Better Understanding of Salmonella in Poultry Presented to US Vets

Ten presentations on Salmonella infections in poultry and the potential they have to be passed on to humans were made to the 150th annual convention of the American Veterinary Medical Association (AVMA) in Chicago earlier this year.
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Epornitic of Salmonella enteriditis Causing Clinical Disease in Commercial Broilers

G. Donald Ritter of Mountaire Farms Inc. reported the study of an epornitic of Salmonella enteriditis (SE) causing clinical disease in commercial broilers on many farms originating from one broiler breeder complex.

Reports of elevated chick mortality associated with specific broiler breeder flocks triggered a vertical origin disease investigation. Early mortality was high on affected farms and morbidity and excess mortality became chronic in many houses with lameness, stunting and septicemia observed. Breeder flock trace-backs revealed that affected broilers were initially from a common group of young breeder flocks, ultimately expanding to include 28 breeder flocks placed over a five-month period.

Typical gross lesions of SE were observed in broilers while implicated hen flocks remained asymptomatic and tested negative via boot swab surveillance for SE. Lab reports confirmed septicaemia with heavy growth of Salmonella Group D that was confirmed as SE by NVSL serotyping.

Antibiotics were administered to SE-positive breeder and broiler flocks to mitigate economic losses.

Dr Ritter added that an epidemiological investigation was unable to determine a common source of SE contamination of SE positive breeder flocks.

Salmonella Trends from 2000 through 2012

Over the last 13 years, more than 290,000 samples have been cultured for Salmonella at the Georgia Poultry Laboratory Network, according to that organisation's Doug Waltman.

The laboratory serogroups every isolate and serotypes representative isolates from every positive accession.

Data were presented showing the trends for the percentage of positive samples, particular serogroups and serotypes for this time period. Salmonella status over time for some participants was also presented at the meeting.

Evaluation of Salmonella Risk Factors on Broiler Farms in North Carolina

Twenty broiler farms were evaluated by Jesica J. Leonard of North Carolina State University and other researchers there an with Wayne Farms LLC to investigate possible correlations between management strategies during the grow-out phase of production and Salmonella incidence at processing.

The farms were selected based on the presence of Salmonella at the hock cutter, pre-chill and post-chill areas of the plant while these farms were being processed over a two-year period (2010-2012).

Ten farms with the highest incidence of Salmonella were selected to represent the 'positive' (POS) farms, and 10 farms with zero positive samples were selected to represent the 'negative' (NEG) farms.

While a few factors such as wind speed and rodent control showed a possible correlation, the factors showing the strongest correlation were the average rate of weight gain and a history of growing broilers for another poultry producer. The POS farms averaged a daily weight gain of 0.1397 pounds, while the NEG farms averaged 0.1341 pounds.

Farm samples using boot swabs were also incorporated into the evaluation and one house per farm was sampled. Of the POS farms, 100 per cent were positive for Salmonella using boot swabs, while 50 per cent of the NEG farms returned boot swabs positive for Salmonella. Within the past two to four years, 70 per cent of the POS farms had grown broilers for another poultry producer.

Because there were variables that were unaccounted for during this evaluation, no statistics were performed, explained Dr Leonard. However, she commented that this evaluation provided a good start to determining the cause and reduction of Salmonella within this particular complex.

Challenge Studies in Vaccinated Commercial Layers to Demonstrate Protection against Salmonella of Food Safety Significance

To help reduce Salmonella colonisation in laying hens, commercial egg producers typically administer a Salmonella enteritidis bacterin to pullets prior to the onset of lay, according to Carl Heeder and colleagues at Pfizer Animal Health (now Zoetis).

Challenge studies designed to understand better the impact various vaccination protocols have on Salmonella protection and food safety were conducted. Groups of pullets were vaccinated at a commercial layer complex. Pullets were vaccinated with various combinations of Poulvac ST, a live ST vaccine, and PoulVac SENDIB, an inactivated SE vaccine. SPAFAS hens were used as an unvaccinated control.

Treatment groups were challenged via oral gavage with Salmonella at Auburn University in isolation cages. One week after the challenge, caecal and internal organ samples were collected and cultured for the presence of Salmonella.

