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Studies on the pathogenesis of bovine ephemeral fever IV: A comparison with the inflammatory events in milk fever of cattle
veterinary microbiology ELSEVIER
Veterinary
Microbiology
46
( 1995) 131-142
Studies on the pathogenesis of bovine ephemeral fever IV: A comparison with the inflammatory events in milk fever of cattle T.D. St. George a,*, G.M. Murphy b, B. Burren b, M.F. Uren ’ a15 Tamarix Street, Chapel Hill, Qld, 4069, Australia b Queensland Department of Primary Industries, Animal Research Institute, Yeerongpilly, Qld, 4105, Australia ’ CSIRO, Division of Tropical Animal Production, Private Bag No. 3, PO, Indooroopilly, Qld, 4068, Australia Accepted 8 March 1995
Abstract The study of ephemeral fever in cattle has defined a range of haematological and biochemical changes in blood which are characteristic of an inflammatory response. One of the clinical signs of ephemeral fever, a temporary paralysis reversible by treatment with calcium borogluconate, is similar to that in milk fever (parturient paresis), a disease of multiparous dairy cows. Three separate groups of cows were studied. Four multiparous cows were observed and sampled repeatedly during calving, three similar cows and one cow calving for the first time in a dairy herd were sampled daily before and after calving; and, in other dairy herds, seven cows with milk fever were sampled during illness. One of the cows under repeated observation during calving developed milk fever. The results showed that all the inflammatory indicators in blood were present in the multiparous cows at calving and that these were essentially similar to those established in ephemeral fever. The similarities in the four cows sampled repeatedly during the periparturient period were: a rectal temperature rise of 1 to 1.2”C; rise in circulating neutrophils to peaks between 5700 and 11200 1K6; disappearance of eosinophils for 1 day; hypocalcaemia (plasma Ca < 2.0 mM 1~ ‘) ; fall of plasma zinc to low levels immediately after calving (plasma Zn < 500 pg l- ’ ) ; fall of inorganic phosphate (plasma P < 0.9 mM 1~ ’ ) ; rises in copper (plasma Cu > 1000 pg I- ‘) and plasma fibrin to > 8.75 g 1-l. Plasma glucose peaked at calving between 5.7 and 8.9 mM l- ’ then fell to levels ranging between 3.4 and 3.8 mM 1~ I. Plasma iron rose in one cow to 1220 /Lg l- ‘, was unchanged in one cow and fell in the other two to 440 and 860 pg l_ 1respectively. The three multiparous cows which were sampled daily and calved normally showed similar haematological, macro and micromineral changes and fibrin response as did the seven milk fever cases. In the periparturient period, milk fever cows differed from multiparous cows calving normally, in degree but not in kind, of inflammatory response. It is postulated that an inflammatory event occurs in the periparturient period of multiparous cows which partially accounts for the falls in plasma calcium. This can precipitate a paralysis and other hypocalcaemic signs similar to that seen in acute ephemeral fever. * Corresponding
author: Tel, (61 7) 3378 5152 Fax: (61 7) 3378 7762
0378-l 135/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIO378-1135(95)00078-X
132
Keywords: paresis
T. George et al. / Veterina~
Bovine
ephemeral
fever; Hypocalcaemia:
Microbiology
46 (1995) 131-142
Inflammation;
Microminerals;
Fibrinogen
; Parturient
1. Introduction Bovine ephemeral fever is caused by infection with a rhabdovirus of the same name. Its clinical expression varies widely in severity. It has two major components. The first of which is expressed as an inflammatory polyserositis plus oedema and the second as a loss of control of smooth and striated muscle associated with hypocalcaemia. As the virus name suggests, clinical expression is ephemeral, usually less than 2-3 days. In more severe cases this paralysis causes deaths in cows, steers and bulls. The inflammatory aspect of the disease was well established by histopathology (Basson et al., 1970) and has since been progressively defined in biochemical terms by St.George et al. (1984). Uren and Murphy (1985), (Murphy et al., 1986, 1989) and Uren et al. ( 1992). the full spectrum of clinical signs can be prevented by treatment with anti-inflammatory drugs (St.George et al., 1986; Uren et al., 1989). However, the treatment of the hypocalcaemia with parenteral calcium borogluconate relieves the paralysis only temporarily unless the anti-inflammatory drugs are also given (St.George et al., 1984). From the biochemical profile and response to treatment, there is no doubt that most of the clinical signs of bovine ephemeral fever are caused by a generalized inflammation with a secondary hypocalcaemia. Milk fever, also known as parturient paresis, most usually develops within the first 2 days after parturition in cows which have had two or more calves. It has rarely been recorded in cows having their first calf. Its prevalence increases with the number of calvings and often recurs in the same animal in successive calvings (Blood and Henderson, 1974). The disease has been known for 250 years but has become more of a problem with the development of the modern high milk yield dairy cow (reviewed Hibbs. 1950). The direct cause of the paresis, which is its major presenting sign, is hypocalcaemia. This was first specifically corrected by Grieg ( 1930) using parenteral treatment with calcium gluconate. This treatment supplanted udder inflation with air or fluids which produced recovery after several hours, by creating back pressure to inhibit milk secretion and consequent drain on calcium. Since that time, research has been focused on the kinetics of calcium transfer within the body of the dairy cow and its hormonal control, as the primary initiators of milk fever in may excellent studies (reviewed Kronfeld. 1971; Ramberg et al., 1984). The current theory is that milk fever is a defect of calcium homeostasis at or near parturition. From personal experience, the nature and response to calcium treatment of clinical signs caused by hypocalcaemia in bovine ephemeral fever and milk fever are similar. However, the diseases have important differences for the clinician. There is a high fever in bovine ephemeral fever and both sexes are affected while in a milk fever cow the rectal temperature is normal, or subnormal in the most acute stage of the disease when the animal is seen by a veterinarian. The present study was undertaken to address these clinical anomalies and to determine whether the range of biochemical dyscrasias that occur in bovine ephemeral fever could
T. George et al. /Veterinary
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133
also be found in multiparous cows at parturition and cow with milk fever. The full range could not be found from a search of the literature.
2. Material and methods Blood samples were obtained from three groups of cattle. The serology, cation analyses (Ca, Mg, Cu, Zn and Fe), blood gas analyses, plasma glucose, inorganic phosphate estimations and haematology, were as described by Uren et al., 1992. 2.1. Group 1: Cows expected to calve norma&
-repeated
observation
Four cows (Nos 14) expected to calve normally with their 3rd, 8th, 10th and 3rd calves respectively were fitted with jugular catheters with a subdermal extension emerging at the withers. This allowed repeated blood sampling without major disturbance (Takken, 1984). Their daily food intake was measured. 2.2. Group 2: Cows expected to calve normally -daily sampling Animals sampled were in a herd of Freisian cattle located 100 km north of Brisbane, Australia. This had been a sentinel herd for ephemeral fever for many years (St.George et al., 1984). The cows grazed on grass pasture with some concentrate supplementation. All cows had experienced ephemeral fever and were immune to the disease. Only four cows were serially sampled because of the dispersal of the herd. Jugular blood samples were collected five to nine times in the 3 weeks prior to calving and daily for 5 to 9 days after calving. 2.3. Group 3: Milk fever cows A jugular blood sample was obtained at the time when clinical milk fever was diagnosed in dairy cows, in commercial herds, before treatment and, when possible, a second blood sample 3 to 5 days later. 2.4. Ephemeral fever: Effects of treatments on plasma microminerals Plasma samples taken during the course of previous experimentation involving treatment of ephemeral fever infected steers with anti-inflammatory drugs (Uren et al., 1989) were analysed for copper and zinc as described by Murphy et al., ( 1986).
