BIOSECURITY AND VACCINATION IN ERADICATING SALMONELLA ENTERITIDIS
Keith Gooderham, Poultry Health Services
10th September 1997

In 1985 we began to see a disease problem in young chicks. Clinically they appeared as birds with "Stunting and Runting " Disease.By about 10 days of age some chicks had stopped growing and had distended abdomens. Post-mortem examination revealed a persistent,inspissated yolk sac and usually pericarditis. Salmonella enteritidis could readily be grown on direct culture on solid medium (Blood Agar,McConkeys Agar,Brilliant Green Agar).

This problem was widespread geographically, under different ownership, in different breeds of chickens both broiler and layer strains, as well as to a limited extent in turkeys and ducks. Whilst, to my knowledge, the origin of the salmonella has not been shown, I believe it originated in a large consignment of a single feed ingredient. By the time the problem was recognised, a secondary wave of the organism was being spread in the feed in Poultry Offal Meal.

A junior health minister of the government was outspoken in stating that all eggs being produced were from salmonella-infected flocks. This was far from true, but public concern was sufficient to result in legislation for testing all flocks (breeding and laying ) and for slaughter of positive flocks. The situation really was that caged egg layers were seldom infected.

Broiler breeder flocks were frequently infected and my estimation in 1989 was that 10% of broiler parent flocks carried the organism.The same probably applied to layer parent flocks as well as barn-egg and free-range layer flocks. Much microbiological testing was carried out and somewhat later,serological testing (when the ELISA kit became available ).

The main effort in my laboratory was directed at broiler parent flocks. Much attention was focussed on feed mills and feed :

• Poultry Offal Meal was no longer used,

• Regular testing of other feed ingredients was increased,

• Ministry compulsory testing of home-produced and imported feed ingredients of animal origin was introduced.

Flock testing over many flocks and many months showed :

• 1)Day-old parents did not carry S.enteritidis,

• 2)Flocks seldom became infected during rear to 18 weeks,

• 3)Flocks frequently became positive on the laying farm, usually either before 30 weeks of age or after 50 weeks of age,

• 4)Cleaning and disinfection of positive laying farms was frequently inadequate for preventing the next flock from becoming infected,

• 5)Hatcheries became contaminated due to receiving hatching eggs from positive flocks,

• 6)More flocks became infected as a result of spread from positive farms and hatcheries,

• 7)Infected mice on laying farms were probably the most important source of
  persistent infection.

There were so many positive flocks detected that the Government Veterinary Department relaxed the slaughter policy. A flock might still be slaughtered, but treatment of the flock or its progeny were offered as alternatives. It was the considerable effort put into eliminating the salmonella that allowed us to learn its characteristics and pattern of spread.

All aspects of biosecurity were considered in detail and, where possible, improved. Cleaning and disinfection of farms between flocks was improved. Rodent control, particularly in preventing their entry into livestock houses was
improved. As a result of all these measures, the incidence of flocks infected with S.enteritidis diminished. Interestingly, the cultural isolation of S. typhimurium and other, less invasive, salmonellae were reduced to an even greater degree.

Competitive Exclusion.

The technique developed by Nurmi in Finland involves supplying a range of cultured bacteria to the bird. The idea is that these organisms establish themselves in the intestine of the bird and compete with salmonella for sites at which to multiply. Experience is varied. My own experience is that it can be extremely effective against horizontal transmission but is limited in its efficacy when transmission is vertical. The value of competitive exclusion should not be ignored but is only one part of the attempted reduction in numbers of salmonella present. Of all the salmonellae which can infect the chicken, S. enteritidis appears to be the most resistent to control by the technique of competitive exclusion.

Despite all efforts, S. enteritidis remained on certain farm premises. Some had experienced 4 successive flocks becoming infected, thus being "positive" for some 4 years. Some Companies had greater problems of persistently contaminated premises than others.

Vaccination

It was at a stage when such positive premises were less than 5% of the total that a vaccine became available for trial purposes. This was an inactivated vaccine administered by injection as two doses given 4 weeks apart.

Initial government trials had been in commercial layers, the vaccine being given at day-old and 4 weeks of age.These were day-old to end of lay trials with groups being taken at intervals for challenge studies.They were considered a success. As broiler parents were not usually found to be challenged during rear, and to speed up the time when vaccinated birds were in lay, it was agreed to vaccinate at 12 and 16 weeks of age. The plan was to vaccinate all flocks sending eggs to a single hatchery so that eventually no eggs came from an unvaccinated flock.This would take about a year from when the vaccine was first available in March 1994.

Our experience was that the vaccine helped in the control of the infection. Of the first 100 flocks vaccinated, 3 became infected and transmitted the infection in their eggs. All 3 flocks were on farms on which the previous flock had been positive. The next cycle of vaccinated flocks on these premises remained negative.

