Serological and bacteriological investigations of chickens from flocks naturally infected with Salmonella enteritidis

Groups of 10 birds were obtained from four flocks which had shown evidence of natural salmonella infection. S enteritidis had been isolated from three flocks and S typhimurium from the fourth. Each bird was housed in a separate cage and blood samples and cloacal swabs were taken weekly to follow the course of natural infection. After four weeks the birds were killed and examined post mortem. The isolation of Salmonella species could not be related to the serological results. In individual birds the rapid slide test and tube agglutination test could not be relied upon to detect infection; the microantiglobulin test and the enzyme-linked immunosorbent assay (ELISA) were more sensitive than the other tests and detected some infected birds that were negative by the rapid slide and tube agglutination tests, and also showed high titres in some birds from which Salmonella species could not be isolated post mortem. Sera obtained from two flocks which had a history of natural S enteritidis infection were evaluated by all the tests; evidence of infection was found with the microantiglobulin and ELISA tests but not with the other tests.     

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.     

Development and application of an ELISA for detecting antibodies to Salmonella enteritidis in chicken flocks.

Enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of IgG antibody to Salmonella enteritidis in poultry flocks. A lipopolysaccharide (LPS) and heat-extracted (HE) antigen for use in the ELISA were evaluated together with the rapid slide test (RST), microagglutination test (MT) and the microantiglobulin (MAG) test. In experimentally infected specific pathogen free chickens, good correlation was seen between all tests although, generally, the MT and MAG detected antibody earlier and titres peaked earlier than the ELISAs. The LPS antigen detected antibody earlier than the HE antigen but the latter gave higher titres in the later stages of infection. Cross reactions were seen between S enteritidis and S typhimurium in the ELISAs although homologous reactions were always much higher. Antisera to S montevideo or S senftenberg gave weak positive reactions in both S enteritidis ELISAs. Serological and bacteriological examinations of representative samples from two commercial chicken flocks were carried out. In flock A the HE-ELISA and MAG test detected antibody in nearly all birds. The LPS-ELISA detected antibody in over 60 per cent of birds, while the MT and RST detected few seropositive birds. The whole blood test using the stained S pullorum antigen on the farm detected antibody in just under 25 per cent of the birds. S enteritidis was isolated from the organs of 25 per cent of the birds. All birds in flock B were seronegative by all tests; no salmonellae were isolated from the organs of these birds.     

Serological response of chickens naturally infected with Salmonella typhimurium detected by ELISA.

Four laying flocks of chickens in Britain, each with a history of Salmonella typhimurium infection, were investigated serologically and bacteriologically. Blood samples were taken from identified birds from a single house on each site and sent to the Central Veterinary Laboratory, Weybridge for serological examination using enzyme-linked immunosorbent assays (ELISA) and rapid slide agglutination test (RST) using stained S. pullorum. The identified birds were taken to the local Veterinary Investigation Centre for bacteriological examination. On site A no salmonellae were recovered from birds in the house chosen for serological examination. Of these birds approximately 20% had antibodies to S. typhimurium in ELISA which used either a lipopolysaccharide (LPS) or heat-extract (HE) antigen from S. typhimurium. S. typhimurium was recovered from birds in one other of the four houses on the same site; these birds were not tested serologically. On site B, S. typhimurium was isolated from 8% of the birds examined. Of the total tested serologically, a third to half were seropositive by S. typhimurium ELISA using the LPS and HE antigen respectively. A small proportion of birds was seropositive by S. enteritidis ELISA and RST. No salmonellae were isolated from the other two sites although about 10% of birds tested on site C were seropositive in S. typhimurium ELISA. Cross-reactions were seen between S. typhimurium antigens in the ELISA and experimentally prepared antiserum to S. enteritidis. The S. enteritidis ELISA was generally more specific although cross-reacting antibodies were detected in sera from birds on sites A and B.     

