|Abstract or Summary
- Hemorrhagic bowel syndrome (HBS) is a devastating disorder affecting high
producing dairy cattle. As an important emerging disease (Animal and Plant Health
Inspection Service, 2003), HBS is now estimated to account for > 2% of the deaths of
dairy cattle (Kirkpatrick et al, 2001). However, little is known about the causative
agents or factors contributing to HBS.
In the first part of this study, I tested the hypothesis that HBS is associated with
an invasive mold: A. fumigatus. To test this hypothesis, I completed a series of
epidemiological studies. They included: an analysis of control and HBS samples
collected from local herds; a broader analysis of control and HBS samples collected
from across the US; and also a collaborative study with the Wisconsin Veterinary
Diagnosis Laboratory. For these studies, I developed a real-time quantitative
polymerase chain reaction (PCR) assay to determine the amount of A. fumigatus DNA
present in blood and tissues of HBS and healthy cows. We also adopted a multiplex
PCR assay to detect C. perfringens toxin genes in the same samples. From the first
study, it was determined that a positive association exists between HBS and A.
fumigatus, but not with C. Perfringens. In the second analysis using blood samples
collected from across the US, I found a high association of A. fumigatus DNA with
HBS (X² = 27.6836, P < 0.00 1) but not with sudden death, downer cows, bloody
diarrhea and other diseases (X² = 0.0442 - 2.42, P < 0.1198 - 0.8335). In the
collaborative experiment with the Wisconsin Veterinary Diagnostic Laboratory
(WVDL), we evaluated the associations between various gastrointestinal diseases
(including HBS) with GI pathogens (A. fumigatus, C. perfringens, Salmonella and
BVDV). A unique aspect of this study was the use of cows dying from other
gastrointestinal diseases as controls. Results indicated there is an association between A.
fumigatus and HBS (X² = 12.15, P [less than or equal to] 0.001) but not between C. perfringens Type A and
HBS (X² =0.532, P [less than or equal to] 0.466).
A. fumigatus has potential to infect immunocompromised individuals. Hence, I
proposed that HBS arises from a combination of A. fumigatus exposure and
immunocompromise. To test whether a combination of A. fumigatus exposure and
immunosuppression could induce HBS, we conducted one sheep study. I also tested the
ability of a commercial feed product to eliminate "HBS" and to regulate innate immune
function. In this study, We failed to develop an HBS model in sheep. However, I
discovered mechanisms-of-action of a commercially-available feed additive. I
determined that dexamethasone injection provides a model for immunosuppression in growing sheep. Specifically, daily injection of dexamthasone reduced concentration of
both L-selectin and IL-1[beta]. The feed additive restored the L-selectin and interleukin- 1[beta]
(IL-1[beta]) expression levels in immune-suppressed sheep. In order to further understand
the mechanisms by which the nutritional product stimulates innate immune function, I
used cDNA microarray analysis to examine gene expression in neutrophils of post-parturient
cows. More than 20 genes, including IL-4 receptor, IL-1 [beta] converting
enzyme, angiopoietin-2, epithelin-1 and -2, nucleoporin, and wilms tumor 1, were
influenced (P<0.05) by addition of the feed product to the diets of dairy cattle.
Independent confirmation of altered expression of these genes used quantitative real-time
RT-PCR (Q-RT-PCR). I found that the feed product up-regulated IL-4 receptor,
IL-1[beta] converting enzyme, upstream stimulatory factor, nucleoporin and down-regulated
angiopoietin, hepatitis B virus-associated factor receptor and forkhead transcription
factor. Many of these gene products are involved in life-expectancy of neutrophils
through control of apoptosis. I propose that the feed product enhances neutrophil
function, in part, via increasing L-selectin and IL-1 [beta] expression and via inhibition of
apoptotic signaling. This should enhance circulation of neutrophils to effectively access
infection sites and kill pathogens.
In summary, I determined that HBS is significantly associated with A. fumigatus. I
also evaluated a commercial feed additive which is currently marketed to prevent HBS.
The additive was able to increase markers of innate immune function in both sheep and
dairy cattle and I propose that these mechanisms account for its efficacy.