Comparative Metabolomics of Mycoplasma bovis and Mycoplasma gallisepticum Reveals Fundamental Differences in Active Metabolic Pathways and Suggests Novel Gene Annotations.

Mycoplasmas are simple, but successful parasites that have the smallest genome of any free-living cell and are thought to have a highly streamlined cellular metabolism. Here, we have undertaken a detailed metabolomic analysis of two species, Mycoplasma bovis and Mycoplasma gallisepticum, which cause economically important diseases in cattle and poultry, respectively. Untargeted gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analyses of mycoplasma metabolite extracts revealed significant differences in the steady-state levels of many metabolites in central carbon metabolism, while 13C stable isotope labeling studies revealed marked differences in carbon source utilization. These data were mapped onto in silico metabolic networks predicted from genome wide annotations. The analyses elucidated distinct differences, including a clear difference in glucose utilization, with a marked decrease in glucose uptake and glycolysis in M. bovis compared to M. gallisepticum, which may reflect differing host nutrient availabilities. The 13C-labeling patterns also revealed several functional metabolic pathways that were previously unannotated in these species, allowing us to assign putative enzyme functions to the products of a number of genes of unknown function, especially in M. bovis. This study demonstrates the considerable potential of metabolomic analyses to assist in characterizing significant differences in the metabolism of different bacterial species and in improving genome annotation. IMPORTANCE Mycoplasmas are pathogenic bacteria that cause serious chronic infections in production animals, resulting in considerable losses worldwide, as well as causing disease in humans. These bacteria have extremely reduced genomes and are thought to have limited metabolic flexibility, even though they are highly successful persistent parasites in a diverse number of species. The extent to which different Mycoplasma species are capable of catabolizing host carbon sources and nutrients, or synthesizing essential metabolites, remains poorly defined. We have used advanced metabolomic techniques to identify metabolic pathways that are active in two species of Mycoplasma that infect distinct hosts (poultry and cattle). We show that these species exhibit marked differences in metabolite steady-state levels and carbon source utilization. This information has been used to functionally characterize previously unknown genes in the genomes of these pathogens. These species-specific differences are likely to reflect important differences in host nutrient levels and pathogenic mechanisms.

Survey of Victorian small ruminant herds for mycoplasmas associated with contagious agalactia and molecular characterisation of Mycoplasma mycoides subspecies capri isolates from one herd.

OBJECTIVE: Regarded as one of the most expensive production diseases of dairy sheep and goats, contagious agalactia (CA) is caused by any of four agents: Mycoplasma agalactiae, M. mycoides subspecies capri (Mmc), M. capricolum subspecies capricolum (Mcc) and M. putrefaciens. Although CA is worldwide in distribution, it has not been reported in Australia, even though studies between the 1950s and 1980s isolated each agent from sheep or goats without any clinical signs associated with it. The aim of this study was to examine sheep and goats in Victoria, Australia, for the presence of CA-associated mycoplasmas and to investigate the evolutionary relationships of these isolates by comparing their genetic differences with their counterparts from other parts of the world. METHODS: A 3-year epidemiological survey of small ruminant populations in Victoria, Australia, was conducted for the presence of CA-associated mycoplasmas and the isolates obtained were genotyped by multilocus sequence typing (MLST). RESULTS: Mmc was the only CA-associated agent isolated from the 1358 samples analysed in the study, but was not associated with CA on the property where it was found. MLST analyses of Mmc strains revealed a distinct clustering of Australian isolates into a novel clade, with the closest relatives being strains from Europe. The distinct clustering is consistent with the absence of clinical disease in Australia. CONCLUSION: The isolation of Mmc indicates that this subspecies persists in Australian small ruminant populations. However, full genome sequencing and in vitro animal experimentation are needed to unequivocally demonstrate the avirulence of Australian strains.

The central role of lipoproteins in the pathogenesis of mycoplasmoses.