Microbiology:
Culture of samples for salmonella is the most sensitive way of detecting infected flocks. Some specific samples are required by law. In addition to these we also test litter samples from flock farms on a regular basis (once monthly).
This is a particularly sensitive test. With suspect positive eggs in the hatchery,the most sensitive sample is to culture from early dead embryos.These can be collected by candling at 10 days of incubation. It is important when examining cultures to realise that the appearance of colonies on the culture plate may not be typical. It is possible to discard as negative what really is a positive isolate.

Serology:
When the ELISA testing kit for S. enteritidis became available, flocks were regularly tested to help identify positive flocks. Results were not entirely accurate, with false positive individuals appearing in negative flocks. Challenges from E.coli could result in some sera "positive" to S.enteritidis. A view of the flock had to be taken to determine whether it was likely to be seroconverting. Further sera could be examined after a few weeks. More importantly, however, a microbiological examination of the flock should be made to confirm its salmonella status.

As an example, a summary was made of all flocks tested for two companies (A and B) each with several flock farms.
Company A had some positive flocks (by culture).
Company B had no positive flocks.

Figure 1. % Prevalence of positive sera according to S/P reporting band

The scatter of mean flock titres against age for the two companies is shown in the slides.

Figure 2, Company A : Mean S/P Bands according to flock age

Figure 3. Company B : Mean S/P bands according to flock age

Note the absence of positive titres during the rearing period. In other companies practicing day-old-to-death flock farm management, salmonella challenge was frequently very early in rear. As flocks became vaccinated, serology was no longer of value in identifying positive flocks.
Serological response to vaccination was, however, monitored.

Age of Vaccination:
As all flocks became vaccinated, it was decided to reduce the age of vaccination. For convenience one company decided to vaccinate at 2 and 6 weeks of age.

This was considered acceptable as the initial studies had shown protection from a day-old and 4-week vaccination programme. It became obvious that the serological response to the early vaccination was less. Was this a problem, indicating lack of protection, or was this useful in protecting the flock but allowing recognition of the difference between vaccinated and challenged flocks? Two early vaccinated flocks became positively infected and egg-transmitted S. enteritidis. It was therefore decided to revert back to vaccinating later, this time at 11 and 15 weeks of age.

Vaccination using the inactivated S.enteritidis vaccine results in a serological response which will interfere with the Pullorum Test. As indicated earlier, any positive serology should be confirmed bacteriologically. Use of the vaccine together with other S. enteritidis control measures would be a far more effective way of controlling Pullorum Disease than using the Pullorum Rapid Plate Test.

Vaccination of a Known S. enteritidis Positive Flock.
A broiler parent chicken flock in two houses was found to be infected and was transmitting S. enteritidis to its progeny. It was decided to vaccinate one house and to treat the other with Enrofloxacin.
Egg transmission was somewhat reduced.
At the end of lay 32 birds were culled from each house (8 birds from each of the 4 pens in each house).
Organs were collected and cultured in pools of 4 . The organs cultured were; liver +gall bladder, intestine including caecal tonsils, oviduct, ovary.
Of the 32 pools of organs cultured from each house, there were 8 positive from each house. The positive organ pools were identical for each house being; liver,1; intestines,2; oviduct,2; ovary,3.
It was concluded that the vaccination of an already positive flock did not help in the control of S. enteritidis.

Opinion:
It is my opinion that the vaccine is an aid to controlling S. enteritidis infection. It would not have worked against the wealth of challenge that existed from 1985 to 1991. Strict attention to biosecurity of farms, rodent control and cleaning and disinfection programmes were vital in reducing the infections to manageable proportions. These remain important. Also important is the continued vigilance in the production of feed. Sourcing of feed ingredients (which are still showing a level of 2% positive samples, but rarely S. enteritidis), heat treatment of all feed production and avoidance of recontamination of finished feed are necessary.

HISTORY:

1984: First appearance of S. enteritidis PT4

1985: Disease in broiler chickens from egg-transmitted S. enteritidis

1985-1988: Increasing infections in broiler parent flocks (10%)

1988 (Dec): Junior Health Minister speaks of problem

1989 (Mar): Legislation.Compulsory testing and slaughter

1989-1994: Attempts to eradicate not totally effective Repeatedly positive farms identified Hatcheries repeatedly contaminated

1994 (Mar): Flock vaccination started

1994-1996: Reduction of positive farms Feed still a potential source of Salmonella

1996: No positive flocks remain (Clients of Poultry Health Services/Hillsdown Veterinary Services)

1997 (Sept): S. enteritidis cultured from the litter of one flock. Enrofloxacin given. Egg transmission has not occurred