Escherichia Coli Bacterin For The Reduction of Clinical Signs of Toxic In Cattle

Dennis E. Van Roekel, DVM* Clinical Assistant Professor University of Wisconsin Madison, Wisconsin 53706

Cooperating Investigator

Perry Clark, PhD University of Wisconsin-River Falls College ofAgriculture Department ofAnimal Science River Falls, Wisconsin 54022

While herd surveys have indicated that 95% of mastitis in California. Similar results were obtained mastitis in cattle is caused by gram-positive bac­ from a single herd in Iowa. 8 teria such as and In 1985-86, was isolated year­ 1 2 , • mastitis caused by coliform round, and was the predominant organism isolated organisms is gaining in recognition. Coliforms causing during the summer in a Wisconsin study.9 Escherichia mastitis include gram-negative, lactose-fermenting or­ coli was the principal cause of mas ti tis of cows in early ganisms of the family Enterobacteriaceae in the genera and late lactation, while nearly equal numbers of E. coli, 2 3 Escherichia, Klebsiella and Enterobacter. • Routine Corynebacterium pyogenes, streptococci and staphylo­ mastitis control measures such as udder washing, teat cocci were isolated at parturition. In this study, 45.5% dipping and milking machine sanitation have reduced (66 _of 145) of the cultures obtained from cows with the incidence of subclinical mastitis caused by the cocci, mastitis and anorexia resulted in the growth of E.coli. but they have not affected the incidence of coliform in­ It was noted that the coliforms caused a more watery fections. 2·4·5 Herds in which mastitis caused by Str. , higher rectal temperatures, less udder swelling agalactiae and S. aureus is under control may still have and increased weakness and anorexia than did other 3 6 problems with clinical mastitis caused by coliforms. · mastitis pathogens. Another investigator10 found that most udders were Prevalence of E.coli Mastitis resistant to E. coli during nonlactating peri­ ods, and that they became susceptible just before Investigators believe that E. coli is the most sig­ parturition. Other reports concur that E. coli mastitis nificant organism causing coliform mastitis because of is most prevalent during early lactation, and declines its wide presence in the barnyard or dairy environ­ during late lactation to an insignificant level in 3 7 2 6 ment, • and studies across the United States have nonlactating periods. , supported this concept.1,3,s.9 In a 1982 study in New York, pathogens were iso­ Pathogenesis and Clinical Signs lated from udder quarters of cows affected with clinical of E.coli Mastitis mastitis which were not responding to intramammary treatment. Forty-four percent (32 of72) of the Clinical mastitis caused by Escherichia coli begins cultures resulted in the discovery of Escherichia coli. 1 with phagocytosis of the with resulting endo­ In a group of studies on eight dairy herds in California, toxin release. Absorption of endotoxin in the mammary 63% of the 158 coliform organisms cultured from cases gland is then believed to cause the subsequent inflam­ of clinical mastitis were E.coli, 10% were Enterobacter matory and systemic reactions. 2 Investigators have aerogenes and 11 % were Klebsiella pneumonia,3 suggest­ shown that endotoxin can be detected in the blood fol­ ing that E. coli is the predominant cause of coliform lowing intramammary infusion, 11 and that acute *Dr. Van Roekel is now a private consultant and veterinarian, Route 2, 640 200th St., Baldwin, Wisconsin 54002.

50 THE BOVINE PRACTITIONER-NO, 28 mastitis with systemic signs occurs after intramammary Also, the following subjective observations of the infusion of endotoxin obtained from E. coli. 12 level of mastitis were made; each parameter was rated in mastitis caused by E.coli is based on a scale from zero to three, with zero used as marked by an early leukopenia mostly due to mass mi­ normal: gration of neutrophils, lymphocytes, monocytes and 7) inflammatory color of udder quarter infused eosinophils to affected mammary quarters.12-14 However, migration is not the only cause of the leukopenia, as 8) hardness of infused quarter one investigation showed that intravenous administra­ 9) degree of udder swelling or increase in size, and tion of endotoxin also causes leukopenia.14 Mammary 10) appearance of milk (degree of watery discoloration quarter inflammation is evidenced by increased redness and presence of clots). and swelling, and by a more watery milk secretion con­ taining clumps of cells. Other clinical laboratory findings The following systemic effects were also noted, and 12 include hyperglycemia, hypercortisolemia12•14 and each was rated using the same zero to three scale with hypocalcemia. 14 Systemic manifestations include fever, zero as normal: depression and anorexia. 2 11) degree of anorexia 12) degree of lethargy Escherichia Coli Bacterin Field Trial 13) degree of dehydration, and Materials and Methods 14) degree oflooseness offeces.

