In vitro screening and in vivo colonization pilot model of Lactobacillus plantarum LP5 and Campylobacter coli DSPV 458 in mice.

The objective of this work was to determine the antibacterial effect of Lactobacillus plantarum strains of pork origin against Campylobacter coli strains, and to conduct experimental colonization pilot models in mice for both microorganisms. Inhibition assays allowed evaluation and selection of L. plantarum LP5 as the strain with the highest antagonistic activity against C. coli and with the best potential to be used in in vivo study. Adult 6-week-old female Balb/cCmedc mice were lodged in two groups. The treated group was administered with 9.4 log10CFU/2 times/wk of L. plantarum LP5. L. plantarum LP5 was recovered from the feces and cecum of the inoculated mice. However, when bacteria stopped being administered, probiotic counts decreased. Experimental colonization with C. coli was carried out in five groups of mice. All animals were treated with antibiotics in their drinking water to weaken the indigenous microbiota and to allow colonization of C. coli. Four groups were administered once with different C. coli strains (DSPV458: 8.49 log10CFU; DSPV567: 8.09 log10CFU; DSPV570: 8.46 log10CFU; DSPV541: 8.86 log10CFU, respectively). After 8 h, mice inoculated with different C. coli strains were colonized because the pathogen was detected in their feces. L. plantarum LP5 tolerated the gastrointestinal conditions of murine model without generating adverse effects on the animals. C. coli DSPV458 colonized the mice without causing infection by lodging in their digestive tract, thus generating a reproducible colonization model. Both models combined could be used as protection murine models against pathogens to test alternative control tools to antibiotics.

Molecular epidemiology of Campylobacter jejuni isolates from the broiler production chain: first report of MLST profiles in Argentina.

Campylobacter jejuni is an important foodborne pathogen with global distribution. We describe a genotyping study of a collection of C. jejuni (n=137) isolated from different broiler farms and from multiple sites along the processing line in a slaughterhouse in Argentina during 2011, 2012 and 2015. The isolates were genotyped using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Based on the PFGE results, the isolates were grouped into 26 pulsotypes. Subsequently, the isolates representing these 26 pulsotypes were chosen for MLST genotyping, which identified 16 different sequence types (STs) and 6 clonal complexes (CCs) (21, 45, 48, 353, 354, 446). Several of the STs (n=7) have not been previously reported in the PubMLST.org database. The most prevalent CCs were 21, 45 (both associated with human campylobacteriosis worldwide) and 353. This study showed high genetic diversity among C. jejuni in the broiler production environment in Argentina with novel MLST genotypes.

Poultry blood from slaughterhouses: development of a biopreservation system to improve microbiological quality prior to transforming blood into by-products.

The aim of this study was to investigate the use of indigenous lactic acid bacteria (LAB) with specific additives as a Biopreservation System (BS) for poultry blood during its storage in slaughterhouses. The BS consisted of two LAB (Enterococcus faecalis DSPV 008SA or Lactobacillus salivarius DSPV 032SA) with 4 additives (lactose 2 g/l, yeast extract 0.4 g/l, ammonium citrate 0.4 g/l and NaCl 1 g/l). After 24 h storage at 30ºC, lower counts of enterobacteria, coliforms, Pseudomonas spp. and Staphylococcus aureus were evident in blood treated with the BS than in untreated blood. The ability of LAB to prevent haemolysis was evident. A decrease in pH was associated with control of spoilage microorganisms but it needed to be regulated to prevent coagulation of proteins. On the basis of these results it is recommended to supplement blood with a BS to avoid undesirable changes during blood storage before processing.

Probiotics and broiler growth performance: a meta-analysis of randomised controlled trials.

1. The aim of this meta-analysis was to investigate the effects of probiotics on the growth performance of broilers. PubMed, Scopus and Scholar Google databases were searched in all languages from 1980 to 2012. The studies in the meta-analysis were only selected if they were randomised and controlled experiments using broilers without apparent disease and the results were published in peer-reviewed journals. 2. A total of 48 and 46 studies were included to assess probiotic effects on body weight gain (BWG) and feed efficiency (FE), respectively. Probiotics increased BWG compared to controls (SMD = 0.661, 95% CI 0.499 to 0.822) and improved FE (SMD = - 0.281, 95% CI -0.404 to -0.157) in the pooled standardised mean difference random effect model, considering the source of heterogeneity and publication biases. However, there are evidences of publication bias and heterogeneity, so the results of this meta-analysis should be considered with caution. Applying the Duval and Tweedie's trim-and-fill methods, the adjusted value for BWG was 0.0594 (95% CI -0.122 to 0.242), and the adjusted value for FE did not show any modifications. 3. The meta-analysis showed that application of probiotics via water resulted in greater BGW and FE than administration through the feed. The effect was not related to the use of mono-strain or multi-strain probiotics, although it may depend on the strain used. The number of broilers and the duration of the experiments had an impact on the outcomes. 4. Additional studies should be conducted with the aim to identify the covariates which can explain the differences in the estimated effect sizes.

