Characteristics of Salmonella enteritidis contamination in eggs after oral,aerosol, and intravenous inoculation of laying hens

Experimental infection models are useful tools for understanding how Salmonella enteritidis is deposited in eggs and for testing potential strategies to control eggborne transmission of disease to humans. Oral inoculation of laying hens is presumed to provide the closest simulation of naturally occurring infections, but alternatives such as intravenous or aerosol inoculation have sometimes been recommended as options to induce higher incidences of egg contamination. The present study compared the frequency, level, and location of S. enteritidis deposition in egg contents after experimental inoculation by three different routes. In two replicate trials, specific-pathogen-free laying hens were infected with an S. enteritidis culture mixture prepared to optimize invasive behavior. Groups of hens received either an oral dose of 10(9) S. enteritidis, an aerosol dose of 10(9) S. enteritidis, or an intravenous dose of 10(5)-10(7) S. enteritidis. Oral inoculation led to the highest incidence of fecal shedding of S. enteritidis, whereas intravenous inoculation produced the highest specific antibody titers. Eggs laid during the first 21 days postinoculation were cultured to detect and enumerate S. enteritidis in the yolk and albumen. No significant differences were observed among the three inoculation routes in the frequencies of isolation of S. enteritidis from either yolk or albumen. For all three routes of administration, S. enteritidis was recovered more often from yolk (at frequencies ranging from 4% to 7%) than from albumen (0 to 2%). Over 73% of contaminated eggs harbored fewer than 1 colony-forming unit (CFU) of S. enteritidis per milliliter, and only 3% of such eggs contained more than 100 CFUs/ml. Significantly higher levels of S. enteritidis contaminants were associated with intravenous inoculation than with the other routes. No advantage of using aerosol or intravenous administration of S. enteritidis as an alternative to oral inoculation for inducing the production of contaminated eggs was evident in this study.     

Experimental Salmonella enteritidis phage type 4 infection and egg transmission in Japanese quails

Japanese quails were inoculated orally with 1 x 10(10) colony-forming units of Salmonella enteritidis phage type 4 isolated from chicks to obtain information concerning S. enteritidis infection and egg transmission. The inoculation resulted in a bacteraemic infection with seeding of the liver, spleen, intestine, peritoneum, ovule, ovary and oviduct. Some infected birds showed diarrhoea, ruffled feathers, depression, loss of appetite and death. However, most infected birds remained clinically normal with normal egg production. Salmonella enteritidis was isolated from the albumen of 7, yolk of 15, shell of 13, and shell membrane of 15 of 164 eggs. The experiment suggests that S. enteritidis phage type 4 is invasive for Japanese quails and the infected eggs laid by S. enteritidis infected quails are probably the result of transovarian infection.     

Deposition of phage type 4 and 13a Salmonella enteritidis strains in the yolk and albumen of eggs laid by experimentally infected hens

Because egg yolk and albumen differ substantially in their abilities to support bacterial growth, the initial level and location of Salmonella enteritidis deposition are critical for determining whether proposed standards for refrigerating eggs are likely to protect public health by preventing extensive microbial multiplication. In the present study, three groups of laying hens were infected with oral doses of approximately 10(9) cells of different S. enteritidis strains (two were phage type 4 and one was phage type 13a) in two replicate trials. For all three S. enteritidis strains, the incidence of yolk contamination (approximately 2.5% overall) was significantly greater than the incidence of albumen contamination (approximately 0.5% overall). The phage type 13a strain was less often isolated from fecal samples at 2 wk post-inoculation than were the phage type 4 strains, but no significant differences between strains were observed in the incidence of egg contamination. Most freshly laid contaminated eggs contained fewer than 1 S. enteritidis cell/ml of egg yolk or albumen, and no sample contained more than 67 S. enteritidis cells/ml.     

Production of Salmonella enteritidis-contaminated eggs by experimentally infected hens

Laying hens of three different ages were experimentally infected with a strain of Salmonella enteritidis by either oral inoculation or contact transmission. Total egg production was depressed in exposed hens of all three age groups. Persistent intestinal shedding was observed in a small number of hens. Eggs with contents contaminated by S. enteritidis were produced by exposed hens at a high frequency, but only during a fairly short period of time that extended through approximately 1 week postinoculation for older hens and through 2 weeks for younger hens. S. enteritidis was recovered from whole yolks and albumen of these eggs at similar frequencies, but not from the content of yolks. Eggs with contaminated shells were also produced, but at a lower frequency. Contaminated eggs were produced by orally inoculated and contact-exposed hens at similar frequencies. S. enteritidis was not isolated from the contents of eggs laid by hens infected with other S. enteritidis strains.     