3. Results 3.1. Group 1 Cows 1, 3 and 4 calved normally on or near the expected date. The placenta was expelled within 3 h. Cow 2 had a false labour 30-35 h before calving. Contractions ceased until 9 h
134
T. George et al. / Veterinary Microbiology
38
-50
-4c.-diL&YYo
46 (1995) 131-142
hFr-_:.
0
10
20
30
-*
HOUR-S Fig. 1. Neutrophils, 3.
lymphocytes
( 10” Lm6) and rectal temperature in relation to calving of normal calving cow
before a still-born calf was delivered with difficulty, at which time the cow briefly showed some signs of milk fever. The signs intensified 9 h later. Treatment with intravenous calcium borogluconate restored full mobility. The haematology and rectal temperatures in the periparturient period for the normal calving of cow 3 and milk fever of cow 2 are shown in Fig. 1 and Fig. 2, and the magnesium and calcium responses in Fig. 3 and Fig. 4. The glucose and phosphate levels for cow 2 are shown in Fig. 5. The microminerals for cow 2 are shown in Fig. 6. The general observations can be summarized: Rumination slowed and ceased for 2-5 h before calving. All cows lost appetite in the 24 h prior to calving but ate avidly within 24 h post calving; cow 2 did not eat until treated. Plasma calcium levels began to fall l-3 days before calving to reach plasma Ca I 2.0 mM-‘. Fibrin levels rose progressively over the post parturient period to > 8.75 g l- ‘, zinc and inorganic phosphate fell (plasma Zn < 500 pg l- I and plasma P < 0.9 mM l- I ) There was a daily periodicity of change in inorganic phosphate of approximately 0.4 mM I- ’ in all four cows. Plasma glucose rose to a peak at parturition between 5.7 and 8.9 mM 1~ ’ and then fell rapidly to 3.4 to 3.8 mM 1-l (Fig. 5).
T. George et al. / Veterinan, Microbiology e
I
-
LymPhoc~tea
-
46 (1995) 131-142
135
NeutroPhll=
Fig. 2. Neutrophils, lymphocytes ( lo3 Ld6) and rectal temperature in cow 2 which developed milk fever following calving and was treated with calcium borogluconate. The first temperature peak coincided with a period of nonproductive labour.
Plasma iron levels showed a variable response in the three normal calving cows. In one animal iron levels fell in a fashion similar to that shown in Fig. 6, another showed little change, while in the remaining cow plasma iron levels rose. Neutrophils peaked at between 5700 and 11200 lm6 close to the peak of rectal temperature. lymphocyte numbers also rose above normal levels for each cow and peaked between 2300 and 11000 116. Eosinophils disappeared for approximately 1 day in the periparturient period.
3.2. Group 2: Cows expected to calve normally All cows in this group calved normally. A summary of the biochemistry relating to these cows in shown in Table 1. The haematology of cow 8 which calved for the first time differed from the multiparous cows in groups 1 and 2. The circulating neutrophils and lymphocyte counts fell in the periparturient period and the eosinophils did not disappear. 3.3. Group 3: Milk fever cases Blood samples were obtained before treatment by a veterinarian from seven cows in different herds with clinical signs of milk fever. The data is shown in Table 2. In five
136
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Calcium
Microbiology
46 (1995) 131-142
--t Magnesium
CALVING 0’
1’ ” -50 -40 -30 -20 -10
0
” 10
20
1’ 30 40
Hours Fig. 3. Plasma calcium and magnesium
levels in relation to calving of normal calving cow 3
instances follow up samples were available when the cows had recovered and were being milked normally. 3.4. Serology In cows in commercial dairy herds ( groups 2 and 3) the serological results excluded the possibility of ephemeral fever recurrence and consequent effect on the biochemistry. 3.5. Ephemeral Fever: Effects of treatment The treatment of experimental cattle given bovine ephemeral fever virus under close observation and treatment with phenylbutazone at intervals and a marked effect on clinical signs, fever and restoration of appetite. There was no difference in the pattern of response of plasma zinc and copper between the treated animals and untreated controls. Plasma zinc fell to I 100 Fg l- ’ by the end of the incubation period of the virus irrespective of treatment while concurrently plasma copper began to rise. By the end of the recovery period 4 to 6 days later, plasma copper levels were 2 1100 pg l- ’ in treated steers. This was similar to the mean level ( 10 10 pg 1~ ’ ) reached in untreated animals. 4. Discussion The study presented here has had a long evolution to bring into harmony two diseases, one with a clinical expression of temporary paralysis originally ascribed to the effects of a
T. George et al. /Veterinary
3-
*
Microbiology
Calcium *
46 (1995) 131-142
137
Magnesium
2.5 -
0
’ -50
I -40
CALVING I I
~ I
I
I
I
-30
-10
0
10
20
-20
~%IO”
Hours Fig. 4. Plasma calcium and magnesium levels in relation to calving of cow 2 which developed milk fever following calving and was treated with calcium borogluconate.
neurotrophic virus and the other of metabolic origin ascribed to hormonal dysfunction. In 1973, valuable cows and bulls which were fully or partially paralysed as the result of naturally acquired ephemeral fever were successfully treated with intravenous calcium borogluconate. The immediate and specific response to the treatment was apparently fully curative in some and temporary in others (St.George et al., 1984). An underlying inflammation was indicated by high plasma fibrin and neutrophilia, so treatment of ephemeral fever infected cattle with nonsteroidal anti-inflammatory drugs was carried out with apparent benefit (St.George et al., 1986). The association between hypocalcaemia and these inflammatory indicators, and aberrant behaviour of plasma microminerals was subsequently demonstrated in the sometimes fatal cutaneous myiasis (flystrike) of sheep (Murphy et al., 1987). The present range of tests developed through this research into ephemeral fever and cutaneous myiasis of sheep has been transferred successfully to applied study of dairy cattle in the periparturient period. The periparturient period is not well defined but for this study is taken to be 2 days before and after calving. The cows in group 1 under repeated observation have given a fuller picture of the train of biochemical events that occur in the periparturient period. The biochemistry of cow 2 with milk fever, was similar to the other three cows in group 1 but with greater departures from expected values. Rises occurred in plasma glucose, copper, fibrin and falls were recorded in calcium, zinc and inorganic phosphate. Iron was variable, with a major fall in cow 2.
