EFFECTS OF CELMANAX LIQUID ON SURVIVAL OF BEEF CALVES WITH ACUTE MYCOTOXIN POISONING AND JEJUNAL HEMORRHAGE SYNDROME AND ON THE CYTOTOXICITY OF FEED EXTRACTS TO ENTEROCYTES IN VITRO**
D. Baines1, Ph.D., C. Elrod, Ph.D., S.Jalukar, Ph.D. and J. Oppy Agriculture and Agri-Foods Canada1, and Varied Industries Corporation, Mason City, IA
Introduction:
Jejunal Hemorrhage Syndrome (JHS, also known as Hemorrhagic Bowel Syndrome or HBS) is a hemorrhagic disease of beef and dairy cattle. It is typically characterized by sudden death which, upon investigation, is found to be caused by massive hemorrhaging of the jejunum and blockage of the intestine with the resulting blood clot. Early work on this disease complex identified two associated factors, moldy feed containing Aspergillus fumigatus and the bacterial pathogen Clostridium perfringens Type A. More recent work from Dr. Baines’ lab expanded the scope of potential causative agents of JHS in dairy cattle to a wider range of mycotoxigenic fungi and to Shigatoxin producing E. coli (also known as STEC. See Vi-COR® Dairy Research Bulletin D-68). STEC is one of the leading causes of food borne pathogen outbreaks in humans, resulting in hemorrhagic colitis and hemolytic uremic syndrome. Cattle are the principal reservoir of these pathogens.
Objective:
The first objective of the current study was to characterize pathogen colonization in hemorrhaged jejunum of beef cattle during natural JHS outbreaks. The second objective was to confirm the association of mycotoxigenic fungi in feeds with the development of JHS. The third objective was to determine the impact of a prebiotic, Celmanax® , on the feed-associated mycotoxin cytotoxicity
in vitro. The final objective was to determine if a prebiotic could modify the progression of disease in beef cattle.
Materials and Methods:
Two beef feedlots in southern Alberta, Canada reported outbreaks of sudden death in receiving cattle averaging 227 Kg (Beef Feedlot 1, BF1) and in finishing cattle averaging 544 Kg (Beef Feedlot 2, BF2). Scientists from Agriculture and Agri-Food Canada (AAFC) were called to assist in the investigation. At BF1, the first morning after introduction of a new hay supply, 50 calves were found dead with evidence of black, tarry feces consistent with gastrointestinal hemorrhage. An additional thirty-four affected calves were transferred to a sick pen where eighteen recovered over the next week. Sixteen calves continued to decline despite antibiotic treatments. Symptoms in the 16 calves included staggering, hind limb paralysis and wasting, suggestive of an acute mycotoxicosis. After a week of further declines, the calves were given an oral drench of 400 mL of Celmanax Liquid. Their condition was monitored by the AAFC scientists. At BF2, fourteen cattle died suddenly over the course of two days, with no apparent change in feedstuffs or management. Samples of jejunum from five calves at BF1, two steers at BF2 and six steers at a local abattoir (to serve as negative controls) were collected. Samples of tissue and bloody digesta were segregated and washed for isolation and characterization of bacteria and fungi. A variety of culture methods, microscopy and polymerase chain reaction (PCR) techniques were utilized to identify and confirm specific serovars of E. coli, Salmonella, Listeria and Clostridia and toxin-related gene expression.
Samples of barley silage and dry hay were collected from the feedlots and evaluated for the presence of mycotoxigenic fungi by microscopic identification and PCR. Potential mycotoxins were extracted with 50% methanol from the forage samples and utilized in a cytotoxicity assay using cultures of intestinal cells. Aliquots of either pure mycotoxins or extracts from the feedlots’ moldy forages were applied to the cultured colon cells in the presence or absence of 0.1% Celmanax and incubated for 4 hours. After washing, the cells were evaluated for viability with Trypan blue stain and treatments rated as either negative, low, moderate or high for cytotoxicity. Reference standards of 0.1 ppm or 1.0 ppm of aflatoxin had (low and high cytotoxicity, respectively.)