Animals used in this field trial consisted of39 preg­ Data collected were averaged within each treat­ nant Holstein heifers which were screened and selected ment group and compared at each interval of on the basis oflow blood titers to E.coli antibodies. The observation. heifers had no known history of mastitis or other dis­ ease, or of vaccination against E. coli. They were Results randomly assigned to vaccinate (21 heifers) and control Average body temperature of heifers in both treat­ (18 heifers) groups. ment groups started rising within two hours of the The product tested was an Escherichia coli challenge, and peaked at six hours post-challenge (Fig­ bacterin*, currently marketed to protect against calf ur_e 1). The control group had consistently higher body scours. The 21 heifers in the vaccinate group received a temperatures than the vaccinates, and the control av- single 2 ml intramuscular dose of the bacterin, and the . erage peaked at 107.0°F while the vaccinates peaked at 18 control heifers received a 2 ml intramuscular dose of 105.3°F. Average pulse and respiratory rates (Figures 2 a sterile saline placebo. and 3, respectively) followed a similar pattern, as val­ All heifers in both treatment groups were then ues for the control group were consistently higher challenged with two virulent cultures of Escherichia coli. although the difference in respiratory rates was not sig­ Using strains B 117 and B44, 40,000 to 70,000 bacteria nificant. were infused into the teat of one rear quarter of each udder at 8-38 days post-parturition, which was 27-122 Figure 1. days after vaccination with the bacterin or placebo. Body Temperature

In the 24-hour period following bacterial challenge, 108.0 ~------, each heifer was observed at two-hour intervals, and the Controls • following data were collected from each heifer: 107.0 1) body temperature (°F) ~ 2) pulse rate co ai 105.0 - Q.. E 3) respiratory rate Cl) I-­ Cl) 4) number of E. coli colonies re-isolated from quar­ 01 C1l ai > ters at 4, 8, 12 and 24 hours post-challenge

*bovine Pili Shield™, Grand Laboratories, Inc., Larchwood, Iowa 51241, U.S. Vet License No. 303

SEPTEMBER, 1994 51 Figure 4 shows the average number of E. coli colo­ in somatic cell counts (SCC) of challenged and unchal­ nies re-isolated from challenged udder quarters at 4, 8, lenged quarters for periods of 7-14 days pre-challenge, 12 and 24 hours post-challenge. On average, less than at time of challenge, and 12 and 24 hours post-challenge. seven colonies could be cultured from the vaccinated The differences between treatment groups were not sig­ heifers while over 100 (too numerous to count) were nificant in either figure, but significant increases in SCC cultured from many of the control heifers. Average num­ occurred in both infected and non-infected quarters in ber of rumen contractions (Figure 5) was similar between both groups post-challenge. the two treatment groups. Figures 6 and 7 show changes

Figure 2. Figure 4.

QJ C> PULSE RATE c:: ~ "§ Average E. Coli Colonies 115.0 u ui 50.00 QJ 11 0.0 a.0 • Controls 'S • Vaccinates c:: "::l ~ ~ 105.0 w 40.00 - ai 0 a. .l(/J QJ ro 100.0 a: a: 30.00 (I) ~ <11 95.0 ro "5 ::, a. a (I) 20.00 C> 90.0 ai ro a. ai <11 > QJ cl: 85.0 'i:: 0 10.00 0 u 80.0 a 0 2 4 6 8 10 12 14 16 18 20 22 24 ai 0.00 .0 0 4 8 12 24 Hours After Challenge E ::, z Hours After Challenge

Figure 3. Figure 5. Respiratory Rate Rumen Contractions

70.0 ------, 2.70 • Controls Controls • 2.52 65.0 2.50 ~ .§ u 2.30 ~ c >, 0 o 50.0 u 2.10 e a ·o. ai <11 .0 (I) E 1.90 a: ::, QJ C> z ro 1.70 ai> 35.0 < 1.50 o 2 4 6 a 1 o 1 2 1 4 1 6 1 a 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24 Hours After Challenge Hours After Challenge

