Veterinary Quarterly
ISSN: 0165-2176 (Print) 1875-5941 (Online) Journal homepage: http://www.tandfonline.com/loi/tveq20
The role of endotoxin in the pathogenesis of acute bovine laminitis R. Boosman , C. W. A. A. M. Mutsaers & A. Klarenbeek To cite this article: R. Boosman , C. W. A. A. M. Mutsaers & A. Klarenbeek (1991) The role of endotoxin in the pathogenesis of acute bovine laminitis, Veterinary Quarterly, 13:3, 155-162, DOI: 10.1080/01652176.1991.9694301 To link to this article: http://dx.doi.org/10.1080/01652176.1991.9694301
Published online: 01 Nov 2011.
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Date: 05 November 2015, At: 14:21
The role of endotoxin in the pathogenesis of acute bovine laminitis
Downloaded by [117.253.161.199] at 14:21 05 November 2015
R. Boosman, C. W. A. A. M. Mutsaers, and A. Klarenbeek SUMMARY. To study the possible role of endotoxin in the pathogenesis of bovine laminitis, local and systemic injections of endotoxin (E. coli 0111 B4) with different doses were given to three groups of four cows each. Clinical and haematologic parameters indicated an acutephase response, including positive plasma ethanol gelation (soluble fibrin), the occurence of fibrin degradation products and decreased thrombocyte counts. Local Shwartzman reactions were not evoked. Clinical examination of the claws and the gait of the animals revealed no signs of laminitis. However, on histopathological examination of the claw corium signs of laminitis such as vacuolisation of the Stratum basale, lymphocyte and leucocyte infiltration and thrombosis were found. These results indicate that endotoxin indeed may be involved in the pathogenesis of laminitis. For the development of a clinical acute laminitis model in cattle
either another dosage, other toxins or factors in addition to the endotoxin used in this experiment are needed.
INTRODUCTION
.
Lactic acid, histamine and endotoxin have been described as contributing to the aetiology and pathogenesis of acute bovine and equine laminitis, although no causal relationship has been established (1, 7, 10, 18, 21, 22). Endotoxin has received special attention because acute bovine laminitis has been reported to occur in diseases (18) associated with endotoxaemia, such as Gramnegative mastitis (12), endometritis (19) and ruminal acidosis caused by carbohydrate overload (4, 7). Also, in horses with experimentally induced laminitis (carbohydrate overload), endotoxaemia was proven (21). Endotoxin may take part in the pathogenesis of laminitis by means of (a-mediated) neurohumoral peripheral vasoconstriction (5, 8) or local/systemic activation of the clotting system (18, 24, 15). Both may lead to hypoxia and tissue damage of the claw corium. A local Shwartzman reaction (LSR), an endotoxin induced local inflammatory reaction (17), was also suggested as being involved in the pathogenesis of laminitis because of the remarkable resemblance in histopathology of the corium of hooves in acute equine laminitis and the LSR in horse skin (20).
In the present study, the effects of different doses of E. coli endotoxin in cattle are reported, with respect to clotting dysfunction, LSR and clinical and histopathologic characteristics of claw and claw corium related to laminitis.
MATERIAL AND METHODS
Animals
Three groups of four cross-bred Holstein-Friesian/Dutch-Friesian cows ranging in age from 2.5 to 5.5 years were used for this study. Their mean bodyweight was 516 kg and the mean daily milk yield was 11.7 1. The animals were housed in a tie stall for at least 3 weeks before and during the experiment. The daily ration consisted of 4-6 kg concentrate, Department of General and Large Animal Surgery, Faculty of Veterinary Medicine, University of Utrecht, Yalelaan 12, 3584 CM Utrecht, The Netherlands. THE VETERINARY QUARTERLY, VOL 13, No. 3, JULY 1991
155
and grass silage with a high fibre content ad libitum. Claws were examined macroscopically, pre-experimentally as well as post mortem, for signs of laminitis. During the experiment indwelling catheters were in the jugular vein, using a 0.1 M sodium
tricitrate-saline solution to keep the catheters patent. The animals in groups I and II were slaughtered 72 h after the experiment started, whereas the cows of group III were kept for another 5 weeks in order to determine if macroscopic signs of laminitis would develop. Endotoxin
Escherichia coli 0111 B4 endotoxin (Difco Laboratories, Detroit, Mich., USA) was administered according to the scheme in Table 1. Intradermal endotoxin injections were given on the thoracic wall divided over six different locations (0.1, 1, 5, 10 (2x) and 20 gig, respectively). Intravenous (iv) endotoxin administration was done via the catheter; infusions were saline diluted. In group II, infusion started with a bolus injection (100 mg)
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of endotoxin. The dosage was progressive; when the animals layed down in lateral recumbancy the infusion rate was diminished. On the third day the same system was
followed. The animals in group III received endotoxin infusions for three days. The dosage was lower than in group II and both the assessment of habitude and thrombocyte count were used as parameters for determining the maximal dosage. The infusion of endotoxin was temporarily stopped when the thrombocyte count was less than 120x109/1. Table I. Dosage schedule of endotoxin administration (mg 0111: B4) (to = intradermal, iv = intravenous, ' bolus injections, 2 infusion between 8 a.m. and 11 p.m.