Effect of prior serial in vivo passage on the frequency of Salmonella enteritidis contamination in eggs from experimentally infected laying hens

Experimental infection models are valuable tools for understanding and preventing the deposition of Salmonella enteritidis inside eggs. Oral inoculation is believed to closely simulate naturally occurring S. enteritidis infections of chickens, but oral infection studies have often generated relatively low frequencies of egg contamination. The present study assessed whether repeated in vivo passage of an S. enteritidis strain could affect its ability to cause egg contamination in experimentally infected hens. The incidence of egg contamination was determined in groups of hens inoculated orally with either a phage type 13a S. enteritidis strain or derivatives of this parent strain that were obtained by three successive rounds of passage and reisolation from tissues of infected hens. Passaged S. enteritidis isolates recovered from ovaries and oviducts induced a significantly higher incidence of egg contamination (16.97%) than was attributed to the parent strain (8.27%). However, passaged S. enteritidis isolates recovered from livers and spleens were not associated with a significantly increased frequency of deposition in eggs. By either inducing or selecting for the expression of relevant microbial properties, passage of S. enteritidis through reproductive tissues of chickens may be useful for improving the efficiency at which experimental infection models produce egg contamination.     

Detection of antibody to Salmonella enteritidis and S typhimurium in the yolk of hens' eggs

Enzyme-linked immunosorbent assays (ELISAs) have been developed to detect IgG antibodies to Salmonella enteritidis and S typhimurium in the yolk of hens' eggs. Better discrimination and more consistent results were obtained between eggs from experimentally infected and uninfected hens by using saline-dilution of yolk rather than chloroform extraction. Threshold absorbance values were determined in three salmonella-free flocks, and on the basis of these results ELISA optical density values greater than 0.25 were considered to be positive for antibodies to salmonella. Four flocks with a history of salmonella infection were examined; three contained birds which were seropositive for S enteritidis by ELISA and from which S enteritidis was isolated, and a large proportion of eggs from these birds contained antibody to S enteritidis. Eggs from the fourth flock had no detectable antibody, although serum antibody was detected in some birds. No salmonellae were isolated from the yolks of the eggs from any of the four flocks.     

Pathogenicity of Salmonella enteritidis phage types 4, 8, and 23 in broiler chicks.

Four hundred fifty day-old Hubbard broiler chicks were subdivided into 15 groups of 30 chicks each. Six groups of chicks received 0.5 ml of broth culture containing 5 x 10(6) colony-forming units (CFU) of Salmonella enteritidis (SE) phage types (PTs) 4, 8, and 23 by crop gavage. Similarly, six other groups received 0.5 ml containing 5 x 10(8) CFU of SE. One group was inoculated with 0.5 ml containing 5 x 10(6) CFU of Salmonella pullorum, and another group received 0.5 ml containing 5 x 10(8) CFU of S. pullorum. A group of 30 chicks were kept as uninoculated controls. Chicks were observed daily for clinical signs and mortality. All birds were weighed at 7, 14, and 21 days postinoculation 21 (DPI). Four chicks were randomly selected from each treatment group, euthanatized, and necropsied at 7 and 14 DPI. Gross lesions were recorded and selected tissues were collected for histopathology. The higher rates of illness and mortality were observed in chicks inoculated with 5 x 10(6) and 5 x 10(8) CFU of S. pullorum, followed by SE PT4 of human origin and SE PT4 of chicken origin. Moderate to high mortality was observed in chicks inoculated with the higher dose of SE isolates that belonged to PT8 and one SE of PT23. Variable mortality was evident in groups inoculated with the lower dose of salmonella. The most consistent gross and histopathologic changes, including fibrinous pericarditis and perihepatitis, were seen in the dead birds from various treatment groups. The lower mean body weights were present in all treatment groups compared with uninoculated controls. No illness or mortality was observed in uninoculated control groups.     

