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In rodent models of experimentally induced fever, the important role of interleukin-6 (IL-6) as a circulating endogenous pyrogen is well established. Studies employing larger animal species and real infections are scarce. Therefore, we assessed bioactive IL-6 in peripheral blood and in broncho-alveolar lavage fluid (BALF) of calves after intra-bronchial inoculation with vital Chlamydia psittaci (Cp), with inactivated Cp, or with BGM cells. Only calves inoculated with vital Cp developed fever (peak at 2-3 days after challenge) and significantly increased IL-6 activity. Controls inoculated with either inactivated Cp or BGM cells also expressed increased bioactive IL-6, but no fever developed. Activity of IL-6 in BALF was significantly higher compared to blood serum. This experimental model of Cp infection revealed no apparent relation between IL-6 in blood and body temperature, but did reveal a relation between IL-6 and other markers of inflammation in BALF. We conclude that a local inflammatory response in the lungs of infected calves caused fever, which developed by mechanisms including other mediators besides IL-6.

Infection of cattle with chlamydiae is ubiquitous and, even in the absence of clinical sequeleae, has a quantifiable negative impact on livestock productivity. Despite recent progress, our knowledge about immune response mechanisms capable of counteracting the infection and preventing its detrimental effects is still limited. A well-established model of bovine acute respiratory Chlamydia (C.) psittaci infection was used here to characterize the kinetics of the local and systemic immune reactions in calves. In the course of two weeks following inoculation, leukocyte surface marker expression was monitored by flow cytometry in blood and bronchoalveolar lavage fluid (BALF). Immune-related protein and receptor transcription were determined by quantitative real-time reverse transcription PCR in blood, BALF and lung tissue. An early increase of IL2RA, IL10 and HSPA1A mRNA expressions was followed by a rise of lymphocytes, monocytes, and granulocytes exhibiting activated phenotypes in blood. Monocytes showed elevated expression rates of CD11b, CD14 and MHC class II. The rates of CD62L expression on CD8hi T cells in blood and on CD4+ T cells in BALF were also augmented and peaked between 2 and 4 dpi. Notably, CD25 antigen expression was significantly elevated, not only on CD8dim/CD62L+ and CD8-/CD62L+ cells in blood, but also on granulocytes in blood and BALF between 2–3 dpi. From 4 dpi onwards, changes declined and the calves recovered from the infection until 10 dpi. The findings highlight the effectiveness of rapid local and systemic immune reaction and indicate activated T cells, monocytes and granulocytes being essential for rapid eradication of the C. psittaci infection.

Background Cumulating reports suggest that acute phase proteins (APPs) do not only play a role as systemic inflammatory mediators, but are also expressed in different tissues as local reaction to inflammatory stimuli. The present study aimed to evaluate presence and changes in luminal lung concentrations of the APPs haptoglobin (Hp), lipopolysaccharide binding protein (LBP), C-reactive protein (CRP), and lactoferrin (Lf) in calves with an acute respiratory disease experimentally induced by Chlamydia (C.) psittaci. Results Intra-bronchial inoculation of the pathogen resulted in a consistent respiratory illness. In venous blood of the infected calves (n = 13), concentrations of plasma proteins and serum LBP were assessed (i) before exposure and (ii) 8 times within 14 days after inoculation (dpi). Increasing clinical illness correlated significantly with increasing LBP—and decreasing albumin concentrations in blood, both verifying a systemic acute phase response. Broncho-alveolar lavage fluid (BALF) was obtained from all 13 calves experimentally infected with C. psittaci at 4, 9 and 14 dpi, and from 6 uninfected healthy calves. Concentrations of bovine serum albumin (BSA), Hp, LBP, CRP and Lf in BALF were determined by ELISA. In infected animals, absolute concentrations of LBP and Hp in BALF correlated significantly with the respiratory score. The quotient [LBP]/[BSA] in BALF peaked significantly in acutely infected animals (4 dpi), showed a time-dependent decrease during the recovery phase (9-14 dpi), and was significantly higher compared to healthy controls. Concentrations of Hp and Lf in BALF as well as [Hp]/[BSA]—and [Lf]/[BSA]-quotients decreased during the study in infected animals, but were never higher than in healthy controls. CRP concentrations and [CRP]/[BSA]-quotient did not express significant differences between infected and healthy animals or during the course of infection. Conclusion In conclusion, absolute concentrations of LBP in blood and BALF as well as the quotient [LBP]/[BSA] in BALF perfectly paralleled the clinical course of respiratory illness after infection. Beside LBP, the suitability of Hp and Lf as local biomarkers of respiratory infections in cattle and their role in the local response to pathogens is worth further investigation, while CRP does not seem to play a role in local defense mechanisms of the bovine lung.

