Immunization against Natural Helicobacter pylori Infection in Nonhuman Primates

Helicobacter pylori infection is widespread in some breeding groups of a rhesus monkey colony (71% H. pylori positive by 1 year), and the rate of seroconversion is also high. As a result, these groups can be used to test the safety and efficacy of an anti-H. pylori vaccine. Nine-month-old female animals were randomized to receive either 8 mg of recombinant urease (rUre) plus 25 μg of Escherichia coli heat-labile enterotoxin (LT) (n = 26) or placebo plus LT (n = 29), given four times at 1-week intervals followed by a booster 1 month later. Ten months after the start of the immunization, the animals were subjected to endoscopy and biopsy samples were obtained. H. pylori negativity was defined as no H. pylori growth by culture and no H. pylori observed at histology. By this criterion, 2 (7%) of 29 animals receiving placebo and 8 (31%) of 26 immunized animals were H. pylori negative (P < 0.035). In addition, antral gastritis score was significantly less in H. pylori-negative immunized monkeys than in H. pylori-positive animals, whether they were given rUre plus LT or placebo plus LT (P < 0.02 or P < 0.01, respectively). Interestingly, antral gastritis was also significantly less in H. pylori-positive animals given rUre plus LT than in H. pylori-positive animals given placebo plus LT (P < 0.02). However, quantitative cultures did not demonstrate significant differences between the two latter groups. It is concluded that oral administration of rUre vaccine plus LT significantly protects nonhuman primates against H. pylori infection while not causing undesirable side effects.     

Yersinia pseudotuberculosis Efficiently Escapes Polymorphonuclear Neutrophils during Early Infection

The human-pathogenic species of the Gram-negative genus Yersinia preferentially target and inactivate cells of the innate immune defense, suggesting that this is a critical step by which these bacteria avoid elimination and cause disease. In this study, bacterial interactions with dendritic cells, macrophages, and polymorphonuclear neutrophils (PMNs) in intestinal lymphoid tissues during early Yersinia pseudotuberculosis infection were analyzed. Wild-type bacteria were shown to interact mainly with dendritic cells, but not with PMNs, on day 1 postinfection, while avirulent yopH and yopE mutants interacted with PMNs as well as with dendritic cells. To unravel the role of PMNs during the early phase of infection, we depleted mice of PMNs by using an anti-Ly6G antibody, after which we could see more-efficient initial colonization by the wild-type strain as well as by yopH, yopE, and yopK mutants on day 1 postinfection. Dissemination of yopH, yopE, and yopK mutants from the intestinal compartments to mesenteric lymph nodes was faster in PMN-depleted mice than in undepleted mice, emphasizing the importance of effective targeting of PMNs by these Yersinia outer proteins (Yops). In conclusion, escape from interaction with PMNs due to the action of YopH, YopE, and YopK is a key feature of pathogenic Yersinia species that allows colonization and effective dissemination.     

Colonization of Cecum Is Important for Development of Persistent Infection by Yersinia pseudotuberculosis

Yersiniosis is a human disease caused by the bacterium Yersinia pseudotuberculosis or Yersinia enterocolitica. The infection is usually resolved but can lead to postinfectious sequelae, including reactive arthritis and erythema nodosum. The commonly used Yersinia mouse infection model mimics acute infection in humans to some extent but leads to systemic infection and eventual death. Here, we analyzed sublethal infection doses of Y. pseudotuberculosis in mice in real time using bioluminescent imaging and found that infections using these lower doses result in extended periods of asymptomatic infections in a fraction of mice. In a search for the site for bacterial persistence, we found that the cecum was the primary colonization site and was the site where the organism resided during a 115-day infection period. Persistent infection was accompanied by sustained fecal shedding of cultivable bacteria. Cecal patches were identified as the primary site for cecal colonization during persistence. Y. pseudotuberculosis bacteria were present in inflammatory lesions, in localized foci, or as single cells and also in neutrophil exudates in the cecal lumen. The chronically colonized cecum may serve as a reservoir for dissemination of infection to extraintestinal sites, and a chronic inflammatory state may trigger the onset of postinfectious sequelae. This novel mouse model for bacterial persistence in cecum has potential as an investigative tool to unveil a deeper understanding of bacterial adaptation and host immune defense mechanisms during persistent infection.     

