todos os isolados foram confirmados por PCR e qPCR. A análise sorológica e o PFGE mostraram um único perfil: Typhimurium. As cepas apresentaram 100% de suscetibilidade à ampicilina, cefotaxima, ciprofloxacina, cloranfenicol, gentamicina e tetraciclina. As carcaças positivas após a refrigeração apresentam um risco direto para o consumidor, uma vez que, após este processo, a carne está pronta para ser comercializada.(AU)

Survey of Salmonella spp. in beef meat for export at slaughterhouses in Brazil / Pesquisa de Salmonella spp. em carcaças bovinas durante o processamento em abatedouros-frigoríficos exportadores

The aim of the present study was to investigate the presence of Salmonella spp. in samples collected from beef meat at three points of the slaughter line (after skinning, washing and cooling) at three slaughterhouses in Brazil that export meat. Detection was based on ISO 6579:2002 and confirmed by PCR and qPCR. The isolates were typified using slide agglutination tests and PFGE. The antibiotic sensitivity profile was determined using the disk diffusion method. Contamination was detected in only one slaughterhouse. The overall frequency of contamination by Salmonella spp. was 6.7% of carcasses (6/90) and 2.6% of carcass surface samples (7/270). All isolates were confirmed by PCR and qPCR. The serological analysis and the PFGE showed a single profile: Typhimurium. The strains demonstrated 100% susceptibility to ampicillin, cefotaxime, ciprofloxacin, chloramphenicol, gentamicin and tetracycline. Positive carcasses after cooling pose a direct risk to consumers, since the meat is considered ready to be marketed after this process.(AU)

O objetivo deste trabalho foi investigar a presença de Salmonella spp. em amostras coletadas de carcaças de bovinos, em três pontos da linha de abate (após a esfola, lavagem e refrigeração) de três frigoríficos exportadores no Brasil. A detecção foi realizada pela ISO 6579:2002, e confirmada por PCR e qPCR. Os isolados foram tipificados por testes de soroaglutinação e PFGE e avaliado o perfil de sensibilidade aos antibióticos pelo método de difusão em disco. A contaminação foi detectada em apenas um abatedouro-frigorífico. As contaminações das carcaças (n=90) e amostras de carne (n=270) por Salmonella spp. foram 6 (6,7%) e 7 (2,6%), respectivamente. Todos os isolados foram confirmados por PCR e qPCR. A análise sorológica e o PFGE mostraram um único perfil: Typhimurium. As cepas apresentaram 100% de suscetibilidade à ampicilina, cefotaxima, ciprofloxacina, cloranfenicol, gentamicina e tetraciclina. As carcaças positivas após a refrigeração apresentam um risco direto para o consumidor, uma vez que, após este processo, a carne está pronta para ser comercializada.(AU)

False-negative reactions to the comparative intradermal tuberculin test for bovine tuberculosis / Reações falso-negativas ao teste cervical comparativo para tuberculose bovina

According to the Brazilian National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis (PNCEBT), the routine tests for the diagnosis of bovine tuberculosis in the country are the simple intradermal tuberculin test (SITT) of the Ministry of Agriculture, Livestock and Food Supply (MAPA), the caudal fold test and the comparative intradermal tuberculin test (CITT). The latter is also used as a confirmatory test. A group of 53 animals from three dairy herds in a focal area for bovine tuberculosis, that were submitted to depopulation in the state of Rio Grande do Sul, were submitted to the CITT. Tissues were cultured and the resulting colonies were confirmed by PCR and DNA sequencing. Among the 53 animals analyzed using the CITT, 32 (60.4%) were negative, 14 (26.4%) were positive and seven (13.2%) results were inconclusive. The CITT detected 11 of the 39 animals with culture-confirmed M. bovis infection as positive. Among the total of 14 uninfected animals based on cultures, the CBT detected eight as negative. Thus, the CITT demonstrated sensitivity of 28.2% and specificity of 57.1% for the population sampled. A total of 24/32 (75.0%) of the animals with negative CITT results were culture positive (confirmed by PCR) and were considered false negatives based on the CITT. The maintenance of these false-negative animals in herds has serious implications for the control of the disease, since they can be a source of infection. The addition of complementary tests could help identify such animals and increase the odds of diagnostic success.(AU)