Group
Day 1
Day 2
Day 3
I II
46.1 ID 46.1 ID 4602 iv
75' iv 2225' iv
63502 iv
1202 iv
710' iv
III
100' iv
Monitoring
During the experiment, the animals were monitored by regular measurements of rectal temperature and respiration and pulse frequencies. The temperature of the digits was estimated by touch and scored semiquantitatively (1 = very cold, 2 = colder than normal, 3 = normal, 4 = warmer than normal, 5 = warm). Whole blood (EDTA-treated) was analysed for white blood cell count (WBC), leucocyte differential and packed cell volume,
heparinised blood Tor blood gases, and heparinised plasma (centrifuged for 10 min, 1500xg, 0° C) was used for the determination of plasma iron (DPC Universal photometer, Vitatron, Dieren, The Netherlands) and zinc (Atomic absorption spectrophotometer 305B, Perkin Elmer, Conn., USA) concentrations. Coagulation tests
An ethanol gelation test, indicating soluble fibrin monomers, was performed with citrated platelet-poor plasma. After incubation for 3 min at room temperature, 0.5 ml plasma was mixed with 0.15 ml of 50% ethanol and put aside for 10 min. Positive plasma samples showed precipitates or gels (11). The remainder of the plasma samples were examined for gelation or precipitation of fibrin monomers after 24 h at 4° C. The following coagulation parameters were studied in two animals of group II and all cows of group III: Fibrin degradation products (FDP) were measured in thrombin activated plasma by latex slide agglutination (Slidex FDP kit, Bio Merieux). According to the manufacturer the sensitivity was 10 tig/m1 plasma. The partial thromboplastin time (PTT) and prothrombin time (PT) were measured in citrated plasma with a semi-automatic apparatus (Option 2, Bio Merieux) which was
calibrated daily with normal and abnormal reference plasma (Uniplasmatrol Normal/ Anormal, Bio Merieux). PTT was determined in cephalin/kaolin-activated plasma (PTT Reagent, Boehringer Diagnostica, Mannheim, Germany) after adding calcium chloride (0.025 M). PT was determined by adding thrombine and calcium (Thrombomat, Bio 156
THE VETERINARY QUARTERLY, VOL. 13, No. 3, JULY 1991
Merieux, Charbonnières les' Baines, France). Values were depicted as a percentage of the coagulation time of normal reference plasma as measured on the respective days.
The trombocyte count was performed on EDTA-treated whole blood, using a cell counter (Sysmex F800 microcellcounter, Toa Medical Electronics, Kobe, Japan). Histology
Skin biopsies for the evaluation of LSR were taken of the l0-pg endotoxin injection sites just before and 8 h after the iv endotoxin injection (biopsy punch 0 4 mm).
Post mortem, claws were sliced and fixed in a 4% phosphate buffered formaldehyde solution. Sections of the claw corium of the dorsal wall, toe area, sole and typical ulceration
place were prepared. Sections were paraffin embedded, cut at 5 im and stained with haematoxylin and eosin, phosphotungstic acid haematoxilin and Van Gieson elastica stain. Skin biopsy specimens were treated similarly (4).
Statistics
Data were expressed as means ± standard deviations (cr.-I), compared with preDownloaded by [117.253.161.199] at 14:21 05 November 2015
experimental values, and analysed by paired t-test or Wilcoxon's signed rank test. Histologic data were ranked and groups were compared with the sign test. RESULTS
Clinical findings
The intradermal injection of endotoxin caused a local inflammatory reaction proportional to the dose of endotoxin (oedematous plaque, 0 5-8 cm for 10 pg endotoxin). Intravenous endotoxin administration 23 h after intradermal injections caused no local Shwartzman reaction. All three groups had significant (P < 0.05) increases in rectal temperature and heart rate after the first iv endotoxin administration. Continued iv administration
caused a significant further increase in body temperature in group II only. Respiration frequency increased after iv endotoxin administration, but due to large fluctuations, these increases were only significant in group II, from 1 to 3 h after first iv administration.
