Results of a Salmonella enteritidis vaccination field trial in broiler-breeder flocks in The Netherlands

From August 1995 until December 1997, the effect of adding Salmonella enteritidis (SE) vaccination to a certified standardized biosecurity program in a situation of increased infection risk was examined in a field trial in The Netherlands. In this field trial, two groups of broiler-breeder flocks with increased infection risk were vaccinated, one group with VAC-T/TALOVAC logSE(group A) and the second group with SALENVAC (group B). The determination of increased infection risk in groups A and B was based on an SE infection history; flocks were either previously infected and treated (PIT) or had other risk factors than previously infected and treated (OPIT). SE infections in both vaccinated groups were assessed by monitoring according to the Dutch salmonella control program. Under field conditions, designation of a vaccinated and a control group on the farm was not possible. In the same period as the vaccinated groups, 608 nonvaccinated flocks (group C) were hatched and monitored according to the Dutch salmonella control program. The flock level occurrence of SE infection in the vaccinated groups was compared with the flock level occurrence of SE infection in the nonvaccinated group on the basis of comparability of infection risk. In group C, whether or not flocks had infection risk PIT was known and for risk factor OPIT, only whether or not a flock had been placed on a previously contaminated farm (= risk of reinfection) was known. The proportion of SE-infected flocks with risk factor PIT in the vaccinated groups was not significantly different from that in the nonvaccinated group C. Only the proportion of SE-infected flocks with a risk of reinfection in the vaccinated group B (0) was significantly lower (P = 0.02) than in the nonvaccinated group C (18%). The fact that no significant result was found in favor of group A is because of the small number of flocks in this part of the study. On the basis of the conditions of the setup of this trial, it can only be concluded that there is an indication that vaccination contributes in the reduction of SE reinfection in broiler breeder flocks.     

Vaccination against Salmonella enteritidis in Dutch commercial layer flocks with a vaccine based on a live Salmonella gallinarum 9R strain: evaluation of efficacy, safety, and performance of serologic Salmonella tests

This study describes a field trial in which 80 commercial layer flocks, with an increased risk of Salmonella enteritidis (SE) infection and placed on farms with a certified Standardized Biosecurity Programme (SBP) or a request for a SBP certificate, were vaccinated with a vaccine based on a live attenuated Salmonella gallinarum (SG) 9R strain. An evaluation is presented of the efficacy of the vaccine against SE infections, the effect on the performance of serologic Salmonella tests, and the spread of the vaccine strain to the egg content. For the efficacy study, assessment of the flock level occurrence of SE infections in the vaccinated group of 80 flocks was compared with that of a nonvaccinated group of 1854 flocks hatched in the same period. This control group was examined according to the compulsory control programme in The Netherlands. An evaluation was done of the performance of serologic Salmonella tests and the spread of the vaccine strain to the inner egg content of five of the vaccinated flocks. Findings demonstrated the flock level occurrence of SE infections in the vaccinated group (2/80 = 2.5%) to be significantly (P = 0.01) lower than that of the nonvaccinated group (214/1854 = 11.5%). Vaccination resulted in 59.0% positive test results in lipopolysaccharide BD enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against Salmonella serogroups B and D and 0% positive test results in the rapid plate agglutination test for detecting antibodies against S. pullorum (SP)/SG. The mean specificities of two blocking ELISAs (gm- and i-double antibody sandwich ELISAs) based on the flagellar antigen of SE and Salmonella typhimurium (ST) on the same sera were 99.6% and 96.1%, respectively. The vaccine strain could not be isolated from any of the 450 pools of 10 eggs. On the basis of these results, we concluded that vaccination with a vaccine based on an attenuated SG 9R strain contributes to the reduction of SE infections in commercial layer flocks. Furthermore, serologic monitoring of SE, ST, and SP/SG can still be carried out on flocks vaccinated with an attenuated SG 9R strain. Additionally, we found no indication of the spread of the vaccine strain to the egg content.     

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.     

Evaluation of the efficacy of Salmonella enteritidis oil-emulsion bacterin in an intravaginal challenge model in hens.

