Highly conserved and extremely variable: The paradoxical pattern of toxin expression revealed by comparative venom-gland transcriptomics of Phalotris (Serpentes: Dipsadidae).

Although non-front fanged snakes account for almost two-thirds of snake diversity, most studies on venom composition and evolution focus exclusively on front-fanged species, which comprise most of the clinically relevant accidents. Comprehensive reports on venom composition of non-front fanged snakes are still scarce for several groups. In this study, we address such shortage of knowledge by providing new insights about the venom composition among species of Phalotris, a poorly studied Neotropical dipsadid genus. Phalotris are known for their specialized venom delivery system and toxic venoms, which can cause life-threatening accidents in humans. We evaluate the venom-gland transcriptome of Phalotris, comparing the following three South American species: P. reticulatus for the Araucaria Pine forests, P. lemniscatus for the Pampa grasslands, and P. mertensi for the Brazilian Cerrado. Our results indicate similar venom profiles, in which they share a high expression level of Kunitz-type inhibitors (KUNZ). On the other hand, comparative analyses revealed substantial differences in the expression levels of C-type lectins (CTL) and snake venom metalloproteinases (SVMP). The diverse set of SVMP and CTL isoforms shows signals of positive selection, and we also identified truncated forms of type III SVMPs, which resemble type II and type I SVMPs of viperids. Additionally, we identified a CNP precursor hosting a proline-rich region containing a BPP motif resembling those commonly detected in viperid venoms with hypotensive activity. Altogether, our results suggest an evolutionary history favoring high expression levels of few KUNZ isoforms in Phalotris venoms, contrasting with a highly diverse set of SVMP and CTL isoforms. Such diversity can be comparable with the venom variability observed in some viperids. Our findings highlight the extreme phenotypic diversity of non-front fanged snakes and the importance to allocate greater effort to study neglected groups of Colubroidea.

Highly conserved and extremely variable: the paradoxical pattern of toxin expression revealed by comparative venom-gland transcriptomics of Phalotris (Serpentes: Dipsadidae)

Although non-front fanged snakes account for almost two-thirds of snake diversity, most studies on venom composition and evolution focus exclusively on front-fanged species, which comprise most of the clinically relevant accidents. Comprehensive reports on venom composition of non-front fanged snakes are still scarce for several groups. In this study, we address such shortage of knowledge by providing new insights about the venom composition among species of Phalotris, a poorly studied Neotropical dipsadid genus. Phalotris are known for their specialized venom delivery system and toxic venoms, which can cause life-threatening accidents in humans. We evaluate the venom-gland transcriptome of Phalotris, comparing the following three South American species: P. reticulatus for the Araucaria Pine forests, P. lemniscatus for the Pampa grasslands, and P. mertensi for the Brazilian Cerrado. Our results indicate similar venom profiles, in which they share a high expression level of Kunitz-type inhibitors (KUNZ). On the other hand, comparative analyses revealed substantial differences in the expression levels of C-type lectins (CTL) and snake venom metalloproteinases (SVMP). The diverse set of SVMP and CTL isoforms shows signals of positive selection, and we also identified truncated forms of type III SVMPs, which resemble type II and type I SVMPs of viperids. Additionally, we identified a CNP precursor hosting a proline-rich region containing a BPP motif resembling those commonly detected in viperid venoms with hypotensive activity. Altogether, our results suggest an evolutionary history favoring high expression levels of few KUNZ isoforms in Phalotris venoms, contrasting with a highly diverse set of SVMP and CTL isoforms. Such diversity can be comparable with the venom variability observed in some viperids. Our findings highlight the extreme phenotypic diversity of non-front fanged snakes and the importance to allocate greater effort to study neglected groups of Colubroidea.

Coalescent-Based Species Delimitation in Herbaceous Bamboos (Bambusoideae, Olyreae) from Eastern Brazil: Implications for Taxonomy and Conservation in a Group with Weak Morphological Divergence Coupled with Low Genetic Diversity.

Species delimitation in herbaceous bamboos has been complex and, in some genera, a great part of its diversity has been confirmed only based on genetic information, as is the case of the genus Raddia. It includes nine species, all occurring in Brazil, but only R. portoi predominates in dry forests of the Northeast associated with the Caatinga phytogeographic domain. This species is morphologically close to R. angustifolia, which is known for a single location in the Atlantic Forest in Southern Bahia, and is considered to be threatened by extinction. Besides problems with taxonomic focus, actions for its conservation are complicated because it is not certain if it must be considered an independent species or included in the more widespread R. portoi. In this study, we used coalescent multispecies (MSC) theory approaches combined with genetic structure analyses in an attempt to delimit these two species. Different analyses were congruent and the species delimitation using MSC inferred distinct lineages supporting their recognition as two species. These results solved the taxonomic doubts and also showed the power of these approaches to delimit species as lineages, even in groups with weak morphological divergence and low genetic variability, and also impacting our knowledge for conservation purposes.