In groups I and III, extremities were cold to the touch from 1 to 5 h after the first iv endotoxin on day 2 (P = 0.06). Group II had cold extremities at the end of day 3 (P = 0.06). The stance and gait of the animals were not indicative of acute laminitis. One animal in group II was recumbent for the last 14 hours of the experiment, deteriorated, and died 6 h later. Necropsy revealed extensive haemorrhages (viscera, pleura and peritoneum) and signs of secondary clostridiosis. One animal in group III was hypersensitive to endotoxin; following an anti-shock treatment (Solu Delta Cortef, Upjohn Co., 3 mg/kg, and Dexadreson, Intervet,
2 times 24 mg) the animal recovered slowly and was withdrawn from the experiment at day 2. Haemotologic findings
Intravenous endotoxin administration caused a rapid and highly significant decrease in WBC (neutropenia). Packed cell volumes and actual base excess did not change significantly during the experiment. In all groups, significant decreases in plasma iron and zinc concentrations were found. The ethanol gelation test was positive by either formation of a precipitate or gel following iv endotoxin administration for all animals except for two cows in group I. All positive samples had a precipitate after 24 h. Fibrin degradation products were found after iv endotoxin administration in all animals tested, with maximum concentrations ranging from 40 to 160 pg/ml. Increased clotting times were found for the two animals in group II. In group III, episodes with significantly increased THE VETERINARY QUARTERLY, VOL 13, No. 3, JULY 1991
157
clotting times were noticed after the first endotoxin administration (Fig. 1).
Thrombocyte counts for the two animals in group II decreased gradually to very low numbers. In group III, thrombocyte counts were decreased significantly from 4 to 24 h after the first iv endotoxin administration, followed by several episodes of decreased thrombocyte counts (Fig. 1). --0-
Group (n=2)
-- Group
II
III
(n=4)
100-
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-75
230 210-
A
e 190 0E 17017
0.
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'°- ------ ..*
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9070
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-
-----
0
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c
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I
15
I
1
I
65 A---A--Time (hours) 25
35
45
55
75
Fig. I. Fibrin degradation products (FDP) Wimp, prothrombin time (PT) and activated partial thromboplastin time (PTT), both as a percentage of a normal reference plasma, and thrombocyte count (109/1) in two cows in group II and four cows in group III (mean ± sd, arrows indicate endotoxin injections and duration of endotoxin infusions).
158
THE VETERINARY QUARTERLY, VOL. 13, No. 3, JULY 1991.
Pathology
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Pre- and postchallenge skin biopsies of the endotoxin injection sites were characterised by eosinophilic leucocyte infiltration. No signs of a local Shwartzman reaction, such as fibrin deposition in or around blood vessels, were found in postchallenge biopsy sections. Pre-experimental claw-trimming revealed no signs related to laminitis, except some petechial haemorrhages in the solar area. Trimming of the claws and cutting them by band-saw after slaughter revealed no signs of laminitis either. Acute changes to be expected in groups I and II were not observed. The claws of the animals in group III were examined for signs of chronic laminitis (e.g. haemorrhages in the sole, ridges on the dorsal wall): none were found. Histologic investigations of the claw corium of the animals in group I showed no signs of acute laminitis. Only very few lymphocytes, plasma cells and leucocytes were seen. The Stratum basale of the epidermis had a regular and dense appearance in two animals; the other two cows had vacuolisation of the Stratum basale cells in the sole area. The animals in group II had mild infiltration of lymphocytes and leucocytes; one animal had marked infiltration of eosinophilic leucocytes. Obvious vacuolisation (Fig. 2) and occasional pyknosis of the Stratum basale cell nuclei were observed in all four animals. Two animals had severe capillary and venular thrombosis (Fig. 3), indicating diffuse intravascular coagulation, accompanied by severe congestion and haemorrhages. The three cows in group III that withstood
the experiment without complications had mild congestion and lymphocyte infiltration in the corium and a marked vacuolation of the Stratum basale cells.
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Vacuolisation of the nuclei in the Stratum basale (Group II, HE, 400x).
THE VETERINARY QUARTERLY, VOL. 13, No. 3, JULY 1991
159
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Table 2.
Semiquantitative score of the histological findings of the claw corium from 12 cows after experimental endotoxaemia. Congestion, thrombosis, lymphocyte and leucocyte infiltration and vacuolisation of the stratum basale were scored semiquantitatively (- = none, ± = within normal range, + = some, ++ = marked and +++ = much.
Group I cow A cow B cow C cow D
Group II cow E cow F cow G cow H
Congestion
Thrombosis
Lymphocyte inf. Leucocyte inf.
Vacuolisation
-.
-
-
-
± ++
± ++
±
++ ++
+ ± +
-
++
++
±
-
-
+ ± ± ±
-
-
+ ++
+
++ ++
+++ +++
++ +
.,
-
-
+
'+
Group III cow I cow J cow K cow L
+ + + ±
-
-
-
-
+
++ .++
The animal that was hypersensitive to endotoxin showed mild infiltration with lymphocytes and plasma cells, and marked vacuolation of the Stratum basale cells (Table 2). In all twelve animals the tonofibrils in the Stratum spinosum appeared regular. No signs of increased mitoses of the Stratum basale cells were seen. No significant differences were found among the histologic parameters of the respective groups. 160
THE VETERINARY QUARTERLY, VOL.
