• Clinical signs
• Diagnostics
• Legislation
• Approach to contaminated farms
• Prevention
• Literature

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The Bacterium

Salmonella Pullorum and S. Gallinarum are gram-negative, non-spore-forming bacteria belonging to the Enterobacteriaceae family.

The Salmonella genus comprises two species and is further subdivided into six subspecies, each with its serotypes and biovars. The serotype is determined based on O and H antigens, and more than 2,500 serotypes have been described. Only one subspecies is associated with clinical signs in mammals, S. enterica. S. enterica includes the subspecies arizonae and enterica, while the latter includes S. Pullorum and S. Gallinarum.

S. Pullorum and S. Gallinarum are biovars of the same serotype (S. enterica subsp. enterica serotype Gallinarum). Since this serotype lacks H antigens (flagellae), S. Pullorum and S. Gallinarum are immobile.

Salmonella can have a wide variety of virulence genes, depending on the specific subtype or serotype. These virulence genes are important for disease progression, as they facilitate cell invasion and colonization.

Generally, the serotypes can be divided into two groups. A limited group of serotypes (including S. Pullorum and S. Gallinarum) causes a 'typhoid-like' disease within a narrow host spectrum. Infection leads to severe symptoms, with bacterial reproduction occurring in various internal organs. These bacteria are especially adept at multiplying in macrophages, favoring the liver and spleen.

Other serotypes share many abilities of S. Pullorum and S. Gallinarum but are typically less likely to cause systemic disease in chickens. However, they spread more readily in the gastrointestinal system and can infect a much broader host spectrum, increasing the risk of human transmission. Significant differences between serovars exist, and for diagnostic purposes, clinical presentation—while useful for early indication—is insufficient. Serovar identification is essential.

Spread

An important route in the epidemiology of S. Gallinarum, S. Pullorum, and S. arizonae is vertical transmission. Hatching eggs can be contaminated both inside the hen (trans-ovarian transmission) and after being laid via shell contamination. Furthermore, when eggs infected with Salmonella hatch, the disease spreads easily via down feathers to other hatching units. Infection of chicks occurs through ingestion, inhalation, eyes, or the navel.

Horizontal contamination occurs through contaminated manure or other organic materials, which can be spread by people, tools, machines, feed, vermin, and insects (mechanical transmission). While carriers (clinically healthy birds carrying the bacterium) may introduce the disease into a new flock, this route plays a limited role in commercial poultry farms due to the all-in/all-out management system but can be a concern in hobby flocks. Spread within a flock occurs through fecal-oral contamination or cannibalism (of dead animals). The incubation period averages 2–3 days before initial signs, with deaths occurring 4–10 days later.

In the wild, S. Pullorum and S. Gallinarum are found in various bird species, particularly fowl. The extent of these Salmonellae in the wild in the Netherlands is unknown. S. Gallinarum was last observed in commercial layers in 2021, while S. Pullorum appeared in backyard poultry in 2023 and in commercial layers in 2021. S. arizonae can occur in wild birds, rats, mice, and reptiles; however, no reports of arizonosis exist in the Netherlands.

Susceptible Animal Species

S. Pullorum and S. Gallinarum have a limited host range, primarily affecting birds. All bird species can carry S. Pullorum, though clinical signs are usually observed in chickens, turkeys, guinea fowl, quail, and pheasants. Symptoms of S. Gallinarum are mostly seen in chickens, turkeys, ducks, pheasants, guinea fowl, peacocks, quail, and grouse.

Public Health

S. Pullorum and S. Gallinarum have minimal zoonotic risk and rarely infect humans. In exceptional cases, they can cause mild intestinal infections in humans. Factors like infectious pressure, route of infection, and immune suppression should be considered when interpreting such rare cases.

Survival

S. Pullorum and S. Gallinarum can persist in animals, resulting in vertical transmission. In poultry manure, Salmonella can survive up to six days at 19°C and 12–25 days at 5–9°C. The survival rate depends heavily on the presence of other microorganisms in the manure, with less competition allowing longer survival. Under favorable conditions, survival can extend up to 300 days.

Disinfecting

Thorough cleaning and disinfection of barns and equipment are essential, along with continuous vermin control. Without proper cleaning, a barn environment can remain contaminated for up to two years.

Effective disinfectants include:

• Products containing chlorine
• Quaternary ammonium compounds
• Formaldehyde/formalin
• Alcohol (70%)
• Sodium hypochlorite (0.025%)

Salmonella can be inactivated in barns by heating to a minimum of 60°C at 100% relative humidity for 24 hours. In feed, Salmonella can be eradicated by compressing pellets to reach temperatures above 80°C. Composting manure at temperatures above 56°C kills Salmonella within weeks.

