Transfer of maternal immunity to piglets is involved in early protection against Mycoplasma hyosynoviae infection.

Mycoplasma hyosynoviae causes arthritis in pigs older than 12 weeks. The role of colostrum in protection of piglets against M. hyosynoviae infection is not clear. Our objective was therefore to investigate whether transfer of maternal immunity to piglets was involved in early protection against the infection. Experimental infections were carried out in three groups of weaners receiving different levels of M. hyosynoviae-specific colostrum components; Group NC derived from Mycoplasma free sows and possessed no specific immunity to M. hyosynoviae. Group CAb pigs, siblings of the NC group, received colostrum with M. hyosynoviae-specific antibodies immediately after birth. Group CCE pigs were born and raised by infected sows and presumably had the full set of colostrally transferred factors, including specific antibodies. When 4½ weeks old, all pigs were inoculated intranasally with M. hyosynoviae. The course of infection was measured through clinical observations of lameness, cultivation of M. hyosynoviae from tonsils, blood and synovial fluid and observation for gross pathological lesions in selected joints. Specific immune status in the pigs was evaluated through detection of antibodies by immunoblotting and measurement of M. hyosynoviae-specific T-cell proliferation. The latter analysis may possibly indicate that M. hyosynoviae infection induces a T-cell response. The CCE piglets were significantly protected against development of lameness and pathology, as well as infection with M. hyosynoviae in tonsils, blood and joints, when compared to the two other groups. Raising the CCE pigs in an infected environment until weaning, with carrier sows as mothers, apparently made them resistant to M. hyosynoviae-arthritis when challenge-infected at 4½ weeks of age. More pigs in group NC had M. hyosynoviae related pathological lesions than in group CAb, a difference that was significant for cubital joints when analysed on joint type level. This finding indicates a partially protective effect of passively transferred M. hyosynoviae-specific colostral antibodies upon development of M. hyosynoviae related pathology. Thus, the level of passive immunity transferred from sow to piglet seems to provide, at least partial, protection against development of arthritis. It cannot be ruled out that the CCE pigs, by growing up in an infected environment, have had the chance to establish an active anti-M. hyosynoviae immune response that complements the maternally transferred immune factors. Evident from this study is that the general absence of M. hyosynoviae arthritis in piglets can be ascribed mainly to their immunological status.

New filtration system for efficient recovery of waterborne Cryptosporidium oocysts and Giardia cysts.

AIMS: To develop a filtration unit for efficient recovery of waterborne Cryptosporidium oocysts and Giardia cysts ((oo-)cysts) in drinking water. METHODS AND RESULTS: This unit utilizes a metallic filter and an ultrasound transducer for eluting (oo-)cysts, with a fixed retentate backwash volume; approx. 400 µl. Changes in the viability was evaluated by seeding wild type (oo-)cysts (1 × 10(4)) followed by sonication for 5, 10, 20 or 40 s (five replicates for each period). Flow cytometry analysis showed negligible increase in the mortality of (oo-)cysts exposed to 5-10 s of sonication. Recovery rate was assessed by seeding ColorSeed(™) (10 replicates) into the filter unit followed by air backwash to a glass slide and counting of (oo-)cysts by epifluorescent microscopy. High recovery rates (mean ± SD) were found: 84·9% ± 4·8 for Giardia cysts and 70% ± 6·5 for Cryptosporidium oocysts. DNA of seeded wild type (oo-)cysts (1 × 10(2); 10 replicates) was successfully amplified using real-time PCR. CONCLUSIONS: The use of a metallic filter, sonication and 'air backwash' were key factors for creating a highly efficient system for recovery of apparently undamaged protozoa. SIGNIFICANCE AND IMPACT OF THE STUDY: This reagent-less system can be used for monitoring of parasite contamination in drinking water.

Cell-mediated immune responses differentiate infections with Brucella suis from Yersinia enterocolitica serotype O:9 in pigs.