Análise dos constituintes de baixa massa molecular no veneno de Lachesis muta / Analysis of the low molecular weigth constituents in the Lachesis muta venom

Snakebite accidents are a serious public health problem neglected in tropical countries. In Brazil, the Northern Region has the largest number of cases and snakes from Lachesis genus are responsible for the most serious accidents and the highest number of deaths. Victims of Lachesis envenoming have bite site damage, bleeding and, in severe cases, hypotension. Proteomic and transcriptomic studies report the presence of bradykinin potentiating peptide (BPPs) in L. muta venom, however, there are no data in the literature describing their purifications and identifications. The objective of this work is to identify and characterize angiotensin-converting enzyme inhibitors in the low molecular weight portion of Lachesis muta venom. For this lyophilized L. muta venom was dissolved in ammonium acetate buffer and filtered by centrifugation for size fractionating, using a 10 kDa centrifugal filter unit. The low molecular weight fraction was then chromatographed by RP-HPLC using a C18 column. Twelve fractions were collected and four were tested in fluorimetric assays using ACE as an enzyme and Abz-FRK(Dnp)P-OH as substrate. These fractions present peaks with retention time similar to the peaks that contained BPPs of other snake venoms. In addition, four fractions presented 100% inhibition of the catalytic activity of ACE over the FRET substrate. Based on these results, and due to other data in the literature describing the presence of BPPs in L. muta venom, is possible that it may contain BPPs. For confirmation of this hypothesis, the next step will be to perform analysis by mass spectrometry aiming to determine the primary sequences of the peptides present in each fraction.

Os acidentes ofídicos são um grave problema de saúde pública negligenciado em países tropicais. No Brasil, a Região Norte apresenta o maior número de casos e as serpentes do gênero Lachesis são responsáveis pelos acidentes mais graves e com maior número de óbitos. Vítimas do envenenamento por Lachesis apresentam danos no local da picada, hemorragias e em casos graves apresentam hipotensão arterial. Estudos proteômicos e trancriptômicos descrevem a presença de peptídeos potenciadores de bradicinina (BPPs) no veneno de Lachesis muta, porém não há dados na literatura descrevendo o isolamento e/ou suas identificações. O objetivo deste trabalho é identificar e caracterizar inibidores da enzima conversora de angiotensina (ECA) na porção de baixa massa molecular do veneno de Lachesis muta. Para isso o veneno liofilizado de Lachesis muta foi dissolvido em tampão acetato de amônio e filtrado por centrifugação para o fracionamento de tamanho, usando uma membrana de corte de 10 kDa. A porção de baixa massa molecular foi fracionada por RP-HPLC usando uma coluna C-18. Doze frações foram coletadas e quatro foram testadas em ensaios fluorimétricos utilizando a ECA como enzima e o Abz-FRK(Dnp)P-OH como substrato. Das quatro frações selecionadas, duas se destacaram devido à alta intensidade dos picos, um em 13,65 minutos de retenção e outro em 14 minutos de retenção, com 44% e 45% concentração de solução B, respectivamente. As frações de interesse apresentam picos eluídos nos mesmos tempos de retenção que os BPPs de outros venenos de serpentes, sugerindo, juntamente com dados da literatura, que eles podem conter BPPs semelhantes. As quatro frações inibiram em 100% a atividade catalítica da ECA sobre o substrato Abz-FRK(Dnp)P-OH. Por essas razões e devido outros dados na literatura descreverem a presença BPPs no veneno de L. muta, acreditamos que as quatro frações selecionadas contenham BPPs. Para a confirmação o próximo passo será realizar a análise por espectrometria de massas.

Phylogenomics and species delimitation of a complex radiation of Neotropical suboscine birds (Pachyramphus).