Abdominal aortic aneurysm infected by Yersinia pseudotuberculosis

Infected aneurysms caused by Yersinia are very uncommon and are principally due to Yersinia enterocolitica. We describe the first case of an infected aneurysm caused by Yersinia pseudotuberculosis in an elderly patient with a history of atherosclerotic cardiovascular disease.     

Characterization of chromosomal regions conserved in Yersinia pseudotuberculosis and lost by Yersinia pestis

The transformation of the enteropathogenic bacterium Yersinia pseudotuberculosis into the plague bacillus, Yersinia pestis, has been accompanied by extensive genetic loss. This study focused on chromosomal regions conserved in Y. pseudotuberculosis and lost during its transformation into Y. pestis. An extensive PCR screening of 78 strains of the two species identified five regions (R1 to R5) and four open reading frames (ORFs; orf1 to orf4) that were conserved in Y. pseudotuberculosis and absent from Y. pestis. Their conservation in Y. pseudotuberculosis suggests a positive selective pressure and a role during the life cycle of this species. Attempts to delete two ORFs (orf3 and orf4) from the chromosome of strain IP32953 were unsuccessful, indicating that they are essential for its viability. The seven remaining loci were individually deleted from the IP32953 chromosome, and the ability of each mutant to grow in vitro and to kill mice upon intragastric infection was evaluated. Four loci (orf1, R2, R4, and R5) were not required for optimal growth or virulence of Y. pseudotuberculosis. In contrast, orf2, encoding a putative pseudouridylate synthase involved in RNA stability, was necessary for the optimal growth of IP32953 at 37°C in a chemically defined medium (M63S). Deletion of R1, a region predicted to encode the methionine salvage pathway, altered the mutant pathogenicity, suggesting that the availability of free methionine is severely restricted in vivo. R3, a region composed mostly of genes of unknown functions, was necessary for both optimal growth of Y. pseudotuberculosis at 37°C in M63S and for virulence. Therefore, despite their loss in Y. pestis, five of the nine Y. pseudotuberculosis-specific chromosomal loci studied play a role in the survival, growth, or virulence of this species.     

Plasmid-determined cytotoxicity in Yersinia pestis and Yersinia pseudotuberculosis.

Yersinia pestis KIM5 was found to be cytotoxic for the IC21 and P388D1 mouse macrophage cell lines, as well as for resident peritoneal macrophages from C57BL/6 mice. Affected cells phagocytosed KIM5 inefficiently, became spherical, detached readily from culture dishes, and retained 51Cr poorly. The cytotoxic effect was dependent on the presence of the 75-kilobase plasmid pCD1. Because this plasmid also encodes the low calcium response (LCR), three Mu d1 insertion mutants previously shown to be LCR- and of reduced virulence in mice were examined for cytotoxicity; all were found to be atoxic. The insertions in these mutants lie within three distinct LCR loci (lcrB, C, and D). Like LCR, cytotoxicity was expressed only at 37 degrees C. Unlike LCR, it was not influenced by Ca2+ concentration, indicating that the V and W antigens are probably not involved. Yersinia pseudotuberculosis was found to have a similar plasmid-dependent cytotoxicity. Thus, biological activity observed as cytotoxicity in vitro may well be a common feature contributing to virulence of the yersiniae.     

Susceptibility to Yersinia pseudotuberculosis Infection is Linked to the Pattern of Macrophage Activation

T helper 1 cells play a crucial role in the clearance of Yersinia pseudotuberculosis infection. By producing cytokines and presenting antigens to T cells, activated macrophages can orientate the adaptive immune response. The pathway used by macrophages to metabolize arginine has been employed as an important parameter to discriminate their activation state. In this study, the pattern of macrophage activation in Y. pseudotuberculosis- infected BALB/ c ( Yersinia -susceptible) and C57BL/6 ( Yersinia -resistant) mice and their immunostimulatory capacity were analysed. In the early phase of infection, macrophages obtained from C57BL/6 mice produced higher levels of NO, lower arginase activity, and larger amounts of IL-12 and TNF-α than macrophages from BALB/ c mice. On the other hand, macrophages derived from BALB/ c mice produced higher levels of IL-10 and TGF-β than C57BL/6 mice. The Y. pseudotuberculosis infection leads to a fall in the macrophage immunostimulatory capacity of both strains of mice, with T-cell proliferation significantly reduced 12 h after infection. Moreover, we observed in the supernatant of co-culture of macrophages from infected mice with T lymphocytes from heat-killed Yersinia -immunized mice lower IFN-γ production by cells from BALB/ c mice than by C57BL/6 mice, and IL-4 was produced only by BALB/ c mice on the first- and third-day post-infection. These results suggest that the pattern of macrophage activation is associated with susceptibility and resistance to Y. pseudotuberculosis infection in BALB/ c and C57BL/6 mice.     

