Residual concentrations of antimicrobial growth promoters in poultry litter favour plasmid conjugation among Escherichia coli.

Considering that plasmid conjugation is a major driver for the dissemination of antimicrobial resistance in bacteria, this study aimed to investigate the effects of residual concentrations of antimicrobial growth promoters (AGPs) in poultry litter on the frequencies of IncFII-FIB plasmid conjugation among Escherichia coli organisms. A 2 × 5 factorial trial was performed in vitro, using two types of litter materials (sugarcane bagasse and wood shavings) and five treatments of litter: non-treated (CON), herbal alkaloid sanguinarine (SANG), AGPs monensin (MON), lincomycin (LCM) and virginiamycin (VIR). E. coli H2332 and E. coli J62 were used as donor and recipient strains, respectively. The presence of residues of monensin, lincomycin and virginiamycin increased the frequency of plasmid conjugation among E. coli in both types of litter materials. On the contrary, sanguinarine significantly reduced the frequency of conjugation among E. coli in sugarcane bagasse litter. The conjugation frequencies were significantly higher in wood shavings compared with sugarcane bagasse only in the presence of AGPs. Considering that the presence of AGPs in the litter can increase the conjugation of IncFII-FIB plasmids carrying antimicrobial resistance genes, the real impact of this phenomenon on the dissemination of antimicrobial resistant bacteria in the poultry production chain must be investigated.

Swine as reservoirs of zoonotic borderline oxacillin-resistant Staphylococcus aureus ST398.

Methicillin resistance mediated by the mecA gene in Staphylococcus aureus, also known as "true MRSA", is typically associated with high oxacillin MIC values (≥8 mg/L). Because non-mecA-mediated oxacillin resistant S. aureus phenotypes can also cause hard-to-treat diseases in humans, their misidentification as methicillin-susceptible S. aureus strains (MSSA) can compromise the efficiency of the antimicrobial therapy. These strains have been refereed as Borderline Oxacillin-Resistant S. aureus (BORSA) but their characterization and role in clinical microbiology have been neglected. Considering the increasing importance of livestock-associated methicillin-resistant S. aureus ST398 (LA-MRSA) as an emerging zoonotic pathogen worldwide, this study aimed to report the genomic context of oxacillin resistance in porcine S. aureus ST398 strains. S. aureus isolates were recovered from asymptomatic pigs from three herds. Oxacillin MIC values ranged from 4 to 32 mg/L. MALDI-TOF-confirmed isolates were screened for mecA and mecC by PCR and genotyped by means of PFGE and Rep-PCR. Seven isolates were whole genome sequenced. None of the isolates harbored the mecA gene or its variants. Although all seven sequenced isolates belonged to one sequence type (ST398), two different spa types (t571 and t1471) were identified. All isolates harbored conserved blaZ gene operon and no mutations on genes encoding for penicillin-binding-proteins were detected. Genes conferring resistance against other drugs such as aminoglycosides, chloramphenicol, macrolide, lincosamide and streptogramin (MLS), tetracycline and trimethoprim were also detected. Isolates also harbored virulence genes encoding for adhesins (icaA; icaB; icaC; icaD; icaR), toxins (hlgA; hlgB; hlgC; luk-PV) and protease (aur). Pigs can serve as reservoirs of non-mecA-mediated oxacillin-resistant ST398 strains potentially pathogenic to humans. Considering that mecA has been the main target to screen methicillin-resistant staphylococci, the occurrence of BORSA phenotypes is probably underestimated in livestock.

Enrichment of the amnion with threonine in chicken embryos affects the small intestine development, ileal gene expression and performance of broilers between 1 and 21 days of age.

The effects of in ovo feeding with threonine (Thr) on intestinal morphology, ileal gene expression and performance of broiler chicken between 1 and 21 d of age (d) were assessed. On day 17.5 of incubation, fertile eggs were randomly allotted to 5 treatments of Thr injection in the amniotic fluid (0; 1.75; 3.5; 5.25; 7%, corresponding to 17.5; 35; 52.5 and 70 mg Thr/mL). After hatch, chicks were given a commercial corn-soybean diet up to 21 d. Daily feed intake (FI), body weight (BW), and food conversion ratio (FCR) were measured from 1 to 7, 14, and 21 d of age. The ileal gene expression of mucin (MUC2), peptide transporter (PepT1), and aminopeptidase enzyme (APN) were evaluated on day of hatch and at 21 d, as well as intestinal morphometric traits. In ovo feeding with threonine significantly increased final weight (FI) and weight gain (WG) and decreased FCR in the period from 1 to 21 d. Threonine levels affected beneficially the villus height, vilo: crypt ratio and villus area on day of hatch and at 21 d. At hatch, all Thr levels increased the expression of MUC2 and PepT1 compared to the control group. APN expression also increased, but for the lowest and the highest threonine levels (1.75 and 7%). At 21 d, there was no effect of threonine on the expression of MUC2, PepT1, and APN. In conclusion, in ovo threonine feeding beneficially affected the morphological and functional development of the intestinal mucosa, which ensured improved performance of chicks at hatch and at 21 d.

