The Influence of Environment Upon Serum Immunoglobulin Levels in Cattle

Br. vet. ]. ( 1971 ), 127, 442

THE INFLUENCE OF ENVIRONMENT UPON SERUM IMMUNOGLOBULIN LEVELS IN CATTLE BY C. C. CuRTAIN

CSIRO Division of Animal Health, Animal Health Research Laboratory, Melbourne, Victoria 3052, Australia

SUMMARY

The sera from 44 Shorthorn-Hereford and 53 Brahman crossbred calves, reared under sub-tropical conditions (Rockhampton), examined by agar-gel electrophoresis to determine the concentrations of the major serum protein groups, and by radioimmunoassay to quantitate the immunoglobulins, IgGr and IgG2, showed significantly lower serum albumin and significantly higher IgGr levels than those found in sera from 58 Herefords reared under cool temperate conditions (Tooradin). The Brahman-crosses had significantly lower IgGr levels and there was a significant negative correlation between bodyweight and IgGr level in both the Brahman crossbred and Shorthorn-Hereford groups. It was suggested that the higher IgGr levels in the Rockhampton animals, particularly those of lower weight, might result from a relatively poorer transfer of maternally-derived colostral antibody shortly after birth, with a consequent early establishment of excessive intestinal parasitism and an excessive or unbalanced growth of the normal saphrophytic intestinal bacteria. INTRODUCTION

During their seven millennia of domestication, cattle have been husbanded in many widely-differing environments, ranging from the cool temperate to the tropics. Like men, cattle have adapted in varying ways to heat and cold stress and to the many diseases characteristic of the different regions of the earth. Most of the studies on bovine adaptability have been on the effects of heat and cold, and extensive work has been done on the relationship of body temperature to sweating, coat type and breed (Findlay & Beackley, I954)· Heat, however, is not the only cause of stress in the tropical environment. As a rule, tropical conditions favour the spread of many infectious diseases which are absent, or minimal, in temperate zones. It is reasonable to expect that this higher intensity of infection will be reflected in both quantitative and qualitative differences in the immune response in individuals of the same species living in and out of the tropics. In man, these differences are well documented. It is known that populations in the tropics have strikingly high serum immunoglobulin levels compared with those in temperate zones, and even in closely related ethnic groups a difference of 2000- 3000 metres in altitude close to the equator will cause an 8 I per cent difference in immunoglobulin level (Curtain, Ig66). In cattle, adaptation of the immune system to tropical conditions has appar-

INFLUENCE OF ENVIRONMENT ON SERUM PROTEINS

443

ently not been investigated. As a location for such a study, Australia is unique in having the largest number of British breed cattle in the world husbanded under tropical conditions (Kelley, I94I) . In recent decades some attempts have been made to improve the adaptation of these cattle to tropical conditions by the introduction of Zebu genes (Kelley, I932, I943)· In this paper a study of the serum proteins, in particular the immunoglobulin levels, of British breed and Brahman crossbred cattle living under tropical conditions, compared with those of Hereford cattle kept under mild temperature conditions in South-eastern Australia, is reported. MATERIALS AND METHODS Cattle serum Blood samples were obtained from three groups of cattle: (I) 23 Shorthorn-Hereford bull calves, derived from reciprocal crosses of Shorthorns and Herefords, born during October-December I966, and 2I born during October-November I967. They were kept at pasture at the C.S.I.R.O. National Cattle Breeding Station, "Belmont", Rockhampton, Queensland (Lat. 23 ° 20' S, sea level). The environment and herd at "Belmont" have been described in detail by Kennedy & Turner (I959). (2) 24 Brahman-cross bull calves born during October-December I966, and 29 born during October-November I967 and also kept at "Belmont". This line has a constitution of 50 per cent Brahman, 25 per cent Hereford, 25 per cent Shorthorn. The animals of both breed groups were run together. The animals born in I 966 had been injected at 2-3 months and 6-8 months with 5 ml of Leptospira bivalent vaccine and 5 ml ofBlackleg bivalent vaccine and, as part of another experiment, with I ml of tetanus antitoxin and 3 ml of influenza virus antigen. Three blood samples were taken from each animal of the group born in 1966 early in August I967, early in September I967 and early in November I967. These animals were all weaned early in August I967 and their weights recorded. The I967 drop were bled and weighed in June I 968 before weaning. (3) 58 Hereford bull calves, kept at the C.S.I.R.O. Division of Animal Health Field Station at Tooradin, Eastern Central Victoria (Latitude 38° 20° S, sea level). These animals came from stock which had been grazed on a mountain snow lease in North-eastern Victoria. Here the cattle are wintered and the calves are born in sheltered lower country, returning to the high plain (I 300I 700 m) for summer grazing. Thus they spend most of their lives at temperatures of I0-20°C. The animals at Tooradin were 6-7 months old and their bodyweights ranged from I80-350 kg at the time of bleeding. In the three groups, blood was collected by puncture of the jugular vein into dry tubes and allowed to clot. The serum was separated by centrifugation and kept at -70 °C. The sera from the "Belmont" group were transported by air to Melbourne, packed in ice.

