14 Listeriosis

Listeriosis occurs in a variety of animals including humans, and most often affects the pregnant uterus, the central nervous system (CNS) or the bloodstream. During pregnancy, infection spreads to the foetus, which will either be born severely ill or die in-utero. In non-pregnant animals, listeriosis usually presents as meningitis, encephalitis or septicaemia. In humans, infection most often occurs in the immunocompromised and elderly, and to a lesser extent the pregnant woman, the unborn, or the newly delivered infant. Infection can be treated successfully with antibiotics, however 20–40% of human cases are fatal.

In domestic animals (especially in sheep and goats) listeriosis usually presents as encephalitis, abortion, or septicaemia.

The genus Listeria comprises seven species of Gram-positive bacteria. Almost all cases of listeriosis are due to Listeria monocytogenes although up to 10% of cases in sheep are due to Listeria ivanovii.

Listeriae are ubiquitous in the environment worldwide, especially in sites with decaying organic vegetable material. Many animals carry the organism in the faeces without serious infection. The consumption of contaminated food or feed is the principal route of transmission for both humans and animals, however other means of transmission occur.

Human listeriosis is rare (<1 to >10 cases per million people in North America and Western Europe), but because of the high mortality rate, it is amongst the most important causes of death from food-borne infections in industrialized countries. In the UK, human listeriosis is the biggest single cause of death from a preventable food-borne disease. Listeriosis in domestic animals is a cause of considerable economic loss. Control measures should be directed towards both to exclude Listeria from food or feed as well as inhibiting its multiplication and survival. Silage which is spoiled or mouldy should not be used, and care should be taken to maintain anaerobic conditions for as long as possible.

Dietary advice is available for disease prevention, particularly targeted at ‘at risk’ individuals to modify their diet to avoid eating specific foods such as soft cheese and pâté.

The bacterium Listeria monocytogenes and the disease listeriosis were first recognized in 1924 as a spontaneous outbreak of infection amongst laboratory rabbits and guinea-pigs in Cambridge (England) by Murray and colleagues. The species name ‘monocytogenes’ was derived from the marked mononuclear leucocytosis shown by these animals. The same bacterium was isolated from infected gerbils in South Africa by Pirie in 1926, and was named Listerella after the surgeon and pioneer of antisepsis Lord Lister. The generic name was changed to Listeria in 1940 for taxonomic reasons. Gill in New Zealand is credited with the first isolation of L.monocytogenes from an infected domestic animal and described a type of ovine encephalitis ‘circling disease’.

L.monocytogenes was isolated from the blood of humans with a mononucleosis-like infection (a rare manifestation of the disease) in Denmark in 1929. In the USA listeriosis was established as a cause of infection during the perinatal period and also as meningitis in adults. Prior to 1926 there are descriptions of disease likely to have been listeriosis, indeed a ‘diphtheroid’ isolated from the cerebrospinal fluid of a soldier in Paris in 1919 was later identified as L.monocytogenes.

During the 1980s the rise in the numbers of both human and animal listeriosis in several countries (including the UK) together with the a series of human food-borne outbreaks in North America and Europe lead to much professional interest in this disease as well as public alarm. The number of cases reduced during the 1990s but have increased again after 2000 in European countries and in North America.

Listeria are coccobacilli or rod-shaped, non-sporing, Gram-positive bacteria with a DNA G+C content of 36–42 mol%. The cell wall is typical for a Gram-positive organism, and contains alanine and glutamic acid cross-linked by meso-diaminopimelic acid: teichoic acids are present. The fatty acids anteiso C_17:0 and anteiso C_15:0 predominate. The metabolism is aerobic and microaerophilic, and both cytochromes and menaquinones are present. Almost all cultures are catalase positive, oxidase negative and motile below 35°C by means of peritrichate flagella. Cultures growing on carbohydrates are weakly fermentative, and lactate but no gas is produced from glucose.

DNA/DNA homology studies indicate that this genus comprises 6 species:

Two further species, L.marthii, and L.rocourtiae were described in 2009. The different species of Listeria can be readily identified by means of a small number of phenotypic or genotypic characters (Table 14.1).

Genotypic data confirms the close relationship between the different species of Listeria, and shows the closet relationship between organisms of the genus Brochothrix which are included in a single family, the listeriaceae, which has a more distant relationship to members of other low G+C Gram-positive genera (i.e. Bacillus, Enterococcus, Lactobacillus, Lactococcus, and Streptococcus).

Almost all cases of human listeriosis are due to L.monocytogenes, although rarely L.seeligeri and L.ivanovii have been implicated. A similar pattern is seen in animals, except up to 10% of cases are due to L.ivanovii. Under experimental conditions using both mice or mammalian tissue culture growing in vitro, only L.monocytogenes and L.ivanovii are pathogenic, and these species share many factors identified with virulence.

The chromosome of L.monocytogenes has been estimated to be about 3,000 Kb in size and a physical and genetic map were established prior to sequencing of the genomes of several different isolates. Complete genomes are available for multiple L.monocytogenes strains together with L.innocua, L.welshimeri, L.grayi and Listeria phages.

Transposons Tn1545, Tn916 and Tn917 (and their derivatives) have been introduced and expressed in L.monocytogenes and have proved extremely useful tools in the understanding of the virulence of this bacterium. A transposon very similar to Tn917 (designated Tn5422) has been recognized in plasmids recovered from L.monocytogenes which also encode resistance to cadmium as well as some antimicrobial agents. Transfer of native listerial plasmids has been demonstrated between stains of L.monocytogenes, between Listeria species, and to other species of bacteria including Bacillus subtilis, Enterococcus faecalis, Streptococcus agalactiae, and Staphylococcus aureus.

Lysogenic phage are commonly carried by Listeria and are generally morphologically similar with isometric heads, long non-contractile tails and correspond to the Myoviridae or Styloviridae families. The phage genomes are linear double stranded DNA of 35–42 Kb in size. Lytic properties of sets of phages have been used for subtyping L.monocytogenes.

