Salmonella in poultry

Salmonella History

Alexander The Great

Alexander the Great died mysteriously in 323 B.C.

In 2001, a group of doctors at the University of Maryland suggested that Salmonella was the cause of death, based on a description of Alexander’s symptoms written by the Greek author Arrian of Nicomedia.

Prince Albert

Prince Albert, the consort of Queen Victoria, died of a Salmonella infection in 1861.

During the Victorian era, an estimated 50,000 cases per year occurred in England.

Jamestown Colony

Scholars working on the history of Jamestown, Virginia, believe that a typhoid outbreak was responsible for deaths of over 6,000 settlers between 1607 and 1624.

Spanish-American War (1898)

In 20,738 recruits contracted the disease, 82% of all sick soldiers, 1,590 soldiers died, yielding a mortality rate of 7.7%.

It accounted for 87% of the total deaths are from disease.

A significant number of these deaths actually occurred at training areas in the southeastern United States.

British Camps During The South African War (1899-1902)

More soldiers suffered from typhoid fever than from battle wounds.

British troops lost 13,000 men to typhoid, as compared to 8,000 battle deaths.

Outbreak was largely due to unsanitary towns and farms throughout Africa, and polluted soil was washed into the network of streams and rivers during the rainy season.

Epidemic potential during a war prominent because of the disposal problems of men’s discharges.

Dr. William Budd (1811-1880)

Most outbreaks that were reported could be traced back to unsanitary water supplies or polluted milk supplies, as polluted water can spread the disease.

Budd urged for more disinfection and water treatment .

The introduction of piped and filtered water supplies, its prominence as a cause of death had diminished.

Salmonella Carriers

Some individuals have natural immunity to Salmonella, they are known as “chronic carriers”

They have only mild or asymptomatic disease, but still carry the bacteria in their body for a long time.

These cases serve as natural reservoir for the disease. • 3% of persons infected with S. typhi • 0.1% of those infected with non-typhoidal salmonellae

become chronic carriers which may last for a few weeks to years.

History: Mary Mallon


“Typhoid Mary” Mallon was the first famous carrier of typhoid fever in the U.S.


She was hired as a cook at several private homes in the New York area in the early 1900’s.


Mary Mallon caused several typhoid outbreaks, moving from household to household, always disappearing before an epidemic could be traced back to the particular household Mary was working in.

She had worked for 7 families, with 22 cases of typhoid and one death.


She was finally overtaken by the authorities in 1907 and committed to an isolation center on North Brother Island, NY.


There she stayed until she was released in 1910, on the condition that she never accept employment involving food handling.


But she was found to work as a cook and to cause typhoid outbreaks again.

She was admitted back to North Brother Island, where she lived until her death in 1938.

History: Recent Outbreaks

Recent Outbreaks

More recently reported outbreaks in the U.S. involve different kinds of Salmonella strains, predominantly S. enteritidis and S. typhimurium.

Recent Outbreaks

In 1985 A salmonellosis (S. typhimurium)

outbreak involving 16,000 confirmed cases in 6 states by low fat milk and whole milk from one Chicago dairy farm.

This is the largest outbreak of food-borne salmonellosis in U.S.

Investigations discovered that raw and pasteurized milk had been accidentally mixed.

Intentional Contaminationof Restaurant Salad Bars

In September 1984 10 area restaurants in the Dalles, Oregon, were involved with outbreaks of S. typhimurium.

Intentional Contaminationof Restaurant Salad BarsJanuary 2000 Infant aged 1 month visited a

clinic with fever and diarrhea.

A stool specimen yielded Salmonella serotype Tennessee.

One week before illness onset, the infant's family moved into a household that contained a bearded dragon (i.e., Pogona vitticeps).

Intentional Contaminationof Restaurant Salad Bars

During June 2002 A child aged 21 months was

admitted to a hospital with fever, abdominal cramps, and bloody diarrhea.

Blood and stool cultures yielded Salmonella serotype Poona (from pet Iguana).

Facts About Salmonella

Study Aim

Many scientists have implicated breeder chickens as vehicles for vertical transmission of Salmonella from the breeder chickens to the fertile egg.