Ana(ysis of sera Electrophoresis. Glass lantern slides, 7·5 em x 2·5 em, coated with a I-mm

444

BRITISH VETERI ARY JOURNAL, 127, g

thick layer of I per cent Difco * agar in pH 8·3, I = o·o5 sodium diethylbarbiturate buffer, were used. The serum samples were applied to 5 x I mm Whatman No. I filter paper wicks which were inserted in the centre of the gel. Five samples were applied per plate. Electrophoretic separation took 45 min at ro v cm- 1 • After drying at 85°C for roo min the slides were immersed for 25 min in a I per cent (wfv) solution of Amidoblack Bt in 6o per cent water, 30 per cent ethanol and ro per cent acetic acid, and washed in three changes of 75 per cent water, 20 per cent ethanol and 5 per cent acetic acid for 24 hours. The slides were then dried for 30 min at 85 °C and scanned in a Beckman "Analytrol" scanner. The system was calibrated by running and scanning a series of known concentrations of bovine IgGr + IgG2 and bovine serum albumin. Q,uantitation of IgGr and IgG2. The IgGr and IgG2 content of the sera were determined by the solid-phase, radio-immune assay of Catt & Tregear ( rg67 ) using specific anti-bovine IgGr and IgG2 and 125 I-labelled IgGr and IgG2. The IgGr and IgG2 were separated from bovine serum by gradient elution chromatography on DEAEt cellulose. The gradient used was from o·or M Ma 2MP0 4 to 0·3 M Na H 2P0 4 and the column was loaded with IO mg protein/ mg of wet DEAE cellulose. The first two peaks of protein eluted from the column were concentrated by pressure dialysis and were each found to give single precipitin lines corresponding to IgG2 and IgGr on immunoelectrophoresis against rabbit, anti-bovine serum. 125I-labelled IgGI and IgG2 were prepared by the method of Hunter & Greenwood (rg62). Antisera to IgGr and IgG2 were prepared by injecting rabbits intramuscularly at weekly intervals with three r o mg samples of each immunoglobulin emulsified in 2·5 ml of Freund's complete adjuvant (Difco) and r ml o·r4 M NaCl. The antisera were made specific by absorption, the anti-IgGI with 3 mgfml of IgG2 and the anti-IgG2 with 3 mgfml of IgGr. Total Protein. The total protein content was determined by diluting the sera r j2oo in o·I4 N NaCl and reading the extinction at 210 mt-t. An E:r::.. of 204 was used to convert absorbance to protein concentration (Tombs, Souter & Maclagan, 1959). The concentration of protein under each peak of the electrophoresis pattern was calculated by dividing the total protein concentration by total number of integration units in the "Analytrol" scan and multiplying by the number of units under each peak. The results of the electrophoresis runs and of the radioimmunoassay were combined and the figures expressed in g of protein per roo ml of serum. RESULTS

The results of serum protein analysis for the various groups of animals are given in Table I. It can be seen that there is no significant difference between the mean values for the three successive bleeds of each of the "Belmont" groups. This constancy was a reflection of constant individual values. The "Belmont" groups, however, differ significantly from each other and from the Tooradin *Difco Laboratories, Detroit, Michigan, U.S.A. tAmidoblack B, DEAE, George T. Gurr, London.

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0° 17 oOlg

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0 °07

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0° 3 0

10lg

0°22

101 5

0' 11

0°22

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1 °37

0 °3 1

1°0 7

0 ° 13

0 °34

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0°21

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1°44 1°3 6

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BRITISH VETERINARY JOURNAL, 127, g