Pathologic and bacteriologic findings in 27-week-old commercial laying hens experimentally infected with Salmonella enteritidis, phage type 4

Two strains of 27-wk-old commercial laying chickens (strain A, brown-egg-laying type and strain B, white-egg-laying type) were inoculated either orally (PO) or intravenously (IV) with a field isolate of Salmonella enteritidis phage type 4. Chickens were sequentially necropsied at regular intervals throughout the 17-wk observation period. Gross and microscopic lesions were most evident between 1 and 14 days postinoculation (DPI). Gross lesions consisted of enlarged livers with white foci, enlarged and mottled white spleens, fibrinous exudate in the peritoneum, and atretic, misshapen ovarian follicles. Microscopic lesions included multifocal coagulative necrosis of hepatocytes and inflammation, fibrinous exudation in vascular sinuses of the spleen, and fibrinosuppurative inflammation of the peritoneum and ovarian follicles. The proportion of reproductive organ infections (ovary and oviduct) in the IV group, 83% (20/24, P = 0.007; 50% and 33% for strains A and strain B birds, respectively), was higher than that of the PO group, 46% (11/24; 29% and 17% for strains A and B, respectively), for the first 16 days of observation postinoculation. The proportion of fecal shedding for the IV group of birds was significantly (P = 0.009) lower, 29% (7/24; 33% and 25% respectively for strain A and strain B birds, respectively), than the PO group, 67% (16/24; 75% and 58% for strain A and strain B birds, respectively). Three (2.6%) of 234 egg pools were culture-positive for group D Salmonella from strain A chickens (1 of 119 pools from the IV group and 2 of 115 pools from the PO group of birds). Chickens infected with the field strain of S. enteritidis phage type 4 harbored the organism in tissues only for a brief time, most clearing within 8 DPI and nearly all within 16 DPI. Overall the percentage of culture-positive birds did not differ significantly (P > 0.05) between birds with and without lesions, but isolation of S. enteritidis tended to be more frequent when lesions were evident. This experiment also demonstrated that brown-egg-laying-type chickens were more susceptible than white-egg-laying-type chickens to S. enteritidis phage type 4 isolated from California based on gross and microscopic lesions and bacteriologic findings.     

A comparative study of detecting Chlamydophila psittaci in pet birds using isolation in embryonated egg and polymerase chain reaction

This study, for the first time in Turkey, investigated the existence of Chlamydophila psittaci and determined the prevalence of its disease, chlamydiosis, in pet birds. Polymerase chain reaction (PCR) was compared with other testing methods that have been typically used in the diagnosis of C. psittaci. Fecal specimens (n =96) of avian origin were tested by PCR and two identification methods, modified Gimenez staining (mGS) and direct fluorescein-conjugated monoclonal antibody staining (FA). The identification methods were implemented by staining the yolk sacs of embryonated chicken eggs inoculated at 6 days of age and harvested between 3 and 10 days after inoculation. Fecal specimens from pet birds were randomly collected from pet shops and homes. These specimens were then used to isolate C. psittaci and to detect its specific DNA. The inocula that were prepared from fecal specimens were then inoculated into yolks of 6-day-old embryonated chicken eggs. The preparations from egg yolk sacs were examined with mGS and direct FA after three blind passages. The PCR method was used to detect specific DNA in feces. In 96 fecal specimens, 33 (34.4%) were positive with PCR, 21 (21.9%) were positive with mGS, and 29 (30.2%) were positive with FA. Among 33 positive specimens with PCR, 28 specimens were positive with FA, and 20 specimens were positive with mGS. The sensitivity and specificity were 59% and 94% between FA and mGS, and 97% and 93% between FA and PCR, respectively.     

Neutralizing antibodies to CELO and avian adenovirus-associated viruses in the albumen of chicken eggs

Neutralizing antibodies to CELO virus and to avian adenovirus-associated virus (A-AV) were detected in the albumen of eggs from four hens inoculated with these viruses. The antibody concentrations of serum, yolk, and albumen were determined before inoculation and at various times postinoculation (PI) by enzyme-linked immunosorbent assay (ELISA) and virus-neutralization (VN) tests. The antibody concentration in albumen was 0.3% to 1.0% of that detected in serum and yolk. Uninoculated hens showed no detectable antibody in serum, yolk, or albumen. It is suggested that the presence of antibody in the egg albumen may play a role in egg-transmission of viruses.     