138
T. Georp
et al. / Veterinan, Microbiology
+ Glucose -
-50
-40
-30
-20
46 (1995) 131-141
Phosphate
-10
0
10
20
Hours Fig. 5. Plasma glucose and inorganic phosphate and was treated with calcium borogluconate.
levels of cow 2 which developed
milk fever following
calving
The results of group 2 animals in a dairy herd (cows 5 to 8) show a similar pattern in the more limited biochemistry and less intense sampling carried out in the early phases of this study. The results from sampling of cows in group 3 (cows 9 to 15) in the acute stage of milk fever, can be judged only against the expected range of values (Table 2). They substantially conform to the cows calving normally, except that the calcium values in blood taken in the acute stages are lower. Some of the changes that we observed in blood parameters other than calcium occurring during the periparturient period have been previously reported from the viewpoint of secondary complications of hypocalcaemia in milk fever or calving, namely hyperglycaemia (Kronfeld, 1971) ; hypophosphataemia (Marr et al., 1955 ); lowered zinc in beef cattle at calving (Dufty et al., 1977) and leucocyte changes of dairy cattle in the periparturient period (Guidry et al., 1976). The l-l .2”C rise in rectal temperature in group 1 cows was in contrast to the 3°C which occurs in ephemeral fever but tends to justify the use of the old name of milk fever over the modem “parturient paresis”. The peak of temperature rise was at or near the peak of neutrophilia. Neutrophilia, lymphocytosis and eosinopaenia occurred in all multiparous cows in groups 1 and 2. This haematological pattern was ascribed to a preceding increase in corticosteroids associated with parturition by Guidry et al. ( 1976). The circulating lymphocyte response in the periparturient period in cows in group 1 was different in one respect to that found in natural and experimental cases of ephemeral fever.
T. George et al. /Veterinary 3000
Microbiology
Copper
+
-
139
46 (1995) 131-142
Zinc + Iron
1 2500.
-50
-40
-30
-PO
-10
0
10
20
Hours Fig. 6. Plasma zinc, copper and iron levels in relation to cow 2 which developed milk fever following calving and was treated with calcium borogluconate.
The circulating lymphocyte numbers fall during the clinical phase of ephemeral fever infection (St.George et al., 1984). In contrast, they rose in the preparturient period in the cows calving under observation. This suggests that an antigen stimulus has occurred some days earlier. The parallels between the haematology and biochemistry of multiparous cows, with or without milk fever, are almost complete with variation being in degree rather than kind. The haematological and biochemical changes that occur in bovine ephemeral fever and the periparturient period of multiparous cows (Fig. l-6) are both characteristic of those found Table 1 The maximum changes of blood constituents during the periparturient period, above ( + ) or below (-) the mean values more than 6 days prior to parturition, from four cows calving normally of different parities (group 2) in a dairy herd Cow Number
5 6 7 8
Age
8 6 5 2
Parity
5 4 3 1
FibgL-’
+5.1 +5.1 +2.1 + 10.0
n&l L-l
Neutrophilia
Wg L-l
Ca
Mg
Zn
Fe
cu
-0.85 -0.23 -0.08 -0.20
+0.43 +0.15 -0.02 -0.01
-450 -331 -305 -400
-1087 -725 -333 -1400
+232 +I00 +202 +400
with band forms
+ + + _
140 Table 2 Biochemistry
T. George et al. /Veterinary
Microbiology
J6 (1995) 131-112
of the blood of cows in dairy herds with clinical milk fever (Group 3).
Serial
Age
parity
9
8
6
10
6
5
11
6
5
12
10
9
13
8
5
14 15 Expected range
5 8
3 7
mM L-’
/I&L
g L ’ Fibrinogen
’
Ca
Mg
Zn
Fe
Cu
0.8 * 1.9** 0.4’ 2.2” 0.9 * 1.7** 1.6’ 1.3” 1.5’ 2.1 ** 3.1 * 1.8’ 2.1 to 2.8
0.4 0.6 1.1 0.7 0.7 1.2 0.9 0.9 1.4 1.0 1.0 1.1 0.6 to 1.2
490 520 380 840 520 500 610 700 470 760 500 400 800 to 1200
1420 610 740 970 I580 1330 600 1290 550 1450 1870 1000 1500 to 2000
650 760 690 1000 8X 780 650 680 640 980 810 720 500 to 1000
‘Blood sampIe taken when cow diagnosed * *Convalescent blood sample.
7.6 7.2 5.0 11.2 4.0 7.0 9.4
Rel Neutrophilia
+
12.6 6.5 12.2 6.8 8.2 to 6
with milk fever.
in the inflammatory response (Van Miert, 1985). It is not certain which changes are part of a general stress response to inflammation. The association occurs in cutaneous myiasis of sheep (Murphy et al., 1987; Depelchin et al., 1985) of hypocalcaemia with inflammatory indicators and in toxic shock syndrome of women (Wagner et al., 1981) as well as in ephemeral fever. The association of inflammation with hypocalcaemia in multiparous cows is plausible. Consequently, inflammation could pay a part in reducing plasma calcium levels in cows in the periparturient period, a time when other calcium demands are increasing, and thereby initiate the cascade of effects. The mechanism by which this occurs is unknown for ephemeral fever, cutaneous myiasis or milk fever. The single first calf cow in the series (number 8) may provide some explanation for the events, as milk fever rarely occurs with first calving. In contrast to all the multiparous cows, the neutrophils and lymphocytes in cow 8 showed a slight reduction, not a rise and eosinophils did not vanish for 24 h though plasma fibrin, macro and microminerals responded similarly to the multiparous cows. This may indicated that some inflammatory stimulus is acquired by repeated calvings and expresses itself when foetal antigens encounter the raw surfaces of the reproductive trace when placenta attachment breaks down before and during calving. The rumen of a cow is a fermentation vessel with a complex bacterial and protozoan population which converts vegetable matter to simpler substances for normal digestion. Rumen motility and regurgitation are essential to normal function. When plasma calcium levels fall, ruminal contractions slow and then cease. A major consequence is a reduced absorption of calcium from the rumen (Kronfeld, 1971). In bovine ephemeral fever the plasma calcium falls before ruminal contractions cease (St.George, 1992). In the cows
T. George et al. /Veterinary
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under repeated observation, this also occurred to a variable extent before calving. On the day of calving, appetite was sharply reduced, especially in cow 2. This effect has been reported by Moodie and Robertson ( 1961) as occurring in multiparous cows but not those having their first calf. Loss of appetite could be secondary to an inflammatory event reducing plasma calcium level and consequently rumen motility. There is a striking difference in mortality between the two diseases, if untreated. Most (97%) lactating cows with ephemeral fever recover, whereas almost all cows with severe milk fever die within l-2 days, if untreated. Lactating cows with ephemeral fever cease secreting milk (Davis et al., 1984). This may be due to a toxic effect of the high levels of circulating interferon which occur during the clinical phase of ephemeral fever (St.George et al., 1986). This effect equates with inflation of the udder with air, an older, slower but effective method of treating milk fever (Hibbs, 1950). Without a reduction in milk secretion, ephemeral fever could be as fatal as milk fever to lactating cows. This study does not negate the regimes that have been evolved to lower the prevalence of milk fever by manipulating diet in late pregnancy to improve calcium availability (Kronfeld, 1971). However, the standard treatment with calcium borogluconate (Blood and Henderson, 1974) with or without other macroelectrolytes, does not effect the other biochemical abnormalities except indirectly by restoring rumen function. Even if anti-inflammatory drugs were found to have clinical value in milk fever as in ephemeral fever, this limitation applies. The indication from treatment of ephemeral fever with anti-inflammatory drugs is that there is striking clinical improvement in the cow with reduction in temperature, neutrophilia and fibrin responses (St.George et al., 1986) but that the underlying micromineral zinc and copper dyscrasias we measured were not affected. This indicates that the drug is having an effect somewhere in the cascade of biochemical events rather than at the root cause. Time and rest are still required for the affected cow to re-establish homeostasis to all disturbed functions in bovine ephemeral fever and milk fever after any symptomatic treatment. The results from thus study imply a general linkage mechanism in the expression of ephemeral fever, milk fever and other inflammatory diseases. Future studies which determine the aetiology of the acute calcium dysfunction during ephemeral fever will shed important light on the mechanism(s) underlying milk fever or other conditions which are characterized by an innate inflammatory process.