Mycoplasmas are a diverse group of pathogens responsible for disease in a wide range of animal species. In recent years there have been considerable advances in knowledge of the proteins and structures involved in adherence in some mycoplasmas, but understanding of the biochemical functions and roles in virulence of another central feature of mycoplasmas, their lipoproteins, continues to develop. The aim of this review is to examine current knowledge of the roles of lipoproteins in the pathogenicity and the evolution of virulence in those mycoplasmas causing disease in domestic animals. Those lipoproteins that have been characterised have roles in adherence, in transport of nutrients into the mycoplasma cell, and in enzymatic interactions with the host. Furthermore they appear to play a prominent role in both inducing the host immune response to infection and in facilitating evasion of this response, particularly through the generation of dramatic levels of antigenic variation on the cell surface. Recent genomic comparisons of several pathogenic mycoplasmas have identified a further level of interaction between lipoproteins and pathogenicity. In several pathogens large scale horizontal gene transfer between distantly related mycoplasma species has resulted in the acquisition of a large number of genes, including those encoding lipoproteins thought to play a role in virulence, by one mycoplasma from another inhabiting the same host species. The interactions between these horizontally transferred genes, their new mycoplasma host and the animal that it infects may be an important contributing factor in the pathogenesis of some mycoplasmoses.

Development of a replicable oriC plasmid for Mycoplasma gallisepticum and Mycoplasma imitans, and gene disruption through homologous recombination in M. gallisepticum.

The genome of Mycoplasma gallisepticum strain R(low) has been sequenced completely, but subsequent genetic studies have been limited by the lack of a replicable vector system. In this study, replicable plasmids were constructed for M. gallisepticum and Mycoplasma imitans using the oriC region upstream from the soj gene. The oriC plasmids of M. gallisepticum (pGTLori) and M. imitans (pMIori) replicated in both species, but Mycoplasma pneumoniae could not support replication of pGTLori. A 180 bp section of the oriC region of M. gallisepticum was found to be the minimal region required for plasmid replication in M. gallisepticum strain S6, the shortest oriC region defined for mycoplasmas. Targeted gene disruption of vlhA1.1 of M. gallisepticum S6 was attempted using these oriC plasmids. Constructs made in pPLoriC7 integrated into the M. gallisepticum genomic oriC region, not into the targeted gene, whereas those made in pMIori disrupted the vlhA1.2 gene, which has 97 % DNA sequence identity with the vlhA1.1 gene. During in vitro passages, antimicrobial selection pressure did not influence the rate of chromosomal integration. These oriC plasmids will thus be useful for genetic studies, including inactivation or expression of selected genes, in M. gallisepticum and M. imitans, and will lead to a better understanding of their molecular biology. They are, to our knowledge, the first replicable plasmids developed for the Pneumoniae phylogenetic group of mycoplasmas.

Disorders of the sternoclavicular joint.

The sternoclavicular joint is vulnerable to the same disease processes as other synovial joints, the most common of which are instability from injury, osteoarthritis, infection and rheumatoid disease. Patients may also present with other conditions, which are unique to the joint, or are manifestations of a systemic disease process. The surgeon should be aware of these possibilities when assessing a patient with a painful, swollen sternoclavicular joint.

Diversity of begomovirus DNA beta satellites of non-malvaceous plants in east and south east Asia.

Two previous analyses of the diversity of begomovirus-associated DNA beta satellites focused predominantly on molecules originating from the Indian sub-continent and southern China. They showed the satellites to group according to the hosts from which they were isolated, either malvaceous or non-malvaceous plants, and then to form sub-groups based upon geographic origin and host. In this study we analysed the diversity of DNA beta satellites in east and south east Asia. Here the satellites group by geographic location and are considerably more diverse than previously indicated. This probably reflects the limited movement of begomovirus/DNA beta complexes in this region and their subsequent diversification from a common ancestor to a variety of hosts.

Cotton leaf curl disease is associated with multiple monopartite begomoviruses supported by single DNA beta.

For bipartite begomoviruses (family Geminiviridae) trans-replication of the DNA B component by the DNA A-encoded replication-associated protein (Rep) is achieved by virtue of a shared sequence, the "common region", which contains repeated motifs (iterons) which are sequence-specific Rep binding sites and form part of the origin of replication. Recently cotton leaf curl disease (CLCuD), a major constraint to cotton production on the Indian subcontinent, has been shown to be caused by a monopartite begomovirus ( Cotton leaf curl Multan virus [CLCuMV]) and a novel single-stranded DNA satellite molecule termed CLCuD DNA beta. The satellite molecule is trans-replicated by CLCuMV but does not possess the iteron sequences of this virus. We have investigated the ability of CLCuD DNA beta to interact with three further clones of monopartite begomoviruses, isolated from cotton, that have distinct Rep binding specificities. All three cloned viruses were capable of trans-replicating the satellite molecule and inducing CLCuD symptoms in cotton, indicating that the interaction between begomovirus and DNA beta is relaxed in comparison to the interaction between DNA A and DNA B components. Field surveys across all the cotton growing regions of Pakistan indicate that dual and multiple infections are the norm for CLCuD with no evidence of synergism. Despite the diversity of begomoviruses associated with CLCuD, only a single class of DNA beta has been detected, suggesting that this satellite has the capacity to be recruited by unrelated begomoviruses.

