YERSINIA PSEUDOTUBERCULOSIS AND YERSINIA ENTEROCOLITICA

HRlSTO najdenski

The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria

The genus Yersinia, family Enterobacteriacea, comprises the three pathogenic species Ypestis, Y.

AETIOLOGY

Both Y. enterocolitica and Y pseudotuberculosis are Gram­negative coccobacilli, motile at 20°C but not at 35°C, and grow well on MacConkey agar. Biochemically they produce catalase and urease, reduce nitrates and produce acid from cellobiose, melibiose, rhamnose and sucrose under aerobic and anaerobic conditions. They are oxidase-negative.

The pathogenic potential of Yersinia is a complex inter­action between the plasmid of virulence (pYV) and specific chromosomal virulence determinants encoded by several genes. The virulence of pathogenic Yersinia is closely asso­ciated with a 64—70 kb plasmid encoding a number of Yersinia outer proteins (Yop) and proteins of the Yersinia type III secretion apparatus whose expression is regulated by the transcriptional regulator VirF. As Yop are immuno­genic in humans and animals, Yersinia infections can be detected by anti- Yop antibodies independent of serovars or biovars(²). Both pathogens are psychrophilic, being able to multiply at low temperatures (2-5°C) and in microaer- ophilic conditions. Yersinia are susceptible to many disin­fectants as well as moist and dry heat. Outside the host, Yersinia can survive for up to 20 days in water and 540 days in soil.

Epidemiology

GEOGRAPHICAL DISTRIBUTION AND HOSTS

Some aspects of the epidemiology of yersiniosis still remain obscure. Yersinia pseudotuberculosis and Y. enterocolitica are ubiquitous and carried subclinically by a range of animal species, including wild mammals, birds and rodents. Out­breaks of disease are usually sporadic, but epizootics causing high mortality have been documented in a wide variety of wild and exotic animals¹-³). Yersiniosis has been recorded in farm and domestic animals (cattle, horses, sheep, goats, pigs, rabbits, cats, dogs, guinea pigs, ham­sters, etc.), in commercially reared fur-bearers (chinchilla, mink and coypu), in wild mammals (European brown and mountain hares, rats, mice, foxes, voles, deer, marmot, beaver, hedgehogs, etc.), and in mammals in zoological gardens (e.g. monkeys, pumas, kangaroos, agoutis). Epiz­ootics have been reported in sheep, farmed wild fur-bearers and farmed deer⁽⁴⁾.

Infectious Diseases of Wild Mammals and Birds in Europe, First Edition. Edited by Dolores Gavier-Widen, J. Paul Duff, and Anna Meredith. © 2012 Blackwell Publishing Ltd. Published 2012 by Blackwell Publishing Ltd.

Disease is relatively frequent in domestic and wild birds. Epizootics have been sporadically reported in turkeys, ducks, stock doves, in aviaries of canaries and finches, and wood pigeons. Sporadic infection has been recorded in more than 50 species of bird.

Infection with Y. enterocolitica has been observed mainly in mammals. Outbreaks have been reported in hares, chin­chillas and captive monkeys, and sporadic infection in dogs, pigs, sheep, guinea pigs, rabbits and cattle⁽³⁾.

Human and animal infection with Y. pseudotuberculosis is essentially of European origin. It is assumed that the appearance of Y. pseudotuberculosis in other parts of the world is attributed to the importation of infected animals from Europe. Moreover, based on the knowledge that the acquired resistance to infection with Ypseudotuberculosis protects against plague, it is suggested that the emergence of the enzootics of Ypseudotuberculosis may have contrib­uted to the disappearance of pandemic plague, and may have curtailed plague in Europe during the third and last pandemic⁽⁵⁾.

