Milk Contamination by Mycobacterium tuberculosis Complex, Implications for Public Health in Amazonas, Brazil.

ABSTRACT: In Brazil, contamination of raw milk with Mycobacterium tuberculosis complex (MTC) has been reported in several states. The highest rate of consumption of raw milk and its derivatives in Brazil occurs in Amazonas. This state also has the highest prevalence of tuberculosis in both humans and livestock. We assessed the contamination of cow's milk and buffalo's milk with MTC in Amazonas, focusing on Mycobacterium bovis, the species most commonly found in cattle and buffalo. In 2019, 250 samples of raw milk (91 from cattle, 159 from buffalo) were collected before processing from three milk plants in the state of Amazonas. The samples were placed into 21 pools and analyzed using shotgun metagenomic sequencing and taxonomic classification with Kraken 2 and MegaBLAST. To confirm the identity of mycobacterial species found, BLASTN was used to identify specific genomic positions in the TbD1 and RD1 regions and flanking RD4 region. MTC genetic material was identified in all pools of raw milk. Genetic material consistent with M. bovis was identified in seven pools of raw milk (1 from cattle, 6 from buffalo). Buffalo's milk had significantly higher MTC reads than did cow's milk. The common practice of consumption of raw milk and its derivatives in Amazonas presents a risk to public health. Urgent measures to prevent transmission of foodborne tuberculosis are needed in the Amazon region. Greater efforts and resources also should be directed toward elimination of bovine tuberculosis in cattle and buffalo herds in Amazonas and the rest of Brazil.

Multiresistance, beta-lactamase-encoding genes and bacterial diversity in hospital wastewater in Rio de Janeiro, Brazil.

AIMS: To investigate the bacterial diversity, antimicrobial resistance patterns and types of beta-lactamase genes in Gram-negative bacteria isolated from a hospital sewage treatment plant. METHODS AND RESULTS: Between July and December 2008, we collected samples from influent, clarifier tank effluent and chlorine contact tank effluent from a sewage treatment plant service of a hospital located in the city of Rio de Janeiro, Brazil. Of the 221 isolates identified, 40% were characterized as extended-spectrum beta-lactamase (ESBL) producers. Nonpathogenic micro-organisms and some pathogenic genera were quantified. The most common ESBL-producing isolates were Klebsiella pneumoniae, Enterobacter cloacae and Escherichia coli. The bla(TEM), bla(SHV) and bla(CTX-M) genes were detected in 82, 48 and 67% of bacterial isolates, respectively. CONCLUSIONS: Results showed that hospital wastewater treatment plant is not suitable systems for the removal of all antibiotic-resistant micro-organisms present in hospital wastewaters. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence that bacteria resistant to multiple antibiotics and their resistance genes that are usually present in the hospital can reach the environment, even after the use of hospital wastewater treatment plants.

Bothrops jararaca venom gland transcriptome: Analysis of the gene expression pattern

Bothrops jararaca is a pit viper responsible for the majority of snake envenoming accidents in Brazil. As an attempt to describe the transcriptional activity of the venom gland, ESTs of a cDNA library constructed from B. jararaca venom gland were generated and submitted to bioinformatics analysis. The results showed a clear predominance of transcripts coding for toxins instead of transcripts coding for proteins involved in cellular functions. Among toxins, the most frequent transcripts were from metalloproteinases (52.6%), followed by serine-proteinases (28.5%), C-type lectins (8.3%) and bradykinin-potentiating peptides (BPPs) (6.2%). Results were similar to that obtained from the transcriptome analysis of B. insularis, a phylogenetically close sister of B. jararaca, though some differences were observed and are pointed out, such as a higher amount of the hypotensive BPPs in B. insularis transcriptome (19.7%). Another striking difference observed is that PIII and PII-classes of metalloproteinases are similarly represented in B. jararaca in contrast to B. insularis, in which a predominance of PIII-class metalloproteinase, which present a more intense hemorrhagic action, is observed. These features may, in part, explain the higher potency of B. insularis venom. The results obtained can help in proteome studies, and the clones can be used to directly probe the genetic material from other snake species or to investigate differences in gene expression pattern in response to factors such as diet, aging and geographic localization.

Internal transcribed spacers (ITS) of Trypanosoma rangeli ribosomal DNA (rDNA): a useful marker for inter-specific differentiation.

