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Log10 SE ml1 egg pool

Quantitation of SE in egg pools The SE multiplied in the egg contents from controlvaccinated hens to a mean density of 8 X 107 SE/ml (Fig. 3) compared with less than 102 SE/ml in similar samples from vaccinated hens (P <0.0001). In a repeat experiment, egg contents from bacterin-vaccinated and control-vaccinated hens were seeded with either SE or P. mirabilis. As in trial 1, the SE replicated well in the egg contents from the control-vaccinated hens, achieving a density of greater than 109 SE/ml (Fig. 3) compared with less than 104 SE/ml in egg contents from birds receiving the bacterin vaccination (P <0.0001). The levels of P. mirabilis were slightly reduced in egg contents from bacterin-vaccinated hens, but the differences were not significant. Effect of dilution of egg contents on inhibition of SE growth Egg contents from control-vaccinated hens were 100% culture positive for SE following overnight incubation at 37C in both trials. Significantly fewer egg contents from bacterin-vaccinated hens were culture positive for SE and diluting the egg contents 1:5 still resulted in significantly fewer culture-positive pools (P < 0.005). Effect of high inoculum dose on inhibition of growth Egg contents from control-vaccinated hens were 100% culture positive for SE following overnight incubation at 37C in both trials. In trial 1, egg contents from bacterinvaccinated hens had significantly fewer positive samples at 24 h when inoculated with 10 SE (P < 0.001) or 100 SE (P < 0.05). In trial 2, significantly fewer egg pools from vaccinated hens inoculated with 10 SE were SEpositive (P < 0.001), but not in those pools receiving a 10-fold higher dose.

In the current study, the pooling of egg contents and incubation of these contents mimicked the egg abuse during food preparation and served as the model for testing whether egg yolk antibodies will affect the growth of SE in these abused eggs. Following 24-hour incubation of egg contents at 37C, there was a 5 log lower level of SE in egg contents from vaccinated hens compared with

10 9 8 7 6 5 4 3 2 1 0 S. enteritidis (a)

10 9 Log10 cells ml1 egg pool 8 7 6 5 4 3 2 1 0

(b)

When examining the utility of hen vaccination to reduce the incidence of human egg-borne SE infections due to product abuse, concentration becomes an important variable. The first variable is the concentration of antibody deposited in the egg. A shipment of eggs received by a food vendor will potentially come from multiple flocks, both vaccinated and nonvaccinated. Inhibition of SE growth was still observed in egg contents from vaccinated hens diluted 1:5 in control eggs, indicating that the eggs could undergo limited pooling and still retain the capacity to inhibit growth of the SE. The second variable is the concentration of challenge organism introduced. Eggs from SE-infected commercial flocks generally contain only low numbers of SE, 10-20 organisms, although, on rare occasions, higher numbers of SE could be found in some eggs. In the current experiment where a 10-fold higher SE inoculation dose was used, the results were mixed.

In one trial, the %SE+ pools receiving the higher challenge were significantly lower than controls, while, in a second trial, no reduction in %SE+ pools were observed. These results indicate that the capacity of egg contents from vaccinated hens to inhibit SE occurs when a moderate SE challenge is present, but may be overwhelmed when higher challenge doses occur. The discovery that egg contents from vaccinated hens inhibit the in vitro growth of SE could have important implications for the egg industry. However, the production of eggs containing this inhibition is fairly short, 8-10 weeks, and to be of much use for industry, this time frame would need to be extended several-fold.

and references please refer to the complete journal article. This research gives a small part of information about the effects of vaccination on egg contents. It is critical to remember that vaccine alone cannot prevent food borne illness created by SE. All aspects of Salmonella reduction must be utilized to reduce Salmonella levels in our food supply. For commercial egg layers, cleaning and disinfection, rodent control, salmonella reduction in feed, and proper monitoring of the program must be conducted on the farm. Once eggs reach the processor, proper egg handling and storage must also be strictly followed to insure a safe product. Then, the ultimate responsibility will fall on the consumer, who must act responsibly in the handling and cooking of eggs and egg products.

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A L O H M A N N A N I M A L H E A LT H N E W S B R I E F V. 2 2 0 0 3

Growth of Salmonella enteritidis (SE) in egg contents from hens vaccinated with an SE bacterin
Edited by Karen E. Burns, D.V.M, M.A.M, diplomate A.C.P.V Technical Services Veterinarian Lohmann Animal Health Gainesville, GA
Peter S. Holt1, Henry D. Stone1, Richard K. Gast1, and Robert E. Porter, Jr21USDA/ARS Southeast Poultry Research Laboratory 934 College Station Road Athens, Georgia 30605, 2Purdue University Animal Disease Diagnostic Lab 1175 ADDL West Lafayette, Indiana 47907-1175 Reprinted with permission from Elsevier Science. For complete article: Food Microbiology, 1996: 417-426.