Level of protection was determined based on the percentage of positive culture from each tissue set. Results were reported to the AVMA meeting.

Salmonella Status of Pullorum Tube Test Reactor Birds

When a reactor, positive test, occurs on the Salmonella Pullorum Tube Test, the bird initiating the reaction is brought into the Georgia Poultry Laboratory for necropsy and isolation of the Salmonella enterica serotype causing the reaction, reported Brenda Glidewell from the Georgia Poultry Laboratory Network.

Since Salmonella Pullorum has not been isolated in commercial poultry in Georgia for many years, if Salmonella is isolated from the submitted bird, it is always another Salmonella enterica serotype. Analysis of the Salmonella enterica serotype isolated and frequency, is compared to the Pullorum Tube test results.

Chicken and the Egg: A Study of Source Attribution Estimates for Salmonella enterica Serotype Enteritidis

To reduce Salmonella Enteritidis (SE) illnesses, FDA published in 2009, 'Prevention of Salmonella Enteritidis in Shell Eggs During Production, Storage, and Transportation', according to Dana J. Cole from Centers for Disease Control and Prevention in Atlanta, Georgia. To measure progress, estimates of the proportion of illnesses attributable to shell eggs are needed. However, different data sources and methods are available, and each produces a different result. Dr Cole reported that she and her group analysed these and designed a new approach to blend different estimates.

The Foodborne Diseases Active Surveillance Network conducted two case-control studies of sporadic (non-outbreak) SE illnesses in 1996 and 2002, respectively. Data from foodborne disease outbreaks are also used to provide estimates based on the proportion of outbreaks associated with each food commodity.

The researchers used newly developed analytic methods to re-analyse these data and calculate new population attributable fractions for each data source and blended these into a single estimate for each commodity.

Re-analysing the case-control studies that previously estimated the proportion of domestically-acquired SE infections associated with chicken, they found between 28 per cent (1996) and 36 per cent (2002), and with eggs, 34 per cent (2002).

Data from outbreaks reported between 1998 and 2009 provides estimates that 18 per cent of foodborne SE outbreaks are associated with chicken, and 62 per cent with eggs.

This new approach standardises these measures and provides preliminary blended estimates of 13 to 18 per cent for chicken and 35 to 40 per cent for eggs.

Dr Cole concluded that source attribution estimates are needed to assess the impact of interventions but the data sources have major limitations. The Atlanta-based group developed an approach to analyse data and blend estimates that limits the biases associated with each data source.

DNAble® Technology for Rapid Detection and Evaluation of Salmonella in Pre-processing Poultry Samples

Salmonella has become an important economic and public safety concern for the commercial poultry industry; contamination of poultry products is the primary vector attributed to Salmonella outbreaks in the United States, reported Nicholas P. Evans from Virginia Tech. Diagnostic testing of raw poultry is now becoming a necessity, he said, and while this strategy may reduce further outbreaks, it does little to circumvent contamination and the potential need for a product recall.

Current methods of Salmonella detection require a minimum of 24 hours to complete. If a rapid detection method could be developed to evaluate the Salmonella load of poultry entering the processing plant, effective intervention strategies could be developed to decrease the potential of foodborne salmonellosis.

New methods incorporating DNAble technology from Envirologix Inc. are currently being investigated because of the potential advantages compared to other diagnostic methods. The DNAble assay can be used with crude sample matrices, requires minimal sample preparation, and has increased sensitivity compared to PCR and culture methods.

To evaluate the incoming Salmonella load, drag swabs were collected from live-haul trailers upon arrival at the processing plant.

Salmonella was detectable as early as six hours after incubation in buffered peptone water with DNAble supplement, Evans reported.

Analysis of incoming Salmonella load and contamination of ground poultry products were presented and implementation of this assay into pre-processing screening were discussed.

Evaluation of a dkgB-linked Intergenic Sequence Ribotyping Method for Assigning Serotype to Salmonella enterica Isolated from Poultry Environmental Samples

The Kauffman White (KW) serotyping method requires more than 250 antisera to characterise more than 2,500 Salmonella serovars. The complexity of serotyping could be overcome using molecular methods.

