31 research outputs found
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 anschlieĂźenden 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 FĂĽr
STUDIE 1 wurden vier Kälber mehrmals unter Allgemeinanästhesie
bronchoskopiert, um die Gewinnung von broncho-alveolärer Lavageflüssigkeit
(BALF), bronchialen BĂĽrstenabstrichen und transbronchialen Lungenbiopsien zu
evaluieren. Für STUDIE 2 & 3 wurden 80 Kälber intrabronchial mit C. psittaci
inokuliert. DreiĂźig 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 zugefĂĽhrt. 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 fĂĽhrte 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
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
Study design.
<p>d ai/h ai = days/hours ante inoculation; dpi = days post inoculation.</p
Results of multiple regression analysis relating IL-6 to other markers of inflammation in broncho-alveolar lavage fluid in all inoculated calves.
<p>Results of multiple regression analysis relating IL-6 to other markers of inflammation in broncho-alveolar lavage fluid in all inoculated calves.</p
Activity of IL-6 (I.U./mL) in blood serum of healthy calves aged 6–8 weeks at two different days within one week.
<p>Activity of IL-6 (I.U./mL) in blood serum of healthy calves aged 6–8 weeks at two different days within one week.</p
Activities of IL-6 (I.U./mL) in BALF and blood serum assessed at the same day in calves.
<p>Activities of IL-6 (I.U./mL) in BALF and blood serum assessed at the same day in calves.</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