Serological detection of chicken flocks naturally infected with salmonella enteritidis,using an enzyme‐linked immunosorbent assay based on monoclonal antibodies against the flagellar antigen

Summary

The Dutch Salmonella enteritidis monitoring and eradication programme for poultry prescribes a periodic examination of all breeding flocks for the presence of S. enteritidis. For the first years of the programme this was done by bacteriological examination of 50 faecal samples per visit per flock.

In this study we compare the results of bacteriological examination of faecal samples taken at 1580 visits from 545 flocks with those of a S. enteritidis enzyme‐linked immunosorbent assay (ELISA) applied on 24 serum samples per visit per flock. Two flocks were found positive for S. enteritidis by bacteriological examination; both flocks were also detected by ELISA. Ten flocks, bacteriologically negative for S. enteritidis were found positive by ELISA. S. enteritidis was isolated from three of these flocks by repeated and extensive bacteriological examination for verification. Verification was not possible in the fourth EL1SA positive flock. S. enteritidis infections were likely in three other flocks because of the farm histories.

On the basis of the results of this study it was decided to use this ELISA, starting from April 1992, as screening technique in the Dutch S. enteritidis programme instead of bacteriological examination of faecal samples. The ELISA is regarded as a flock test; an extensive, confirmatory bacteriological investigation for S. enteritidis is carried out in ELISA positive flocks to decide whether the flocks are truly infected.     

Prevalence of Salmonella spp. in Austrian broiler flocks in the context of the EU-wide baseline survey 2005-2006

In Austria an EU-wide baseline survey on the prevalence of Salmonella spp. in broilers organized by the EU commission was conducted from October 2005 to September 2006. The aim of this study was to produce comparable data on the prevalence of Salmonella in broiler flocks and holdings for all member states and for the EU-Commission to set EU-wide targets for the control of Salmonella in the broiler populations. A randomised sampling plan was designed according to EU-commission parameters (p = 50%; CI = 95%, a = 5%). Sampling was carried out regularly throughout the whole year. On every farm one flock was sampled with five pairs of boot swabs and analysed in the lab according to appendix D of ISO 6579 (2002). In Austria, 363 flocks on farms consisting of at least 5000 broilers each were tested. 28 flocks (7.7%) showed infections with Salmonella spp., eight flocks (2.2%) had either S. Enteritidis (six flocks) or S. Typhimurium (two flocks). In detail, S. Enteritidis (1.7%), S. Typhimurium (0.6%), S. Montevideo (4.1%), S. Infantis 0.6%, S. Senftenberg, S. Tennessee and S. Virchow (0.3% each) have been found. Data indicated that the risk of vertical transmission of Salmonella spp. to broiler flocks has almost been kept at bay; however, the risk of horizontal transmission still needs attention. Contamination of feeding stuff, possible persistence, spreading between barns of a farm as well as introduction of Salmonella spp. through individuals or materials are important factors for future control strategies.     

Salmonella contamination of poultry flocks in The Netherlands

The contamination of poultry in the Netherlands with Salmonella enteritidis was tested. For this, different methods (detection of S. enteritidis in faecal samples of 25 g; detection of S. enteritidis in cloacal swabs; detection of S. enteritidis by serological testing of antibodies in serum) were compared for their efficiency to detect S. enteritidis in flocks of poultry. Testing of faecal samples clearly yielded the best results. This method was used in a transmission study, in which 14 flocks descending from a contaminated primary mother flock were screened for the presence of S. enteritidis. The method was also used for screening 49 flocks of laying hens and 52 flocks of broiler chickens throughout the Netherlands. From the transmission study it became clear that S. enteritidis, phage type 2 (Dutch phage set) was isolated both from the mother flock and from five of the descendent flocks. Screening of poultry flocks for the presence of salmonella revealed that salmonella was present in 47% of the layer flocks and in 94% of the broiler flocks. S. enteritidis was isolated from 15% of the flocks screened.     

A national survey to estimate the prevalence of Salmonella species among Canadian registered commercial turkey flocks.