No Brasil, segundo o Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose Animal (PNCEBT), do Ministério da Agricultura, Pecuária e Abastecimento (MAPA), os testes de rotina para o diagnóstico de tuberculose bovina são o teste cervical simples (TCC), o teste da prega caudal (TPC) e o teste cervical comparativo (TCC), sendo que o último também é utilizado como teste confirmatório. Um grupo de 53 animais oriundos de três rebanhos leiteiros de área de foco para tuberculose bovina que foram submetidos a vazio sanitário no Rio Grande do Sul foi submetido ao TCC. Os tecidos destes animais foram cultivados e as colônias resultantes confirmadas por PCR e sequenciamento de DNA. Dos 53 animais analisados no TCC, 32 (60,4%) foram negativos, 14 (26,4%) positivos e sete (13,2%) inconclusivos, com base no PNCEBT. O TCC detectou como positivos 11 dos 39 animais com infecção por M. bovis confirmada por cultivo. Do total de 14 animais não infectados, baseado na cultura, o TCC detectou oito como negativos. Assim, o TCC apresentou, para a população amostrada, sensibilidade de 28,2% e especificidade de 57,1%. Um total de 24/32 (75,0%) dos animais negativos ao TCC foi positivo no cultivo (confirmado por PCR), sendo considerados falso-negativos ao TCC. A manutenção destes animais falso-negativos nos rebanhos tem sérias implicações para o controle da enfermidade, já que os mesmos podem ser fonte de infecção. A adição de testes complementares poderia auxiliar na identificação destes animais, aumentando a cobertura diagnóstica.(AU)

False-negative reactions to the comparative intradermal tuberculin test for bovine tuberculosis / Reações falso-negativas ao teste cervical comparativo para tuberculose bovina

According to the Brazilian National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis (PNCEBT), the routine tests for the diagnosis of bovine tuberculosis in the country are the simple intradermal tuberculin test (SITT) of the Ministry of Agriculture, Livestock and Food Supply (MAPA), the caudal fold test and the comparative intradermal tuberculin test (CITT). The latter is also used as a confirmatory test. A group of 53 animals from three dairy herds in a focal area for bovine tuberculosis, that were submitted to depopulation in the state of Rio Grande do Sul, were submitted to the CITT. Tissues were cultured and the resulting colonies were confirmed by PCR and DNA sequencing. Among the 53 animals analyzed using the CITT, 32 (60.4%) were negative, 14 (26.4%) were positive and seven (13.2%) results were inconclusive. The CITT detected 11 of the 39 animals with culture-confirmed M. bovis infection as positive. Among the total of 14 uninfected animals based on cultures, the CBT detected eight as negative. Thus, the CITT demonstrated sensitivity of 28.2% and specificity of 57.1% for the population sampled. A total of 24/32 (75.0%) of the animals with negative CITT results were culture positive (confirmed by PCR) and were considered false negatives based on the CITT. The maintenance of these false-negative animals in herds has serious implications for the control of the disease, since they can be a source of infection. The addition of complementary tests could help identify such animals and increase the odds of diagnostic success.(AU)

No Brasil, segundo o Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose Animal (PNCEBT), do Ministério da Agricultura, Pecuária e Abastecimento (MAPA), os testes de rotina para o diagnóstico de tuberculose bovina são o teste cervical simples (TCC), o teste da prega caudal (TPC) e o teste cervical comparativo (TCC), sendo que o último também é utilizado como teste confirmatório. Um grupo de 53 animais oriundos de três rebanhos leiteiros de área de foco para tuberculose bovina que foram submetidos a vazio sanitário no Rio Grande do Sul foi submetido ao TCC. Os tecidos destes animais foram cultivados e as colônias resultantes confirmadas por PCR e sequenciamento de DNA. Dos 53 animais analisados no TCC, 32 (60,4%) foram negativos, 14 (26,4%) positivos e sete (13,2%) inconclusivos, com base no PNCEBT. O TCC detectou como positivos 11 dos 39 animais com infecção por M. bovis confirmada por cultivo. Do total de 14 animais não infectados, baseado na cultura, o TCC detectou oito como negativos. Assim, o TCC apresentou, para a população amostrada, sensibilidade de 28,2% e especificidade de 57,1%. Um total de 24/32 (75,0%) dos animais negativos ao TCC foi positivo no cultivo (confirmado por PCR), sendo considerados falso-negativos ao TCC. A manutenção destes animais falso-negativos nos rebanhos tem sérias implicações para o controle da enfermidade, já que os mesmos podem ser fonte de infecção. A adição de testes complementares poderia auxiliar na identificação destes animais, aumentando a cobertura diagnóstica.(AU)

Estimate of the economic impact of mastitis: A case study in a Holstein dairy herd under tropical conditions.