Invasin expression in Yersinia pseudotuberculosis.

A 3.2-kb region on the chromosome of Yersinia pseudotuberculosis, called inv, encodes invasin, a 103-kDa protein of the bacterial outer membrane. Invasin mediates bacterial entry into cultured animal cells. Six Y. pseudotuberculosis strains isolated from animal or human infections were analyzed for the presence of inv-related sequences with a radiolabeled inv clone, pRI203. We found that inv-specific sequences were present in all strains studied. Strains cured of virulence plasmid pYV were studied by Western immunoblot analysis with a monoclonal antibody directed against invasin. All but one strain produced invasin, but some strains produced more invasin than others. A strong correlation was found between the level of invasin production by these strains and their ability to enter into HEp-2 or CHO cells. The virulence of these strains was assessed in a murine model by measuring the number of bacteria in the spleen after intravenous challenge or in the mesenteric lymph nodes after intragastric challenge. The capacities of strains to invade cultured mammalian cells and to colonize the spleen were strongly correlative. In contrast, the ability of strains to translocate from the intestinal lumen to the mesenteric lymph nodes after intragastric inoculation did not correlate with their in vitro invasiveness.     

Yersinia pseudotuberculosis enteritis in four calves

A Yersinia pseudotuberculosis enteritis in four yearling calves is described. This is the first report of this organism causing enteric disease in domestic cattle in North America.     

Lack of a Major Role of Staphylococcus aureus Panton-Valentine Leukocidin in Lower Respiratory Tract Infection in Nonhuman Primates

Panton-Valentine leukocidin (PVL) is a two-component cytolytic toxin epidemiologically linked to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, including serious invasive infections caused by the epidemic clone referred to as strain USA300. Although PVL has long been known to be a S. aureus virulence molecule in vitro, the relative contribution of this leukotoxin to invasive CA-MRSA infections such as pneumonia remains controversial. We developed a nonhuman primate model of CA-MRSA pneumonia and used it to test the hypothesis that PVL contributes to lower respiratory tract infections caused by S. aureus strain USA300. The lower respiratory tract disease observed in this monkey model mimicked the clinical and pathological features of early mild to moderate S. aureus pneumonia in humans, including fine-structure histopathology. In this experiment using a large sample of monkeys and multiple time points of examination, no involvement of PVL in virulence could be detected. Compared with the wild-type parental USA300 strain, the isogenic PVL deletion-mutant strain caused equivalent lower respiratory tract pathology. We conclude that PVL does not contribute to lower respiratory tract infection in this nonhuman primate model of human CA-MRSA pneumonia.Staphylococcus aureus is the most abundant cause of serious bacterial infections in the United States.^1–4 In addition, the organism acquires antibiotic resistance readily and methicillin-resistant S. aureus (MRSA) strains have been a major concern in health care facilities for decades.^3–7 The pathogen causes a wide spectrum of infections, ranging from mild skin problems to fatal invasive diseases.¹ The mortality rate associated with invasive MRSA infections is approximately 20%, the majority of which are health care– or hospital-associated.¹ Inasmuch as health care–associated S. aureus infections occur in individuals with predisposing risk factors, the health status of the patient plays a prominent role. In contrast, community-associated MRSA (CA-MRSA) strains generally cause infections in otherwise healthy individuals.CA-MRSA emerged unexpectedly in the 1990s and is now epidemic in the United States.^2,8,9 The prototype CA-MRSA strain in the United States, known as pulsed-field type USA400 (MW2), caused fatal pneumonia in children in the Midwest.^10,11 Whole-genome sequencing of a USA400 strain revealed the presence of a novel methicillin-resistance element SCCmecIV and lukS-PV and lukF-PV (lukS/F-PV) genes encoding a two-component cytolytic toxin known as Panton-Valentine leukocidin (PVL).¹² Although strains of USA400 remain a significant cause of CA-MRSA infections, especially in Canada,^13,14 they have been nearly replaced in the United States by the pulsed-field type USA300 epidemic clone (USA300).^9,15,16 Notably, SCCmecIV and PVL genes are also present in the genome of USA300,¹⁷ and thus, they are strongly linked by epidemiology to CA-MRSA infections in the United States. The great majority of USA300 infections involve skin and soft tissue, accounting for 75% to 95% of CA-MRSA infections.^8,16,18,19 However, this pathogen also has the ability to cause severe invasive disease, including cases of fatal pneumonia.^20,21Several lines of evidence suggest that PVL may be an important virulence factor in the pathogenesis of S. aureus pneumonia. Lina and colleagues first described an association of PVL with S. aureus strains that cause primary community-acquired pneumonia, some of which were fatal cases of necrotizing pneumonia.²² Subsequently, PVL-producing S. aureus, mostly methicillin-susceptible S. aureus strains, were reported to be associated with necrotizing pneumonia in young immunocompetent individuals.²³ Recent reports also document that PVL-positive strains of USA300 can cause necrotizing pneumonia, albeit these infections are infrequent and often associated with influenza or parainfluenza virus coinfection.²⁴Despite the epidemiological linkage, there is currently little direct experimental evidence that PVL is a primary virulence factor for CA-MRSA pneumonia. One study suggested that PVL promotes S. aureus pneumonia in a mouse infection model.²⁵ However, subsequent work by several investigators using wild-type USA300 and USA400 and isogenic PVL deletion mutant strains failed to support the idea that PVL contributes to experimental CA-MRSA pneumonia in mice or rats.^26–29Elucidating whether PVL has a role in pneumonia in humans is critical because the leukotoxin is widely considered to be a possible determining factor in CA-MRSA pneumonia, and therefore, it has become a target for antistaphylococcal therapeutics and vaccines.³⁰ We developed a nonhuman primate model of S. aureus lower respiratory tract infection and used it to test the hypothesis that USA300 wild-type and isogenic PVL mutant strains differ in their ability to cause CA-MRSA pneumonia.     