Effect of probiotic Lactiplantibacillus plantarum and chestnut flour (Castanea sativa mill) on microbiological and physicochemical characteristics of dry-cured sausages during storage.

The effect of chestnut flour (Castanea sativa Mill) on L. plantarum viability and physicochemical characteristics in a dry-cured sausage (Longaniza de Pascua) during storage is discussed. Four batches were prepared: CL with 3% chestnut flour added; CPL with 3% chestnut flour and 8.5 log CFU/g L. plantarum added; PL with 8.5 log CFU/g L. plantarum added and L, the batch control. The sausages were stored at 4 °C and 20 °C, and vacuum packed for 43 d. L. plantarum viability was affected by storage time (P < 0.001). However, higher L. plantarum counts at the final of storage were reached due to chestnut flour addition (P < 0.001). At room storage, chestnut flour caused a higher increase in TBARS values (P = 0.022). Nevertheless, all lipid oxidation treatments were in the range of accepted values at the sensory detection level. In conclusion, Longaniza de Pascua can be kept at 4 °C or 20 °C for 43 d without causing any rancidity problems.

Omega-3 enriched egg production: the effect of α -linolenic ω -3 fatty acid sources on laying hen performance and yolk lipid content and fatty acid composition.

1. Diets high in total lipids, saturated fatty acids, trans fatty acids, and having high ω-6:ω-3 fatty acid ratios, have been shown to be related to increased instances of coronary heart disease, while diets high in ω-3 fatty acids have been shown to decrease the risk. 2. Feeding ω-3 fatty acid diets to laying hens has been shown to improve the quality of eggs produced in terms of saturation and ω-3 content. 3. A study was undertaken to determine if the ω-3 fatty acid source, when fed to hens, influences the amount transferred to eggs. 4. Flaxseed and flaxseed oil, along with chia seed and chia seed oil, were the two main sources of ω-3 fatty acid examined during the 84 d trial. 5. All α-linolenic enriched treatments yielded significantly higher ω-3 fatty acid contents per g of yolk and per yolk, than the non-α-linolenic enriched diets. Chia oil and chia seed yielded 54·5 and 63·5% more mg of ω-3 fatty acid per g of yolk for the 56 d test period, and 13·4 and 66·2% more for the 84 d test period, than flaxseed oil and flaxseed, respectively. 6. The differences in omega-3 content were significant, except for the chia oil compared with the flax oil, at the end of the trial. 7. This trial has shown that differences in conversion exist among ω-3 fatty acid sources, at least when fed to hens, and indicates that chia may hold a significant potential as a source of ω-3 fatty acid for enriching foods, thereby making these foods a healthier choice for consumers.

Prevalence, associated risk factors, and antimicrobial resistance profiles of non-typhoidal Salmonella in large scale swine production in Córdoba, Argentina.

Non-typhoidal Salmonella is considered a major public health concern. The growing relevance of pigs as reservoir of Salmonella spp. has prompted several countries to set up surveillance and control programs to fight Salmonella infection in swine and reduce public health risk. In the last decade, pork production in Córdoba increased significantly to become one of the most important pig production provinces in Argentina. The aim of this study was to estimate Salmonella spp. prevalence and associated risk factors in large scale-farms in this province. Mesenteric lymph nodes (MLN) of 580 pigs from 20 finishing large-scale farms were collected between 2014 and 2015 to estimate Salmonella infection. A prevalence of 41.5% (95%CI: 37.6-45.6%) was observed. Two major risk factors were significantly associated with Salmonella infection, both related to the pre-slaughter period (distance from the farm to the slaughterhouse and lairage time), highlighting the need to pay special attention to pre-slaughter practices in the province. Shortening transport times and complying with national regulations for lairage time at slaughter may help to reduce the prevalence of infection. Sixteen different serovars were identified, being S. Anatum and S. Typhimurium the most prevalent ones. Moreover, two isolate of the monophasic variant of Salmonella Typhimurium (I 4,5,12:i:-) resistant to enrofloxacin and which also displayed multidrug resistance was isolated for first time from pigs in Córdoba. The moderate to high levels of antimicrobial resistance detected for antibiotics commonly used in the pig sector suggested the need for implementing a plan to limit their use in the province.

