introduction to the enterobacteriales or coliforms

The Enterobacteriales are ubiquitous, gram-negative, non-spore forming, mostly motile, oxidase-negative, facultative anaerobic  rods or coccobacilli. They are collectively known as “coliforms”. The most important pathogenic members of this enormous Order are Escherichia coli and Salmonella enterica.

Below is a phylogenetic tree that focuses on pathogenic genera of the Class Enterobacteriales

Diseases caused by members of the Enterobacteriales

Known pathogens of animals in this order include serovars of Escherichia coli, Salmonella species, Yersinia spp., Klebsiella spp., and Proteus species. (In humans 4 species of Shigella are known to cause enteric disease). All members of this family have endotoxin as part of their cell wall and therefore have the potential to cause endotoxaemia. Serratia an environmental bacterium is a known nosocomial (hospital-acquired) pathogen. Coliforms, especially E. coli,  are amongst the most common group of bacteria idetified in veterinary diagnostic laboratories.

The Table below summarises the more common diseases in animals caused by members of the Enterobacteriales.

Natural habitats of the enterobacteriales

Most pathogenic members are found in the gastrointestinal tracts of animals where they reside as intestinal commensals. The most well-known enteric coliform of terrestrial animals – Escherichia coli- when cultured from fresh water is considered to provide evidence of faecal contamination of this water. Some Enterobacteriales are also found in organically-rich environments such as muddy paddocks, and are known to contaminate washing water, soaps etc. An example is Serratia species which is a known nosocomial agent. Klebsiella species are found in the intestinal tract and in moist environments rich in rotting woody material i.e. wood shavings bedding. Mastitis in cattle caused by Klebsiella species is often associated with wood shavings being used as bedding.

Characteristics of the Enterobacteriales that allow the identification of pathogenic members

Since these bacteria can vary in shape from a coccobacillus to a long filament, they cannot be distinguished morphologically. They are easily cultured in air at 35-42°C on most laboratory non-selective agars. The two important pathogens in this Class can be differentiated on selective agars (see pictures below)  MacConkey agar which contains bile salts and crystal violet is one of the agars designed to selectively allow the growth of all members of this Class. E. coli is a lactose fermenter on this agar.

Salmonella enterica is a non-lactose fermenter on MacConkey agar  and reduces iron salts to hydrogen sulphide (black colonies) on XLD agar. Furthermore, unlike E. coli, it does not produce indole and is citrate positive. Because of the pathogenic and zoonotic significance in humans and animals, selective media has been developed to isolate Salmonella enterica. In veterinary laboratories selective enrichment with either Selenite or Rappaport-Vassaliades broths is done followed by isolation on either xylose-lysine-deoxycholate (XLD) or XLT agar medium.

The different genera and species in this order are identified based on how they differentially utilise different substrates (nutrients). One such test panel is known as the API20E.

These tests are not able to distinguish non-pathogenic (harmless colonisers) and pathogenic (disease-causers) of the same species. Thus other tests are required.

In addition, various typing schemes are used and include the following:

1. Serotyping: Antigens present in/on the lipopolysaccharide of the outer cell membrane (“O-antigen”); flagella (“H-antigen”) and Capsule (“K-antigen”) are typed. Only smooth colony types of Escherichia coli on MacConkey agar (those with a thick outer membrane) can be serotyped. Serotyping is still the basis of typing most pathogens in this order. Note that serovars identifying a bacterium are written in normal script with the first letter in Capitals i.e. Salmonella enterica serovar Dublin.
2. Biotyping. This is when related bacteria are characterised by an arrays of biochemical and other phenotypic tests. An example are the poultry pathogens – Salmonella enterica biotype gallinarum and Salmonella enterica biotype pullorum. Both belong to the same serovar.
3. Phage typing: Certain pathogens such as Salmonella serovar Typhimurium or Salmonella serovar Enteritidis are further typed using bacteriophage typing. (Lytic viruses that specifically attack a strain of Salmonella – a classic example is Salmonella Typhimurium defective phage type 104). The aim of this is to trace outbreaks and determine the spread of virulent and multidrug resistant strains.
4. Genotyping: Specific genes encoding for virulence factors are detected i.e. Shigatoxin-producing strains of E. coli. This determines the presence of pathogenic strains.
5. Genetic fingerprinting. Used to trace the spread of a bacterial clone in disease outbreaks. Typically pulsed-field gel electrophoresis is used and results deposited in the database “PulseNet” managed by the CDC. More recently whole genome sequencing is being used to fingerprint these bacteria.

