Paper 3.22

The biochemistry of ephemeral fever in cattle.

Proceedings Fourth Symposium Arbovirus Research in Australia, May 6-9, 1986,

CSIROIQIMR, Brisbane, Australia. (St. George, ToO., Kay B.H., and Blok, J. Eds)

ppo307-313 (1986).

by

Murphy, G.M., 5t.George, T.O., Uren, M.F. and Collins, R.G.

The study of the effects of infection with bovine ephemeral fever under field conditions

has severe limitations. Accordingly, I arranged for cattle to be held in an air-conditioned

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1 chemistry. Dr St.George and Dr Uren provided veterinary supervision on a round-the-

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Paper 3.22

THE BIOCHEMISTRY OF EPHEMERAL FEVER IN CA TfLE

G M Murphy*, T D St.Georget, M F Urent and R G Collins*

*Queensland Department of Primary Industries, Animal Research Institute,Yeerongpill}J, QLD 4105, AUSTRALIA.

tCSIRO, Division of Tropical Animal Science, Long Pocket Laboratories, Private BagNo.3 PO, Indooroopilly, QLD 4068, AUSTRALIA.

Ephemeral fever is a disease of cattle causedby bovine ephemeral fever (BEF) virus and canbe reproduced experimentally. Previously, wehave made limited comment on the biochemicalchanges occurring during natural ephemeralfever infection (BEF) of cattle (S.George et at1984; Uren and Murphy 1985).

A consistent observation was the marked dropin total serum calcium (C~) levels on the daythat clinical signs were most pronounced. Falls ofmore than 0.2 mM L -I C~ were common andfrank hypocalcaemia (Ca<2.0 mM L -I) occurredin an unpredictable percentage of affectedanimals.

Paper 3.22

308 ARBOVIRUS RESEARCH IN AUSTRALIA-PROCEEDINGS 4TH SYMPOSIUM

.Moseley and Axfor~ (1.973) considered stress TABLE 1Induced hypocalcaemIa m sheep in terms of ...increased lipolytic activity of adipose tissue. Biochemical and haematologIc.a1 measurementsThey demonstrated a highly significant, nega- --performed at 2 hourly mtervals.tive correlation betweeI! plasma CaT and -~

non-esterified fatty acid (NEF A) levels. Prime,fat cattle are frequently noted to be worst affec-ted during natural outbreaks of BEF (St. Georgeet aI1986). In this paper we examine the role ofadipose tissue as a Ca 'sink' during experiment-al infection, as well as the behaviour ofultrafiltrable calcium (Cau) during the clinicalphase of ephemeral fever when viraemia is.con-sistently present (St.George et al 1986).

A wide range of inflammatory conditions areknown to evoke marked changes in circulatinglevels of zinc (Zn), copper (Cu) and iron (Fe) inmammals (Underwood 1977; Beisel 1977). Sincecattle affected with BEF frequently exhibitneutrophilia and elevated fibrinogen levels, bothconsistent with an inflammatory event, we madedetailed observations on the changes occurringin circulating Zn, Cu and Fe. Comparable obser-vations were also made in animals treated with aspecific cyclo-oxygenase inhibitor (phenyl-butazone) (MF Uren, TD St. George and HZakrzewski unpub data).

Unit

mML-1

mML-1J.lgL-1J.lgL-1J.lgL-1

mML-1gL -I

J.lmL-1UL-IUL-I

x 109 L-I

BiochemicalCalcium -Total (CaT) and

Ultrafiltrable (Cau)*Magnesium -Total (M~) and

Ultrafiltrable (M~)*ZincCopperIron*Inorganic phosphateProtein -Total and AlbuminNEFA* (non-esterified fatty acid)Alkaline PhosphataseCreatine Kinase

HaematologicalWCC -Total and Differential

* = Measurements commenced within 2 h of thestart of fever, other measurements commenced36h after inoculation.

and frequency were adequate to maintain rectaltemperature below 39°C. Throughout the exper-imental period, these cattle remained loose in anisolation unit. Blood was obtained by venipunc-ture. The protocols for physical and physiologi-cal monitoring were similar to, but less intensivethan, those for group 1 animals. Labour con-straints prevented the following measurements:plasma Ca. and Mg. and serum NEFA.