Quantification of FITC-labelled probiotic Lactobacillus salivarius DSPV 001P during gastrointestinal transit in broilers.

The knowledge related to the fate of probiotics in the complex environment of the intestinal microbiota in broilers is just beginning to be elucidated; however, it is not yet well understood. A good method to investigate the mechanisms by which probiotics mediate their effects is to mark probiotic bacteria and trace them. The aim of this research was to develop a new method to estimate in vivo fluorescein isothiocyanate (FITC)-labelled Lactobacillus salivarius DSPV 001P counts during passage through the gastrointestinal tract (GIT) of broilers. Forty-five, 1 d old Cobb broilers were used in this trial. Programmed necropsies were performed 30 min, 6 h, and 12 h after the administration of the probiotic bacterium, and samples of liver, crop, duodenum, caecum, and bursa of fabricius were collected. To determine the spatial and temporal transit of L. salivarius DSPV 001P in broilers, the number of bacteria as well as its respective fluorescent signal produced by FITC were measured. In order to observe the relationship between the variables, a logistic regression analysis was applied. The amount of fluorescence could be used as an indicator of fluorescent probiotic bacteria in the crop and duodenum 30 min after probiotic bacterium supplementation. In addition, the fluorescent signal could be used to estimate bacterial counts in caecum 6 and 12 h after L. salivarius DSPV 001P administration. To the best of our knowledge, this research is the first in vivo trial to employ the bacterial FITC-labelling technique in order to enumerate probiotic bacteria during gastrointestinal transit in broilers.

Probiotic effect on calves infected with Salmonella Dublin: haematological parameters and serum biochemical profile.

The aim of this study was to evaluate the effect of a probiotic/lactose inoculum on haematological and immunological parameters and renal and hepatic biochemical profiles before and during a Salmonella Dublin DSPV 595T challenge in young calves. Twenty eight calves, divided into a control and probiotic group were used. The probiotic group was supplemented with 100 g lactose/calf/d and 1010 cfu/calf/d of each strain of a probiotic inoculum composed of Lactobacillus casei DSPV318T, Lactobacillus salivarius DSPV315T and Pediococcus acidilactici DSPV006T throughout the experiment. The pathogen was administered on day 11 of the experiment, at an oral dose of 109 cfu/animal (LD50). Aspartate aminotransferase (AST), gamma glutamyl transpeptidase (GGT), urea, red blood cells, haemoglobin, haematocrit, mean cell haemoglobin (MCH), mean corpuscular volume, mean corpuscular haemoglobin concentration (MCHC), white blood cells, lymphocytes, neutrophils, band neutrophils, monocytes, eosinophils, basophils and the neutrophils/lymphocytes ratio were measured on days 1, 10, 20 and 27 of the experiment. In addition, animals were necropsied to evaluate immunoglobulin A (IgA) production in the jejunal mucosa. The most significant differences caused by the administration of the inoculum/lactose were found during the acute phase of Salmonella challenge (9 days after challenge), when a difference between groups in neutrophils/lymphocytes ratio were detected. These results suggest that the probiotic/lactose inoculum administration increases the calf's ability to respond to the disease increasing the systemic immune response specific. No differences were found in haemoglobin, haematocrit, MCH, MCHC, AST, urea, GGT, band neutrophils, eosinophils, monocytes and IgA in the jejunum between the two groups of calves under the experimental conditions of this study. Further studies must be conducted to evaluate different probiotic/pathogens doses and different sampling times, to achieve a greater understanding of the effects of this inoculum on intestinal infections in young calves and of its mechanism of action.

Quantitative Risk Assessment of Human Trichinellosis Caused by Consumption of Pork Meat Sausages in Argentina.