group in a number of respects. Overall, the "Belmont" cattle had significantly higher (P < o·oi) IgGI and significantly lower albumin levels than the Tooradin cattle. At "Belmont" the Shorthorn-Hereford groups had significantly lower mean albumin and significantly higher mean IgGI levels, (2·6g and 1·68 g/ Ioo ml) than the Brahman-cross group (3 ·02 and 1·39 gjioo ml). The other fractions, including the IgG2, remained fairly constant between the different groups. Correlation co-efficients were calculated for the IgGI levels and the weights of the "Belmont" Brahman-cross and Shorthorn-Hereford groups and the Tooradin Hereford Group. A coefficient of -o·8g was found for the Brahmancross, of -o ·87 for the "Belmont" Shorthorn-Hereford calves and o·og for the Tooradin Herefords. A positive correlation was found between the serum albumin and weight of both Belmont groups (r = o·78 for Brahman cross and o·g4 for Shorthorn-Herefords) but there was no correlation between the albumin and weight of the Tooradin group. The slope of the regression line of the Brahman-cross albumin/weight plot was significantly less (P < o·o5 ) than that of the Shorthorn-Hereford albumin plot. The immunoglobulin and albumin levels of the various groups are shown plotted against weight in Fig. I, 2 and 3· DISCUSSION

The increased immunoglobulin and decreased albumin levels of the "Belmont" compared with the Tooradin cattle parallels the situation with tropical and temperate human populations (Curtain, Ig66). However, unlike human IgG, the serum IgG of cattle is divisible into two electrophoretically-distinct subclasses, IgGI and IgG2, with markedly different biological properties. Both

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BRITISH VETERINARY JOURNAL, 127, g

contain precipitating antibodies, but only lgGI binds complement and takes part in the homologous passive cutaneous reaction (Anderson, personal communication), and it is a major immunoglobulin of colostrum and probably of the other exocrine secretions (Pierce, I967). Further, Curtain, Clark & Dufty (I 97 I ) have shown that practically all of the plasma cells occurring in the lamina propria and at the base of the villi the respiratory, alimentary and of genital mucosae of cattle contain lgGI only. lgGI was found to be the predominating immunoglobulin in the mucus secretions. Curtain & Anderson (I 97 I) have found that helminth infestation in sheep is accompanied by a significant increase in the number of lgG I -containing plasma cells in the mucus of the alimentary tract. However, attempts to correlate serum lgGI levels with degree of helminth infestation have been inconclusive (Curtain & Anderson, unpublished). In this connection our finding that the increase in immunoglobulin in the tropical group is entirely due to a selective increase in lgGI is of considerable interest, particularly as it appears that this increase is greater in the low-weight animals. Over the two months in which the I 966 drop of "Belmont" cattle were under observation the individual serum protein values remained remarkably constant, suggesting that the lgGi jlgG2 had been fixed before 7-8 months of age. The significant negative correlation between body weight and lgGI levels in both the "Belmont" Shorthorn-Hereford and Brahman-cross groups suggests that the influences leading to a higher lgGI level in the tropical animals pressed most heavily on the animals with lowest weaning weight. Possibly the low weight animals had been deprived nutritionally early in life, owing either to a diminished supply of maternal milk, or to the effect of heat stress on suckling. If this deprivation occurred during the postnatal 24 hours oflife when the calf receives its colostral lgGI, then it is conceivable that this deficiency of antibody might allow the establishment of an early and high level of parasitic infection and perhaps lead to excessive, or unbalanced, growth of the saphrophytic intestinal flora. Since immunoglobulins of the lgGI class seem to be largely concerned with the local immune response in the alimentary tract in ruminants, this condition would lead to an excessive number oflymphoid cells committed to lgGI production and this in turn would be reflected in the high level of lgGI in the serum. The Brahman-cross group are presumably less affected in the postnatal period by heat and this better start is reflected by higher serum albumin and lower lgGI levels. There is some evidence that in man, high levels and rates of synthesis of serum immunoglobulins, attained during childhood in the tropics, tend to persist in adult life, even when the apparent causes of the hypergammaglobulinaemia are removed, either by improved hygiene or removal to a temperate climate (Schofield, I 95 7; Cohen & McGregor, I 963) . Possibly a similar situation may occur in cattle, where a poor start will be reflected in increased immunoglobulin levels through life. ACKNOWLEDGEMENTS

The author is grateful to Mr R. Camp for skilled technical assistance with the

INFLUENCE OF ENVIRONME T ON SERUM PROTEINS

449

laboratory aspects of this work and Mr H. G. Turner, Officer-in-Charge, CSIRO Division of Animal Genetics, Rockhampton, Mr J. Kennedy, Officerin-Charge of the National Cattle Breeding Station, "Belmont", Rockhampton, and Mr J. H. Dufty, CSIRO Animal Health Research Laboratory, Melbourne, for the provision of sera and data from the animals used in the study. REFERENCES