Effect of l-glutamic acid supplementation on performance and nitrogen balance of broilers fed low protein diets.

The aim of this study was to evaluate the effect of protein reduction and supplementation of l-glutamic acid in male broiler diets. A total of 648 chicks of the Cobb 500 strain were distributed in a completely randomized design with six treatments and six replications with eighteen birds per experimental unit. The study comprised pre-starter (1-7 days), starter (8-21 days), growth (22-35 days) and final (36-45 days) phases. The first treatment consisted of a control diet formulated according to the requirements of essential amino acids for each rearing phase. The second and third treatments had crude protein (CP) reduced by 1.8 and 3.6 percentage points (pp) in relation to the control diet respectively. In the fourth treatment, l-glutamic acid was added to provide the same glutamate level as the control diet, and in the last two treatments, the broilers were supplemented with 1 and 2 pp of glutamate above that of the control diet respectively. The reduction in CP decreased the performance of broilers and the supplementation of l-glutamic acid did not influence performance when supplied in the diets with excess of glutamate. The lowest excreted nitrogen values were observed in the control diet, and treatments 2 and 3, respectively, in comparison with treatments with the use of l-glutamic acid (5 and 6). Retention efficiency of nitrogen was better in the control diet and in the treatment with a reduction of 1.8 pp of CP. It was verified that the serum uric acid level decreased with the CP reduction. A reduction in CP levels of up to 21.3%, 18.8%, 18.32% and 17.57% is recommended in phases from 1 to 7, 8 to 21, 22 to 35 and at 36 to 42 days, respectively, with a level of glutamate at 5.32%, 4.73%, 4.57%, 4.38%, also in these phases.

Substituição de minerais inorgânicos por orgânicos na alimentação de poedeiras semipesadas / Substitution of inorganic minerals for organic minerals in the diet of semi heavy layers

Avaliou-se o efeito da suplementação dietética de fontes orgânicas de microminerais para galinhas poedeiras sobre o desempenho e a qualidade do ovo. Foram utilizadas 216 aves de linhagem comercial, distribuídas num delineamento inteiramente ao acaso, com quatro tratamentos e nove repetições, com seis aves por parcela. O experimento teve duração de 140 dias, divididos em cinco ciclos de 28 dias. Os tratamentos consistiram de uma dieta basal formulada para atender às exigências nutricionais das aves, suplementada com minerais na forma inorgânica ou porcentagens de minerais orgânicos: T1 - Basal + 100% minerais inorgânicos; T2 - Basal + 100% minerais orgânicos; T3 - Basal + 66% minerais orgânicos; T4 - Basal + 33% minerais orgânicos. A produção de ovos, peso do ovo, massa de ovo, conversão alimentar por massa e dúzia de ovo, porcentagem de albúmen, gravidade específica, espessura de casca e margem bruta relativa apresentaram melhores resultados quando os minerais inorgânicos são substituídos em 66% pelos orgânicos.

This study evaluated the effect of the supplementation with organic microminerals on performance and egg quality of semi-heavy layers. Dekalk Brown layers (n=216) were distributed according to a completely randomized design into four treatments, nine repetitions and six birds per repetition. The experiment lasted 140 days and was comprised of five 28 day cycles. The basal diet was formulated to fulfill the nutritional requirements and was supplemented with organic or inorganic minerals, resulting in four treatments: T1 - basal + 100% inorganic minerals; T2 - basal + 100% organic minerals; T3 - basal + 66% organic minerals; T4 - basal + 33% organic minerals. Egg production, egg weight, egg mass, feed conversion for dozen and mass, albumen percentage, specific gravity, shell thickness and relative gross margin have better results when the inorganic minerals are replaced with 66% organic.

Estudo comparativo da proteína do feno de maniçoba em relação à proteína do feno de alfafa na ração de coelhos / Comparative study of maniçoba hay crude protein in relation to alfafa hay crude protein in the diet of rabbits

O presente experimento foi conduzido com o objetivo de avaliar a substituição parcial da proteína bruta (PB) do feno da alfafa (FA) pela PB do feno de maniçoba (FM) na alimentação de coelhos em crescimento, bem como o valor nutricional da proteína bruta do feno de maniçoba. Foram estudados os parâmetros de desempenho, digestibilidade, rendimento de carcaça e dos cortes nobres submetidos às dietas experimentais. Os tratamentos consistiram em quatro níveis de substituição (0, 25, 50 e 75%) da proteína do feno de alfafa pelo feno de maniçoba. A substituição crescente dos níveis de feno maniçoba resultou em um aumento linear no consumo de ração e no ganho de peso de forma quadrática aos 83 dias, mostrando que esse ingrediente pode ser utilizado como substituto ao feno de alfafa na dieta de coelhos. A proteína do feno da alfafa pode ser substituída parcialmente pela proteína do feno de maniçoba.

