Occurrence of Aflatoxin M1 and Estimate of Dietary Exposure in Cheeses from Organic and Conventional Production Systems.

This study aimed to compare AFM1 occurrence in different cheese types produced by organic and conventional systems; and to evaluate the risk of food exposure to AFM1. A total of 176 commercial cheeses of 17 types were analyzed, 84 of organic and 92 of conventional production. Determination of AFM1 was performed by high- performance liquid chromatography (HPLC), being detected in 30.5% of samples, with 4.8% of organic cheese samples presenting quantifiable AFM1 values between 0.88 and 1.50 µg/kg. On the other hand, 4.3% of conventional cheese samples with values between 0.79 and 6.70 µg/kg. Two conventional cheese samples were above the limit of AFM1 allowed for cheeses by the Brazilian legislation. No statistical difference were found between organic and conventional cheeses regarding the occurrence (p = 0.1780) and concentration of AFM1 (p = 0.1810), according to the Chi-square and the T test, respectively. Estimated daily intake (EDI) and hazard index (HI) of dietary exposure to AFM1 were 0.26 ng/kg/day and 1.28 ng/kg/day, respectively, for conventional cheese samples, and 0.09 ng/kg/day and 0.47 ng/kg/day for organic samples, with no statistical difference for EDI (p = 0.1729) and HI (p = 0.1802) between the two production systems. Comparison between several cheese types from conventional and organic systems indicated that AFM1 is an obstacle to dairy production. Control and prevention of AFM1 contamination, as well as detoxification methods in the final products, are necessary. In the case of organic products, additional research is needed in order to determine which control and detoxification methods should be allowed in this production system.

Molecular detection and phylogenetic analysis of Cyprinid herpesvirus 3 in Brazilian ornamental fish.

Cyprinid herpesvirus 3 has a worldwide distribution and presents high mortality rates in species of Cyprinus carpio, causing serious economic loss to the global aquaculture industry. The description of this infection in other ornamental fish species is still limited. For this purpose, 100 ornamental fish from 24 different species were tested by PCR for Cyprinid hespesvirus 3 and the positive samples represented 6% of the tested samples. Phylogenetic reconstruction, based on the Thymidine Kinase gene, revealed the existence of two distinct clades. One clade grouped a Brazilian sample with European and Asian genotypes of CyHV-3 and a second clade, containing only Brazilian sequences described in this study. All of the Brazilian sequences showed identity values greater than 97.7% when compared to each other. This is the first report of the occurrence of Cyprinid herpesvirus 3 in ornamental fish species in Brazil. These results in association with further studies of viral isolation and characterization can help in establishing effective surveillance and disease control program.

Norovirus GII and astrovirus in shellfish from a mangrove region in Cananéia, Brazil: molecular detection and characterization.

In recent years, annual cases of gastroenteritis have been reported in the world at high rates, suggesting an association with the consumption of shellfish with enteric viruses in their tissues. Anthropic activities are considered a source of environmental pollution and the main responsible for contamination by pathogenic microorganisms in aquatic environments. The objective of this study was to evaluate, by RT-semi-nested PCR, the presence of astrovirus (AstV) and norovirus genogroup II (NoV GII) in mussels (Mytella falcata) and oysters (Crassostrea brasiliana) collected in two sites of the Lagunar Complex of Cananéia, State of São Paulo, Brazil. A total of 150 samples of mussels and oysters (75 samples each) were analyzed. AstV was not identified in any shellfish sample. NoV GII was detected in 21 samples (14%), 8 mussel samples (38%), and 13 oyster samples (62%). From the 21 positive samples, 16 were analyzed by nucleotide sequencing. The molecular characterization revealed that Brazilian samples were grouped into clades along with other sequences from Brazil, Japan, and Mexico. There was 93.8-100% amino acid sequence similarity among the samples in this study and > 94.9% when compared with the strains isolated from clinical cases in Brazil. The screening of shellfish for the presence of health-significant enteric viruses can help prevent outbreaks among consumers and contribute to the improvement of the estuarine environment.

Detection and molecular characterization of Lymphocystivirus in Brazilian ornamental fish.

The aim of this study is to report the occurrence of Lymphocystivirus in Brazilian ornamental fish. From 25 ornamental fish species submitted for molecular diagnosis, only one sample (Pomacanthus imperator) was positive, and its viral nucleotide sequence obtained clustered with sequences of genotype VII. To our knowledge, this is the first report on the genetic characterization of Lymphocystivirus in Brazil.

Genome analysis of Ranavirus frog virus 3 isolated from American Bullfrog (Lithobates catesbeianus) in South America.