Acknowledgements We acknowledge the assistance of Dr B.F. Sheahan and Dr C.J. McCaughan for providing blood samples and T. and W. Hunt for providing cows for serial sampling. S.S. Davis, R.G. Collins, T. Richter, J.A. Connell and D. Ouwerkerk for technical assistance and E.J. Harris for illustrations.
References Basson, P.A.. Pienaar. J.G. and Van Der Westhuizen, B., 1970. The pathology experimental disease in cattle. J. Sth. Afr. Vet. Med. Ass., 40: 385-397.
of ephemeral fever: a study of the
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Blood, D.C. and Henderson, J.A., 1974. In “Veterinary Medicine” 4th Ed. Bailliere, Tindall, London: 683-702. Davis, S.S., Gibson, D.S. and Clark. R., 1984. The effect of bovine ephemeral fever on milk production. Aust. Vet. J., 64: 128-129. Depelchin. B.O.. Bloden. S., Hooremans. M.. Noirfalise, A. and Ansay, M.. 1985. Clinical and experimental modifications of plasma iron and zinc concentrations in cattle. Vet. Rec.. 116: 519-521. Dufty, J.H., Bingely, J.B. and Cove, L.Y.. 1977. The plasma zinc concentration of nonpregnant and parturient Hereford cattle. Aust. Vet. J.. 53: 519-522. Grieg, J.R., 1930. Calcium gluconate as a specific in milk fever. Vet. Rec., 10: 115-120. Guidry, A.J., Paape. M.J. and Pearson, R.E., 1976. Effects of parturition and lactation on blood and milk cell concentrations, cotticosteroids and neutrophile phagocytons in the cow. Am. J. Vet. Res. 37: 1195-l 200. Hibbs, J.W., 1950. Milk fever (parturient paresis) in dairy cows -a review. J. Dairy Sci., 33: 758-789. Kronfeld, D.S., 1971. Parturient hypocalcemia in dairy cows. Adv. in Vet. Sci.. 15: 133-158, Marr, A., Moodie, E.W. and Robertson. A., 1955. Some biochemical and clinical aspects of milk fever. J. Comp. Path., 65: 347-365. Moodie. E.W. and Robertson. A., 1961. Dietary Intake of the parturient cow. Res. Vet. Sci.. 2: 217-226. Murphy. GM., St.George, T.D., Uren, M.F. and Collins. R.G., 1986. The biochemistry of ephemeral fever in cattle. In T.D. St.George, B.H. Kay and J. Blok (ed.), Arbovirus Research in Australia. Proc 4th Sym., p. 307-313. Murphy, G.M.. St.George, T.D., Guerrini, V.H., Collins, R.G.. Broadmeadow, A.C.C., Uren M.F. and Doolan. D.L., 1987. Trace element and macroelectrolyte behaviour during inflammatory disease. Proc. VI Int. Symp. on Trace Element Metab. in Man and Animals (TEMA-6). Pacific Grove, California, USA, 403404. Murphy, G.M., St.George, T.D. and Uren, M.F.. 1989. Ephemeral fever -a biochemical model for inflammatory disease in cattle and sheep. M.F. Uren, J. Blok and L.H. Manderson ted.), Arbovirus Research in Australia. Proc. 5th Symp.. p. 268-274. Remberg, C.F., Johnson, E.K., Fargo, R.D. and Kronfeld, D.S.. 1984. Calcium homeostasis in cows. with special reference to parturient hypocalcaemia. Am. J. Physiol., 246: R699-704 St.George, T.D., 1992. The natural history of ephemeral fever of cattle. Bovine ephemeral fever and related rhabdoviruses. Proc. 1st Int. Sym. (No. 44) Aust. Centre Int. Ag. Res., Canberra, Australia. p. 13-19. St.George, T.D., Cybinski, D.H. Murphy, G.M. and Dimmock, C.K., 1984. Serological and biochemical factors in bovine ephemeral fever. Aust. J. Biol. Sci., 37: 341-349. St.George, T.D., Uren, M.F. and Zakrzewski. H.. 1986. The pathogenesis and treatment of bovine ephemeral fever. In T.D. St.George, B.H. Kay and J. Blok (ed.), Arbovirus Research in Australia, Proc. 4th Symp, p. 303-307. Takken, A., 1984. A long-term venous catheter for cattle. Proc. Aus. Sot. Anim. Prod., 15: 756759. Uren, M.F. and Murphy, G.M.. 1985. Studies on the pathogenesis of bovine ephemeral fever in sentinel cattle. II. Haematological and biochemical data. Vet. Microbial., 10: 505-5 15. Uren, M.F., St.George, T.D. and Zakrzewski, H.. 1989. The effect of anti-inflammatory agents on the clinical expression of bovine ephemeral fever. Vet. Microbial., 19: 99-111. Uren, M.F., St. George, T.D. and Murphy, G.M., 1992. Studies on the pathogenesis of bovine ephemeral fever in experimental cattle. Virological and biochemical data. Vet. Microbial., 30: 297-307. Van Miert, A.S.J.P.A.M., 1985. Fever and associated haematologic and blood biochemical changes in the goat and other animal species. Vet. Q,, 7: 2@216. Wagner, M.A., Batts, D.H. Colville, J.M. and Lauter, C.B.. 1981. Hypocalcaemia and toxic shock syndrome, Lancet 1: 1208.