13, No, 3, JULY 1991
DISCUSSION
Acute laminitis is not a common disease in cattle, attempts to induce laminitis experimentally have been only partially successful (18, 23, 4). In the present study,
the effects of endotoxaemia on the claw corium and factors related to the pathogenesis of laminitis were investigated. The knowledge gained in this experiment may help in the future development of a bovine laminitis model. Studies on local Shwartzman reactions in cattle have not been reported before. An evident LSR was not induced, neither clinically nor histologically. Therefore, a LSR is not likely to be involved in the pathogenesis of acute bovine laminitis. Activation of the coagulation system occurred at the lowest dosage of endotoxin administration (positive ethanol gelation). With higher doses additional data were collected, indicating episodes of coagu-
lopathy. Thrombosis of the hoof and claw vasculature is considered a major hallmark of acute laminitis. Despite systemic activation of the clotting system and Downloaded by [117.253.161.199] at 14:21 05 November 2015
thrombosis of the claw corium in two animals in the present study, no acute laminitis was evoked. For that reason we conclude that systemic activation of the clotting system may be involved in the pathogenesis of acute laminitis, but cannot evoke acute laminitis. Histopathologic examination revealed lesions indicative of acute laminitis in the corium and epidermis (2, 14, 16, 18), such as vacuolation of the Stratum basale
cells, lymphocyte and in one case polymorphonuclear leucocyte infiltration, congestion and haemorrhages. These results indicate that endotoxin may be involved in the pathogenesis of laminitis. The endotoxin doses used in the experiment are known to cause a full acute-phase reaction in cows accompanied by an obvious sympatico-adrenal reaction (5, 6). In this study an additional activation of the coagulation system was proven. All
these reactions have been associated with the aetiology and pathogenesis of laminitis and have been reported to occur in the equine laminitis model (13). Nevertheless, we did not evoke clinical acute laminitis. This negative result may be due to a low susceptibility to a LSR and (therefore) acute laminitis in cattle when compared to horses. The time period during which the endotoxin stimulus was given (48-72 h) is not likely to be too short for induction of laminitis in cattle, since in the equine laminitis model laminitis developed within 40 h following carbohydrate overload (9). In addition, the sort of endotoxin may have differed from the naturally occurring endotoxins in the equine laminitis model. Moreover, spontaneously occurring laminitis is often alleged to be associated with mastitis and/or endometritis (18) in which Gram-positive bacteria may also be involved. Toxins of Gram-negative and Gram-positive bacteria can strongly potentiate each other (3). In conclusion, for the development of a clinical acute laminitis model in cattle either another dosage, other toxins or factors in addition to the endotoxin used in this experiment are needed. ACKNOWLEDGEMENTS
The authors would like to thank Prof. E. Gruys and Prof. F. Németh for their supervision.
REFERENCES I. Akerblom E. Ober die Atiologie und Pathogenese der Futterrehe beim Pferde. Scand Arch Physiol 2.
1934; 68: Suppl: 1-168. Andersson LA, Bergman A. Pathology of bovine laminitis especially as regards vascular lesions. Acta Vet Scand 1980; 21: 559-66.
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3.
Blomster-Hautamaa DA, Schlievert PM. Non-enterotoxic staphylococcal toxins. In: Handbook of natural toxins. Vol 4 Bacterial toxins. Eds: Hardegree MC, Tu AT. Marcel Dekker Inc. NY
4.
Boosman R. Bovine laminitis. Histopathologic and arteriographic aspects and its relation to
1988; ch 10: 297-330.
endotoxaemia. Thesis (chapter 6, pp. 75-94), University of Utrecht, 1990. 5. Boosman R, Mutsaers CWAAM, Dieleman SJ. Sympathico-adrenal aspects of endotoxaemia in cattle. Vet Rec 1990; 127: 11-4. 6. Boosman R, Niewold TA, Mutsaers CWAAM, Gruys E. Serum amyloid A concentration in cows given endotoxin as an acute phase-stimulant. Am J Vet Res 1989; 50: 1640-4. 7.
Dougherty RW, Coburn KS, Cook HM, Allison MJ. Preliminary study of appearance of
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endotoxin in circulatory system of sheep and cattle after induced grain engorgement. Am J Vet
Res 1975; 36: 831-2. 8. Elmes PJ, Eyre P. Vascular reactivity of the bovine foot to neurohormones, antigens, and chemical mediators of anaphylaxis. Am J Vet Res 1977; 38: 107-12. 9. Garner HE, Coffman JR, Hahn AW, Hutcheson DP, Myron E, Tumbleson ME. Equine laminitis of alimentary origin: An experimental model. Am J Vet Res 1975; 36: 441-4. 10. Garner HE, Hutcheson DP, Coffman JR, Hahn AW, Salem C. Lactic acidosis: a factor associated with equine laminitis. J Anim Sci 1977; 45: 1037-41. 11. Godal HC, Abilgaard U, Kierulf P. Ethanol gelation and fibrin monomers in plasma. Scand J Haemotol 1971; S 13: 189-91. 12. Hakogi E, Shimada Y, Kume T, Tabuchi K. Perchloric acid treatment and use of chromogenic substrate in the Limulus test; application to veterinary diagnosis. Vet Microbiol 1984/1985; 10: 33-42.