Ranaviruses (family Iridoviridae) cause important diseases in cold-blooded vertebrates. In addition, some occurrences indicate that, in this genus, the same virus can infect animals from different taxonomic groups. A strain isolated from a Ranavirus outbreak (2012) in the state of Sao Paulo, Brazil, had its genome sequenced and presented 99.26% and 36.85% identity with samples of Frog virus 3 (FV3) and Singapore grouper iridovirus (SGIV) ranaviruses, respectively. Eight potential recombination events among the analyzed sample and reference FV3 samples were identified, including a recombination with Bohle iridovirus (BIV) sample from Oceania. The analyzed sample presented several rearrangements compared to FV3 reference samples from North America and European continent. We report for the first time the complete genome of Ranavirus FV3 isolated from South America, these results contribute to a greater knowledge related to evolutionary events of potentially lethal infectious agent for cold-blooded animals.

Genetic diversity of bovine Picobirnavirus, Brazil.

Picobirnaviruses (PBVs) are emerging and opportunistic viruses with possible zoonotic potential. In this study, we present the detection, molecular characterization, and genotypic differentiation of PBVs from genogroup I in bovine stool samples from different Brazilian regions. A high proportion of PCR-positive samples (23.4%) was detected in a total of 77 analyzed. Nucleotide identity, alignment, and phylogenetic analyses revealed high diversity among the studied sequences. The results obtained indicate, for the first time, the circulation of bovine PBVs belonging to genogroup I in different Brazilian states, with heterogeneous phylogenetic-clustering profiles.

Molecular detection and phylogenetic analysis of megalocytivirus in Brazilian ornamental fish.

Megalocytiviruses have a worldwide distribution, causing serious economic loss to the global aquaculture industry. They also present a threat to ornamental fish trade because megalocytiviral infections have unspecified symptoms, making early diagnosis difficult. In this study, 100 ornamental fish from 24 different species were tested by PCR for megalocytivirus, with a 47% positive rate being identified. Phylogenetic reconstruction, based on the major capsid protein (MCP) gene, clustered all Brazilian samples into a single clade, showing identity values ranging from 99% to 100% when compared to each other. This is the first report of megalocytivirus infection in some ornamental fish species in Brazil.

Cleaning and disinfection programs against Campylobacter jejuni for broiler chickens: productive performance, microbiological assessment and characterization.

Detailed cleaning and disinfection programs aims to reduce infection pressure from microorganisms from one flock to the next. However, studies evaluating the benefits to poultry performance, the sanitary status of the facilities, and the sanitary quality of the meat are rarely found. Thus, this study was designed to evaluate 2 cleaning and disinfecting programs regarding their influence on productive performance, elimination of Campylobacter, and characterization of Campylobacter jejuni strains when applied to broiler chickens' facilities. Two subsequent flocks with 960 birds each were distributed into 32 pens containing 30 birds each. In the first, the whole flock was inoculated with a known strain of Campylobacter jejuni in order to contaminate the environment. In the second flock, performance and microbiological evaluations were done, characterizing an observational study between 2 cleaning and disinfection programs, regular and proposed. The regular program consisted of sweeping facilities, washing equipment and environment with water and neutral detergent. The proposed cleaning program consisted of dry and wet cleaning, application of 2 detergents (one acid and one basic) and 2 disinfectants (250 g/L glutaraldehyde and 185 g/L formaldehyde at 0.5% and 210 g/L para-chloro-meta-cresol at 4%). Total microorganism count in the environment and Campylobacter spp. identification were done for the microbiological assessment of the environment and carcasses. The positive samples were submitted to molecular identification of Campylobacter spp. and posterior genetic sequencing of the species identified as Campylobacter jejuni. The birds housed in the facilities and submitted to the proposed treatment had better performance when compared to the ones in the regular treatment, most likely because there was a smaller total microorganism count on the floor, walls, feeders and drinkers. The proposed program also resulted in a reduction of Campylobacter spp. on floors, drinkers and birds. Moreover, it was possible to identify 6 different Campylobacter jejuni strains in the facilities. The proposed treatment resulted in a positive influence on the birds' performance and reduction of environment contamination for broiler chickens.

Molecular characterization and genetic diversity of bovine Kobuvirus, Brazil.