Yersinia enterocolitica has a wider geographical distribu­tion and is found not only in Europe, but also on other continents. The highest concentration of human and animal cases has been found in France, Belgium, the UK, Sweden and Finland. In France, 300 isolates of Y. entero­colitica and related Yersinia species were isolated by testing 1307 specimens from a terrestrial ecosystem⁽⁶⁾. In another study in France on regional prevalence and intestinal dis­tribution, Y. enterocolitica was isolated from 5.7% of 3533 small wild mammals⁽⁷⁾. In Norway, Sweden and Finland 24 isolates of Y. enterocolitica were recovered from 551 small wild rodents⁽⁸⁾. In Sweden, Y enterocolitica was iso­lated in 5.6% and Y. pseudotuberculosis in 0.6% of 468 faecal samples from 57 different species of migratory bird⁽⁹⁾. In this study Y pseudotuberculosis was detected in song thrush (Turdus philomelos) and redwing (Turdus iliacus), and Y. enterocolitica in barnacle goose (Branta leucopsis) (in highest frequency), redshank ( Tringa totanus), dunlin (Calidris alpina), redstart (Phoenicurus phoenicu- rus), blackcap ( Sylvia atricapilla) and rough-legged buzzard (Buteo lagopus). Of the 29 Y. enterocolitica isolates, 10 belonged to bioserotype 3/O:3 (more often associated with human disease), three belonged to 1A/O:5, and one to 1A/O:3. In Finland, a large population of deer and hares on Ahvenanmaa Island were presumable subclinical carri­ers and reservoirs of Y. pseudotuberculosis⁽⁹’¹⁰⁾. Twenty-six Y. enterocolitica strains were isolated from 178 brown rats (Rattus norvegicus) and black rats (Rattus rattus) captured in pig houses in the former Czechoslovakia¹-¹¹).

In the UK, 321 Y pseudotuberculosis strains have been isolated from 72 different animal species, of which 101 were from wild animals living in captivity, and 68 from free-living wild animals⁽³⁾. Of the latter, 28 strains were isolated from six species of mammal: coypu (Myocastor coypus), brown hare (Lepus europeus), mouse (Mus muscu- lus), rabbit ( Oryctolagus cuniculus), red fox ( Vulpes vulpes) and field vole (Microtus agrestis). Sporadic infection has been found in the Scottish blue or mountain hare (L epus timidus scoticus)ⁱ'^v4.

Forty Y. pseudotuberculosis strains have been isolated from 21 species of wild bird: blackbird (Turdus merula), eider (Somateria mollisima), fieldfare ( Turduspilaris), great tit (Parus major), greenfinch ( Chloris chloris), green wood­pecker (Picus viridis), hedge sparrow (Prunella modularis), hoopoe (Upupa epops), house martin (Delichon urbica), magpie (Pica pica), oystercatcher (Haematopus ostralegus), partridge (Perdix perdix), pheasant (Phasianus colchicus), pied wagtail (Motacilla alba), redwing (Turdus musicus), swallow (Hirundo rustica), swift (Apus apus), stock dove (Columba oenas), tree sparrow (Passer montanus), wood pigeon (Columba palumbus) and wren (Troglodytes troglo- dytes)⁽³⁾. Other British wild birds in which the disease has been reported include the coot (Fulica atra), chough (Pyr- rhocorax pyrrhocorax), goldfinch ( Carduelis carduelis), jackdaw ( Corvus monedula), pied flycatcher (Ficedula hypo- leuca), puffin (Fratercula arctica), rook ( Corvus frugilegus), starling (Sturnus vulgaris), skylark (Alauda arvensis), spar­rowhawk (Accipiter nisus), song thrush ( Turdus ericetorum), swallow (Hirundo rustica) and willow warbler (Phylloscopus trochilur)⁽¹⁵⁾.

Of wild animals, hares (Lepus spp.) are probably most susceptible to yersiniosis. They appear to be equally sus­ceptible to Y. enterocolitica infection as to Y. pseudotubercu­losis infection. In France and Germany, Y. pseudotuberculosis accounts for more leporine deaths than any other bacterial cause, having been isolated from up to 60% of hares in some areas. Similarly high percentages of affected hares have also been found in the UK⁽³⁾. Anti-Yersinia antibodies were found in 55% of hare sera from Northern Germany⁽¹⁶⁾ and Y. pseudotuberculosis was isolated from 13% of shot or found dead hares and Y enterocolitica in 4%⁽¹⁷⁾. In a Belgian survey, 14% of hares were found to be carriers⁽¹⁸⁾.