The efficacy of Salmonella enteritidis (SE) oil-emulsion bacterin (a commercially available vaccine) was evaluated in an intravaginal challenge model in hens producing a high rate of SE-contaminated eggs. Hens were vaccinated at 38 wk of age. A second (booster) bacterin injection was administered 4 wk later. Two weeks after the second vaccination, all hens were challenged intravaginally with 10(7) colony-forming units of SE. After challenge, 36 of 189 eggs (19.0%) in the vaccinated hens were positive for SE, and this contamination rate was significantly (P < 0.01) lower than that in the unvaccinated hens (61 of 165 eggs, 37.0%). SE was highly recovered from the cloacal and vaginal swabs of the unvaccinated and vaccinated hens, but the number of SE from the cloaca of the vaccinated hens was significantly (P < 0.05) lower than that in the unvaccinated hens at 7 days post-challenge (PC). The recoveries of SE from the spleen and ovary in the vaccinated hens were significantly (P < 0.05) lower than those in the unvaccinated hens at 7 days PC. At necropsy, SE was recovered from 2 of 15 forming eggs (13.3%) taken from the oviducts of the unvaccinated hens, whereas no SE was recovered from 17 forming eggs in the vaccinated hens. After vaccination, serum antibodies for SE in the vaccinated hens were significantly higher than those in the unvaccinated hens. Antibodies from the oviductal washing, especially immunoglobulin G isotype, in the vaccinated hens were higher than those in the unvaccinated hens after challenge. This intravaginal challenge model produced frequent contaminated eggs and clearly demonstrated the ability of the bacterin to protect against egg contamination. The present model may be a useful tool for further studies to evaluate the protective effect against SE contamination of eggs by potential vaccine candidates.     

The spread of Salmonella gallinarum 9R vaccine strain under field conditions.

A live vaccine based on an attenuated Salmonella gallinarum 9R strain is in use in a Salmonella enteritidis control program in commercial layer flocks in The Netherlands. In a field study, the potential spread of the vaccine strain from vaccinated flocks to nonvaccinated flocks has been studied after both the primary and the booster injection at four different rearing farms and at one layer farm. The vaccinated and the nonvaccinated flocks were monitored at regular intervals by bacteriologic and serologic examination. In this field study, no evidence was found for the fecal spread of the vaccine strain.     

Persistence of immunity in commercial egg-laying hens following vaccination with a killed H6N2 avian influenza vaccine

The California poultry industry experienced an outbreak of H6N2 avian influenza beginning in February 2000. The initial infections were detected in three commercial egg-laying flocks and a single noncommercial backyard flock but later spread to new premises. The vaccination of pullet flocks with a commercially prepared, killed autogenous vaccine prior to their placements on farms with infected or previously infected flocks was used as a part of the eradication programs for some multiage, commercial egg production farms. The purpose of this study was to follow three vaccinated flocks on two commercial farms to track the immune responses to vaccination. The antibody-mediated responses of the three flocks followed in this study were markedly different. One flock achieved 100% seroconversion at 12.5 wk of age, but by 32 wk of age, all of the hens were seronegative by agar gel immunodiffusion (AGID). In contrast, at 32 wk of age, flocks from the other farm (flocks 2A and 2B) were 95% and 72% seropositive by AGID, respectively. Of the differences that were identified between the vaccination protocols on the two farms, the distinction that could explain the level of disparity between responses is the delivery of the second dose of vaccine with a bacterin on the first farm, which may have interfered with the persistence of immunity in this flock. Hens from flocks 2A and 2B were experimentally challenged at 25 wk of age with H6N2 avian influenza virus. Hens from flock 2A did not transmit virus to naive contact-exposed hens, but hens from flock 2B did. At 34 wk of age, hens from flock 2A were again challenged and naive contact-exposed hens were infected in this second trial. These challenge experiments served to demonstrate that despite detectable antibody responses in flocks 2A and 2B, the birds were protected from infection for less than 21 wk after the second vaccination.     

The effect of killed Salmonella enteritidis vaccine prior to induced molting on the shedding of s. enteritidis in laying hens

Effects of administering killed Salmonella enterica serovar enteritidis (SE) vaccines to laying hens prior to induced molting on egg production and on shedding of SE were investigated. Forty hens were vaccinated with one of two SE vaccines available commercially in the United States and Japan. Twenty-five days after vaccination, feed was withdrawn for 2 wk from 20 vaccinated plus 10 unvaccinated hens to induce molt. Four days after molt induction, all hens were challenged with a dose of 2.4 X 10(9) of SE. For the 25 days following administration of the SE bacterins, egg production in vaccinated hens showed approximately a 15% decrease. After molt induction, egg production in molted hens ceased and then returned to normal levels 8 or 9 wk postvaccination. Through the 3-mo experimental period, the decreases in numbers of eggs laid in the unvaccinated/molted group and two vaccinated/molted groups were 225 (26.2%), 245 (28.4%), and 274 (31.9%), respectively, compared with 860 in the unvaccinated/unmolted group. There was no significant difference in egg lay at the P < 0.05 level among the former three groups. Hens in the vaccinated/molted groups shed about two logs less SE than hens in the unvaccinated/molted group 3 14 days postchallenge (P < 0.05 or 0.01). These results indicate that vaccination prior to induced molting might be effective in preventing the exacerbation of SE problems within flocks in which the potential for SE contamination may exist.     