General pathogenesis of the enterobacteriales

Diseases caused by the Enterobacteriales fall into three categories:

1. Enteric disease with diarrhoea
2. Septicaemia
3. Localised pyogenic infections

Since these bacteria, even the animals pathogens, are ubiquitous, disease only occurs when the predisposing factors associated with the animal host, environment and agent occur.

Predisposing factors to disease

1. Host factors:

• Age susceptibility: Neonatal animals are more susceptible to E. coli infections than older animals
• Genetic: The presence of receptor sites for pathogenic E. coli is genetically encoded i.e. F4 in piglets
• Immunity: Neonatal animals with no passive immunity are highly susceptible to E. coli infections
• Stress. Physiological (nutrition, recent transport) and pyschological (bullying, new environment) increases disease susceptibility. This is especially true for Salmonella infections.
• Concurrent infections: Viral and parasitological infections can predispose animals to Enterobacteriales infections.

2. Environmental factors:

• Climatic temperature: chilling can lead to E. coli infections in neonates
• Overcrowding and faecal-rich soils: increase exposure to the pathogens and cause stress.

3. Agent factors:

• Only those members that are able to adhere to the intestinal tract and produce toxins or invade the host will cause disease.

Transmission

Generally these bacteria enter the body via the faecal-oral route. Occasionally the respiratory and urogenital route and wounds act as portals of entry. Of special note is the umbilicus in a neonatal mammal where entry via this route leads to a localised infection with spread to the rest of the body via the blood vessels (haematogenous).

Pathogenesis of septicaemia

Members of the Gammaproteobacteriaceae all have lipopolysaccharide (LPS) as a component of their outer cell membrane. The pathogens have factors that allow them to attach, invade, overcome the host’s immune defenses and multiply within the host. Thus animals who have not received sufficient maternal antibodies or who have not developed a strong adaptive immunity are prone to these infections. For Escherichia coli there is an age-related susceptibility where only neonatal animals are susceptible. Septicaemia when it develops is usually an endotoxaemia as once the bacteria have proliferated they die and release their LPS which sets off the systemic inflammation cascade.

Escherichia coli infections “colibacillosis”

Escherichia coli is the cause of colibacillosis (diarrhoea or septicaemia) in primarily neonatal calves, lambs, kids, piglets, crias (young of alpacas), puppies and chicks. It is also known to cause opportunistic infections in animals, such as environmental mastitis in cows and sows; urinary tract infections in dogs and sows, ear infections in dogs and endometritis in mares. A related bacterium known as Escherichia albertii can cause attaching and effacing lesions in humans and birds. (Disease syndromes that you must know, but are not in this section are linked to the workbook where the syndrome is discussed).

In this section only colibacillosis in livestock and urinary tract infections in dogs will be discussed.

colisepticaemia

Colisepticaemia caused by virulent strains of E. coli is more common in neonatal animals who have not received sufficient maternal or passive immunity. (Refer to the General pathogenesis sub-heading in the Introduction to read about this pathogenesis).

Watery mouth in lambs (Colisepticaemia)

Describes a prominent clinical sign noted in neonatal lambs between 12 to 72 hours of age that are suffering from colisepticaemia. These lambs often born in dirty camps, come from multiple births and have usually failed ingest sufficient colostrum in time. The E. coli after ingestion proliferates in the intestines releasing endotoxins which result in intestinal hypomotility allowing the endotoxins to be taken up systemically. The lambs appear dull, have a wet mouth due to excessive salivation and the eyes appear puffy. The abdomen later distends.

Watery mouth is diagnosed by culturing E. coli from a whole blood sample. Treatment with fluids, glucose, anti-inflammatory drugs and bacteriostatic antibiotics early in the disease may be successful.

E. coli systemic infections in poultry

Of intestinal origin, a large variety of O-antigen types of E. coli attach to the respiratory epithelium and invade into the circulatory system of poultry. They can cause airsacculitis, polyserositis, septicaemia and other extraintestinal infections. In mature hens, pathogenic genotypes cause a salpingitis-peritonitis syndrome. Opportunistic infections caused by pathogens in this species can cause huge losses to the poultry industry. In some countries where antibiotic use is less restricted than Australia, a large variety of oral (in-feed/in-water) antibiotics are used to treat it resulting in multi-drug resistant E. coli.

enteritis caused by Escherichia coli strains

Enteritis in the neonatal animal occurs in animals that have failed to receive maternal immunity and is caused by different pathogenic strains of E. coli. 