As well as the use of transposon mutagenesis, plasmid complementation experiments together with the behaviour of L.monocytogenes either in mammalian tissue culture or in experimentally infected mouse models has lead to a great increase in the understanding of the genes involved with the virulence of this organism. Techniques to generate in-frame point mutations by allelic exchange, introduce genetic material by electroporation, and express L.monocytogenes genes in L.innocua and Bacillus subtilis have now also been applied with much success to the analysis of this bacterium (see later sections).

L.monocytogenes is widely distributed in the environment, and has been isolated from numerous sites including soil, sewage, water and decaying plant material (especially poorly fermented silage): its viability is remarkable with survival in soil or silage for >2 years. It is found in excreta from apparently healthy animals (including humans) although carriage in the gut is likely to be transitory.

Many types of raw, processed, cooked and ready to eat foods contain L.monocytogenes, albeit usually at low levels (<10 organisms/g). The properties of the organism favour food as an agent in transmission of listeriosis. L.monocytogenes grows in a wide range of foods having relatively high water activities (A_w >0.95) and over a wide range of temperatures (<0°C to 45°C). Growth occurs at refrigeration temperatures, although this is relatively slow with a maximum doubling time of about 1–2 days at 4°C. Multiplication in food is somewhat restricted to the pH range 5 to 9, and L.monocytogenes are not sufficiently heat resistant to survive mild heating including pasteurization. The tolerance of the bacterium to sodium chloride and sodium nitrite, and the ability to multiply (albeit slowly) in foods at refrigeration temperatures makes L.monocytogenes of particular concern as a post processing contaminant in refrigerated foods. Even when present at high levels in foods, spoilage or taints are not generally produced. The widespread distribution of L.monocytogenes and the ability to survive on dry and moist surfaces favours post-processing contamination of foods from both raw product and factory sites.

Prior to the mid 1980s, ‘cold enrichment’ utilizing the ability of Listeria to outgrow competing organisms at refrigeration temperatures in non-selective broths was principally used for selective isolation. However, because of the lack of specificity of this method and its slowness (some workers incubated broths for up to 6 months), procedures have been much improved.

Media have been developed which rely on a number of selective agents, including: acriflavin, lithium chloride, colistin, ceftazidime, cefotetan, fosfomycin, moxolactam, nalidixic acid, cycloheximide, and polymyxin. Furthermore, chromogenic agar based on phospholipase and glucosidase activity are now available which allows differentiate of L.monocytogenes colonies from other Listeria species on solid selective agar. Methods for identification of Listeria species are also now widely available including PCR based techniques for detection in liquid enrichment broths. These techniques have resulted in the widespread ability of microbiology laboratories (especially those involved with the examination of foods) to detect, selectively isolate and enumerate L.monocytogenes as well as other Listeria species.

Listeriosis is an opportunistic infection which most often affects those with severe underlying illness, the elderly, pregnant women and both unborn and newly delivered infants. However, patients without these risk factors can also become infected. Individuals at greatest risk of contracting listeriosis include those with malignant neoplasms, or who are undergoing immunosuppressive therapy. Other predisposing conditions include those with: agents to reduce stomach acid, AIDS, alcoholism, alcoholic liver disease, diabetes, and recipients of prosthetic heart valves or articulation joints. Patients >60 years of age with concurrent pathologies are now the most common group affected in England and Wales (Fig. 14.1).

Listeriosis most often affects the pregnant uterus, the central nervous system or the blood stream. The recent increase in human listeriosis in England and Wales has been almost exclusive to those >60 years of age with bacteraemia which now comprises the most common presentation (Fig. 14.1). The rate of listeriosis in the >60 age group has more than tripled between 1990 and 2009 in England and Wales. In non-pregnant individuals, listeriosis most frequently presents septicaemia without involvement of the central nervous system, or, to a lesser extent, as meningitis (with or without septicaemia). The former is generally confined to immunocompromised individuals and rarely has identifiable foci of infection. Listeric meningoencephalitis and encephalitis occur less commonly.

In the pregnant woman, listeriosis is most often recognized with one or more self-limiting influenza-like episodes during or after the latter half of the second trimester, although infection can occur throughout gestation. Maternal listeriosis usually presents with pyrexia and other non-specific symptoms, although some individuals may be asymptomatic. Maternal listeric meningitis during pregnancy is very rare. During pregnancy, infection spreads from the maternal circulatory system to the foetus, probably via the placenta, although this is not inevitable. Foetal infection developing before the third trimester usually results in intra-uterine death. The foetus has severe and overwhelming multisystem infection involving internal organs, with the widespread formation of granulomatous lesions, especially in the liver and placenta, and is named granulomatosis infantiseptica. Infection of the infant during the third trimester results in either intra-uterine death, or the delivery of a severely ill neonate (early onset infection).

Early-onset sepsis is characterized by non-specific signs of infection and prematurity. Cutaneous lesions may be present (sometimes with granulomas) and the neonate may have convulsions. Most early-onset cases are septicaemic, some with meningitis, however some infants appear infected only at superficial sites. The degree of severity may be partially dependant on the gestational age at infection. Surviving infants can exhibit long term sequelae, especially those delivered prematurely or with involvement of the central nervous system.

Late-onset neonatal sepsis typically occurs after uncomplicated full term pregnancies, and usually presents as meningitis about 10 days after delivery. L.monocytogenes is acquired either from maternal sites during or shortly after delivery (possibly during passage through the birth canal) or from the post-natal environment, including from direct or indirect contact with an early onset case of neonatal listeriosis.

Focal infections caused by L.monocytogenes are relatively rare, and are primarily confined to immunocompromised individuals. Deep seated infections with or without abscess formation occurs in a wide variety of sites. Listeric endocarditis also occurs, and is usually confined to patients with underlying cardiac lesions or with prosthetic heart valves. Diarrhoeal disease has also been described, although this does not appear in all cases and may be specific to some L.monocytogenes strains.