In 1990-1991, a national survey was conducted to estimate the prevalence of Salmonella species among Canadian commercial turkey flocks. Two hundred and seventy flocks were randomly selected across Canada. The proportion sampled from each province was selected according to each province's share of the national turkey market. Samples, consisting of 12 pooled litter and four pooled dust samples, were used to determine the Salmonella status of the environment of each flock. Additionally, a one kilogram sample of feed was taken from each flock premise. Salmonella was recovered from environmental samples in 234/270 (86.7%) of flocks and from feed samples in 26/266 (9.8%) of flocks. Forty-eight different Salmonella serovars were isolated from flock environmental samples. The most prevalent serovars were S. anatum, S. hadar, S. agona, S. heidelberg and S. saintpaul which were isolated from 53/270 (19.6%), 49/270 (18.1%), 49/270 (18.1%), 42/270 (15.6%) and 34/270 (12.6%) flocks, respectively.     

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.     

Evaluation of a French ELISA for the detection of Salmonella Enteritidis and Salmonella Typhimurium in flocks of laying and breeding hens

In France, the regular and compulsory detection of Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) in flocks of breeding and laying hens is based on bacteriological examination of environmental swabs and faeces samples. The aim of this study was to compare this bacteriological examination with a serological method (ELISA) developed in our laboratory. This ELISA was first evaluated by use of artificially infected hens. During these experimental infection studies, several groups of hens were inoculated with SE, ST, different vaccines and different Salmonella serovars to calculate the experimental parameters of our ELISA. Then, in a field study, 43 flocks were followed monthly using two bacteriological samples (environmental swab and pool of faeces) and 20 serological samples (sera or yolks). Twenty-seven flocks without SE or ST gave a negative serological response throughout their surveillance. Among the 10 various serovars different from SE and ST isolated in this study, S. Heidelberg, S. Agona and S. Hadar gave seropositive results in seven flocks. Consequently, this ELISA was not specific of SE and ST as it detected serovars sharing or not common antigens with SE and ST. Seropositive results were also obtained each month for two flocks where no Salmonella could be isolated. Finally, in seven flocks found infected with SE or ST, the positive ELISA results appeared later than the bacteriological detection. Therefore, for the detection of chicken flocks recently infected with SE or ST, bacteriological examination currently used in France seems to be more appropriate than this ELISA.     

Field observations with Salmonella enteritidis bacterins

A study involving 11 commercial layer flocks was conducted to determine the efficacy of Salmonella enteritidis bacterins (autogenous or federally licensed). The criterion for evaluation of vaccine efficacy was the presence or absence of S. enteritidis in the environment, the organs of the bird (including ovary and oviduct), and eggs. Environmental, rodent, and organ specimens from dead birds as well as eggs were cultured throughout the life of the flock. All layers were obtained from pullet sources that were negative for S. enteritidis, as determined by organ and environmental cultures. Despite the use of S. enteritidis vaccination, 63.6% of the houses had S. enteritidis-positive environmental cultures and 100% of the flocks had S. enteritidis organ-culture-positive birds. The range of positive cultures for S. enteritidis in the environment in vaccinated flocks was between 0 and 45.5%. Birds in vaccinated flocks were organ-culture positive for S. enteritidis between 10% and 40% of the time. The unvaccinated portion of flocks in the same house and the unvaccinated flock in a complex had similar results compared with the vaccinated portion of the flocks.     

Salmonella enteritidis and other Salmonella in laying hens and eggs from flocks with Salmonella in their environment.