The internal transcribed spacers (ITS) flanking the 5.8S subunit of the ribosomal RNA genes (rDNA) of Trypanosoma rangeli strains isolated from distinct geographical regions and hosts were studied. The results revealed the sequence variability of the ITS spacers showing the presence of microsatellite repeats and single nucleotide polymorphisms (SNP), which were also observed within the 5.8S rDNA sequence. ITS-2 spacer was the most phylogenetically informative region due the presence of a higher number of parsimonious sites in both inter- and intra-specific analysis. Sequence analysis of both ITS spacers plus the 5.8S rDNA of T. rangeli strains allowed a clear inter-specific differentiation from Trypanosoma cruzi strains representative of the parasite zymodemes.

The use of ITS1 rDNA PCR in detecting pathogenic African trypanosomes.

There are 11 different pathogenic trypanosomes in trypanosomiasis endemic regions of Africa. Their detection and characterisation by molecular methods relies on species-specific primers; consequently several PCR tests have to be made on each sample. Primers ITS1 CF and ITS1 BR, previously designed to amplify the internal transcribed spacer (ITS1) of rDNA, have been evaluated for use in a universal diagnostic test for all pathogenic trypanosomes. Blood was collected from 373 cattle and 185 camels. The primers gave constant PCR products with the stocks of each taxon tested. Members of subgenus Trypanozoon (T. brucei brucei, T. evansi, T. b. rhodesiense and T. b. gambiense) gave a constant product of approximately 480 bp; T. congolense, savannah 700 bp, T. congolense kilifi 620 bp and T. congolense forest 710 bp: T. simiae 400 bp, T. simiae tsavo 370 bp, T. godfreyi 300 bp and T. vivax 250 bp. The sensitivity of the test ranged from 10 pg for Trypanozoon, T. congolense clade and T. vivax to 100 pg for T. simiae and T. godfreyi. The primers detected cases of multi-taxa samples, although the sensitivity was reduced with an increase in the combinations. A better detection rate of trypanosome DNA was recorded with buffy coats than from direct blood. With the field samples, the diagnostic sensitivity was close to the sensitivity obtained using single reactions with species-specific primers for Trypanozoon 38/40 (95%) and T. congolense savannah 30/33 (90.9%) but was lower with T. vivax 25/31 (77.4%). The primers offer promise as a routine diagnostic tool through the use of a single PCR; however, further evaluation is recommended.

Detection and characterization of rabies virus in Southern Brazil by PCR amplification and sequencing of the nucleoprotein gene.

Due to the medical and socio-economical importance of both human and animal rabies infection, several studies have suggested the use of molecular techniques such as RT-PCR and DNA sequencing for diagnosis and phylogenetic studies of the rabies virus. Considering the conservancy of the nucleoprotein (N) gene of the virus, we herein describe a RT-PCR assay for rabies diagnosis and characterization. A total of 75 samples obtained from a variety of animal species in the state of Santa Catarina (SC), Southern Brazil, were comparatively studied by fluorescence antibody test (FAT), mouse inoculation test (MIT), cell infection assay and RT-PCR, which revealed itself to be as sensitive as FAT and MIT and less time-consuming than MIT. Direct sequencing of the 5' end of the N gene allowed the clustering of the SC samples with samples from the vampire bat-related or sylvatic cycle through comparative sequence analysis.

Enzootiology of Trypanosoma evansi in Pantanal, Brazil.

In order to better understand the enzootiology of trypanosomiasis caused by Trypanosoma evansi in the Brazilian Pantanal we examined domestic and wild mammals by microhematocrit centrifuge technique (MHCT), immunofluorescence antibody test (IFAT) and polymerase chain reaction (PCR). T. evansi infection was detected in all species sampled with exception of the sheep and the feral pig. High parasitemias were observed in capybaras (5/24), coatis (18/115), horses (31/321) and dogs (3/112). Among these species, only the capybaras did not develop anemia. Low parasitemias, only detected by PCR, were found in buffaloes (18/43), bovines (29/331), marsupials (1/4), small rodents (14/67), bats (7/18), and one armadillo (1/8). The highest prevalence of T. evansi infection was recorded in horses (73%), although no neurological signs in infected horses were observed. Diagnosis through standard parasitological tests and IFAT should be used with caution since they may overlook comprovedly infected horses. The relationship between ranch management and T. evansi infection in horse was investigated. The importance of other transmission mechanisms apart from the tabanids and reservoir hosts are discussed.