First Case of Postaneurysmal Prosthetic Vascular Infection Due to a Nonsuperantigenic Yersinia pseudotuberculosis Strain

Among Yersinia spp., Y. enterocolitica is the species most frequently isolated from infected aneurysms. This report describes the first case of postaneurysmal prosthetic vascular infection due to a superantigen-negative Yersinia pseudotuberculosis strain, showing a potential affinity of this species for endovascular tissue.     

Recognition of ESAT-6 Sequences by Antibodies in Sera of Tuberculous Nonhuman Primates

Previous work in our laboratory showed that the ESAT-6 protein of Mycobacterium tuberculosis and Mycobacterium bovis induces strong antibody responses in a large proportion (~90%) of experimentally or naturally infected nonhuman primates. Here, the antibody response to ESAT-6 in tuberculous monkeys was characterized at the epitope level by measuring antibodies to overlapping, synthetic peptides spanning the ESAT-6 sequence. The antibody response against the COOH-terminal portion of the protein was the strongest in both experimentally and naturally infected animals. Moreover, these antibodies became detectable the earliest during experimental infection, suggesting an ordered expansion of ESAT-6-specific B-cell clones in the course of infection. The data support use of synthetic peptides in lieu of the full-length ESAT-6 protein in diagnostic antibody detection assays.     

Protection of Nonhuman Primates against Two Species of Ebola Virus Infection with a Single Complex Adenovirus Vector