It is suggested that Bovine kobuvirus (BKV) is involved in the etiology of gastroenteric diseases especially among calves; however, this association remains unknown. This study evaluated 216 fecal samples from cattle with and without diarrhea symptoms obtained from different regions of Brazil. A 216 bp fragment of the BKV 3D gene was amplified by RT-PCR in 14.4 % (31/216) of the studied samples, and 17 samples were subjected to nucleotide sequencing. All positive samples were obtained from animals aged less than 5 months, and most of animals presented diarrhea (p < 0.05). Phylogenetic analyses showed that the obtained sequences were grouped within the genogroup 2 of BKV forming subclades specific for each Brazilian municipality sampled. In addition, the alignment of the sequences revealed differences of nucleotides between sequences from different locations. Our results indicate for the first time that there is a regional genotypic differentiation of BKV in Brazil.

Microencapsulated jabuticaba (Myrciaria cauliflora) extract added to fresh sausage as natural dye with antioxidant and antimicrobial activity.

The aim was to evaluate the addition of microencapsulated jabuticaba extract (MJE) to fresh sausage as natural dye with antioxidant and antimicrobial activity. Fresh sausages without dye, with cochineal carmine and with addition of 2% and 4% MJE were evaluated for chemical, microbiological and sensory properties during 15days of refrigerated storage. TBARS values were lower (P<0.05) throughout the storage period in sausages with 2% and 4% MJE (below 0.1mg of malondialdehyde/kg sample) than in control and carmine treatments (from 0.3 to 0.6mg of malondialdehyde/kg sample). T2% and T4% also showed lower microbial counts on storage days 4 and 15 for APCs. The addition of 4% MJE negatively influenced (P<0.05) sensory color, texture and overall acceptance attributes. On the other hand, T2% presented similar (P>0.05) sensory acceptance to control and carmine treatments in most of the attributes evaluated except for a decrease in color. Thus, addition of 2% MJE to fresh sausage can be considered as a natural pigment ingredient.

Biofilm-producing ability and efficiency of sanitizing agents against Prototheca zopfii isolates from bovine subclinical mastitis.

The objectives of the present study were to evaluate (1) the capacity of the microalga Prototheca zopfii isolated from subclinical bovine mastitis cases to form biofilms; and (2) the resistance of these isolates to sanitizing agents. Ten isolates of P. zopfii from cows with subclinical mastitis (somatic cell count>200×10(3) cells/mL), distributed in 5 dairy farms, were evaluated for their capacity to form biofilms in polystyrene microplate assays and stainless steel coupons, at 25°C and 37°C±1°C. Prototheca zopfii were isolated from milk samples via microbiological culture and analyzed by 18S rRNA gene sequencing. Biofilm formation on the coupons was observed by scanning electron microscopy. The resistance to sanitizing agents was assessed using the biofilm-forming P. zopfii isolates in stainless steel coupon assays, which were subjected to 3 sanitizers: peracetic acid, sodium hypochlorite, and iodine solution. To evaluate resistance to the sanitizers, the minimum inhibitory concentration (MIC) technique was performed using decreasing concentrations of the sanitizing agents (20, 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039, and 0.019 g/L). After inoculating the isolates, all concentrations were evaluated at 3 distinct incubation periods (24, 48, and 72 h) to assess the effect of incubation time on the MIC. Using the polystyrene microplate assays, 1 isolate showed weak biofilm production, 5 moderate, and 4 strong, when incubated at 25°C±1. For isolates incubated at 37°C±1, 6 showed weak biofilm production and 4 moderate. All P. zopfii isolates (n=10) had the capacity to form biofilms on stainless steel coupons. The longer the incubation period of the P. zopfii isolates at different dilutions, the greater the concentrations of sanitizer needed to prevent growth of the microalgae under the tested conditions. We detected a significant effect of sanitizer and time of incubation (24, 48, and 72 h) on MIC values against P. zopfii isolates. The isolates were sensitive in vitro to peracetic acid (MIC90≥0.019 g/L), sodium hypochlorite (MIC90≥0.312 g/L), and iodine solution (MIC90≥0.625 g/L), after 24 h of incubation (where MIC90=concentration needed to inhibit 90% of isolates). Of the tested sanitizers, peracetic acid had the greatest efficiency against P. zopfii. We conclude that P. zopfii isolates are capable of biofilm production, which may contribute to their persistence in a milking and dairy environment.

Molecular detection and phylogenetic analysis of bovine astrovirus in Brazil.

Bovine astrovirus (BoAstV) is associated with gastroenterical disorders such as diarrhea, particularly in neonates and immunocompromised animals. Its prevalence is >60 % in the first five weeks of the animal's life. The aim of this study was to detect and perform a phylogenetic analysis of BoAstV in Brazilian cattle. A prevalence of 14.3 % of BoAstV in fecal samples from 272 head of cattle from different Brazilian states was detected, and 11 samples were analyzed by nucleotide sequencing. The majority of positive samples were obtained from diarrheic animals (p < 0.01). Phylogenetic analysis revealed that Brazilian samples were grouped in clades along with other BoAstV isolates. There was 74.3 %-96.5 % amino acid sequence similarity between the samples in this study and >74.8 % when compared with reference samples for enteric BoAstV. Our results indicate, for the first time, the occurrence of BoAstV circulation in cattle from different regions of Brazil, prevalently in diarrheic calves.