I n north- eastern Germany, 62.6 % of the wild boars were found positive for anti-Yop antibodies⁽¹⁹⁾. In the UK, 1% of 3,000 coypus (Myocastor coypus) live trapped or otherwise caught and killed in Norfolk and Suffolk over a 6-year period were recorded as having yersiniosis⁽²⁰⁾. In wild foxes only one case, from which Y pseudotuberculosis was isolated from many organs, has been recorded in the UK⁽²¹⁾. There are few reports of infection in wild rabbits (Oryctolagus cuniculus). The first epizootic among farmed chinchilla ( Chinchillidae family) imported from California and caused by Y enterocolitica was reported in Germany⁽²²⁾. In Continental Europe the disease has also been observed in roe deer (Capreolus capreolus), wild fallow deer (Dama dama), mink (Mustela lutreola), martens (Martes martes), moles (Talpa europaea), hedgehogs (Erinaceus europaeus) and marmots (Marmota marmota).

ENVIRONMENTAL FACTORS AND EPIDEMIOLOGICAL ROLE OF WILD ANIMALS

The principal reservoirs of Y. pseudotuberculosis are rodents and birds. Rats and mice are resistant to natural disease and for this reason play an important epidemiological role in the spread of infection.

Human infection with Y. pseudotuberculosis and Y. ente- rocolitica is acquired by direct or indirect contact with domestic animals, wild animals, birds or consumption of food and water contaminated with the bacteria.

TRANSMISSION

Yersiniosis is principally transmitted by the faeco-oral route by ingestion of contaminated food and water, at pasture or watering places. Transmission of Y. enterocolitica appears to be similar to that of Y. pseudotuberculosis. The creation of new interfaces between livestock and wildlife is the most important factor in disease transmission⁽²⁴⁾. It is accepted that contaminated soil, green crops, feeds and water sources are important sources of infection for farm and wild animals, rodents and birds. The role of insect vectors remains unclear. Although the rat flea (Xenopsylla cheopis) has been found to be a carrier of Y. pseudotuberculosis, its capability of transmitting the infection to a susceptible guinea pig was not proved⁽²⁰⁾. Milk-borne spread is a pos­sibility from yersinial mastitis. Venereal transmission is possible through semen derived from an infected repro­ductive tract. Transplacental spread to the fetus has been recorded in several species. Vertical transmission is a pos­sibility in the turkey at least, as seen by the detection of infected eggs⁽²⁵⁾. Experimental intranasal infection has been reproduced in guinea pigs and mice^(26,27). It has been suggested that insectivorous birds and mammals may acquire the disease from insects that have fed on the drop­pings of infected rodents and birds⁽²⁸⁾.

Although faecal shedding occurs as long as clinical signs persist (usually 2-3 weeks), prolonged sub- clinical carriage has also been reported. The faeces of wild animals should therefore be considered a potentially important source of Yersinia spp.

PATHOGENESIS, PATHOLOGY AND IMMUNITY

Following ingestion, Y enterocolitica and Y. pseudotuber­culosis attach to the intestinal mucosa, and Y. enterocolitica produces an enterotoxin that induces diarrhoea. Both pathogens are able to cross the gut epithelium and prolif­erate locally in the underlying tissue. The bacteria selec­tively enter via the M (microfold) cells, and reach the intestinal lymphoid aggregates, the Peyer’s patches⁽²⁹⁾. The chromosomally encoded Inv protein enables the organism to invade Peyer’s patch M cells. Entry into the Peyer’s patches leads to an enormous recruitment of phagocytic polymorphonuclear leucocytes, with formation of micro- abcesses comprising extracellular Yersinia, appearance of dead apoptotic cells and, eventually, complete destruction of the cytoarchitecture of the Peyer’s patches⁽³⁰⁾. Mono­cytes infiltrate the Peyer’s patches and mature into inflam­matory macrophages to produce cytokines such as interleukin-12, gamma interferon (IFN-γ) and tumour necrosis factor alpha (TNF-Ol), which aid in development of the immune response⁽³¹⁾. Professional phagocytes sig­nificantly restrict the rate at which Yersinia multiplies in the host tissues, thereby allowing the host to develop a specific protective immunity. However, Yersinia can respond by impairing phagocytosis, inhibiting killing by phagocytes, triggering apoptosis and suppressing the normal release of TNF- α and other cytokines. Once established in the Peyer’s patches, the bacteria can dis­seminate to the mesenteric lymph nodes and eventually to the liver, spleen and lung⁽³²⁾.

In the host, cytosol effector Yop inhibit several innate immune mechanisms, like phagocytosis, synthesis of pro- inflammatory signalling molecules and activation of the adaptive immune system⁽³³⁾. Yop also play a role in induc­ing apoptosis⁽³⁴⁾. CD4 and CD8 T cells play an essential role in the adaptive immune response directed at Yersinia infection⁽³⁵⁾.