Editors Comments:
The previous article was reprinted in a summarized form to allow for key information to be presented. To find complete materials, methods, results

reduced in vaccinated hens challenged with SE. High antibody titers to the organism were achieved in the serum of vaccinated birds. Antibodies are deposited in the egg yolk during egg formation and the yolk antibody repertoire closely mirrors that found in serum. This phenomenon has been exploited experimentally by a number of investigators for use in disease prevention strategies. Egg yolk antibodies have been used as a noninvasive means of serologically determining the SE status of a laying flock. Serum antibodies generated against a particular microorganism can elicit a number of deleterious effects on that organism and similar effects could also occur with yolk. As pooled eggs had been implicated as the vehicle in a number of food-borne outbreaks of human SE, pooled egg contents from SE-vaccinated and controlvaccinated hens were chosen as the medium for examining SE growth. The objective of the current study was to examine the impact of antibodies deposited in eggs during

vaccination on the in vitro growth of SE in egg contents from vaccinated and control birds.

Materials and Methods Introduction

With the recognition of intact table eggs as a significant vehicle for food-borne outbreaks of Salmonella enteritidis (SE), on-farm intervention schemes were developed to reduce the incidence of eggs contaminated with the organism. One method employed was vaccination of laying hens with a killed SE bacterin since experimental evidence indicated that hens immunized with a SE bacterin were at least partially protected against a SE infection. In those experiments, SE fecal shedding, dissemination of the organism to internal organs, and eggs contaminated with SE were
Gainesville, GA 30501 1146 Airport Parkway

Production of the bacterin has been previously described in Gast et.al 1992. The bacterin or the control, mineral oil emulsion lacking the killed SE, were administered subcutaneously in the nape of the neck, 0.5 ml per hen. Eggs were collected daily and labeled individually. Monday through Friday eggs were used for the SE growth studies; Saturday and Sunday collections were used in yolk ELISA tests. Blood was collected weekly for serum anti-SE titer by ELISA. Eggs from the same 10 birds in each trial were used to study the impact of vaccination on the ability of SE to grow in egg contents and, in trial 2, eggs from these same 10 birds in each vaccination group were used weekly to ascertain egg yolk antibody levels. The remainder of the eggs from each week in trial 2 (weeks 2, 3, 4, and 6) were saved and used for the different SE growth experiments described below.
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Discussion
Hens vaccinated with killed preparations of SE elicit strong serum antibody responses to SE, and, as shown in Fig. 2, concomitant with the serum antibody titer rise, levels of anti-SE antibodies in the yolks of eggs from these birds also rise. Epidemiological studies of egg-borne outbreaks of human SE infections showed that abuse of the eggs or egg products were responsible for a number of the outbreaks. Vaccination with an SE bacterin, with its resultant anti-SE antibodies deposited in the egg yolks, could potentially serve as a means to intercede and block SE replication during the incidences of improper egg handling.

S. enteritidis Inoculum

P. mirabilis

Figure 3. Quantitation of bacterial growth in egg contents from bacterin-vaccinated (filled bars) and control-vaccinated hens (open bars). (a) Growth of SE in egg contents from pool of eggs from multiple hens. (b) Growth of SE and Proteus mirabilis in egg contents from individual birds.

those of sham-vaccinated controls. This inhibition appeared to be antigen specific as a similar inhibition was not observed against P. mirabilis, providing evidence that anti-SE antibodies were responsible.

In this issue of avian insight:

Growth of Salmonella enteritidis (SE) in egg contents from hens vaccinated with an SE bacterin ...............................................P.1

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For further information: 770.532.3627 800.655.1342 www.lahinternational.com

From the president...............P.4

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Egg contents from weekly collections for each bird were deposited into individual sterile stomacher bags. The egg pools were homogenized for 30 seconds. 100 ml of the homogenate was transferred to a sterile specimen container. Each pool received 1 ml of a 10-8
TRIAL 1
Vaccination One vaccination

TRIAL 2
2 vaccinations, 5 weeks apart 32 9 weeks, starting 2 weeks p.v.