Variation between pathogenic serovars within Salmonella pathogenicity islands.

Although four of the five Salmonella pathogenicity islands (SPIs) have been characterized in detail for Salmonella enterica serovar Typhimurium, and the fifth has been characterized for Salmonella enterica serovar Dublin, there have been limited studies to examine them in detail in a range of pathogenic serovars of S. enterica. The aim of this study was to examine these regions, shown to be crucial in virulence, in pathogenic serovars to identify any major deletions or insertions that may explain variation in virulence and provide further understanding of the elements involved in the evolution of these regions. Multiple strains of each of the 13 serovars were compared by Southern blot hybridization using a series of probes that together encompassed the full length of all five SPIs. With the exception of serovar Typhimurium, all strains of the same serovar were identical in all five SPIs. Those serovars that differed from serovar Typhimurium in SPI-1 to SPI-4 and from serovar Dublin in SPI-5 were examined in more detail in the variant regions by PCR, and restriction endonuclease digestion and/or DNA sequencing. While most variation in hybridization patterns was attributable to loss or gain of single restriction endonuclease cleavage sites, three regions, in SPI-1, SPI-3, and SPI-5, had differences due to major insertions or deletions. In SPI-1 the avrA gene was replaced by a 200-base fragment in three serovars, as reported previously. In SPI-5, two serovars had acquired an insertion with similarity to the pagJ and pagK genes between pipC and pipD. In SPI-3 the genes sugR and rhuM were deleted in most serovars and in some were replaced by sequences that were very similar to either the Escherichia coli fimbrial operon, flanked by two distinct insertion sequence elements, or to the E. coli retron phage PhiR73. The distribution of these differences suggests that there have been a number of relatively recent horizontal transfers of genes into S. enterica and that in some cases the same event has occurred in multiple lineages of S. enterica. Thus, it seems that insertion sequences and retron phages are likely to be involved in continuing evolution of the pathogenicity islands of pathogenic Salmonella serovars.

Universal primers for the PCR-mediated amplification of DNA 1: a satellite-like molecule associated with begomovirus-DNA beta complexes.

DNA 1 is a single-stranded DNA molecule of approximately 1370 nucleotides. It is associated with monopartite geminiviruses of the genus Begomovirus, which require a DNA beta component for symptomatic infection. The DNA 1 molecule requires the helper begomovirus for movement in plants, but is capable of self-replication. We designed two abutting primer pairs (DNA101/DNA102 and UN101/UN102) to conserved sequences of DNA 1. This allowed polymerase chain reaction-mediated amplification of the full-length molecule from total nucleic acid extracts produced from various host plants from geographically distinct, worldwide locations. These primers are useful both as diagnostic probes and for producing full-length infectious clones for in planta studies.

Universal primers for the PCR-mediated amplification of DNA beta: a molecule associated with some monopartite begomoviruses.

DNA beta is an approx 1350 nucleotide, single-stranded DNA molecule which has been shown to be associated with some monopartite geminiviruses of the genus Begomovirus. This component requires the helper begomovirus for replication in the cells of host plants and for insect transmission, possibly by trans-encapsidation. Sequence comparisons of the two available DNA beta sequences has identified a highly conserved region upstream of a predicted hairpin structure. Abutting primers designed to this conserved region allows PCR-mediated amplification of the full-length DNA beta component from total nucleic acid extracts isolated from infected plants originating from a variety of geographically distinct sources and host plants.

Induction of enzootic pneumonia in pigs by the administration of an aerosol of in vitro-cultured Mycoplasma hyopneumoniae.

The aim of this study was to establish whether enzootic pneumonia could be induced reliably in piglets by administering an aerosolised culture of Mycoplasma hyopneumoniae. Groups of five M hyopneumoniaefree Landrace x Large White piglets weaned at 11 to 14 days of age were exposed to aerosols of in vitro cultures of a virulent strain of M hyopneumoniae. In three separate trials, 14 of 15 pigs exposed to the bacteria developed pneumonia, but pigs exposed to the culture medium alone did not develop the disease. Lung pathology, both gross and histological, indicated acute disease. Ten of the pigs were tested for seroconversion by Western blot and they were all positive. The growth rates of the infected pigs were significantly reduced and the water consumption of the infected groups was also depressed. M hyopneumoniae was recovered from eight of the 15 infected pigs.