Control of ovine gastrointestinal helminthiasis by the use of 'clean' grazing and strategic dosing in the field

Despite the widespread adoption of clean grazing systems in lowland sheep flocks, detailed parasitological investigations had not previously been carried out on such flocks. A trial was therefore conducted on two commercial flocks: a traditional permanent pasture flock (A) and one operating a system of clean grazing (B), and on an East of Scotland College flock (C) which had operated a clean grazing system for eight years. Ewe and lamb worm egg output, pasture larval levels and lamb liveweight gains were monitored and tracer lambs were grazed during July and August on each farm. Under clean grazing conditions on farm C all parasitological parameters were lower than on both commercial farms. However, in the commercial flocks comparable contamination was evident from midsummer onwards and tracer lambs grazed during August on farm B had significantly greater worm burdens than on the other two farms. The differences observed between the flocks were thought to be due to greater residual contamination by overwintered larvae in both commercial flocks while the higher worm burdens in August on farm B probably resulted partly from incomplete control of the periparturient rise in ewe faecal egg output and partly to autoinfection of the lamb crop. It was concluded that farm C grazing was the cleanest. Considerable contamination was present on farm A while farm B occupied an intermediate position which resulted in considerable worm burdens in lambs grazing during the latter part of the season.     

The efficacy of three commercial Mycoplasma gallisepticum vaccines in laying hens

The efficacy of three commercial Mycoplasma gallisepticum (MG) immunizing agents-a bacterin, a recombinant fowlpox-MG vaccine, and a live F-strain vaccine-was compared in specific-pathogen-free hens in egg production. Three groups of 25 chickens were vaccinated with one of the vaccines at 10 wk of age and 25 birds were not vaccinated. At 25 wk of age (and approximately 50% egg production), 20 birds from each of the three vaccinated groups and 15 nonvaccinated controls were challenged with virulent R-strain via aerosol; the birds were necropsied and evaluated at 10 days post-challenge. The MG bacterin and live F-strain vaccinations were both protective and resulted in significant differences in air sac lesions, tracheal lesions, and ovarian regression compared to the nonvaccinated controls and the recombinant fowlpox-MG vaccine (P < or = 0.05). The evaluation of ovarian regression is a useful method of testing the efficacy of MG vaccines in laying hens.     

Identification of Avibacterium paragallinarum serovar B-1 from severe infectious coryza outbreaks in Panama

The isolation and identification of Avibacterium paragallinarum serovar B-1 from severe infectious coryza outbreaks in broiler breeders in Panama is reported for the first time. Infectious coryza was absent for over a decade in the breeder farms area. Disease outbreaks were characterized by an up to 45% drop in egg production and increased mortality. Use of a commercial trivalent bacterin and a strengthened biosecurity program prevented outbreaks in susceptible flocks in the farm.     

An epidemiological analysis of Salmonella enteritidis contamination in a rat-infested chicken layer farm, an egg processing facility, and liquid egg samples by pulsed-field gel electrophoresis

In order to determine the epidemiological link between the Salmonella Enteritidis contamination in a rat-infested chicken layer farm, an attached egg processing facility and liquid egg samples, several S. Enteritidis isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and bacteriophage typing. A total of 33 S. Enteritidis strains were isolated from a total of 4,081 samples. Similar pulsed-field patterns were generated by S. Enteritidis isolates from liquid eggs, rats and effluent water. Additionally, only two phage types were detected among the S. Enteritidis isolates, PT 1b and PT 6. These results suggest that S. Enteritidis isolates from rats, egg processing facility, and liquid eggs are genetically related. Furthermore, S. Enteritidis infection in rats in layer farms poses a serious public health concern and should be included in future epidemiological studies.     

Mycoplasma gallisepticum vaccination: further studies on egg transmission and egg production

Leghorn hens vaccinated twice with an inactivated Mycoplasma gallisepticum (MG) bacterin before egg production and subsequently challenged with virulent MG were protected against transmission of MG through the egg. Unvaccinated control hens transmitted MG through the egg at a high rate. When unvaccinated hens were vaccinated with MG bacterin 2 weeks after challenge with MG, there was no significant decrease in egg transmission. Hens vaccinated twice before laying did not suffer as severe egg-production drops as unvaccinated hens did when challenged with virulent MG. In a natural MG outbreak in a leghorn breeder flock, 50 hens were separated from the remainder of the flock and vaccinated with MG bacterin approximately 3 weeks after initial exposure. The unvaccinated hens transmitted MG through the egg at a rate three times higher than the rate of transmission of the vaccinated hens.     