ELISA titers

agar for P. mirabilis. One ml of each aliquot was also transferred to 9 ml of buffered peptone water. The plates and the BPW enrichments were incubated overnight at 37C and the bacterial colonies were enumerated. For any samples with no growth, 100 ml of the BPW enrichment were spread plated onto the proper medium. The plates were incubated overnight at 37C and were then evaluated for the organism. The theoretical level of detection was 1 organism/ml. Variables affecting inhibition of SE growth in eggs Experiments were conducted to explore which factors affect the antibody-mediated inhibition of SE growth. The first experiment examined the effect of diluting the egg contents from bacterin-vaccinated hens 1:5 (V:V) in egg contents from control-vaccinated hens. The growth

Birds per group Egg Collection

15 Weeks 2 and 3 post vaccination

of 1 ml of a 10-8 dilution of SE in this group was compared with that in samples of undiluted egg from bacterin-vaccinated and from control-vaccinated birds. The second experiment examined the growth of a 10-fold higher SE inoculum (1 ml of a 10-7 dilution of the overnight broth culture) in egg contents from bacterinvaccinated and control-vaccinated hens. In all experiments, the egg contents were incubated at 37C for 24 hours and sampled by direct plating onto 2 BGN plates. The plates were incubated for 24 hours at 37C and examined for SE. Determination of anti-SE antibody levels in plasma in plasma and egg yolk Weekly blood samples were collected with heparinized syringes, then centrifuged to obtain plasma. The antibodies in egg yolks were extracted by diluting each individual yolk, 1:5 (v:v) in PBS; vortexing each mixture; and then allowing the mixture to sit undisturbed overnight at 4C. An aliquot was removed from each sample. Both plasma and yolk extracts were assayed by ELISA as described previously by Holt and Porter (1993). Statistical analysis The numbers of SE/ml were converted to log10, and then means were calculated. Significant differences between the mean log10 SE/ml and rates of recovery of SE in pooled egg samples from bacterin-vaccinated vs shamvaccinated birds were determined via pooledvariance t-test with significances expressed at P < 0.05.

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from the president

New LAHI Breeder Vaccine Finding Success
We have seen a remarkable response to our new FC4 Gold by US broiler breeder companies. The new vaccine came about as a result of customer demand to improve cholera bacterin form. These are the issues addressed with the new vaccine:
Dave Zacek, President of Lohmann Animal Health, Gainesville, Georgia, USA

savings in cost of syringes is possible if the customer is using LAHI vaccines with it as well.

Autogenous vaccine growth

% SE positive contents

9 ELISA titers 8 7 6 5 4 1 2 3 4 5 6 7 8 9 10 Week post vaccination

60 50 40 30 20 10

* *
2 3

Two experiments were conducted to examine the levels achieved by SE in egg contents from the vaccine groups: Experiment 1: Contents from 10 eggs from each treatment group were pooled by group and homogenized. Five 25-ml aliquots from each group were transferred into sterile tubes and each aliquot received 1 ml of a 10-8 dilution of the overnight SE broth culture. The samples were incubated overnight at 37C. Serial 10fold dilutions were made of each aliquot and 100 ml of each dilution was spread-plated onto two BGN plates. The plates were incubated overnight at 37C and SE colonies were then enumerated. The theoretical level of detection of SE was 100 SE/ml. Experiment 2: Contents from 3 eggs/bird, 8 birds/group, were pooled by individual bird and the contents were homogenized. Two 25-ml aliquots were transferred to sterile tubes and 1 tube received 1 ml of a 10-8 dilution of the SE culture. To examine the specificity of the inhibition, the second tube was inoculated with 1 ml of a 10-8 dilution of an overnight broth culture of a poultry isolate of Proteus mirabilis. The aliquots were incubated overnight at 37C and then serial dilutions were made of each culture. 100 microliters of the undiluted sample and the dilutions were spread plated onto BGN for the SE and hektoen enteric

% SE positive contents

dilution (approximately 10 organisms as determined by dilution analysis) of a phage type 14b SE isolate (S10072-2, Maryland Department of Agriculture). This strain grows well in egg contents and was a heterologous challenge for the vaccine. The pools were incubated at 37C for 24 hours and were then cultured by direct plating onto brilliant green agar containing 20 mg novobiocin/ml (BGN). The plates were incubated for 24 hours at 37C and examined for SE.

