Use of a phospholipase-C assay, in vivo pathogenicity assays and PCR in assessing the virulence of Listeria spp.
Introduction
Listeriosis is an important bacterial disease caused by infection with pathogenic species of the genus Listeria. In particular, Listeria monocytogenes accounts for approximately 98% of human and 85% of animal cases of listeriosis (McLauchlin, 1987). In animals, listeriosis causes abortion, endometritis, repeat-breeding, encephalitis, septicaemia and mastitis (Radostits et al., 2000) and Listeria ivanovii is a frequent cause of abortion in sheep with infection rates ranging from 1.65% to 12% (Sergeant et al., 1991, Alexander et al., 1992). Listeriosis in animals arises mainly from the ingestion of contaminated food and water, and is particularly common in ruminants fed silage (Low and Donachie, 1997).
An array of virulence-associated genes associated with the pathogenicity of Listeria spp., include plcA encoding phosphatidylinositol phospholipase-C (PI-PLC), plcB encoding phosphatidycholine phospholipase-C, hlyA encoding a haemolysin, mpl encoding a metalloprotease and actA encoding the surface actin polymerisation protein actA. All of these genes are physically linked in a 9 kb chromosomal island referred to as Listeria pathogenicity island-1 (LIPI-1) (Vazquez-Boland et al., 2001). The enzyme PI-PLC, expressed by plcA gene, is reported to be only associated with pathogenic Listeria spp. such as L. monocytogenes and L. ivanovii (Notermans et al., 1991b) and a correlation between the activity of this enzyme and bacterial virulence has been demonstrated by in vivo pathogenicity assays (Shakuntala et al., 2006, Kaur et al., 2007, Rawool et al., 2007).
Recently, the assessment of the virulence of L. monocytogenes and L. ivanovii has been assisted by the development of a multiplex PCR assay targeting the virulence genes of these organisms and a PI-PLC activity assay (Kaur et al., 2007, Rawool et al., 2007). The use of these methods has the potential to assist in the rapid assessment of the virulence of clinical isolates. In the present study we used such an approach to assess the virulence of Listeria bacteria isolated from animals with various reproductive disorders.
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Standard bacterial isolates
The standard strains of L. monocytogenes 4b (MTCC 1143), Staphylococcus aureus (MTCC 1144) and Rhodococcus equi (MTCC 1135) were obtained from the Microbial Type Culture Collection and Gene Bank (MTCC) at The Institute of Microbial Technology (IMTECH), Chandigarh, India. The strains of L. monocytogenes (4b [NCTC 11994], 1/2a [NCTC 7973] and 1/2b [NCTC 10887]), L. ivanovii (NCTC 11846), Listeria innocua (NCTC 11288), Listeria seeligeri (NCTC 11856), Listeria grayi (NCTC 10812) and Listeria
Isolation of Listeria spp.
Of the six isolates resembling Listeria spp. cultured from the 50 cases of reproductive disorder in cattle, five were identified as L. seeligeri (three from genital and two from faecal swabs). The remaining isolate was L. welshimeri and was recovered from a faecal swab. Of the 60 samples from buffaloes with a history of abortion, one isolate of L. seeligeri and one of L. welshimeri were cultured from genital swabs. Seven isolates of L. seeligeri (four from genital and three from faecal swabs)
Discussion
L. monocytogenes and L. ivanovii are established causes of abortion, encephalitis and septicaemia in animals (McLauchlin, 1987, Radostits et al., 2000). Diagnosis of listeriosis is enhanced by the detection of virulence markers of isolated Listeria spp. using both molecular techniques and by in vitro expression assays (Notermans et al., 1991b). In the current study the isolation rates of non-pathogenic Listeria spp. from cases of reproductive disorders in cattle and buffaloes were comparable to
Conclusions
The findings of this study of a range of in vitro and in vivo assays to assess the virulence of Listeria spp. isolated from animals with a history of reproductive disorder and from other sources suggest that the PI-PLC and PCR assays are potential alternative assessment methods to in vivo pathogenicity tests, and may result in fewer experimental animals being used in such procedures.