Phylogeographic studies within the Neotropics continue to uncover hidden diversity, the extent of which remains poorly known. In birds, molecular studies are producing evidence that species-level diversity is substantially underestimated. Many avian taxa comprise large complexes of subspecies that often represent species-level taxa by various criteria. One such group of Neotropical suboscine birds, the becards (Pachyramphus), ranges from Argentina through northern Mexico. Their taxonomic limits have been complex and controversial as the genus has bounced around a number of suboscine families. Additionally, the phylogenetic relationships within Pachyramphus are unresolved due to insufficient sampling of taxa and populations across species' ranges. We used target capture of ultraconserved elements for 62 individuals representing 42 taxa, and sequenced two mitochondrial genes and two nuclear introns covering 265 individuals of 51 taxa, including all recognized species, resulting in the most densely and completely sampled phylogenetic hypothesis for Pachyramphus to date. We delimited species using a traditional taxonomic approach and then tested them under a Bayesian multi-species coalescent framework. In doing so, we provide evidence for multiple young, previously undetected evolutionary lineages within Pachyramphus. Deep, well-supported branches and a high number of intraspecific lineages across the tree suggest that at least 50% of species diversity may be unrecognized.

The modular nature of bradykinin-potentiating peptides isolated from snake venoms.

Bradykinin-potentiating peptides (BPPs) are molecules discovered by Sergio Ferreira - who found them in the venom of Bothrops jararaca in the 1960s - that literally potentiate the action of bradykinin in vivo by, allegedly, inhibiting the angiotensin-converting enzymes. After administration, the global physiological effect of BPP is the decrease of the blood pressure. Due to this interesting effect, one of these peptides was used by David Cushman and Miguel Ondetti to develop a hypotensive drug, the widely known captopril, vastly employed on hypertension treatment. From that time on, many studies on BPPs have been conducted, basically describing new peptides and assaying their pharmacological effects, mostly in comparison to captopryl. After compiling most of these data, we are proposing that snake BPPs are 'modular' peptidic molecules, in which the combination of given amino acid 'blocks' results in the different existing peptides (BPPs), commonly found in snake venom. We have observed that there would be mandatory modules (present in all snake BPPs), such as the N-terminal pyroglutamic acid and C-terminal QIPP, and optional modules (amino acid blocks present in some of them), such as AP or WAQ. Scattered between these modules, there might be other amino acids that would 'complete' the peptide, without disrupting the signature of the classical BPP. This modular arrangement would represent an important evolutionary advantage in terms of biological diversity that might have its origins either at the genomic or at the post-translational modification levels. Regardless of the modules' origin, the increase in the diversity of peptides has definitely been essential for snakes' success on nature.

Species delimitation of the blue-spotted spiny lizard within a multilocus, multispecies coalescent framework, results in the recognition of a new Sceloporus species.

Species delimitation is a major topic in systematics. Species delimitation methods based on molecular data have become more common since this approach provides insights about species identification via levels of gene flow, the degree of hybridization and phylogenetic relationships. Also, combining multilocus mitochondrial and nuclear DNA leads to more reliable conclusions about species limits. Coalescent-based species delimitation methods explicitly reveal separately evolving lineages using probabilistic approaches and testing the delimitation hypotheses for several species. Within a multispecies, multilocus, coalescent framework, we were able to clarify taxonomic uncertainties within S. cyanostictus, an endangered lizard that inhabits a narrow strip of the Chihuahuan Desert in Mexico. We included, for the first time in a phylogenetic analysis, lizards from the three populations of S. cyanostictus recognized so far (East Coahuila, West Coahuila and Nuevo León). Phylogenetic analysis corroborates the hypothesis of two separately evolving lineages, i.e. the East and West Coahuila populations, as proposed in a previous study. We also found a distant phylogenetic relationship between the lizards from Nuevo León and those of East and West Coahuila. Finally, based on the species delimitation results, we propose and describe a new species of Sceloporus: S. gadsdeni sp. nov.

In the shadows: Phylogenomics and coalescent species delimitation unveil cryptic diversity in a Cerrado endemic lizard (Squamata: Tropidurus).

The recognition of cryptic diversity within geographically widespread species is gradually becoming a trend in the highly speciose Neotropical biomes. The statistical methods to recognise such cryptic lineages are rapidly advancing, but have rarely been applied to genomic-scale datasets. Herein, we used phylogenomic data to investigate phylogenetic history and cryptic diversity within Tropidurus itambere, a lizard endemic to the Cerrado biodiversity hotspot. We applied a series of phylogenetic methods to reconstruct evolutionary relationships and a coalescent Bayesian species delimitation approach (BPP) to clarify species limits. The BPP results suggest that the widespread nominal taxon comprises a complex of 5 highly supported and geographically structured cryptic species. We highlight and discuss the different topological patterns recovered by concatenated and coalescent species tree methods for these closely related lineages. Finally, we suggest that the existence of cryptic lineages in the Cerrado is much more common than traditionally thought, highlighting the value of using NGS data and coalescent techniques to investigate patterns of species diversity.