EXPERIMENTAL METHODS

Measurements were made on 2 groups ofcattle: group 1 comprised 4 prime Hereford year-lings (mean live weight 275 kg). Cattle were fittedwith novel bilateral jugular catheters (Takken1984) several days prior to inoculation with BEFvirus. Throughout the experiment, cattle werehoused in a controlled temperature environmentin individual metabolism crate~. These were fit-ted with heavy rubber mats to prevent skeletaland muscle damage in recumbent animals.Intensive monitoring commenced 36 h after i. v.inoculation with known virulent BEF virus(strain M6). Vital signs (respiration, pulse,rumen function, reflexes) were manuallyrecorded each hour for the next 5 days. Appro-priate blood samples were taken at 2 hourlyintervals throughout the same period. The analy-ses performed are listed in Table I. Plasmaultrafiltrate preparations were commencedimmediately after sample collection. SerumNEFA analyses were performed within 4 h ofsample collection using a proprietary kit (W AKOChemical Co., Japan). All cation analyses (Ca,Mg, Cu, Zn and Fe) were carri~d out by atomicabsorption spectroscopy (AAS). Other metab-olite and enzyme assays were performed byappropriate autoanalyser techniques (GilfordIMP ACT 400). Haematological assays were car-ried out as described previously (Uren andMurphy 1985).

Group 2 consisted of 12 weaners assigned to 1of 3 treatment groups: untreated controls; earlytreated; and late treated. Treated groups receivedi.m. phenylbutazone therapy (PBZ) eitherimmediately after inoculation (early treated) or 6h after the temperature rise (late treated). Dosage

RESULTSThe individual reaction patterns for selected

parameters of all animals in group I are illus-trated in Figure 1. All measurements are ad-justed to the onset of fever (t = 0) as the incu-bation period varied from animal to animal. Theanimals are numbered 1 to 4 commencing withthe upper graph in each subset. Significant differ-ences are readily apparent. Animals 2 and 3 showa single fever reaction of less than 24 h duration(Fig. lc). Neither animal showed appreciableneutrophil response. Conversely animals I and 4show a triphasic fever reaction of up to 72 h dur-ation with marked bursts of neutrophilia.

Prolonged periods of hyperventilation (RR =40 min-l) were features of numbers I and 4;maximal rates were in excess of 100 min-l. Prob-ably the most visible difference between animalshowever, was that of sternal recumbency with aninability to rise. This was not seen in numbers 2and 3, whereas both 1 and 4 showed this syn-drome. The condition was apparent in both ani-mals within 12 h of the initial temperature rise.Animal I recovered some 40 h after the initialepisode; animal 4 failed to'recover, lapsed intofull lateral recumbency (44 h), and was eutha-nased 4 days later.

Histopathological examination of animal 1showed very severe axonal swelling andWallerian degeneration in the white matter ofthe anterior cervical cord. All funiculi were affec-

Paper 3.22

ARBOVIRUS RESEARCH IN AUSTRALIA-PROCEEDINGS 4TH SYMPOSIUM

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Fig/Ire 1. Cattle infected with ephemeral fever virus: changes after the onset of fever (t -0) in (a) plasmairon (Fe) and zinc (Zn); (b) serum albumin, and total serum and ultrafiltrable plasma calcium (CaT' Cau); (c)rectal temperature, circulating neutrophils and hyperventilation (respiration rate ~ 40 min-'); and (d) totalserum and ultrafiltrable plasma magnesium (MgT, Mg.), Animals are numbered I to 4 commencing with theupper graph in each subset.'\ \ \\ \ \ indicates rectal temperature =< 39.C;!!','.!/ indicates respiration rate =< 40 min-i.

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ted with the ventral being worst. Areas of haem-orrhage were present in the grey matter.

Clinical hypocalcaemia (Ca;- < 2.0 mM Lo')was not evident in any of the 4 animals. As indi-cated in Figure I b plasma Ca- fluctuated mark-edly (= 0.2 mM Lo') during 'the course of theviraemia: and fluctuations were most pro-nouncedin animals I and 4. By comparison with

c~, the changes in Ca are even more dramatic.In relative tenus. Cau (re,quently fell to less than35% of Ca,- (nonual range = 50%). Relevant datafor plasma Mg (MgT and Mg ) are shown in Fig-ure I d. There is little discernible evidence of Carelated Mg compensation.