Veterinary
Microbiology
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( 1995) 131-142
Studies on the pathogenesis of bovine ephemeral fever IV: A comparison with the inflammatory events in milk fever of cattle T.D. St. George a,*, G.M. Murphy b, B. Burren b, M.F. Uren ’ a15 Tamarix Street, Chapel Hill, Qld, 4069, Australia b Queensland Department of Primary Industries, Animal Research Institute, Yeerongpilly, Qld, 4105, Australia ’ CSIRO, Division of Tropical Animal Production, Private Bag No. 3, PO, Indooroopilly, Qld, 4068, Australia Accepted 8 March 1995
Abstract The study of ephemeral fever in cattle has defined a range of haematological and biochemical changes in blood which are characteristic of an inflammatory response. One of the clinical signs of ephemeral fever, a temporary paralysis reversible by treatment with calcium borogluconate, is similar to that in milk fever (parturient paresis), a disease of multiparous dairy cows. Three separate groups of cows were studied. Four multiparous cows were observed and sampled repeatedly during calving, three similar cows and one cow calving for the first time in a dairy herd were sampled daily before and after calving; and, in other dairy herds, seven cows with milk fever were sampled during illness. One of the cows under repeated observation during calving developed milk fever. The results showed that all the inflammatory indicators in blood were present in the multiparous cows at calving and that these were essentially similar to those established in ephemeral fever. The similarities in the four cows sampled repeatedly during the periparturient period were: a rectal temperature rise of 1 to 1.2”C; rise in circulating neutrophils to peaks between 5700 and 11200 1K6; disappearance of eosinophils for 1 day; hypocalcaemia (plasma Ca < 2.0 mM 1~ ‘) ; fall of plasma zinc to low levels immediately after calving (plasma Zn < 500 pg l- ’ ) ; fall of inorganic phosphate (plasma P < 0.9 mM 1~ ’ ) ; rises in copper (plasma Cu > 1000 pg I- ‘) and plasma fibrin to > 8.75 g 1-l. Plasma glucose peaked at calving between 5.7 and 8.9 mM l- ’ then fell to levels ranging between 3.4 and 3.8 mM 1~ I. Plasma iron rose in one cow to 1220 /Lg l- ‘, was unchanged in one cow and fell in the other two to 440 and 860 pg l_ 1respectively. The three multiparous cows which were sampled daily and calved normally showed similar haematological, macro and micromineral changes and fibrin response as did the seven milk fever cases. In the periparturient period, milk fever cows differed from multiparous cows calving normally, in degree but not in kind, of inflammatory response. It is postulated that an inflammatory event occurs in the periparturient period of multiparous cows which partially accounts for the falls in plasma calcium. This can precipitate a paralysis and other hypocalcaemic signs similar to that seen in acute ephemeral fever. * Corresponding
author: Tel, (61 7) 3378 5152 Fax: (61 7) 3378 7762
0378-l 135/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIO378-1135(95)00078-X
132
Keywords: paresis
T. George et al. / Veterina~
Bovine
ephemeral
fever; Hypocalcaemia:
Microbiology
46 (1995) 131-142
Inflammation;
Microminerals;
Fibrinogen
; Parturient
1. Introduction Bovine ephemeral fever is caused by infection with a rhabdovirus of the same name. Its clinical expression varies widely in severity. It has two major components. The first of which is expressed as an inflammatory polyserositis plus oedema and the second as a loss of control of smooth and striated muscle associated with hypocalcaemia. As the virus name suggests, clinical expression is ephemeral, usually less than 2-3 days. In more severe cases this paralysis causes deaths in cows, steers and bulls. The inflammatory aspect of the disease was well established by histopathology (Basson et al., 1970) and has since been progressively defined in biochemical terms by St.George et al. (1984). Uren and Murphy (1985), (Murphy et al., 1986, 1989) and Uren et al. ( 1992). the full spectrum of clinical signs can be prevented by treatment with anti-inflammatory drugs (St.George et al., 1986; Uren et al., 1989). However, the treatment of the hypocalcaemia with parenteral calcium borogluconate relieves the paralysis only temporarily unless the anti-inflammatory drugs are also given (St.George et al., 1984). From the biochemical profile and response to treatment, there is no doubt that most of the clinical signs of bovine ephemeral fever are caused by a generalized inflammation with a secondary hypocalcaemia. Milk fever, also known as parturient paresis, most usually develops within the first 2 days after parturition in cows which have had two or more calves. It has rarely been recorded in cows having their first calf. Its prevalence increases with the number of calvings and often recurs in the same animal in successive calvings (Blood and Henderson, 1974). The disease has been known for 250 years but has become more of a problem with the development of the modern high milk yield dairy cow (reviewed Hibbs. 1950). The direct cause of the paresis, which is its major presenting sign, is hypocalcaemia. This was first specifically corrected by Grieg ( 1930) using parenteral treatment with calcium gluconate. This treatment supplanted udder inflation with air or fluids which produced recovery after several hours, by creating back pressure to inhibit milk secretion and consequent drain on calcium. Since that time, research has been focused on the kinetics of calcium transfer within the body of the dairy cow and its hormonal control, as the primary initiators of milk fever in may excellent studies (reviewed Kronfeld. 1971; Ramberg et al., 1984). The current theory is that milk fever is a defect of calcium homeostasis at or near parturition. From personal experience, the nature and response to calcium treatment of clinical signs caused by hypocalcaemia in bovine ephemeral fever and milk fever are similar. However, the diseases have important differences for the clinician. There is a high fever in bovine ephemeral fever and both sexes are affected while in a milk fever cow the rectal temperature is normal, or subnormal in the most acute stage of the disease when the animal is seen by a veterinarian. The present study was undertaken to address these clinical anomalies and to determine whether the range of biochemical dyscrasias that occur in bovine ephemeral fever could
T. George et al. /Veterinary
Microbiology
46 (1995) 131-142
133
also be found in multiparous cows at parturition and cow with milk fever. The full range could not be found from a search of the literature.
2. Material and methods Blood samples were obtained from three groups of cattle. The serology, cation analyses (Ca, Mg, Cu, Zn and Fe), blood gas analyses, plasma glucose, inorganic phosphate estimations and haematology, were as described by Uren et al., 1992. 2.1. Group 1: Cows expected to calve norma&
-repeated
observation
Four cows (Nos 14) expected to calve normally with their 3rd, 8th, 10th and 3rd calves respectively were fitted with jugular catheters with a subdermal extension emerging at the withers. This allowed repeated blood sampling without major disturbance (Takken, 1984). Their daily food intake was measured. 2.2. Group 2: Cows expected to calve normally -daily sampling Animals sampled were in a herd of Freisian cattle located 100 km north of Brisbane, Australia. This had been a sentinel herd for ephemeral fever for many years (St.George et al., 1984). The cows grazed on grass pasture with some concentrate supplementation. All cows had experienced ephemeral fever and were immune to the disease. Only four cows were serially sampled because of the dispersal of the herd. Jugular blood samples were collected five to nine times in the 3 weeks prior to calving and daily for 5 to 9 days after calving. 2.3. Group 3: Milk fever cows A jugular blood sample was obtained at the time when clinical milk fever was diagnosed in dairy cows, in commercial herds, before treatment and, when possible, a second blood sample 3 to 5 days later. 2.4. Ephemeral fever: Effects of treatments on plasma microminerals Plasma samples taken during the course of previous experimentation involving treatment of ephemeral fever infected steers with anti-inflammatory drugs (Uren et al., 1989) were analysed for copper and zinc as described by Murphy et al., ( 1986).