Non-systemic cutaneous and ocular listeriosis resulting from contact with infected animals or animal material has been described, the most common being lesions on the hand and arm after attending a bovine abortion. These superficial lesions may develop to serious systemic infection.

Since L.monocytogenes is common in the environment individuals must be exposed to the organism through food alone on a frequent basis: it is possible that the majority of incidents of listeriosis are subclinical. This is also supported by the relatively mild forms of the disease presenting as cutaneous infections or as bacteraemia in the pregnant woman.

During infection of the foetus, the pregnant woman often exhibits a series of pyrexial influenza-like episodes resulting from the same strain of L.monocytogenes invading the maternal blood stream. However in both humans and animals it is very rare for the pregnant individual with an infected foetus to develop serious infection (such as meningitis or encephalitis) despite invasion of the blood stream.

Since the clinical symptoms of human listeriosis are not sufficiently characteristic to establish a diagnosis, it is necessary to isolate and identify L.monocytogenes from the patient’s body sites. Special culture media and procedures are not usually required when examining samples from normally sterile sites.

Diagnosis of septicaemia or meningitis is made by culturing blood or cerebrospinal fluid. L.monocytogenes is sometimes seen in stained cerebrospinal fluid smears collected from patients with meningitis together with a moderate leucocyte reaction (usually lymphocytic), elevated protein levels and depressed sugar concentrations. Despite the species name ‘monocytogenes’, monocytosis during septicaemia or meningitis is rarely observed.

During pregnancy, L.monocytogenes can be cultured from maternal blood, especially when collected during febrile episodes. Before delivery, an abnormal visual appearance of the amniotic fluid may give an early suggestion of listeriosis, especially when discoloured by meconium. At birth L.monocytogenes is present in high numbers both on multiple sites of the infected infant and in the maternal genital tract, and can be seen in stained smears. This bacterium can be readily cultured from: the placenta and lochia, the infant’s blood, cerebrospinal fluid, respiratory and gastric tract, skin and other surface sites, and the maternal high vagina. Amniotic fluid and meconium should be cultured when available.

Following intra-uterine death and expulsion of the foetus, necropsy should be carried out. Material taken from the infant’s liver, spleen, brain and other internal organs should be examined for L.monocytogenes. Culturing maternal high vaginal swabs may also be useful in retrospective diagnosis of intra- uterine infection within several weeks after delivery.

Methods to detect specific nucleic acid sequences by PCR have been described. Procedures to detect specific antibody have also been described, however interpretation is problematic and these have not led to useful diagnostic tests.

Histological changes in human listeriosis are similar to that observed in animals. Infection causes necrosis followed by proliferative activity of cells in the reticuloendothelial system resulting in miliary granuloma formation and focal necrosis with supporation of the affected tissues. The bacterium is often present in the necrotic foci: the numbers and extent of the lesions varies with the sites infected as well as between patients.

Meningitis is characterized by supporative inflammation of the meninges with granuloma and necrosis of cerebral tissue. A thick purulent exudate may be found in the subarachnoid space. During encephalitis, gross lesions may be absent, or present in the pons and medulla oblongata. In cases of encephalomyelitis, submiliary to miliary nodules are often found in the leptomeninges.

In the newborn, the disease is characterized by a massive involvement of the liver with disseminated lesions in numerous other organs including the spleen, adrenal glands, lungs, oesophagus, posterior pharyngeal wall, and the tonsils. Cutaneous foci are often seen, especially on the back and lumbar region.

In the placenta, multiple white or grey necrotic areas occur within the villous parenchyma and decidua, the largest usually in the basal villi and decidua basalis. This gross appearance may allow a presumptive diagnosis of listeriosis. The necrotic foci are identical to those in the foetal organs. Gram-positive rods are usually seen within the necrotic centres of the villous and decidual abscesses, as well as within the membranes, umbilical cord, and surface of the foetus. The necrotic foci typically contain collections of polymorphonuclear leucocytes and are found between the trophoblast and stroma. Inflamed or necrotic chorionic villi are enmeshed in intervillous inflammatory material and fibrin.

In vitro studies on the activities of various antimicrobial agents show that L.monocytogenes is almost always uniformly sensitive to ampicillin, penicillin, and erythromycin, although up to 10% may be resistant to tetracycline. L.monocytogenes is uniformly highly resistant to cephalosporins, and fluoroquinolones have insufficient activity to be recommended for the treatment of listeriosis.

There is general agreement that a combination of ampicillin or penicillin plus an aminoglycoside is superior to using either drug alone. For central nervous system listeriosis in adults, treatment with intravenous ampicillin plus an aminoglycoside for 3 to 4 weeks is recommended. During pregnancy, 2 weeks treatment with ampicillin plus an aminoglycoside should be given intravenously if amnionitis is present, or amoxicillin if not present. In cases of serious allergy to ampicillin, trimethoprime plus sulfamethoxazole may be given intravenously.

Neonatal listeriosis should be treated with intravenous ampicillin plus an aminoglycoside. Two weeks of treatment is recommended, but a longer course should be considered if a diagnosis of meningitis has been made. With late onset neonatal listeriosis, meningitis is commonly present and ampicillin plus gentamicin is recommended. The length of treatment is variable; if prompt clinical improvement occurs and the bacterium is absent from the CSF, 2 weeks treatment may be adequate. However 4 to 6 weeks should generally be considered.