The modular nature of bradykinin-potentiating peptides isolated from snake venoms

Abstract Bradykinin-potentiating peptides (BPPs) are molecules discovered by Sergio Ferreira who found them in the venom of Bothrops jararaca in the 1960s that literally potentiate the action of bradykinin in vivo by, allegedly, inhibiting the angiotensin-converting enzymes. After administration, the global physiological effect of BPP is the decrease of the blood pressure. Due to this interesting effect, one of these peptides was used by David Cushman and Miguel Ondetti to develop a hypotensive drug, the widely known captopril, vastly employed on hypertension treatment. From that time on, many studies on BPPs have been conducted, basically describing new peptides and assaying their pharmacological effects, mostly in comparison to captopryl. After compiling most of these data, we are proposing that snake BPPs are modular peptidic molecules, in which the combination of given amino acid blocks results in the different existing peptides (BPPs), commonly found in snake venom. We have observed that there would be mandatory modules (present in all snake BPPs), such as the N-terminal pyroglutamic acid and C-terminal QIPP, and optionalmodules (amino acid blocks present in some of them), such as AP or WAQ. Scattered between these modules, there might be other amino acids that would complete the peptide, without disrupting the signature of the classical BPP. This modular arrangement would represent an important evolutionary advantage in terms of biological diversity that might have its origins either at the genomic or at the post-translational modification levels. Regardless of the modules origin, the increase in the diversity of peptides has definitely been essential for snakes success on nature.

The modular nature of bradykinin-potentiating peptides isolated from snake venoms

Bradykinin-potentiating peptides (BPPs) are molecules discovered by Sergio Ferreira - who found them in the venom of Bothrops jararaca in the 1960s - that literally potentiate the action of bradykinin in vivo by, allegedly, inhibiting the angiotensin-converting enzymes. After administration, the global physiological effect of BPP is the decrease of the blood pressure. Due to this interesting effect, one of these peptides was used by David Cushman and Miguel Ondetti to develop a hypotensive drug, the widely known captopril, vastly employed on hypertension treatment. From that time on, many studies on BPPs have been conducted, basically describing new peptides and assaying their pharmacological effects, mostly in comparison to captopryl. After compiling most of these data, we are proposing that snake BPPs are 'modular' peptidic molecules, in which the combination of given amino acid 'blocks' results in the different existing peptides (BPPs), commonly found in snake venom. We have observed that there would be mandatory modules (present in all snake BPPs), such as the N-terminal pyroglutamic acid and C-terminal QIPP, and optionalmodules (amino acid blocks present in some of them), such as AP or WAQ. Scattered between these modules, there might be other amino acids that would 'complete' the peptide, without disrupting the signature of the classical BPP. This modular arrangement would represent an important evolutionary advantage in terms of biological diversity that might have its origins either at the genomic or at the post-translational modification levels. Regardless of the modules' origin, the increase in the diversity of peptides has definitely been essential for snakes' success on nature.(AU)

The modular nature of bradykinin-potentiating peptides isolated from snake venoms

Bradykinin-potentiating peptides (BPPs) are molecules discovered by Sergio Ferreira - who found them in the venom of Bothrops jararaca in the 1960s - that literally potentiate the action of bradykinin in vivo by, allegedly, inhibiting the angiotensin-converting enzymes. After administration, the global physiological effect of BPP is the decrease of the blood pressure. Due to this interesting effect, one of these peptides was used by David Cushman and Miguel Ondetti to develop a hypotensive drug, the widely known captopril, vastly employed on hypertension treatment. From that time on, many studies on BPPs have been conducted, basically describing new peptides and assaying their pharmacological effects, mostly in comparison to captopryl. After compiling most of these data, we are proposing that snake BPPs are 'modular' peptidic molecules, in which the combination of given amino acid 'blocks' results in the different existing peptides (BPPs), commonly found in snake venom. We have observed that there would be mandatory modules (present in all snake BPPs), such as the N-terminal pyroglutamic acid and C-terminal QIPP, and optionalmodules (amino acid blocks present in some of them), such as AP or WAQ. Scattered between these modules, there might be other amino acids that would 'complete' the peptide, without disrupting the signature of the classical BPP. This modular arrangement would represent an important evolutionary advantage in terms of biological diversity that might have its origins either at the genomic or at the post-translational modification levels. Regardless of the modules' origin, the increase in the diversity of peptides has definitely been essential for snakes' success on nature.(AU)

The diversity and abundance of phytase genes (ß-propeller phytases) in bacterial communities of the maize rhizosphere.