3. Results 3.1. Group 1 Cows 1, 3 and 4 calved normally on or near the expected date. The placenta was expelled within 3 h. Cow 2 had a false labour 30-35 h before calving. Contractions ceased until 9 h
134
T. George et al. / Veterinary Microbiology
38
-50
-4c.-diL&YYo
46 (1995) 131-142
hFr-_:.
0
10
20
30
-*
HOUR-S Fig. 1. Neutrophils, 3.
lymphocytes
( 10” Lm6) and rectal temperature in relation to calving of normal calving cow
before a still-born calf was delivered with difficulty, at which time the cow briefly showed some signs of milk fever. The signs intensified 9 h later. Treatment with intravenous calcium borogluconate restored full mobility. The haematology and rectal temperatures in the periparturient period for the normal calving of cow 3 and milk fever of cow 2 are shown in Fig. 1 and Fig. 2, and the magnesium and calcium responses in Fig. 3 and Fig. 4. The glucose and phosphate levels for cow 2 are shown in Fig. 5. The microminerals for cow 2 are shown in Fig. 6. The general observations can be summarized: Rumination slowed and ceased for 2-5 h before calving. All cows lost appetite in the 24 h prior to calving but ate avidly within 24 h post calving; cow 2 did not eat until treated. Plasma calcium levels began to fall l-3 days before calving to reach plasma Ca I 2.0 mM-‘. Fibrin levels rose progressively over the post parturient period to > 8.75 g l- ‘, zinc and inorganic phosphate fell (plasma Zn < 500 pg l- I and plasma P < 0.9 mM l- I ) There was a daily periodicity of change in inorganic phosphate of approximately 0.4 mM I- ’ in all four cows. Plasma glucose rose to a peak at parturition between 5.7 and 8.9 mM 1~ ’ and then fell rapidly to 3.4 to 3.8 mM 1-l (Fig. 5).
T. George et al. / Veterinan, Microbiology e
I
-
LymPhoc~tea
-
46 (1995) 131-142
135
NeutroPhll=
Fig. 2. Neutrophils, lymphocytes ( lo3 Ld6) and rectal temperature in cow 2 which developed milk fever following calving and was treated with calcium borogluconate. The first temperature peak coincided with a period of nonproductive labour.
Plasma iron levels showed a variable response in the three normal calving cows. In one animal iron levels fell in a fashion similar to that shown in Fig. 6, another showed little change, while in the remaining cow plasma iron levels rose. Neutrophils peaked at between 5700 and 11200 lm6 close to the peak of rectal temperature. lymphocyte numbers also rose above normal levels for each cow and peaked between 2300 and 11000 116. Eosinophils disappeared for approximately 1 day in the periparturient period.
3.2. Group 2: Cows expected to calve normally All cows in this group calved normally. A summary of the biochemistry relating to these cows in shown in Table 1. The haematology of cow 8 which calved for the first time differed from the multiparous cows in groups 1 and 2. The circulating neutrophils and lymphocyte counts fell in the periparturient period and the eosinophils did not disappear. 3.3. Group 3: Milk fever cases Blood samples were obtained before treatment by a veterinarian from seven cows in different herds with clinical signs of milk fever. The data is shown in Table 2. In five
136
T. George et al. /Veterinary *
Calcium
Microbiology
46 (1995) 131-142
--t Magnesium
CALVING 0’
1’ ” -50 -40 -30 -20 -10
0
” 10
20
1’ 30 40
Hours Fig. 3. Plasma calcium and magnesium
levels in relation to calving of normal calving cow 3
instances follow up samples were available when the cows had recovered and were being milked normally. 3.4. Serology In cows in commercial dairy herds ( groups 2 and 3) the serological results excluded the possibility of ephemeral fever recurrence and consequent effect on the biochemistry. 3.5. Ephemeral Fever: Effects of treatment The treatment of experimental cattle given bovine ephemeral fever virus under close observation and treatment with phenylbutazone at intervals and a marked effect on clinical signs, fever and restoration of appetite. There was no difference in the pattern of response of plasma zinc and copper between the treated animals and untreated controls. Plasma zinc fell to I 100 Fg l- ’ by the end of the incubation period of the virus irrespective of treatment while concurrently plasma copper began to rise. By the end of the recovery period 4 to 6 days later, plasma copper levels were 2 1100 pg l- ’ in treated steers. This was similar to the mean level ( 10 10 pg 1~ ’ ) reached in untreated animals. 4. Discussion The study presented here has had a long evolution to bring into harmony two diseases, one with a clinical expression of temporary paralysis originally ascribed to the effects of a
T. George et al. /Veterinary
3-
*
Microbiology
Calcium *
46 (1995) 131-142
137
Magnesium
2.5 -
0
’ -50
I -40
CALVING I I
~ I
I
I
I
-30
-10
0
10
20
-20
~%IO”
Hours Fig. 4. Plasma calcium and magnesium levels in relation to calving of cow 2 which developed milk fever following calving and was treated with calcium borogluconate.
neurotrophic virus and the other of metabolic origin ascribed to hormonal dysfunction. In 1973, valuable cows and bulls which were fully or partially paralysed as the result of naturally acquired ephemeral fever were successfully treated with intravenous calcium borogluconate. The immediate and specific response to the treatment was apparently fully curative in some and temporary in others (St.George et al., 1984). An underlying inflammation was indicated by high plasma fibrin and neutrophilia, so treatment of ephemeral fever infected cattle with nonsteroidal anti-inflammatory drugs was carried out with apparent benefit (St.George et al., 1986). The association between hypocalcaemia and these inflammatory indicators, and aberrant behaviour of plasma microminerals was subsequently demonstrated in the sometimes fatal cutaneous myiasis (flystrike) of sheep (Murphy et al., 1987). The present range of tests developed through this research into ephemeral fever and cutaneous myiasis of sheep has been transferred successfully to applied study of dairy cattle in the periparturient period. The periparturient period is not well defined but for this study is taken to be 2 days before and after calving. The cows in group 1 under repeated observation have given a fuller picture of the train of biochemical events that occur in the periparturient period. The biochemistry of cow 2 with milk fever, was similar to the other three cows in group 1 but with greater departures from expected values. Rises occurred in plasma glucose, copper, fibrin and falls were recorded in calcium, zinc and inorganic phosphate. Iron was variable, with a major fall in cow 2.