Serum NEFA levels were significantly elev-ated in all animals throughout the viraemia.

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Paper 3.22

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310 ARBOVIRUS RESEAl

Levels in all animals rose from < 50 uM L-' (pre-experimental) to lie within a range of 400 to 600uM L-' during viraemia.

Serum albumin levels are depicted in FigureI b. Again there are obvious differences betweenanimals according to the severity of theviraemia. Whereas animals 2 and 3 (least affec-ted) show a frank, but short lived, hypoalbum-inaemia within 20 h of the onset of fever, num-bers I and 4 (severely affected) show little changein albumin levels until 40 to 44 h after the start offever.

Plasma Zn and Fe fell in all animals (Fig. la).The extent of the fall and its duration mimic theseverity of the clinical disease. Again animals Iand 4 show the greatest response. Plasma Culevels rose in all animals about 36 h after the startof the febrile response. Animals 2 and 3 showedlittle increase « 200 ~g L-') from the initial meanlevel of 980 :t 30 ~g L-I, whereas significantincreases (= 600 ~g L-I) were found in numbers Iand 4.

Plasma alkaline phosphatase (AP) and cre-atine kinase (CK) activities both reflect the sev-erity of clinical signs. AP showed little change inanimals 2 and 3 but fell to less than 30% of initialactivities (66 and 45 UL-' in numbers I and 4respectively. CK activity remained below 50UL-' in numbers 2 and 3 throughout the course ofviraemia but significant increases occurred inboth numbers 1 and 4 (Fig. 2). In animal numberI, CK began to rise 52 h after the onset of feverand 4 h prior to it regaining its feet. CK rose from32 UL-' to I 500 UL-' within 10 h before falling tonear normal (140 UL"), 86 h later. In animal 4(the animal that had to be euthanased), CK

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Selected results for a typical reactor from eachof the 3 treatments in group 2 (PBZ experiment)are portrayed in Figure 4. Fever was effectivelycontrolled by PBZ treatment but a neutrophilresponse to viral challenge remained.

Plasma C~ fluctuated markedly during the48 h following the onset of fever. irrespectiveof treatment (Fig. 4b). Minimum values of 2.06and 2.07 mM L -I are compared with pre-

experimental levels of 2.57 and 2.55 nM L-Ifor the early and late treated animals respect-ively. Frank, clinical hypocalcaemia (1.95 mML") was evident in the control animal at 48 hafter the start of fever. No compensatory changesoccurred in plasma M~.

Significant decreases occurred in plasma Znin all 3 animals (Fig. 4b). Maximum depressionappears coincident with peak neutrophilia.

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CH IN AUSTRALIA-PROCEEDINGS 4TH SYMPOSIUM

began to rise 40 h after the onset of fever and 4 hprior to its collapse into complete lateral recum-bency. Values rose to = I 500 UL-' within 24 h

but then commenced to fall until, at euthanasia(100 h after initial rise), CK activity was < 500UL-'.

Feed and water intake of marginal reactors(numbers 2 and 3) showed little if any deviationfrom pre-experimental patterns. In contrast, sev-ere reactors (numbers I and 4) stopped drinkingand eating at or about 30 h after the start offever-A typical reaction pattern is shown in Figure 3.Cessation of food and water intake appeared tocoincide with loss of the swallowing reflex. Thiswas accompanied by a variety of signs of varyingintensity and duration. These included: excess-ive lachrymation, nasal discharge, profusedrooling of saliva, bloat, ruminal stasis and

recumbency.

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Paper 3.22

ARBOVIRUS RESEARCH IN AUSTRALIA-PROCEEDINGS 4TH SYMPOSIUM 311

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DISCUSSION

This experimental evidence confirms ourearlier observations (St. George et a11984; Urenand Murphy 1985) that transient falls of morethan 0.25 mM L.I in serum C~ are a significantfeature ofBEF affected cattle. Although classicalhypocalcaemia (C~ < 2.0 mM L.I) was evidentin only 1 animal during the viraemia (group2:control), serum Ca levels fluctuated markedlyin all animals monito~edforthis indice (group 1).The duration, frequency and magnitude of thefalls in Ca.. paralleled the severity of the diseaseand were expressed clinically as tachycardia,muscle tremor, ruminal stasis, inability toswallow, and recumbency. This is consistentwith the response to calcium therapy reported bySt.George et al (1984): we are unaware of anysimilar studies demonstrating this facet of aviraemia.