ALVAC-SIV-gag-pol-env-based vaccination and macaque major histocompatibility complex class I (A*01) delay simian immunodeficiency virus SIVmac-induced immunodeficiency.

T-cell-mediated immune effector mechanisms play an important role in the containment of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) replication after infection. Both vaccination- and infection-induced T-cell responses are dependent on the host major histocompatibility complex classes I and II (MHC-I and MHC-II) antigens. Here we report that both inherent, host-dependent immune responses to SIVmac251 infection and vaccination-induced immune responses to viral antigens were able to reduce virus replication and/or CD4+ T-cell loss. Both the presence of the MHC-I Mamu-A*01 genotype and vaccination of rhesus macaques with ALVAC-SIV-gag-pol-env (ALVAC-SIV-gpe) contributed to the restriction of SIVmac251 replication during primary infection, preservation of CD4+ T cells, and delayed disease progression following intrarectal challenge exposure of the animals to SIV(mac251 (561)). ALVAC-SIV-gpe immunization induced cytotoxic T-lymphocyte (CTL) responses cumulatively in 67% of the immunized animals. Following viral challenge, a significant secondary virus-specific CD8+ T-cell response was observed in the vaccinated macaques. In the same immunized macaques, a decrease in virus load during primary infection (P = 0.0078) and protection from CD4 loss during both acute and chronic phases of infection (P = 0.0099 and P = 0.03, respectively) were observed. A trend for enhanced survival of the vaccinated macaques was also observed. Neither boosting the ALVAC-SIV-gpe with gp120 immunizations nor administering the vaccine by the combination of mucosal and systemic immunization routes increased significantly the protective effect of the ALVAC-SIV-gpe vaccine. While assessing the role of MHC-I Mamu-A*01 alone in the restriction of viremia following challenge of nonvaccinated animals with other SIV isolates, we observed that the virus load was not significantly lower in Mamu-A*01-positive macaques following intravenous challenge with either SIV(mac251 (561)) or SIV(SME660). However, a significant delay in CD4+ T-cell loss was observed in Mamu-A*01-positive macaques in each group. Of interest, in the case of intravenous or intrarectal challenge with the chimeric SIV/HIV strains SHIV(89.6P) or SHIV(KU2), respectively, MHC-I Mamu-A*01-positive macaques did not significantly restrict primary viremia. The finding of the protective effect of the Mamu-A*01 molecule parallels the protective effect of the B*5701 HLA allele in HIV-1-infected humans and needs to be accounted for in the evaluation of vaccine efficacy against SIV challenge models.

Association of a Monopartite Begomovirus Producing Subgenomic DNA and a Distinct DNA Beta on Croton bonplandianus Showing Yellow Vein Symptoms in Pakistan.

The recent discovery that monopartite begomoviruses on ageratum and cotton essentially require a DNA satellite called DNA ß (2,4) is leading to identification of several other hosts that have similar disease complexes. A weed species (Croton bonplandianus) belonging to the family Euphorbiaceae is one such example. C. bonplandianus is widely distributed on wastelands throughout the Punjab Province in Pakistan. It very often shows yellow vein symptoms indicating infection by a begomovirus. To detect a begomovirus, both symptomatic and asymptomatic plants were collected from several widely separated locations in the Punjab Province. Total DNA was isolated from these samples by the cetyltrimethylammoniumbromide (CTAB) method, resolved in an agarose gel, and blotted on a nylon membrane (2). A full-length clone of DNA A of Cotton leaf curl virus (CLCuV) labeled with 32PdCTP was used as a probe in Southern hybridization (2). The probe detected hybridizing bands only in symptomatic plants, confirming the presence of a begomovirus. In addition to hybridizing bands of the expected sizes, smaller bands were also detected, suggesting the presence of subgenomic molecules derived from DNA A. Universal polymerase chain reaction (PCR) primers for dicot-infecting geminiviruses (1) were used in PCR for amplification of DNA A of the begomovirus associated with the disease. The use of these primers in PCR was expected to result in amplification of full-length DNA A. In addition to a product of the expected size (2.7 to 2.8 kb), another product of approximately 1.4 kb was amplified. The presence of subgenomic DNAs that are derived from DNA A is an indicator of the monopartite nature of begomoviruses, because in bipartite begomoviruses subgenomic DNAs are derived solely from DNA B. The presence of a DNA ß, a DNA satellite associated with certain monopartite begomoviruses, was suspected because of symptoms and the possible monopartite nature of the virus. Universal primers for amplification of DNA ß (3) were used in PCR for amplification of a putative DNA ß. The PCR reaction yielded a product of expected size (≈1.4 kb). A probe from the amplified product was made by the oligolabeling method. The probe detected hybridizing bands in all symptomatic samples collected from three locations, confirming the association of a DNA ß with the disease. A duplicate blot when hybridized with a DNA ß associated with ageratum yellow vein disease did not hybridize to these samples. These results confirm that yellow vein disease on this weed is associated with a monopartite begomovirus and a distinct DNA ß. References: (1) R. W. Briddon et al. Mol. Biotechnol. 1:202, 1994. (2) R. W. Briddon et al. Virology 285:234, 2001. (3) R. W. Briddon et al. Mol. Biotechnol. In press. (4) K. Saunders et al. Proc. Natl. Acad. Sci. U S A 97:6890, 2000.

