The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal indivuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research.

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Listeria pathogenesis and molecular virulence determinants.

Epidemiology of Cryptosporidium: transmission, detection and identification.

There has been an explosion of descriptions of new species of Cryptosporidium during the last two decades. This has been accompanied by confusion regarding the criteria for species designation, largely because of the lack of distinct morphologic differences and strict host specificity among Cryptosporidium spp. A review of the biologic species concept, the International Code of Zoological Nomenclature (ICZN), and current practices for Cryptosporidium species designation calls for the establishment of guidelines for naming Cryptosporidium species. All reports of new Cryptosporidium species should include at least four basic components: oocyst morphology, natural host specificity, genetic characterizations, and compliance with the ICZN. Altogether, 13 Cryptosporidium spp. are currently recognized: C. muris, C. andersoni, C. parvum, C. hominis, C. wrairi, C. felis, and C. cannis in mammals; C. baileyi, C. meleagridis, and C. galli in birds; C. serpentis and C. saurophilum in reptiles; and C. molnari in fish. With the establishment of a framework for naming Cryptosporidium species and the availability of new taxonomic tools, there should be less confusion associated with the taxonomy of the genus Cryptosporidium. The clarification of Cryptosporidium taxonomy is also useful for understanding the biology of Cryptosporidium spp., assessing the public health significance of Cryptosporidium spp. in animals and the environment, characterizing transmission dynamics, and tracking infection and contamination sources.

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Cryptosporidium Taxonomy: Recent Advances and Implications for Public Health

There has been an explosion of descriptions of new species of Cryptosporidium during the last two decades This has been accompanied by confusion regarding the criteria for species designation, largely because of the lack of distinct morphologic differences and strict host specificity among Cryptosporidium spp A review of the biologic species concept, the International Code of Zoological Nomenclature (ICZN), and current practices for Cryptosporidium species designation calls for the establishment of guidelines for naming Cryptosporidium species All reports of new Cryptosporidium species should include at least four basic components: oocyst morphology, natural host specificity, genetic characterizations, and compliance with the ICZN Altogether, 13 Cryptosporidium spp are currently recognized: C muris, C andersoni, C parvum, C hominis, C wrairi, C felis; and C cannis in mammals; C baileyi, C meleagridis, and C galli in birds; C serpentis and C saurophilum in reptiles; and C molnari in fish With the establishment of a framework for naming Cryptosporidium species and the availability of new taxonomic tools, there should be less confusion associated with the taxonomy of the genus Cryptosporidium The clarification of Cryptosporidium taxonomy is also useful for understanding the biology of Cryptosporidium spp, assessing the public health significance of Cryptosporidium spp in animals and the environment, characterizing transmission dynamics, and tracking infection and contamination sources

Cryptosporidium Taxonomy: Recent Advances and Implications for Public Health (Review)

At least 325 water-associated outbreaks of parasitic protozoan disease have been reported. North American and European outbreaks accounted for 93% of all reports and nearly two-thirds of outbreaks occurred in North America. Over 30% of all outbreaks were documented from Europe, with the UK accounting for 24% of outbreaks, worldwide. Giardia duodenalis and Cryptosporidium parvum account for the majority of outbreaks (132; 40.6% and 165; 50.8%, respectively), Entamoeba histolytica and Cyclospora cayetanensis have been the aetiological agents in nine (2.8%) and six (1.8%) outbreaks, respectively, while Toxoplasma gondii and Isospora belli have been responsible for three outbreaks each (0.9%) and Blastocystis hominis for two outbreaks (0.6%). Balantidium coli, the microsporidia, Acanthamoeba and Naegleria fowleri were responsible for one outbreak, each (0.3%). Their presence in aquatic ecosystems makes it imperative to develop prevention strategies for water and food safety. Human incidence and prevalence-based studies provide baseline data against which risk factors associated with waterborne and foodborne transmission can be identified. Standardized methods are required to maximize public health surveillance, while reporting lessons learned from outbreaks will provide better insight into the public health impact of waterborne pathogenic protozoa.

