• 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 2 species and is further subdivided into 6 subspecies, each with their own serotypes and biovars. The serotype is determined on the basis of O and H antigens, and more than 2500 serotypes have been described. Only 1 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). As this serotype does not have 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 the disease progression, due to their facilitation of cell invasion and colonisation, for example.

Generally speaking, the serotypes can be subdivided into two different groups. A limited group of serotypes (including S. Pullorum and S. Gallinarum) gives a ‘typhoid-like’ disease within a narrow host spectrum. Infection gives severe symptoms with bacterial reproduction taking place in different internal organs. The bacteria are especially adept at multiplying in macrophages, giving them a predisposition for the liver and spleen.

While the other serotypes share a lot of the abilities of S. Pullorum and S. Gallinarum they are typically less likely to cause this systemic disease in chicken. They do however spread more readily in the gastrointestinal system and are able to infect a much broader broader host spectrum, which increases the risk of spreading to humans, among others. Large differences between serovars exist in this regard though. For diagnostic purposes the clinical presentation, while useful for an early indication, is insufficient and 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 the egg has been laid by soiling of the shell. Furthermore, when eggs infected with salmonella hatch, the disease is very simply spread via down feathers to other hatching units. Infection of chicks takes place via ingestion, inhalation, via the eyes or via the navel.

Horizontal contamination takes place through contaminated manure (particles) or other contaminated organic materials which can be spread via people, tools, machines, feed, vermin and insects (mechanical transmission). While carriers (clinically healthy birds that carry the bacterium) may introduce the disease in a new flock, this route plays a limited role in commercial poultry farms thanks to the all-in/all-out management system, but it can be a factor of concern in hobby flocks. Spread within the flock takes place through faecal-oral contamination or through cannibalism (of dead animals). The incubation period averages 2-3 days prior to the first signs, and it will take 4 to 10 days before deaths are seen.

In the wild, S. Pullorum and S. Gallinarum are found in various bird species and particularly in fowl. We do not know the extent of these salmonellae in the wild in the Netherlands. S. Gallinarum was last seen in commercial layers in 2021, S. Pullorum in backyard poultry in 2023 and in commercial layers in 2021. S. arizonae can occur in wild birds, rats, mice and reptiles; there are no known reports of arizonosis in the Netherlands.

Susceptible animal species

S. Pullorum and S. Gallinarum have a limited host range, and are almost exclusively seen in birds. All bird species can be carriers of S. Pullorum, though clinical signs are only reported in chickens, turkeys, guinea fowl, quail and pheasants. Symptoms of S. Gallinarum are mainly seen in chickens, turkeys, ducks, pheasants, guinea fowl, peacocks, quail and grouse.

Public health

S. Pullorum and S. Gallinarum are not very good at infecting humans and these pathogens have an extremely limited zoonotic risk. In exceptional cases S. Pullorum and S. Gallinarum can cause a (mild) intestinal infection in humans. Infectious pressure, the route of infection and immune suppression of the patient should be considered when interpreting such rare cases.

Survival

S. Pullorum and S. Gallinarum can survive as a persistent infection in animals, resulting in vertical transmission. In poultry manure, Salmonella can survive up to 6 days at 19°C and for 12-25 days at 5-9°C. The survival rate also depends very much on any other micro-organisms already present in the manure. Salmonella will survive longer if there is less competition. The survival period can even run up to 300 days under favourable ambient conditions.

Disinfecting

Diligent cleaning and disinfecting of barns and equipment is extremely important, as is effective and continuous vermin control. If not cleaned and disinfected, a barn environment can remain contaminated for up to two years.

Effective disinfectants are:

• products containing chlorine;
• quaternary ammonium compounds;
• formaldehyde/formalin;
• alcohol (70%);
• sodium hypochlorite (0.025%).