UNLABELLED: The ecology of microbial communities associated with organic phosphorus (P) mineralization in soils is still understudied. Here, we assessed the abundance and diversity of bacteria harbouring genes encoding ß-propeller phytases (BPP) in the rhizosphere of traditional and transgenic maize cultivated in two Brazilian soils. We found a soil-dependent effect towards a higher abundance of phytase genes in the rhizosphere, and an absence of any impact of plant genotype. Phylogenetic analyses indicated members of the genera Pseudomonas, Caulobacter, Idiomarina and Maricaulis, close to 'uncultured bacteria', to constitute the dominant bacteria hosting this gene. The results obtained validate a methodology to target bacteria that are involved in the organic P cycle, and depict the responsiveness of such bacteria to the rhizosphere, albeit in dependency of the soil in which maize is cultivated. The data also identified the major bacterial groups that are associated with the organic P mineralization function. SIGNIFICANCE AND IMPACT OF THE STUDY: Micro-organisms play a key role in nutrient balance in soil ecosystems that are essential to life on the planet. However, some processes such as organic phosphorus mineralization, an important source of phosphorus supply in soil, is poorly studied mainly due the absence of an efficient methodology to assess the phytase-producing micro-organisms. In this study, a method to assess beta-propeller phytase (BPP)-carrying bacteria in soil was validated. This method may contribute to the knowledge of how these micro-organisms behave in the environment and contribute for plant growth promotion.

New proline-rich oligopeptides from the venom of African adders: Insights into the hypotensive effect of the venoms.

BACKGROUND: The snakes from the Bitis genus are some of the most medically important venomous snakes in sub Saharan Africa, however little is known about the composition and effects of these snake venom peptides. Considering that the victims with Bitis genus snakes have exacerbate hypotension and cardiovascular disorders, we investigated here the presence of angiotensin-converting enzyme modulators on four different species of venoms. METHODS: The peptide fractions from Bitis gabonica gabonica, Bitis nasicornis, Bitis gabonica rhinoceros and Bitis arietans which showed inhibitory activity on angiotensin-converting enzyme were subjected to mass spectrometry analysis. Eight proline-rich peptides were synthetized and their potencies were evaluated in vitro and in vivo. RESULTS: The MS analysis resulted in over 150 sequences, out of which 32 are new proline-rich oligopeptides, and eight were selected for syntheses. For some peptides, inhibition assays showed inhibitory potentials of cleavage of angiotensin I ten times greater when compared to bradykinin. In vivo tests showed that all peptides decreased mean arterial pressure, followed by tachycardia in 6 out of 8 of the tests. CONCLUSION: We describe here some new and already known proline-rich peptides, also known as bradykinin-potentiating peptides. Four synthetic peptides indicated a preferential inhibition of angiotensin-converting enzyme C-domain. In vivo studies show that the proline-rich oligopeptides are hypotensive molecules. GENERAL SIGNIFICANCE: Although proline-rich oligopeptides are known molecules, we present here 32 new sequences that are inhibitors of the angiotensin-converting enzyme and consistent with the symptoms of the victims of Bitis spp, who display severe hypotension.

Evaluation and implementation of good pratices in main points of microbiological contamination in milk production / Avaliação e implantação de boas práticas nos principais pontos de contaminação microbiológica na produção leiteira

In order to improve the microbiological quality of Brazilian milk and dairy products some measures have been implemented in the milk-producing sector throughout the years, such as milk refrigeration and bulk collection. Refrigeration is a very efficient procedure, however allows psychrotrophics multiplication, which are largely found in milk produced in poor hygienic conditions. To assure and improve the microbiologic quality of milk turns out to be necessary the implementation of hygienic measures, known as Good Manufacturing Practices (GMP). In this study, several hygienic practices were tested in the main contamination points in milk production, previously determined: teats (different chlorine concentrations pre-dipping), cans and bulk tanks (hygienic techniques) and residual water (elimination). The recommended practices were picked up considering their efficiency in microbial reduction, practiced and economic viability. When the practices were all applied, reduction in microbial counts was observed. After 48 hours the final and refrigerated milk showed a reduction of 11.95x106 CFU/mL to 12.48x103 CFU/mL of mesophilic aerobes, and a reduction of 18.10x106 CFU/mL to 5.38x103 CFU/mL of psychrotrophics. The results show that the procedures proposed by LIPOA is efficient, viable and easily adopted by milk farmers, representing an important alternative in producing high quality