Due to almost identical lipopolysaccharide (LPS) O-antigens, infections with Yersinia enterocolitica serotype O:9 (YeO:9) cause false positive serological reactions (FPSR) in tests for Brucella and thus cause problems in National Brucella surveillance programs. As LPS are strong inducers of antibody responses it was hypothesized that cell-mediated immune responses to non-LPS antigens of the two bacteria can be used to separate immune responses to these two biologically very different infections. Following subclinical experimental infections with Brucella suis biovar 2, high interferon-gamma (IFN-gamma) assay responses with a commercial Brucella melitensis antigen preparation (Brucellergene OCB) preceded the development of antibodies. High IFN-gamma responses in the seven B. suis inoculated pigs with serological evidence of infection were consistent throughout a 20-week post-inoculation observation period. In contrast, IFN-gamma responses in two B. suis inoculated pigs without bacteriological or serological evidence of infection were below a cut-point of 25pg/ml at all samplings. IFN-gamma responses in repeated samplings from 5 uninfected control pigs and 18 pigs experimentally infected with YeO:9 were all negative, except for solitary false positives in 3.7% of the samples from both the experimentally YeO:9 infected pigs and control pigs. Skin tests using the same commercial Brucella antigen confirmed the ability of cell-mediated immune responses to differentiate between the two infections. In addition, a field evaluation of the diagnostic use of cell-mediated immune responses by IFN-gamma assay and skin test to resolve serological suspicions of Brucella was conducted in an YeO:9 infected pig herd. Following a screening of 200 pigs 39 pigs were identified with false positive serological Brucellosis reactions. While 36 of the 39 FPSR pigs were also FPSR in a second test, none of the pigs were test positive in whole blood IFN-gamma assay or Brucellergene OCB skin test. In conclusion, use of IFN-gamma assay and skin test as measurements of cell-mediated immune responses to non-LPS Brucella antigens were specific and sensitive in discriminating subclinical experimental infections with B. suis from both natural and experimental infections with YeO:9.

Differentiation between serological responses to Brucella suis and Yersinia enterocolitica serotype O:9 after natural or experimental infection in pigs.

False-positive serological reactions (FPSR) due to infections with Yersinia enterocolitica serotype Oratio9 (YeOratio9) are a problem in tests for brucellosis. In the present study, FPSR in classical and novel tests for brucellosis following experimental infections of pigs with YeOratio9 were compared with responses of B. suis biovar 2-inoculated pigs. FPSR were limited to 2-9 weeks post-YeOratio9 inoculation, while B. suis-infected pigs were test-positive throughout the 21-week period of investigation. Although YeOratio9-inoculated pigs exhibited FPSR in Brucella tests for a limited period of time, the serological responses in a YeOratio9-purified O-antigen indirect ELISA did not decrease accordingly. Analysis of available cross-sectional serum samples from pig herds naturally infected with YeOratio9 or B. suis biovar 2 confirmed that the observed difference in the duration of the serological responses between the two infections could be used to discriminate between herds infected with B. suis biovar 2 and YeOratio9.

Seroprevalence of brucellosis, tularemia, and yersiniosis in wild boars (Sus scrofa) from north-eastern Germany.

Brucellosis and tularemia are classical zoonotic diseases transmitted from an animal reservoir to humans. Both, wildlife and domestic animals, contribute to the spreading of these zoonoses. The surveillance of the animal health status is strictly regulated for domestic animals, whereas systematic disease monitoring in wildlife does not exist. The aim of the present study was to provide data on the prevalence of anti-Brucella, anti-Francisella and anti-Yersinia antibodies in wild boars from North-Eastern Germany to assess public health risks. A total of 763 sera of wild boars from Mecklenburg-Western Pomerania hunted in 1995/1996 were tested using a commercially available Brucella suis ELISA, an in-house lipopolysaccharide (LPS)-based Francisella ELISA, and commercially available Western blot kits for the detection of anti-Francisella and anti-Yersinia antibodies. The Yersinia enterocolitica O:9 LPS is able to induce serological cross-reactions indistinguishable from brucellosis due to a similar immunodominant epitope in the Brucella O-polysaccharide. The Yersinia Western blot assay was, therefore, based on five recombinant Yersinia outer proteins which have been proved to be specific for the serodiagnosis of yersiniosis. Anti-Brucella, anti-Francisella and anti-Yersinia antibodies were detected in 22.0%, 3.1%, and 62.6% of the wild boars, respectively. The high seroprevalence of tularemia and brucellosis in wild boars indicates that natural foci of these zoonoses are present in wildlife in Germany. However, the impact of transmission of zoonotic pathogens from wildlife to livestock is unknown. Only careful and systematic monitoring will help to prevent the (re)emergence of these zoonotic diseases in domestic animals and consequently human infection.