Ebola viruses are highly pathogenic viruses that cause outbreaks of hemorrhagic fever in humans and other primates. To meet the need for a vaccine against the several types of Ebola viruses that cause human diseases, we developed a multivalent vaccine candidate (EBO7) that expresses the glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus-based vector (CAdVax). We evaluated our vaccine in nonhuman primates against the parenteral and aerosol routes of lethal challenge. EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination. These results demonstrate the feasibility of creating a robust, multivalent Ebola virus vaccine that would be effective in the event of a natural virus outbreak or biological threat.The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), cause outbreaks of severe hemorrhagic fever disease in humans, with case-fatality rates that range up to 90%. Among the Ebolavirus genus, there are four distinct species: Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Reston ebolavirus (REBOV), and Cote d''Ivoire ebolavirus (CIEBOV) (10), with a possible fifth species identified in a recent outbreak in the Bundibugyo region of Western Uganda (34). Of these, ZEBOV and SEBOV are known to cause lethal disease in humans. The persistence of these viruses in nature is not well understood. Sporadic outbreaks due to EBOV have been occurring in Central Africa since the 1970s, but since the mid-1990s, the incidence of outbreaks has increased more than 4-fold (6, 7, 8, 42-45), and EBOV has spread aggressively throughout the great ape sanctuaries of West and Central Africa, decimating wild populations of gorillas and chimpanzees (2). While the filoviruses infect both humans and great apes, due to the high mortality rates of the infection, neither is thought to serve as reservoirs for these viruses but only as accidental hosts (16). Recent findings suggest that African fruit bats may serve as a reservoir host for filoviruses (2, 22); however, little is known about the nature of transmission to humans and nonhuman primates from bats or the likelihood of other reservoir species. In outbreak situations, filoviruses are believed to transmit from person to person mainly through contact with bodily fluids from infected patients. However, recent studies of Ebola outbreaks in wild apes have suggested that there could be other modes of transmission, including aerosol (2, 36). Studies in nonhuman primates have shown that EBOV and MARV can be spread through aerosolized droplets under controlled laboratory conditions (18, 21). So, despite the low incidence of infections globally, the lethality and potential airborne transmission of filoviruses in heavily populated areas makes them a significant biological threat, resulting in their placement on the Centers for Disease Control and Prevention list of Category A Bioterrorism Agents and the Department of Health and Human Services (DHHS) list of select agents and toxins. Concern is further compounded by the potential for these agents to be obtained from the wild (2, 22). In a biological threat scenario, aerosol transmission will likely be the main mode of viral dissemination, and protection against aerosolized EBOV would be of utmost importance (3). However, most previous vaccine candidates have only been evaluated for efficacy against intramuscular or intraperitoneal challenge and not against an aerosol challenge in nonhuman primates.At present, there are no licensed vaccines or specific antiviral treatments available for EBOV or MARV infections. However, significant progress has been made over the past few years in developing vaccine candidates that can protect nonhuman primates (NHPs) from lethal EBOV and MARV challenges (11, 17, 20, 23, 32, 40, 41). Most of the candidates utilize recombinant vaccine approaches that direct the protective immune response toward the surface glycoprotein (GP) of a single species of EBOV. Importantly, each species of EBOV is antigenically distinct, based on the sequences of the viral GP (10), and therefore, vaccines targeted against the GP of one species of the virus will not provide cross-protection against infection by another (19). Unique among the vaccine candidates is the recombinant complex adenovirus vaccine (CAdVax) system, which provides multivalent protection of NHPs against multiple species of filoviruses (33). The CAdVax vaccine platform is based on a complex, replication-defective adenovirus 5 (Ad5) vector (28-30, 37, 38) that allows for the incorporation of multiple gene inserts into the vector''s genome. Using this design, a bivalent vaccine vector (EBO7) was developed that expresses modified GP gene sequences of SEBOV and ZEBOV. When included in a novel pan-filovirus vaccine formulation, this vaccine was 100% protective in NHPs against two species of EBOV (ZEBOV and SEBOV) and two different strains of MARV (Musoke and Ci67) (33).In the study presented here, we further tested the protective efficacy of the CAdVax-based EBO7 vaccine in macaques by comparing aerosol to parenteral challenge. Aerosol challenge is potentially even more lethal than parenteral infection, because it induces hemorrhagic pneumonia. This is particularly true of SEBOV aerosol challenge, against which protection has not previously been demonstrated. In our studies, we have found that for either route of infection, the vaccine-induced bivalent anti-EBOV responses were protective against lethal challenge with either SEBOV or ZEBOV. This is the first report of a vaccine that is capable of protecting against aerosol SEBOV challenge. In addition, we found that EBO7 was also capable of protecting macaques with preexisting immunity to adenovirus against ZEBOV challenge. These results provide further insight into the feasibility of developing a fully protective multivalent EBOV vaccine using the CAdVax vaccine platform.     

