The ease of translocation of Salmonella enteritidis through the eggshell wall: an immunocytochemical/ultrastructural study

Evidence is presented to indicate that: a) The cuticular layer of the shell is rarely present as an even covering at any stage in the laying year. So, its role as a first line of defence is questionable. b) The shell membranes do inhibit bacterial transfer to some degree, even when they are structurally disrupted; however, if the challenge is great enough, then their function as effective barriers is reduced. c) In the absence of the shell membranes, Salmonella enteritidis Phage type 4 does not move freely across the shell, but it is either facilitated or inhibited in its passage by structural variation in the true shell, particularly at the level of the mammillary layer. Statistical data support in most instances a significant and positive correlation between the presence of structural defects and bacterial transfer. d) In a three tier battery system, a tier effect exists with respect to ease of translocation of microoganisms, with eggs from the top tier being more susceptible, i.e. structurally inferior. e) The results confirm earlier work that shell quality declines with age, and extends this finding to show that this morphological deterioration is accompanied by a decreased resistance to bacterial movement. f) Patent gas exchange pores, whilst obvious portals for bacterial ingress, are in this respect of secondary importance to structural defects within the shell. Evidence is also provided to substantiate the assumption that birds, irrespective of strain, display diverse shell structural quality. One of the strains evaluated (strain B) was structurally better than the other (strain A), at the beginning and middle of lay, and was also more capable of withstanding bacterial challenge in all three laying periods tested. 4. The housing system can influence shell quality; thus Barn and Battery eggs were structurally superior to their Range counterparts, at the end of lay. 5. The tagging of Salmonella with immunogold markers proved to be a valuable technique, which allowed a more precise localisation of the bacteria within the shell's ultrastructure, as viewed by the Scanning Electron Microscope (S.E.M.). This method gave support to other findings in this work, confirming that bacterial transfer was specifically encouraged by late fusion and alignment of the mammillae and pitting occurrences, with the cone layer probably implicated in the process of penetration in vivo. (now derestricted

SEROVARS AND ANTIMICROBIAL RESISTANCE OF Salmonella spp. ISOLATED FROM TURKEY AND BROILER CARCASSES IN SOUTHERN BRAZIL BETWEEN 2004 AND 2006

Salmonella spp. causes diseases in fowls, when species-specific serovars (Salmonella Pullorum and S.Gallinarum) are present in flocks, and public health problems, when non-typhoid serovars are isolated, as well as possible bacterial resistance induced by the preventive and therapeutic use of antimicrobials in animal production. This study describes the serovars and bacterial resistance of 280Salmonella spp. strains isolated from turkey and broiler carcasses in Southern Brazil between 2004 and 2006. SalmonellaEnteritidis was the most prevalent serovar (55.7%), followed by Heidelberg (5.0%), Agona (4.3%), Bredeney (3.9%), Hadar (3.2%), and Typhimurium (2.9%). Tennessee and S. Enterica subspecies enterica(O: 4.5) were isolated only in turkeys, and Hadar (18.6%) was the most prevalent serovar in this species. Antimicrobial susceptibility tests were performed in 178 isolates (43 from turkeys and 135 from broilers). All isolates were sensitive to amoxicillin + clavulanic acid, polymyxin B, ciprofloxacin, and norfloxacin, and were resistant to bacitracin and penicillin. Broiler carcass isolates showed resistance to nalidixic acid (48.9%), nitrofurantoin (34.3%), neomycin (9.6%), tetracycline (5.2%), and kanamycin (8.9%); and turkey carcass isolates were resistant to nalidixic acid (62.8%), tetracycline (34.9%), and neomycin (30.2%), with a significant difference in turkeys when compared to broiler carcass isolates. These results indicate the need for judicious use of antimicrobials in livestock production, given that the serovars identified are potential causes of food poisoning

Número mais provável (NMP) de Salmonella sp. em cecos de frangos de corte e correlação com a população linfocitária bursal

A contaminação de produtos avícolas por Salmonella tem sido um real desafio à avicultura desde o reconhecimento da carne de frango e ovos como importantes fontes de transmissão de salmonelas para humanos. O estado imune das aves tem um papel crítico na defesa contra diferentes patógenos. Lotes de aves com imunossupressão sofrem aumento da incidência de infecções secundárias e têm seu desempenho afetado negativamente. Entre os patógenos virais aviários que têm capacidade imunossupressora encontra-se o vírus da doença infecciosa da bursa ou doença de Gumboro. Neste estudo, avaliou-se a população linfocitária bursal através de exame histopatológico e a contagem de Salmonella em cecos de frango de corte através do método do número mais provável (NMP) buscando-se correlacionar estes fatores. Foram coletados os cecos e as bolsas de Fabricius (BF) de 347 frangos de corte com 28 dias de idade. Os cecos foram congelados e analisados pela microbiologia convencional para determinar a presença ou ausência de Salmonella sp e as amostras positivas submetidas à técnica de contagem pelo NMP. Foram identificadas duas amostras positivas (0,58%) para Salmonella Enteritidis e estas apresentaram 0 e 21 NMP/g, enquanto os cecos correspondentes apresentaram entre 60-70% de depleção linfocitária. Outros gêneros bacterianos identificados foram Providencia rettgeri, Proteus mirabilis e E. coli. Os escores histopatológicos evidenciaram depleção linfocitária moderada a severa nas bursas, porém o isolamento de Salmonella Enteritidis em duas amostras não possibilitou maiores inferências a respeito do envolvimento da Doença de Gumboro quanto à persistência de salmonelas no ceco das aves analisadas, sob a presente metodologia