Study Design

In 1991, Cox et al. evaluated 6 commercial broiler breeder hatcheries for the presence and level of salmonellae in; 1. Egg fragments2. Paper pads from chick boxes3. Chick fluff

Results

42; 11%

338; 89%

Salmonella Positive Salmonella Negative

Results

Samples from Number of samples Positive Samples %

Egg fragments 145 22 15.2%

Chick fluff 100 5 5%

Paper pads 125 15 12%

Results

Hatcheries 1 2 3 4 5 6

Salmonella Positive Samples % 1.3% 5% 22.5% 11.4% 36% 4.3%

Results

140 random samples selected for enumeration, salmonellae were found in 11 samples. 4 samples had greater than 10 log 3 salmonellae per sample. 3 samples between 10 log 2 - 10 log 3 4 samples less than 10 log 2

Conclusion

The authors found that, the incidence and extent of salmonellae-positive samples found in the breeder hatcheries were MUCH LESS THAN That previously found in broiler hatcheries

Meaning that the industry is reducing Salmonella in breeder chicken populations.

Breeders and Salmonella


Challenging breeder hen with 10 log 6 Salmonella cells caused the ovary and oviduct to become infected.

Timoney et al. (1989) reported that oral inoculation of laying hens resulted in infection of the reproductive tract.


Cox et al. (2000) observed that the egg production rate for infected chickens was unaffected, and Salmonella was not detected in all fecal samples; therefore breeders infected with salmonellae may not always be easily detectable on the farm.

For the contaminated breeder hens, the yolks of 10% of the eggs laid were contaminated with S. enteritidis.


However, when hens were inoculated with Salmonella at levels of 10 log 8 cells, a noticeable drop in egg production and signs of pathogenesis occurred (Cox et al., 2000).

Salmonella Serotypes in Breeders House and in Hatcheries


Breeders Farm Hatcheries Previous

FlockCurrent

FlockCarcass at Processing

6% 98% 24% 60% 7%

7% 98% 26% 22% 36%

Trial 1

Trial 2


In the first trial, 7 different Salmonella serotypes were identified.

In the second trial, 12 different serotypes were identified (Bailey et al. 2002).

Interestingly, for both trials, there was poor correlation between the serotypes found in the breeder farms and those found in the hatchery.

Role of Males in vertical Transmission

Role of Males in Vertical Transmission

In 1995 Reiber et al. conducted three experiments to determine the bacteriological quality of rooster semen.


The most frequently isolated genera of bacteria from rooster semen included:1. Escherichia2. Staphylococcus3. Micrococcus4. Enterococcus5. Salmonella


Most of the isolated bacteria were endemic to poultry and were commonly found in the environment of chickens (Reiber et al., 1995).

Thus, during mating, female breeders may become inoculated with Salmonella during semen transmission.

The Effect Of Disinfection And Rodents


In a study to determine the effect of disinfection on Salmonella in breeders.


3 broiler breeder Houses at three different locations Sampled before and after cleansing and disinfection (Davies

and Wray, 1996).

None of the farms were able to achieve total elimination of Salmonella Enteritidis from the poultry house environment.


The authors concluded that, in each of the three breeder houses, failure to eliminate mice from the house that was infected with S. Enteritidis was likely to be the most important hazard for transmission to the next flock (Davies and Wray, 1996).

Chicks Early Infection


Inoue et al. (2008) stated that young poultry are very susceptible to Salmonella Enteritidis (SE) infections because of:1. Absence of complete intestinal flora colonization 2. Immature immune system in baby chicks


The authors conducted a study to evaluate the role of passive immunity on the resistance of young birds against early infections caused by SE.


The progeny of:Vaccinated broiler breeders Were compared to the progeny of Unvaccinated breeders .

The efficacy of the vaccine was determined by challenging birds at Days 1 and 14 with SE.

After challenge at 1 day of age;

Liver Spleen Cecal Contents0

1

2

3

4

5

6

7

2.21 2.312.852.76 3.02

6.03

Salmonella Number (log10)

Progeny of Vaccinated Birds Progeny of Unvaccinated Birds

3 Days After Challenge

Salmonella Positive0%

10%20%30%40%50%60%70%80%90%

100%

28%

100%Salmonella Positive

Progeny of Vaccinated Birds Progeny of Unvaccinated Birds

Conclusion

Birds that were challenged at 14 days of age showed a lower number of positive samples compared with those challenged at 1 day of age.