Salmonella can be inactivated in the barn by heating to a minimum of 60°C at 100% RH for 24 hours.
In feed, Salmonella can be eradicated by compressing the pellets (reaching a temperature of >80°C). Salmonella will be killed within a relatively short period of time (weeks) in composting manure if the temperature increases above 56°C.

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Clinical signs

Clinical signs in chicks

Following vertical transmission, chicks may already die in the egg. The disease is then immediately visible upon collection. When chicks only become infected in the hatchery, it may take a week before the problems become apparent.

Clinical signs can be extremely severe in chicks:

• poor hatching (in case of vertical contamination);
• general illness;
• weak, pumping breathing;
• little appetite;
• slimy faeces;
• white coating on the cloaca;
• inflammation of joints;
• blindness (S. Pullorum);
• pale comb and lobes (S. Gallinarum);
• mortality can be as high as 90 to 100%.

The peak of clinical signs is seen at 7 to 10 days following the initial signs in the flock. Surviving chicks are straggly, poorly feathered and sometimes lame (runting and stunting).

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

• lethargy and weakness;
• shaking;Anorexia;
• diarrhoea;
• paresis/paralysis;
• torticollis;
• blindness/enlarged eyeballs as a result of cloudiness of the cornea and accumulation of cheesy material in the eye.

In adult animals

Infection with S. Pullorum at a later age can often subclinical, although occasional outbreaks in mature birds are reported. In those rare cases a drop in production and increased mortality may be seen and post mortem findings in laying hens include peritonitis and abnormally shaped ovaries. Infection with S. Gallinarum generally starts with an sharp increase in mortality, often with a low morbidity due to the rapid (acute to peracute) development of the disease in individual birds. Sometimes a reduced feed intake, egg production, fertilisation and hatching, are noted whereby the animals are lethargic and have ruffled feathers.

Turkeys infected with S. Gallinarum are not only anorexic and lethargic, with ruffled feathers, but also thirsty and showing greenish diarrhoea. In turkeys too, mortality can rise quickly.

In quail, an S. Gallinarum infection gives signs comparable to those in turkeys, with losses running up to 55%.

Morbidity/mortality

S. Pullorum

In chicks younger than 3 weeks in particular, morbidity and mortality caused by S. Pullorum can be high (up to 100%). Chicks who survive early infection can become (asymptomatic) carriers and can be sero-negative. The bacteria can start multiplying and spreading again once the carriers reach reproductive age. While these birds often still show no or few clinical signs they can excrete the bacteria, potentially leading to vertical spread of S. Pullorum, and show increasing sero-response. An infection of S. Pullorum in older chicks can also result in illness and mortality, though to a lesser degree. S. Pullorum may result in mortality in (young) adult turkeys.

S. Gallinarum

Mortality as a result of S. Gallinarum is particularly observed in adult animals. However, losses exceeding 20% have also been observed in young chicks (up to a month old). Mortality rates can increase even further if they are infected on day one. Following such an early infection, losses often remain overly high during the rearing period. The mortality among adult animals varies greatly, from 10-70%, and is influenced by factors such as vaccination status of the flock, breed (heavy brown layer breeds are often more susceptible) and virulence of the S. Gallinarum strain.

S. arizonae

In adult animals, S. arizonae rarely results in clinical signs, and there are also virtually no losses as the result of an infection. Mortality can run from 32% to 70% in turkey chicks, and is particularly visible during the first three weeks, though this can persist until five weeks.

Excretion of the bacteria

Vertical transmission of S. Pullorum, S. Gallinarum and S. arizonae takes place trans-ovarially. Up to 1/3 of the eggs of infected hens can become infected in this way. Upon hatching, this may result in ‘explosions’ of bacteria and massive mortality of the chicks in the next few days. The most important form of transmission within a flock takes place via excretion with the faeces. Survivors and animals infected later on in the rearing process may be non-excreting carriers. These carriers are an important source of infection, particularly for S. Pullorum. Excretion via faeces and eggs often suddenly increases once the hens enter lay, possibly relating to hormonal changes and/or stress factors.