On-farm risk factors for Salmonella Enteritidis contamination

Forty layer farms from 2 states participated in a study to examine the risk factors and incidence of Salmonella Enteritidis from multiple samples, including environmental drag swabs from the bird areas, feed, water, flies, rodents, live rodent traps, and environmental swabs from areas occupied by other livestock. Twenty-four of these farms had between 3,000 and 31,000 bird flocks (medium-sized flocks) and 16 had less than 3,000 birds (small-sized flocks). All were housed in cage-free production systems. Twenty-two farms included outside pasture areas for the birds. Most of the participants had just come under the FDA Egg Rule and had not yet tested their flocks (flocks under 3,000 birds are exempt) for Salmonella Enteritidis. Many, however, obtained their pullets from commercial Salmonella Enteritidis-clean breeder sources hatched in National Poultry Improvement Plan hatcheries. Vaccination against Salmonella Enteritidis was performed on 21 of the 40 farms (combination of live and killed vaccines). Salmonella Enteritidis was detected on 7 out of the 40 farms, primarily in rodents, their feces, or from swabs taken inside live traps. Of these 7 Salmonella Enteritidis-positive farms, 3 farms that had vaccinated their pullets with live Salmonella Typhimurium vaccine and killed-Salmonella Enteritidis vaccine; no Salmonella Enteritidis was isolated from the environmental drag swabs taken from the bird area or from the eggs on these farms. However, on the farms that had not vaccinated for Salmonella Enteritidis, the organism was isolated from 4 environmental drag swabs and 3 egg pools. The last 4 farms had flocks under 3,000 birds. No Salmonella Enteritidis was isolated from any of the samples of feed, flies, water, or swabs taken from other livestock areas. Based on the initial findings in this study, we suggest the 2 most important risk factors for Salmonella Enteritidis contamination inside the bird area and in the eggs in these small- and medium-sized flocks are the presence of infected rodents and the absence of an Salmonella Enteritidis vaccination program.     

Mycoplasma gallisepticum vaccination: effects on egg transmission and egg production

The effects of Mycoplasma gallisepticum (MG) vaccination on egg transmission of MG and egg production were evaluated. Leghorn hens vaccinated with live MG (strain F), with strain F plus MG bacterin, with one dose of MG bacterin, or with two doses of MG bacterin all transmitted MG through the egg at a significantly lower level than unvaccinated controls. Hens vaccinated with two doses of MG bacterin had the longest lag before detectable transmission of MG through the egg. All vaccinated groups were protected against the egg-production drop seen in unvaccinated hens challenged with virulent MG.     

Bovine genital campylobacteriosis (vibriosis): vaccination of experimentally infected bulls

The therapeutic efficacy of a Campylobacter fetus subsp venerealis bacterin was determined in experimentally infected bulls. Ten of twelve 5-year-old Angus bulls became infected after being infused intrapreputially with C fetus subsp venerealis. Of the 10 bulls, 6 were vaccinated with 5 ml of C fetus subsp venerealis vaccine on 2 occasions 4 weeks apart. Preputial washings of the vaccinated bulls were culturally negative by the 8th week after primary vaccination. None of the 18 heifers exposed to the vaccinated bulls became infected. The 4 infected, nonvaccinated bulls remained culturally positive to C fetus (P less than 0.002), and each bull infected at least 1 heifer (P less than 0.001). Two noninfected, nonvaccinated bulls remained culturally negative and did not infect any heifer. The 4 infected, nonvaccinated bulls were then vaccinated. Two bulls remained infected 9 weeks after primary vaccination, as determined by the virgin heifer test and cultural examination of preputial washings. Serologic data from 7 sampling periods were different (P less than 0.001) for vaccinated vs nonvaccinated bulls at 4 (against K antigen) or 6 (against O antigen) weeks after primary vaccination. Vaccination was effective in eliminating the infection in most of the infected bulls, but cannot be recommended as the sole measure of control in infected herds.     

Evaluation of the efficacy of an oil-emulsion bacterin for protecting chickens against Salmonella enteritidis.