The aim of this study was to evaluate pulmonary dysfunction induced by experimental infection with Parachlamydia acanthamoebae in calves. Intra-bronchial inoculation with P. acanthamoebae was performed in 31 calves aged 2–3 months old at two different challenge doses of 108 and 1010 inclusion-forming units (IFU) per animal. Control animals received heat-inactivated bacteria. The effects on pulmonary gas exchange were determined by arterial blood gas analysis and haemoximetry during the 7 days post inoculation (DPI). For pulmonary function testing (PFT), impulse oscillometry, capnography, and measurement of O2 uptake were undertaken in spontaneously breathing animals 7 and 3 days before inoculation, and were repeated until 10 DPI. In the early phase after challenge (1–3 DPI), mild hypoxaemia occurred, which was accompanied by a significant reduction in both tidal volume and alveolar volume (each related to bodyweight, BW). In parallel, expiratory flow rate and specific ventilation (i.e. minute ventilation related to O2 uptake) were significantly increased. Minute ventilation and alveolar ventilation (each related to metabolic BW) increased significantly, due to higher respiratory rates, lasting until 4 and 5 DPI, respectively. Oxygen uptake was slightly reduced during the first 2 days after challenge, but increased significantly during the recovery phase, from 4 to 8 DPI. No deterioration in respiratory mechanics or acid-base balance was observed. Respiratory infection with 1010 IFU P. acanthamoebae per calf induced mild respiratory dysfunction, mainly characterised by hypoxaemia. The study findings do not indicate severe pathophysiological consequences of P. acanthamoebae infection on pulmonary function in the bovine host.

Ziel der Studie war die Charakterisierung von Phanotyp und Aktivierungsstatus boviner Lymphozyten und Granulozyten auf systemischer und lokaler Ebene im etablierten Modell der akuten respiratorischen Chlamydia psittaci (Cp)-Infektion. Dreisig Kalber (6 – 8 Wochen alt) wurden intrabronchial mit Cp inokuliert. Alle Tiere zeigten ab dem 2. Tag nach Inokulation (dpi) deutliche Symptome einer respiratorischen Erkrankung. Fur die durchflusszytometrischen Untersuchungen leukozytarer Oberflachenmarker wurden venoses Blut (vor Inokulation sowie 1, 2, 3, 5, 7, und 10 dpi) und broncho-alveolare Lavageflussigkeit (BALF; 4, 9 und 14 dpi) gewonnen. Im Blut zeigte die beim Rind uberwiegend aus γδ T-Zellen bestehende CD8αdim/CD62Lhi- Lymphozytenpopulation einen signifikanten Anstieg der CD25-Expression im Zeitraum schwerster klinischer Erscheinungen (2 3 dpi). Die CD62L-Expression dieser Population blieb unverandert. Im Gegensatz dazu wiesen zwei Populationen der αβ T-Zellen (CD8αhi/CD62Lhi und CD4+/CD62L+) einen moderaten Abfall der CD25-Expression und einen hochsignifikanten Anstieg der CD62L-Expression auf, wobei die Maxima in beiden Populationen 3 dpi erreicht wurden und bis 10 dpi ein langsamer Abfall erfolgte. Die gleiche Kinetik zeigte sich fur die CD62L-Expression in CD4+/CD62L+ BALF Lymphozyten, wahrend die CD25 Expression 9 dpi maximal und 14 dpi noch immer signifikant erhoht war. Neutrophile Granulozyten, die die Wirtsantwort auf eine Cp-Infektion dominieren, zeigten in Blut und BALF nach dem Eintreten klinischer Symptome eine signifikante Erhohung der Expression sowohl des Adhasionsmolekuls CD62L als auch des kurzlich als Biomarker fur akute Infektionsgeschehen in Rindern vorgeschlagenen Oberflachenmarkers CD25. Die Parallelitat der Expressionsanderung von Oberflachenantigenen auf bovinen Leukozyten mit dem klinischen Verlauf impliziert eine Beteiligung der entsprechenden Zellpopulationen an der effektiven Kontrolle einer Cp-Infektion beim Kalb.

Antimicrobial treatment of chlamydial infections is known to be of limited efficacy. In this study, effects of doxycycline (D), usually the drug of choice, were compared with the combined therapy of doxycycline and rifampicin (R) in a bovine model of respiratory Chlamydia psittaci infection. After intrabronchial inoculation of the pathogen, 30 animals were assigned to five groups (n = 6 per group): untreated controls, monotherapy with D (5 mg kg(-1)day(-1) or 10 mg kg(-1)day(-1)), and combination therapy of D and R (600 mg day(-1)). Treatment continued until day 14 post inoculation (d.p.i.). Clinical signs, inflammatory markers, and pathological findings confirmed successful infection in all animals. Reisolation of the pathogen was possible in 4/6 untreated animals and in 4/12 animals treated with D alone until 4 d.p.i., but in none of the calves of the two D + R groups. Pathogen detection was possible in all animals without significant differences among groups. Severity of disease and time course of its resolution, assessed by clinical and pathological findings as well as inflammatory parameters, were not significantly different between untreated controls and calves receiving D alone or in combination with R. Regardless of the treatment regimen, all groups recovered clinically and cleared the infection within 2 weeks.

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals. The aim was to establish in vivo sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions. Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host.

