An outbreak of Salmonella München in Germany associated with raw pork meat.

In summer 2001, an outbreak of Salmonella München occurred in Germany. We conducted descriptive epidemiology and hypothesis-generating interviews among case patients, two retrospective cohort studies, and a case-control study of suboutbreaks. We performed pulsed-field gel electrophoresis (PFGE) from selected patient isolates and a limited trace-back investigation for analytical purposes. Four states were consecutively affected: Saxonia (SX), Brandenburg (BB), Berlin (BE), and Baden-Württemberg (BW). Although hypothesis-generating interviews failed to identify a plausible food item, descriptive data and investigations of the suboutbreaks suggested pork meat as a probable source in three states (SX, BB, and BE) but not in BW. The PFGE profiles from isolates of case patients in the first three states were indistinguishable but differed from PFGE profiles of case patients in BW. Trace-back investigation suggested that contamination of pork meat occurred early in the rearing-production chain. This outbreak demonstrates how contamination early in the production process that can yield different end products may complicate multistate outbreaks. Investigation of suboutbreaks and use of the trace-back method as investigational tools may be useful adjuncts in solving the problem of multistate outbreaks.

Cytokine and chemokine secretion by human peripheral blood cells in response to viable Echinococcus multilocularis metacestode vesicles.

In human alveolar echinococcosis, asexually proliferating metacestodes of Echinococcus multilocularis progressively infiltrate host tissues and cause serious pathology to the affected organs. This study employed an in vitro culture of E. multilocularis and examined the production of cytokines and chemokines by peripheral blood cells from echinococcosis patients in response to viable proliferating E. multilocularis metacestode vesicles (Em-vesicles). A significant interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) production was elicited in echinococcosis patients when their cells were cocultured with viable Em-vesicles and autologous immune sera. Furthermore, in echinococcosis patients, substantial amounts of cytokines were detected; and the levels of IL-12 and IL-13 found in patients correlated with the actual state of clinical disease. These observations suggest that viable E. multilocularis vesicles will induce significant cellular production of cytokines and chemokines in patients, and that such immune mediators may activate and enhance antibody-dependent cellular effector mechanism against proliferating metacestodes of E. multilocularis.

Subclass-specific serological reactivity and IgG4-specific antigen recognition in human echinococcosis.

Immunoglobulin (Ig) subclass-specific antibody responses and isotype-specific recognition of E. multilocularis (Em) and E. granulosus (Eg) antigens (Ag) were evaluated in both alveolar echinococcosis (AE) and cystic echinococcosis (CE). AE patients were divided into 3 groups by clinical and therapeutic criteria according to their actual state of infection, i.e. elimination of parasite, and regression or progression of disease, CE patients were either before or after surgery, of in continuous chemotherapy due to parasite persistence. Total IgE was highly elevated in progressive AE cases (7/11), but not in the cases with eliminated infection or regression. In AE patients with active disease, EmAg-specific IgE, total IgG, IgG1, IgG2 and IgG4 were particularly high. Similarly, in 9 of 30 CE patients, total IgE was raised above reference values, indicating progressive disease. CE patients" sera antibody cross-reacted with crude EmAg, and detectable Ig levels of the same isotype were also measured by ELISA. In both AE and CE, parasite-specific antigen recognition was dominated by IgG1 and IgG4. In AE patients with progressive disease, IgG4 distinctively recognized low molecular weight EmAg of Ar 26 kD, 18 kD, 16 kD and 12 kD. As prominent IgG4 and IgE responses develop with chronic helminth infections only, these serological parameters may indicate successful parasite infestation and severe outcome of disease. In summary, analyses of immunoglobulin isotype responses in AE patients by ELISA in combination with immunoblotting are a useful approach for post-treatment follow-up of patients at risk of developing recrudescent disease.

The diverse expression of immunity in humans at distinct states of Onchocerca volvulus infection.