Using PCR for unraveling the cryptic epizootiology of livestock trypanosomosis in the Pantanal, Brazil.

Trypanosoma vivax and Trypanosoma evansi are livestock parasites of economic importance in Africa, Asia and South America. In the Pantanal, Brazil, they cause economic losses in both cattle and equines. Little is known of their maintenance and spread in nature, particularly in terms of reservoirs and means of mechanical transmission. Here we report for the first time the use of PCR for the detection of T. vivax and T. evansi in bovines, buffaloes and sheep. Whereas parasitological diagnosis detected only two T. vivax infections, one in buffalo and another in a cow, PCR detected infections in 34.8% buffaloes, 44.7% bovines and 37.3% sheep. Trypanozoon primers detected 41.8% infections in buffaloes and 8.1% in cattle. PCR revealed 6.9% mixed infections in buffaloes and 5.3% in cattle. The potential role of cattle and buffaloes as hosts and reservoirs of T. vivax is discussed, as well as the implications of possible extravascular foci in the maintenance of livestock trypanosomosis.

Applications of PCR-based tools for detection and identification of animal trypanosomes: a review and perspectives.

This paper aims to review the applications of the polymerase chain reaction (PCR) for the detection and identification of trypanosomes in animals. The diagnosis of trypanosomes, initially based on microscopic observations and the host range of the parasites, has been improved, since the 1980s, by DNA-based identification. These diagnostic techniques evolved successively through DNA probing, PCR associated to DNA probing, and currently to PCR alone. Several DNA sequences have been investigated as possible targets for diagnosis, especially multi-copy genes such as mini-exon, kinetoplastid mini-circles, etc., but the most favoured target is the nuclear satellite DNA of mini-chromosomes, which presents the advantages, and the drawbacks, of highly repetitive short sequences (120-600 bp). Several levels of specificity have been achieved from sub-genus to species, sub-species and even types. Random priming of trypanosome DNA has even allowed "isolate specific" identification. Other work based on microsatellite sequences has provided markers for population genetic studies. For regular diagnosis, the sensitivity of PCR has increased with the advancement of technologies for sample preparation, to reach a level of 1 trypanosome/ml of blood, which has brought to field samples a sensitivity two to three times higher than microscopic observation of the buffy coat. Similarly, PCR has allowed an increase in the specificity and sensitivity of diagnosis in vectors such as tsetse flies. However, because of the diversity of Trypanosoma species potentially present in a single host, PCR diagnosis carried out on host material requires several PCR reactions; for example, in cattle, up to five reactions per sample may be required. Research is now focusing on a diagnosis based on the amplification of the internal transcribed spacer-1 (ITS-1) of ribosomal DNA which presents the advantages of being a multi-copy locus (100-200), having a small size (300-800 bp), which varies from one taxon to another but is conserved in size in a given taxon. This may lead to the development of a multi-species-specific diagnostic protocol using a single PCR. By reducing the cost of the PCR diagnosis, this technique would allow a greater number of field samples to be tested in epidemiological studies and/or would increase the variety of Trypanosoma species that could be detected. Further investigations are required to develop and optimise multi-species-specific diagnostic tools for trypanosomes, which could also serve as a model for such tools in other pathogens.

Coagulopatia devido à infecçäo aguda por Trypanosoma evansi em um cäo / Coagulopathy due to Trypanosoma evansi acute infection in a dog

O Trypanosoma evansi é um tripanosoma da secçäo salivaria pertencente ao subgênero Trypanozoon. Ele causa a "surra" no velho mundo e o "mal de caderas" na área subtropical da Argentina e no Pantanal, Brasil. Há similaridades entre as lesöes e patogenia causadas por T. brucei, T. evansi e T. equiperdum, inclusive no consumo de plaquetas na coagulaçäo intravascular disseminada (DIC). O presente estudo mostra os valores de APTT, PT e contagem de plaquetas evidenciando a DIC no primeiro relato de infecçäo natural pelo T. evansi em cäo no Pantanal, Brasil