138
T. Georp
et al. / Veterinan, Microbiology
+ Glucose -
-50
-40
-30
-20
46 (1995) 131-141
Phosphate
-10
0
10
20
Hours Fig. 5. Plasma glucose and inorganic phosphate and was treated with calcium borogluconate.
levels of cow 2 which developed
milk fever following
calving
The results of group 2 animals in a dairy herd (cows 5 to 8) show a similar pattern in the more limited biochemistry and less intense sampling carried out in the early phases of this study. The results from sampling of cows in group 3 (cows 9 to 15) in the acute stage of milk fever, can be judged only against the expected range of values (Table 2). They substantially conform to the cows calving normally, except that the calcium values in blood taken in the acute stages are lower. Some of the changes that we observed in blood parameters other than calcium occurring during the periparturient period have been previously reported from the viewpoint of secondary complications of hypocalcaemia in milk fever or calving, namely hyperglycaemia (Kronfeld, 1971) ; hypophosphataemia (Marr et al., 1955 ); lowered zinc in beef cattle at calving (Dufty et al., 1977) and leucocyte changes of dairy cattle in the periparturient period (Guidry et al., 1976). The l-l .2”C rise in rectal temperature in group 1 cows was in contrast to the 3°C which occurs in ephemeral fever but tends to justify the use of the old name of milk fever over the modem “parturient paresis”. The peak of temperature rise was at or near the peak of neutrophilia. Neutrophilia, lymphocytosis and eosinopaenia occurred in all multiparous cows in groups 1 and 2. This haematological pattern was ascribed to a preceding increase in corticosteroids associated with parturition by Guidry et al. ( 1976). The circulating lymphocyte response in the periparturient period in cows in group 1 was different in one respect to that found in natural and experimental cases of ephemeral fever.
T. George et al. /Veterinary 3000
Microbiology
Copper
+
-
139
46 (1995) 131-142
Zinc + Iron
1 2500.
-50
-40
-30
-PO
-10
0
10
20
Hours Fig. 6. Plasma zinc, copper and iron levels in relation to cow 2 which developed milk fever following calving and was treated with calcium borogluconate.
The circulating lymphocyte numbers fall during the clinical phase of ephemeral fever infection (St.George et al., 1984). In contrast, they rose in the preparturient period in the cows calving under observation. This suggests that an antigen stimulus has occurred some days earlier. The parallels between the haematology and biochemistry of multiparous cows, with or without milk fever, are almost complete with variation being in degree rather than kind. The haematological and biochemical changes that occur in bovine ephemeral fever and the periparturient period of multiparous cows (Fig. l-6) are both characteristic of those found Table 1 The maximum changes of blood constituents during the periparturient period, above ( + ) or below (-) the mean values more than 6 days prior to parturition, from four cows calving normally of different parities (group 2) in a dairy herd Cow Number
5 6 7 8
Age
8 6 5 2
Parity
5 4 3 1
FibgL-’
+5.1 +5.1 +2.1 + 10.0
n&l L-l
Neutrophilia
Wg L-l
Ca
Mg
Zn
Fe
cu
-0.85 -0.23 -0.08 -0.20
+0.43 +0.15 -0.02 -0.01
-450 -331 -305 -400
-1087 -725 -333 -1400
+232 +I00 +202 +400
with band forms
+ + + _
140 Table 2 Biochemistry
T. George et al. /Veterinary
Microbiology
J6 (1995) 131-112
of the blood of cows in dairy herds with clinical milk fever (Group 3).
Serial
Age
parity
9
8
6
10
6
5
11
6
5
12
10
9
13
8
5
14 15 Expected range
5 8
3 7
mM L-’
/I&L
g L ’ Fibrinogen
’
Ca
Mg
Zn
Fe
Cu
0.8 * 1.9** 0.4’ 2.2” 0.9 * 1.7** 1.6’ 1.3” 1.5’ 2.1 ** 3.1 * 1.8’ 2.1 to 2.8
0.4 0.6 1.1 0.7 0.7 1.2 0.9 0.9 1.4 1.0 1.0 1.1 0.6 to 1.2
490 520 380 840 520 500 610 700 470 760 500 400 800 to 1200
1420 610 740 970 I580 1330 600 1290 550 1450 1870 1000 1500 to 2000
650 760 690 1000 8X 780 650 680 640 980 810 720 500 to 1000
‘Blood sampIe taken when cow diagnosed * *Convalescent blood sample.
7.6 7.2 5.0 11.2 4.0 7.0 9.4
Rel Neutrophilia
+
12.6 6.5 12.2 6.8 8.2 to 6
with milk fever.
in the inflammatory response (Van Miert, 1985). It is not certain which changes are part of a general stress response to inflammation. The association occurs in cutaneous myiasis of sheep (Murphy et al., 1987; Depelchin et al., 1985) of hypocalcaemia with inflammatory indicators and in toxic shock syndrome of women (Wagner et al., 1981) as well as in ephemeral fever. The association of inflammation with hypocalcaemia in multiparous cows is plausible. Consequently, inflammation could pay a part in reducing plasma calcium levels in cows in the periparturient period, a time when other calcium demands are increasing, and thereby initiate the cascade of effects. The mechanism by which this occurs is unknown for ephemeral fever, cutaneous myiasis or milk fever. The single first calf cow in the series (number 8) may provide some explanation for the events, as milk fever rarely occurs with first calving. In contrast to all the multiparous cows, the neutrophils and lymphocytes in cow 8 showed a slight reduction, not a rise and eosinophils did not vanish for 24 h though plasma fibrin, macro and microminerals responded similarly to the multiparous cows. This may indicated that some inflammatory stimulus is acquired by repeated calvings and expresses itself when foetal antigens encounter the raw surfaces of the reproductive trace when placenta attachment breaks down before and during calving. The rumen of a cow is a fermentation vessel with a complex bacterial and protozoan population which converts vegetable matter to simpler substances for normal digestion. Rumen motility and regurgitation are essential to normal function. When plasma calcium levels fall, ruminal contractions slow and then cease. A major consequence is a reduced absorption of calcium from the rumen (Kronfeld, 1971). In bovine ephemeral fever the plasma calcium falls before ruminal contractions cease (St.George, 1992). In the cows
T. George et al. /Veterinary
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under repeated observation, this also occurred to a variable extent before calving. On the day of calving, appetite was sharply reduced, especially in cow 2. This effect has been reported by Moodie and Robertson ( 1961) as occurring in multiparous cows but not those having their first calf. Loss of appetite could be secondary to an inflammatory event reducing plasma calcium level and consequently rumen motility. There is a striking difference in mortality between the two diseases, if untreated. Most (97%) lactating cows with ephemeral fever recover, whereas almost all cows with severe milk fever die within l-2 days, if untreated. Lactating cows with ephemeral fever cease secreting milk (Davis et al., 1984). This may be due to a toxic effect of the high levels of circulating interferon which occur during the clinical phase of ephemeral fever (St.George et al., 1986). This effect equates with inflation of the udder with air, an older, slower but effective method of treating milk fever (Hibbs, 1950). Without a reduction in milk secretion, ephemeral fever could be as fatal as milk fever to lactating cows. This study does not negate the regimes that have been evolved to lower the prevalence of milk fever by manipulating diet in late pregnancy to improve calcium availability (Kronfeld, 1971). However, the standard treatment with calcium borogluconate (Blood and Henderson, 1974) with or without other macroelectrolytes, does not effect the other biochemical abnormalities except indirectly by restoring rumen function. Even if anti-inflammatory drugs were found to have clinical value in milk fever as in ephemeral fever, this limitation applies. The indication from treatment of ephemeral fever with anti-inflammatory drugs is that there is striking clinical improvement in the cow with reduction in temperature, neutrophilia and fibrin responses (St.George et al., 1986) but that the underlying micromineral zinc and copper dyscrasias we measured were not affected. This indicates that the drug is having an effect somewhere in the cascade of biochemical events rather than at the root cause. Time and rest are still required for the affected cow to re-establish homeostasis to all disturbed functions in bovine ephemeral fever and milk fever after any symptomatic treatment. The results from thus study imply a general linkage mechanism in the expression of ephemeral fever, milk fever and other inflammatory diseases. Future studies which determine the aetiology of the acute calcium dysfunction during ephemeral fever will shed important light on the mechanism(s) underlying milk fever or other conditions which are characterized by an innate inflammatory process.