Seven Canadian layer flocks with Salmonella enteritidis in their environment were investigated to determine the numbers of hens infected with S. enteritidis, the localization of S. enteritidis in organs of infected hens and the numbers of S. enteritidis-infected eggs produced by two affected flocks. By a microagglutination test (MAT) using S. pullorum antigens, these flocks had more seropositive hens (mean 51.9 +/- 16.9%) than two Salmonella-free flocks (mean 13.0 +/- 4.2%). Culture of tissues of 580 hens (433 seropositive) from the seven flocks detected 26 (4.5%) S. enteritidis-infected hens from two flocks. In one flock, 2/150 hens were infected with S. enteritidis phage type (PT) 8, which was confined to the ceca, and no Salmonella spp. were isolated from 2520 eggs (one day's lay). In the second flock, where 24/150 hens were infected with S. enteritidis PT13, extraintestinal infection was found in nine hens and involved the ovaries and/or oviduct in two hens. Salmonella enteritidis PT13 was isolated from one sample of egg contents and from one sample of cracked shells from among 14,040 eggs (one day's lay) from this flock. The overall prevalence of S. enteritidis-contaminated eggs from the two flocks with infected hens was less than 0.06%. Other Salmonella spp. isolated were S. heidelberg from 58 hens (10%), and S. hadar, S. mbandaka and S. typhimurium from one hen (0.2%) each. The MAT with antigens of S. pullorum had a sensitivity of 81% and a specificity of 24% for detecting S. enteritidis-infected hens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of vaccination and other preventive methods for Salmonella enteritidis on commercial laying chicken farms

The effect of introducing vaccinated commercial laying chickens on to farms, which previously had laying flocks that were infected with Salmonella Enteritidis, was investigated by sampling faeces and environmental samples, and in some cases spent hens. In 15 of 17 free-range flocks vaccination eliminated any evidence of infection. In 11 barn egg production flocks, vaccination produced similar results in four flocks on one farm but infection persisted in seven flocks on other farms. Vaccination of two consecutive cage layer flocks led to a gradual disappearance of the infection, but in 18 other flocks there was evidence of infection after vaccination. In one continuously occupied cage layer house, treatment by competitive exclusion was followed by a gradual disappearance of S Enteritidis in faeces and a substantial reduction in its levels in the environment. On four barn egg production sites disinfection with a formaldehyde, glutaraldehyde and quaternary ammonium compound disinfectant eliminated Salmonella species even though birds housed subsequently were not vaccinated. In three flocks that had been vaccinated for four years, S Enteritidis was still present. In most cases the poor performance of the vaccine was associated with severe rodent control problems and a poor standard of cleaning and disinfection.     

Assessment of cleaning and disinfection in Salmonella-contaminated poultry layer houses using qualitative and semi-quantitative culture techniques

Salmonella infection of laying flocks in the UK is predominantly a problem of the persistent contamination of layer houses and associated wildlife vectors by Salmonella Enteritidis. Methods for its control and elimination include effective cleaning and disinfection of layer houses between flocks, and it is important to be able to measure the success of such decontamination. A method for the environmental detection and semi-quantitative enumeration of salmonellae was used and compared with a standard qualitative method, in 12 Salmonella-contaminated caged layer houses before and after cleaning and disinfection. The quantitative technique proved to have comparable sensitivity to the standard method, and additionally provided insights into the numerical Salmonella challenge that replacement flocks would encounter. Elimination of S. Enteritidis was not achieved in any of the premises examined although substantial reductions in the prevalence and numbers of salmonellae were demonstrated, whilst in others an increase in contamination was observed after cleaning and disinfection. Particular problems with feeders and wildlife vectors were highlighted. The use of a quantitative method assisted the identification of problem areas, such as those with a high initial bacterial load or those experiencing only a modest reduction in bacterial count following decontamination.     

Evaluation of radiolabeled and colorimetric DNA probes in comparison with an antigen screening assay for the detection of Salmonella from poultry farms

A total of 48 environmental drag-swab samples from various poultry farms were tested for the presence of Salmonella spp. by culture, an enzyme-linked immunosorbent assay-based Salmonella antigen screening (SAS) assay, and two DNA probes (radiolabeled and colorimetric). The radiolabeled DNA probe was allowed to hybridize with culture-positive samples (n = 8) and was found to detect Salmonella spp. in all cases (100%). Both of the probes, subsequently hybridized with culture-negative samples (n = 8), were observed to yield good agreement (91%) with the culture findings. The remaining samples (n = 32) were tested by the SAS assay, and where there was no agreement between the culture and SAS, samples were further examined by the DNA probes. Results using both probes agreed with those obtained by culturing the samples but did not agree with the SAS assay result when the ratio of samples tested to samples positive (S/P) cutoff value used was 0.5.     