Results:
Of the sixteen calves with prolonged illness which were provided with Celmanax Liquid, eleven returned to good health within forty-eight hours and had no further health issues. Five calves, all of which had suffered hind limb paralysis within twenty-four hours of consuming the moldy hay, never regained mobility and eventually died. The 400 mL dose of Celmanax Liquid appears to have reversed the course of the disease complex and aided in the return to feed consumption and good health in 69% of the calves treated.
Jejunal tissue samples from five receiving cattle dying of JHS had common pathologies including: inflamed Peyer’s Patches, severe jejunal hemorrhage, bloody digesta, complete loss of mucosal structure in the hemorrhaged regions of the jejunum, large blood clots and edema. Jejunal samples from two finishing cattle had similar symptoms, though the blood clots were generally smaller and there was more edema evident. Jejunal samples from the six steers collected at the abattoir had no hemorrhaging, edema or inflamed Peyer’s Patches.
The primary bacterial pathogen isolated was E. coli, with both O157 and non-O157 Shigatoxin producing variants present. All E. coli isolates expressed the Stx1 and Stx2 genes, which are key virulence traits associated with the more severe forms of animal and human disease. The finishing cattle had 10-fold higher CFUs of the STEC pathogens than the younger, smaller receiving cattle. Samples from the control cattle had no isolates of Shigatoxin or other E. coli (Figure 1).
Figure 1. Log CFU of STEC in Jejunal Tissue or Digesta of Cattle
There were no detectable fungi in the tissues or digesta of the cattle sampled. However, in the case of the receiving cattle feedlot, moldy hay had been removed over two weeks before the calves died and tissues from them were obtained. Fungi of the Fusarium, Aspergillus and Penicillium genera were isolated from the various forages as shown in Table 1.
Table 1. Percent of forage samples positive for mycotoxigenic fungi from beef feedlots(n=5-10 samples/forage source)
Mycotoxigenic Fungi |
BF1 Barley Silage |
BF1 Hay |
BF2 Barley Silage |
Fusarium poae |
0 |
0 |
100 |
Fusarium verticilloides |
100 |
100 |
0 |
Fusarium sporotrichiodes |
100 |
0 |
0 |
Aspergillus fumigatus |
0 |
100 |
0 |
Penicillium roqueforti |
100 |
100 |
100 |
For BF1, the 2008 barley silage had a higher cytotoxicity score (1) than the 2007 barley silage (0) which had no cytotoxic activity. The hay from BF1, which had initially lead to cattle deaths, had a cytotoxicity score of 3, similar to forages which had previously contributed to JHS cases in local dairies (See Vi-COR Dairy Research Bulletin D-68). All other feed components on BF1 had no cytotoxic activity. The barley silage from BF2, where the JHS outbreak had been much less severe, had a moderate cytotoxicity score of 2. Inclusion of 0.1% Celmanax Liquid in the incubation with these forage extracts resulted in a complete elimination of the cytotoxic effects (Figure 2).
Figure 2. Cytotoxicity score of extracts of moldy forages, with or
without 0.1% Celmanax. (n=6)
Conclusion:
From digesta and tissues of beef cattle dying from JHS, both O157:H7 and non-O157:H7 serovars of Shigatoxin producing E. coli were isolated. No pathogenic Salmonella, Listeria or Clostridia were found. Several mycotoxigenic fungi, including Aspergillus fumigatus, Penicillium roqueforti and several Fusarium species were identified in forages from the affected feedlots. It appears that the development of JHS requires the dual insults of a pathogenic bacteria and a mycotoxin challenge. These challenges are not limited to Aspergillus fumigatus and Clostridium perfringens Type A as previously thought. Celmanax, at 0.1%, was able to completely eliminate the cytotoxic effects of extracts from these forages in vitro. A single dose of 400 mL of Celmanax Liquid was able to reverse the course of disease in 69% of calves with advanced and progressive mycotoxicosis and presumptive JHS.
**Based on published work: Baines, et al. BMC Veterinary Research 2011, 7:24