Foetal infection and death during early gestation is a recognized complication of maternal infection, but this is not inevitable. Once infection of the pregnant uterus has occurred, successful pre-partum treatment of the mother with antimicrobial agents has resulted in the birth of apparently healthy infants. In late gestation, death of the foetus may occur, although it is more common to result in the birth of a severely ill infant. Early-onset neonatal listeriosis has a high fatality (>35%), and in those that survive, sequelae such as neurodevelopmental handicaps, hydrocephalus, ptosis, and strabismus may occur. Late-onset neonatal listeriosis also has a high mortality rate, although generally not as high as early-onset infection. The long-term prognosis for infants after late onset sepsis or meningitis has not been well studied.

Mortality rates for adult and juvenile listeriosis varies from 10 to >50%, with poor prognostic indicators including: age (>50 years), pre-existing disease, early convulsions (in cases of meningitis), and the needs for cardiovascular renal or ventilatory support. Residual disabilities may occur. Relapses of infection, some greater than two years after the original episodes, have been described.

Amongst domestic animals, sheep and goats are most susceptible, although infection also takes place in cattle (Low and Donachie 1997). Infection has been recognized in >40 other species of feral and domesticated animals, although this account will principally deal with sheep and cattle.

Listeriosis presents as a wide range of disorders which parallel much of what has already been outlined for humans, although there are some differences. L.monocytogenes is the major pathogen, however some of the cases of abortion or septicaemia in sheep (but less commonly in cattle) are accounted for by L.ivanovii.

There are six main manifestations of the disease: abortion, septicaemia, encephalitis, diarrhoea, mastitis, and ocular infections. However the disease varies depending on the species involved. Listeriosis in primates manifests similarly to that in humans.

In sheep, goats and cattle, abortion is recognized late in pregnancy and is rarely accompanied by severe systemic disease in the dam. Aborting animals may excrete the organism in the milk without evidence of mastitis. Septicaemia in young animals occurs in the first few weeks of life: some with diarrhoea, but there is no specific symptomology. Diarrhoea and septicaemia also occur in older animals (principally ewes).

Unlike human listeriosis, the most common form of listeriosis in animals is as an encephalitis. In ruminants, this takes the form of a unilateral (or less commonly bilateral) cranial nerve paralysis effecting the eye, eyelid, ear, and lips (with consequent dropping of cud), which is often followed by ataxia, and moving in circles: hence the name circling disease. The affected animals are dull with the head standing to one side sometimes with food or cud hanging from the mouth and, because of the partial paralysis of the pharynx, saliva is often drooled. Animals sometimes stand still pressing against a fixed object, or appear recumbent. The course of the disease in bovines is quite prolonged (4–14 days), but is much more acute in sheep where death can occur within 4 to 48 hours. In sheep, the disease can resemble pregnancy toxaemia (ketosis). Encephalitis occurs in older animals (in sheep most often during late pregnancy or soon after lambing) as well as in the young.

Abortion, septicaemia and encephalitis are usually sporadic in cattle, but can occur as outbreaks amongst flocks of sheep where losses may be heavy. During outbreaks, septicaemia and abortion may occur together with cases of encephalitis, but is unusual. Experimental infection indicates the septicaemia can develop in a few days after consumption of contaminated feed, but the incubation period for encephalitis is likely to be much longer (20–30 days).

L.monocytogenes causes mastitis in cows and sheep, where large numbers of the bacterium can be shed into milk. All four quarters or only one may be affected, and the disease severity can vary markedly, sometimes appearing subclinical. Excretion of the organism into milk can persist for >3 years.

Keratoconjunctivits together with iritis occurs in both sheep and cattle. These conditions are usually unilateral. In cases of conjunctivitis, other bacterial or viral pathogens may also be present on the conjunctiva.

Listeric abortion, septicaemia and encephalitis has been recognized in pigs, horses, dogs and cats, but this is rare.

Listeriosis occurs in rodents and >20 species of birds, although is probably rare. Infection is most often recognized in those birds more commonly farmed, i.e. chickens, turkeys and ducks. Septicaemia and myocardial necrosis are the most common manifestation, and these have been suggested to be often secondary to other infections.

Although less commonly associated with predisposing factors (as described for humans) listeriosis in animals shows characteristics of an opportunistic pathogen. The unborn and newly delivered are more susceptible to infection, and encephalitis occurs most often in the adult pregnant animal during the later stages of gestation or shortly after delivery. Outbreaks have been associated with climatic stress (sudden drops in temperature, snow falls, drought, and shortage of food), and cases most often occur in the spring when animals may be in a poor condition. Increases in susceptibility of animals to experimental infection have been demonstrated by malnutrition, immunosuppression, viral infection, and other uncharacterized stress factors.

As with humans, the majority of animal listeriosis cases are assumed to be acquired via contaminated feed (a particular risk factor is the consumption of poor quality silage).

Diagnosis of abortion is usually achieved by culturing foetal organs (liver or spleen) or stomach contents. The culturing of the organism from placenta may be difficult to interpret in the absence of histological analysis where necrosis and abscess formation will be observed. The placental lesions are pin-point, yellowish necrotic foci involving the tips of the cotyledonary villi, with a focal to diffuse intercotyledonary placentitis covered in a red/brown exudate. The foetus is usually autolytic with miliary necrotic foci scattered throughout the liver and spleen, although these are not always present. Septicaemia is often accompanied by focal hepatic necrosis (sawdust liver).

Anti-mortem diagnosis of listeric encephalitis is problematic since there are no satisfactory diagnostic tests, and listeric encephalitis may mimic other diseases. Prior to death, L.monocytogenes may be cultured from the brain, but in some cases is absent: the organism is invariably only isolated from the brain and not from other organs. Necropsy samples show a diagnostic pattern of histological changes and this has proved to be the only definitive means of diagnosis. It is essential that histological examination is performed to exclude the possibility of other diseases causing the condition. The typical listeric parenchimal lesions are confined to the brain stem and medulla and are composed of microabscesses which begin with the collection of neutrophils or microglial cells. Gross pathological lesions are rarely observed. The glial nodules often persist and become infiltrated by macrophages. Adjacent to the lesions is heavy perivascular cuffing composed mainly of lymphocytes and histocytes in addition to occasional neutrophils and eosinophils. Bacteria, either singly or in small clumps are sometimes seen near the periphery of the lesions, but not in the perivascular cuffs. Meningitis (affecting the cerebellum and anterior cervical cord) probably developing secondarily to the parenchimal lesions and neuritis of the trigeminal nerve may also be present. A correlation between the degree of cell mediated immunity and brain lesion suggests that immunopathological reactions are important components of this condition. L.monocytogenes has been isolated from brains of apparently healthy sheep, but it is not clear how commonly this occurs.