11 10

110 100 90 80 70 60 50 40 30 20 10 0

(b)

80 70

(a)

LAHI has grown to be the most significant supplier of autogenous vaccines in the USA for poultry. This segment of our business has now grown to a point where we will soon be limiting new Application by intramuscular method: customers in order to remain able to Other bacterins on the market are As a consequence, weve seen usage serve our current customers in the best labeled only for subcutaneous injection. grow month after month. After only four way possible. We will make every This method of application has months, it has become one of the largest effort to accommodate new customers, inherent risks of misinjection; either selling cholera bacterins in the USA. but those who inquire early are more into the neck muscles of the bird or likely to be successful. self-injection into LAHI has the ability to crewmembers hands. FC4 LAHI listened to the requirements of utilize a variety of Gold is licensed for use either subcutaneously our customers and responded with a adjuvants and organism growth strategies to or intramuscularly. When optimize the final product used intramuscularly in state of the art vaccine. for its intended use. As a the breast, it provides consequence, we are now greater ease of applicaproducing a large number of vaccines tion, reduced bird injury and potenDouble dose syringe in this category. tially fewer crewmembers needed.

110 100 90 80 70 60 50 40 30 20 10 0

Results
(b)

allows greater flexibility

* *
2

* * *

*
3

Growth of SE in pooled egg contents A small inoculum of SE, 10 organisms, was able to grow to detectable levels following 24 h incubation at 37C in a high percentage of egg contents from control-vaccinated hens (Fig. 1). In a repeat experiment where egg culturing was performed over a more extended period of time, similar results were observed. The SE was detected in a high percentage of the pooled contents of eggs obtained from control-vaccinated hens (Fig. 1). Anti-SE antibody titers in plasma and egg yolks following vaccination Antibody titers against the SE flagella antigen rose to high levels by week 2 postvaccination and remained high throughout the experiment (Fig. 2). Egg yolks from these birds showed anti-SE titers peaking at week 2 postvaccination and gradually declined in successive

% SE positive contents

Figure 2(a). Anti-SE antibody levels in serum (squares) and egg yolks (circles) in vaccinated (filled figures) and control (open figures) hens. Results represent the ln ELISA titers of 10 birds per treatment group per time period. (b) Comparison of anti-SE antibody levels over time in egg yolks from vaccinated hens (line), derived from Figure 2(a), and percent SE-positive egg contents from these birds (filled bar), derived from Figure 1(b). Arrows indicate the times of vaccine administration.

weeks, though still remaining 3-4 titer units above control levels. A plot of yolk antibody titers and percent SE+ egg pools from bacterin-vaccinated hens showed that the reduced percentage of SE+ pools corresponded with elevated antibody titers following primary vaccination (Fig. 2).

Dose size has been reduced to 0.25 ml. Using proprietary concentration techniques and a proprietary adjuvant, LAHI is able to combine the four Pasteurella strains and provide significant efficacy all packed into a 0.25 ml dosage. The net result is a vaccine that is easier on the bird and is less likely to be evident at slaughter when compared to older adjuvants that require 0.5ml dosage. LAHI listened to the requirements of our customers and responded with a state of the art vaccine. It is the only USDA licensed Fowl Cholera vaccine which utilizes the special WOW adjuvant and is concentrated to allow 0.25 ml dosage.

4 5 6 7 8 Week post vaccination

10

Figure 1. (a) Percent SE- positive egg contents from vaccinated hens (filled bars) and control hens (open bars) in trial 1. (b) Percent SE-positive egg contents from vaccinated hens (filled bars) and control hens (open bars) in trial 2. * = significantly different from control levels at P < 0.05. Arrows indicate the times of vaccine administration.

LAHI serves as an outlet for an Italian designed and manufactured syringe that has multiple injection heads available. The applicator tips provided with the syringe allow use of two different vaccines applied at the same time with one plunger motion. Two needles keep the vaccines separate at injection sites. This cuts down on a crew member and saves some handling time as well. (Also included in the syringe kit is a single needle head, which is fed from both vaccine sources). The vaccine tips can be independently set to deliver 0.25 ml or 0.5 ml (or another dosage) on both or either side independent of one another. This syringe is offered for sale to all who need it. Significant

Due to the very nature of autogenous vaccine usage, customers requirements are intermittent. So, even though we are currently nearing capacity, we anticipate the ability to add new customers in the near term. We are pleased producers have given us a leadership role in developing and manufacturing inactivated vaccines and their application. LAHI is the premier supplier of vaccines to the table-egg-layer producers in the US and a significant supplier to the breeder and turkey segment. In the broiler area, we are pleased with the success of our new IBD vaccine, ViBursa CE and our Conn-type IB vaccine.

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