13. Hood DM, Stephens KA. Physiopathology of equine laminitis. Comp Cont Ed 1981; 3: S454S459.
14. Maclean CW. The histopathology of laminitis in dairy cows. J. Comp Pathol 1971; 81: 563-70. 15. McClure JR, McClure JJ. Intravascular coagulopathies associated with alimentary induced acute laminitis in the pony. Proc. of the 1st Equine Endotoxaemia-Laminitis Symp. Newsletter Am Assoc Equine Pract 1982; 2: 124-7. 16. Mgasa MN, Mortensen K, Mbassa GK. The occurence of bovine lameness in Tanzania extensive and intensive dairy production systems. Proc. of the 15th World Buiatric Congress, Palma de Mallorca, Spain. 1988; vol II: 1089-94. 17. Movat HZ, Burrowes CE. The local Shwartzman reaction: endotoxin-mediated inflammatory and thrombo-hemorrhagic lesions. In: Handbook of endotoxin Vol 3 Cellular biology of endotoxin. Ed.: Berry LJ. Elsevier Publishers, Amsterdam 1985; 206-302. 18.
Nilsson SA. Clinical, morphological and experimental studies of laminitis in cattle. Acta Vet Scand 1963; 4 Suppl I.
19.
Peter AT, Bosu WTK, Luker CW. Plasma endotoxin and concentrations of stable metabolites of prostacyclin, thromboxane A2, and prostaglandin E2 in postpartum dairy cows. Prostaglandins 1987; 34: 15-28.
20. Sprouse R, Garner H, Eyestone W. The Shwartzman phenomenon in equine species. Exp Pathol
1982; 22: 165-71.
21. Sprouse RF, Garner HE, Green EM. Plasma endotoxin levels in horses subjected to carbohydrate induced laminitis. Equine Vet J 1987; 19: 25-8. 22. Suber RL, Hentges JF, Gudat JC, Edds GT. Blood and ruminal fluid profiles in carbohydratefoudered cattle. Am J Vet Res 1979; 40: 1005-8. 23. Takahashi K, Young BA. Effects of grainoverfeeding and histamine injection on physiological responses related to acute bovine laminitis. Jpn J Vet Sci 1981; 43: 375-85. 24. Thomson GW, Mc Sherry BJ, Valli VEO. Endotoxin induced disseminated intravascular coagulation in cattle. Can J Comp Med 1974; 38: 457-66.
162
THE VETERINARY QUARTERLY, VOL 13, No. 3, JULY 1991
ISSN: 0165-2176 (Print) 1875-5941 (Online) Journal homepage: http://www.tandfonline.com/loi/tveq20
The role of endotoxin in the pathogenesis of acute bovine laminitis R. Boosman , C. W. A. A. M. Mutsaers & A. Klarenbeek To cite this article: R. Boosman , C. W. A. A. M. Mutsaers & A. Klarenbeek (1991) The role of endotoxin in the pathogenesis of acute bovine laminitis, Veterinary Quarterly, 13:3, 155-162, DOI: 10.1080/01652176.1991.9694301 To link to this article: http://dx.doi.org/10.1080/01652176.1991.9694301
Published online: 01 Nov 2011.
Submit your article to this journal
Article views: 147
View related articles
Citing articles: 8 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tveq20 Download by: [117.253.161.199]
Date: 05 November 2015, At: 14:21
The role of endotoxin in the pathogenesis of acute bovine laminitis
Downloaded by [117.253.161.199] at 14:21 05 November 2015
R. Boosman, C. W. A. A. M. Mutsaers, and A. Klarenbeek SUMMARY. To study the possible role of endotoxin in the pathogenesis of bovine laminitis, local and systemic injections of endotoxin (E. coli 0111 B4) with different doses were given to three groups of four cows each. Clinical and haematologic parameters indicated an acutephase response, including positive plasma ethanol gelation (soluble fibrin), the occurence of fibrin degradation products and decreased thrombocyte counts. Local Shwartzman reactions were not evoked. Clinical examination of the claws and the gait of the animals revealed no signs of laminitis. However, on histopathological examination of the claw corium signs of laminitis such as vacuolisation of the Stratum basale, lymphocyte and leucocyte infiltration and thrombosis were found. These results indicate that endotoxin indeed may be involved in the pathogenesis of laminitis. For the development of a clinical acute laminitis model in cattle
either another dosage, other toxins or factors in addition to the endotoxin used in this experiment are needed.
INTRODUCTION
.