Biofilm formation by Salmonella enteritidis at different incubation temperatures

Background: The genus Salmonella, associated with poultry products, is considered the leading cause of foodborne outbreaks in humans in many countries. In Brazil, Salmonella Enteritidis (SE) is the serovar remains as one most frequently isolated from humans, and it is also a major serovar found in animals, food, animal feed, and environmental samples, despite all the efforts to control this pathogen. Also, the bacterium is able to form biofilms on different surfaces, protecting cells from both cleaning and sanitizing procedures in the food industries. This study aimed to verify the ability of Salmonella Enteritidis isolates to form biofilm on polystyrene at different incubation temperatures. Materials, Methods & Results: A total of 171 SE samples were isolated from foodborne outbreaks (foods and stool cultures) and poultry products between 2003 and 2010. The biofilm-forming ability of samples was measured at four different temperatures (3°C, 9ºC, 25ºC, and 36ºC), for 24 h, simulating temperatures usually found in poultry slaughterhouses. Later, 200 μL of each bacterial suspension was inoculated, in triplicate, onto 96-well, flat-bottomed sterile polystyrene microtiter plates, washed, after that, the biofilm was fixed with methanol. The plates were dried at ambient temperature, stained with 2% Hucker’s crystal violet. Afterwards, absorbance was read using an ELISA plate reader and the optical density (OD) of each isolate was obtained by the arithmetic mean of the absorbance of three wells and this value was compared with the mean absorbance of negative controls (ODnc). The following classification was used for the determination of biofilm formation: no biofilm production, weak biofilm production, moderate biofilm production and strong biofilm production. Results demonstrated all isolates from stool cultures and foods involved in foodborne outbreaks, at least one of the four temperatures tested, were able to form biofilm, even at 3°C, undescribed as possible for the growth of SE. SE strains from poultry products also formed biofilm at least at one of the temperatures. Discussion: The prevention of biofilms formation is very important, once they can be difficult to remove from utensils and food equipment surfaces, becoming a chronic source of microbial contamination of foods, possible dissemination of diseases, and increase of resistance to cleaning and sanitization procedures. A high ability for biofilm formation on plastic surfaces was observed. We may consider that Salmonella has the capacity to bind to surfaces, with relevant impacts on public health. Although biofilm formation could be affected by temperature, most of the SE isolates analyzed in our study were strong biofilm producers at all temperatures, including at 3°C, a temperature used for food preservation and until then not acknowledged as worrisome regarding the development of Salmonella spp. There is a common sense that maintenance of food at low temperatures, particularly below 5°C, is safer to consumers as low temperatures reduce microbial multiplication. However, our results show that the growth of SE in its sessile form is possible under refrigeration. These findings lead to the assumption that the ability of SE to form biofilms, especially at low temperatures, is related to its endurance in inhospitable environments, eventually infecting humans, and that may be one of the factors associated with the high prevalence of this serovar in outbreaks of foodborne diseases. To our knowledge, this is the first publication about biofilm formation by Salmonella Enteritidis at 3ºC