Mucosal AIDS vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques.

Given the mucosal transmission of HIV-1, we compared whether a mucosal vaccine could induce mucosal cytotoxic T lymphocytes (CTLs) and protect rhesus macaques against mucosal infection with simian/human immunodeficiency virus (SHIV) more effectively than the same vaccine given subcutaneously. Here we show that mucosal CTLs specific for simian immunodeficiency virus can be induced by intrarectal immunization of macaques with a synthetic-peptide vaccine incorporating the LT(R192G) adjuvant. This response correlated with the level of T-helper response. After intrarectal challenge with pathogenic SHIV-Ku2, viral titers were eliminated more completely (to undetectable levels) both in blood and intestine, a major reservoir for virus replication, in intrarectally immunized animals than in subcutaneously immunized or control macaques. Moreover, CD4+ T cells were better preserved. Thus, induction of CTLs in the intestinal mucosa, a key site of virus replication, with a mucosal AIDS vaccine ameliorates infection by SHIV in non-human primates.

Complementation of bipartite begomovirus movement functions by topocuviruses and curtoviruses.

The DNA A components of bipartite species of the genus Begomovirus (family Geminiviridae) encode all viral functions required for replication and encapsidation but require gene products encoded on DNA B for cell-to-cell movement in plants. We demonstrate that geminiviruses of the genera Curtovirus and Topocuvirus are able to trans-complement efficient movement of DNA A for two bipartite begomoviruses in the absence of their corresponding DNA B. It previously has been shown that DNA A can, at low efficiency and without inducing symptoms, spread in the absence of DNA B. The spread of DNA A independent of DNA B, following Agrobacterium-mediated inoculation, is shown to require coat protein whereas trans-complemented spread of DNA A can occur independent of the coat protein encoded on DNA A. The significance of these findings to our understanding of the geminiviral infection cycle is discussed.

Efflux-mediated drug resistance in Gram-positive bacteria.

Gram-positive bacteria express numerous membrane transporters that promote the efflux of various drugs, including many antibiotics, from the cell to the outer medium. Drug transporters can be specific to a particular drug, or can have broad specificity, as in so-called multidrug transporters. This broad specificity can be a consequence of the hydrophobic nature of transported molecules, as suggested by recent structural studies of soluble multidrug-binding proteins. Although the functions of drug transporters may involve both the protection of bacteria from outside toxins and the transport of natural metabolites, their clinical importance lies largely in providing Gram-positive pathogens with resistance to macrolides, tetracyclines and fluoroquinolones. A number of agents, discovered in recent years, that inhibit drug transporters can potentially be used to overcome efflux-associated antibiotic resistance.

HV(MNE), a novel lymphocryptovirus related to Epstein-Barr virus, induces lymphoma in New Zealand White rabbits.