Pathogenesis of Salmonella enteritidis infection in laying chickens. I. Studies on egg transmission, clinical signs, fecal shedding, and serologic responses

Laying hens were inoculated orally, intracloacally (IC), or intravenously (IV) with Salmonella enteritidis phage type 8 isolates from a human (E700-87) eggs (Y-8P2), or the ovary of a hen (27A). Oral or IV inoculation of 2 x 10(8) to 4 x 10(8) colony-forming units (CFU) of E700-87 caused depression, anorexia, reduced egg production, diarrhea, and some mortality. Lower doses resulted in milder clinical signs. S. enteritidis was cultured from the shells of a few eggs but not from egg contents. Fecal shedding persisted for up to 6 weeks in some birds. Isolate Y-8P2 (10(6) CFU) also caused anorexia, diarrhea, and a drop in egg production. Hens inoculated orally or IC were less severely affected than those inoculated IV. Fecal shedding was intermittent and lasted up to 18 days. Eggshells from the IC-inoculated birds had the highest rate of contamination, and S. enteritidis was isolated from the albumen of 11 and yolk of three of 726 eggs. Oral inoculation of 10(6) CFU of isolate 27A resulted in a bacteremic infection with seeding of the liver, spleen, peritoneum, ovule, and oviduct. However, the birds remained clinically normal with normal egg production. S. enteritidis was cultured from the yolk and albumen of a small number of eggs until 11 days postinfection. Antigen prepared from S. enteritidis detected antibody in more sera than did commercially available S. pullorum antigen in agglutination tests.     

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.     

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.     

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.     

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.     

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)     

Detection and screening of Salmonella enteritidis-infected chickens with recombinant flagellin.

Screening and identification of Salmonella enteritidis in commercial poultry flocks have assumed principal roles in preventing transmission of this pathogen to humans from hen eggs. Serologic diagnosis of S. enteritidis infection in commercial flocks currently relies on laboratory-based tests for detection of antibodies to the lipopolysaccharide, whole flagella, and bacteria. We amplified a sequence from the g,m flagellin of S. enteritidis, followed by cloning, expression, and purification of the protein. The recombinant protein was first characterized by western blot and subsequently evaluated as enzyme-linked immunosorbent assay (ELISA) antigen for detection of S. enteritidis infection. A total number of 49 positive sera and 40 negative sera were tested for ELISA validation. A cutoff value of 0.14 was shown to be sufficient to discriminate the negative and positive sera. Results obtained by testing sera raised against different bacterial strains/serotypes further confirmed that this recombinant flagellin-based ELISA was indeed specific for the detection of S. enteritidis. Both sensitivity and specificity of the developed ELISA test were comparable with a commercially available test, indicating that it is a highly promising and reliable diagnostic tool for S. enteritidis infection.     

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.     

Characterisation of monoclonal antibodies against a fimbrial structure of Salmonella enteritidis and certain other serogroup D salmonellae and their application as serotyping reagents

A panel of 13 monoclonal antibodies from different hybridomas was produced against a novel salmonella fimbrial antigen expressed predominantly by Salmonella enteritidis strains. The specificity of the monoclonal antibodies to this antigen (SEF14) was confirmed by enzyme-linked immunosorbent assay (ELISA) using purified SEF14, immune electron microscopy and, with 11 monoclonal antibodies, the identification of a repeating protein subunit (14,300kDa) on the antigen. Blocking-ELISA with the monoclonal antibodies identified epitopes in at least three, non-overlapping clusters which appeared evenly distributed on SEF14 in immune electron microscopy. The use of the monoclonal antibodies in direct-binding ELISA on a range of salmonella serotypes suggested that the epitopes on SEF14 are highly conserved and were expressed by all the S enteritidis strains examined; some strains of S dublin and the only strain of S moscow available were the only other serotypes that expressed SEF14. A latex agglutination reagent based on a monoclonal antibody was developed and used to test for SEF14 on 280 strains (representing 120 serotypes in 24 serogroups of salmonellae) that had been grown on Sensitest agar for 18 hours at 37 degrees C. All S enteritidis strains (64) and most S dublin strains (28 of 33) produced SEF14 as did the two strains representing S blegdam and S moscow. SEF14 was not detected in any other strains of serotypes from serogroup D or from any other serogroup examined.     

The relationship between the magnitude of the specific antibody response to experimental salmonella enteritidis infection in laying hens and their production of contaminated eggs.