Organic egg quality during fifty weeks of laying

Atualmente, a produção de ovos em sistemas alternativos vem crescendo, porém não há informações relativas aos seus critérios de qualidade. Com o objetivo de analisar características físico-químicas de ovos orgânicos, associadas ao avanço da idade de postura das aves, avaliou-se peso do ovo, altura de albúmen, espessura da casca, índice e porcentagem de gema, porcentagem de albúmen e de casca e unidade Haugh (UH). Quinzenalmente, seis ovos frescos foram avaliados, durante a 28a e 80a semana de postura. A qualidade externa e interna dos ovos foi afetada pela idade. Peso do ovo e espessura da casca resultaram em efeito cúbico e quadrático (P<0,001), respectivamente. Avaliação da UH e da porcentagem de gema e albúmen resultaram em comportamento cúbico (P<0,05). Índice de gema teve comportamento linear decrescente. O aumento da idade das aves ocasionou diminuição de indicadores de qualidade interna. Apesar da redução na qualidade, ovos de sistema orgânico mantiveram os padrões adequados para consumo, ao longo do ciclo produtivo. Com o avanço da idade, os ovos ficaram mais pesados, porém com redução da espessura de casca, da unidade Haugh e do índice e porcentagem de gema.Currently, the production of eggs in alternative systems has been increasing, but there is no information regarding its quality criteria. Aiming to analyze the physical and chemical characteristics of organic eggs, associated with the advancing age of the laying, egg weight, albumen height, shell thickness, yolk index and percentage, albumen and shell percentage and Haugh Unit (HU) has been evaluated. Every two weeks, six fresh eggs were evaluated, during the 28th and 80th week of laying. The external and internal eggs quality was affected by the age of the birds. Egg weight and shell thickness resulted in cubic and quadratic effect (P<0.001), respectively. HU evaluation and yolk and albumen percentage resulted in cubic behavior (P<0.05). The yolk index had a decreasing linear behavior. The increase in the age of laying hens caused the decrease in indicators of internal quality. Despite the reduction in quality, eggs from the organic production system maintained adequate standards for consumption throughout the production cycle. With advancing age laying, the eggs got heavier, however with reduced shell thickness, Haugh unit, index and yolk percentage

Detection and quantification of Campylobacter spp. in Brazilian poultry processing plants

Introduction: Campylobacteriosis is considered the most common bacteria-caused human gastroenteritis in the world. Poultry is a major reservoir of Campylobacter. Human infection may occur by consumption of raw and undercooked poultry or by contamination of other foods by these items. The aim of this study was to assess the prevalence of Campylobacter spp. in poultry processing plants with conventional culture method and real-time PCR. Methodology: A total of 108 poultry processing plant samples were collected to test with conventional microbiology and qPCR. Sampling included cloacal swabs, swabs of transport crates (before and after the cleaning and disinfection process) and carcasses (after the chiller, cooled at 4°C and frozen at −12°C). Results: Positivity in cloacal swabs indicated that poultry arrived contaminated at the slaughterhouse. Contamination in transport cages was substantially increased after the cleaning process, indicating that the process was ineffective. The detection of Campylobacter on carcasses was higher than that on cloacal swabs, which could indicate cross-contamination during the slaughtering process. Conventional microbiology and molecular methods revealed a prevalence of 69.4% and 43.5%, respectively. Lower detection by qPCR can be attributed to the high specificity of the kit and to biological components that could inhibit PCR reactions. Conclusions: Our results indicate that poultry arrive contaminated at the slaughterhouse and that contamination can increase during the slaughtering process due to cross-contamination. The isolation of Campylobacter in cooled and frozen carcasses corroborates the bacterial survival even at temperatures considered limiting to bacterial growth which are routinely used for food preservation

Salmonella Enteritidis forma biofilme sob baixas temperaturas em diferentes superfícies da indústria de alimentos

We evaluated the influence of temperature on the ability of Salmonella Enteritidis (SE) to form biofilms on stainless steel, polyethylene, and polyurethane surfaces under different hygiene procedures. These materials were placed on SE culture and incubated at 42±1 ºC, 36±1 ºC, 25±1 ºC, 9±1 ºC, and 3±1 ºC for 4, 8, 12, and 24 h. Hot water at 45 ºC and 85 ºC, 0.5% peracetic acid solution, and 1% quaternary ammonia were used for hygienization. Biofilm formation occurred at all temperatures evaluated, highlighting at 3 ºC which has not been reported as an ideal temperature for the adhesion of SE to these materials. The SE adhered more often to polyethylene surfaces than to polyurethane and stainless steel surfaces (P<0.05). Peracetic acid and water at 85 ºC had similar hygienization efficiency (P<0.05) followed by quaternary ammonia whereas water at 45 ºC was not effective. SE adhered to these materials under low temperatures which to date have been deemed safe for food preservation.Avaliou-se o efeito da temperatura na capacidade de Salmonella Enteritidis (SE) formar biofilme em superfícies de aço inoxidável, polietileno e poliuretano e diferentes processos de higienização. Corpos de prova destes materiais foram postos frente a culturas de SE e incubados a 42±1 ºC, 36±1 ºC, 25±1 ºC, 9±1 ºC e 3±1 ºC por 4, 8, 12 e 24 horas. Para a higienização foram testados água aquecida a 45ºC e 85 ºC e soluções de ácido peracético 0,5% e amônia quaternária 1%. Verificou-se a formação de biofilmes em todas as temperaturas avaliadas, ressaltando-se a 3 ºC, ainda não citada como propícia para adesão de SE. Houve maior adesão ao polietileno do que ao poliuretano e ao aço inoxidável (P<0.05). Para higienização, o ácido peracético e a água a 85 ºC tiveram ação semelhante (P<0.05), seguidos por amônia quaternária, enquanto que a água a 45 ºC não foi eficaz. Todos os materiais avaliados propiciaram a aderência de SE, mesmo sob temperaturas baixas, consideradas até então seguras para a conservação dos alimentos