Namely

1. Enterotoxigenic E. coli (ETEC) – most common
2. Enteropathogenic E. coli (EPEC)
3. Enterohaemorrhagic or Shigatoxin producing E. coli (STEC)

1. Enterotoxigenic E. coli (ETEC)

E. coli strains with the fimbrial antigens: F4 (was K88), F5 (was K99), F6 (was 987P) and F41 adhere to the brush border of the intestinal cells and produce heat stable (ST) or heat labile (LT) toxins. These toxins are internalised in the cells and adhere to guanylate cyclase and adenyl cyclase receptors respectively increasing the production of cyclic guanosine monophosphate (cGMP) and cyclic adenosine phosphate (cAMP). Both affect the ion channels within the cell membrane by increasing ion loss and reducing ion intake. This results in the intestinal lumen having a high osmolality than the cells with water lost into the intestinal lumen by osmosis. This acts like a water pump drawing water from the interstitium, through the intestinal epithelium and into the intestinal lumen across a water gradient. Thus dehydration and a metabolic acidosis results that in untreated animals can lead to death. There is no structural damage of the intestinal cells. Clinical signs in animals include a whitish watery diarrhoea (white scours) and severe dehydration.

A special case- ETEC in piglets

Intensively-reared piglets are especially prone to colibacillosis. There are 2 ages where they are most susceptible (see table below):

1. In the neonatal period. This is usually in pigs who have not received sufficient maternal immunity and those subjected to chilling. The mortality rate is up to 100% in affected piglets.

2. In the post-weaning period (4 to 8 weeks of age). This is when the piglets have lost their passive immunity and are stressed by the weaning process. Pigs with food allergies are more susceptible to colibacillosis. Since these pigs are more robust than neonatal piglets, the mortality drops to about 10%. This is the more common form of colibacillosis in pigs.

2. Enteropathogenic E. coli (EPEC)

EPEC is a lesser-known cause of diarrhoea in calves, piglets and puppies. Since the colon is usually more severely affected, some animals will develop dysentery. It is a common infection in children. It affects neonatal and animals up to 2 months of age. The lesions are more severe when there is a concurrent parasitic (Cryptosporidium) or viral infection.

This group of E. coli produce an adhesion protein known as intimin (Type III secretion system) that adhere tightly with the enterocytes and stimulate actin reorganisation of the enterocyte cytoskeleton to form pedestals. These pedestals improve contact of the bacterium with the cell. This decreases the absorptive surface and interferes with ionic exchange. Furthermore, the presence of the bacterium stimulates an inflammatory response. Whilst most of the intestinal tract is affected, the colon has the most severe lesions.

Most strains of EPEC will also produce Shigatoxin. However, the effect of Shigatoxin is more important in people (see the following E. coli type: EHEC).

3.  Shiga-toxin E. coli (STEC) (also called Enterohemorrhagic E. coli (EHEC))

Disease caused by Shigatoxin producing strains in humans can cause a serious haemorrhagic colitis. The most well-known of these E. coli is E. coli 0157:H7. It originates from farm animals and can cause haemorrhagic uraemic syndrome (HUS) in children. This zoonotic disease was at one time common in children who visited petting zoos. This is now much less due to improved hand hygiene, yard sanitation and animal nutritional management on these zoos.

EHEC strains produce intimin leading to the typical attaching and effacing lesions (see EPEC) as well as Shigatoxin (formerly verotoxin). Genes present in lysogenic phages encode the two Shigatoxin types, STx1 and STx2, and both have the same mode of action. After attachment, E. coli produces Shigatoxin, an A-B toxin that is taken up by the cell via endocytosis.  The vesicle moves to the endoplasmic reticulum, where the “A” portion moves out of the vesicle, attaches to the 28S Ribosome, and removes an adenine residue from the ribosome, preventing it from interacting with Elongation factor 1. This halts protein manufacture and the cell dies.

A special Shigatoxin producing E. coli causing Oedema Disease in pigs

Oedema disease is a sporadic, global disease in commercial weaners caused by the Shigatoxin, ST2e-producing strains of E. coli. It affects up to 30% of the best piglets, with an outbreak heralded by deaths in some piglets and nervous signs in others. Clinical signs usually appear within 10 days after weaning. Death in severely affected pigs follows within 4 to 36 hours after the onset of clinical signs.