Environmental effects on steroid hormone concentrations in laying hens’ eggs

Abstract

The hormone content of a birds’ egg may reflect the environmental conditions of the bird. In this study we measured hormone concentrations of eggs from hens living under different housing conditions. Eggs from 16 floor-housed and 16 singly caged hens were analysed for androstenedione and estradiol. The concentrations of these hormones were highest in the yolk of eggs from floor-housed hens (P≤0.05). The concentration of estradiol in the albumen of eggs was highest for caged birds (P≤0.0001). In caged hens, the concentrations of both hormones varied significantly over days in the egg yolk, but not in the albumen. As the concentration of androstenedione and estradiol in the yolk of chicken eggs is environmentally dependent, these hormones may provide a mechanism by which the hen signals the state of the environment to her progeny.     

The serologic response to Salmonella enteritidis and Salmonella typhimurium in experimentally infected chickens,followed by an indirect lipopolysaccharide enzyme-linked immunosorbent assay and bacteriologic examinations through a one-year period

Three groups of 100 individually marked salmonella-free chickens were followed for a period of 53 wk. The chickens were infected as day olds by crop instillation of 10(8) colony-forming units: one group with Salmonella enteritidis and a second group with Salmonella typhimurium. A third group was kept uninfected as controls. The groups were monitored bacteriologically by examination of cloacal swabs and organs and serologically by examination of serum and egg yolk by a lipopolysaccharide enzyme-linked immunosorbent assay throughout the period. Within the first week, 100% of birds in both infected groups were excreting salmonella bacteria in the feces. However, the number of fecal excretors declined rapidly with time, down to 6% in 16 wk for S. typhimurium and down to a similar level within the first 8 wk for S. enteritidis. For the latter, relapses with up to 40% positive birds were observed at the onset of egg production. For both S. typhimurium and S. enteritidis, positive bacteriologic cultures were obtained by sampling from internal organs at the end of the experiment, more than 1 yr from the time of infection. At the age of 6-7 wk, 50% of the chickens in the two infected groups showed a measurable serologic response in serum samples. The response persisted throughout the study in both serum and egg yolk samples. The inclusion of serologic methods is a valuable additional tool in the detection of salmonella in poultry, but serology should be used in conjunction with bacteriologic methods in surveillance programs, in particular to detect flocks in early stages of infection before a measurable serologic response has been raised.     

不同周龄母鸡鸡蛋及不同蛋壳质地鸡蛋的蛋品质比较

本文比较了同一品种不同周龄(26、40周龄和64周龄)母鸡所产蛋的蛋壳品质和蛋品质,包括蛋重、蛋壳相对重、蛋形指数、蛋壳强度、蛋壳厚度、蛋黄颜色、蛋黄指数、蛋黄比率、哈氏单位、蛋清pH值。之后,还比较了该鸡种不同蛋壳质地鸡蛋(正常蛋和砂壳蛋)之间上述指标的差异。结果表明:在不同周龄母鸡所产蛋之间,蛋重、蛋黄指数、蛋黄比率、哈氏单位等均存在显著差异(p<0.05),而蛋壳厚度之间差异不显著(p>0.05)。此外,蛋重、蛋形指数、蛋黄颜色、蛋黄比率随着母鸡周龄的增加呈上升趋势,而蛋壳强度、蛋壳相对重、哈氏单位、蛋黄指数、蛋清pH值呈下降趋势。最后,蛋壳质地不同的鸡蛋(正常蛋与砂壳蛋)之间,蛋重、蛋壳相对重、蛋壳强度、蛋清pH值之间存在极显著差异(p<0.01),而其他参数之间差异不显著(p>0.05)。     

Differences in abilities to colonize reproductive organs and to contaminate eggs in intravaginally inoculated hens and in vitro adherences to vaginal explants between Salmonella enteritidis and other Salmonella serovars