Age influenced the susceptibility of birds to SE infections.

Transmission By Egg Contamination


Many opportunities exist for Salmonella to be transferred from contaminated eggs to uninfected baby chicks during the hatching process, Cox et al. (2000)


Salmonella may be found in:1. The nest boxes where breeders lay eggs.2. The cold storage egg room at the breeder farm.3. The truck that transports baby chicks to the grow out houses.4. The hatchery environment


One mechanism for natural contamination of the eggs is when moist, freshly laid eggs are cooled from the body temperature of the hen to the air temperature, the internal contents of the egg shrink, pulling bacteria into the shell through pores (Cox et al., 2000).Once transferredSalmonella is carried on the surface of the shell or just beneath the shell if it is able to penetrate the shell.


All of these situations may cause horizontal contamination of the eggs with Salmonella.

Salmonellosis

Salmonellosis

• Salmonella overview• Etiology• Transmission• Clinical signs • Post mortem lesions• Treatment

Road Map

Salmonella Overview

Salmonellosis is any of several bacterial infections caused by species of Salmonella, ranging from mild to serious infections.

Salmonella Overview

“Salmonella”, the name, derived from Dr. Daniel Salmon, a U.S. veterinary surgeon, who discovered and isolated the strain enterica or choleraesuis from the intestine of a pig in 1885.

Salmonella Overview

Rod-shaped Gram-negative Facultative anaerobe Non-spore-forming It belongs to the same family

as Escherichia, which includes the species E.coli.

Salmonella Overview

Predominantly motile Has peritrichous flagella (flagella

that are all around the cell body) Presence of fimbriae projections

that aid attachment to the gut. Ability to survive on diverse carbon

sources. Ability to synthesize aromatic amino

acids.

Salmonella Overview

Refrigeration prevents growth but does not kill bacteria.

Heating at 57-60°C has shown to be effective in killing the bacteria.

Optimal growth: 37°C

Salmonella Overview

Bacterium of 2501 identified strains, as of 2001. Many different diseases are caused by more than 1,400

serotypes of this bacteria genus.

Salmonella Overview

One major problem of Salmonella is its colonization without disease, which leads to contamination of meat prior to or at slaughter.

Caeca are the main sites of colonization, up to 10 log 10 CFU per gram of fecal material may occur.

Persistence may occur for several months and be accompanied by fecal shedding.

Transmission through flocks appear due to coprophagic behavior (coprophagy is the consumption of feces).

Salmonellosis


Road Map

Avian Salmonellosis

Caused by serovars of salmonella enterica.

Types Of Salmonella Infection In Poultry

1. Colonization of GIT + little or no systemic invasion. E.g. S. Hadar, S. Infantis

2. Colonization of GIT + transient systemic infection + occasional vertical transmissionE.g. S. typhimurium, S. Enteritidis

3. Systemic infection + little or no colonization of GIT + vertical transmission. E.g. S. Gallinarum, S. Pullorum.

Systemic Disease Of Poultry Specific Serovars

S. Gallinarum Causes sever systemic disease of all age (fowl typhoid). Fowl Typhoid has a mortality rate around 60%.

Systemic Disease Of Poultry Specific Serovars

S. Pullorum Causes sever systemic disease in chicks with mortality up to

90%. Characterized by white diarrhea. Disease is less sever in older birds. A carrier state leading to reproductive tract infection may

occur in hens with S. Pullorum leading to vertical transmission.

Salmonellosis


Road Map

Transmission

Salmonella transmission may be;1. Horizontal 2. Vertical

Transmission - Horizontal

The bacteria is shed from an infected bird in:1. Nasal and or ocular secretions.2. Fecal material.3. Feather dust.

Transmission - Horizontal

The organism remains stable outside the host body and dries as a dusty substance.

This dust or aerosol contaminates the air that is then inhaled by another possible host.

Susceptibility as well as the amount of contamination determine whether or not the new host becomes infected with the disease.

Transmission may be primarily through the air. Salmonella spp. are mainly transmitted by the fecal-oral

route.

Transmission - Vertical

Occurs in birds, with contamination of the vitelline membrane, albumen and the yolk of eggs.