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Clinical Signs

Clinical Signs in Chicks

Following vertical transmission, chicks may already die in the egg. The disease becomes immediately visible upon collection. When chicks are infected in the hatchery, it may take up to a week before symptoms appear.

Clinical signs can be extremely severe in chicks:

• Poor hatching (in case of vertical contamination)
• General illness
• Weak, pumping breathing
• Reduced appetite
• Slimy faeces
• White coating on the cloaca
• Inflammation of joints
• Blindness (S. Pullorum)
• Pale comb and lobes (S. Gallinarum)
• Mortality can reach 90% to 100%

The peak of clinical signs occurs 7 to 10 days after initial symptoms appear in the flock. Surviving chicks may be straggly, poorly feathered, and occasionally lame (runting and stunting).

Turkey chicks infected with S. arizonae may exhibit the following clinical signs:

• Lethargy and weakness
• Shaking; anorexia
• Diarrhoea
• Paresis or paralysis
• Torticollis
• Blindness or enlarged eyeballs due to corneal cloudiness and cheesy material accumulation in the eye

Clinical Signs in Adult Animals

Infection with S. Pullorum at a later age is often subclinical, though occasional outbreaks in mature birds have been reported. In rare cases, production declines and increased mortality are observed. Post-mortem findings in laying hens may include peritonitis and abnormally shaped ovaries. Infection with S. Gallinarum often begins with a sharp rise in mortality, usually accompanied by low morbidity due to the rapid (acute to peracute) disease progression in individual birds. Other signs may include reduced feed intake, egg production, fertilisation, and hatching, with affected birds appearing lethargic and ruffled.

Turkeys infected with S. Gallinarum may show anorexia, lethargy, ruffled feathers, excessive thirst, and greenish diarrhoea. Mortality rates can rise quickly in turkeys.

In quail, S. Gallinarum infection causes similar signs, with losses reaching up to 55%.

Morbidity and Mortality

S. Pullorum

In chicks younger than 3 weeks, morbidity and mortality caused by S. Pullorum can be as high as 100%. Survivors often become asymptomatic carriers and may be seronegative. The bacteria can start reproducing again when carriers reach reproductive age, potentially leading to vertical transmission. Birds often exhibit no or few clinical signs while excreting bacteria. Infections in older chicks may result in milder illness and lower mortality rates. S. Pullorum infections in adult turkeys may also lead to mortality.

S. Gallinarum

Mortality due to S. Gallinarum is most commonly observed in adult animals. However, young chicks (up to 1 month old) can experience losses exceeding 20%, especially if infected on the first day of life. Early infections often result in prolonged high mortality rates during the rearing period. In adults, mortality rates range from 10% to 70%, influenced by factors such as vaccination status, breed (heavier brown layer breeds are more susceptible), and the virulence of the S. Gallinarum strain.

S. Arizonae

In adult animals, S. arizonae infections are usually asymptomatic and rarely cause losses. In turkey chicks, mortality rates range from 32% to 70%, with the first three weeks being the most critical period. Mortality may persist until five weeks.

Excretion of Bacteria

Vertical transmission of S. Pullorum, S. Gallinarum, and S. arizonae occurs trans-ovarially, with up to one-third of a hen’s eggs becoming infected. Upon hatching, bacterial “explosions” can result in massive mortality within a few days. Within a flock, excretion through faeces is the primary mode of transmission. Non-excreting carriers infected during rearing may later become significant infection sources, especially for S. Pullorum. Excretion often increases suddenly when hens begin laying, potentially triggered by hormonal changes or stress.

Differential Diagnostics

Young Chicks

• Other Salmonella types (S. Enteritidis, S. Typhimurium)
• Aspergillus (mortality during the first week from hatchery infection)
• Navel infections
• Breeding errors
• Feed or water deficits
• E. coli
• Clostridium perfringens
• Botulism
• Carbon monoxide or dioxide poisoning
• Avian Encephalomyelitis (epidemic tremor)
• Avian Influenza
• Newcastle Disease

Older Chicks

Possible differential diagnoses include:

• Mycoplasma gallisepticum (joint or tendonitis)
• Mycoplasma synoviae
• Marek’s Disease (heart abnormalities with white nodules)
• Pasteurella multocida (local infections)
• Streptococcal infections
• Other Salmonella species

Adult Animals

In cases of acute mortality, differential diagnoses include:

• E. coli
• Staphylococcus aureus
• Pasteurella multocida
• Erysipelothrix rhusiopathiae (erysipelas)
• Avian Influenza
• Newcastle Disease

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Diagnosis

Clinical and post-mortem signs are not specific.