Prevalence and distribution of pathogenic genes in Campylobacter jejuni isolated from poultry and human sources

Introduction: Campylobacter jejuni is one of the most common bacterial causes of human gastroenteritis. Despite its public health importance, the virulence factors and mechanisms underlying C. jejuni pathogenesis remain poorly understood and the relationships between these genes and the sources of the strains are not clear. We aimed to determine the virulence profiles of C. jejuni isolated from poultry and human cases of Campylobacteriosis. Methodology: A total of 50 strains of C. jejuni isolated from poultry and human cases of Campylobacteriosis were screened by polymerase chain reaction (PCR) for the presence of six pathogenic genes (flaA, iam, wlaN, cdtA, cdtB, cdtC). Results: A total of 40% (10/25) of the human isolates presented only one virulence marker. In contrast, 64% (16/25) of the poultry-derived strains showed four or five virulence markers. cdtA and flaA occurred more frequently in poultry-derived strains than in human strains. Ten different virulence profiles were observed among the human isolates and 11 among the poultry strains. Only four profiles were common to both sources: profiles 3 (flaA, cdtA, cdtB, and cdtC), 5 (cdtA and cdtB), 7 (flaA and cdtB), and 10 (iam, flaA, cdtA, cdtB, and cdtC). The human-derived strains had a higher Shannon diversity index (1.9396) and Simpson index (0.8367), indicating a more diversified population than found in poultry (1.7742 and 0.7333, respectively). Conclusions: We found variations in the genetic profiles of the circulating strains based on the isolation source and genes that are potentially pathogenic to humans were detected in poultry-derived strains

Antimicrobial activity of poultry hatch baskets containing copper inserts

Commercial poultry hatcheries provide ideal conditions for the multiplication and spread of microorganisms. Formaldehyde is widely used as a disinfectant; however, it is harmful to human health and can cause abnormal morphology in chicks. An alternative microbiological control is the use of copper, a metallic antimicrobial agent for contact surfaces. The antimicrobial efficacy of copper surfaces has been established in healthcare environments. However, its use in the poultry chain is still limited. This study aimed to compare the antimicrobial activity of common polypropylene hatch baskets with hatch baskets composed of copper (polypropylene hatch baskets covered by copper hatch baskets; polypropylene hatch baskets with solid copper plates on the bottom; polypropylene hatch baskets covered by copper hatch baskets and with solid copper plates on the bottom). To simulate a hatching environment with high contamination, the eggs and hatching cabinet were not fumigated. Microbiological analysis of the hatching cabinet environmental, surface of hatch basket, and fluff were performed. The results indicated that the absence of bottom holes resulted in a higher volume of organic matter that interfered with the copper's antimicrobial activity. The presence of copper in the hatch baskets did not decrease microbial contamination under the conditions evaluated, confirming that the metal should only be used as a complement to standard hygiene and not as a substitute for surface disinfectants. Further analysis will evaluate the antimicrobial activity of hatch baskets composed only of copper and the ability of nanoparticles to remove the biofilms formed by bacteria isolated from the poultry environment

Occurrence and severity of white striping in broilers until 50d of age fed with high and low-energy diets : body weight, histopathological changes and meat quality

White striping (WS) is a condition characterized by the occurrence of white striations parallel to the muscle fibers on breast, thigh, and tender muscle of broilers. This study was aimed to evaluate the occurrence and severity of white striping and histopathological changes in breast fillets from 10 to 50 d broilers disregarding the effect of diet. Birds (n=572) were randomly assigned to a high- (HED) or low-energy (LED) diet (11 replicates of 26 birds/ dietary treatment) and were processed at 10, 20, 30, 40 and 50 d of age to evaluate occurrence of white striping, BW, histological analysis, and meat quality. The results showed that at 10 d of age, 2.27% of the birds showed some degree of WS and at 20 and 30 d of age the occurrence of WS (%) was higher in birds fed HED than birds fed LED diets. There was no differences (P>0.05) in the L*, a*, and b* values for meat or among different degrees of WS as well as between dietary treatments; however, carcass characteristics varied by age or carcass weight. Histologically, the severity of WS causes changes in myofibers, with muscle fibers showing sarcoplasmic reticulum vacuolization, a higher intercapillary distance and a reduced capillary to fiber ratio, suggesting loss of integrity of the cell structure, that was seen in birds fed both diets (low and high nutrient densities) after 30 d of age and lower oxygenation