In this study reported by Bwalya Lungu from the Aviagen Veterinary Laboratory in Elkmont, Alabama, a dkgB-linked intergenic sequence ribotyping (ISR) method that generates sequence occurring within and around one ribosomal region of the Salmonella enterica genome was applied.

A group of 248 suspected Salmonella isolates obtained from poultry environmental samples was evaluated.

The ISR method correctly assigned serotype to 228/248 (91.9 per cent) of the isolates, and this group included 21 isolates (8.5 per cent) that could not be assigned serotype by KW. Twenty of the 248 (8.1 per cent) of the isolates were not Salmonella.

Of the 227 isolates assigned serotype by ISR, 151/227 (67 per cent) isolates had matching serotypes between the ISR and KW methods. However, 76/227 (33 per cent) isolates had disagreement.

Conflicts between ISR and KW suggest the cultures from which the test isolates were obtained may have been mixed at time of storage. Storing samples by freezing appeared to impact results in two ways, namely by increasing the number that could not be serotyped by the KW method and the incidence at which mixtures may yield an alternative serotype.

In addition to high specificity and sensitivity, ISR is faster, cheaper and less time-consuming than traditional serotyping.

Overall, concluded Lungu, the results suggest that the ISR method is an alternative tool to the Kauffman White method for serotyping and differentiating between the various Salmonella enterica serotypes isolated from the poultry environment.

Use of Intragenic Sequence Ribotyping for Serotyping Salmonella Obtained from Commercial Birds and Poultry Environment in Mississippi and Determination of Their Antibiotic Resistance Patterns

The PCR method called Intergenic Sequence Ribotyping (ISR) uses sequence data from the ribosomal region neighbouring the gene, dkgB, from the end of the 23S rRNA to the start of tRNA aspU to assign serotype to Salmonella enterica.

The purpose of this research, explained M. Pulido from the National University of Colombia, was to compare the results obtained by ISR with those obtained with the traditional Kauffman-White-Le Minor (KWL) scheme (Performed by NVSL, Ames, Iowa) for the characterisation and serotyping of Salmonella isolates from commercial birds and their environment included in the bacterial collection of the Poultry Research and Diagnostic Lab in Mississippi.

Antimicrobial resistance of Salmonella from poultry was also evaluated because resistance is a serious public health issue.

In this study the Antibiotic Resistance Patterns (ARP) of selected S. enterica isolates (Enteritidis, Kentucky, Typhimurium, Montevideo, Mbandaka, 4,5,12 i:-, Bredeney and Saintpaul) were determined by Minimum Inhibitory Concentration (Sensititre Microbiologic Systems).

ISR assigned serotype to 50 Salmonella enterica isolates and results were in agreement with KWL in more than 92 per cent of the cases. Mixtures of serotypes were also identified, Pulido reported. The antibiotic susceptibility test showed differences between Salmonella serotypes and within isolates of the same serotype.

Whole Genome Sequences and Comparative Genotypic and Phenotypic Analysis of Multiple Salmonella enterica serovar Enteritidis Isolates from Poultry and Poultry Environments

Twenty Salmonella enterica serovar Enteritidis (SE) isolates from poultry and poultry environments were selected for whole genome sequencing using Ion Torrent semiconductor technology, explained Ceva Animal Health's John El-Attrache.

The SE isolates were selected from different geography, environment and time sources. Comparative genomic analysis was performed and included evaluation of virulence plasmids, Salmonella pathogenicity islands (SPI 1 and 2), repetitive elements as well as regulatory, core, flagella, fimbrial and biofilm genes. Phenotypic analysis included phage typing, biochemistries, motility, and biofilm potential.

Genetic and phenotypic analysis has allowed for a more refined grouping of these SE isolates and these groupings will be further assessed via chick pathogenicity analysis, El-Attrache reported. He added that compilation of final genotype and phenotype characteristics will be used to validate key genomic signatures across the whole microbial genome that identify potential vaccine candidates.

Further Reading

Find out more information on salmonellosis in poultry by clicking here.

December 2013

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