Resistance to synthetic pyrethroids in South Australian populations of sheep lice (Bovicola ovis)

SUMMARY Lice were collected from 71 flocks detected as Infested at market Inspection in 1990 and 1991, from 16 flocks where resistance was suspected and from 31 flocks from Kangaroo Island. Susceptibility to cypermethrin was measured by a treated surface technique and survival of lice at 5 ppm or greater was taken as an indication of resistance. The prevalence of resistance was 34% in louse populations from the market inspection sample, 50% in flocks in which resistance was suspected and 68% in flocks from Kangaroo Island. Most resistance factors were in the range 1 to 20 although one louse population with a resistance factor of 91 was found on Kangaroo Island.     

Salmonellosis in breeds of pigs with latent infections and in salmonellosis foci

The occurrence of salmonellae in pigs in Slovakia is described for the period from 1971 to 1980. On the whole, 1430 strains (11 serological types) of salmonellae were isolated in stocks with latent infections. The proportions of the serological types were as follows: S. agona 0.69%, S. anatum 0.14%, S. arizona 0.07%, S. bareilly 0.14%, S. decatur 0.07%, S. enteritidis 1.12%, S. give 0.28%, S. heidelberg 0.07%, S. choleraesuis 93.71%, S. panama 0.07% and S. typhimurium 2.45%. In 1315 salmonellosis foci 1333 strains (six serological types) of salmonellae were isolated. The proportions of the serological types were as follows: S. agona 0.37%, S. anatum 0.07%, S. bareilly 0.22%, S. enteritidis 1.20%, S. choleraesuis 90.59% and S. typhimurium 5.30%. The annual pattern of the occurrence of the most frequent serological types is described.     

Bayesian estimation of flock-level sensitivity of detection of Salmonella spp., Enteritidis and Typhimurium according to the sampling procedure in French laying-hen houses

A study was carried out to estimate the prevalence of flocks infected by Salmonella spp., S. Enteritidis and S. Typhimurium in 521 French laying-hen farms from October 1st 2004 to September 30th 2005 as part of a European Union-wide baseline study to define targets for Salmonella reduction in member states. The sampling scheme prescribed and financed by the European Commission to detect Salmonella in laying-hen flocks was based on 2 dust-samples and 5 faeces-samples per farm. A latent-class Bayesian approach for correlated tests was used to estimate the sensitivity of detection of reduced sampling schemes corresponding to the 16 combinations of 2 dust- and 5 faeces-samples. For each model the full sampling scheme (7 samples) and the reduced protocol were considered as two correlated tests, the biological principle being identical and the reduced protocol being a subset of the full sampling scheme. As the observed apparent prevalence in cage flocks was higher than in other systems (barns, outdoor, or organic) these two sub-populations were considered separately. Bayesian estimation of posterior medians with 95% probability intervals for true prevalence in cage flocks were 0.34 (0.29; 0.39) and 0.13 (0.10; 0.18) for Salmonella spp. and Salmonella Enteritidis+Typhimurium respectively. In alternative flocks posterior medians with 95% probability intervals for true prevalence were 0.09 (0.06; 0.13) and 0.05 (0.03; 0.08) for Salmonella spp. and Salmonella Enteritidis+Typhimurium, respectively. In cage flocks Bayesian estimation of posterior distributions for sensitivity indicated that at least 5 samples, including 2 dust samples were necessary to attain comparable sensitivity levels to the full sampling scheme. In alternative flocks and for Salmonella spp. 6 samples were required to ensure a comparable sensitivity level to the full sampling scheme. Detection sensitivity was improved by increasing the number of dust samples in cage farms and by increasing the total number of samples whatever their type in alternative farms.     