Detecting infected laying flocks is a vital part of many efforts to control egg-associated transmission of Salmonella enteritidis to humans. The relationship between the development of a specific antibody response in infected hens and the deposition of S. enteritidis in eggs is important for establishing the epidemiologic relevance of serologic testing methods. In two trials, laying hens were infected with large oral doses of phage types 13a and 14b isolates of S. enteritidis. Approximately 38% of all infected hens produced at least one contaminated egg, at an overall incidence of 5.2%, between 3 and 23 days postinoculation. As determined by enzyme-linked immunosorbent assay with an S. enteritidis flagellar antigen, 91.7% of inoculated hens produced specific serum antibodies. Although hens with very high antibody titers were associated with a significantly elevated frequency of egg contamination, a consistently direct relationship was not evident between the magnitude of the antibody responses of individual hens and the frequency at which they laid contaminated eggs. Accordingly, although serologic tests can be valuable screening tools for preliminary detection of S. enteritidis infections in poultry, the magnitude of the antibody responses detected in individual hens may not predict the overall risk of egg contamination associated with particular laying flocks.     

Incidence of Salmonella infection in healthy dogs in Gifu Prefecture,Japan

A total of 1,013 feces samples and 8 mesenteric lymphonodus samples obtained from apparently healthy dogs were examined for the incidence of salmonella infection. One strain of S. typhimurium (ST) was isolated from feces of one dog, and S. enteritidis (SE) was isolated from the mesenteric lymphonodus of one dog. Sera obtained from 330 apparently healthy dogs were examined for Salmonella antibodies using an ELISA with heated whole cells of SE and ST. Fifty-one of the 330 serum samples were considered to be positive for salmonella antibodies, including 12 which were SE-positive and 39 which were ST-positive. These results indicate that dogs cause possible environmental problems as Salmonella carriers.     

Influence of enrofloxacin and chloramphenicol on the level of IgY in serum and egg yolk after immunostimulation of hens with Salmonella enteritidis antigens

In the chicken, maternal antibodies are transferred into the egg and subsequently transported into the developing embryo. IgG (called IgY) is the primary immunoglobulin isotype of the egg yolk. Their level in serum depends on the correct function of immunological system in laying hens. Many factors have a direct or indirect influence on antibody level in fowl. One of them is a commonly used antibiotic, but its influence on avian immune system is still unknown. The objective of the study was to determine the effect of enrofloxacin and chloramphenicol on the level of IgY antibody in serum and egg yolk after immunostimulation of hens with living cells of Salmonella enterica subsp. enterica serovar Enteritidis and lipopolisaccharide. Forty adult egg-laying Arbor Acres and Isa 215 hens (32 and 50 weeks old) from the reproductive flocks and 1640 of their eggs were used for the investigation. No clinical symptoms of any diseases were observed in birds during the entire breeding period. Additionally the birds were checked as free from Salmonella spp. in the beginning of the experiment. The birds were divided into 6 experimental and 2 control groups (5 birds in one group). The hens in the experimental groups were immunized with S. Enteritidis antigens: living bacteria and lipopolisaccharide and treated with enrofloxacin or chloramphenicol. Antibiotics were administered in drinking water for 10 days (from 3rd to 13th day of experiment). To indicate anti-S. Enteritidis, antibodies in sera and egg yolk were used indirectly on ELISA based on lipopolisaccharide from S. Enteritidis. As conjugate these were applied anti-chicken IgY with horseradish peroxidase and ABTS with H2O2 as obtained. Additionally, to detect antibody in serum, a rapid slide test was used with Pullognost and Enterognost standard antigens made in the laboratory. The study revealed that both antibiotics tested decreased the level of specific IgY in laying hens immunized with living bacteria and lipopolisaccharide. It seems that antibiotics have a suppressive effect on the immunological system. The strongest immunosuppressive effect was exerted by chloramphenicol.     

Serologic reactions against Salmonella in samples from broiler parent stock with and without preceding colibacillosis: a case-control study.

