Search

A method is described allowing the selective determination of four cathepsins (B, H, K, and L) in live cells. Adherently growing cells are incubated with partially selective substrates for each cathepsin (peptidic derivatives of 4-methoxy-β-naphthylamine) in microtiter plates together with nitrosalicylaldehyde. Using an appropriate reader, accumulating fluorescent products may be detected continously or by end point measurement. Selectivity is achieved by running parallel assays containing inhibitors that are partially selective for each of the cathepsins (in case of cathepsin H, the nonlysosomal aminopeptidases are inhibited by bestatin). Individual cathepsin activities can then be calculated by the difference between the uninhibited and the inhibited assay. The method was validated by measurements in cells isolated from cathepsin B-/--, K-/--, and L-/-- mice. This strategy suggests that the combination of two partially selective reaction partners, substrate and inhibitor, can yield selective cathepsin assays.

Einleitung: Chlamydia psittaci (Cp)-Infektionen konnen bei Mensch und Tier respiratorische Erkrankungen unterschiedlichen Schweregrades verursachen. Unter Verwendung eines etablierten Kalber-Modells [1, 2] waren die Ziele der vorliegenden Studie 1.) die pathophysiologische Charakterisierung der pulmonalen Reaktionsmechanismen sowie 2.) der klinische Vergleich mit naturlich infizierten Kalbern. Methoden: Je 21 Kalbern wurden 108 Einschluss-bildende Einheiten Cp oder Zellsuspension intrabronchial inokuliert. 1 – 2 Tage nach Inokulation (dpi) wurden drei gesunde, naive Sentinel-Kalber mit den Cp-infizierten Tieren vergesellschaftet. Ausgangswerte wurden ab einer Woche vor Inokulation gewonnen. Analysiert wurden Ergebnisse des klinischen Scorings, der nicht-invasiven Lungenfunktionsdiagnostik (Spirometrie, Kapnovolumetrie, Impulsoszillo-resistometrie) und der Blutproben (Zellkomposition, Konzentration des Lipopolysaccharid-bindenden Proteins (LBP), Gehalt chlamydialer DNA, Antikorper Bildung). Wahrend der Sektionen (n = 3 an 2, 3, 4, 7, 10, 14 & 35 dpi) wurde broncho-alveolare Lavageflussigkeit (BALF) gewonnen und zytologisch untersucht. Ergebnisse: Nach experimenteller Inokulation erreichte die fieberhafte, von respiratorischen Symptomen gepragte Allgemeininfektion ihren Hohepunkt 2 – 3 dpi und klang im Laufe einer Woche ab, ohne das Ausgangsniveau wieder zu erreichen. Dieser Verlauf spiegelte sich auch in der LBP-Konzentration wider. Trotz kurzen, flachen Atmungsmusters war 2 – 3dpi das Atemminutenvolumen erhoht. Anderungen der Atmungsmechanik resultierten aus Obstruktionen und Restriktionen, die bis 11dpi nachweisbar waren. Der Gehalt an Lymphozyten (L) im Blut sank nach Cp Inokulation signifikant (bis 4 dpi), wahrend stab- und segmentkernige neutrophile Granulozyten (N) anstiegen (2 dpi Leukozytose). Beide Zellfraktionen wurden zum Entzundungsherd Lunge rekrutiert (BALF: ↑N: 2 – 7 dpi, ↑L : 7 – 10 dpi). Nach Erregerkontakt war chlamdiale DNA bei 67% der Cp-exponierten Kalber im Blut nachweisbar, wobei der Gehalt in der experimentell infizierten Gruppe signifikant hoher war als bei den Sentinels, deren Infektion auch deutlich milder verlief. In beiden Gruppen fanden sich geringe DNA-Mengen bis zum Studienende (35 dpi) im Blut. Eine humorale Immunantwort war nur bei 67% der experimentell inokulierten Tiere nachweisbar, begann fruhestens 10 dpi und sank teilweise schon zum Studienende hin wieder ab. Diskussion: Die Freiheit von Symptomen nach Cp-Infektion erlaubt keinen Ruckschluss bezuglich der Elimination des Erregers oder dem Aufbau einer humoralen Immunantwort. Literatur: [1] Ostermann C et al. Vet J 2013; 196:351 – 359. [2] Reinhold P et al. PLoS One. 2012;7:e30125.7.

Chlamydia (C.) psittaci is the causative agent of psittacosis, a zoonotic disease in birds and man. In addition, C. psittaci has been repeatedly found in domestic animals and is, at least in calves, also able to induce respiratory disease. Knowledge about transmission routes in cattle herds is still deficient, and nothing is known about differences in host response after either experimental or natural exposure to C. psittaci. Therefore, our recently developed respiratory infection model was exploited to evaluate (i) the presence of the pathogen in blood, excretions and air, (ii) the possibility of transmission and (iii) clinical symptoms, acute phase and immune response until 5 weeks after exposure. In this prospective study, intrabronchial inoculation of 10(8) inclusion-forming units of C. psittaci (n = 21 calves) led to reproducible acute respiratory illness (of approximately one week), accompanied by a systemic inflammatory reaction with an innate immune response dominated by neutrophils. Excretion and/or exhalation of the pathogen was sufficient to transmit the infection to naïve sentinel calves (n = 3) co-housed with the infected animals. Sentinel calves developed mild to subclinical infections only. Notably, excretion of the pathogen, predominantly via feces, occurred more frequently in animals naturally exposed to C. psittaci (i.e. sentinels) as compared to experimentally-inoculated calves. The humoral immune response was generally weak, and did not emerge regularly following experimental infection; however, it was largely absent after naturally acquired infection.

This study aimed to establish and evaluate a bovine respiratory model of experimentally induced acute C. psittaci infection. Calves are natural hosts and pathogenesis may resemble the situation in humans. Intrabronchial inoculation of C. psittaci strain DC15 was performed in calves aged 2-3 months via bronchoscope at four different challenge doses from 10(6) to 10(9) inclusion-forming units (ifu) per animal. Control groups received either UV-inactivated C. psittaci or cell culture medium. While 10(6) ifu/calf resulted in a mild respiratory infection only, the doses of 10(7) and 10(8) induced fever, tachypnea, dry cough, and tachycardia that became apparent 2-3 days post inoculation (dpi) and lasted for about one week. In calves exposed to 10(9) ifu C. psittaci, the respiratory disease was accompanied by severe systemic illness (apathy, tremor, markedly reduced appetite). At the time point of most pronounced clinical signs (3 dpi) the extent of lung lesions was below 10% of pulmonary tissue in calves inoculated with 10(6) and 10(7) ifu, about 15% in calves inoculated with 10(8) and more than 30% in calves inoculated with 10(9) ifu C. psittaci. Beside clinical signs and pathologic lesions, the bacterial load of lung tissue and markers of pulmonary inflammation (i.e., cell counts, concentration of proteins and eicosanoids in broncho-alveolar lavage fluid) were positively associated with ifu of viable C. psittaci. While any effect of endotoxin has been ruled out, all effects could be attributed to infection by the replicating bacteria. In conclusion, the calf represents a suitable model of respiratory chlamydial infection. Dose titration revealed that both clinically latent and clinically manifest infection can be reproduced experimentally by either 10(6) or 10(8) ifu/calf of C. psittaci DC15 while doses above 10(8) ifu C. psittaci cannot be recommended for further studies for ethical reasons. This defined model of different clinical expressions of chlamydial infection allows studying host-pathogen interactions.