A bovine model of a respiratory Parachlamydia acanthamoebae infection

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: 108 and 1010 bacteria per animal. Controls received 108 heat-inactivated bacteria. Challenge with 108 viable Parachlamydia resulted in a mild degree of general indisposition, whereas 1010 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 interactio

Evaluierung antimikrobieller Behandlungsstrategien gegen Chlamydia psittaci in einem bovinen respiratorischen Infektionsmodell

Background Chlamydiae are obligate intracellular pathogens that can cause a variety of diseases in many different mammalian and avian hosts. The successful antimicrobial treatment of chlamydial infections is still an unresolved issue in both, human and veterinary medicine. Aim and design of the project The project was designed to evaluate regimens commonly used for the treatment of chlamydial infections (i.e., tetracyclines, macrolides and quinolones), and to compare them to the most promising treatment regimens that were identified in vitro (i.e., combination of the antimicrobial substances with rifampicin). As animal model we chose a recently developed bovine model of an acute respiratory C. psittaci infection. In order to perform the treatment studies in the afore mentioned animal model, bronchoscopic sampling methods had to be adapted for the use in calves aged 6-10 weeks and for the use under experimental conditions in a biosafety level 2. Animals, material and methods For STUDY 1, four male Holstein-Friesian calves were repeatedly bronchoscoped under general anesthesia beginning at the age of 6 weeks. The following methods were evaluated: bronchial brushing, broncholaveolar lavage and transbronchial lung biopsy. For STUDY 2 and STUDY 3, a total of 80 conventionally raised, male Holstein-Friesian calves were intrabronchially inoculated with 108 inclusion forming units C. psittaci strain DC15. At 30 h post inoculation (pi), the animals were assigned either to the untreated control groups or to one of 11 different treatment groups. All animals were clinically examined on a daily basis. Results were summarized using a clinical scoring system. Venous blood was sampled throughout the study. Bronchial brushings, bronchoalveolar lavage fluid (BALF) and pharyngeal swabs were sampled under general anesthesia. At the end of the study on 14 dpi, all animals were euthanized and necropsied. Levels of antimicrobial substances were determined. The presence of the inoculated pathogen in treated and untreated animals was assessed by recultivation from bronchial brushings and by quantitative real-time PCR testing of blood, lung and mediastinal lymph node and swabs (i.e., nasal, conjunctival, pharyngeal, and rectal swabs). The hosts’ reaction was characterized by the clinical scores and signs of local and systemic inflammation. Results The intrabronchial inoculation with C. psittaci led to acute respiratory disease with fever in all animals that resolved until 10 dpi. The established protocol of obtaining bronchial brushings, BALF and transbronchial lung biopsies proved suitable for the use under experimental conditions. Sufficient antibiotic levels were detected in in blood and tissue samples of all treated animals. Recultivation results revealed that viable Chlamydiae could more often be isolated from untreated than from treated animals. Single drug therapy inhibited chlamydial growth in the same extent as combination therapy with rifampicin. Clinical score, white blood cell count, LBP concentration in the blood, and BALF cell count revealed acute respiratory and systemic disease in all animals, but again, no differences were visible between treated and untreated animals. All 80 infected animals included in the project regained clinical health by the end of the study, regardless if they were treated or not.Einleitung Chlamydien sind gram-negative Bakterien mit einer obligat intrazellulären Lebensweise, die in eine Vielzahl unterschiedlichster Erkrankungen bei Säugern und Vögeln involviert sein können. Antimikrobiellen Behandlungen chlamydialer Infektionen sind mit Tetrazyklinen, Chinolonen oder Makroliden beschrieben, allerdings sind die dokumentierten Effekte dieser antichlamydialen Therapieversuche oft nicht zufriedenstellend. Studien in Zellkulturmodellen konnten zeigen, dass der Zusatz von Rifampicin die antichlamydialen Effekte der zuvor genannten Wirkstoffgruppen steigert. Ziel und Projektdesign Das Ziel dieses Projektes war es, die aus In vitro- Experimenten abgeleitete Hypothese zur Kombinationstherapie chlamydialer Infektionen in einem etablierten bovinen Infektionsmodell auf ihre Validität in vivo zu testen. Dafür mussten zunächst diverse bronchoskopische Methoden an 6 10 Wochen alte Kälber unter S2-Bedingungen adaptiert werden (STUDIE 1). Die sich anschließenden Therapiestudien erfolgten in zwei Abschnitten. In STUDIE 2 wurden die Behandlungseffekte von Tetrazyklinen, allein oder in Kombination mit Rifampicin, untersucht. STUDIE 3 diente zur Dokumentation der Behandlungseffekte von Chinolonen und Makroliden, als Monotherapie oder in Kombination mit Rifampicin. Tiere, Studiendesign, Material und Methoden Für STUDIE 1 wurden vier Kälber mehrmals unter Allgemeinanästhesie bronchoskopiert, um die Gewinnung von broncho-alveolärer Lavageflüssigkeit (BALF), bronchialen Bürstenabstrichen und transbronchialen Lungenbiopsien zu evaluieren. Für STUDIE 2 & 3 wurden 80 Kälber intrabronchial mit C. psittaci inokuliert. Dreißig Stunden nach Inokulation wurden die Tiere der unbehandelten Kontrollgruppe oder einer von elf Behandlungsgruppen zugeteilt und bis zum Ende der Studie mit den entsprechenden Antibiotika behandelt. Zu Versuchsende nach 14 Tagen wurden alle Tiere der Sektion zugeführt. Als Parameter zur Dokumentation der Behandlungserfolge dienten: » Befunde des täglich durchgeführten klinischen Untersuchungsganges, » Erregeranzucht aus bronchialen Bürstenabstrichen und Lungengewebe, » Nachweis chlamydialer DNA mittels PCR in Rachentupfern, Kottupfern sowie Lungen- und Lymphknotengewebe, » Leukozytenzahl, Differentialblutbild und Konzentration von Lipopolysaccharid bindendem Protein im venösen Blut , » Gesamtzellzahl, Differentialzellbild und Proteinkonzentration in der BALF, » Ausmaß und Charakter der Lungenveränderungen und » Konzentration der applizierten antimikrobiellen Wirkstoffe im Blut und im Lungen-, Muskel- und Lebergewebe. Ergebnisse STUDIE 1: Das erarbeitete Protokoll zur bronchoskopischen Probengewinnung erwies sich unter den gegebenen experimentellen Umständen als praktikabel. STUDIEN 2 & 3: Die Inokulation mit C. psittaci führte bei allen Tieren zu einer fieberhaften akuten respiratorischen Erkrankung, welche ihren Höhepunkt um den Tag 3 erreichte und innerhalb von 10 Tagen abklang. Bei allen behandelten Tieren wurden therapeutisch wirksame Konzentrationen der entsprechenden Wirkstoffe in Blut und Gewebe nachgewiesen. Die Re-Isolation des inokulierten Pathogens C. psittaci gelang häufiger aus unbehandelten Tieren als aus behandelten – jedoch ohne signifikanten Einfluss des angewandten Therapieschemas. Sämtliche anderen untersuchten Parameter unterschieden sich zu keinem Zeitpunkt signifikant zwischen behandelten und unbehandelten Tieren

