Louise Kruse Jensen

Non-lethal infection parameters in mice separate sheep Type II Toxoplasma gondii isolates by virulence

The zoonotic protozoan parasite Toxoplasma gondii can infect all warm-blooded animals, but virulence of isolates has previously been characterised mainly by the ability to kill mice after experimental infections. In the present study, 15 Type II strains of T. gondii, isolated from five adult sheep, six sheep abortions, two pigs, one cat and one fox were examined for their virulence to young mice by less dramatic parameters. Clinical disease of inoculated mice, directly evidenced by reduced weight gain, was correlated to increase in serum level of haptoglobin and level of specific antibodies. Although Type II T. gondii strains are non-virulent to mice by lethality studies, significant differences in mouse virulence were observed between the strains of T. gondii isolated either from adult sheep or from sheep abortions. It was not possible to characterise strains isolated from sheep abortions as being more or less virulent than strains isolated from adult slaughter sheep.

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.

Monoclonal antibodies to Toxoplasma gondii strain 119 identify recently isolated Danish strains as one group

Four mAb raised against the Danish Toxoplasma gondii strain 119, were selected by screening hybridoma supernatants by indirect immunofluorescence against tachyzoites of the RH strain in order to obtain strain-restricted markers. Strain restriction extended beyond discrimination of the 119 and RH strains, as demonstrated on a further six T. gondii reference strains [BK and GT1 (group A), NTE and 561 (group B), and NED and C56 (group C)]. The bradyzoite-specific mAb, 4.3, reacted to the GT1, NTE and 561 strains, but not to the BK, NED or C56 strains. The tachyzoite-specific mAb, 4.25, reacted to all strains tested except the RH strain, while mAb 5.1 reacted to tachyzoites of strains NTE and 561, but not to those of the BK, GT1, NED or C56 strains. Monoclonal antibody 5.15 reacted with the same strain restriction as monoclonal antibody 5.1, but to bradyzoites as well as tachyzoites. A T. gondii strain collection representative for a small geographic area (Denmark) was established within a short time span from a variety of animal species. Using the mAb as typing reagents to this Danish strain collection, all 36 animal and two human strains were identified as having the same reaction pattern as strains 119, NTE and 561.

Effects of Implant-associated Osteomyelitis on Cefuroxime Bone Pharmacokinetics: Assessment in a Porcine Model

BACKGROUND The prolonged antibiotic therapy that is often needed for successful management of osteomyelitis may be related to incomplete penetration of antibiotics into the target site. The objective of this study was to assess the effects of implant-associated osteomyelitis on cefuroxime penetration into bone. METHODS Implant-associated osteomyelitis using a Staphylococcus aureus strain was induced in the right tibia in ten pigs. After five days and following administration of 1500 mg of cefuroxime, measurements of cefuroxime were obtained using microdialysis for eight hours in the implant-related bone cavity, in the adjacent infected cancellous bone and infected subcutaneous tissue, and in healthy cancellous bone and subcutaneous tissue in the contralateral leg. Measurements of the corresponding free plasma concentrations were also obtained. The extent of the infection was assessed by postmortem computed tomography (CT) scans and cultures of blood, swabs, and bone specimens. RESULTS Bone destruction was found in the implant cavities. No structural bone changes in the adjacent infected cancellous bone were visible on CT scans. S. aureus was grown on culture of specimens from all implant cavities and from eight of ten swabs and seven of ten bone samples from the infected bone. The areas under the concentration-time curves for the different tissues differed significantly, with the lowest area under the curve found in the implant cavity (analysis of variance; p < 0.001). Although not as notable as for the implant cavity, cefuroxime penetration into infected cancellous bone was incomplete but comparable with that in healthy bone. Despite poorer tissue penetration, slightly increased time with concentrations above the minimal inhibitory concentration (MIC) was achieved in the implant cavity up to MICs of 2 mg/L compared with the other tissues, but the time was shorter for higher MICs. CONCLUSIONS Cefuroxime penetration into infected cancellous bone was incomplete but comparable with that in healthy bone. The destructive bone processes associated with acute osteomyelitis reduced cefuroxime penetration further. CLINICAL RELEVANCE These findings support the general clinical perception that fast diagnosis and early initiation of antibiotics before the development of implant-associated cavities is important in nonsurgical management of acute osteomyelitis.

Functional Characterization of a Porcine Emphysema Model

BackgroundLung emphysema is a central feature of chronic obstructive pulmonary disease (COPD), a frequent human disease worldwide. Cigarette smoking is the major cause of COPD, but genetic predisposition seems to be an important factor. Mutations in surfactant protein genes have been linked to COPD phenotypes in humans. Also, the catalytic activities of metalloproteinases (MMPs) are central in the pathogenesis of emphysema/COPD. Especially MMP9, but also MMP2, MMP7, and MMP12 seem to be involved in human emphysema. MMP12−/− mice are protected from smoke-induced emphysema. ITGB6−/− mice spontaneously develop age-related lung emphysema due to lack of ITGB6-TGF-β1 regulation of the MMP12 expression.MethodsA mutated pig phenotype characterized by age-related lung emphysema and resembling the ITGB6−/− mouse has been described previously. To investigate the emphysema pathogenesis in this pig model, we examined the expression of MMP2, MMP7, MMP9, MMP12, and TGF-β1 by quantitative PCR (qPCR). In addition, immunohistochemical stainings of the lungs with SP-B, SP-C, MMP9, and MMP12 antibodies were performed. The haematologic/immunologic status of the pigs also was studied.ResultsThe qPCR study showed no difference between pigs with and without emphysema, and no systemic differences were indicated by the haematologic and immunologic studies. However, the immunohistochemical stainings showed an increased expression of MMP9 and MMP12 in older, mutated pigs (with emphysema) compared with normal and young mutated pigs (without emphysema).ConclusionsThe pig model is comparable to human emphysema patients and the ITGB6−/− mouse model with respect to both morphology and functionality.