Lactic acid, histamine and endotoxin have been described as contributing to the aetiology and pathogenesis of acute bovine and equine laminitis, although no causal relationship has been established (1, 7, 10, 18, 21, 22). Endotoxin has received special attention because acute bovine laminitis has been reported to occur in diseases (18) associated with endotoxaemia, such as Gramnegative mastitis (12), endometritis (19) and ruminal acidosis caused by carbohydrate overload (4, 7). Also, in horses with experimentally induced laminitis (carbohydrate overload), endotoxaemia was proven (21). Endotoxin may take part in the pathogenesis of laminitis by means of (a-mediated) neurohumoral peripheral vasoconstriction (5, 8) or local/systemic activation of the clotting system (18, 24, 15). Both may lead to hypoxia and tissue damage of the claw corium. A local Shwartzman reaction (LSR), an endotoxin induced local inflammatory reaction (17), was also suggested as being involved in the pathogenesis of laminitis because of the remarkable resemblance in histopathology of the corium of hooves in acute equine laminitis and the LSR in horse skin (20).
In the present study, the effects of different doses of E. coli endotoxin in cattle are reported, with respect to clotting dysfunction, LSR and clinical and histopathologic characteristics of claw and claw corium related to laminitis.
MATERIAL AND METHODS
Animals
Three groups of four cross-bred Holstein-Friesian/Dutch-Friesian cows ranging in age from 2.5 to 5.5 years were used for this study. Their mean bodyweight was 516 kg and the mean daily milk yield was 11.7 1. The animals were housed in a tie stall for at least 3 weeks before and during the experiment. The daily ration consisted of 4-6 kg concentrate, Department of General and Large Animal Surgery, Faculty of Veterinary Medicine, University of Utrecht, Yalelaan 12, 3584 CM Utrecht, The Netherlands. THE VETERINARY QUARTERLY, VOL 13, No. 3, JULY 1991
155
and grass silage with a high fibre content ad libitum. Claws were examined macroscopically, pre-experimentally as well as post mortem, for signs of laminitis. During the experiment indwelling catheters were in the jugular vein, using a 0.1 M sodium
tricitrate-saline solution to keep the catheters patent. The animals in groups I and II were slaughtered 72 h after the experiment started, whereas the cows of group III were kept for another 5 weeks in order to determine if macroscopic signs of laminitis would develop. Endotoxin
Escherichia coli 0111 B4 endotoxin (Difco Laboratories, Detroit, Mich., USA) was administered according to the scheme in Table 1. Intradermal endotoxin injections were given on the thoracic wall divided over six different locations (0.1, 1, 5, 10 (2x) and 20 gig, respectively). Intravenous (iv) endotoxin administration was done via the catheter; infusions were saline diluted. In group II, infusion started with a bolus injection (100 mg)
Downloaded by [117.253.161.199] at 14:21 05 November 2015
of endotoxin. The dosage was progressive; when the animals layed down in lateral recumbancy the infusion rate was diminished. On the third day the same system was
followed. The animals in group III received endotoxin infusions for three days. The dosage was lower than in group II and both the assessment of habitude and thrombocyte count were used as parameters for determining the maximal dosage. The infusion of endotoxin was temporarily stopped when the thrombocyte count was less than 120x109/1. Table I. Dosage schedule of endotoxin administration (mg 0111: B4) (to = intradermal, iv = intravenous, ' bolus injections, 2 infusion between 8 a.m. and 11 p.m.
Group
Day 1
Day 2
Day 3
I II
46.1 ID 46.1 ID 4602 iv
75' iv 2225' iv
63502 iv
1202 iv
710' iv
III
100' iv
Monitoring
During the experiment, the animals were monitored by regular measurements of rectal temperature and respiration and pulse frequencies. The temperature of the digits was estimated by touch and scored semiquantitatively (1 = very cold, 2 = colder than normal, 3 = normal, 4 = warmer than normal, 5 = warm). Whole blood (EDTA-treated) was analysed for white blood cell count (WBC), leucocyte differential and packed cell volume,
heparinised blood Tor blood gases, and heparinised plasma (centrifuged for 10 min, 1500xg, 0° C) was used for the determination of plasma iron (DPC Universal photometer, Vitatron, Dieren, The Netherlands) and zinc (Atomic absorption spectrophotometer 305B, Perkin Elmer, Conn., USA) concentrations. Coagulation tests
An ethanol gelation test, indicating soluble fibrin monomers, was performed with citrated platelet-poor plasma. After incubation for 3 min at room temperature, 0.5 ml plasma was mixed with 0.15 ml of 50% ethanol and put aside for 10 min. Positive plasma samples showed precipitates or gels (11). The remainder of the plasma samples were examined for gelation or precipitation of fibrin monomers after 24 h at 4° C. The following coagulation parameters were studied in two animals of group II and all cows of group III: Fibrin degradation products (FDP) were measured in thrombin activated plasma by latex slide agglutination (Slidex FDP kit, Bio Merieux). According to the manufacturer the sensitivity was 10 tig/m1 plasma. The partial thromboplastin time (PTT) and prothrombin time (PT) were measured in citrated plasma with a semi-automatic apparatus (Option 2, Bio Merieux) which was
calibrated daily with normal and abnormal reference plasma (Uniplasmatrol Normal/ Anormal, Bio Merieux). PTT was determined in cephalin/kaolin-activated plasma (PTT Reagent, Boehringer Diagnostica, Mannheim, Germany) after adding calcium chloride (0.025 M). PT was determined by adding thrombine and calcium (Thrombomat, Bio 156
THE VETERINARY QUARTERLY, VOL. 13, No. 3, JULY 1991
Merieux, Charbonnières les' Baines, France). Values were depicted as a percentage of the coagulation time of normal reference plasma as measured on the respective days.