The underlying mechanism(s) responsible forthis aberrant calcium metabolism is not clear.Starvation and/or impaired digesta flow per seare not considered to be primary factors. Markedfalls in plasma Ca.. and serum C~ occurredwithin 18 h of the onset of fever, whereas theseverely reacting animals (group 1 :numbers 1and 4) continued to eat and drink for a further 12h, at which time (30 h) ruminal stasis was firstobserved. Likewise, a hypoalbuminaemia re-lated hypocalcaemia does not provide a satis-factory rationale for the behaviour of serum C~.Transitory hypoalbuminaemia (albumin < 25 gL -I) did occur in mild reactors (group 1 :numbers2 and 3) at or about 24 h after the temperaturebegan to rise. However, it did not occur in severereactors (group I:numbers 1 and 4) for a further36 h. At this time, serum C~ had begun to rise

although Cau continued to fluctuate signifi-cantly, in both absolute and relative terms.

The experimental data from group 1 doessuggest 2 factors which could account for at leastpart of the behaviour shown by C~ and Ca dur-ing the viraemia. Firstly, prolonged hyperp"noeaincreases blood pH and promotes formationof the albumin/calcium complex at the expenseof ionizable calcium pool. Respiratory dis-tress did occur in all animals; and the degree andduration closely followed the severity of theclinical signs (Fig. 1 c). Thus fever relatedhyperventilation with attendant alkalemia couldaccount for the fluctuations in Ca .However, inthe absence of blood gas/pH meas"urements, thisproposal remains conjectural. Secondly, in-creased lipolytic activity of adipose tissue isaccompanied by a significant rise in plasma freefatty acids (Akgun and Rudman 1969). In sheepsubjected to a variety of stressful stimulii (minorsurgery, restrictive handling and adrenalininfusion), Moseley and Axford (1973) demon-strated a 3-fold increase in plasma NEF A levels(= 500 JlM L-l). This was accompanied by a 0.25mM L-t drop in serum Ca,.. In vitro studies withadipose tissue supported their hypothesis 'thatunder the effect of stressful conditions. calciumis transferred from plasma into adipose tissueand this transference of calcium may be pro-portional to the degree and duration of stress'.We found plasma NEF A concentrations in-creased in all animals during the viraemia. Thegreatest increases (= 550 IJ.M L") occurred in theworst affected.

Infection. bacterial products such as endo-toxins, and many inflammatory agents. in-crease plasma Cu (Underwood 1977) and de-crease plasma Zn and Fe levels (Beisel 1977).

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Paper 3.22

312 ARBOVIRUS RES EAR

These changes are considered b~neficialresponses~ since Fe availability determInes thegrowth rate of many bacteria (Bullen 1981), andZn is known to inhibit phagocytosis (Sugarman1983). The increase in Cu reflects de novo syn-thesis of ceruloplasmin. This is proposed to have

an essential role as an aqueous phase scavengerof free oxygen radicals produced by phagocyticleukocytes (Clark et al 1985). Current opinionidentifies these trace metal changes as part of areaction cascade mediated via rnterleukin-l(Dinarello 1984; Oppenheim et al 1986).Analagous behaviour of Zn, Fe and Cu show dur-ing BEF infection suggest that an Interleukin-1cascade might be involved in the physiologicalexpression of the disease.

Several aspects of the available experimentaldata support this possibility. The inflammatorynature of the disease is well documented(Mackerras et a11940; Basson et a11970; Youngand Spradbrow 1980; Young and Chung 1986;St. George et al 1986). More importantly, theneutrophilia and trace element behaviour inPBZ treated cattle (group 2) show that theseresponses are independent of fever per se. This isin complete agreement with the results ofSobrado et al (1983). They demonstrated thatsuppression of fever via a comparable specificcyclooxygenase inhibitor (ibuprofen) did notaffect whole body trace metal, haematological, orhepatic acute-phase-induced responses toleukocytic pyrogen or endotoxin. Likewise Scottet al (1983) found indomethacin preventedsymptoms but did not suppress the neutrophiliaand elevated corticosteroids or prevent the fall inplasma zinc seen in human volunteers "giventoxic levels of a2 interferon.