The experiment was conducted to evaluate the nutritional value and the partial substitution of crude protein (CP) of alfalfa hay (FA) with CP hay maniçoba (FM) in diets for growing rabbits. The performance, digestibility, carcass yield and prime cuts parameters submitted to experimental diets were studied. Treatments consisted of four levels (0, 25, 50 and 75%) of protein alfalfa hay and maniçoba hay. The increasing substitution levels of maniçoba hay resulted in a linear increase in feed intake and weight gain quadratically at 83 days, showing that this ingredient can be used as a substitute for alfalfa hay in the diet of rabbits. The protein of alfalfa hay can be partially replaced by the maniçoba protein hay.

Protective effect of mannan oligosaccharides against early colonization by Salmonella Enteritidis in chicks is improved by higher dietary threonine levels.

AIMS: To evaluate mannan oligosaccharide (MOS) and threonine effects on performance, small intestine morphology and Salmonella spp. counts in Salmonella Enteritidis-challenged birds. METHODS AND RESULTS: One-day-old chicks (1d) were distributed into five treatments: nonchallenged animals fed basal diet (RB-0), animals fed basal diet and infected with Salmonella Enteritidis (RB-I), animals fed high level of threonine and infected (HT-I), birds fed basal diet with MOS and infected (MOS-I), birds fed high level of threonine and MOS and infected (HT+MOS-I). Birds were inoculated at 2d with Salmonella Enteritidis, except RB-0 birds. Chicks fed higher dietary threonine and MOS showed performance similar to RB-0 and intestinal morphology recovery at 8 dpi. Salmonella counts and the number of Salmonella-positive animals were lower in HT+MOS-I compared with other challenged groups. CONCLUSION: Mannan oligosaccharides and threonine act synergistically, resulting in improved intestinal environment and recovery after Salmonella inoculation. SIGNIFICANCE AND IMPACT OF THE STUDY: Nutritional approaches may be useful to prevent Salmonella infection in the first week and putative carcass contamination at slaughter. This is the first report on the possible synergistic effect of mannan oligosaccharides and threonine, and further studies should be performed including performance, microbiota evaluation, composition of intestinal mucins and immune assessment.

Milk adulteration: Detection of bovine milk in bulk goat milk produced by smallholders in northeastern Brazil by a duplex PCR assay.

The aim of this study was to investigate the adulteration of goat milk produced by smallholders in semiarid northeastern Brazil with bovine milk as an adulterant. The study was requested by the association of smallholder producers in the region to investigate and to inhibit adulteration practices as a need to ensure the quality and safety of goat milk. A duplex PCR assay has been developed and standardized. Further validation was performed in 160 fresh bulk goat milk samples. The detection limit of the duplex PCR was 0.5% bovine milk in goat milk and the results indicated that 41.2% of the goat milk presented to market was positive for bovine milk. Making the test available to the association of producers, together with extension activities, have been applied to reduce adulteration in goat milk sold to small-scale dairy plants and to ensure the species origin for goat milk in the state of Paraíba.

Sodium levels for Japanese quail at initial phases.

The aim of this research was to determine the nutritional requirements of sodium for Japanese quail (Coturnix coturnix Japonica) during the periods of 1 to 21 d and 22 to 40 d of age, as well as to evaluate the residual effect on egg production and densitometry bone traits from 41 to 63 d. Two experiments were developed. Experiment 1: 360 Japanese quail were used, from 1 to 21 d of age. Treatments consisted of 5 sodium levels (0.06, 0.12, 0.18, 0.24, and 0.30%). Experiment 2: 240 Japanese quail were used, from 22 to 40 d. Treatments consisted of 5 sodium levels (0.04, 0.12, 0.20, 0.28, and 0.36%). In both experiments, weight gain, final weight, and feed conversion presented a quadratic trend, whereas water intake presented a linear trend. Treatments did not affect the densitometry of bone traits, although they presented a quadratic influence on tibia ash, calcium, and calcium:phosphorus ratio. Therefore, the nutritional requirement of sodium for Japanese quail from 1 to 21 d and from 22 to 40 d is 0.222% and 0.253%, respectively.

Risk factors associated with selected indicators of milk quality in semiarid northeastern Brazil.

