Proceedings of the 10th International Colloquium on Paratuberculosis

#95 Role <strong>of</strong> nitric oxide production in dairy cows naturally infected with Mycobacterium avium
subsp. paratuberculosis
Judith R. Stabel, Mohammad S. Khalifeh, Ahmad M Al-Majali
USDA-ARS-NADC, USA; Jordan University <strong>of</strong> Science and Technology, Jordan
Nitric oxide (NO) is a crucial mediator in host defense and is one <strong>of</strong> <strong>the</strong> major killing mechanisms within macrophages.
Its induction is highly affected by <strong>the</strong> types <strong>of</strong> cytokines and <strong>the</strong> infectious agents present. In <strong>the</strong> current
study, NO production was evaluated after in vitro infection <strong>of</strong> unfractionated peripheral blood mononuclear
cells (PBMCs) with Mycobacterium avium subsp. paratuberculosis (MAP) after 8 hr, 3 and 6 days <strong>of</strong> culture for
cows in different stages <strong>of</strong> disease. In addition, <strong>the</strong> effects <strong>of</strong> in vitro exposure to inhibitory cytokines such as
interleukin-10 (IL-10) and transforming growth factor β (TGF-β) as well as <strong>the</strong> pro-inflammatory cytokine IFN-γ
were correlated with <strong>the</strong> level <strong>of</strong> NO production. Nitric oxide production was consistently higher in cell cultures
from subclinically infected animals at all time points. An upregulation <strong>of</strong> NO production was demonstrated in
unfractionated cell cultures from healthy control cows after exposure to MAP infection as compared to noninfected
cell cultures. A similar increase in NO due to <strong>the</strong> addition <strong>of</strong> MAP to cell cultures was also noted for clinically
infected cows. NO level among subclinically infected cattle was greater at all time points tested and was
fur<strong>the</strong>r boosted with <strong>the</strong> combination <strong>of</strong> both in vitro MAP infection and IFN-γ stimulation. Finally, <strong>the</strong> in vitro
exposure to inhibitory cytokines such as IL-10 and TGF-β prior to MAP infection or LPS stimulation resulted
in <strong>the</strong> downregulation <strong>of</strong> this inflammatory mediator (NO) in all experimental groups at all time points. In summary,
a higher level <strong>of</strong> NO production was associated with cows in <strong>the</strong> subclinical stage <strong>of</strong> MAP infection.
#127 Multiplication <strong>of</strong> Mycobacterium avium subsp. paratuberculosis is higher in
macrophages induced by M-CSF than GM-CSF
Kazuhiro Yoshihara, Reiko Nagata, Yasuyuki Mori
National Institute <strong>of</strong> Animal Health, National Agriculture and Food Research Organization, Japan
Mycobacterium avium subsp. paratuberculosis (MAP) strain ATCC 19698 was grown in Middlebrook 7H9 broth
supplemented with 0.2% glycerol, 2 micrograms/ml mycobactin J and 12.5% OADC Enrichment for several
weeks. One milliliter <strong>of</strong> bacteria culture was collected in a microtube and centrifuged. After removal <strong>of</strong> <strong>the</strong>
supernatant, <strong>the</strong> pellet was suspended in 0.5 ml PBS, and <strong>the</strong>n <strong>the</strong> bacteria were dispersed for 5 seconds with
a homogenizer. The microtubes were allowed to settle for 3 minutes, and several suspensions were pooled
and passed through a 5-micrometer-pore-size filter. Twenty milliliters <strong>of</strong> bovine blood was infected with MAP
(3x10 8 cells). After 90 minutes <strong>of</strong> incubation, peripheral blood mononuclear cells (PBMC) were isolated by use
<strong>of</strong> Ficoll-Paque PLUS density gradient centrifugation. Monocytes were collected from <strong>the</strong> PBMC by a magnetic
cell separation system (MACS) with anti human CD14 antibody- coupled microbeads. Monocytes were
incubated at 2x10 5 /well in 96-well tissue culture plates with 10% FCS RPMI1640 including ei<strong>the</strong>r M-CSF or
GM-CSF. One, two and three weeks later, macrophages induced by M-CSF or GM-CSF were collected into
microtubes by using 0.2% EDTA and 5% FCS in PBS and genomic DNAs were extracted with an UltraClean
Microbial DNA Isolation kit. Multiplication <strong>of</strong> MAP was estimated by <strong>the</strong> amount <strong>of</strong> MAP DNA measured by
real- time PCR. The amount <strong>of</strong> MAP DNA phagocytosed by monocytes in blood was 14.5pg/well (day 0). The
amounts <strong>of</strong> MAP DNA within macrophages incubated with M-CSF were 40.1 pg/well (day 7), 48.3 (day 14) and
23.1 (day 14). Meanwhile, DNA volumes <strong>of</strong> MAP in GM-CSF-stimulated macrophages were 29.5 pg/well (day
7), 23.7 (day 14) and 8.9 (day 21). These results suggest that MAP multiplies in high number in macrophages
induced by M-CSF as compared to GM-CSF. For <strong>the</strong> next step, we will investigate cytokine pr<strong>of</strong>iles <strong>of</strong> <strong>the</strong>se
macrophages.
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