Conflict of interest statement
None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
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Comparison of recombinant and synthetic listeriolysin- O peptide- based indirect ELISA vis-à-vis cultural isolation for detection of listeriosis in caprine and ovine species
2021, Journal of Microbiological MethodsCitation Excerpt :There has been a continuous search for such virulence markers of Listeria spp. capable of eliciting the antibody response during listeric infection; some of them include listeriolysin-O (LLO), the haemolysin (hly) (Churchill et al., 2006); internalins (InlA, InlB, InlC, InlC2, InlJ etc.) (Das et al., 2013); the leucine rich repeat (LRR) proteins (Boerlin, 2003); the actA gene-encoded actin (Travier and Lecuit, 2014.) ; two phospholipases- C namely, phosphoinositide phospholipase- C (PI-PLC) and phosphatidylcholine phospholipase- C (PC-PLC) encoded by plcA and plcB, respectively (Chaudhari et al., 2004; Kaur et al., 2010); and the autolysin p60 protein (Hess et al., 1996). Among all the virulence-associated antigens, LLO has widely been employed as an immunodominant antigen (Hernández-Flores and Vivanco-Cid, 2015) in the development of enzyme-linked immune sorbent assay (ELISA) for the serodiagnosis of listeriosis in cattle (Barbuddhe et al., 2000; Boerlin, 2003), buffaloes (Chaudhari et al., 2001), goats (Bhanu et al., 2006; Kulesh et al., 2019), sheep (Shoukat et al., 2013; Ibrahim et al., 2015), and humans (Berche et al., 1990; Suryawanshi et al., 2014).
Comparative whole genome analysis of Listeria monocytogenes 4b strains reveals least genome diversification irrespective of their niche specificity
2017, Gene ReportsCitation Excerpt :These strains had different virulence potential, wherein BHU2 and 3 were pathogenic contrary to BHU1. We observed BHU1to harbor nucleotide mutation from A to G in the major virulence genes i.e. hemolysin D and listeriolysin O which is in accordance with the previous reports suggesting that mere presence of virulence associated genes cannot be correlated with the virulence of L. monocytogenes (Soni et al., 2015b; Kaur et al., 2010). Further, the literature survey also indicates that some naturally virulence attenuated L. monocytogenes strains often contain mutations in prfA, hlyA, actA, and inlA genes, and this attribute possibly results in the expression of truncated or non-functional PrfA, LLO, ActA and InlA proteins (Orsi et al., 2007; Shen et al., 2013).
Comparative diagnostic efficacy of recombinant LLO and PI-PLC-based ELISAs for detection of listeriosis in animals
2017, Journal of Microbiological MethodsCitation Excerpt :Amongst all the known virulence factors of listeriae, LLO has been well characterized as an immunodominant antigen (Hernández-Flores and Vivanco-Cid, 2015). Currently, the purified LLO-based ELISA remains the most reliable and acceptable serodiagnostic test worldwide for assessment of the listeric infection in humans (Berche et al., 1990;Gholizadeh et al., 1996; Kaur et al., 2010) and animals (Barbuddhe et al., 2002; Shakuntala et al., 2007; Shoukat et al., 2013; Ibrahim HA Abd El-Rahim et al., 2015), however, prior adsorption of test sera with SLO is necessary to avoid false positive results (Berche et al., 1990; Kaur et al., 2010; Shoukat et al., 2013). The process of production of purified LLO from L. monocytogenes strains is quite tedious and time-consuming and it often ends up with low yields (Baetz and Wesley, 1995).
Epidemiology and risk management of listeriosis in India
2012, International Journal of Food MicrobiologyCitation Excerpt :The prevalence of L. monocytogenes and other Listeria spp. was found to be 4.4 and 7.4%, respectively from buffaloes with a history of reproductive disorders (Shakuntala et al., 2006). L. ivanovii was recovered from one of the 54 samples from the 18 ewes with reproductive disorders (Kaur et al., 2010). L. ivanovii was isolated from 7.5, 5.56 and 14.52% goats of those aborted, mastitic and apparently healthy, respectively (Elezebeth et al., 2007).
Practical relevance of methodologies for detecting and tracing of Listeria monocytogenes in ready-to-eat foods and manufacture environments - A review
2011, LWTCitation Excerpt :Simultaneous detection of pathogens, as it was performed with L. monocytogenes, Salmonella typhimurium and Escherichia coli O157:H7, was demonstrated down to 10 cells/25 g of liquid whole egg (Germini, Masola, Carnevali, & Marchelli, 2009). PCR assays for the detection of Listeria and rapid assessment of their virulence (Kaur, Malik, Bhilegaonkar, Vaidya, & Barbuddhe, 2009) most commonly target the hlyA gene (Listeriolysin O, a pore forming toxin; β-hemolysin) (Khelef et al., 2006), the iap gene (invasion-associated gene) (Liu, 2006), the inl gene (group of internalins), the prfA gene (regulator protein for activation of virulence cluster) (Liu, 2006; Rossmanith, Krassnig, Wagner, & Hein, 2006) and also 16S RNA (Aznar & Alarcón, 2002). Amplification of polymorphic virulence genes is more useful when compared to housekeeping genes.