The ST2e associated damage of the arterioles results in widespread oedema. The piglets have swelling of the eyelids, snout and ears. Their voices becomes squeaky due to laryngeal oedema. Brain oedema results in apparent blindness, head-pressing and in-coordination. Later on the piglets will lie down and show paddling movements. The disease may be confused with salt poisoning or water deprivation in pigs. Oedema is also present in the greater curvature of the stomach, between the mucosa and the muscle layers. Jelly-like areas of oedema may be present in the mescocolon, larynx and the kidney capsule. Pigs that take longer to die have evidence of encephalomalacia and degeneration and proliferative arteriopathy.

The typical clinical signs and pathology are highly suggestive of oedema disease. Confirm the disease by the culture of a beta-haemolytic E. coli that has genes encoding for F18 and ST2e. ST2e detection in the intestines and bloodstream.  Use only fresh carcasses for toxin identification. Most ST2e E. coli have O138, O139 or O141 antigens.

Water medication with ampicillin, tetracyclines, neomycin or potentiated sulphonamides. The disease is limited by adding zinc oxide to the diet, restricting the feed intake or increasing the roughage of pigs just post weaning, and acidifying the feed. Good hygiene of the sow and pigs will decrease the environmental load. Eradicate the agent by depopulation, environmental cleaning and disinfection, and restocking with uninfected pigs after 3 weeks. Some piglets are genetically resistant.

Uropathogenic E. coli (UPEC) in dogs

Like human women, female dogs are susceptible to infections with uropathogenic E. coli. Intestinal commensal E. coli colonises the vaginal and periurethral tissue and moves up the urethra to the bladder (ascending infection). These bacteria have special adherence fimbriae, the most important being P-fimbriae, that attach to the bladder epithelium of dogs. Bacteria growing on the surface of the bladder will form biofilms that protects them against flush out and antibiotic destruction. Fusiform vesicles produced by the umbrella cells of the bladder allow the bladder to stretch and return to normal size. These vesicles assist in the internalisation of adherent E. coli into the cells. These vesicles are recognised as part of the cell, so don’t fuse with lysosomes. The E. coli in these vesicles can multiply and persist in the bladder epithelium. However, when they start to divide rapidly, they stimulate an inflammatory response that results in cystitis and increase the bladder cell turnover. Uropathogenic strains produce haemolysins and thus are beta-haemolytic on blood agar.

A further adaption of E. coli in the bladder is in response to beta-lactam antibiotics where some strains are not killed, but fail to produce cross walls and daughter cells. These bacteria become elongated forming long filaments.

salmonellosis

In mammals and birds, Salmonella enterica and occasionally salmonellae of reptile origin are responsible for salmonellosis. Salmonellosis is characterised clinically by septicaemia, acute enteritis, chronic enteritis and extraintestinal infections such as abortion or joint ill. This global disease affects all vertebrates, but is particularly severe in stressed animals that are kept in high densities i.e. intensive farming. Salmonellae of animal-origin are zoonotic and cause food-associated infections in people.

Additional epidemiological factors associated with salmonellosis

Agent factors

Salmonella enterica is only present in low numbers in animals. Since a lower infection dose is required for disease and the fact that all strains of Salmonella are potentially pathogenic, they have been further characterised into serovars based on “O- and H-antigen” typing . At least 2000 serovars have been identified, but only a few are highly pathogenic.

Another way of characterising these serovars is to group them based on their host specificity. Note that all mammal and bird salmonellae belong to one species, namely Salmonella enterica.

• Host-specific where they will usually only infect one animal species. Examples are Salmonella biovar gallinarum the cause of fowl typhoid in layers and breeders and Salmonella biovar pullorum the cause of pullorum disease in chicks. Salmonella Typhi and S. Paratyphi infect only humans causing typhoid and paratyphoid fever respectively.
• Host-adapted where they predominately infect one host species but can infect other species. Examples are Salmonella Dublin in cattle and Salmonella Choleraesuis in pigs.
• Ubiquitous where serovars can infect a wide species range. Most of the common and more virulent zoonotic salmonellae are in this group i.e. Salmonella Typhimurium and Salmonella Enteritidis. Note that S. Enteritidis is found primarily in poultry.