Molecular Identification of Entamoeba spp. in Captive Nonhuman Primates

This study describes the molecular identification of 520 Entamoeba-positive fecal samples from a large and diverse population of captive nonhuman primates (NHP). The results revealed the presence of Entamoeba histolytica (NHP variant only), E. dispar, E. moshkovskii, E. hartmanni, E. coli, and E. polecki-like organisms.Various Entamoeba species are frequently found in the stools of both captive (15, 22) and wild (5, 7) nonhuman primates (NHP). Although the majority of these Entamoeba spp. are considered to be harmless, care should be taken when E. histolytica, the causative agents of amoebiasis, is involved. Infection with this gastrointestinal parasite in NHP may cause hemorrhagic dysentery (6, 28) and extraintestinal pathologies (e.g., liver abscesses) and death (12, 16). Moreover, amoebiasis is of major concern in public health, resulting in similar pathologies in humans and causing up to 100,000 deaths worldwide each year (19). Currently, little is known about the occurrence of E. histolytica in NHP, and the role of these animals as a potential reservoir for zoonotic transmission remains unclear. Most of the previous studies were based on the detection of cysts or trophozoites in stools by using light microscopy. However, differentiation between E. histolytica and other Entamoeba spp. (such as E. coli, E. hartmanni, and E. polecki-like organisms) based on morphological features is difficult (8, 27) and when E. dispar or E. moshkovskii is involved, it can even be impossible (4). For this purpose, molecular methods are more appropriate. Furthermore, recent molecular analyses of E. histolytica indicate genetic differences between human and NHP isolates (21, 23, 24). Although these differences may contribute to the elucidation of zoonotic transmission pathways, little is known about the distribution of these E. histolytica variants in both humans and NHP. Therefore, the objective of the present study was to identify the Entamoeba spp. in a large and diverse population of captive NHP, including differentiation between the human and NHP variants of E. histolytica.A total of 520 stool samples containing Entamoeba cysts were selected for further molecular identification. These samples were obtained from previous epidemiological surveys (10; unpublished data) and were stored at −20°C. The animals were housed in nine zoological gardens and one sanctuary in Belgium and the Netherlands, representing 58 NHP groups belonging to 36 animal species (see Table S1 in the supplemental material). None of the animals showed clinical signs associated with gastrointestinal disorders. DNA was extracted using the QIAamp stool minikit according the instructions of the manufacturer (Qiagen) and the adaptations described previously (9). The identification of E. histolytica, E. dispar, E. moshkovskii, E. hartmanii, E. coli, and E. polecki-like organisms was based largely on a previously described PCR-reverse line hybridization blot (PCR-RLHB) protocol targeting the small-subunit rRNA gene (26). This assay was preferred since it allows the simultaneous detection of various Entamoeba species. The amplification reactions were performed in a volume of 25 μl containing 2.5 μl DNA, 0.5 μl of each primer (10 μM), 1 μl MgCl₂ (25 mM), 5 μl GoTaq Flexi buffer, 14.875 μl PCR-grade H₂O, and 0.125 μl GoTaq Flexi DNA polymerase. For E. histolytica, a novel probe (5′-YAT TRA ATR AAT TGG CCA TTT TGT A-3′) was designed based on the gene sequences of the human variant (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"X64142","term_id":"296694","term_text":"X64142"}}X64142) and the NHP variant (GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"AB197936","term_id":"134026420","term_text":"AB197936"}}AB197936 and {"type":"entrez-nucleotide","attrs":{"text":"AB282657","term_id":"145698413","term_text":"AB282657"}}AB282657) to ensure the detection of both variants. In each PCR-RLHB run, control DNA samples from E. histolytica (both variants), E. dispar, E. moshkovskii, E. hartmanni, E. coli, and E. polecki-like organisms were included. Samples showing hybridization with the E. histolytica probe were retained for additional differentiation between the human and the NHP variants by using novel variant-specific reverse primers (human variant primer, 5′-CAT TTC TAG AAA CTT TAC TTA CAT-3′; NHP variant primer, 5′-CAT TTC TAG AAA CTT TAC TTA TGC-3′) designed from sequences with the GenBank accession numbers mentioned above. The amplification conditions remained unchanged. In each PCR run, control DNA samples from both the human variant and the NHP variant of E. histolytica were included. PCR products were run on agarose gels, stained with ethidium bromide, and detected upon UV transillumination. Samples reacting only with the general Entamoeba probe and not with any of the species-specific probes were retained for further sequence analyses. To this end, the PCR preceeding the RLHB assay was repeated with unlabeled primers. The obtained PCR products were purified with QIAquick purification columns (Qiagen, Germany) and cloned into the pGEM-T Easy vector according to the instructions of the manufacturer (Promega, Madison, WI). Clones containing the expected amplicon of approximately 550 bp were sequenced using the BigDye Terminator kit (Applied Biosystems). Sequence reactions were analyzed with an ABI-3730xl sequencer (Applied Biosystems), and sequences were assembled using Seqman II (DNAstar, Madison, WI).The RLHB analysis revealed the presence of Entamoeba DNA in 372 (71.5%) of 520 samples. The distribution of the different Entamoeba spp. within these 372 samples is described in Table ​Table1.1. E. hartmanni (present in 51.9% of samples) was the most prevalent species, followed by E. polecki-like organisms (in 42.7% of samples), E. histolytica (in 36.0% of samples), and E. coli (in 21.5% of samples). E. dispar (present in 2.4% of samples) and E. moskovskii (present in 1.9% of samples) were found in only a small number of samples. Most samples (51.9%) carried mixed infections. A large proportion of the samples (18.8%) hybridized with the general Entamoeba probe but could not be assigned to any of the known Entamoeba spp. The E. histolytica variant-specific PCR revealed solely the NHP variant in 124 of the 132 E. histolytica-positive samples. For the remaining 8 samples, no amplification was found in either PCR protocol.