The aim of this study was to estimate the economic impact of mastitis at the herd level and the weight (percent) of the components of this impact in a Holstein dairy herd under tropical conditions. Three estimates of the economic impact of mastitis were performed. In estimates 1 and 2 the real production and economic indices from February 2011 to January 2012 were considered. In the estimate 1, indices for mastitis classified as ideal were considered, whereas in the estimate 2, the mastitis indices used were those recorded at the farm and at Holstein Cattle Association of Minas Gerais State database (real indices). Ideal mastitis indices were bulk milk somatic cell counts less than 250,000 cells/mL, incidence of clinical mastitis less than 25 cases/100 cows/year, number of culls due to udder health problems less than 5% and the percentage of cows with somatic cell counts greater than 200,000 cells/mL less than 20%. Considering the ideal indices of mastitis, the economic impact was US$19,132.35. The three main components of the economic impact were culling cows (39.4%) and the reduction in milk production due to subclinical and clinical mastitis (32.3% and 18.2%, respectively). Estimate 2 using real mastitis indices showed an economic impact of US$61,623.13 and the reduction in milk production due to mastitis (77.7%) and milk disposal (14.0%) were the most relevant components. The real impact of culling cows was approximately 16 times less than the weight that was considered ideal, indicating that this procedure could have been more frequently adopted. The reduction in milk production was 27.2% higher than the reduction in Estimate 1, indicating a need to control and prevent mastitis. The estimate 3 considered the same indices as estimate 2, but for the period from February 2012 to January 2013. Its economic impact was US$91,552.69. During this period, 161 treatments of cows with an intramammary antibiotic were performed to eliminate Streptococcus agalactiae, and eight cows chronically infected with Staphylococcus aureus were culled. The reduction in milk production due to mastitis was the main component of the economic impact (54.9%). The culling of cows with chronic infection was associated with an increase in the economic impact of mastitis and a reduction in the average productivity per cow. At the herd level reduction in milk production was the component that presented the largest weight in the economic impact of the disease.

Cooled semen for fixed-time artificial insemination in beef cattle.

This study evaluated the use of cooled semen in a fixed-time artificial insemination (FTAI) program compared with frozen-thawed semen to improve pregnancy rates in beef cattle. Ejaculates of three bulls were collected and divided into two treatments: (1) frozen-thawed semen and (2) cooled semen. Egg-yolk extender without glycerol was used for the cooled semen treatment. Straws (25×106 spermatozoa) were submitted to cooling for preservation at 5°C for 24h, after which FTAI was performed. Nelore cows (n=838) submitted to FTAI were randomly inseminated using frozen-thawed semen or cooled semen. There was a 20% increase in the pregnancy per AI (P AI-1) using cooled semen compared with frozen-thawed semen (59.9±4.7 vs 49.4±5.0%; P<0.005). There was no difference in P AI-1 among the bulls (P=0.40). The frozen-thawed semen had fewer functional spermatozoa than did the cooled semen when evaluated by sperm motility (61.7 vs 81.0%), slow thermoresistance test (41.7 vs 66.7%) and hypoosmotic swelling test (38.3 vs 53.7%; P<0.05). The percentage of sperm abnormalities did not differ between the freeze-thawing and cooling processes (18.6 vs 22.1%; P>0.05). Because there was less damage to spermatozoa and improvement in P AI-1, the use of cooled semen instead of frozen-thawed semen is an interesting approach to increase reproductive efficiency in cattle submitted to a FTAI protocol.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR.

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Neglected tropical diseases: prevalence and risk factors for schistosomiasis and soil-transmitted helminthiasis in a region of Minas Gerais State, Brazil.

BACKGROUND: Among the neglected tropical diseases (NTDs), schistosomiasis and the three main soil-transmitted helminthiases (STHs), i.e., ascariasis, trichuriasis and hookworm infection, represent the most common infections in developing countries. In Brazil, there is a lack of epidemiological data in many parts of the country, which favors the unawareness of the real situation concerning these diseases. Due to this, we investigated the occurrence of schistosomiasis and STHs in a region of Minas Gerais State, Brazil. METHODS: One stool sample was collected from 503 individuals, whose ages ranged from 0.1 to 91.2 years, and screened using both the Kato-Katz and the Formol-Ether methods. In parallel, a malacological survey was carried out in the main water bodies of the district, and Biomphalaria susceptibility assays and kernel-based techniques were also performed. RESULTS: No individual was found infected with Ascaris lumbricoides or hookworm. Schistosoma mansoni was the most common parasite found (1.6%). The prevalence was higher in males and the chance of acquiring the disease increased by 43.35 times with contact with a body of water. None of the Biomphalaria tenagophila and B. glabrata specimens were found naturally infected, but B. glabrata was highly susceptible to infection with Schistosoma mansoni. Using kernel-based techniques, clusters of Biomphalaria were found near the households where the infected individuals lived. CONCLUSIONS: Schistosomiasis was the most prevalent parasitic infection found. Our findings show that the occurrence of this disease has been underestimated by the local health care service, and highlight the importance of epidemiological surveillance in areas of low prevalence for schistosomiasis.