In Experiment 1, mature laying hens were inoculated intravaginally with 10(6) colony-forming units of Salmonella enterica serovar enteritidis (S. enteritidis), Salmonella typhimurium, Salmonella infantis, Salmonella hadar, Salmonella heidelberg, or Salmonella montevideo to compare their abilities to colonize the reproductive organs of chickens and to contaminate eggs. Salmonella enteritidis was more frequently recovered (from 11 of 40 eggs, 27.5%) than the other serovars, and especially the inner shell was contaminated with these organisms in 10 of 40 eggs (25.0%). The contamination rates and the viable counts in cloaca were significantly (P < 0.05) higher in hens inoculated with S. enteritidis than in those inoculated with the other serovars at 4 days postinoculation (PI). In the vagina, the positive rates were 90%-100% in hens inoculated with S. enteritidis, and the viable counts of the organisms in this portion were significantly (P < 0.05) higher than those of the other serovars at 2, 4, and 7 days PI. The ceca were colonized similarly by each serovar at 7 days PI. The spleen and ovary were infected with S. enteritidis in three and one hen, respectively. No Salmonella was recovered from liver and peripheral blood in any hen. Salmonella enteritidis was recovered from other oviductal portions than the vagina (10%-20%), whereas no forming egg was contaminated in the oviduct. In Experiment 2, the in vitro adherence of these six serovars to the vaginal epithelium was compared with vaginal explants. The mean number of S. enteritidis attaching to the secondary villi in the vaginal lumen was significantly (P < 0.05) higher than those of the other serovars. These results suggest that S. enteritidis has a specific advantage over the other Salmonella serovars by its capacity to colonize the vaginal tissues of hens, and this higher affinity of S. enteritidis to the vagina may play a significant role in the production of many S. enteritidis-contaminated eggs.     

Genetic Relations between Reproduction,Chick Weight and Maternal Egg Composition in a White Leghorn Line

Selection is a viable way to increase egg dry matter, an important trait for the egg processing industry, but simultaneously a change in the embryonic environment takes place. The main purpose of this study was to estimate genetic parameters for the percentage of fertile eggs of eggs set (FERT), percentage of eggs hatched of fertile eggs (HATCH), percentage of live chicks 2 weeks post-hatch of hatched eggs (SURV/h) and of fertile eggs (SURV), and their genetic correlations with egg weight, albumen weight, yolk weight, yolk proportion, albumen dry matter and mean chick weight in a White Leghorn selection line. Direct and maternal heritabilities were low for FERT, HATCH and SURV, and non-existent for SURV/h. All direct-direct genetic correlations between reproduction traits and egg composition traits were not significant. Maternal-direct genetic correlations between HATCH and egg weight, albumen weight, yolk weight, yolk proportion and albumen dry matter were - 0.22, - 0.30, 0.28, 0.52 and 0.26, respectively. Similar correlations were found between SURV and egg composition traits. No significant genetic relationship was found between chick weight and post-hatch survival. In conclusion, within the range of values for egg composition in this study, selection for an increased egg dry matter should not impair reproduction.     

鸡蛋暗斑对蛋品质及抗氧化能力的影响

[目的]研究鸡蛋暗斑对蛋品质及其抗氧化能力的影响。[方法]从饲喂同种日粮、饲养环境相同、同日龄褐壳蛋鸡同一批次所产的褐壳蛋中,用灯光照射法挑选出24枚典型正常蛋、24枚典型暗斑蛋进行试验,并分别对正常蛋和暗斑蛋进行蛋品质、蛋黄抗氧化指标的测定及比较。[结果]蛋品质比较:暗斑蛋与正常蛋相比,其蛋形指数、蛋壳强度、蛋重、哈氏单位、蛋白高度、鸡蛋等级、蛋黄颜色、蛋黄重、蛋壳重、蛋清重、蛋壳厚度、蛋黄比重、蛋壳比重、蛋清比重均差异不显著(P>0.05);暗斑蛋蛋壳颜色极显著深于正常蛋(P<0.01)。抗氧化指标比较:暗斑蛋蛋黄的总抗氧化能力(T-AOC)显著低于正常蛋蛋黄的总抗氧化能力(P<0.05),暗斑蛋蛋黄的丙二醛(MDA)含量与正常蛋蛋黄的丙二醛含量相比差异不显著(P>0.05)。[结论]与正常蛋相比,暗斑蛋的蛋壳颜色较深,总抗氧化能力较差。     

Double-yolked pheasant eggs provide an insight into the control of albumen secretion in bird eggs