L.monocytogenes is probably rare as a cause of mastitis, but should be considered during cultural procedures as part of the investigation of this condition. The condition presents with abnormal milk secretion and swelling of the affected quarter(s).

A marked monocytosis is commonly observed in infected rodents, together with focal hepatic necrosis from which the organism can be readily cultured. A diffuse myocardial necrosis is often observed in guinea pigs.

Listeriosis in birds is characterized by conspicuous lesions of myocardial degeneration and necrosis, with necrotic foci found in the liver, spleen, and lungs from which the organism can be readily cultured. Involvement of the central nervous system also sometimes occurs.

Because of the disease severity and rapid onset of clinical symptoms, treatment of infected sheep or cattle is rarely attempted: infected animal may be destroyed on humanitarian grounds. During outbreaks, mortality rates are often 100%, and those surviving can exhibit permanent central nervous system disorders.

As is found with humans, the pregnant dam with an intra-uterine infection is rarely accompanied by severe systemic disease so it is not necessary to attempt treatment. A listeric abortion does not seem to effect the possibility of subsequent conceptions.

The response to antibiotic treatment in cows with listeric mastitis has been poor and the organism can be excreted for extended periods of time. Hence it is recommended that such animals should not be used for milk production and culling should be considered.

The production of an experimental keratoconjunctivitis (Anton’s eye test) performed in either guinea-pigs or rabbits by instilling a live bacterial suspension into the conjunctiva has been used to demonstrate the virulence of L.monocytogenes for the past 60 years. Mice and rabbits are now more frequently used and they suffer an acute fatal infection one to seven days after a sufficient dose of a virulent strain (usually >10⁴ bacteria) is given either intravenously or intraperitoneally. Virulence can be measured by LD₅₀, by the kinetics of growth of the bacterium in tissues, or by the extent of survival in the liver and spleen. Intraperitoneal carrageenin or mineral oil may be given prior to inoculation to increase susceptibility to infection.

Ovine encephalitis accompanied by the characteristic histological features has been experimentally achieved by inoculating the organism into the dental pulp. Histological encephalitis was evident after 6 days, but the onset of clinical neurological disease varied between 20 to 40 days.

Oral inoculation of mice, rats and guinea pigs have been reported. Infection showed most consistency in gnotobiotic animals and interference of colonization by the microflora of the gastrointestinal tract has been suggested. Differences have been reported between both strains of L.monocytogenes, and growth condition used for the bacteria prior to inoculation which were not apparent when using the intravenous or intraperitoneal route. Oesophageal inoculation of 10⁶  L.monocytogenes to juvenile rats showed about a 50% infection rate in the liver or spleen. A reduction in the acidity of the stomach by cimetidine treatment reduced the infective dose.

Feeding trials in cynomologous monkeys have been reported, and only those animals receiving 10⁹ cells showed fever, septicaemia, loss of appetite, irritability, and occasional diarrhoea. Those animals fed ≥10⁷ bacteria (some of which were completely asymptomatic) shed the organism in the faeces for up to 21 days.

In chick embryos injected by the intra-allantoic route, the LD₅₀ is around 10² organisms. Lesions occur in the chorio-allantoic membrane, liver, and heart, and the bacterium can be readily cultured from these sites.

Chronic mastitis can be induced experimentally in cows by intramammary injection. Bacteraemia could not be detected in these animals and typically 10³ to 10⁴  L.monocytogenes/ml was shed into milk for 9 to 12 months of the remaining lactation period.

L.monocytogenes is able to infect a range of mammalian cell types growing in vitro, including enterocytes, macrophages, and fibroblasts. The use of such models has contributed much to an understanding of the factors involved in intracellular invasion and growth (see later description).

It is a characteristic of the natural disease in both humans and animals in that there is usually a low attack rate. The susceptibility to infection may be increased by external factors, some of which have already been mentioned. However other factors (such as other infectious agents or products of the metabolism of other microorganisms) may yet prove to be of importance. L.monocytogenes is a somewhat marginal pathogen. Hence experimental models reflecting the natural infection may work poorly, and relatively large numbers of animals may be needed for a small proportion of these to produce clinical symptoms of disease.

There is evidence supporting the role of antacid therapy in increasing susceptibility of some patients, and in experimental animal infection. The buffering capacity of some food types may also be of importance in facilitating the survival of the organism, which may then invade at sites further along the gastrointestinal tract, although other routes of infection may occur. Experimental septicaemia in animals can be achieved via the respiratory route, and further evidence supporting this possibility comes from one of the cases (septicaemia and aspiration pneumonia) which developed after eating contaminated coleslaw salad in the 1981 Canadian outbreak.

Histopathological analysis suggests that the intestinal tract can act as the site of invasion and the M cells overlying the Peyer’s patches may act as the site of penetration. Although the observation that L.monocytogenes (as well as L.ivanovii) can readily invade various epithelial and fibroblast cell types growing in vitro suggests that there may be multiple routes by which this bacterium initially invades the hosts’ cells. In the caecum and colon of animals following oral inoculation, the bacteria can be observed together with an inflammatory reaction in phagocytic cells present in the underlying lamina propria. Following this phase, invasion of the uterine contents or central nervous system (for patients with shorter incubation periods), may occur probably via the circulatory system.