Plasmid analysis and epidemiology of Salmonella enteritidis infection in three commercial layer flocks.

Ninety-six S. enteritidis isolates obtained from three commercial layer flocks in 1988-90 were examined following DNA extraction, restriction enzyme digestion, and gel electrophoresis for plasmid size profiles and restriction fragment length polymorphisms (RFLPs). The S. enteritidis isolates from the three flocks had three, eight, and two different plasmid profiles, respectively. Only four isolates from one flock lacked plasmids. A 36-megadalton (mDa) (54-kilobase) plasmid was present in 73% of the isolates, either alone or in combination with other plasmids. Isolates with only the 36-mDa plasmid had identical RFLPs. The diversity of plasmid profiles was greater than that of phage-types among isolates from the three flocks: 12 unique plasmid profiles vs. four phage-types. Mixed infections with S. enteritidis strains having distinct plasmid profiles occurred in all three flocks. Reinfection of these flocks in 1990 with one or more of the strains obtained earlier was evident, because some of the original isolates and the 1990 isolates had matching plasmid profiles and were of the same phage-types. Isolates from both environmental and tissue samples, examined from one flock, were found to share the same plasmid profile and phage-type.     

Detection of specific antibodies against deflagellated Salmonella Enteritidis and S. Enteritidis FliC-specific 9kDa polypeptide

Specific antibody levels of laying hens and young chickens experimentally infected with Salmonella Enteritidis and vaccinated farm flocks were evaluated by enzyme-linked immunosorbent assays (ELISAs) with two different antigens, deflagellated S. Enteritidis whole cell (DEWC) and S. Enteritidis FliC-specific 9kDa polypeptide (SEP9). Infected laying hens excreted S. Enteritidis throughout the experimental period, and the specific antibody titers in DEWC-ELISA, were significantly higher than the uninfected group. It suggests that this DEWC-specific antibody will serve as an effective indicator of S. Enteritidis infection, especially for non-vaccinated laying flocks. SEP9-specific antibodies were detected in spray-inoculated young chickens but not in oral-inoculated young chickens. Compared with greatly high SEP9-specific antibody levels of vaccinated farm flocks, no response was observed in orally infected hens. These results indicate that S. Enteritidis discontinues expressing SEP9 once it has crossed the intestinal barrier, and that SEP9-ELISA will serve as a valuable monitoring tool for the status of S. Enteritidis vaccination on a flockwide basis, independent of stable S. Enteritidis infections.     

The role of litter beetles as potential reservoir for Salmonella enterica and thermophilic Campylobacter spp. between broiler flocks

We evaluated the role of beetles infesting broiler chicken rearing facilities as potential reservoirs for Salmonella enterica infections between successive broiler flocks. In addition, their role as potential reservoirs for thermophilic Campylobacter spp. was also investigated. Fourteen broiler houses located at 11 different farms were included in the study. The houses were nonrandomly selected on the basis of their salmonella status; nine were persistently contaminated with salmonella whereas five were salmonella negative. For each broiler house, two consecutive broiler flocks (i.e., 28 broiler flocks in all) as well as beetles collected during both rotations of production and in the empty period (after cleaning and disinfection) between these flocks were monitored for the presence of salmonella. Examinations for the presence of campylobacter in the same sample materials were also performed. Beetles sampled during production were positive for salmonella or campylobacter or both. Furthermore, in one house, the occurrence of Salmonella indiana in two consecutive broiler flocks coincided with the presence of S. indiana-contaminated beetles in the empty period between the flocks. The genotype of the identified S. indiana was in all cases identical when analyzed by pulsed-field gel electrophoresis. However, our results also suggest that salmonella from beetles may not always be transmitted to the chickens and that beetles living in contaminated houses can remain free of infection. All cases of campylobacter-positive beetle samples were detected in connection with a positive chicken flock; in no case was campylobacter isolated from beetles taken from the empty period between rotations. Four beetle species were identified during this study. Alphitobius diaperinus was found in all houses and was relatively abundant in most. Typhaea stercorea and Ahasverus advena were found in eight and nine houses, respectively, and were abundant in most of these. Carcinops pumilio was found in small numbers in eight houses. No other insect species was identified. These investigations have shown that beetles in broiler houses infrequently are positive for salmonella. However, transmission of S. indiana between two consecutive broiler flocks can coincide with the presence of salmonella-contaminated beetles in the empty period, indicating that the beetles were the reservoir of S. indiana between the two flocks. Concerning campylobacter, the results suggest that beetles do not play a significant role as a reservoir of campylobacter from one rotation to the next.     

