Kapil K. Chousalkar

Effect of Egg Washing and Correlation between Eggshell Characteristics and Egg Penetration by Various Salmonella Typhimurium Strains

Salmonella is an important foodborne pathogen, causing an estimated 11,992 cases of infection in Australia per year. Egg or egg product related salmonellosis is a major concern for the egg industry. Worldwide, S. Typhimurium is one of the most common serovars identified in Salmonella food poisoning cases. The current study investigated the ability of five S. Typhimurium strains to penetrate washed and unwashed eggs using whole egg and agar egg penetration methods. All S. Typhimurium strains were able to penetrate eggshells and survive in egg albumen (at 20°C) according to whole egg penetration results. Polymerase Chain Reaction results demonstrated that S. Typhimurium strain 2 (103 and 105 CFU/mL), and strain 5 (103 and 105 CFU/mL) egg penetration was significantly higher (p<0.05) in washed eggs when compared to unwashed eggs. Statistical analysis of the agar penetration experiment indicated that S. Typhimurium was able to penetrate washed eggs at a significantly higher rate when compared to unwashed eggs (p<0.05). When compared to unwashed eggs, washed eggs also had significantly damaged cuticles. Statistical analysis also indicated that eggshell penetration by S. Typhimurium was related to various eggshell ultrastructural features such as cap quality, alignment, erosion, confluence, Type B bodies and cuticle cover.

Association between Indoor Environmental Contamination by Salmonella enterica and Contamination of Eggs on Layer Farms

ABSTRACT This study involves longitudinal and point-in-time surveys of Salmonella carriage and environmental contamination on two commercial cage layer farms positive for Salmonella enterica subsp. enterica serovar Typhimurium (flock A age, 32 weeks; flock B age, 34 weeks). Salmonella-positive fecal, egg belt, and dust samples were all unconditionally associated with eggshells testing positive for Salmonella. The odds of an eggshell testing positive for Salmonella were 91.8, 61.5, and 18.2 times higher when fecal, egg belt, and dust samples, respectively, tested positive for Salmonella. The agreement between the culture-based methods and real-time PCR on preenriched broths for detecting Salmonella was almost perfect for eggshell (observed agreement, 99.19%; kappa coefficient, 0.94) and egg belt samples (observed agreement, 95%; kappa coefficient, 0.88), and it was substantial for fecal (observed agreement, 87.14%; kappa coefficient, 0.47) and floor dust samples (observed agreement, 80.61%; kappa coefficient, 0.58). A 1-log increase in the load of Salmonella detected in the fecal, egg belt, and floor dust samples resulted in 35%, 43%, and 45% increases, respectively (P < 0.001), in the odds of an eggshell testing positive for Salmonella. The multilocus variable-number tandem-repeat analysis (MLVA) patterns of the S. Typhimurium strains isolated from flock A were distinct from those of flock B. S. Typhimurium strains detected from human food poisoning cases exhibited an MLVA pattern similar to those of the strains isolated from flocks A and B.

Shedding of Salmonella in single age caged commercial layer flock at an early stage of lay

The shedding of Salmonella in a single age commercial egg layer flock was investigated at the onset of lay (18weeks) followed by two longitudinal samplings at 24 and 30weeks. At the age of 18weeks, when the first sampling was performed, the prevalence of Salmonella in faeces was 82.14% whereas all egg belt and dust samples were Salmonella positive by culture method. In later samplings, at the age of 24 and 30weeks, the prevalence of Salmonella in faeces was significantly reduced (p<0.001) to 38.88% and 12.95% respectively, however all egg belt and dust samples remained positive by culture method. The prevalence of Salmonella in faeces collected from the low tier cages was significantly higher (p=0.009) as compared with samples from the high tier cages. In all types of samples processed by culture method, S. Mbandaka was the most frequently (54.40%) isolated serovar followed by S. Worthington (37.60%), S. Anatum (0.8%), and S. Infantis (0.8%). All samples were also tested by real-time PCR method. The observed agreement between culture method and real-time PCR in detecting Salmonella-positive dust and egg belt samples was 100%. There was almost perfect agreement (observed agreement=99.21%) for the detection of Salmonella-positive eggshells. Observed agreement between culture method and real-time PCR for detecting Salmonella-positive shoe cover and faecal samples was, however, moderate (80%) and low (54.27%) respectively. Real-time PCR results showed that there was a significant increase in the load of Salmonella on egg belt, dust and shoe cover samples at the 24 and 30weeks of lay as compared to the 18weeks of lay. Real-time PCR provided a more rapid and reliable method of detection of Salmonella on all dry sample types whereas the traditional culture method proved much more reliable when trying to detect Salmonella in wet faecal samples.