Salmonellosis


Road Map

Clinical Signs

General symptoms of Salmonella include:1. Lethargy.2. Anorexia.3. Diarrhea. In chronic cases4. Arthritis may be present. With high dose infections:5. Excessive thirst.6. Conjunctivitis.7. Indications of liver, spleen, kidney or heart damage can occur.

Salmonellosis


Road Map

Post-mortem lesions

In acute disease there may be few lesions.1. Dehydration.2. Enteritis.3. Focal necrotic intestinal lesions.4. Foci in liver.5. Unabsorbed yolk.6. Cheesy cores in caecae.7. Pericarditis.8. Perihepatitis.9. Misshapen ovules in the ovaries in S.E. infection.

Salmonellosis


Road Map

Treatment

Treatment of salmonella infections are more successful if salmonella species is first determined.

Once the particular species of salmonella has been identified, the appropriate antibiotic can be administered.

The frequently found Salmonella strains are sensitive to many commonly available antibiotics.

Treatment

Antibiotics Antidiarrheal

Salmonella Vaccines

Ideal Salmonella Vaccine

The aim of vaccination as part of a complex control programs for Salmonella infections in poultry is to: 1. Reduce or prevent the intestinal colonization resulting in

reduced fecal shedding and egg shell contamination. 2. Prevent systemic infection resulting in a diminished

localization in the reproductive tissues.

Ideal Salmonella Vaccine Strain

1. High degree of protection against systemic and intestinal infection.

2. High protective potential against a variety of important serovars (serogroups)

3. Adequate attenuation for poultry, other animal species, humans and the environment as well as animal welfare issues

4. The inactivated and live vaccines should not affect growth of the animal

5. Vaccine strains should not be resistant to antibiotics


6. Vaccines should be easy to administer and need to have markers enabling the differentiation from salmonella wild-type strains.

7. Application of vaccines should not interfere with salmonella detection methods.

8. Humoral antibody response after vaccination should be distinguishably from a salmonella wild-type response to allow the use of serological detection methods

9. Attenuated live salmonella vaccine strains should be able to induce the colonisation inhibition effect between salmonella organisms.


10. Salmonella vaccine strains should have preserved the ability to invade the gut as prerequisite to induce the invasion inhibition effect between salmonella organisms

Salmonella Vaccines

• Types of salmonella vaccines– Live attenuated– Inactivated– SubunitRoad Map

Live Virus Attenuation

The aim of attenuation is to diminish the virulence of the pathogen, but retaining its immunogenicity.

Live Attenuated VaccinesRisk Factors

Several risks are associated with live vaccines, especially in immunocompromised individuals:

1. Some of the structural components of the microorganism used may contain immunosuppressive antigens.

2. Attenuated strains may still have some residual virulence due to an incomplete inactivation.

3. Attenuated strains, after being released into the environment, can recover their virulence in other hosts, or can acquire genes from other microorganisms by natural genetic transfer.

Live Attenuated VaccinesRisk Factors

Under these points of view, despite their extended use, the restrictions for the use of attenuated modified organisms in vaccination are becoming more and more stringent.

Live Attenuated VaccinesAdvantages Over Inactivated Ones

1. Easy administration.2. Ability to carry heterologous antigens.3. Capacity to induce cellular and humoral immune responses.

Live Attenuated Vaccines

Empirical Attenuation Methods Attenuated Mutations

Metabolic functions

Virulence factors

Methods of Attenuation



Metabolic functions

Virulence factors

Live Attenuated VaccinesEmpirical Attenuation Methods

The empirical attenuation methods developed by Pasteur and Koch at the end of the twentieth century.

Many successful live viral and bacterial vaccines are produced by repetitive in vitro passage in cell culture.



Metabolic functions

Virulence factors

Live Attenuated VaccinesAttenuated Mutations

The knowledge we have nowadays about genomics allows us to selectively knock out concrete virulence genes.


Salmonella has around 4,500 genes.A change in any gene (mutation) can lead to an alteration in protein.This mutation may change the structure and function of the bacterium.Some mutation may have no effect.

Target Mutagenesis

Specific alteration by genetic manipulation of the genome; Insertion, deletion or framshift changes alter the gene

structure or function.Deletion and insertion frequently used experimentally and

in potential vaccines.

Deletion

A gene or part of it is knocked out. The gene is no longer transcribed, no

RNA and no protein is synthesized.

A B C

A C

Insertion

A foreign piece of DNA is inserted into the targeted gene.