Buscando a melhoria da qualidade microbiológica de leite e derivados produzidos no Brasil, nos últimos anos algumas medidas vêm sendo implantadas na produção leiteira como a refrigeração e coleta a granel. A refrigeração é um procedimento eficiente no controle de aeróbios mesófilos, mas permite multiplicação de psicrotróficos, encontrados em altas contagens em leite produzido com pouca higiene. Para garantia e melhoraria da qualidade microbiológica do leite é necessária a implantação de medidas higiênicas, conhecidas como Boas Práticas de Produção (BPP). Neste estudo, diversas práticas higiênicas foram testadas nos principais pontos de contaminação na produção leiteira, previamente determinados: tetos (pré-dipping com diversas concentrações de cloro), latões e refrigeradores (técnicas de higienização), e água residual (eliminação). As práticas recomendadas foram escolhidas quanto à eficiência na redução de microrganismos, praticidade e viabilidade econômica. Quando as práticas foram aplicadas conjuntamente, obteve-se reduções consideráveis nas contagens microbianas. Após 48 horas de refrigeração do leite, as contagens foram reduzidas de 11,95x106 CFU/mL para 12,48x103 CFU/mL de aeróbios mesófilos, e de 18,10x106 CFU/mL para 5,38x103 CFU/mL de psicrotróficos. Os resultados mostram que as práticas propostas são eficientes, viáveis e facilmente adotáveis por produtores leiteiros,

Evaluation and implementation of good pratices in main points of microbiological contamination in milk production / Avaliação e implantação de boas práticas nos principais pontos de contaminação microbiológica na produção leiteira

In order to improve the microbiological quality of Brazilian milk and dairy products some measures have been implemented in the milk-producing sector throughout the years, such as milk refrigeration and bulk collection. Refrigeration is a very efficient procedure, however allows psychrotrophics multiplication, which are largely found in milk produced in poor hygienic conditions. To assure and improve the microbiologic quality of milk turns out to be necessary the implementation of hygienic measures, known as Good Manufacturing Practices (GMP). In this study, several hygienic practices were tested in the main contamination points in milk production, previously determined: teats (different chlorine concentrations pre-dipping), cans and bulk tanks (hygienic techniques) and residual water (elimination). The recommended practices were picked up considering their efficiency in microbial reduction, practiced and economic viability. When the practices were all applied, reduction in microbial counts was observed. After 48 hours the final and refrigerated milk showed a reduction of 11.95x106 CFU/mL to 12.48x103 CFU/mL of mesophilic aerobes, and a reduction of 18.10x106 CFU/mL to 5.38x103 CFU/mL of psychrotrophics. The results show that the procedures proposed by LIPOA is efficient, viable and easily adopted by milk farmers, representing an important alternative in producing high quality

Buscando a melhoria da qualidade microbiológica de leite e derivados produzidos no Brasil, nos últimos anos algumas medidas vêm sendo implantadas na produção leiteira como a refrigeração e coleta a granel. A refrigeração é um procedimento eficiente no controle de aeróbios mesófilos, mas permite multiplicação de psicrotróficos, encontrados em altas contagens em leite produzido com pouca higiene. Para garantia e melhoraria da qualidade microbiológica do leite é necessária a implantação de medidas higiênicas, conhecidas como Boas Práticas de Produção (BPP). Neste estudo, diversas práticas higiênicas foram testadas nos principais pontos de contaminação na produção leiteira, previamente determinados: tetos (pré-dipping com diversas concentrações de cloro), latões e refrigeradores (técnicas de higienização), e água residual (eliminação). As práticas recomendadas foram escolhidas quanto à eficiência na redução de microrganismos, praticidade e viabilidade econômica. Quando as práticas foram aplicadas conjuntamente, obteve-se reduções consideráveis nas contagens microbianas. Após 48 horas de refrigeração do leite, as contagens foram reduzidas de 11,95x106 CFU/mL para 12,48x103 CFU/mL de aeróbios mesófilos, e de 18,10x106 CFU/mL para 5,38x103 CFU/mL de psicrotróficos. Os resultados mostram que as práticas propostas são eficientes, viáveis e facilmente adotáveis por produtores leiteiros,