Host factors

Once infected, disease may or may not occur. However, infected as well as recovered humans and animals can either eliminate the bacteria or remain long-term carriers. This is especially true of host-specific and host-adapted strains of Salmonella where they remain in the bile ducts, GALT (gut associated lymphoid tissue) and mesenteric lymph nodes.

In salmonellosis 3 types of carrier states may occur:

1. Active carriers: these are individuals that are infected with the bacteria, which is multiplying and being shed either in the stool or in the eggs of birds or reptiles. Note that animals can still shed salmonellae for a period after recovery from disease. This is dependent on bacterial strain type and animal species. Asymptomatic bacteraemia in birds leads to salmonellae being sequested in the eggs during egg manufacture within the oviduct.
2. Latent carriers: these are carriers that are infected with salmonellae but the bacteria are inactive. In periods of stress the bacteria may activate and start shedding. Sites where latent bacteria can be found include the intestinal lymphatic tissue, lymph nodes and epithelium of the bilary tract.
3. Passive carriers: The uninfected individuals pass the bacteria intact through the intestines. They will not shed once removed from the source of bacteria.

Humans, animals and their environment which includes food sources are linked. The web in the picture below shows just how salmonellae can spread to different animals and people. For example, a vegan may contract zoonotic salmonellosis. He/she may have eaten greens that had been organically grown, using improperly treated chicken litter or the fields were plagued with high wild bird numbers.

The environment

Although the bacterium colonises and multiplies in the intestinal tract, it can survive for several weeks in a dry environment and months in a moist environment. Ultra-violet light and heating to 60°C for 90 minutes will destroy it.

Specific pathogenesis of Salmonella

Transmission is generally faecal-oral and the incubation period is 1 to 4 days. Salmonella uses flagella move to and locate suitable adhesion sites and then fimbrae to attach to the small and large intestinal cells and “M-cells”. The presence of a capsule in some virulent serotypes which prevents the binding of IgM and phagocytosis by immune cells. It uses a molecular syringe (see YouTube video) to inject proteins into the cell. It is a Type III secretion system. These proteins effect a rearrangement of the cell actin proteins so that projections are formed to engulf the bacterium in a macropinosome (vacuole used for collecting excess extracellular fluid). They survive in the vacuole by delaying maturation and also making their outer membrane more resistant to attack. One of the ways they do this is by the production of uperoxide dimutases. These vacuoles not being phagocytic are less subject to fusion with lysosomes. Bacteria then divide in the vacuoles. Thus salmonellae are able to persist in cells without stimulating a strong immune response. The vacuolated bacteria eventually moving to the basement membrane end of the cell where they are released into the interstitium. Released bacteria are engulfed in the same manner by macrophages and neutrophils and are transported to the reticuloendothelial system. Dendritic cells can directly engulf the salmonellae from the intestinal lumen.

Serotypes causing an enteritis also initiate a secretory diarrhoea using Shigatoxins and stimulate intestinal inflammation with migration of neutrophils into the intestinal lumen. Thus, the enteritis is generally more severe than that coused by E. coli. Salmonella causes a necrotising fibrinous enteritis. Lesions are most severe in the lower ileum and the large intestine and vary from shortening of villi with loss of the epithelium to complete loss of intestinal architecture. There is a neutrophilic reaction in the lamina propria, and thrombi may be seen in blood vessels in this region.

Some specific diseases caused by Salmonella enterica in animals

Salmonella Dublin, S. Bovismorbificans and S. Typhimurium infection in cattle and sheep

In Australia outbreaks of gastroenteritis and septicaemia in dairy calves from two- to six-weeks of age due to salmonellosis is common.  Organ localisation of the bacteria after the bacteraemic phase occurs in older calves with a subsequent polyarthritis, pneumonia or meningitis. Rarely vasculitis of the extremities occur resulting in loss of the tail, ears or feet.  Stressed adult animals may also be affected. Cows and ewes develop fever, lose their appetite, have decreased milk production and diarrhoea which varies from haemorrhagic to pasty. Abortions are common. Up to 75% of untreated cows and ewes die.

Even after recovery, cattle and sheep can remain long-term carriers of Salmonella Dublin. Cattle and sheep are also susceptible to serovars which may not be in the vaccine. For example, a recent survey in New Zealand indicated that Salmonella Brandenburg was the most common diagnosed cause of abortion in sheep.