This study examined the development and persistence of immunity in humans presenting defined states of Onchocerca volvulus infection, i.e. in exposed endemic control individuals without microfilaridermia and clinical disease, in patients with patent or post-patent onchocerciasis, and in patients concurrently infected with Mansonella perstans. Onchocerca volvulus antigen (OvAg)-specific cellular reactivity was significantly diminished in microfilariae (mf)-positive patients, while the highest reactivity was measured in exposed but mf-negative endemic controls, those being free of any clinical signs of onchocercal disease. In patients who became post-patent, responses to OvAg were significantly augmented, but did not approach entirely the magnitude observed in endemic controls. In onchocerciasis patients with concurrent mansonelliasis, cellular unresponsiveness to OvAg persisted, even when mf of O. volvulus were eliminated permanently by repeated ivermectin therapy. Cells from mf-positive onchocerciasis patients produced significantly less interferon-gamma (IFN-gamma) (P < 0.01) and interleukin-5 (IL-5) (P < 0.05) in response to OvAg than those taken from endemic controls or post-patent individuals in whom IFN-gamma and IL-5 production was similarly high. In contrast, both OvAg-driven as well as spontaneous IL-10 secretion was higher in mf-positive patients than in endemic controls or post-patent cases. In all individuals examined, serological recognition of OvAg by immunoglobulins was dominated by IgG4; in mf-positive patients OvAg of 205,000-12,000 molecular weight (MW) were strongly bound. In post-patent individuals, and similarly in endemic controls. OvAg recognition by IgG4 varied from intense (with numerous antigens being recognized) to weak or absent antigen binding. Significantly elevated OvAg-specific IgG isotypes were measured in mf-positive onchocerciasis patients in comparison with endemic controls or post-patent individuals (with the exception of IgG3). IgG1, IgG2 and IgE were higher, but IgG4 was lower in endemic controls compared with post-patent onchocerciasis patients. The ratios of IgG4/IgG1 differed (P < 0.001) between endemic controls and mf-positive or post-patent onchocerciasis patients, with IgG4/IgG1 ratios of R < 3.0 being characteristic for endemic controls and post-patent O. volvulus infection. In conclusion, this cross-sectional immunoepidemiological investigation showed that distinct states of O. volvulus infection correlate with a particular cellular and humoral immune response. The mf-free condition appeared to be associated with a vigorous parasite-specific cellular reactivity and a particular cytokine production profile, while concurrent M. perstans infection depressed OvAg-specific cellular responsiveness. Antibody responses, in all likelihood, reflected the intensity and state of infection, and not the degree of acquired immunity protective against parasite aggregation.

Ivermectin-facilitated immunity in onchocerciasis; activation of parasite-specific Th1-type responses with subclinical Onchocerca volvulus infection.

The present study examined the quantitative and qualitative changes registered in the parasite-specific antibody response, cellular reactivity and cytokine production profile in onchocerciasis patients repeatedly treated with ivermectin over a period of 8 years. The densities of Onchocerca volvulus microfilariae (mf) in treated patients remained significantly reduced, whereas the number of permanently amicrofilaridermic patients (subclinical infection) increased with repeated treatments. In vitro cellular responses to O. volvulus antigen (OvAg) were highest (P < 0.01) in untreated control individuals exposed to infection, but negative for mf of O. volvulus (endemic normals). Cellular reactivity in repeatedly treated patients was higher at 84 than at 36 months post initial treatment (p.i.t); furthermore, the proliferative responses to OvAg, mycobacterial purified protein derivative (PPD) and streptococcal SL-O were greater (P < 0.05) at 84 months p.i.t. in amicrofilaridermic than in microfilaria-positive onchocerciasis patients. In amicrofilaridermic patients such reactivity approached the magnitude observed in endemic normals. Peripheral blood mononuclear cells (PBMC) from patients and endemic normals produced equivalent amounts of IL-2, IL-4 and interferon-gamma (IFN-gamma) in response to mitogenic stimulation with phytohaemagglutinin (PHA); in response to OvAg, however, significantly more IL-2 and IFN-gamma were produced by PBMC from subclinical amicrofilaridermic patients or endemic normals than by mf-positive patients. OvAg-specific production of IL-4 by PBMC from treated patients was lower at 84 than at 36 months p.i.t. At three months p.i.t. the titres of circulating OvAg-specific IgG1-3 had increased (P < 0.05), but they then continuously declined with repeated treatments. Only IgG1 and IgG4 bound to OvAg of mol. wt 2-12 kD at 1 month p.i.t., while recognition of OvAg of mol. wt 10-200 kD by IgG1, IgG2 and IgG4 reached a maximum intensity at 3-6 months p.i.t., with the overall intensity of binding to OvAg gradually weakening thereafter. These results suggest that onchocerciasis-associated immunosuppression is reversible following ivermectin-induced permanent clearance of microfilariae from the skin; and that a vigorous parasite-specific cellular reactivity and a sustained production of IL-2 and IFN-gamma in amicrofilaridermic individuals may contribute to controlling O. volvulus infection.

Ivermectin-facilitated immunity in onchocerciasis. Reversal of lymphocytopenia, cellular anergy and deficient cytokine production after single treatment.