52 THE BOVINE PRACTITIONER-NO. 28 Average degree of change in inflammatory color of Both the degree of anorexia (Figure 12) and de­ infused quarters (Figure 8), average degree of quarter gree oflethargy (Figure 13) were greater in the control hardness (Figure 9) and average degree of udder swell­ group after six hours post-challenge. While one heifer ing (Figure 10) were all consistently greater in the in the vaccinated group showed slight dehydration, av­ control animals. Although appearing similar up to eight erage dehydration in the vaccinated group was negligible hours post-challenge, afterwards the milk obtained from (Figure 14), and only slightly higher in the control group. the control heifers appeared more watery and had more Average consistency offeces (Figure 15) was erratic, and clots than did the milk from the vaccinated heifers (Fig­ did not show a difference between treatment groups. ure 11).

Figure 6. Figure 8. E. Coli Challenge Trial sec Levels Color of Quarter Infected Quarters: Control VS Vaccinate 2.50 --.------10.0 --.------c::, C ontrols • Vaccinates • • Controls 0 • Vaccinates a:; 2.00 - E 8.0 - 1.50 0 6.0 ~ en u C 0 a, 1.00 ~ Q) ~ 4.0 o .E: Q) Cl () () ~ 0.50 (/) 2.0 Cl) cii ;;,,

SEPTEMBER, 1994 53 Figure 10. Figure 13. Degree of Udder Swelling Degree of Lethargy

2.00 2.00 Controls• (Q) S S Controls • 6 6 Vaccinates • 1 .75 -0 n -0 (I) (I) 0 ro iii "'O 1.50 s 1 .50 '-< s >, '"i Cl Cl ...... :§ 1.25 ro (JQ ai -= ~ ::: ai ..-+- (f) 1.00 ...J 1.00 0 0 > (I) (I) 8 (I) 0.75 (I) (D a> a> ...... '"i Q) (I) (") 0 0 0.50 0.50 ~ (I) (I) Cl Cl ~ (1) ca Q) 0.25 aj > > 00> <( <( 00 0 0 2 4 6 8 10 12 14 16 18 20 22 24 4 6 8 10 12 14 16 18 20 22 24 (")...... Hours After Challenge Hours After Challenge a...... 0 ~ 0 1-i; Figure ll. Figure 14. to 0 Appearance of Milk Degree of Dehydration <...... S ~ 0 (D "C 2.00 S 0.30 (I) ~ • Controls 6 iii • Controls '"i • Vaccinates -0 ~ s (I) 0.25 (") iii ..-+- en 1.60 ...... 0 s ..-+-...... u 0.20 0 "C .2 c:: iii ~ (1) (D 1.20 -5 '"i c:: 0.15 00 .Q ;:!" ai iii 0 0 0 "'O 0 0.80 0 (D u (I) en (I) ~ 0 a> (I) 0.05 ~ >, Cl (") (") Q) 0.40 (I) iii Cl 0.0 (D ;: (1) 00 cii 00 > 0 <( 0.. (I) 0.00 -0.05 ...... (I) 00 O> 0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24 ..-+- (I) ...... '"i 0 Hours After Challenge Hours After Challenge cr' I= ..-+-...... 0p Figure 12. Figure 15. Anorexia Consistency of Feces

1.80 S 1.00 6 Contois • M' 1.60 "C Vaccinates • 0 (I) 0,81 -a iii 0.80 G) 1.40 s iv en ~ a, G) u u (I) C LL. 0.60 Ill 1.00 0 >, ! (.) Q. C Ill 0.80 a, .5 vi 0.40 0.60 'cii 0 C 0 0.40 () *c, a, 0.20 ~ Olca 0.20 aj > <( IJ.00 0.00 0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24 Hours After Challenge Hours After Challenge