1. The possible role of the presence of the yolk in stimulating secretion of albumen was investigated. 2. Double-yolked and single-yolked pheasant (Phasianus colchinus) eggs were opened to determine the masses of the shell, albumen and yolk(s). 3. In double-yolked eggs, the two yolk masses were not significantly different. Albumen mass was increased above that expected from an egg with a single-yolk of comparable size but below that expected from an egg having a mass of the combined yolks. The mass of shell per unit area reflected the mass of the initial mass of the egg irrespective of the number of yolks. 4. The additional mass of albumen is unrelated to yolk or initial egg mass. It is postulated that in double-yolked eggs the oviduct is mechanically stimulated by the presence of both yolks, which empties the stores of water-soluble albumen proteins in the magnum wall. Such stores are insufficient to provide the same amount of protein for the two yolks.     

Morphology and chemical composition of turkey eggs

The objective of this study was to evaluate selected morphological characteristics and the chemical composition of turkey eggs of different weight classes. The percentage of eggs weighing ≥100 g increased to 46.66% in wk 23. Eggs weighing 95 and 110 g had lower yolk content (3.05 and 4.86%) and higher albumen content (by 3.16 and 5.21%) compared with eggs weighing 80 g. The total protein content of the albumen increased (from 9.93 to 10.71%) and the water content of the albumen decreased (from 88.30 to 87.59%) with an increase in egg weight. Albumen weight per gram of yolk increased from 1.87 to between 2.18 and 2.42 g in larger eggs. The energy density of the egg decreased from 5.68 to 4.90 kJ/g with an increase in egg weight, and the energy density of the yolk remained at a similar level in all egg weight classes. The other analyzed yolk and shell parameters were not affected by egg weight. The highest energy and total protein content of the yolk was observed in wk 14 of the laying season. No correlations were found between the progress of the laying cycle and eggshell percentage and the water content of the yolk. The values of the remaining egg parameters varied over the laying season.     

Experimental transmission of turkey viral hepatitis to day-old poults and identification of associated viral particles resembling picornaviruses.

Turkey viral hepatitis (TVH) was experimentally reproduced in two experiments in 1-day-old poults. In the first experiment, an infectious inoculum was prepared from filtered yolk materials harvested from dead embryonating chicken eggs (ECE) previously inoculated with suspensions of liver and pancreas tissues collected from TVH-affected birds in commercial turkey flocks. One-day-old poults given a yolk-sac inoculation or oral gavage with this preparation developed lesions in the liver and pancreas characteristic of TVH at 20 days postinoculation (PI) in 60% and 14% of the experimentally infected birds, respectively. With the identical inoculum, embryo mortality occurred at 8 and 10 days PI in embryonating turkey eggs (ETE) inoculated into the yolk sac. In the second experiment, an infectious inoculum was prepared from filtered yolk materials from dead ETE harvested in the first experiment. One-day-old poults given a yolk-sac inoculation with this filtered yolk material developed lesions in the liver and pancreas within 5 days PI. At 20 days PI, 67% of the experimentally infected birds had similar lesions. With the inoculum given to these poults, embryo mortality occurred at 6, 8, and 10 days PI in ETE inoculated into the yolk sac. Virus particles 26-28 nm in diameter with icosahedral morphology typical of picornaviruses were identified by EM in the yolk sacs of ETE that died in both experiments, and inoculated ETE that died following passage of filtered suspensions of pancreatic tissues collected from affected birds in the first experiment.     

Effects of housing system (outdoor vs cages) and age of laying hens on egg characteristics

1. Effects of two housing systems (cages vs outdoor) on external and internal egg characteristics were investigated. 2. In total 785 eggs from three different lines in cages and 268 eggs from outdoor-housed layers were examined for egg weight, albumen, yolk and shell content, albumen height and pH, and albumen and yolk dry matter content. 3. Interactions between layer age and housing systems were found for egg weight, eggshell content, albumen height, albumen pH, and dry matter content of the albumen and yolk. This was mainly due to the greater variation with age in the outdoor layers, compared to the caged layers. 4. Irrespective of age eggs from outdoor layers were relatively broader than eggs from the caged layers. Yolk colour was considerably darker in the outdoor group (11.0 vs 9.3). 5. We concluded that it is more difficult to maintain constant external and internal egg quality in an outdoor housing system than in a battery cage system. Factors that determine the greater fluctuations in internal egg quality need to be investigated.