Acknowledgements We acknowledge the assistance of Dr B.F. Sheahan and Dr C.J. McCaughan for providing blood samples and T. and W. Hunt for providing cows for serial sampling. S.S. Davis, R.G. Collins, T. Richter, J.A. Connell and D. Ouwerkerk for technical assistance and E.J. Harris for illustrations.
References Basson, P.A.. Pienaar. J.G. and Van Der Westhuizen, B., 1970. The pathology experimental disease in cattle. J. Sth. Afr. Vet. Med. Ass., 40: 385-397.
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Blood, D.C. and Henderson, J.A., 1974. In “Veterinary Medicine” 4th Ed. Bailliere, Tindall, London: 683-702. Davis, S.S., Gibson, D.S. and Clark. R., 1984. The effect of bovine ephemeral fever on milk production. Aust. Vet. J., 64: 128-129. Depelchin. B.O.. Bloden. S., Hooremans. M.. Noirfalise, A. and Ansay, M.. 1985. Clinical and experimental modifications of plasma iron and zinc concentrations in cattle. Vet. Rec.. 116: 519-521. Dufty, J.H., Bingely, J.B. and Cove, L.Y.. 1977. The plasma zinc concentration of nonpregnant and parturient Hereford cattle. Aust. Vet. J.. 53: 519-522. Grieg, J.R., 1930. Calcium gluconate as a specific in milk fever. Vet. Rec., 10: 115-120. Guidry, A.J., Paape. M.J. and Pearson, R.E., 1976. Effects of parturition and lactation on blood and milk cell concentrations, cotticosteroids and neutrophile phagocytons in the cow. Am. J. Vet. Res. 37: 1195-l 200. Hibbs, J.W., 1950. Milk fever (parturient paresis) in dairy cows -a review. J. Dairy Sci., 33: 758-789. Kronfeld, D.S., 1971. Parturient hypocalcemia in dairy cows. Adv. in Vet. Sci.. 15: 133-158, Marr, A., Moodie, E.W. and Robertson. A., 1955. Some biochemical and clinical aspects of milk fever. J. Comp. Path., 65: 347-365. Moodie. E.W. and Robertson. A., 1961. Dietary Intake of the parturient cow. Res. Vet. Sci.. 2: 217-226. Murphy. GM., St.George, T.D., Uren, M.F. and Collins. R.G., 1986. The biochemistry of ephemeral fever in cattle. In T.D. St.George, B.H. Kay and J. Blok (ed.), Arbovirus Research in Australia. Proc 4th Sym., p. 307-313. Murphy, G.M.. St.George, T.D., Guerrini, V.H., Collins, R.G.. Broadmeadow, A.C.C., Uren M.F. and Doolan. D.L., 1987. Trace element and macroelectrolyte behaviour during inflammatory disease. Proc. VI Int. Symp. on Trace Element Metab. in Man and Animals (TEMA-6). Pacific Grove, California, USA, 403404. Murphy, G.M., St.George, T.D. and Uren, M.F.. 1989. Ephemeral fever -a biochemical model for inflammatory disease in cattle and sheep. M.F. Uren, J. Blok and L.H. Manderson ted.), Arbovirus Research in Australia. Proc. 5th Symp.. p. 268-274. Remberg, C.F., Johnson, E.K., Fargo, R.D. and Kronfeld, D.S.. 1984. Calcium homeostasis in cows. with special reference to parturient hypocalcaemia. Am. J. Physiol., 246: R699-704 St.George, T.D., 1992. The natural history of ephemeral fever of cattle. Bovine ephemeral fever and related rhabdoviruses. Proc. 1st Int. Sym. (No. 44) Aust. Centre Int. Ag. Res., Canberra, Australia. p. 13-19. St.George, T.D., Cybinski, D.H. Murphy, G.M. and Dimmock, C.K., 1984. Serological and biochemical factors in bovine ephemeral fever. Aust. J. Biol. Sci., 37: 341-349. St.George, T.D., Uren, M.F. and Zakrzewski. H.. 1986. The pathogenesis and treatment of bovine ephemeral fever. In T.D. St.George, B.H. Kay and J. Blok (ed.), Arbovirus Research in Australia, Proc. 4th Symp, p. 303-307. Takken, A., 1984. A long-term venous catheter for cattle. Proc. Aus. Sot. Anim. Prod., 15: 756759. Uren, M.F. and Murphy, G.M.. 1985. Studies on the pathogenesis of bovine ephemeral fever in sentinel cattle. II. Haematological and biochemical data. Vet. Microbial., 10: 505-5 15. Uren, M.F., St.George, T.D. and Zakrzewski, H.. 1989. The effect of anti-inflammatory agents on the clinical expression of bovine ephemeral fever. Vet. Microbial., 19: 99-111. Uren, M.F., St. George, T.D. and Murphy, G.M., 1992. Studies on the pathogenesis of bovine ephemeral fever in experimental cattle. Virological and biochemical data. Vet. Microbial., 30: 297-307. Van Miert, A.S.J.P.A.M., 1985. Fever and associated haematologic and blood biochemical changes in the goat and other animal species. Vet. Q,, 7: 2@216. Wagner, M.A., Batts, D.H. Colville, J.M. and Lauter, C.B.. 1981. Hypocalcaemia and toxic shock syndrome, Lancet 1: 1208.