54 THE BOVINE PRACTITIONER-NO. 28 Discussion References

In 1982, the National Mastitis Council estimated 1. Stem, E.S. et al: Isolation of Mastitis Pathogens From Quarters of that mastitis in beef and had cost produc­ Cows Recently Treated WithAntimicrobialAgents. JAVMA 184:161- 163; 1984. 2. Eberhart, R.J.: Coliform Mastitis. JAVMA ers in the United States over $2 billion annually each 170:1160-1163; 1977. 3. Jasper, D.E. et al: Herd Studies on Coliform of the previous 10 years. 15 As mentioned earlier, while Mastitis. JAVMA 166:778-780; 1975. 4. Kingwill, R.S. et al: The routine mastitis control measures are used to success­ Effect of a Mastitis Control System on Levels of Subclinical and Clini­ fully reduce subclinical mastitis caused by the cal Mastitis inTwo Years. Vet. Rec. 87:94-100; 1970. 5. Natzke, R.P.: Long Term Effect of a Teat Dip-Dry Cow Treatment Program. In Pro­ gram-positive pathogens, they do not reduce the inci­ ceedings, 13th Ann. Meeting, National Mastitis Council, 1974:74-76; 2 6 dence or significance of coliform organisms. - However, 197 4. 6. Eberhart, R.J.; Buckalew, J.M.: Intramammary Infections newer procedures such as pre-dipping and proper dry­ in a Dairy Herd With a Low Incidence of Streptococcus agalactiae ing of teats pre-milking, do help reduce the incidence of and Staphylococcus aureus Infections. JAVMA 171:630-634; 1977. 7. Howell, D.: Survey on Mastitis Caused by Environmental coliform mastitis. An effective vaccine could add to pro­ Bacteria. ¼t. Rec. 90: 654-657; 1972. 8. McDonald, T.J. et al:Aero­ tection against coliform mastitis. bic Gram-Negative Rods Isolated from Bovine Udder Infections. In this challenge trial, results indicate that a single AJVR 31:1937-1941; 1970. 9. Jones, G.F.; Ward, G.E.: Cause, Oc­ dose of Pili Shield™ can reduce the severity of clinical currence, and Clinical Signs of Mastitis and Anorexia in a Wisconsin Study. JAVMA 195:1108-1113; 1989. 10. Bramley, A.J.: The coliform mastitis in a dairy herd. Eleven of the 14 pa­ Aetiology and Control of Coliform Mastitis in Dairy Cattle. Ph.D. rameters used to monitor the level of mastitis showed Thesis, University of Reading, England, 1974. 11. Ziv, G. et al: the beneficial effects of the bacterin; the only param­ Endotoxin in Blood and Milk and Enzymes in the Milk of Cows Dur­ eters that did not show significant differences were the ing Experimental Escherichia coli Endotoxin Mastitis. Theriogenology 6:343-352; 1976. 12. Paape, M.J. et al: Plasma number of rumen contractions, consistency offeces and Corticosteroid, Circulating Leukocyte and Milk Somatic Cell Re­ . sponses to Escherichia coli Endotoxin Induced Mastitis. Proc Soc Another investigator17 found that pre- and post­ Exp Biol Med 145:553-559; 1974. 13. Schalm, O.W.: Pathologic partum injections of a mutant (strain J5) E. coli vaccine Changes in the Milk and Udder of Cows with Mastitis. JAVMA 170:1137-1140; 1977. 14. Griel, L.C. et al: Clinical and Clinico­ reduced the rate of clinical coliform mas ti tis from 12.8% Pathological Effects of Escherichia coli Endotoxin in Mature Cattle. to 2.6%. Other field studies18 have concurred that ad­ Can J Comp Med. 39:1-6; 1975. 15. National Mastitis Council, ministration of bovine Pili Shield™ may reduce the Udder Topics, February, 1982. 16. Grand Laboratories, Inc., Bul­ incidence of clinical coliform mastitis. letin on bovine Pili Shield™. 17. Gonzalez, R.N.; Cullor, J.S.; Jasper, D.E. et al: Prevention of Clinical Coliform Mastitis in Dairy Cows by a Mutant Escherichia coli Vaccine. Can J Vet Res. 53:301; 1989. 18. Peter, A T.; Clark, P.W.; Van Roekel, D. E.; Luker, C.W.; Gaines, J.D.; Bosu, W.T.K.: Temporal Changes in Metabolites of Prostanoids in Milk of Heifers After lntramammary Infusion of Escherichia coli Organisms. Prostaglandins 39:451; 1990.

Studies on virulence factors of Escherichia Coli isolated from cows with acute mastitis.

C. Valente, P. Cardaras, A. Ciorba, and B. Tesei. Arch. Vet. Ital. (1988) 39, 254-260.

Escherichia coli isolated from udders with acute mastitis E. coli isolated from calves with enteric syndromes. E. were identified biochemically and serologically. The bac­ coli from the udder were a serologically heterogeneous teria were then tested for mannose haemagglutination, group without the characteristics that are usually cor­ en terotoxin production, serum resistance and related with E. coli of intestinal origin. invasiveness, characteristics normally associated with

SEPTEMBER, 1994 55