The aim of this study was to gain information on quality traits, mainly bacterial and somatic cell counts of bulk milk, produced by small- and medium-scale producers in a semiarid northeastern region of Brazil and to identify and characterize possible risk factors associated with those quality traits. A cross-sectional study was performed on 50 farms. Bulk milk samples were collected for bacterial and somatic cell counts. Additionally, information about farm demographics, general management practices, hygiene, and milking procedures was also obtained. Multivariable analysis using logistic regression was performed with predictors previously identified by univariate analysis using a Fisher's Exact test. Aerobic mesophilic bacteria counts varied from 3.59 log to 6.95 log cfu/mL, with geometric mean of 5.27 log cfu/mL. Mean total coliform count was 3.27 log (1.52 log to 5.89 log) most probable number (MPN)/mL, whereas mean thermotolerant coliforms was 2.38 log (1.48 log to 4.75 log) MPN/mL. A high positive correlation was observed between aerobic mesophilic bacteria and coliform counts. Although most farms met the standard for the current regulations for total bacteria (88%) and somatic cell counts (94%), nearly half of the producers (46%) would have problems in achieving the 2012 threshold limit for total bacteria count if no improvement in milk quality occurs. Mean value for staphylococci was 3.99 log (2.31 log to 6.24 log) cfu/mL, and Staphylococcus aureus was detected in 33 (66%) farms. Premilking teat-end wash procedure (odds ratio=0.191) and postmilking teat dip (odds ratio=0.67) were associated with lower aerobic mesophilic bacteria and Staphylococcus aureus counts in bulk milk, respectively. Considering that the farm characteristics in this study are representative of the semiarid northeastern region, these findings encourage further investigations for supporting intervention measures intended to improve the quality of milk produced by smallholders.

Simultaneous detection of Brachyspira hyodysenteriae, Brachyspira pilosicoli and Lawsonia intracellularis in porcine faeces and tissue samples by multiplex-PCR.

Diarrhoea in growing and finishing pigs is usually caused by infectious agents and laboratory diagnosis is a prerequisite for efficient therapy. Cultivation of Brachyspira hyodysenteriae or Brachyspira pilosicoli and detection of Lawsonia intracellularis by means of immunofluorescence tests (IFT) are time-consuming and in some cases lack sensitivity. A multiplex-PCR was designed to detect simultaneously these three pathogens in faeces and tissue samples, allowing the differential diagnosis of dysentery, intestinal spirochaetosis and proliferative enteropathy. Detection limits for B. hyodysenteriae, B. pilosicoli and L. intracellularis were 10(4), 10(2) and 10(3) copies respectively. Agreement between multiplex-PCR and nested-PCR or cultivation was considered substantial to almost perfect. Agreement between multiplex-PCR and IFT in detecting L. intracellularis was only moderate, which was probably related to false-positive results given by IFT. The multiplex-PCR described herein is a valuable tool for the rapid and simultaneous detection of three different pathogens in porcine samples causing enteric diseases.

Fluorescent amplified fragment length polymorphism genotyping of human and animal Staphylococcus aureus isolates from dairy farms with manual milking.

Fluorescence amplified fragment length polymorphism (fAFLP) was used to assess the genetic relatedness of 40 Staphylococcus aureus strains isolated from human and animal skin samples in seven dairy farms with manual milking. S. aureus was isolated from 11 out of 30 (36%) human skin samples and from 29 out of 100 (29%) teat skin samples from apparently healthy cows. Genomic DNA from each isolate was double-digested with EcoRI and MseI and complementary oligonucleotide adaptors were ligated to the restriction fragments. Pre-selective and selective amplification reactions were performed, the amplified fragments were separated by electrophoresis in an ABI377 sequencer and analysed using GeneScan 3.1 and Genotyper 2.5. Three single isolates (a-c), a predominant cluster with 35 isolates (d) and another cluster with two isolates (e) were identified. Both clusters d and e included human and animal isolates genetically related, because the profiles had 90-100% homology. Since no cluster was comprised uniquely of human or animal isolates and given the close genetic relatedness among human and animal samples in the farms, the present findings support the hypothesis that dairy workers can spread S. aureus through manual milking.

Effect of heat exposure on the thermoregulatory responses of selected naked neck chickens

The variation in cloacal temperature, body weight loss and expression of the 70 kDa heat shock protein (Hsp70) in three naked neck broiler genotypes during heat stress were studied. Twelve birds of each genotype (Na/Na, Na/na and na/na) were reared to market weight (approximately 2.1kg) at thermoneutral temperature. Six birds from each group served as controls and the remaining six underwent gradual heat stress (from 28oC to 36oC). Cloacal temperature and body weight were measured before and after exposure to heat. Liver samples were collected and Hsp70 levels were quantified using western blotting with monoclonal anti-chicken Hsp70 antibody. Heterozygous (Na/na) birds had a significantly lower cloacal temperature variation and less body weight loss during heat stress than the other genotypes. There was no significant difference in the Hsp70 levels among the genotypes. Heterozygous birds (Na/na) appeared to have a slight advantage over the other genotypes during gradual heat stress, perhaps because of a heterotic effect