Comparative phenotypic and genotypic virulence of Salmonella strains isolated from Australian layer farms

There are over 2500 Salmonella enterica serovars that circulate globally. Of these, serovars those classified into subspecies I are the most common cause of human salmonellosis. Many subspecies I Salmonella serovars are routinely isolated from egg farm environments but are not frequently associated with causing disease in humans. In this study, virulence profiles were generated for 10 strains of Salmonella enterica isolated directly from egg farm environments to investigate their potential public health risk. Three virulence parameters were assessed including in vitro invasion, in vivo pathogenicity and characterization of genomic variation within five specific pathogenicity islands. These 10 Salmonella strains exhibited significant differences in invasion into the human intestinal epithelial cell line, Caco2. Low, moderate, and high invasion patterns were observed and the degree of invasion was dependent on bacterial growth in a nutritive environment. Interestingly, two Salmonella strains, S. Adelaide and S. Bredeney had consistently low invasion. The S. Typhimurium definitive types and S. Virchow exhibited the greatest cell invasion following growth in Luria Bertani broth. Only the S. Typhimurium strains caused disease in BALB/c mice, yet the majority of serovars were consistently detected in feces over the 21 day experiment. Genomic comparison of the five specific pathogenicity islands has shown that variation in virulence is likely multifactorial. Sequence variability was observed primarily in strains with low virulence. In particular, genes involved in forming the structures of the SPI-1 and SPI-2 type 3 secretion systems as well as multiple effector proteins were among the most variable. This variability suggest that serovars with low virulence are likely to have both invasion and within host replication defects that ultimately limit their pathogenicity.

Pathogenicity of Salmonella Strains Isolated from Egg Shells and the Layer Farm Environment in Australia

ABSTRACT In Australia, the egg industry is periodically implicated during outbreaks of Salmonella food poisoning. Salmonella enterica serovar Typhimurium and other nontyphoidal Salmonella spp., in particular, are a major concern for Australian public health. Several definitive types of Salmonella Typhimurium strains, but primarily Salmonella Typhimurium definitive type 9 (DT9), have been frequently reported during egg-related food poisoning outbreaks in Australia. The aim of the present study was to generate a pathogenicity profile of nontyphoidal Salmonella isolates obtained from Australian egg farms. To achieve this, we assessed the capacity of Salmonella isolates to cause gastrointestinal disease using both in vitro and in vivo model systems. Data from in vitro experiments demonstrated that the invasion capacity of Salmonella serovars cultured to stationary phase (liquid phase) in LB medium was between 90- and 300-fold higher than bacterial suspensions in normal saline (cultured in solid phase). During the in vivo infection trial, clinical signs of infection and mortality were observed only for mice infected with either 103 or 105 CFU of S. Typhimurium DT9. No mortality was observed for mice infected with Salmonella serovars with medium or low invasive capacity in Caco-2 cells. Pathogenicity gene profiles were also generated for all serovars included in this study. The majority of serovars tested were positive for selected virulence genes. No relationship between the presence or absence of virulence genes by PCR and either in vitro invasive capacity or in vivo pathogenicity was detected. Our data expand the knowledge of strain-to-strain variation in the pathogenicity of Australian egg industry-related Salmonella spp.

Egg quality and age of laying hens: implications for product safety

Eggs were collected from commercial caged layer flocks in early, mid, late and very late lay. Eggs were candled and scored for translucency. Cuticle cover was estimated using MST Cuticle Stain and a Konica Minolta hand-held spectrophotometer. Traditional measures of egg quality were determined using specialised equipment. Shell ultrastructural features were scored following plasma ashing of shell samples and viewing under a benchtop scanning electron microscope. Translucency score was significantly higher in late lay than for all other age groups. Shell quality declined with increasing flock age. However, the extent of cuticle cover on the egg shell was not significantly different among flock age groups. The incidence of shell ultrastructural features associated with good quality shells was lower for older flocks and incidence of ultrastructural features associated with poorer quality shells was higher for older flocks. Translucency score had a low correlation with the ultrastructural features of the mammillary layer.

Effects of egg shell quality and washing on Salmonella Infantis penetration

The vast majority of eggs in Australia are washed prior to packing to remove dirt and fecal material and to reduce the microbial contamination of the egg shell. The egg contents can be an ideal growth medium for microorganisms which can result in human illness if eggs are stored improperly and eaten raw or undercooked, and it is estimated that egg-related salmonellosis is costing Australia

Effect of egg washing and correlation between cuticle and egg penetration by various Salmonella strains.