HV(MNE) is a novel Epstein-Barr (EBV)-like virus isolated from a Macaca nemestrina with CD8(+) T-cell mycosis fungoides-cutaneous T-cell lymphoma. Here it is demonstrated that intravenous inoculation of irradiated HV(MNE)-infected T cells or cell-free virus from the J94356(PBMC) cell line in New Zealand White rabbits results in seroconversion to the viral capsid antigen (VCA) of EBV; all animals that seroconverted to VCA developed malignant lymphoma within months of inoculation. In contrast, control rabbits, inoculated with heat-inactivated culture supernatants from the same cell line, failed to seroconvert to VCA and did not develop disease. Disseminated lymphoma cells of mixed origin were detected in most vital organs, including the spleen, liver, lungs, kidneys, and heart of the affected rabbits. Neoplastic infiltrates were also observed in lymph nodes, thymus, skin, and subcutaneous tissues. HV(MNE) DNA and EBV-like RNA expression was demonstrated in the lymphomatous organs and in 2 transformed T-cell lines, one established from the lymph node and the other from the blood of the 2 lymphomatous animals. Analysis of one of these T-cell lines demonstrated the persistence of HV(MNE) DNA, expression of an LMP1-like protein, and acquisition of interleukin-2 independence, and constitutive activation of the Jak/STAT pathway. Thus, HV(MNE) in rabbits provides a valuable animal model for human T-cell lymphoma whereby genetic determinants for T-cell transformation by this EBV-like animal virus can be studied.

Identification of dna components required for induction of cotton leaf curl disease.

Cotton leaf curl disease (CLCuD) is a major constraint to cotton production in Pakistan. Infectious clones of the monopartite begomovirus cotton leaf curl virus (CLCuV), associated with diseased cotton, are unable to induce typical symptoms in host plants. We have identified and isolated a single-stranded DNA molecule approximately 1350 nucleotides in length which, when coinoculated with the begomovirus to cotton, induces symptoms typical of CLCuD, including vein swelling, vein darkening, leaf curling, and enations. This molecule (termed DNA beta) requires the begomovirus for replication and encapsidation. The CLCuV/DNA 1/DNA beta complex, together with a similar complex previously identified in Ageratum conyzoides, represent members of an entirely new type of infectious, disease-causing agents. The implications of this finding to our understanding of the evolution of new disease-causing agents are discussed.

A conformational C4 peptide polymer vaccine coupled with live recombinant vector priming is immunogenic but does not protect against rectal SIV challenge.

The conserved, immunogenic CD4 binding site on the viral envelope is an attractive HIV or SIV vaccine candidate. Polymerization of an 18 amino acid segment derived from the C4 domain of SIV gp120 produced a peptide polymer or "peptomer," having an alpha-helical conformation possibly mimicking a proposed structure of the C4 domain in the unbound native protein. The SIV peptomer and native gp120 were compared as subunit boosts following two adenovirus type 5 host range (Ad5hr)-SIVenv recombinant priming immunizations. Both vaccine regimens successfully elicited SIV-specific CTL responses in five of six immunized macaques. Peptomer-boosted macaques exhibited significantly higher envelope-specific T cell proliferative responses than either the gp120-boosted macaques or controls. Peptomer immunization also elicited peptomer and SIV gp120-specific binding antibodies, but only native gp120 boosting elicited SIV neutralizing antibodies. Upon intrarectal challenge with SIVmac32H, all nine macaques became infected. The solely envelope-based vaccine conferred no protection. However, changing the boosting immunogen to the C4 peptomer did not improve protective efficacy in spite of its elicitation of humoral and cellular immune responses, including robust T-helper activity. In spite of the peptomer's strong immunogenicity and potential for induction of broadly protective immune responses, it was not effective as a subunit vaccine.

Molecular epidemiology of Salmonella Heidelberg in an equine hospital.

From 1992 to 1997, multi-drug resistant (MDR) Salmonella Heidelberg isolates were cultured from a number of horses hospitalised in a veterinary hospital in Victoria, Australia. To examine the relationships between the cases, 28 isolates from the hospital were compared by pulsed field gel electrophoresis (PFGE), IS200 element profiles, antimicrobial resistance patterns, plasmid profiles and phage typing. The PFGE patterns following digestion with XbaI and BlnI restriction endonucleases showed that the isolates from the veterinary hospital originated from a common source. These isolates also had indistinguishable IS200 profiles. However, PFGE was more discriminatory than IS200 profiles. All the veterinary hospital isolates and one independent isolate had the same antimicrobial resistance pattern and had at least one plasmid in common. Localisation of antimicrobial resistance genes indicated that the veterinary hospital isolates had more than one plasmid carrying resistance genes and that the genes encoding sulphathiazole and trimethoprim resistance were not on these plasmids. Phage typing was ineffective as 22 of the 28 isolates were untypeable. In conclusion, the combination of different methods used for epidemiological studies suggested that a single strain of MDR S. Heidelberg was isolated from horses admitted to the hospital for 6 years and caused salmonellosis in susceptible horses within that period with no apparent correlation between the antimicrobials used and retention of its MDR phenotype.