Salmonella Abortusovis the cause of abortions in sheep is not present in Australia. It is a Notifiable Disease.

Additional reading: Mohler VL, Izzo MM, House JK. 2009. Salmonella in Calves. Vet. Clinics NA: Large Animal Practice. 25: 37-54

Salmonellosis in horses

Horses of all ages are especially susceptible to Salmonella infection. Adult horses often have had a history of recent invasive surgery or transport over long distances.  Even though all serovars can infect and cause colic and diarrhoea in horses, the most serious is usually Salmonella Typhimurium. Adult horses typically develop a fever, neutropaenia and acute enteritis, they become severely dehydrated and untreated horses can die within 24 hours after the onset of diarrhoea. It is especially a problem in foals on breeding farms  and equine hospitals where it is a major cause of nosocomial infections.

Some additional readings are provided:

Ekiri AB, Morton AJ, Long MT, MacKay RJ, Hernandez JA. 2010. Review of the epidemiology and infection control aspects of nosocomial Salmonella infections in hospitalised horses. Equine Veterinary Education., 12:631-64

Feary DJ, Hassel DM. 2006. Enteritis and Colitis in Horses. Vet Clin Equine 22:437–479

The only host-adapted salmonella of horses, known as Salmonella Abortusequi is not present in Australia and is a notifiable disease. It is the cause of abortion in horses and is rarely reported in the world.

Salmonellosis in poultry

1. Salmonella Enteritidis in poultry. Present in Australia, Salmonella Enteritidis is an important egg-borne zoonosis. This bacterium rarely causes disease in poultry. However, it can cause septicaemic disease in young chicks and pullets going into lay. It is a Notifiable Disease.
2. Salmonella enterica biovar gallinarum infection (Fowl Typhoid) in layers (Not present in Australia). Fowl typhoid is a septicaemic disease of mainly broiler hens and layers caused by Salmonella enterica biovar gallinarum. This disease is absent from Australia. In countries where it is present, it is a major cause of mortalities in hens and pullets. Birds are either infected orally or via the egg. Morbidity is 10 to 100% and mortality can reach 100% in endemic areas. The bacterium can be cultured from affected organs and is identified by being non-motile and by biochemical tests. Serology using the plate agglutination test is used to detect positive layer birds. It is a Notifiable Disease.
3. Salmonella enterica biovar pullorum, Pullorum Disease, ‘Bacillary White Diarrhoea’ in chicks. This bacterium is related to S. biovar gallinarum giving the same serotype, however, it causes septicaemia and diarrhoea in chicks and other birds up to 3 weeks of age. In Australia, it is absent from commercial flocks, but may rarely cause disease in non-commercial flocks.

Additional reading: OIE Terrestrial Manual 2015, Chapter 2.3.11 “Fowl typhoid and Pullorum disease”

Other animals

Outbreaks in other animal species such as pigs, dogs, cats and wildlife is rare and is often associated with concurrent infection, overcrowding or other stresses. Wild animals recently introduced into captivity are especially susceptible i.e. salmonellae are the most common cause of diarrhoea in captive macropods (kangaroos and wallabies). The source of salmonellae in these cases is often contaminated feed.

Reptiles, especially snakes, carry reptile-associated salmonellae in their intestinal tract. Snakes feed on rodents, a major source of salmonellae. Thus wound-associated infections and septicaemia caused by Salmonella species is often encountered in captive reptiles.

Diseases caused by Yersinia, Klebsiella, Proteus and serratia

Yersinia

Yersinia pseudotuberculosis and Yersinia entercolitica cause infection of the jejunum in young animals. Although chronic enteritis may develop i.e. terminal ileitis in lambs, and European brown hares, most infected animals carry the agent in their intestinal tract without evidence of disease. Intestinal and septicaemic disease due to Y. pseudotuberculosis appears to be more common in temperate climates where disease is often precipitated by a cold snap and heavy rainfall. Thus in the 2021-2022 rainfall season, there was a spike in seasonal cases in Canberra and NSW in ruminants and wild birds. Humans become infected from drinking contaminated water or milk or eating contaminated meat products. In humans the infection is characterized by diarrhoea, fever and abdominal pain.