In Argentina, there are three known species of genus Trichinella; however, Trichinella spiralis is most commonly associated with domestic pigs and it is recognized as the main cause of human trichinellosis by the consumption of products made with raw or insufficiently cooked pork meat. In some areas of Argentina, this disease is endemic and it is thus necessary to develop a more effective programme of prevention and control. Here, we developed a quantitative risk assessment of human trichinellosis following pork meat sausage consumption, which may be used to identify the stages with greater impact on the probability of acquiring the disease. The quantitative model was designed to describe the conditions in which the meat is produced, processed, transported, stored, sold and consumed in Argentina. The model predicted a risk of human trichinellosis of 4.88 × 10(-6) and an estimated annual number of trichinellosis cases of 109. The risk of human trichinellosis was sensitive to the number of Trichinella larvae that effectively survived the storage period (r = 0.89), the average probability of infection (PPinf ) (r = 0.44) and the storage time (Storage) (r = 0.08). This model allowed assessing the impact of different factors influencing the risk of acquiring trichinellosis. The model may thus help to select possible strategies to reduce the risk in the chain of by-products of pork production.

TBA test by an extractive method applied to 'paté'.

The aim of this work was to study the influence of different conditions on the thiobarbituric acid (TBA) test, determined by the extractive method, as a measure of lipid oxidation in 'paté'. Different extracting agents (trichloroacetic acid and trichloroacetic acid in 2 M phosphoric acid), different concentrations of these acids (10, 15 and 20%), different reaction times and temperatures (35 min at 100 °C and 900 min at room temperature) and the effect of sulfanilamide additions were evaluated. All the samples were measured by HP 8451A Diode ARRAY spectrophotometer at 532 nm. Significant differences between 35 and 900 min of MA-TBA reaction time were found. Highest TBA numbers were found in samples with a reaction time of 35 min in a thermostatically controlled waterbath at 100 °C, rather than 900 min at room temperature. Lowest TBA numbers were found in samples with the addition of sulfanilamide rather than without it. The 10% TCA solution gave the best recovery percentages for 'paté', making it the preferred extractant.

Modification of physico-chemical and colour parameters during the marketing of 'paté'.

The evolution of different physico-chemical parameters (iodine number, TBA test, residual nitrite level, pH and moisture) and CIE L (∗)a (∗)b (∗) colour evolution, in pork liver 'pate', with and without sodium ascorbate addition, were studied. The samples were stored in an illuminated display cabinet at different lux intensities and in the dark. The regression equations were obtained for each one of the parameters under study, from the first day of exposure to light. The sodium ascorbate showed a protective effect on the photo-oxidation mechanism (measured by TBA test), but it did not have an influence on the samples in darkness. In both types of paté exposed to light, fading takes place and the most important variations in colour parameters are produced during the first 24 h of exposure to light.

[5 years of experience in arterial vascular surgery (January 1972-December 1977)]. / Cinque anni di esperienza in chirurgia vascolare arteriosa (Periodo gennaio 1972 - dicembre 1977).

The Authors report their own experience in arterial vascular surgery in the Hospital of Città di Castello from the 1st January 1972 to the 31st December 1977. 87 surgical interventions have been carried out (50 thromboendarterectomy, 15 by-passes, 9 thromboembolectomy and 15 various). The indications were atherosclerosis, thrombosis, aneurysms, arteriovenous fistulas, hypertrophic sthenosis and injuries. They discuss the indications, the surgical technics used, the early and late complications and the results. The long-term results good in only 64% of the cases have to be correlated with an enlargement of the indication to surgical operation.

Quantitative risk assessment of human campylobacteriosis by consumption of salad cross-contaminated with thermophilic Campylobacter spp. from broiler meat in Argentina.

Here, we developed a quantitative risk assessment for thermophilic Campylobacter spp. related to the consumption of salad prepared alongside broiler meat. The assessment considered initial contamination levels, cross-contamination and decontamination events during the broiler slaughter process and distribution, and storage and consumption patterns in Argentina and other Latin American countries. The model predicted an infection risk of 3.32×10(-4) per serving. This estimation was variable according to the dose-response model used. Considering the number of chickens slaughtered annually in Argentina, the estimated number of people who could suffer campylobacteriosis related to poultry meat consumption was, on average, 484,304. The risk of human campylobacteriosis was most sensitive to the probability of infection from a Campylobacter (r=0.72), the number of Campylobacter spp. per serving (r=0.40), the frequency of washing the cutting board (r=-0.31), the preparation of raw poultry before salad using the same cutting board (r=0.14), and the frequency of hand washing (r=-0.14). The most sensitive stages of the process identified through the risk assessment can be used as a basis for measures of risk management. Public campaigns on hygiene habits during food preparation at home should focus on the importance of washing the cutting board before preparing raw and ready-to-eat foods and of washing the hands during food preparation.