Systemic inflammatory response and local cytokine expression in porcine models of endocarditis

The knowledge of systemic inflammation and local cytokine expression in porcine endocarditis models is limited, though it could provide valuable information about the pathogenesis and comparability to human endocarditis. Analyses of bacteriology and hematology were performed on blood samples from pigs with non‐bacterial thrombotic endocarditis (NBTE, n = 11), Staphylococcus aureus infective endocarditis (IE, n = 2), animals with S. aureus sepsis without endocarditis (n = 2) and controls (n = 2). Furthermore, immunohistochemistry was used to examine the local expression of IL‐1β and IL‐8. Bacterial blood cultures were continuously positive in IE pigs from inoculation to euthanasia, and negative in all other pigs at all times. The total white blood cell counts and total neutrophil counts were massively elevated in pigs with IE. Local IL‐1β and IL‐8 expression in IE pigs were moderate to high, and high, respectively. In addition, slight local expression of IL‐1β and IL‐8 was present in some NBTE pigs. In the IE model, both the systemic inflammatory response and the high local expression of IL‐8 were comparable to the human disease. Furthermore, the results indicate IL‐1β and IL‐8 as important contributors in the endocarditis pathogenesis.

A novel porcine model of implant associated osteomyelitis: a comprehensive analysis of local, regional and systemic response

Pigs are favorable experimental animals for infectious diseases in humans. However, implant‐associated osteomyelitis (IAO) models in pigs have only been evaluated using high‐inoculum infection (>108 CFU) models in 1975 and 1993. Therefore, the aim of this paper was to present a new low inoculum porcine model of human IAO based on 42 experimental pigs. The model was created by drilling an implant cavity in the tibial bone followed by insertion of a small steel implant and simultaneous inoculation of Staphylococcus aureus bacteria (n = 32) or saline (n = 10). The infected pigs were either inoculated with 104 CFU (n = 26) or 102 and 103 CFU (n = 6). All animals were euthanized 5 days after insertion of implants. Pigs receiving the high‐inoculum infections showed a significantly higher volume of bone lesion, number of neutrophils around the implant, concentrations of acute phase proteins in serum, and enlargement of regional lymph nodes. A positive correlation was present between a high number of surrounding neutrophils and high values of all other parameters. Furthermore, a threshold of 40 neutrophils per 10 high power fields for the histopathological diagnosis of high grade IAO was defined. In conclusion: This paper describes a novel low‐inoculum S. aureus porcine model of IAO which was demonstrated to be reliable, reproducible and discriminative to human IAO, and represents a requested and valuable tool in orthopedic research.

A study of virulence parameters for Toxoplasma gondii infections in mice.

Abstract With the aim of establishing assessments of the virulence of Toxoplasma gondii isolates for mice, we investigated weight loss, serum levels of haptoglobin, and serum levels of tumor necrosis factor-alpha (TNF-α) as alternative parameters to mouse mortality. Groups of BALB/c mice were inoculated i.p. with increasing parasite doses (5 × 101, 104, and 106) of isolate 119 and with a low dose (5 × 101) of isolates GT1 and NED. The inoculation dose was inversely correlated with the interval to the onset of weight loss (starting at days 9, 7, and 5, respectively) and the onset of increase in serum haptoglobin (starting at about days 8, 6, and 4, respectively). The GT1 strain (inducing 100% mortality at day 9) also gave the fastest response in terms of weight loss (onset by day 8) and increase in serum haptoglobin (onset by day 6), which occurred 1–2 days before these parameters were affected in the other low-dose groups. The low-dose NED and 119 inoculations were distinguished by continued weight loss, which lasted until day 20 in the former group (max. average weight loss 2.9 g as compared with 0.4 g).

Porcine Models of Biofilm Infections with Focus on Pathomorphology

Bacterial biofilm formation is one of the main reasons for a negative treatment outcome and a high recurrence rate for many chronic infections in humans. The optimal way to study both the biofilm forming bacteria and the host response simultaneously is by using discriminative, reliable, and reproducible animal models of the infections. In this review, the advantages of in vivo studies are compared to in vitro studies of biofilm formation in infectious diseases. The pig is the animal of choice when developing and applying large animal models of infectious diseases due to its similarity of anatomy, physiology, and immune system to humans. Furthermore, conventional pigs spontaneously develop many of the same chronic bacterial infections as seen in humans. Therefore, in this review porcine models of five different infectious diseases all associated with biofilm formation and chronicity in humans are described. The infectious diseases are: chronic wounds, endocarditis, pyelonephritis, hematogenous osteomyelitis, and implant-associated osteomyelitis (IAO).

Whole-Genome Sequence of Staphylococcus aureus S54F9 Isolated from a Chronic Disseminated Porcine Lung Abscess and Used in Human Infection Models

ABSTRACT We obtained a draft genome sequence of Staphylococcus aureus strain S54F9, which was isolated from a chronic disseminated porcine lung abscess and used in porcine infection models. Genes coding for a number of toxins, including enterotoxins and superantigen, were demonstrated in this strain.