Yersinia pestis is the agent of plague a disease that destroyed almost a third of Europe’s and the UK’s population in the Middle Ages. The human body louse and flea were the primary vectors of plague (black death). Today, infections are rare, (1000 to 3000 human cases per year), with an endemic focus in the USA. Rodents are the natural hosts where they are infected by the bites from fleas. In the USA the prairie dog, a herbivorous rodent, is the natural host of Y. pestis. The domestic cat may also be infected by fleas or by handling infected rodents and in turn can infect humans. In cats, and sometimes dogs, there is a swelling under the jaw, but it can be elsewhere like in the inguinal region. It is accompanied by fever, anorexia and lethargy. It can be misdiagnosed as a cat fight abscess.

The most common form of plaque in people is bubonic plague, where a lymph node becomes swollen “a bubo”, 2 to 6 days after infection and is accompanied by fever, chills, headache and tiredness. If untreated the bacterium can spread haematogenously to rest of the body, including the brain and lungs, and result in severe disease and even death. Pneumonic plague occurs when the lower respiratory tract is infected. I in 7 plague cases in the USA are fatal.

Klebsiella

Klebsiella species (K. pneumoniae and K. oxytoca) are lactose fermenting coliforms found in the intestinal tract, fresh surface waters and in moist woody environments. They are recognised by having a very thick capsule. They are a common cause of opportunistic infections of the respiratory, the mammary, the reproductive and urinary tracts in humans and animals. In horses they are a cause of sexually transmitted endometritis (See Chapter on Non-fermentative bacteria). In humans respiratory tract infections are often associated with alcoholism.

Proteus

Proteus species are common intestinal inhabitants that are associated with opportunistic infections. In dogs, the bacterium is a common cause of urinary tract infections, otitis externa/media and pododermatitis.

Serratia and Enterobacter

Environmental bacteria that cause opportunistic infections. Multi-drug resistant strains are associated with nosocomial (hospital-acquired) infections where they can be present and grow in soaps, expired disinfectants, multiuse injectables, catheters, endotracheal tubes and other tubing.

Diagnosis of infections caused by the Enterobacteriales

Whilst the case history and clinical picture can provide a suspect diagnosis, always confirm the suspect diagnosis. You will find that the differential diagnosis for the disease syndrome you are investigating may still be too long to pinpoint a cause.

Confirm the diagnosis by tests used to detect the bacterium that include:

1. Bacterial culture and identification using phenotypic array tests or mass spectrophotometry (MALDITOF). The specific identification is usually followed up by serotyping, phagetyping and genetic fingerprinting of the isolated bacterium (E. coli, Salmonella, Klebsiella).
2. When a specific bacterial cause is suspected. Salmonellosis in horses or oedema disease in pigs: a PCR to detect Salmonella-specific genes can be done.

Samples:

Living animals: Fresh faeces in cases of enteritis, or swabs of lesions. Fresh blood in EDTA (PCR and culture) or heparin (Culture only) for septicaemic disease. Always collect blood if salmonellosis is suspected. Fresh urine, preferably by cystocentesis in suspect cystitis.

 At post-mortem examination collect fresh tissues: Biopsies of lesions; tied-off ileum and contents; ileo-caecal lymph nodes and other intestinal lymph nodes. In the case of septicaemia: liver, spleen, kidneys, lung and brain., the detection of carrier

Salmonellosis : A special case.

For control purposes:

1. Detection of Salmonella carriers. Collect daily faecal samples for Salmonella culture. Up to 7 daily faecal samples may be required in equine salmonellosis.
2. Use sterile kitchen wipes of walls, and floors. Sterile surgical disposable overshoes are used in poultry houses. Collect bedding.

To isolate salmonellae that are in low numbers in faecal and environmental samples, Use the following steps:

1. Incubation in buffered peptone water for 24 hours (enrichment)
2. Incubation in a selective broth (selenite, Rappaport-Vassiliades) for 24 hours – selective enrichment
3. Incubation of a selective agar (XLD/XLT) – selective
4. Biochemical array, latex antigen agglutination test for polyclonal O-antigen of Salmonella – Identification to genus and species level
5. Serotyping to determine serotype
6. If necessary, phagetyping and genetic fingerprinting using whole genome sequencing

Control of diseases caused by the Enterobacteriales

Management of affected animals

Being a zoonosis as well as contagious, infected animals should be isolated into completely separate pens and biosanitary measures carried out.