Pathology

Chicks: S. Pullorum and S. Gallinarum

• Yolk sac infection
• Swollen organs with signs of shock
• Pneumonia
• Enteritis
• Caecal cores
• Fatty areas in lungs, air sacs, heart, abdominal wall, intestines, spleen, pancreas, and liver
• Swollen joints with yellow exudate (S. Pullorum)
• Inflammation of the anterior eyeball chamber (S. Pullorum)
• Splenomegaly and hepatitis with multifocal areas of necrosis and inflammation (S. Gallinarum)

Older Animals: S. Gallinarum

Acute sepsis is the most striking feature, with hepatomegaly being prominent in all recent cases. Heavy brown laying hens are often overrepresented. Other observations include:

• Bronze-green livers with haemorrhaging and necrosis
• Affected serosal surfaces, including peritonitis and pericarditis
• Swollen kidneys and splenomegaly
• In turkeys, lung abnormalities and necrosis of the heart muscle

Older Animals: S. Pullorum

Hallmark lesions include septicaemia, hepatomegaly, and splenomegaly. Other findings include:

• Oophoritis with deformed or stalked egg follicles
• Lesions resembling E. coli peritonitis

S. Arizonae

• Enlarged yellow liver with white foci
• Duodenum congestion
• Yolk sac infection
• Cheesy material in intestines/caeca
• Inflammation of oviduct/peritoneum
• Eye lesions (exudate in the anterior eyeball chamber/vitrium)
• Purulent exudate in the brain

Isolation of the Bacteria

S. Pullorum and S. Gallinarum are immobile Salmonellae without flagella. General culture procedures targeting motile Salmonellae are not suitable for their detection. Recommended methods include:

• Accumulation in selenite-cystine or RVS without pre-accumulation for manure or similar samples
• GD testing using manure, down samples, or dead animals via RVS accumulation, followed by PCR or serotyping and biochemistry

S. Arizonae, being motile, can be diagnosed with general culture procedures, but its colonies on XLD plates differ from other Salmonellae. Specific communication is required if testing for S. Arizonae.

In clinical disease cases, S. Pullorum, S. Gallinarum, or S. Arizonae can be cultivated from abnormal organs of diseased or recently dead birds. Suitable media include Sheep Blood Agar (SBA) or MacConkey agar. Characteristics include:

• Small colony size for S. Pullorum and S. Gallinarum
• Smaller Gram-stain size compared to other Salmonellae

Distinguishing vaccine strains from field isolates of S. Gallinarum can be challenging. Biochemical or genetic tests (e.g., PFGE/WGS) may help, though grey areas remain.

PCR

General Salmonella PCR can detect S. Pullorum and S. Gallinarum. GD offers PCR for these species from down samples.

Serology

As highly invasive and contagious pathogens, antibodies against S. Pullorum and S. Gallinarum are usually detected within weeks of infection. Serological monitoring is critical, particularly at the start of hatching egg production when seroconversion is most likely in subclinically infected breeding flocks.

Key tests and considerations include:

• Rapid Plate Agglutination (RPA) test for monitoring
• Temporary cross-reactions from inactivated S. Enteritidis vaccines or infections
• Rare reactions from live S. Gallinarum vaccines
• Serology may fail in acute mortality cases (S. Gallinarum) or infections with variant strains

There are no serological tests available for S. Arizonae.

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Legislation

Dutch Legislation

Under the Dutch Animal Health Decree, Royal GD is the authorised body for the compulsory monitoring of S. Gallinarum, S. Pullorum, and S. Arizonae.

Key points regarding monitoring:

• Monitoring of S. Pullorum and S. Gallinarum is mandatory for commercially raised chickens, turkeys, guinea fowl, quail, pheasants, partridge, and ducks intended for breeding purposes.
• Monitoring of S. Arizonae is required only for turkeys.
• Monitoring occurs at the beginning of production, typically around 22 weeks for breeding chickens and 34 weeks for breeding turkeys.

Hatching eggs may not be collected from breeding flocks that test positive for S. Pullorum, S. Gallinarum, or S. Arizonae.

European Legislation

The European Animal Health Regulation (AHR), which came into force on 21 April 2021, governs the prevention and control of animal diseases. The Dutch government website provides guidance on these changes for farmers or transporters of poultry, day-old chicks, hatching eggs, or show birds.

Relevant details from European regulations:

• According to the European Animal Health Regulation (EU) 2016/429, S. Gallinarum, S. Pullorum, and S. Arizonae are categorized as category D diseases.
• Category D diseases impose requirements for the international trade of poultry and eggs, specifically for farmers producing hatching eggs.
• Delegated Regulation (EU) 2019/2035 mandates that hatcheries submit down/meconium samples and weak chicks or dead animals for S. Pullorum/S. Gallinarum (SP/SG) testing at least every six weeks.
• Additional testing is required at the commencement of each production period of hatching eggs.