Clark et al (1985) have argued that tissue dam-age described as 'inflammation' should be con-sidered as a consequence of the release of freeradical species generated by invading phagocyticleukocytes. lnterleukin-l promotes extravascu-lar infiltration of polymorphonuclear leukocytes(Oppenheim et al 1986; Lewis and Granger1986) and this is a prominent feature in BEFaffected cattle (Young and Spradbrow 1980;Young and Chung 1986). The clinically signifi-cant, though relatively short lived elevations inCK levels in severe reactors (group 1 :numbers 1and 4) are consistent with such an occurrence.

Basson et al (1970) found discrete, localisedareas of muscle necrosis accompanied byneutrophil infiltration of the sarcoplasm in sev-ere BEF reactors, killed at a late stage of disease.Unfortunately they did not measure the CKlevels in these animals.

Other factors may have contributed to theelevated CK activities in this present study. Theextensive lesions in the anterior cervical cord ofthe euthanased animal (group l:number 4) aresimilar to those diagnosed as the cause of con-tinuing ataxia and/or paralysis in refractory fieldcases of BEF (Hill and Schultz 1977). Grosslyelevated CK activities (= 20 x 103 UL-I) areexpected in such "downer animals in whichextensive pressure induced muscle damage can

CH IN AUSTRALIA-PROCEEDINGS 4TH SYMPOSIUM

occur in as little as 6 h (Cox et a/ 1982). Howeve..even though animal number 4 was recumbent f~;4 days, CK activity did not exceed 1.6 x 103 UL-\and in fact had fallen to < 500 UL-' at eutha-nasia. Likewise the behaviour of CK activity inthe other severe reactor (group l:number I) isatypical of that normally seen in "downer" cattle(Cox et a/ 1982).

Overall, many of the clinical signs and bio-chemical changes occurring during BEF areanalagous to those seen in toxaemias with achemical or bacterial origin. We have confirmedthat, as is the case with staphlococcal toxic shocksyndrome in humans (Chesney et a/ 1983),uncompensated hypocalcaemia is intimatelyconnected with the clinical signs of the disease.The fact that the biochemical aberrations occur-ring during the viraemia are not dependent onfever has important implications for the shortterm management of infected cattle. Treatmentwith nonsteroidal anti-inflammatory drugs suchas phenylbutazone, indomethacin (Scott et a/1983) and ibuprofen (Sobrado et a/ 1983) willproduce afebrile, asymptomatic animals. Ourresults demonstrate quite clearly that despite"treatment", these animals remain biochemic-ally dysfunctional.

Further studies are needed to determine if thebiochemical sequelae of ephemeral feverinvolves an Interleukin-l mediated cascade. Dis-turbed calcium homeostasis should also be inves-tigated as an intrinsic component of the patho-genesis of other disease states characterised by aninflammatory response.

Acknowledgments

We wish to acknowledge the contribution ofDenise Doolan and John Steiner to the analysesand the technical assistance of T J Daniel, E JHarris, S S Davis, M S O'Bryan, P G Allingham.

REFERENCES

Akgun S and Rudman D (1969) Relationships betweenmobilisation of free fatty acids from andipose tissue. andthe concentrations of calcium in the extracellular fluidand in the tissue. Endocrinology 84: 926-930.

Basson P A. Pienaar JG and van der Westhuizen B ( 1970) Thepathology of ephemeral fever: a study of the experimentaldisease in cattle. J S Afr Vet Med Assn 40: 385-397.

Beisel WR (1977) Magnitude of host nutritional response toinfection. Am J Clin Nutr 30: 1236-1247.

Bullen 1J (1981) The significance of iron in infection. RevInfect Dis 3: 1127-1138.

Chesney RW, McCarron DM. Haddad JG. Hawker CD,DiBella FP, Chesney P J and Davis JP (1983) Pathogenicmechanisms of the hypocalcemia of the staphylococcaltoxic-shock syndrome. 1 Lab Clin Med 101: 576-585.