Relação entre psicrotróficos e frações de caseína do leite longa vida durante o armazenamento / Relationship between psychrotrophic and casein fractions of long life milk during storage

As bactérias psicrotróficas podem afetar a qualidade de diversos produtos lácteos. A relação entre a contagem de psicrotróficos no leite pasteurizado e as frações de caseína do leite longa vida foi avaliada. O processo ultra alta temperatura foi conduzido de acordo com a seguinte sequência: pasteurização a 72-75 ºC por 15 s, pré-aquecimento a 85º C, esterilização a 142-145 ºC por 2 s por injeção direta de vapor ao leite, remoção da água condensada, resfriamento a 70 ºC, homogeneização, resfriamento a 20 ºC, envase asséptico em embalagens cartonadas de 1L e armazenamento à temperatura ambiente por 120 dias. As contagens de psicrotróficos foram determinadas no leite pasteurizado por contagem padrão em placas. As frações de caseína (as1, as2, beta e k) foram avaliadas no leite longa vida nos dias 8 e 120 de armazenamento por cromatografia líquida de alta eficiência. Foi observado aumento na quantidade de as2-caseína e redução da as2-caseína como porcentagem da caseína total ao final do armazenamento (P<0,05). Houve correlação positiva (P<0,05) no dia 120 de armazenamento entre psicrotróficos e a fração k-caseína.

Psycrothrophic bacteria can affect the quality of several dairy products. The relationship between psycrothrophic counts in pasteurized milk and casein fractions of ultra-high-temperature (UHT) milk was evaluated. The UHT process was performed according to the following sequence: pasteurization at 72-75 oC for 15 s, pre-heating at 85 oC, sterilization at 142-145 ºC for 2 s by direct steam injection into milk, removing of the condensed water, cooling to 70 ºC, homogenization, cooling to 20 ºC, bottling aseptically in 1-L sterile carton boxes and storage at room temperature for 120 days. Psycrothrophic counts were determined in pasteurized milk by standard plate count. Casein fractions (as1, as2, beta and k) were analyzed in UHT milk on days 8 and 120 by high performance liquid chromatography. It was observed increase in quantity of as2-casein and reduction of as2-casein as a percentage of total casein at the end of storage period (P<0,05). There was a positive correlation (P<0,05) on day 120 of storage between psychrotrophic and the k-casein fraction (P<0.05).

Composition, functional properties and sensory characteristics of Mozzarella cheese manufactured from different somatic cell counts in milk

In the present study, composition, functional properties and sensory characteristics of Mozzarella cheese produced from milk with somatic cell counts (SCC) at low (<200,000 cells/mL), intermediate (≈400,000 cells/mL) and high (>800,000 cells/mL) levels were investigated. Three batches of cheese were produced for each SCC category. The cheeses were vacuum packed in plastic bags and analysed after 2, 9, 16, 23 and 30 days of storage at 4ºC. SCC level did not affect the moisture, fat, total protein and ash content, mesophilic and psychrotrophic bacteria, and sensory parameters of Mozzarella cheese. However, meltability increased in cheese manufactured from high SCC milk. Results indicated that raw milk used to produce Mozzarella cheese should not contain high SCC (>800,000 cells/mL) in order to avoid changes in the functional properties of the Mozzarella cheese.

No presente estudo foram investigadas a composição, as propriedades funcionais e as características sensoriais do queijo Mussarela produzido a partir de leite com contagens de células somáticas (CCS) em níveis baixos (<200.000 CS/mL), intermediários (≈400.000 CS/mL) e altos (>800.000 CS/mL). Foram produzidos 3 lotes de queijo para cada CCS. Os queijos foram embalados a vácuo e analisados após 2, 9, 16, 23 e 30 dias de armazenamento a 4ºC. O nível de CS não afetou a umidade, os teores de gordura, proteína total e cinzas, os níveis de bactérias mesófilas e psicrotróficas, e os parâmetros sensoriais do queijo Mussarela. Entretanto, houve aumento da capacidade de derretimento no queijo fabricado com leite de alta CCS. Os resultados indicam que o leite cru utilizado para a produção de queijo Mussarela não deve conter níveis de CS acima de 800.000/mL, para evitar alterações nas propriedades funcionais do queijo Mussarela.