A longitudinal investigation has been conducted into the cell-mediated immune responses of onchocerciasis patients after a single-dose treatment with ivermectin. Untreated patients tested for delayed cutaneous hypersensitivity (DCH) to seven recall antigens showed lower responses than infection-free control individuals (P less than 0.01), but 6 and 14 months after treatment DCH reactions increased to similar levels to those seen in the controls. The in vitro cellular reactivity to Onchocerca volvulus-derived antigen (OvAg) was reduced in untreated patients as compared with controls, and the lymphocyte blastogenic responses to OvAg and streptolysin-O clearly improved up to 14 months after treatment. Peripheral blood mononuclear cells (PBMC) from untreated patients produced IL-1 beta, tumour necrosis factor-alpha (TNF-alpha) and IL-6 in response to mitogenic stimulation with phytohaemagglutinin (PHA), only low levels of IL-1 beta, IL-2 and TNF-alpha in response to OvAg, but higher amounts of IL-4 and interferon-gamma (IFN-gamma) in response to OvAg than control individuals. After ivermectin treatment, the OvAg-induced production of IL-1 beta and TNF-alpha increased significantly 1 and 14 months after treatment. The PHA-induced production of IL-2 and IL-4 increased 1 month after treatment and remained significantly elevated until 14 months after treatment, whereas the OvAg-specific secretion of IL-2, IL-4 and IFN-gamma did not change after ivermectin treatment. Flow cytometric analysis of lymphocyte-subsets in the peripheral blood of untreated patients revealed a relative and absolute (P less than 0.01) diminution of CD4+ cells and a significantly smaller CD4+/CD8+ cell ratio as compared with controls. By 4 weeks after treatment and thereafter, CD4+ T cells increased relatively and absolutely (P less than 0.01); likewise there was an absolute increase in T-helper-inducer cells (CD4+CD45RO+) and a temporarily improved CD4+/CD8+ cell ratio (P = 0.001). The expression of the low-affinity receptor for IgE (CD23) on total lymphocytes decreased from 14% to 7% by 14 months after treatment. The CD8+ cells and CD3+TCR gamma delta + cells were higher in patients than in controls and both remained elevated until 14 months after treatment. These results suggest a distinctly improved cellular immunity in human onchocerciasis that was facilitated by ivermectin therapy.

Experimental onchocerciasis in chimpanzees. Antibody response and antigen recognition after primary infection with Onchocerca volvulus.

Nine of 18 chimpanzees inoculated with 250 infective third-stage larvae (L3) each developed patent (i.e., positive for microfilariae) Onchocerca volvulus infection. Four of 6 infected chimpanzees that received 200 micrograms/kg ivermectin at 28 days postinfection (pi) became patent, whereas, when ivermectin was given concurrently with L3 challenge only 1 of 6 infected animals developed patent infection. The antibody response to O. volvulus adult worm-derived antigens (OvAg) showed clear differences between patent and nonpatent chimpanzees. Three months pi, all sera detected several OvAg in the range of M(r) 35-120 k. Sera collected 6 mo pi from later patent animals recognized increasing numbers of OvAg, especially in the lower MW range of M(r) 13 to 33 k. Beginning 10 months pi Onchocerca-antigens of M(r) 21, 24, 26, and 28 k were detected only by patent chimpanzee's sera. The antibody response in nonpatent chimpanzees consistently recognized fewer OvAg, most of which were limited to the higher M(r) range (35-120 k). The reactivity of sera from infected chimpanzees to a low molecular weight fraction (LMW) of total OvAg doubled within 6 months pi, and increased continuously in patent animals from 13 until 30 months pi. Serological reactivity of nonpatent animals to LMW-OvAg remained low. The titers of circulating IgG directed against total OvAg increased in all infected chimpanzees, and continued to rise with patency. In nonpatent chimpanzees the antibody production gradually returned to preinfection values. Total and OvAg-specific IgE increased in patent and nonpatent chimpanzees. Also, during prepatency the granulocyte and antibody-mediated in vitro killing of microfilariae of O. volvulus increased in subsequently patent chimpanzees. The in vitro immobilization of L3 remained low.

Experimental onchocerciasis in chimpanzees. Cell-mediated immune responses, and production and effects of IL-1 and IL-2 with Onchocerca volvulus infection.

Nine of eighteen chimpanzees inoculated with infective third-stage larvae of Onchocerca volvulus developed patent infection with microfilariae in skin biopsies. In all infected chimpanzees the in vitro cellular reactivity to O. volvulus adult worm-derived Ag (OvAg) increased significantly after exposure to third-stage larvae. However, during prepatency the in vitro cellular responses to OvAg decreased gradually in subsequently mf positive (patent) animals, and returned with patency to values not different to those before infection. In non-patent chimpanzees cellular responses remained significantly higher than before infection. Stimulation of PBMC in vitro with bacterial Ag and mitogen did not show any differences between the experimental groups through 20 months p.i. The addition of exogenous IL-2 did not restore the impaired responses of PBMC to OvAg in patent animals. Exogenous IL-2 elicited an additive increase of the cellular response to OvAg in nonpatent, and a mitogenic effect to OvAg in patent animals. Selective depletion of adherent, suppressor/cytotoxic (CD8+), NK cells (CD16+) and the use of autologous serum had no effect on antigenic and mitogenic cellular responsiveness. OvAg-induced IL-2 production decreased after patency, whereas, IL-1 production was significantly greater in both patent and nonpatent than in control chimpanzees. In summary, these data demonstrate that experimental O. volvulus infection in chimpanzees stimulated a substantial cell-mediated immune response. In patent chimpanzees an OvAg-specific cellular hyporesponsiveness occurred before onset of patency, possibly due to decreased IL-2 production and responsiveness.