• Quarantine and infectious control procedures. Waste from these animals should also be treated as infectious and destroyed with drainage from the isolation pens separate to other pens.
• Once the animals have recovered or moved, dispose bedding, thorough cleaning and disinfection of pens, stables etc. Cement is highly porous providing an anchor for pathogens. Floor and walls have to be scrubbed to remove pathogens. Exposure to sunlight. To ensure that the bacteria have been destroyed kitchen towel swabs of the stalls/pens/stables can be collected and tested (see diagnosis section).
• Milk from infected cows should be discarded (not fed to calves as what happens to a lot of mastitis milk) and other milk pasteurised.
• Calf handlers should always work with the youngest animals first and lastly with the sick animals.
• NB. Oral electrolyte mixes should be administered.  Valuable animals, including horses, should receive intravenous fluid therapy.
• Therapy with the correct antibiotics (Trimethoprim-sulfonamide, ampicillin, fluoroquinolones, or third-generation cephalosporins) is effective in treating bacteraemia and those animals with non-enteric infections. Antibiotic treatment of enteric infections is controversial as it may promote carriers and will harm normal intestinal antimicrobial flora. Horses suffering from salmonellosis and endotoxaemia are often treated with sub-therapeutic concentrations of polymixin B. The reason for this being that polymixin B binds Lipid A = antiendotoxic.
• Alternative therapies: probiobics (harmless intestinal microflora), probiotics (fatty acids and other nutrients that promote the growth of normal intestinal microflora) and specific bacteriophage therapy. Creep-feed management for recently weaned piglets.
• In the case of Salmonella, recovered animals usually become long-term carriers of host-adapted strains. Many of the infected calves become “poor-doers” and should never be allowed to mingle with the younger calves.

Management of susceptible animals 

• Very young mammals can be protected by the feeding of colostrum within 6 hours of birth.
• Keep all young animals away from possible infected animals and their waste and limit access of non-essential personnel. Make sure they are in clean pens.
• During an outbreak, avoid cross-fostering of piglets.
• Good pen and worker hygiene on intensive farms. An all-in-all-out system with thorough cleaning of pens between pigs groups will reduce disease incidence.
• Wash sow before she enters the farrowing pen and ensure that the pen is cleaned and disinfected.
• Young animals fed milk replacers that are correctly formulated, fresh and not over-fed.
• Pig E. coli vaccines: Vaccination of the mother with a bacterin vaccine containing the infecting serovars will reduce the prevalence and severity of disease but not the agent.

Antibiotic resistance in the enterobacteriales

Being enteric bacteria that live in a diverse community, the Enterobacteriales are able to transfer genetic material, mainly by conjugation and transduction, not only within their own species but also between other members of the family. This means that they can acquire virulence and antibiotic resistance genes that are located in plasmids and transposons.

Certain strains of zoonotic bacteria such as Salmonella Typhimurium DT104 have acquired antibiotic resistance in their R-plasmid (Integron 1) to ampicillin, streptomycin, kanamycin, chloramphenicol, tetracycline, and sulphonamides, all of which are commonly used to treat salmonellosis in people and animals. The clinical outcome of patients with these infections is poor.

Extended-spectrum beta-lactamases (ESBL) are common. This means that patients infected with resistant bacteria cannot be treated with most of the beta-lactam drugs (penicillins & cephalosporins) and for some ESBLS it extends to the carbapenems. ESBLs can be screened for by testing for susceptibility to the 3^rd generation cephalosporins; ceftazidime and cefopodoxime. There are a large number of genes coding for different types of beta-lactamase resistance. Carbapenems (impenem and meropenem) are not registered for animal use and only used when multidrug resistant isolates have been obtained from a dogs or cat. However, there have been rare reports of carbapenemase resistant strains of E. coli and K. pneumoniae in animals. This is of major public health concern. The source of these bacteria are rarely if ever, determined.

For human patients infected with ESBL- and multi-drug resistant Enterobacteriales, colistin has become the last resort. However, colistin-resistant strains of E. coli have emerged in human patients. In Europe, pigs were accused as being the source of this resistance as oral colistin is used to treat colibacillosis in piglets. Colistin is not used for animals in Australia. However, the related antibiotic polymixin B is used to treat endotoxaemia in horses and ear infections caused by Pseudomonas aeruginosa in dogs.

Multi-drug or pan-drug resistance is more common in Enterobacteriales infections of companion animals. The main reason being that antimicrobial use in food-animals is legally restricted whereas infections in pets are often treated with antibiotics.

END OF CHAPTER