The aim of this study was to evaluate the pathogenicity of Parachlamydia (P.) acanthamoebae as a potential agent of lower respiratory tract disease in a bovine model of induced lung infection. Intrabronchial inoculation with P. acanthamoebae was performed in healthy calves aged 2-3 months using two challenge doses: 10(8) and 10(10) bacteria per animal. Controls received 10(8) heat-inactivated bacteria. Challenge with 10(8) viable Parachlamydia resulted in a mild degree of general indisposition, whereas 10(10) bacteria induced a more severe respiratory illness becoming apparent 1-2 days post inoculation (dpi), affecting 9/9 (100%) animals and lasting for 6 days. The extent of macroscopic pulmonary lesions was as high as 6.6 (6.0)% [median (range)] of lung tissue at 2-4 dpi and correlated with parachlamydial genomic copy numbers detected by PCR, and with bacterial load estimated by immunohistochemistry in lung tissue. Clinical outcome, acute phase reactants, pathological findings and bacterial load exhibited an initial dose-dependent effect on severity. Animals fully recovered from clinical signs of respiratory disease within 5 days. The bovine lung was shown to be moderately susceptible to P. acanthamoebae, exhibiting a transient pneumonic inflammation after intrabronchial challenge. Further studies are warranted to determine the precise pathophysiologic pathways of host-pathogen interaction.

Einleitung: Aufgrund von widerspruchlichen Berichten zur Wirksamkeit von Doxyzyklin bei der Behandlung von Chlamydieninfektionen wurde der Effekt von Doxyzyklin auf eine respiratorische Chlamydia psittaci (Cp) Infektion in einem bovinen Modell uberpruft. Die Dosierung wurde an die Zielspezies Mensch und Rind angepasst. Methoden: Achtzehn Kalber im Alter von 6 – 8 Wochen wurden wie zuvor beschrieben [1] mit Cp inokuliert. Nach Auftreten der ersten klinischen Symptome (30h nach Inokulation) wurden zwei Gruppen a 6 Tiere uber 13 Tage oral mit Doxyzyklin behandelt (Dosis: entweder 5 mg/kg/Tag oder 10 mg/kg/Tag). Als Kontrolltiere fungierten 6 infizierte Kalber, die unbehandelt blieben. Alle Tiere wurden taglich klinisch untersucht. An Tag 5 und 9 nach Inokulation wurde die Lunge eines jeden Tieres bronchoskopisch lavagiert und am Tag 14 post inoculationem wurden alle Tiere euthanasiert und seziert. Ergebnisse: Die Behandlung mit Doxyzyklin hat den klinischen Verlauf nicht verbessert. In der broncho-alveolaren Lavageflussigkeit wurden keine signifikanten Unterschiede hinsichtlich Zytologie, der Konzentration an Totalprotein oder dem Gehalt an Eicosanoiden gefunden. Bei der Sektion unterschieden sich die Gruppen weder durch die Ausdehnung noch durch die Morphologie der Lungenveranderungen. Die mit der quantitativen real-time PCR nachgewiesene Menge der Genomkopien in den veranderten Lungenarealen unterschied sich nicht signifikant. Schlussfolgerung/Ausblick: Die Ergebnisse sprechen nicht fur eine erfolgversprechende Behandlung von akuten Chlamydieninfektionen mit Doxyzyklin. Finanzierung: Bundesministerium fur Bildung und Forschung. Quellennachweis: [1] Reinhold et al. (2012) PLoS ONE 7, 1:e30125

Chlamydia psittaci is a zoonotic bacterium with a wide host range that can cause respiratory disease in humans and cattle. In the present study, effects of treatment with macrolides and quinolones applied alone or in combination with rifampicin were tested in a previously established bovine model of respiratory C. psittaci infection. Fifty animals were inoculated intrabronchially at the age of 6-8 weeks. Seven served as untreated controls, the others were assigned to seven treatment groups: (i) rifampicin, (ii) enrofloxacin, (iii) enrofloxacin + rifampicin, (iv) azithromycin, (v) azithromycin + rifampicin, (vi) erythromycin, and (vii) erythromycin + rifampicin. Treatment started 30 hours after inoculation and continued until 14 days after inoculation (dpi), when all animals were necropsied. The infection was successful in all animals and sufficient antibiotic levels were detected in blood plasma and tissue of the treated animals. Reisolation of the pathogen was achieved more often from untreated animals than from other groups. Nevertheless, pathogen detection by PCR was possible to the same extent in all animals and there were no significant differences between treated and untreated animals in terms of local (i.e., cell count and differentiation of BALF-cells) and systemic inflammation (i.e. white blood cells and concentration of acute phase protein LBP), clinical signs, and pathological findings at necropsy. Regardless of the reduced reisolation rate in treated animals, the treatment of experimentally induced respiratory C. psittaci infection with enrofloxacin, azithromycin or erythromycin alone or in combination with rifampicin was without obvious benefit for the host, since no significant differences in clinical and pathological findings or inflammatory parameters were detected and all animals recovered clinically within two weeks.