Clark IA. Cowden WB and Hunt NH (1985) Free radical.induced pathology. Med Res Rev 5: 297-332.

Cox VS, McGrath CJ and Jorgensen SE (1982) The role ofpressure damage in pathogenesis of the downer cow syn-drome. Am 1 Vet Res 43: 26-29.

Dinarello CA (1984) Interleukin-1 and the pathogenesis ofthe acute-phase response. N Engl 1 Med 311 :1413-1418.

Hill MWM and Schultz K (19i7) Ataxia and paralysis associ.ated with bovine ephemeral fever infection- Aust Vet 1.~ ' 1-" I,.>: -1-_- .

Paper 3.22

ARBOVIRUS RESEARCH IN AUSTRALIA-PROCEEDIJ

Lewis ~E. and Granger HJ (1986) Neutrohil-dependentmedIatIon of microvascular permeability. Fed Proc 45:109-113.

Mackerras 1M. Mackerras MJ and Burnett FM (1940)E,perimental studies of ephemeral fever in AustralianCattle. Aust Commonw Counc Sci Ind Res Bull No. 136.

Moseley G and Axford RFE (1973) The effect of stress onthe redistribution of calci um in sheep. J Agric Sci 81:

403-409.Oppenheim,JJ, Kovacs, EJ, Matsushima K and Ourum SK

(1986) There is more than one Interieukin-i. ImmunolToday 7: 45-56.

Scott GM, Ward RJ, Wright 01, Robinson JA, Onwubalili1K and Gauci CL (1983) Effects of cloned interferon al,innormal volunteers: febrile reactions and changes in circu-lating corticosteroids and trace metals. AntimicrobAgents Chemother 23: 589-592.

Sobrado 1, Moldawer LL, Bistrian BR, Oinarello CA andBlackburn GL (1983) Effect of ibuprofen on fever andmetabolic changes induced by continuous infusion ofleukocytic pyrogen (Interieukin-l) or endotoxin. InfectImmlln 42: 997-1005.

St.George TO, Cybinski OH, Murphy GM and Oimmock

DISCUSSION -SESSION 7BChairman: D Hoffmann

Ephemeral Fever Pathogenesis

Q Cox. How long after the onset of this injecteddose of BEF virus did you post mortem thatanimal? Q Young. The animal with im-munoperoxidase staining? Q Cox. No, an ani-mal with muscle degeneration where oedemahad spilled over from the muscle planes. AYoung. Because there is a variable incubationperiod, I standardised the second day of clinicaldisease to autopsy the animals. The first day ofclinical disease is one in which there is fever, theanimal is off colour and looks sick. The secondday is one in which lameness starts and muscleoedema appears.

Q Osburn. What do you consider as pathogno-monic lesions of ephemeral fever. Q Young. Youmean histological lesions? Osburn. There aremany things which can lead to similar types oflesions as you have discussed. A Young. There isnot a pathognomonic lesion of ephemeral fever.If an animal has fever, lameness and neutrophilsin the joint fluids, then this is a good indicationthat the lesion of the disease is significant.

Q P. Doherty. I suspect that my colleagues inANU would interpret this as a typical incident offree radical induced pathology. You would saythat because there are lots of neutrophils, thereare free oxygen radicals produced in the lesionsites which cause vascular permeability changes.You should look and find, for instance, productsof lipid peroxidation. Have you looked at thataspect at all? A Murphy. The proposition refers torecent work that Ian Clarke, ANU, Canberra hasdone. They have reviewed the concept of the sortof biochemical changes that we are seeing here,of the inflammation process in a wide varietv ofmodels. These can, in fact, be considered interms of increased peroxidation of free radicals(oxygen and hydroxyl radicals). These are known

to be detoxified to peroxide which is then brokendown by peroxidase. This could be happening inphagocytic cells and could eventuate in all sortsof things ego muscle damage. We have not lookedat that yet. I db plan to, because we are also usingthis as a mod~l to look at inflammation in otherdisease states.

Q P. Doherty. If I recall correctly, hydroxyureawas used as a free radical scavenger. A Murphy.Yes, well the interesting thing here is that it posesa possible explanation for the host response indropping iron levels as well, because I of thepositive factors of decreasing plasma iron duringthis acute reaction phase would be to slow downthe autocatalysis of those radicals, or generationof those radicals.