44 million per year. Egg shell characteristics such as shell thickness, amount of cuticle present, and thickness of individual egg shell layers can affect the ease with which bacteria can penetrate the egg shell and washing could partially or completely remove the cuticle layer. The current study was conducted to investigate the effects of egg washing on cuticle cover and effects of egg shell quality and cuticle cover on Salmonella Infantis penetration of the egg shell. A higher incidence of unfavorable ultrastructural variables of the mammillary layer such as late fusion, type B bodies, type A bodies, poor cap quality, alignment, depression, erosion and cubics were recorded in Salmonella penetrated areas of egg shells. The influence of egg washing on the ability of Salmonella Infantis on the egg shell surface to enter the egg internal contents was also investigated using culture-based agar egg penetration and real-time qPCR based experiments. The results from the current study indicate that washing affected cuticle cover. There were no significant differences in Salmonella Infantis penetration of washed or unwashed eggs. Egg shell translucency may have effects on Salmonella Infantis penetration of the egg shell. The qPCR assay was more sensitive for detection of Salmonella Infantis from egg shell wash and internal contents than traditional microbiological methods. The agar egg and whole egg inoculation experiments indicated that Salmonella Infantis penetrated the egg shells. Egg washing not only can be highly effective at removing Salmonella Infantis from the egg shell surface, but also allows subsequent trans-shell and trans-membrane penetration into the egg. Consequently, it is important to prevent recontamination of the egg after washing.

Survey of Enterobacteriaceae contamination of table eggs collected from layer flocks in Australia

In Australia, Europe and the United States, eggs and egg products are frequently associated with Salmonella food poisoning outbreaks. Many of the egg-associated Salmonella outbreaks have been due to the products such as mayonnaise, ice-cream and cold desserts which are eaten without cooking following the addition of raw egg. The ability of four Salmonella isolates (one each of S. Singapore, S. Adelaide, S. Worthington and S. Livingstone) to penetrate washed and unwashed eggs using whole egg and agar egg penetration methods was investigated in the current study. The results of the agar penetration experiment indicated that all the isolates used in the present study have the capacity to penetrate the eggshell. Eggshell penetration by the S. Worthington isolate was higher but not significant (p=0.06) in washed eggs compared to unwashed eggs. However, for all other isolates (S. Singapore, S. Adelaide and S. Livingstone), there was no significant difference in penetration of washed and unwashed eggs. Statistical analysis indicated that cuticle score was a significant linear predictor of Salmonella eggshell penetration. Whole egg penetration results showed that all of the Salmonella isolates used in the present study were capable of surviving on the eggshell surface after 21days of incubation (at 20°C) following a high dose of inoculation (10(5)CFU/mL). The combined data of all isolates demonstrated that, the survival rate of Salmonella on eggshells (inoculated with 10(5)CFU/mL) was significantly higher (p=0.002) at 20°C as compared to 37°C. S. Singapore, S. Worthington, and S. Livingstone were not detected in egg internal contents whereas S. Adelaide was detected in one eggs internal contents.

Prevalence of antibodies to Mycoplasma synoviae in laying hens and possible effects on egg shell quality

In the present study, eggs from commercial caged layer flocks at different stages of lay in Australia were collected. Enterobacteriaceae populations from eggshell surface and eggshell pore were enumerated and these populations characterized using API® Rapid 20E strips. The eggshell surface, eggshell pore and egg internal content samples were also processed for the isolation of Salmonella and these isolates were tested for the presence or absence of several virulence genes (prgH, sopB, spiC, orfL, invA, sifA, sitC, misL). Results indicated that there was no significant difference in total Enterobacteriaceae count on the eggs of the flock from early, mid or late lay flocks. Enterobacteriaceae isolates were of 11 different genera which included: Cedecea, Citrobacter, Enterobacter, Escherichia, Klebsiella, Kluyvera, Leclercia, Pantoea, Salmonella, Serratia and Yersinia. Out of all 153 identified Enterobacteriaceae isolates, the Escherichia genus was reported most frequently (60.78%). Results also indicated that overall there were 4.51% (14/310) Salmonella positive pooled samples. In this study, 14 Salmonella strains were isolated, serotyping confirmed that 12 out of them were Salmonella Infantis and the 2 others were Salmonella enterica subsp. enterica serovar 4,12:d: Polymerase chain reaction results indicated that all Salmonella Infantis isolates harboured invA, misL, orfL, prgH, sifA, sitC, sopB and spiC genes which suggests that Salmonella Infantis strains isolated from eggshell surface may have the capacity to invade and survive in macrophages.