In the liver, the organism is cleared from the blood by the phagocytic Kupffer cells. In their non-activated state, some bacteria will survive, escape to the cell cytoplasm, and subsequently spread to hepatocytes using the process described in the following section. Formation of localized lesions occur in the liver and also in the spleen.

Intrauterine infection of the foetus results from haematogenous spread from the mother. Abscess formation takes place in the placenta, and this may spread via the umbilical vein or the amniotic fluid to the foetal internal organs. The series of pyrexial episodes observed in the mother may result from re-invasion of maternal blood stream from placental sites. L.monocytogenes is unusual in that it is able to survive and grow in amniotic fluid, and aspiration of this leads to the pathological changes in the foetal respiratory tracts. The presence of high numbers of the organism in amniotic fluid results in widespread contamination of neonatal and maternal surface sites at delivery as well as the postnatal environment and may result in cases of neonatal cross-infection.

Experimental and field studies suggest that encephalitis in sheep and cattle results from L.monocytogenes reaching the base of the brain along cranial nerves, particularly the trigenimal nerve. It is assumed that animals eat contaminated feed, particularly silage, and the organism enters the nerves after penetrating the oral mucus membrane or through pre-existing areas of trauma such as tooth root scars (which are prominent in sheep during the spring). The mechanism for travel along nerves is not well understood

Listeria genes involved with invasion and intracellular movement in mammalian cells are shown in Fig. 14.2. The cell surface listerial protein named internalin is involved with initial stages of invasion and binds to mammalian cell surface proteins including E-cadherin. Subsequently L.monocytogenes becomes encapsulated in a membrane bound compartment which dissolves by the action of a thiol activated haemolysin (hly gene). L.monocytogenes enters the host cell cytoplasm where it multiplies and becomes surrounded by polymerized host cell actin, which becomes preferentially polymerized at the older pole of the bacterium by the ActA cell surface protein. The actin polymerization confers intracellular motility to the bacterium which allows invasion of an adjacent mammalian cell. The bacterium is then encapsulated in a double membrane bound compartment which is dissolved by the action of a lecithinase which is activated by a metalloprotease (the haemolysin and a phospholipase may also contribute in this process) and the whole process is repeated. The genes are all regulated by the positive regulation factor (prfA gene) and are located in two operons located quite closely together on the bacterial chromosome.

It is of note that all the above mentioned genes (or a very similar set) are present in L.ivanovii, which follows the same general pattern of cellular invasion and movement. Unlike L.monocytogenes, L.ivanovii does not cause plaque formation in fibroblasts growing in vitro, and it has been suggested that the lower virulence of the latter species may be related to lack of as yet uncharacterized other factors. There is evidence supporting the presence of these genes in L.seeligeri, although it is only weakly invasive to mammalian cells. It is not clear if L.seeligeri too lacks additional virulence factors or if these genes are poorly functional. An alternative explanation may be that L.seeligeri is adapted to survive in quite different eukaryotic environments.

Regulation of the genes involved with the virulence of L.monocytogenes is under the control of the positive regulation factor (prfA) gene product, and other promoters have been identified which interact with this protein (Fig. 14.3), including a promoter for its own production. Listeriolysin is one of the major extracellular proteins produced by L.monocytogenes under conditions of heat shock, and there is evidence to suggest that some of the above mentioned genes are also regulated by the stage of the cell cycle and temperature. The disaccharide cellobiose represses the expression of the listeriolysin and phosholipase genes by an as yet uncharacterized mechanism. It is tempting to speculate that the absence of this environmentally ubiquitous plant-derived molecule allows the induction of genes as a pathogenic response to the environment of eukaryotic cells.

L.monocytogenes is an intracellular parasite, and it is in this environment that the pathogen gains protection and evades some of the hosts defences. However the host has a number of strategies to deal with such parasites and is beyond the scope of this chapter.

The widespread distribution of L.monocytogenes provides numerous potential ways in which the disease may be transmitted to both animals and humans. Although there has been much current interest in infection via the oral route, this is not the only mode of transmission (Fig. 14.2). The peak in the incidence of human listeriosis most often occurs in the end of the summer and early in the autumn. The reasons for this are not understood. Animal listeriosis principally occurs in the Spring. This is probably not only because of the physiology and condition of the animals, but also because of the provision of poorly prepared feed stuffs (see later notes).

The reported incidence of human listeriosis varies between countries from <1 to >10 cases per million of the total population. Although these in part may reflect differences in surveillance systems, they probably represent true differences in incidence. There are similar differences in the incidence of animal listeriosis in different regions: for example in the UK, listeriosis in sheep is a particular problem in Scotland and the North of England. These differences may in part be due to the ability to produce good quality feed.

Listeriosis may be transmitted by direct contact with infected animals or animal material. In such cases the disease occurs as papular or pustular cutaneous lesions usually on the upper arms or wrists of farmers or veterinarians 1–4 days after attending bovine abortions. Since listeric infection is more common in sheep than cows, it is remarkable that such infections do not occur in association with sheep. However the duration of manipulation and extent of skin exposure is greater when dealing with bovines abortions (McLauchlin and Low 1994).

L.monocytogenes is widespread in the environment and superficial infections in humans from other sources are rare. This observation together with the likelihood that extremely high levels of the L.monocytogenes (10⁸cfu/ml) occurring in infected bovine amniotic fluid as are found during intra-uterine infection in humans, suggests that the infective dose for human cutaneous listeriosis is high.

Although cutaneous listeriosis in adults are invariably a mild infection with a successful resolution (even without antimicrobial therapy), systemic involvement is suggested in some cases by fever and tenderness of the axillary lymph nodes. Indeed brucellosis has been considered as a differential cause in some cases. Furthermore, cases have also been described of acute meningitis in farmers after assisting at bovine abortions, although in these instances the route of infection is unclear.

Conjunctivitis in poultry workers has also been reported.