The trombocyte count was performed on EDTA-treated whole blood, using a cell counter (Sysmex F800 microcellcounter, Toa Medical Electronics, Kobe, Japan). Histology
Skin biopsies for the evaluation of LSR were taken of the l0-pg endotoxin injection sites just before and 8 h after the iv endotoxin injection (biopsy punch 0 4 mm).
Post mortem, claws were sliced and fixed in a 4% phosphate buffered formaldehyde solution. Sections of the claw corium of the dorsal wall, toe area, sole and typical ulceration
place were prepared. Sections were paraffin embedded, cut at 5 im and stained with haematoxylin and eosin, phosphotungstic acid haematoxilin and Van Gieson elastica stain. Skin biopsy specimens were treated similarly (4).
Statistics
Data were expressed as means ± standard deviations (cr.-I), compared with preDownloaded by [117.253.161.199] at 14:21 05 November 2015
experimental values, and analysed by paired t-test or Wilcoxon's signed rank test. Histologic data were ranked and groups were compared with the sign test. RESULTS
Clinical findings
The intradermal injection of endotoxin caused a local inflammatory reaction proportional to the dose of endotoxin (oedematous plaque, 0 5-8 cm for 10 pg endotoxin). Intravenous endotoxin administration 23 h after intradermal injections caused no local Shwartzman reaction. All three groups had significant (P < 0.05) increases in rectal temperature and heart rate after the first iv endotoxin administration. Continued iv administration
caused a significant further increase in body temperature in group II only. Respiration frequency increased after iv endotoxin administration, but due to large fluctuations, these increases were only significant in group II, from 1 to 3 h after first iv administration.
In groups I and III, extremities were cold to the touch from 1 to 5 h after the first iv endotoxin on day 2 (P = 0.06). Group II had cold extremities at the end of day 3 (P = 0.06). The stance and gait of the animals were not indicative of acute laminitis. One animal in group II was recumbent for the last 14 hours of the experiment, deteriorated, and died 6 h later. Necropsy revealed extensive haemorrhages (viscera, pleura and peritoneum) and signs of secondary clostridiosis. One animal in group III was hypersensitive to endotoxin; following an anti-shock treatment (Solu Delta Cortef, Upjohn Co., 3 mg/kg, and Dexadreson, Intervet,
2 times 24 mg) the animal recovered slowly and was withdrawn from the experiment at day 2. Haemotologic findings
Intravenous endotoxin administration caused a rapid and highly significant decrease in WBC (neutropenia). Packed cell volumes and actual base excess did not change significantly during the experiment. In all groups, significant decreases in plasma iron and zinc concentrations were found. The ethanol gelation test was positive by either formation of a precipitate or gel following iv endotoxin administration for all animals except for two cows in group I. All positive samples had a precipitate after 24 h. Fibrin degradation products were found after iv endotoxin administration in all animals tested, with maximum concentrations ranging from 40 to 160 pg/ml. Increased clotting times were found for the two animals in group II. In group III, episodes with significantly increased THE VETERINARY QUARTERLY, VOL 13, No. 3, JULY 1991
157
clotting times were noticed after the first endotoxin administration (Fig. 1).
Thrombocyte counts for the two animals in group II decreased gradually to very low numbers. In group III, thrombocyte counts were decreased significantly from 4 to 24 h after the first iv endotoxin administration, followed by several episodes of decreased thrombocyte counts (Fig. 1). --0-
Group (n=2)
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Fig. I. Fibrin degradation products (FDP) Wimp, prothrombin time (PT) and activated partial thromboplastin time (PTT), both as a percentage of a normal reference plasma, and thrombocyte count (109/1) in two cows in group II and four cows in group III (mean ± sd, arrows indicate endotoxin injections and duration of endotoxin infusions).
158
THE VETERINARY QUARTERLY, VOL. 13, No. 3, JULY 1991.