There is an increasing amount of evidence suggesting that the O antigen of lipopolysaccharide (LPS) is required for virulence in pathogenic Yersinia, including coloniza­tion of host tissues, resistance to complement- mediated killing and resistance to innate immunity¹-³⁶). A case of co­infection was found in a dead mouflon ( Ovis musimon) in which Y. pseudotuberculosis O:3 was isolated from the heart and Y. enterocolitica O:5 from the faeces⁽¹³⁾. Mixed infec­tion is also reported in brown hares from Northern Germany⁽¹⁷⁾. Yersinia enterocolitica was isolated mainly from tonsils and tongue, but Y. pseudotuberculosis from lung, spleen, heart and kidney of rabbit, wild boar (Sus scrofa scrofa), Asiatic jackal (Canis aureus), red fox ( Vulpes vulpes), European river otter (Lutra lutra), beech marten (Martes foina), polecat (M ustela putorius) and wild cat (Felis silvestri-).

Gross pathologic changes vary among different host species. Infection by Y pseudotuberculosis and Y enterocol- itica causes lesions primarily in the gastrointestinal tract and associated lymphoid tissues mostly in liver, spleen, mesenterial lymph nodes and peritoneum, but may occur in the lungs, kidneys and other organs. Intestinal conges­tion, acute fibrino-necrotizing (fulminant yersiniosis) and granulomatous enteritis and mesenterial lymphadenitis with enlargement and congestion of mesenteric lymph nodes, ulcers or nodular lesions of the duodenum, splenomegaly, are common findings. Following dissemina­tion numerous granulomatous nodules, small necrotic foci and focal caseous abscesses are found in the liver, spleen, lung, mammary glands and other organs.

Histopathologically there are a large number of intral- esional Gram-negative coccobacilli, micro-abscessation or diffuse suppurative or pyogranulomatous inflammation of the intestinal mucosa. Multifocal suppurative mesenterial lymphadenitis and hepatitis are detected too. As an inci­dental finding or in animals with Yersinia infection in other organs, caseous mesenteric lymphadenitis with pyo- granulomas containing microcolonies of Yersinia sur­rounded by neutrophils and giant cells may be detected. Lesions may also be observed in other organs. Yersinia infection may cause sporadic pneumonia and septicaemia with lesions in various organs.

Experimental infection of ground squirrels (Citellus cytellus) was followed by hyperplasia of the peribronchial lymph tissue and defined catarrhal pneumonia. Small non­reactive necrotic foci were found in the spleen⁽³⁷⁾. Splenitis, lymphadenitis and necrotic foci were seen in the spleen and liver. In experimentally orally infected pigs, purulent meningoencephalitis with leucocytic infiltration of the white brain substance and accumulation of lymphoid cells around the blood vessels were established⁽³⁸⁾. Miliary necrotic foci around the lympho-reticular tissue of tonsils, catarrhal pneumonia and lymphoid cell proliferation were well demonstrated in the Peyer’s patches.

CLINICAL SIGNS AND TREATMENT

Both Y. pseudotuberculosis and Y enterocolitica infections may provoke the same clinical picture¹-^16,39). The most common clinical manifestations of yersiniosis are entero­colitis, fever, diarrhoea, abdominal pain, nausea, vomiting (in humans), mesenteric adenitis and/or terminal ileitis, and leucocytosis. Acute yersiniosis takes the form of a fulminating septicaemia involving visceral organs (e.g. liver and spleen), enteritis, and death within 1 to 3 days. In the subacute and chronic forms, the disease is character­ized by loss of weight over a period of days or weeks due to necrotizing or ulcerative enteritis, increasing listlessness, anorexia, severe diarrhoea, respiratory distress, muscular weakness and incoordination⁽⁴⁰⁾.

In wild birds the disease is usually fatal and often takes a peracute course with catarrhal or nodular lesions of the duodenum. The acute and subacute forms of the disease are characterized by diarrhoea and lameness or stiffness of gait.

Interstitial pneumonia and myocarditis in farmed deer infected with Y. pseudotuberculosis, as well as abortions and epididymo-orchitis in sheep and cows have been observed too^(4,41). Latent infections of wild animals are also possible⁽¹³⁾.