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Approach to Contaminated Farms

Vaccination

There is a vaccine available in the Netherlands for S. Gallinarum, with assumed cross-immunity against S. Pullorum, although this has not been conclusively proven. The vaccine is suitable for administration to chickens from the age of 6 weeks. Various studies also describe cross-immunity from S.E. vaccines against S. Gallinarum and from S. Gallinarum vaccines against S.E..

Antibiotics

Non-zoonotic salmonella are not controlled diseases in broiler and layer chickens. Antibiotic treatment is desirable in some cases, for instance in the event of increased mortality. Keep in mind that antibiotic treatment will not cure the infection and that birds with carrier status will remain in the flock. Mortality and production declines may therefore be recurrent. Given that vertical transmission also occurs, the only option for ensuring that the spread of S. Pullorum or S. Gallinarum is stopped in breeding flocks is generally a cull.

According to the old formulary (KNMvD, 2012), oxytetracycline is a treatment option for laying hens. According to the current formulary (KNMvD, 2022), the options are tiamulin and tylosin.

To obtain a picture of the sensitivity of Salmonella Pullorum to antibiotics, tests were carried out on nineteen isolates from Dutch poultry farms (commercial and small-scale) from 2011 to 2024.

Based on the efficacy as reported in the literature plus the sensitivity of isolates from Dutch poultry farms to antibiotics, oxytetracycline is the first-choice drug for laying birds in the event of infection with S. Pullorum. Using sulphonamides is not recommended for laying hens because of their potential effect on shell formation. When younger chickens and broilers are infected with a Salmonella spp., trimethoprim + sulfamethoxazole and doxycycline are the first-choice drugs.

Aminoglycosides are another possible treatment option. The options within this group of antibiotics are apramycin and neomycin (in which the susceptibility of the Dutch isolates is good, at 0% resistant), and to a lesser extent streptomycin (0% resistant, 32% intermediate sensitivity), but not spectinomycin (5% resistant and 95% with intermediate sensitivity). However, aminoglycosides that are administered orally are only absorbed from the intestine to a very limited extent and are therefore unsuitable for treating systemic infections with S. Pullorum. Because Enterobacteriaceae, the family that the salmonella genus belongs to, are considered to be intrinsically resistant to tiamulin and tylosin, those agents are not an option for treatment.

Given the low incidence of Salmonella Pullorum outbreaks in the Netherlands, it is recommended that the pathogen should be isolated in all cases so that the antibiotic susceptibility can be determined. The results of that determination can be used for updating the overview whenever there is an outbreak.

Table Percentages of sensitive (S), intermediate (I) and resistant (R) Salmonella Pullorum isolatesfrom Dutch poultry, 2011 to 2024



*Trimethoprim/Sulfamethoxazole was tested at a concentration ratio of 1:19.

Other Measures

Detecting and Disposing of Infected Animals

In breeding poultry, S. Pullorum, S. Gallinarum, and S. Arizonae are reportable diseases due to the risk of vertical transmission. Hatching eggs from positive flocks may not be traded, and in practice, all animals at positive breeding farms are culled.

General Measures

S. Pullorum, S. Gallinarum, and S. Arizonae can persist in the barn environment. Effective cleaning and disinfecting are therefore crucial, with special attention given to:

• Insect and blood lice control
• Vermin management
• Disinfectant application (salmonellae are susceptible to most disinfectants)

Biocontainment is critical to preventing disease spread. Recommendations include:

• Restricting access (lock-up measures)
• Making the farm the last visit of the day
• Using dedicated farm clothing and equipment
• Showering and disinfecting upon entry and departure
• Implementing one-to-one transport and disposable trays

Public Health Measures

S. Pullorum and S. Gallinarum pose a minimal risk to public health. Actions to limit their incidence are primarily driven by their significant impact on animal health.

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Prevention

Preventing infection with non-zoonotic salmonellae relies on the use of hatching eggs from Salmonella-free flocks that are hatched in a Salmonella-free hatchery. Monitoring includes:

• Blood testing at the beginning of the laying period
• Subsequent clinical inspections

In cases of S. Pullorum, S. Gallinarum, and S. Arizonae in young animals, it is critical to initiate a tracing investigation. This ensures the identification and mitigation of infection sources.

In addition to securing Salmonella-free sources, implementing strict hygiene measures is vital to prevent the disease’s introduction. Following an outbreak of S. Gallinarum, vaccination of subsequent flocks is strongly recommended.

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Literature

• Barrow, P., Nair, V., Baigent, S., Atterbury, R., & Clark, M. (Eds.). (2021). Poultry Health: A Guide for Professionals. CABI.
• Swayne, D. E. (2019). Diseases of Poultry. John Wiley & Sons.
• Daigle, F. (2021). Special Issue: Salmonella: Pathogenesis and Host Restriction. Micro-organisms, 9(2), 325.