In utero infection with porcine reproductive and respiratory syndrome virus modulates leukocyte subpopulations in peripheral blood and bronchoalveolar fluid of surviving piglets.

It is well known that piglets congenitally infected with porcine reproductive and respiratory syndrome virus (PRRSV) can be viremic at birth, and that preweaning mortality due to secondary infections often increases during acute outbreaks of PRRS. Therefore, an immunosuppressive effect of in utero infection has been suggested. The aim of the present study was to characterise the changes of leukocyte populations in piglets surviving in utero infection with PRRSV. A total of 27 liveborn uninfected control piglets and 22 piglets infected transplacentally with a Danish strain of PRRSV were included. At 2 and 4 weeks of age, 21 of 22 (96%) and 7 of 14 (50%) examined infected piglets were still viremic, whereas PRRSV could not be detected in the six infected piglets examined at 6 weeks of age. Flow cytometry analysis was used to determine the phenotypic composition of leukocytes in peripheral blood and bronchoalveolar lavage fluid (BALF) of 2-, 4- and 6-week-old infected piglets and age-matched uninfected controls. The key observation in the present study is that high levels of CD8(+) cells constitute a dominant feature in peripheral blood and BALF of piglets surviving in utero infection with PRRSV. In BALF, the average high level of CD8(+) cells in 2-week-old infected piglets (33.4 +/- 12.6%) was followed by a decline to 7.3 +/- 3.0 and 11.1 +/- 3.0% at 4 and 6 weeks of age. BALF of control piglets contained 1.6 +/- 0.9, 2.3 +/- 1.8 and 1.9 +/- 0.5% CD8(+) cells, only. In peripheral blood, however, the average number of CD8(+) cells remained at high levels in the infected piglets throughout the post-natal experimental period (2.8 +/- 1.9, 2.9 +/- 1.8 and 3.2 +/- 1.7 x 10(6) CD8(+) cells/ml at 2, 4 and 6 weeks, respectively). In the controls, the average levels of CD8(+) cells were 0.9+/-0.2, 1.9 +/- 1.7 and 1.6 +/- 0.5 x 10(6)/ml, respectively. Furthermore, the numbers of CD2(+) , CD4(+)CD8(+) and SLA-classII(+) cells, respectively, in peripheral blood, together with the levels of CD2(+) and CD3(+) cells in BALF were increased in the infected piglets infected in utero compared to the uninfected controls. The kinetic analyses carried out in the present study reflect that in utero infection with PRRSV modulates immune cell populations in peripheral blood and BALF of surviving piglets. The observed changes are characterised by high levels of CD8(+) cells supporting an important role of these cells in PRRSV infection. The present results, however, do not support the existence of post-natal immunosuppression following in utero infection with PRRSV.

Aerosol challenge of calves with Haemophilus somnus and Mycoplasma dispar.

The aim of the study was to examine the ability of Haemophilus somnus and Mycoplasma dispar to induce pneumonia in healthy calves under conditions closely resembling the supposed natural way of infection, viz. by inhalation of aerosol droplets containing the microorganisms. The infections were investigated by recording clinical data, cytokine expression of peripheral blood cells and pathology. Twelve calves were included in the study: Three animals were exposed to H. somnus only, and two to M. dispar only, whereas five were challenged to M. dispar followed by exposure to H. somnus 11-14 days later. Also, one calf was exposed to M. dispar followed by exposure to a sterile saline solution 11 days later, and one calf was only exposed to a sterile saline solution. Just one animal, only challenged with H. somnus, developed a focal necrotizing pneumonia, from which H. somnus was isolated. Thus, the ability of H. somnus and M. dispar to act as primary pathogens under these conditions were minimal and inconsistent.However, a transient rise in body temperature, a marked granulocytosis and increased levels of interleukin-8 in peripheral blood after inoculation with H. somnus indicated a clear systemic response, probably as a consequence of the natural non-specific local and systemic defence mechanisms acting in healthy calves.

Pathogenicity of selected Toxoplasma gondii isolates in young pigs.