Differential diagnostics

Young chicks

• other types of salmonella (S. Enteritidis, S. Typhimurium);
• Aspergillus (mortality in first week, following infection in hatchery);
• navel infection;
• breeding errors;
• feed or water deficit;
• E. coli;
• Clostridium perfringens;
• botulism;
• CO, CO2;
• Avian Encephalomyelitis (epidemic tremor);
• Avian influenza;
• Newcastle Disease.

Older chicks

Joint/tendinitis

• Mycoplasma gallisepticum;
• Mycoplasma synoviae.

Heart abnormalities (white nodules)

• Marek’s disease

Local infections

• Pasteurella multocida;
• Streptococci;
• other types of Salmonella.

Adult animals in the event of acute mortality:

• 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 small areas of necrosis and inflammation (S. Gallinarum).

Older animals, S. Gallinarum

The most striking impression is that of acute sepsis, with hepatomegaly being the most prominent finding in all recent cases reported in the Netherlands and heavy brown laying hens being strongly overrepresented. Livers can be bronze-green with haemorrhaging and necrosis. Serosal surfaces can be affected, with peritonitis and pericarditis, and other organs can be involved too, including but not limited to lesions such as swollen kidneys and splenomegaly.

In turkeys, an S. Gallinarum infection has also been observed to cause lung abnormalities and necrosis of the heart muscle.

Older animals, S. Pullorum

Hallmark lesions are signs of septicaemia (often with hepatomegaly and/or splenomegaly) and oophoritis with deformed and/or stalked egg follicles and in some outbreaks lesions can appear similar to those seen in E. coli peritonitis.

S. arizonae

• enlarged yellow liver with white foci;
• duodenum congestion;
• yolk sac infection;
• cheesy material in intestinal/caeca;
• inflammation of oviduct/peritoneum;
• eye lesions (exudate in anterior eyeball chamber/vitrium);
• purulent exudate in the brain.

Isolation of the bacteria

S. Pullorum and S. Gallinarum are Salmonellae without a flagellum and are therefore immobile. As the general culture procedure is aimed at the motile Salmonellae, it is not suitable for the discovery of S. Pullorum or S. Gallinarum. Accumulation in selenite-cystine or RVS without pre-accumulation is the designated method for samples in which many ambient bacteria may be expected (manure samples, for example). GD can test for SP/SG using manure/down/dead animals. This takes place via accumulation in RVS, after which further identification is based on PCR (down) or serotyping and biochemics (manure/dead animals).

S. arizonae is motile and can be diagnosed using the general culture procedure, though it is important to take account of the colonies on the XLD plate deviating from the other salmonellae. If testing for S. arizonae is required, this must be communicated beforehand.

In the case of clinical disease, S. Pullorum, S. Gallinarum or S. arizonae can simply be cultivated from abnormal organs from diseased or recently dead birds. Culture from femoral bone marrow from chickens that died during an outbreak was show to be a good sample. Standard media such as Sheep Blood Agar (SBA) or MacConkey agar are suitable for this purpose. The colonies of S. arizonae very much resemble those of the zoonotic salmonellae. The colonies of S. Pullorum and S. Gallinarum are generally a lot smaller. Also in a gram stain, S. Pullorum and S. Gallinarum are smaller than the other salmonellae.
It can be difficult to distinguish between vaccine and field isolates of S. Gallinarum. Clinical infections have been described with isolates showing the same characteristics as the vaccine strain. In certain situations, biochemical or genetic testing (PFGE/WGS), can identify a field isolate, but a grey area remains in which vaccine and field isolates cannot be distinguished.

PCR

A general salmonella PCR can also detect (the presence of) S. Pullorum and S. Gallinarum. GD has a PCR available for the identification of S. Pullorum and S. Gallinarum from down samples.