Yersiniosis is often self-limiting. Antibiotics may shorten the duration and severity of clinical signs when adminis­tered orally or parenterally — for example, aminoglicosides, doxycycline, potentiated sulphonamides or seftriaxone.

DIAGNOSIS

Laboratory diagnosis of infection, with or without clinical signs, includes isolation of Y. enterocolitica or Y. pseudotu­berculosis from faeces, throat swabs, mesenterial lymph nodes, peritoneal fluid or blood. Faecal cultures are gener­ally positive during the first two weeks of illness. Positive Yersinia serology by agglutination test or enzyme- linked immunosorbent assay (ELISA) are of diagnostic support, as there can be difficulties in bacteriological isolation of Yersinia. Polymerase chain reaction (PCR)-positive identi­fication of several key genes (ail, inv, virF, yopB and yopH) indicate Yersinia infection. A western blot kit is commer­cially available — recomBlot Yersinia IgG/IgA (Microgen, Martinsried, Germany). The kit is based on five recom­binant Yersinia outer proteins, which are specific for the serodiagnosis of yersiniosis. By using the more specific protein-based western blot strips, cross-reactivity with tularaemia and brucellosis can be excluded. The kit has been successfully applied for detection of anti- Yersinia antibodies in a study of the prevalence of yersiniosis in wild boars from north-eastern Germany⁽¹⁹⁾.

MANAGEMENT, CONTROL AND REGULATIONS

Surveillance for yersiniosis is strictly regulated for domestic animals in EU countries, where National Food Agencies monitor this food- borne disease. In addition, investiga­tions of case reports and outbreaks of yersiniosis are con­ducted to control them and to learn more about how to prevent this infection. Council Directive 92/117/EC, also referred to as the Zoonoses Directive, introduces and out­lines the requirements for the surveillance, monitoring and reporting of certain zoonoses on a national and EU level, e.g. yersiniosis.

No specific and effective preventive measures are known. Normal hygienic measures, regular disinfection and effec­tive rodent control programmes in animal farms, slaugh­terhouses, zoological collections, etc. help to prevent transmission and diminish the risk of incidence of yers- iniosis. Additional protection may be provided by preven­tion of access to food supplies for humans and animals by rodents, other small wild mammals and birds.

Infection may be introduced into hitherto yersiniosis- free areas and countries by the importation of animals — for example, for re-stocking hunting reserves or for breeding purposes⁽³⁾.

PUBLIC HEALTH CONCERN

As a zoonosis, yersiniosis is of concern to the economy and public health. The highest rates are reported in cooler climates — Northern Europe and Scandinavia, where Y. enterocolitica is the third most important food-borne path­ogen. The infection is more frequent and severe in chil­dren. Approximately 65% of Y. enterocolitica infections occur in infants and young children, and the 5—20 age group accounts for 75% of Y. pseudotuberculosis infections. In a small proportion of cases, complications such as skin rash, joint pains or septicaemia can occur. Risk factors for human infections are related to local disease ecology, including animal reservoirs. Wildlife can be the source of human disease transmitted directly or indirectly via domestic animals. Ingestion of contaminated food and water, contact with wild and domestic animals, including household pets, are potential routes of exposure and infection. Pigs are the main sub-clinical reservoir for path­ogenic Yersinia strains, being isolated predominantly from the tonsils(⁴²). Consumption of raw pork and pork prod­ucts including cold cuts presents an increased risk of infec­tion by Y. enterocolitica because of its ability to multiply under refrigeration and vacuum packaging. The health risks associated with the consumption of game meat are unknown.

SIGNIFICANCE AND IMPLICATIONS FOR ANIMAL HEALTH

Yersiniosis is a relatively uncommon disease that generally affects the intestinal tract, where the inflammation may be self-limiting. In some animals there is a more protracted course with extra- intestinal infection, which may spread to other tissues. This dissemination of infection is usually fatal and may develop in elderly, stressed and immuno­compromised animals. The significance and implications of yersiniosis for animal health are likely to grow with increased densities and range expansion of susceptible wild species. The disease prevalence in wildlife is also likely to increase where there is an increased risk of contact with livestock. The impact ofyersiniosis on wildlife is not clearly known; however, this disease is a matter of concern in domestic livestock, semi- domestic herds, farmed cervids and as a food-borne infection in humans.