This knocks out the gene function although transcribed RNA no longer encodes correct protein.

Here gene B in an inserted. Insertions are less stable than

deletions, but easier to perform.

A C

A CB


Advantages of this strategy:1. Some important antigenic determinants can be retained

by attenuated strains that are able to elicit both antibody and cellular immunity.

2. Growing capacity of these attenuated vaccines provides prolonged exposure of antigens to the immune system, resulting in the production of long-lasting memory cells.



Metabolic functions

Virulence factors

Live Attenuated VaccinesAttenuated Mutations/Metabolic Functions

The most widely studied metabolically attenuated strains include mutants deficient in:The biosynthesis of:

1. Aromatic amino acids (i.e. aroA, or aroC and aroD) 2. Purines (purA, purE)

Or in the production of: 3. Adenylate cyclase (cya) 4. Cyclic AMP receptor protein (crp).

Live Attenuated VaccinesAttenuated Mutations/Metabolic Functions

In general, auxotrophy can interfere with bacterial replication within the host whenever the required metabolites are:

1. Absent.2. Present in amounts insufficient for bacterial growth in the

compartment where the bacteria reside.



Metabolic functions

Virulence factors

Live Attenuated VaccinesAttenuated Mutations/Virulence Factor

The two more extensively characterized virulence-attenuated vaccine strains have mutations in the two-component regulatory system phoP/phoQ or in Salmonella pathogenicity island 2 (SPI2) loci.

Salmonella Vaccines


Killed Whole Organisms

To avoid the risk of live vaccines, the use of killed organisms was introduced as safer vaccines.

These vaccines are made from the entire organism but inactivated (killed) by physical or chemical agents.

Killed Whole Organisms

However, protection induced by bacterins in poultry is generally modest .

The limitations of these kinds of vaccines are:1. Their immunogenicity usually has to be enhanced by co-

administration with adjuvants. 2. Multiple doses are necessary for obtaining long-term

protective immunity; besides, as live vaccines, they may contain immunosuppressive antigens.

Salmonella Vaccines


Subunit Vaccines

Subunit vaccines contain immunodominant components of the bacteria.

Subunit vaccines may be:1. Crude or purified extracts of the pathogen.2. Synthetic peptides. 3. Obtained by the use of recombinant DNA technology, pure

DNA or RNA.

Subunit Vaccines

The primary goal of this approach is: 1. To identify the individual antigens of the pathogen that

are involved in inducing protection.2. Avoiding the immunosuppressive ones.

Subunit Vaccines

Combining genomics with our understanding of pathogenesis, it is possible to identify specific proteins from most pathogens that are critical in inducing the right protective immune responses.

Subunit Vaccines

The potential advantages are 1. Vaccine safety.2. The potential abilities to target the vaccines to the site

where immunity is required.3. To differentiate vaccinated animals from the infected ones

through the right selection of the components.

Subunit Vaccines

In spite of these data, low levels of resistance against salmonellosis can be induced by administration of flagella, porins or polysaccharide fractions.

The potency of these vaccines is often poor when administered without adjuvant and/or a delivery system.

Subunit Vaccines

New generation adjuvants are designed to induce minimal side effects, enhance the duration of the immune response, and concurrently stimulate humoral and cellular immune responses.

Ideal vaccine properties Attenuated Inactivated

Subunit Classical Adjuvant (Alum)

New adjuvants (DDS)

I +++ + + +++

II +++ + + +++

III + ++ ++ +++

IV + + + +++

V + +++ + +++

Strong, protective and long lasting immune response

Right sort of immune responses.

Safe

Single dose of vaccine should confer robust, long-lived immunity. Affordable

I

A good vaccine should stimulate a strong, protective and long lasting immune response. Through the induction of strong, long-lived immunological specific T and B cell memory cells. Measurement of the specific subsets elicited by immunization may guide vaccine development.

II

A good vaccine should induce the right sort of immune responses. The protective immune responses against extracellular pathogens seem to be mediated by long-lived humoral immune responses through the production of antibodies. However, in the control of intracellular infection, cellular immune responses have been shown to be crucial in mediating protection. Therefore, the development of a successful vaccine against those diseases will be facilitated by a thorough understanding of how cellular immune responses are generated and maintained in vivo.