Evaluierung antimikrobieller Behandlungsstrategien gegen Chlamydia psittaci in einem bovinen respiratorischen Infektionsmodell

Background Chlamydiae are obligate intracellular pathogens that can cause a variety of diseases in many different mammalian and avian hosts. The successful antimicrobial treatment of chlamydial infections is still an unresolved issue in both, human and veterinary medicine. Aim and design of the project The project was designed to evaluate regimens commonly used for the treatment of chlamydial infections (i.e., tetracyclines, macrolides and quinolones), and to compare them to the most promising treatment regimens that were identified in vitro (i.e., combination of the antimicrobial substances with rifampicin). As animal model we chose a recently developed bovine model of an acute respiratory C. psittaci infection. In order to perform the treatment studies in the afore mentioned animal model, bronchoscopic sampling methods had to be adapted for the use in calves aged 6-10 weeks and for the use under experimental conditions in a biosafety level 2. Animals, material and methods For STUDY 1, four male Holstein-Friesian calves were repeatedly bronchoscoped under general anesthesia beginning at the age of 6 weeks. The following methods were evaluated: bronchial brushing, broncholaveolar lavage and transbronchial lung biopsy. For STUDY 2 and STUDY 3, a total of 80 conventionally raised, male Holstein-Friesian calves were intrabronchially inoculated with 108 inclusion forming units C. psittaci strain DC15. At 30 h post inoculation (pi), the animals were assigned either to the untreated control groups or to one of 11 different treatment groups. All animals were clinically examined on a daily basis. Results were summarized using a clinical scoring system. Venous blood was sampled throughout the study. Bronchial brushings, bronchoalveolar lavage fluid (BALF) and pharyngeal swabs were sampled under general anesthesia. At the end of the study on 14 dpi, all animals were euthanized and necropsied. Levels of antimicrobial substances were determined. The presence of the inoculated pathogen in treated and untreated animals was assessed by recultivation from bronchial brushings and by quantitative real-time PCR testing of blood, lung and mediastinal lymph node and swabs (i.e., nasal, conjunctival, pharyngeal, and rectal swabs). The hosts’ reaction was characterized by the clinical scores and signs of local and systemic inflammation. Results The intrabronchial inoculation with C. psittaci led to acute respiratory disease with fever in all animals that resolved until 10 dpi. The established protocol of obtaining bronchial brushings, BALF and transbronchial lung biopsies proved suitable for the use under experimental conditions. Sufficient antibiotic levels were detected in in blood and tissue samples of all treated animals. Recultivation results revealed that viable Chlamydiae could more often be isolated from untreated than from treated animals. Single drug therapy inhibited chlamydial growth in the same extent as combination therapy with rifampicin. Clinical score, white blood cell count, LBP concentration in the blood, and BALF cell count revealed acute respiratory and systemic disease in all animals, but again, no differences were visible between treated and untreated animals. All 80 infected animals included in the project regained clinical health by the end of the study, regardless if they were treated or not