Detection of Mycobacterium bovis in bovine and bubaline tissues using nested-PCR for TbD1.

In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR, 97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analytical specificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-related Actinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positive results in the comparative intradermal tuberculin test (CITT), as well as from those with lesions compatible with tuberculosis (LCT) that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals with CITT- results, with no visible lesions (NVL) and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in the detection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals than the culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay to detect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

ELISA based on recombinant MPB70 and P27 for detection of antibodies against Mycobacterium bovis

A tuberculose bovina é uma importante enfermidade causada pela bactéria Mycobacterium bovis. Testes de tuberculinização intradérmica e abate de animais infectados levaram à redução da incidência da tuberculose bovina em muitos países. Entretanto, são necessários métodos maispráticos e eficientes com maior sensibilidade e especificidade. O objetivo do presente estudo foidesenvolver um teste imunoenzimático (ELISA), utilizando as proteínas recombinantes MPB70 e p27 de M. bovis, que possibilitasse detectar anticorpos contra esta bactéria em bovinos. A sensibilidade e especificidade observadas foram, respectivamente, de 88,7por cento e 94,6por cento para o ELISA-MPB70 e de 98,1por cento e 91,9por cento para o ELISA-p27. O uso de testes sorológicos, como o ELISA com MPB70 e p27 recombinantes, juntamente com testes celulares, pode resolver algunsproblemas relacionados ao diagnóstico da tuberculose bovina tais como os resultados inconclusivos e a ausência de detecção de animais anérgicos em estágios avançados da infecção.

Genotyping did not evidence any contribution of Mycobacterium bovis to human tuberculosis in Brazil.

The contribution of Mycobacterium bovis to the global burden of tuberculosis (TB) in man is likely to be underestimated due to its dysgonic growth characteristics and because of the absence of pyruvate in most used media is disadvantageous for its primary isolation. In Brazil Mycobacterium culture, identification and susceptibility tests are performed only in TB reference centers, usually for selected cases. Moreover, solid, egg-based, glycerol-containing (without pyruvate supplementation) Löwenstein-Jensen (L-J) or Ogawa media are routinely used, unfavouring M. bovis isolation. To determine the importance of M. bovis as a public health threat in Brazil we investigated 3046 suspected TB patients inoculating their clinical samples onto routine L-J and L-J pyruvate enriched media. A total of 1796 specimens were culture positive for Mycobacterium spp. and 702 TB cases were confirmed. Surprisingly we did not detect one single case of M. bovis in the resulting collection of 1674 isolates recovered from M. bovis favourable medium analyzed by conventional and molecular speciation methods. Also, bacillary DNA present on 454 sputum smears from 223 TB patients were OxyR genotyped and none was recognized as M. bovis. Our data indicate that M. bovis importance on the burden of human TB in Brazil is marginal.

Low-grade central osteosarcoma of the mandible: a case study report.

Low-grade central osteosarcoma is a rare type of osteosarcoma with peculiar clinical radiographic and microscopic features. The aim of this article is to report and discuss a case of low-grade central osteosarcoma in the mandible of a 42-year-old woman. The patient reported sensing mild pain and tooth mobility for a period of 4 years, despite continuous dental treatment. Radiographic evaluation showed a mixed radiopaque/radiolucenct lesion in the body, ramus, coronoid process, and condyle of the left side of the mandible. Destruction of the mandibular cortex in the area was also observed. After incisional biopsy, the patient underwent hemimandibulectomy. Microscopic findings showed a tumor exhibiting spindle cells with nuclear hyperchromasia and no mitotic activity, irregular osteoid formation, and soft tissue infiltration. The immunohistochemical analysis of the expression of Ki-67, Cyclin B1, and PCNA proteins (cellular proliferation markers) revealed a very low Ki-67+ and Cyclin B1+ cell index (mean 7% and 3%, respectively), but a moderate number of PCNA+ cells (mean 49%). The 2 years of clinical and imaging postoperative follow-up showed no evidence of recurrence. Clinicians should be aware of these lesions, because histopathologicially low-grade central osteosarcoma may be misinterpreted as fibrous dysplasia.