III

An ideal vaccine should be safe. Despite the success of vaccination in eliminating disease and death, the public acceptance of even minor side effects of vaccination is very low. One major challenge faced in developing new vaccines is to achieve strong immunogenicity without increasing reactogenicity.

IV

A single dose of vaccine should confer robust, long-lived immunity. Only a few live vaccines have achieved this goal. In contrast to the results with live vaccines, it has been difficult to promote long lived immunity with a single dose of non-living antigen vaccines. One goal of vaccine development is to rectify this using new adjuvants and antigen delivery systems.

V

An ideal vaccine should be affordable by the population at which they are aimed and should be formulated to resist high and low temperatures to facilitate distribution. This is a main problem for alive attenuated vaccines

IMMUNOADJUVANTS

Immunoadjuvants

Adjuvants They are defined as a group of structurally heterogenous compounds that enhance or modulate the immunogenicity of the associated antigens.

Immunoadjuvants

Despite the recognition of many different types of adjuvant, however, little is known about their mode of action.

Janeway called adjuvants “the immunologists dirty little secret”, because their mode of action is poorly understood.

Immunoadjuvants

The events triggered by these immunomodulators appear to come from one or the combination of several of the following effects:

Immunoadjuvants

Adjuvant Effect

Depot Effect Effect On Antigen Presenting Cells (APC)

Nonspecific Immunostimulanting Effect

Particulated Carrier Systems

Immunoadjuvants Depot Effect

If antigen is present in a solution, it is mostly quickly removed by neutrophils and macrophages.

Subsequently, antigens are unable to prime naive T cells.

Following that, the antigens disappear and the immune response is hardly detectable.


Adjuvants, such as:1. Oil emulsions 2. Antigen absorbing aluminium salts

Retain antigen at the injection site, Then, it is released in minute quantities over a prolonged period of time.

These compounds mainly stimulate the production of antibodies by the induction of Th2-lymphocytes.


In the case of use of alum, the mechanism of action seems to be due to Formation of a depot of free alum that will induce the

recruitment and activation of immune cells to the site of inoculation.

Immunoadjuvants

Adjuvant Effect




Immunoadjuvants Effect On Antigen Presenting Cells (APC)

The adjuvant induced enhancement of an immune response may be related to the improved delivery of antigens into the draining lymph nodes.

This may be achieved by:1. Facilitating the antigen uptake by APCs.2. Increasing the influx of APCs into the injection site.

Immunoadjuvants Effect On Antigen Presenting Cells (APC)

This results in:1. Increase in the provision of antigen-loaded APCs, and in turn,

effective priming of specific T cells.2. Promoting the activation state of APCs by upregulating

costimulatory signals or MHC expression. 3. This results in the corresponding cytokine release, enhancing

the speed, magnitude and duration of the specific immune response.

Immunoadjuvants

Adjuvant Effect




Immunoadjuvants Nonspecific Immunostimulating Effect

Some agents can stimulate the non-specific component of the immune system.

Numerous microorganisms contain “alert signals”, the so called “microbial or pathogen associated molecular patterns” (MAMPs or PAMPs, respectively), which is not present in mammalian cells.

These structures activate immune cells through interaction with specific receptors (toll like receptors, TLRs).

Immunoadjuvants Nonspecific Immunostimulating EffectSome examples are: 1. Lipopolisaccharide (LPS)2. Monophosphoryl lipid A (MPL)3. Flagellin4. Lipoproteins5. Muramil dipeptide (MDP)6. Trehalose dimycolate (TDM), 7. Cpg DNA, among others.Besides, the special chemical nature of some polymers used in the formulation of vaccine delivery systems may also be recognized as scavenger ligands for the APCs.

Immunoadjuvants

Adjuvant Effect

Depot EffectEffect On Antigen Presenting Cells

(APC)

Nonspecific Immunostimulantin

g Effect


Immunoadjuvants Particulated Carrier Systems

Particulated Carrier Systems have been proposed to improve the mucosal bioavailability of antigens.These carriers protect labile molecules from degradation in

the gastrointestinal tract. Nanoparticles are submicron-sized colloidal systems that may

protect antigen against chemical, enzymatic or immunological degradation, and facilitates targeting and presentation of antigens to inductive sites of mucosal immune system.

Salmonella in poultry

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