The pathogenicity in 7-week-old pigs to five different Toxoplasma gondii strains of various host species origin was compared after i.v. inoculation of 10(4) tachyzoites. Additionally, one group of pigs was inoculated i.v. with 10(6) tachyzoites of the reference strain, SSI 119. In response to the infection a significant effect of T. gondii tachyzoite inoculation dose as well as differences among strains could be observed in several parameters. The 10(6)-dose inoculated pigs showed variable degrees of clinical illness and recurrent episodes of fever 4-17 days p.i., while pigs of four of the 10(4) tachyzoite inoculated groups experienced a short-lived rise in body temperature from day 6-8 p.i. without any apparent illness or inappetence. Control pigs and pigs infected with the least pathogenic strain had normal body temperature throughout the experiment. In all inoculated pigs, T. gondii-specific IgM and IgG antibodies appeared from day 8-10 and 10-17 p.i., respectively. Serum levels of alkaline phosphatase and the acute phase protein haptoglobin were decreased or increased, respectively, in response to the infection. Differential leukocyte count on peripheral blood revealed a significant lymphocytopenia on day 6 p.i. equal to both CD4+ and CD8+ T-cells, but shifting towards a reduced ratio of CD4+/CD8+ T-cells from day 8-14 p.i. In the 10(6)-dose inoculated pigs a considerable increase in zymosan induced and spontaneous oxidative burst capacity of peripheral blood leukocytes was observed from 6 days p.i. compared with control pigs. Oxidative burst capacity was not examined for other pigs. In conclusion, several useful parameters to identify differences in T. gondii pathogenicity other than mortality were identified. Furthermore, even at low doses, significant differences between recently collected Danish T. gondii field isolates were demonstrated after i.v. inoculation in young pigs.

Interaction between Salmonella typhimurium and phagocytic cells in pigs. Phagocytosis, oxidative burst and killing in polymorphonuclear leukocytes and monocytes.

Interactions between Salmonella typhimurium and peripheral blood leucocytes from healthy, Salmonella-free pigs were investigated in vitro. Both granulocytes and monocytes phagocytized FITC-labelled heat-killed Salmonella bacteria as shown by flow cytometry. Phagocytosis in whole blood and isolated leucocytes was measured as acquired fluorescence in the leukocytes and was both time and dose related. Living, serum-opsonized Salmonella bacteria induced a dose-dependent oxidative burst in PMNs and monocytes as measured by luminol-enhanced chemiluminescence (LC). When opsonized in normal serum the Salmonella bacteria, in the range of 2-5 x 10(7) cfu, induced a LC response in monocytes comparable to the level of responses induced by phorbol myristate acetate (PMA) and opsonized zymosan, and the Salmonella-induced response was only marginally reduced by superoxide dismutase (SOD). Intracellular killing of Salmonella by monocytes was assessed from plate colony counts of lysed monocytes and showed that Salmonella typhimurium was able to survive and proliferate in adherent monocytes in vitro despite a reduction in intracellular cfu during the first hour's incubation in cells from some pigs. Experiments with the exhaustion of oxidative burst in non-adherent monocytes were performed by prestimulation with PMA, heat-killed Salmonella or buffer. Prestimulation with PMA led to a strong reduction in oxidative burst induced by living opsonized Salmonella bacteria, whereas prestimulation with heat-killed bacteria gave rise to an enhanced response. In these experiments intracellular killing of the added living Salmonella gave variable results, in that monocytes from two out of three pigs showed no essential change in intracellular bactericidal activity, but with cells from one pig a less pronounced bactericidal activity was found after prestimulation with PMA.

Comparison of adherent and non-adherent staphylococci in the induction of polymorphonuclear leukocyte activation in vitro.

The ability to consume complement and activate neutrophils was investigated for staphylococci adherent to silicone surfaces and non-adherent staphylococci. Staphylococcus epidermidis strain ATCC 14990 and Staphylococcus aureus strain E 2371 were used in this study. The bacteria were allowed to adhere to silicone catheter segments for 2 h at 37 degrees C. Complement consumption was measured by reduction in serum haemolytic activity against sheep red blood cells. The induction of chemiluminescence was measured after opsonization of the staphylococci in 20% AB-positive human serum for 60 min at 37 degrees C. The bacteria consumed complement to approximately the same extent when adherent to the catheter segments, but more slowly in comparison with planktonic bacteria. When planktonic bacteria were compared, complement was consumed more quickly by S. epidermidis than by S. aureus. Measuring the induction of chemiluminescence by planktonic bacteria, S. epidermidis induced a lower response than S. aureus, while when adherent to the catheter segments the bacteria induced similar responses. These responses were only 15 to 20% of those induced by planktonic bacteria and only slightly higher than the spontaneous chemiluminescence by the neutrophils. Inter-strain variation was found, but all strains induced about the same low chemiluminescence when adherent to the catheter segments. The reduction in inflammatory response caused by adherence of staphylococci to catheter segments may interfere with phagocytosis and elimination of S. epidermidis during the early establishment of a foreign body infection.