Pathology
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Pre- and postchallenge skin biopsies of the endotoxin injection sites were characterised by eosinophilic leucocyte infiltration. No signs of a local Shwartzman reaction, such as fibrin deposition in or around blood vessels, were found in postchallenge biopsy sections. Pre-experimental claw-trimming revealed no signs related to laminitis, except some petechial haemorrhages in the solar area. Trimming of the claws and cutting them by band-saw after slaughter revealed no signs of laminitis either. Acute changes to be expected in groups I and II were not observed. The claws of the animals in group III were examined for signs of chronic laminitis (e.g. haemorrhages in the sole, ridges on the dorsal wall): none were found. Histologic investigations of the claw corium of the animals in group I showed no signs of acute laminitis. Only very few lymphocytes, plasma cells and leucocytes were seen. The Stratum basale of the epidermis had a regular and dense appearance in two animals; the other two cows had vacuolisation of the Stratum basale cells in the sole area. The animals in group II had mild infiltration of lymphocytes and leucocytes; one animal had marked infiltration of eosinophilic leucocytes. Obvious vacuolisation (Fig. 2) and occasional pyknosis of the Stratum basale cell nuclei were observed in all four animals. Two animals had severe capillary and venular thrombosis (Fig. 3), indicating diffuse intravascular coagulation, accompanied by severe congestion and haemorrhages. The three cows in group III that withstood
the experiment without complications had mild congestion and lymphocyte infiltration in the corium and a marked vacuolation of the Stratum basale cells.
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THE VETERINARY QUARTERLY, VOL. 13, No. 3, JULY 1991
159
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Table 2.
Semiquantitative score of the histological findings of the claw corium from 12 cows after experimental endotoxaemia. Congestion, thrombosis, lymphocyte and leucocyte infiltration and vacuolisation of the stratum basale were scored semiquantitatively (- = none, ± = within normal range, + = some, ++ = marked and +++ = much.
Group I cow A cow B cow C cow D
Group II cow E cow F cow G cow H
Congestion
Thrombosis
Lymphocyte inf. Leucocyte inf.
Vacuolisation
-.
-
-
-
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± ++
±
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±
-
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-
-
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+
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.,
-
-
+
'+
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+ + + ±
-
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The animal that was hypersensitive to endotoxin showed mild infiltration with lymphocytes and plasma cells, and marked vacuolation of the Stratum basale cells (Table 2). In all twelve animals the tonofibrils in the Stratum spinosum appeared regular. No signs of increased mitoses of the Stratum basale cells were seen. No significant differences were found among the histologic parameters of the respective groups. 160
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DISCUSSION
Acute laminitis is not a common disease in cattle, attempts to induce laminitis experimentally have been only partially successful (18, 23, 4). In the present study,
the effects of endotoxaemia on the claw corium and factors related to the pathogenesis of laminitis were investigated. The knowledge gained in this experiment may help in the future development of a bovine laminitis model. Studies on local Shwartzman reactions in cattle have not been reported before. An evident LSR was not induced, neither clinically nor histologically. Therefore, a LSR is not likely to be involved in the pathogenesis of acute bovine laminitis. Activation of the coagulation system occurred at the lowest dosage of endotoxin administration (positive ethanol gelation). With higher doses additional data were collected, indicating episodes of coagu-
lopathy. Thrombosis of the hoof and claw vasculature is considered a major hallmark of acute laminitis. Despite systemic activation of the clotting system and Downloaded by [117.253.161.199] at 14:21 05 November 2015
thrombosis of the claw corium in two animals in the present study, no acute laminitis was evoked. For that reason we conclude that systemic activation of the clotting system may be involved in the pathogenesis of acute laminitis, but cannot evoke acute laminitis. Histopathologic examination revealed lesions indicative of acute laminitis in the corium and epidermis (2, 14, 16, 18), such as vacuolation of the Stratum basale
cells, lymphocyte and in one case polymorphonuclear leucocyte infiltration, congestion and haemorrhages. These results indicate that endotoxin may be involved in the pathogenesis of laminitis. The endotoxin doses used in the experiment are known to cause a full acute-phase reaction in cows accompanied by an obvious sympatico-adrenal reaction (5, 6). In this study an additional activation of the coagulation system was proven. All
these reactions have been associated with the aetiology and pathogenesis of laminitis and have been reported to occur in the equine laminitis model (13). Nevertheless, we did not evoke clinical acute laminitis. This negative result may be due to a low susceptibility to a LSR and (therefore) acute laminitis in cattle when compared to horses. The time period during which the endotoxin stimulus was given (48-72 h) is not likely to be too short for induction of laminitis in cattle, since in the equine laminitis model laminitis developed within 40 h following carbohydrate overload (9). In addition, the sort of endotoxin may have differed from the naturally occurring endotoxins in the equine laminitis model. Moreover, spontaneously occurring laminitis is often alleged to be associated with mastitis and/or endometritis (18) in which Gram-positive bacteria may also be involved. Toxins of Gram-negative and Gram-positive bacteria can strongly potentiate each other (3). In conclusion, for the development of a clinical acute laminitis model in cattle either another dosage, other toxins or factors in addition to the endotoxin used in this experiment are needed. ACKNOWLEDGEMENTS
The authors would like to thank Prof. E. Gruys and Prof. F. Németh for their supervision.
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