Occurrence of thermotolerant Campylobacter spp. at different stages of the poultry meat supply chain in Argentina.

AIMS: The objectives of this study were to investigate the occurrence and concentration of thermotolerant Campylobacter spp. at different stages of the poultry meat supply chain in Argentina. METHODS: Three integrated poultry companies were sampled. Each supply chain was considered at different stages from the reproductive farm to chicken meat at a retail market. The stages sampled were: (a) hens from breeder flocks, (b) eggs in the incubator, (c) broiler chickens in flocks (aged <1 week and >5 weeks), (d) chickens at a slaughterhouse, and (e) chicken meat at a retail market. The chickens sampled along each supply chain were in the same batch. Samples collected were: (a) cloacal samples from hens and chickens on the farms, (b) fertile eggs, (c) feed, water and litter from flocks, (d) chicken carcasses from the slaughterhouse and retail market, and (e) caeca and livers from the slaughterhouse. Samples obtained were examined for Campylobacter spp. The isolates were biotyped and the genus and species identified by PCR. Campylobacter spp. on chicken carcasses at slaughterhouse and retail market were enumerated. RESULTS: The highest proportions of Campylobacter positive samples were observed in carcasses at retail (25/30, 83.3%) and faecal samples from breeding hens (27/45, 60.0%). Only 3.3% (3/90) samples collected from broiler chickens aged <1 week were positive, but the percentage of positive samples had risen to 28.9% (26/90) by the end of the rearing period. The proportions of Campylobacter positive carcasses and caecal contents at the slaughterhouse were both 33.3% (10 of 30 samples each). The concentration of Campylobacter contamination observed on carcasses at retail markets ranged from no bacteria/carcass to 3.71 log10 cfu/carcass. CONCLUSIONS: The data obtained provide essential information for future quantitative risk assessments aiming to estimate the probability of a person contracting campylobacteriosis following consumption of broiler meat in Argentina. CLINICAL RELEVANCE: The proportions of Campylobacter-positive samples found in this preliminary study indicate that a large proportion of the cases of human gastroenteritis in Argentina may be due to this pathogen. Human cases of gastroenteritis should be studied in greater detail and measures should be developed to reduce the proportion of poultry products that are contaminated by Campylobacter species.

Impact of probiotic administration on the health and fecal microbiota of young calves: a meta-analysis of randomized controlled trials of lactic acid bacteria.

Before weaning, dairy calves are susceptible to many pathogens which can affect their subsequent performance. The use of lactic acid bacteria (LAB) has been identified as a tool to maintain the intestinal microbial balance and to prevent the establishment of opportunistic pathogenic bacterial populations. However, a consensus has not been reached as to whether probiotics may be effective in reducing the prevalence of gastrointestinal diseases in young calves. The aim of this meta-analysis was to assess the effect of probiotics on diarrhea incidence and the intestinal microbial balance. LAB supplementation has been shown to exert a protective effect and to reduce the incidence of diarrhea (relative risk, RR=0.437, 95% confidence interval (CI) 0.251-0.761). In the subanalysis, this protective effect of the probiotics against diarrhea was observed only in trials that used whole milk (RR=0.154, 95% CI 0.079-0.301) and trials that used multistrain inocula (RR=0.415, 95% CI 0.227-0.759). Probiotics did not improve the fecal characteristics (standardized mean difference, SMD=-0.4904, 95% CI -1.011-0.035) and were unable to change the LAB:coliforms ratio (SMD=0.016, 95% CI -0.701-0.733). Probiotics showed a beneficial impact on the LAB:coliforms ratio in the subanalysis that included trials that used whole milk (SMD=0.780, 95% CI 0.141-1.418) and monostrain inocula (SMD=0.990, 95% CI 0.340-1.641). The probability of significant effects (probiotic positive effect) in a new study was >0.70 for diarrhea and fecal consistency. Whole milk feeding improved the action of the probiotic effect on the incidence of diarrhea and LAB:coliforms ratio. The probability to find significant effects in the diarrhea frequency and LAB:coliforms ratio was higher (P>0.85) if the new studies were conducted using whole milk to feed calves. This paper defines the guidelines to standardize the experimental designs of future trials. LAB can be used as growth promoters in calves instead of antibiotics to counteract the negative effects of their widespread use.