In the Danish Salmonella Control Program, eggs from broiler parent flocks are surveyed by serologic analysis every 4 wk for antibodies against Salmonella lipopolysaccharide O-antigens 1, 4, 5, 9, and 12 (Mix-enzyme-linked immunosorbent assay [ELISA]) and 6 and 7 (Infantis-ELISA). The antibody response is measured in percentage optical density (OD%) of a strong positive reaction, and the cutoff value has been determined to be 40 OD%. Two or more reactors above 40 OD% will place the parent flock under suspicion. There has been concern about possible cross-reactions between Salmonella spp. and other Enterobacteriaceae, e.g., Escherichia coli, because a high specificity of a Salmonella antibody test is desirable. Moreover, false-positive Salmonella results have economic consequences and impede planning the production. A case-control study based on cases of clinical E. coli infections (colibacillosis) from two Danish hatcheries, supplying about 62% of the Danish broiler production, is described. In order to eliminate a possible bias from age and season, the controls were matched on age of the birds and on time of submitting the samples. This study shows that flocks with preceding colibacillosis did not have higher salmonella reactions than matched flocks without a preceding colibacillosis. This observation was confirmed in longitudinal studies.     

Seroprevalence of Histomonas meleagridis in pullets and laying hens determined by ELISA

Serum samples from 1120 layers from 56 flocks and 400 pullets from 20 flocks were tested by an indirect sandwich ELISA to investigate the prevalence of antibodies to Histomonas meleagridis in chickens kept in alternative husbandry systems. The overall prevalence of antibodies to H meleagridis in layers was 37.3 per cent, and positive birds were identified in 50 flocks. This was significantly higher than in pullets, where only 8.3 per cent of the birds tested positive. Optical density (OD) values obtained from pullet sera were much lower than the OD values from layers; however, positive birds were detected in half of the pullet flocks. In particular, all birds from an organic pullet flock were found to be positive, with high OD values. Overall, the highest prevalence of positive sera was obtained from birds kept in free-range flocks. Attempts to reisolate live histomonads from birds in 18 layer flocks were unsuccessful.     

Diagnostic utility of egg yolk for the detection of avian metapneumovirus antibodies in laying hens

Surveillance and diagnosis of avian metapneumovirus (AMPV) infection typically involve measurement of serum antibodies. In the current study, eggs instead of serum samples were used for the detection of AMPV antibodies in egg-laying chicken hens by enzyme-linked immunosorbent assay (ELISA). AMPV-free commercial layer hens were experimentally challenged with AMPV strain SC1509 through intravenous or oculonasal administration. Antibody levels were determined by ELISA. AMPV antibodies were detected in egg yolks from challenged hens by 7 days postinoculation (dpi), with the peak titer at 16 dpi. Antibody levels in eggs laid at 28 dpi correlated well (r = 0.93) with sera taken 28 dpi from the same hens. In a field trial of the yolk ELISA, six broiler breeder farms were surveyed, and all tested positive for AMPV antibodies in hen eggs, although positivity varied from farm to farm. Abnormal discolored eggs collected from outbreak farms had significantly higher titers of AMPV yolk antibodies than normal eggs from the same farm, unlike clinically healthy farms, where normal and abnormal eggs had similar antibody titers. These results indicate that diagnosis of AMPV infection by yolk ELISA to detect anti-AMPV antibodies may be a suitable alternative to serologic testing.     

Salmonella infection sources in poultry flocks in northern Greece]

From 10 egg production poultry farms 1516 samples were collected and examined for the presence of salmonella. The samples were: 201 chicken, 36 sparrows, 35 rats, 35 pools of 20 flies each, 450 eggs, 60 mattresses, 188 feces, 425 feedstuffs and 86 water samples. Salmonellae were isolated only from 163 (10.8%) samples. From the 146 (89.6%) of these S. gallinarum was isolated. From the rest 17 (10.4%) the following mobile salmonella strains were isolated: two strains of S. virchow and Salmonella of subgroup II, four strains of S. typhimurium var. Copenhagen, seven strains of S. Livingstone, one S. enteritidis and one S. infantis The S. gallinarum was isolated from dead or sick chicken (46%), eggs (10.4%), rats Rattus norvegicus (14.3%) and mattresses 1.6%. The mobile salmonellae were isolated from feedstuffs (2%), flies (14.3%), rats (2.8%), feces (1%). From the present study, it seems that rats, chicken and eggs are important for the salmonella dissemination.