An economic analysis of the impact of subclinical (mild) necrotic enteritis in broiler chickens

Costs to broiler producers associated with subclinical (mild) necrotic enteritis (SNE) were estimated using published information on impacts on body weight and feed conversion rate (FCR) associated with SNE and costs and revenues associated with broiler production. Estimates were expressed in U.S. dollars from the perspective of poultry producers. SNE was estimated to result in a 12% reduction in body weight and a 10.9% increase in FCR compared with healthy birds. For the purposes of this analysis, we considered scenarios involving hypothetical flocks of 20,000 birds raised to final body weights ranging from 4.63 to 7.94 lb. The incidence of SNE was assumed to occur at 20% based on the literature. For flocks raised for the length of time required to reach these target weights, SNE resulted in a loss to producers ranging from US$878.19 to US$1480.52 per flock. When feed costs required to obtain SNE flocks having a total live body weight equal to equivalent healthy flocks at market age were calculated, the increased cost to producers ranged from US$370.49 to US$739.38 per flock. SNE has the potential to cause a significant negative economic impact in broiler flocks. Strategies to reduce the incidence of SNE may help to increase the profitability of broiler production.     

Isolation of avian influenza virus in Texas

An avian influenza virus with surface antigens similar to those of fowl plague virus (Hav 1 Nav 2) was isolated in 1979 from 2 commercial turkey flocks in Central Texas. Two flocks in contact with these infected flocks developed clinical signs, gross lesions, and seroconversion but yielded no virus. This was the first recorded incidence of clinical avian influenza in Texas turkeys and only the second time that an agent with these surface antigens was isolated from turkeys in U.S.     

Monitoring the Immune Status of Broilers Against Reoviruses Using Challenge and Serologic Data

Reoviruses are an important cause of suboptimum performance in commercial broilers worldwide. Integrators use the enzyme-linked immunosorbent assay against the S1133 antigen for monitoring serum of breeders for indicating pullet vaccine success. However, without correlating serology to reovirus challenge, it is difficult to determine whether titers reflect protective immunity. We developed a broiler challenge test against 2 common reovirus isolates (2408 and S1133) to evaluate the efficacy of reovirus pullet vaccine programs. Two reovirus serologic and challenge studies were undertaken using chicks from broiler integrators from the southeastern United States. Breeder flocks, from which the chicks were obtained, received at least 1 live and 2 inactivated reovirus vaccines during their pullet phase. One-day-old progeny were collected from 6 breeder flocks. At 1 d of age, 20 chicks from each broiler flock were bled, and serum was analyzed for antibodies. At 3 to 4 d of age, 20 progeny per flock were challenged with the 2408 reovirus by intratracheal route. At 10 to 14 d of age, another 20 birds per flock were challenged with the S1133 reovirus by footpad. Twenty birds per flock were used as nonchallenged controls. At 3 wk of age, all birds were killed and weighed. Percentage of protection was calculated for each flock based on the absence of gross lesions. Flocks with at least 50% protection were considered well protected. Most flocks were well protected against both viruses. The percentage of protection correlated with day-old enzyme-linked immunosorbent assay titers. Chicks from younger hens had higher titers and the best protection against challenge. Producers, whose hen flocks were monitored herein, were doing a good job of immunizing pullets against reovirus. They are now using reovirus progeny challenge studies along with breeder antibody titers to determine vaccination success of their pullets.