To assess the potential protective efficacy of a Salmonella enteritidis bacterin, an acetone-killed oil-emulsion vaccine was prepared from a phage type 13a S. enteritidis strain and administered subcutaneously to hens in two experiments. Hens were housed individually, and every other hen was vaccinated (at 23 weeks of age in one experiment and at 45 weeks in the other). A second (booster) bacterin injection was administered 6 weeks later in both experiments. Three weeks after the second vaccination, all hens were challenged with an oral dose of approximately 10(9) cells of a heterologous (phage type 14b) S. enteritidis strain. In both trials, S. enteritidis was isolated from fewer internal organs (spleens, ovaries, and oviducts) and pools of egg contents from vaccinated hens than from unvaccinated control hens. Vaccination did not, however, affect the percentage of hens that shed S. enteritidis in feces in either experiment.     

Campylobacter spp. in broiler flocks at farm level and the potential for cross-contamination during slaughter

Screening of broiler flocks for their Campylobacter carriage on farm level and consequently the spread of Campylobacter spp. during slaughtering can help to identify hygiene control points. Therefore, between December 2001 and August 2002 in total 51 broiler flocks from three farms of different geographical regions in Germany were analysed for thermophilic Campylobacter. Campylobacter spp. were isolated from 45% of the broiler flocks examined. Subsequently, 1101 samples were taken from 22 flocks during different stages of processing. Samples were collected from: transport crates before and after cleaning/disinfection, evisceration, post-scalded and post-chilled carcasses and endproducts. Additionally, 45 selected Campylobacter isolates of droppings were genotyped by pulsed-field gel electrophoresis (PFGE). Campylobacter carriage of flocks showed seasonal variation, with the highest contamination rate during the period of June to August. No evidence was found for a horizontal transmission from one broiler flock to the next via a persistent house-contamination. In each positive flock, one to three different genotypes were found. One or two clones dominated isolations obtained from the farm level. The fact that in different flocks indistinguishable isolates of clonal origin were detected during the same rearing period suggested a transmission between the broiler flocks or an intermittent common external source. In one case, isolates of clonal origin were detected in various farms during different rearing periods. Sampling during processing confirmed that the entrance of a positive flock resulted in contamination of the abattoir environment. Campylobacter spp. were isolated from all sampling stages along the processing line, with a percentage of 91.1-100 of isolates at different stages of slaughtering.     

Growth of Salmonella typhimurium and Salmonella enteritidis in egg yolks from highly immunized hens.

This experiment was conducted to ascertain whether the growth of Salmonella Enteritidis (SE) or Salmonella Typhimurium (ST) would be suppressed in the presence of antibodies contained in egg yolks. Specific pathogen-free chickens (102 days of age) were subcutaneously immunized with oil-adjuvanted bacterin of SE or ST, twice within a four-week interval. During 160 to 170 days of age, eggs were collected, the yolks were removed and mixed with an equal volume of physiological buffered saline, inoculated with ten colony forming units (CFU) of SE or ST, and incubated at 37 degrees C or 20 degrees C for 23 hr. The growth of organisms in each yolk solution was examined. The egg yolk derived from non-immunized hens was examined in the same manner as the controls. There was no difference in the growth titer between the antibody-positive yolk and the negative yolk. The result suggests that the antibodies in the yolk do not influence the growth of each organism, even if the hens are highly immunized.     

Henhouse feeding style and Salmonella Enteritidis contamination in unvaccinated flocks of egg farms, April 1994-March 2001

From April 1994 to March 2001, monitoring tests (surveillance) for contamination of Salmonella Enteritidis (SE) at commercial egg farms and farm traceback inspections for cases of SE foodborne illness were carried out in the Himeji Livestock Hygiene Service Center jurisdiction of Hyogo Prefecture, Japan. SE vaccination was not performed before the outbreak of SE-associated foodborne illness, and SE contamination of a farm was recognized. In the surveillance, the year average percentages of identified SE-positive farms were 6.1% in farms with floor feeding in an open-type henhouse (F-OH) and 12.7% in farms with cage feeding in a windowless-type henhouse (C-WH), but 0% in farms with only cage feeding in an open-type henhouse (C-OH). The highest identified proportion of SE-positive farms among all styles of farms was found (4.3%) in April 1997-March 1999. Farm traceback inspections were performed in 2 of 7 farms feeding by F-OH and in 5 of 9 farms feeding by C-WH in this period. Easier contamination with SE was found for C-WH or F-OH than for C-OH. Ninety percent of the birds (3,632,000 birds at 70 farms) are fed at these easier contamination farms by C-WH (89% of birds in 13% of farms) or F-OH (1% of birds at 10% of farms). Integrated sanitary requirements and SE vaccinations are especially necessary on farms feeding by F-OH or C-WH. Since 1999, these countermeasures have been performed, and SE foodborne illnesses and affected patients in Hyogo Prefecture have gradually decreased.