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Waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt.

Cryptosporidium wrairi was isolated from guinea pigs during a spontaneous outbreak of cryptosporidiosis. Despite the morphological and antigenic similarities to C. parvum, C. wrairi displayed a different host range and site of infection and may represent a separate species or sub-species. We used the polymerase chain reaction to clone two distinct 550 bp-long DNA fragments, Wc-I and Wc-II, of the gene encoding the Cryptosporidium oocyst wall protein (COWP) of C. wrairi, which showed 98% identity to the C. parvum homologue. Within Wc-I, polymorphic RsaI restriction sites were used to develop a polymerase chain reaction-restriction fragment length polymorphism method able to distinguish C. wrairi from C. parvum and to identify two groups of C. parvum isolates differentially associated with animal and human infections.

PCR-RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene discriminates between C. wrairi and C. parvum, and between C. parvum isolates of human and animal origin

A technique based on the analysis of banding patterns obtained by SDS-PAGE Western-blotting of an oocyst wall antigen obtained from faeces has been evaluated to subtype Cryptosporidium parvum Tyzzer, 1912. This technique appears to have sufficient stability to recognise multiple types of this parasite. A similar Western-blotting technique has also been used to assess antibody responses to cryptosporidial antigens in human sera. Two systems were developed: one against three antigens of apparent molecular weights 6, 14 and 17 kDa; the second against oocyst wall antigens of apparent molecular weights 57, 69, 75, 89, 128, 151 and 173 kDa. Antibodies to three antigens of apparent molecular weights 6, 14 and 17 kDa were most successful as diagnostic markers in that they were found in >88% of convalescent phase sera from confirmed cryptosporidiosis patients and were uncommon (greater than or equal to 7%) in control subjects. Faecal samples from human and animal sporadic cases yielded a wide range of cryptosporidial antigen banding patterns. Samples from patients in a water-borne outbreak in South Devon (England) in 1995 also yielded a wide range of banding patterns including members within individual household family groups. These results are in contrast with those from samples collected from other defined geographical areas, including some from a second water-borne outbreak where much more homogeneous banding patterns were obtained. Sera collected for other purposes from apparently uninfected individuals 9 months after the South Devon 1995 outbreak were examined. Antibodies to the three antigens of molecular weights 6, 14 and 17 kDa were detected in 32-49% of individuals resident in the outbreak water supply area, and in 15-21% of those resident in an adjacent water supply area. The significance of these findings is discussed in relation to data obtained from epidemiological field studies.

The epidemiology of cryptosporidiosis: application of experimental sub-typing and antibody detection systems to the investigation of water-borne outbreaks.

Forty-three Listeria ivanovii isolates were collected in the UK between 1991 and 1997 from: 35 animal infections; two human infections; five foods; and one environmental source. A further two type strains of L. ivanovii (subsp. ivanovii and subsp. londoniensis) were obtained from a culture collection. These bacteria were characterised by conventional phenotypic methods and by pulsed-field gel electrophoresis (PFGE) using ApaI and SmaI. Forty-two of the isolates from the UK were identified as L. ivanovii subsp. ivanovii and the remaining culture as L. ivanovii subsp. londoniensis. Six and four PFGE profiles were obtained using ApaI and SmaI digestion respectively; six composite profiles were obtained combining the results for both enzymes. The PFGE profile of the UK L. ivanovii subsp. londoniensis (isolated from processed shrimps) was similar to the type strain of this subspecies and differed from all of the L. ivanovii subsp. ivanovii tested. The majority of isolates (38 out of 45) belonged to one profile showing that the UK population of this bacterium is much less genetically diverse than similar studies have shown for Listeria monocytogenes.

James McLauchlin

Papers

PCR-RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene discriminates between C. wrairi and C. parvum, and between C. parvum isolates of human and animal origin

The epidemiology of cryptosporidiosis: application of experimental sub-typing and antibody detection systems to the investigation of water-borne outbreaks.

Characterisation of Listeria ivanovii isolates from the UK using pulsed-field gel electrophoresis