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and <em>In Vivo</em> Sampling Techniques

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

Is doxycycline the right choice to treat an acute respiratory chlamydia psittaci infection?

Introduction: Although tetracycline has been known as a front-line antibiotic for treatment of Chlamydia infections, information about its efficacy is contradictory. Objectives: The aim of this study was to assess the effects of doxycycline treatment in a bovine model of respiratory Chlamydia psittaci (Cp) infection. To address aspects of both human and veterinary medicine, doses were adjusted to humans and cattle. Animals & Methods: Eighteen calves aged 6-8 weeks were inoculated with Cp as described previously [1]. With appearance of the first clinical signs (about 30 hours after inoculation), doxycycline was applied orally for 13 days (dosis: either 5 mg/kg/day or 10 mg/kg/day). In addition to the two treatment groups (each n=6), six infected calves served as untreated controls. All animals were clinically examined on a daily basis. Broncho-alveolar lavage was performed at days 5 and 9 after inoculation (dpi). At 14 dpi, all animals were sacrificed. Results: Treatment with doxycycline did not improve clinical outcome. In BALF, no significant differences in cytology, total protein or eicosanoids were found between the groups at any time point. At necropsy, neither the percentage of lung affected nor the morphology of pulmonary lesions differed significantly between the groups. Using quantitative real-time PCR, the amount of chlamydial genome copies detected in the affected lung areas did not differ significantly. In conclusion, results do not support the use of doxycycline for successful antimicrobial treatment of acute Chlamydia psittaci infection

Circulating and broncho-alveolar interleukin-6 in relation to body temperature in an experimental model of bovine Chlamydia psittaci infection.

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

Parachlamydia acanthamoebae as a respiratory pathogen: Challenge dose titration in a bovine model

Background: The majority of community acquired pneumonia (CAP) in humans fails to be diagnosed aetiologically. Besides Chlamydia spp., Chlamydia-like organisms are thought to be causative in CAP. Pneumopathogenic potential has been described for Parachlamydia (P.) acanthamoebae, a well studied agent among the Chlamydia-like organisms. Objective: This study aimed to evaluate the pathogenicity of Parachlamydia (P.) acanthamoebae as a potential agent of lower respiratory tract disease. A bovine model of induced lung infection was introduced for translational reasons. Method: Intrabronchial inoculation with P. acanthamoebae was performed in healthy calves aged 2-3 months (n=30) using two different challenge doses: 108 and 1010 inclusion-forming units (ifu) per animal. Controls received heat inactivated pathogen. Results: While the challenge with 108 ifu of viable Parachlamydia resulted in a mild degree of clinical illness, the dose of 1010 ifu induced a respiratory desease that became clinically apparent 1-2 days after challenge. Parachlamydiae were detected in neutrophils and alveolar macrophages within pulmonary lesions by immunohistochemistry. This was in accordance with molecularbiological findings as well as with re-isolation of Parachlamydia. Clinical outcome, acute phase reactants, pathological findings and antigen load exhibited an initial dose-dependent effect of severity. Conclusion: The bovine lung was shown to be susceptible for P. acanthamoebae. Recommending the challenge dose of 1010 ifu, this large animal model is suitable to elucidate host-pathogen interactions

Study design.

<p>d ai/h ai = days/hours ante inoculation; dpi = days post inoculation.</p

Kinetics of Local and Systemic Leucocyte and Cytokine Reaction of Calves to Intrabronchial Infection with Chlamydia psittaci.

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