Q P.Doherty. Considering these vascular per-meability changes, did you look at CNS to seewhether there was leakage into it? A Young. No.There were no vascular permeability changes inCNS, in either the brain or the spinal cord.

Q Woo/cock. Bone marrow: are techniques sensi-tive enough, do you think, to detect low titrevirus in the tissues? A Young. The technique thatDr Young Chung has developed (immunoper-oxidase technique using monoclonals), certainlygave a much better result with the buffy coat pre-parations than we have ever seen before. We be-lieve the technique that is becoming availableis much more sensitive than previously, so wehope that we can detect small quantities of virus.Dr Young Chung also plans to do immunoelec-tron-microscopy of the samples he has collected,so that will give another guide as to where theviru~ replicating. Comment St. George. I take itfrom that you are wondering whether the virus ispersisting in bone marrow. Well even if it were,

IOS 4TH SYMPOSIUM 313

CK (1984) Serological and.bioc~emical factors in bovineephemeral fever. Aust J BIoi SCI 37: 341-349.

St. George TO, Uren MF and Zakrzewski H (1986) Patho~en-esis and treatment of bovine ephemeral fever. ArbovIrusResearch in Australia, Proc 4th Symp 303-307.

Sugarman B (1983) Zinc and infection. Rev. Infect. Ois. 5:137-148.

Takken A (1984)A long-term venous catheter for cattle. Proc.Aust Soc Anim Prod 15: 756.

Underwood EJ (1977) In: Trace Elements in Human andAnimal Nutrition. 4th Edition, Academic Press. NewYork, San Francisco and London.

Uren.MF and Murphy GM (1985) Studies on the pathogen-esIs of bovine ephemeral fever in sentinel cattle. II.Haematological and biochemical data. Vet Microbiol10: 505-515.

Young PL and Spradbrow PB (1980) The role ofneutrophilsin bovine ephemeral fever virus infection of cattle. JInfect Ois 142: 50-55.

Young PL and Chung Y (1986) The pathology of ex peri men-tal bovine ephemeral fever. Arbovirus Research in Aus-tralia, Proc 4th Symp 301-302.

Paper 3.22

ARBOVIRUS RESEARCH IN AUSTRALIA-PROCEEDI~

Q Francis. Would some of them, especially thosethat were thrashing around, be more stressed? Ai'.1urphy. Yes.

St. George. We do not have any controls. I do notthink C 1 is looked at in downer milk fever cases?A Hill. Only in extreme circumstances. I thinkthe point that ought to be made here is that a cer-vicallesion is a very unusual sequela to ephem-eral fever. Weare not proposing that thisWallerian degeneration of the spinal cord andlesions are usually responsible for animals goingdown with ephemeral fever.

Q Spradbrow. The component of this interactionwhich we know so little about is the virus. Wehave a parasite that is not obviously killing cellsin the cattle host. It certainly is not killing any ofthe cultured bovine cells where we have beenlooking for it. Do we know where it is going afterthe infection? What organs is it in? What is itdoing there? A Young. No. I do not think weknow much more now than we did 5 years ago.Dr Young Chung has in progress some exper-im~"t-" which mav 2ive the answer. The suot that

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we are thinking about more and more now isbone marrow. We have seen the virus inneutrophils, both in fluids, in body cavities andthe blood. We do not propose that the virus isactually replicating in the neutrophils, because Ido not think that neutrophils have the where-withal to support viral replication. It is possiblethat replication does occur in some stem cell andthe virus ends up being carried around the bodyin neutrophils. Certainly this is I of the greatunknowns of BEF. Where does the virusreplicate?Q O.\"burn. What types of interferon were asayedfor: was it total interferon? A Sl.George. Totalinterferon, and I must stress that I could not con-trol the tests with synthetic interferon becausethere was none available in Australia at the time.Up to the present I am unable to get hold ofbov-ine interferon, although Genetech has offeredsome. We did characterise the interferon and itappears to be partly heat labile and partly pHlabile. These criteria of classifying interferonsseem to be going by the board. We appear to havea mixed bag of interferons.