Effect of treatment with methicillin and gentamicin in a new experimental mouse model of foreign body infection.

A new mouse model of foreign body infection has been developed. Intraperitoneal placement of a silicone catheter followed by injection of 10(8) Staphylococcus aureus organisms resulted in a reproducible, localized foreign body infection. The infection persisted as an intra-abdominal abscess surrounding the catheter for at least 30 days. Treatment with up to nine doses of methicillin or gentamicin or both was started 3 days after infection. The treatment showed a significant effect (P < 0.05), measured as reduction of bacteria on the foreign body, for all three regimens with a reduction of up to 2 log units, but no synergism was observed. The result of the treatment was poor, despite the facts that the local concentrations of methicillin were greater than the MIC for at least 72 h and that nine peak concentrations of gentamicin of > 13 micrograms/ml were obtained. The poor result of the treatment was not caused by development of antibiotic resistance or influenced by protein concentration, pH, or local presence in the pus of inhibitors of antibiotics. Both antibiotics showed good effects in time-kill studies in vitro on bacteria on catheters taken out of infected mice and catheters infected in vitro. During treatment, the proportion of intracellular bacteria increased in all treated mice to 60 to 75% compared with 20 to 30% in nontreated mice (P < 0.05). This indicates that intracellular survival of staphylococci may influence the outcome of the treatment in foreign body infections.

Induction of oxidative burst response in human neutrophils by adherent staphylococci. Comparison between Staphylococcus epidermidis and Staphylococcus aureus.

The ability of staphylococci adherent to silicone surfaces to induce superoxide anion (O2-) production by polymorphonuclear leukocytes (PMNs) was investigated and compared with the same activity induced by planktonic bacteria. The responses to Staphylococcus aureus strain E 2371 and Staphylococcus epidermidis strain ATCC 14990 were compared. The staphylococci were allowed to adhere to silicone catheters for 2 h at 37 degrees C. After opsonization of adherent bacteria in 30% human AB-positive serum, the induction of superoxide anion production by PMNs was measured in a cytochrome C reduction assay. Both bacterial strains, when adhered to the surfaces, were able to induce superoxide anion production by PMNs to about the same extent. Comparing adherent and planktonic bacteria with these two bacterial strains, it was found that planktonic S. epidermidis induced one to three times higher superoxide anion production than the adherent bacteria, whereas planktonic S. aureus induced four to seven times higher superoxide anion production than the adherent bacteria. Interstrain variation between the response to adherent and planktonic staphylococci was found. The lower phagocytic response to adherent staphylococci as compared to the response to planktonic organisms may interfere with the killing process and thereby contribute to poor clearance of these bacteria when adherent to foreign bodies such as catheters.

Neutrophil chemotactic activity of peptidoglycan. A comparison between Staphylococcus aureus and Staphylococcus epidermidis.

The ability of peptidoglycan from Staphylococcus epidermidis and Staphylococcus aureus to generate in human serum chemoattractant for peripheral blood neutrophils was studied. It was shown that PG from the two bacteria was able to induce chemotactic activity in normal human serum. Sonication of PG was required to generate this activity. Very little or no activity was generated in heat-treated or C5-deficient human serum by PG, indicating that PG treatment of serum resulted in generation of chemoattractants by activation of complement. Kinetics studies employing C2-deficient or MgEGTA-chelated serum revealed that S. epidermidis induced chemotactic activity by activating the alternative complement pathway. The alternative complement activation induced by S. epidermidis occurred rapidly and was completed after 15 min, whereas S. aureus activated the alternative pathway much more slowly, with activation reaching a maximum at 60 min. The rapid activation of the alternative complement pathway by S. epidermidis PG may partly explain why this bacterium does not normally cause infections in healthy individuals.