Hospital cross-infection between newborn infants occurs and these show a common pattern of an infant born with congenital listeriosis (onset within 1 day of birth). In the same hospital, and within a short period of time, an apparently healthy (or more rarely more than one) neonate is born who typically develops late onset listeriosis between the 5th and 12th day after delivery. The same strain of L.monocytogenes is isolated from both infants and the mother of the early onset case, but not from mother of the late onset case. In most of the episodes, the cases are either delivered or nursed in the same or adjacent rooms, and consequently staff and equipment are common to both. Two larger series have been described occurring in Sweden and Costa Rica, where 4 and 7 cases respectively resulted from single early onset cases. The likely routes of transmission here involved a contaminated rectal thermometer and a mineral oil bath. In one episode, true person to person transmission occurred where, 3 days after delivery, the mother of an early onset case was nursed in an open ward and handled a neonate from an adjacent bed who subsequently developed late onset listeriosis.

As previously outlined, L.monocytogenes is able to infect the pregnant uterus where extremely high levels of the organism occur (10⁸/ml in amniotic fluid). At delivery, very large numbers of the organism are present on the newborn, in the maternal birth canal, and on sites, personnel and instruments contaminated during delivery in the postnatal environment. The very large numbers of organisms present during early onset neonatal infection clearly cause infection to further late onset neonatal cases.

There is little or no evidence for cross-infection or person to person transmission outside the neonatal period.

The consumption of contaminated foods is the principal route of transmission for this disease and microbiological and epidemiological evidence supports an association with many food types (dairy, meat, vegetable, fish and shellfish) in both sporadic and epidemic listeriosis (Table 14.2). Although diverse in their constituents and manufacturing processes, foods associated with transmission often show the common features of:

One food (alfalfa tablets), was quite different in that it was a dry product in which L.monocytogenes would not be able to grow. The most common food type associated with cases of listeriosis in the UK has been pre-prepared sandwiches served in hospitals.

The incubation period varies widely between individuals from 1 up to 90 days, with an average for intra-uterine infection of around 30 days. It is not known if these differences after oral ingestion are dose or strain dependent, or perhaps reflect unknown differences in host susceptibility. Based on data from a very small number of cases, high levels of L.monocytogenes (10³ to >10⁷/g) have been found in foods consumed by patients prior to infection, suggesting that the infective dose is high. However much caution is needed here since there is likely to be much variation in susceptibility between individuals. In addition, suspect foods are generally only available for examination for relatively short periods and hence will be more likely to be collected from patients showing short incubation periods. These observations, together with the ability of L.monocytogenes to multiply in foods (even under ideal storage conditions) means that there are considerable difficulties in ascribing a ‘safe’ level of L.monocytogenes in food.

As has already been stated, L.monocytogenes is widespread in the environment, including food, although it is generally present in relatively low numbers. This feature together with the properties and types of foods associated with transmission of infection (Table 14.2), also support a dose response with very much greater risk following exposure to high numbers of the bacterium through food.

Outbreaks of human listeriosis involving >100 individuals have been recognized, however most cases are probably sporadic. Outbreaks have occurred over durations of 6 months to 5 years, and this is likely to represent a long term colonization of specific sites in the food manufacturing environment as well as the long incubation periods shown by some patients. L.monocytogenes has been shown to survive well in a variety of environments where food is manufactured, particularly those that are moist with organic material, and it is from such sites that contamination of food occurs during processing. Given the properties and distribution of L.monocytogenes, cases related by a common source may be very widely distributed both temporally and geographically.

Abortion and septicaemia in newborn animals results from intra-uterine infection acquired from the dam, and parallels abortion and early onset neonatal infection in humans. Septicaemia (also with meningitis in some animals) in lambs in the first few weeks of life has been attributed to umbilical infection acquired in lambing pens, possibly from contaminated soil or feed: this corresponds to late-onset neonatal infection in humans.

Iritis and keratoconjunctivitis usually occur during the winter in silage fed sheep and cattle. These may occur by direct introduction of contaminated feed into the eye, and have been particularly associated where feed is provided in holders or racks at eye level.

As in human infection, the majority of cases of animal listeriosis are assumed to be acquired via the oral route. There is a strong association between the feeding of silage and all manifestations of listeriosis in sheep and cattle, although cases do occur where this has not been used. However, the exact mechanism in which silage feeding leads or predisposes to listeriosis is not clear. Under normal conditions it is impossible to produce silage free of Listeria: the organism has been isolated from silage with a pH of <4, albeit in very low numbers. However, where poor quality silage has been produced and a low pH and anaerobic conditions are not achieved, proliferation of Listeria takes place and very high numbers can be found. Poor quality is often also due to insufficient herbage quality, or to contamination by soil or faeces. The change to production of silage in polythene bales (‘big bale’ silage) corresponded to increases in ovine listeriosis in the UK. Although the big bale method is more economical than the traditional use of clamps, these are more prone to spoilage and growth of Listeria: high numbers of the organism are often associated with sites where the damage to the bags has occurred or at the tied end. The peak in the numbers of animal listeriosis in the spring may reflect a decrease in the quality of silage used for feed. The observation that listeriosis is often associated with poor quality silage where high numbers of L.monocytogenes occur suggests that there is a similar dose response to humans.

The possible routes of infection in both human and animal disease are represented in Fig. 14.2. For both humans and animals the majority of infection probably results from ingestion of food or feed where excessive proliferation of L.monocytogenes has occurred.

It is not clear if there are differences in virulence between strains of L.monocytogenes and L.ivanovii in the environment, and why L.ivanovii is such a very rare pathogen for humans. Although some non-pathogenic cultures of L.monocytogenes have been identified, experimental infection of animals (albeit with rather ‘unnatural’ models) have indicated limited variation in virulence. However, the distribution of ‘types’ of L.monocytogenes usually varies between cultures isolated from the environment and to those causing disease. The proportion of L.monocytogenes types is significantly different between syndromes in both humans and animals. In addition, the majority of the strains responsible for the apparently unrelated large food-borne outbreaks of human listeriosis have been unexpectedly similar. The significance of these three observations is not clear, however they may be reflecting adaptions by strains (or species) to different environmental niches which effects virulence. For public health purposes, all strains of L.monocytogenes should be regarded as potentially pathogenic and capable of causing disease.