Serology

As S. Pullorum and S. Gallinarum are clearly invasive and are extremely contagious within a flock, antibodies will soon be detected in some of the animals fairly rapidly (a number of weeks) after infection. Serology therefore plays an important role in monitoring. The start of the hatching egg production is the most suitable time for testing for monitoring purposes. This is precisely the point at which seroconversion often occurs in subclinically infected breeding flocks.

The rapid plate agglutination (RPA) test is used for monitoring purposes. Cross-infections can occur with inactivated S. Enteritidis vaccines or S. Enteritidis infections. Cross-infections with inactivated S. Enteritidis vaccines are of a temporary nature. The live S. Gallinarum vaccine very rarely gives reactions in the S. Pullorum and S. Gallinarum antibodies RPA. An infection may not be serologically detected in the event of (per-)acute mortality (the animals do not have time for seroconversion), particularly in S. Gallinarum) or in an infection with a variant strain.

There are no serological tests available for S. arizonae.

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Legislation

Dutch legislation

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

Monitoring of S. Pullorum and S. Gallinarium is compulsory for commercially raised chickens, turkeys, guinea fowl, quail, pheasants, partridge and ducks for breeding purposes. Monitoring of S. arizonae is only compulsory for turkeys. The time of monitoring is at the beginning of production (around 22 weeks for breeding chickens, 34 weeks for breeding turkeys).

Hatching eggs may not be collected from breeding flocks which have tested positive for S. Pullorum, S. Gallinarum or S. arizonae.

European legislation

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

Based on this European Animal Health Regulation (EU) 2016/429, S. Gallinarum, S. Pullorum and S. arizonae have been designated as category D diseases. This means that requirements are in place regarding the international trade in poultry and eggs. These requirements apply to farmers which produce hatching eggs, and are established in the delegated regulation (EU) 2019/2035. This states that hatcheries must submit down/meconium samples and weak chicks/dead animals for SP/SG testing at least every 6 weeks. Samples are also taken upon commencement of the production period(s) 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 proven), which 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

These salmonellae do not have compulsory notification and eradication in broilers and layers. Antibiotic treatment reduces the clinical signs and losses, but flocks often remain carriers, and the losses and production decline may be recurrent. Antibiotic treatment may prove useful in animals laying eggs for human consumption. In the event of an S. Pullorum or S. Gallinarum infection, oxytetracycline is the first choice of treatment.

In broilers infected with Salmonella spp., TMP/S is the first choice, possibly replaced by oxytetracycline.

With a view to vertical transmission, culling the flock is generally the only option in breeding flocks, in order to completely stop the spread of S. Pullorum or S. Gallinarum.

Other measures

Detecting/disposing of infected animals

In breeding poultry, S. Pullorum, S. Gallinarum and S. arizonae are compulsory 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 are easily retained in the barn environment. Diligent cleaning and disinfecting is therefore required, with extra attention for insects, blood lice and vermin control. Salmonella is susceptible to most disinfectants. Biocontainment is also important in preventing spread of the disease: if applicable, think in terms of lock-up, making the farm the last visit of the day, using specific farm clothing, showering and disinfecting upon (entry to and) departure from the farm, one-to-one transport, use of disposable trays.

Public health measures

S. Pullorum and S. Gallinarum only form a very limited risk to public health. All actions undertaken to limit their incidence are linked to the fact that this is a very serious disease among animals.

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Prevention

In preventing infection with non-zoonotic salmonellae, it is essential to use hatching eggs from Salmonella-free flocks, hatched in a Salmonella-free hatchery. Monitoring takes place by means of blood testing at the beginning of the laying period, and subsequently via clinical inspections. It is therefore important that a tracing investigation is instigated in cases of S. Pullorum, S. Gallinarum and S. arizonae in young animals. Besides a Salmonella-free source, hygiene measures are important in order to prevent introduction of the disease. Following an outbreak of S. Gallinarum, it is recommended that subsequent flocks are vaccinated.

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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.