TABLE 1.

Numbers of mono- and mixed infections with Entamoeba spp. in 372 samples based on a PCR-RLHB protocol targeting the small-subunit rRNA gene

Multiple outbreaks of Yersinia pseudotuberculosis infections in Finland

During 2001, 89 culture-confirmed cases of Yersinia pseudotuberculosis were reported in Finland; 55 (62%) were serotype O:1, and 34 (38%) were serotype O:3. Four major pulsed-field gel electrophoresis profiles were identified. A case-control study of 25 case patients and 71 healthy controls identified eating outside the home as a risk factor for infection.     

Human platelet aggregation by Yersinia pseudotuberculosis is mediated by invasin.

Plasmid-free strains of Yersinia pseudotuberculosis induce aggregation of human platelets in vitro. It appears that this phenomenon is mediated by invasin (Inv), a 103-kDa outer membrane protein that permits bacteria to penetrate mammalian cells, since (i) an isogenic inv-deficient mutant failed to aggregate platelets compared with the parental strain; (ii) a monoclonal antibody directed against invasin inhibited platelet aggregation; (iii) Inv+ Escherichia coli HB101 promoted platelet aggregation. Platelet receptors for invasin were identified by using a panel of anti-platelet glycoprotein monoclonal antibodies in a bacterial adhesion assay. We found that bacteria bind to platelet membrane glycoproteins Ic and IIa. Electron microscopic study of bacterium-platelet interactions also revealed that bacteria expressing invasin attach to and are phagocytized by thrombocytes, in contrast to inv-deficient bacteria, indicating that these anucleated cells are able to internalize bacteria in vitro after specific interaction with invasin.     

NKT cells-containing inflammatory lesions induced by Yersinia pseudotuberculosis glycolipids.

Valpha14-expressing NKT (invNKT) cells are a population of non-conventional T lymphocytes (TL) that bridge mammalian innate and adaptive immunity. Their role in infectious diseases and inflammatory processes is still largely ununderstood. A previous report has shown that an acute granulomatous-like reaction can be elicited by sub-cutaneous injection of Mycobacterium tuberculosis glycolipids in mice, and that recruitment of invNKT cells at the injection site is instrumental in this process. Here, we describe the mouse response to enterobacterium Yersinia pseudotuberculosis glycolipids extracts during the first week post injection. The cellular reaction is an acute inflammatory infiltrate where TL are abundant from early times on. InvNKT cells are present in the lesions, detectable as early as day 1 post injection. They compose all of the Valpha14-expressing TL, although conventional T cells expressing non-Valpha14 alpha-chains can be detected. The reaction is strictly dependent on ester-linked fatty acids as mild alkaline treatment of the extract prior to injection results in the absence of analysable lesions. Thus, glycolipids from Yersinia induce inflammatory lesions comparable to those induced by mycobacteria glycolipids, in spite of the totally different cell wall composition in the two genera. Moreover, the present findings show that invNKT cell response is not unique to mycobacterial glycolipids.