Apart from human infection acquired as a direct result of attending infected animals or from food directly contaminated from an infected animal (as may be the case with milk from an animal with listerial mastitis), it is unclear what, if any, are the connections between human and animal listeriosis. Analysis of strains causing ‘sporadic’ human and animal listeriosis indicates a wide range of ‘types’ in both groups which do not generally appear related. The seasonal peaks in human and animal listeriosis do not coincide, also suggesting that these two groups are not causally related. This perhaps should not be unexpected of a group of marginal pathogens which are probably not host adapted and occur widely in the environment as many different types.

L.monocytogenes and L.ivanovii are members of a group of bacteria which are adapted to saprophytic environments in soil or water and have additionally adapted to survive in eukaryotic intracellular environments. Quite how these additional factors have evolved is not clear. It may be that these factors evolved for survival in mammals, or for a free living multicellular eukaryotes living in the soil.

Manufacturers of food, and food processing equipment should be aware of the properties of Listeria. The design of the factory and equipment should facilitate cleaning and sanitizing to reduce the possibility of contamination and colonization with L.monocytogenes. Producers, shippers and retailers of perishable foods must utilize good refrigeration and shelf life control, together with adequate packaging and consumer advice including ‘sell by’ and ‘consume by’ date and proper storage conditions. Application of a HACCP (hazard analysis critical control point) programme or a similar comprehensive assessment and control scheme is advised for both the safety and quality of the final product. Over the past decade, the food industry in Europe and North America has been active in investigating Listeria in foods and the factory environment, in implementing hazard analysis, and establishing codes of practice, and there is evidence to suggest that some foods have improved in microbiological quality with respect to contamination by L.monocytogenes.

L.monocytogenes can multiply (albeit slowly) at refrigeration temperatures, hence it is not only important to exclude the organism from foods, but also to inhibit its multiplication and survival. Because L.monocytogenes is a potential pathogen, and the detection of other species of Listeria can indicate environmental contamination (especially in those foods which have undergone a listericidal process), the ideal should be to exclude all Listeria from food, and this should be actively pursued. The achievement of this objective is probably impractical for all foods, and is clearly unattainable for raw foods or those which have not undergone a listericidal process. EU legislation (EC No. 2073/2005, Microbiological criteria for foodstuffs) defines different criteria for three categories of foods (25g samples) by:

Foods were defined as unable to support the growth of L.monocytogenes in this legislation by:

pH ≤4.4; a_w ≤ 0.92; pH ≤5.0 and a_w ≤ 0.94; shelf life less than 5 days

To avoid listeriosis and other food-borne infections, the general public should be educated about the use of good food and personal hygiene practices from the time the food is purchased to consumption. This includes avoiding cross-contamination between raw and cooked foods, properly maintaining refrigerators, and not over-extending the normal shelf life of foods. Since Listeria are widely distributed in the environment, total avoidance and complete elimination of L.monocytogenes from all food is not possible, and it is likely that all individuals will be exposed to Listeria at some time. However, pregnant women and immunocompromised individuals are at increased risk of contracting listeriosis. In a number of countries (including the UK, USA, France, New Zealand and parts of Australia) ‘at risk’ individuals are advised to take special precautions. Processed and ready-to-eat foods such as soft and surface-ripened cheese and pâté have been identified as being particularly hazardous, and general advice has been issued in England and Wales for vulnerable individuals not to consume these. Further general advice was issued in the USA to avoid other types of soft cheese (feta and Mexican-style), to avoid delicatessen type foods and to reheat ‘cold cut’ type meats. In the light of the recent outbreaks in Australasia, it might also be prudent to include similar advice to avoid types of cooked and ready to eat fish and seafood.

Susceptible individuals should also avoid consuming raw or inadequately cooked meat and seafood, wash fruits and vegetables to be consumed raw, and thoroughly reheat all pre-cooked and ‘leftover’ foods before consumption, especially highly processed ready-to-eat meals.

Unexplained influenza-like illness in pregnant women and the immunocompromised should be medically investigated by the culturing of blood to establish a diagnosis of listeriosis. In addition, those individuals attending infected animals should be aware of the possibility of cutaneous or ocular listeriosis, and should also seek medical attention if suspect lesions develop. It may also be prudent to advise the pregnant and immunosuppressed not to help with lambing, milking ewes that have recently given birth, touch the afterbirth, or come into contact with newborn lambs. This clearly represents a potential route of infection, although with the exception of the cutaneous and ocular lesions, significant associations have not been observed between listeriosis cases and rural backgrounds.

To prevent cross-infection during the neonatal period, strict infection control measures must be instigated at the time of delivery. Such measures include adherence to barrier nursing, use of heat sterilized or single use equipment, single use barriers and alcohol wipes for surfaces, and the wearing of gloves and aprons which are changed prior to hand-washing and attending other patients.

Because human listeriosis is a rare disease, surveillance schemes and epidemiological investigations of cases are essential to identify clusters of patients related by common source and vehicles of infection. The use of analytical studies to investigate outbreaks of listeriosis has had mixed success, and common source outbreaks may only be recognized by collection of isolates from infected patients and identification of common strains. Since L.monocytogenes is widespread in the environment (including foods), it is important that comparisons of isolates are made using discriminatory typing schemes.

To prevent listeriosis in animals attention should be paid to feeds, especially to silage, and this has already been discussed. Live attenuated vaccines have been developed for use in animals and it is claimed that this offers some protection although results of field trials are equivocal.