GATT, K. & TREGEAR G. W. ( I967). Science, 158, I570. CoHEN, S. & McGREGOR, I. A. (I963). In Immunity to Protozoa, ed. P. C. C. Garnham, A. E. Pierce & I. Roitt. Oxford: Blackwell. CuRTAIN, C. C. (I966). Papua New Guin. med. ]. 9, I45· CuRTAIN, C. C. & ANDERSON, N. ( I97I). Clin. exp. Immun. 8, I5!. CuRTAIN, C. C., CLARK, B. L. & DuFTY, J. (I97I). Clin. exp. Immun. (in press). FINDLAY,]. D. & BEACKLEY, W. R. (I954). Prog. Physiol. Fm. Anim., ed.J. Hammond, 1 , 252. London: Butterworth. HuNTER, W. M. & GREENWOOD, F. C. (I962). Nature, Land. 194, 495· KELLEY, R. B. (I932). Pamph. Coun. scient. ind. Res. Aust. No. 27. KELLEY, R. B. (I94I). Emp. ]. exp. Agric. g, I53· KELLEY, R. B. (I943), Bull. Coun. scient. ind. Res., Melb. No. I72. KENNEDY, J. F. & TuRNER, H. G. ( I959) Div. Rep. Div. Animal Hlth. Prod. C.S.I.R.O. Aust., No. 8 (Ser. S.W. -3). PIERCE, A. E. (I967). Proc. XVIIIth Int. vet. Gong., Paris, 1 , 407. ScHOFIELD, F. D. (I957)· Trans. R. Soc. trop. Med. Hyg. 51, 332. ToMBS, M.P., SouTER, F. & MAcLAGAN, N. F . ( I959). Biochem. ]. 73, I67. (Accepted for publication 27 April I 97 I ) L'influence de l'environnement sur les niveaux de serum immuno-globulines dans le betail

{Curtain) Resume. Les serums de 44 veaux Shorthorn-Hereford et 53 Brahman de races croisees eleves dans des conditions sub-tropicales (Rockhampton), qui avaient ete examines avec l'agar-gelelectroforese pour determiner les concentrations des groupes proteines de major serum et avec Ia radio-immuno-essai pour quantifier les immuno-globulines, IgGI et IgG2, exprimaient des significatifs plus bas niveaux de serum albumine et des niveaux de IgGI significativement plus eleves que ceux trouves dans les serums de 58 Herefords eleves dans des conditions fraiches temperees (Tooradin). Les Brahman-croises avaient des plus bas niveaux de IgGI et il y avait la une significative correlation negative entre le poids corpore! et le niveau de IgGI dans les deux groupes Brahmancroises et Shorthorn-Hereford. II avait suggere que les plus hauts niveaux de IgGI parmi les animaux de Rockhampton, en particulier ceux avec un poids plus bas, pouvaient resulter d'un relatif et plus limite transfert des anticorps colostraux d'origine maternelle, de suite apres la naissance, avec un consequent precoce etablissement d'un excessif parassitisme intestinal et d'une excessive ou instable croissance des normales bacteries saprofitiques intestinales. Der Einfluss des umgebenden Milieus auf die Iuununoglobulinkonzentration im Serum von Rindern

(Curtain) Zusammenfassung. Die Sera von 44 Shorthorn-Hereford Rindern und von 53 gernischtrassigen Brahman Kiilbern, die unter subtropischen Bedingungen aufgezogen wurden (Rockhampton), wurden mittels der Agar-Gel-Elektrophorese untersucht, urn die Konzentration der wichtigeren Serumproteingruppen festzustellen, und rnittels Radiurnimmunopriifung, urn die Menge des Immunoglobulins festzustellen: IgGr und IgG2. Man fand bei den Brahman Kiilbern signifikant niedrigere Serumalbuminwerte und signifikant hohere IgGI Konzentrationen als in den Seren von 58 Herefords, die bei kiihleren Temperaturen aufgezogen worden waren (Tooradin).

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BRITISH VETERINARY JOURNAL, 127, 9

Die Brahmankreuzungen hatten signifikant niedrigere IgGr Konzentrationen und man fand ausserdem cine signifikante negative Korrelation zwischen Gewicht und IgGr Konzentration bei heiden Gruppen, den Brahman Kreuzungen und den Shorthorn-Herefords. Es wird angenommen, class die hoheren IgGr Konzentrationen bei den Rockhampton-Tieren, besonders bei denen mit geringem Gewicht, von einer relativ schlechteren Ubernahme von Antikorpern aus dem miitterlichen Kolostrum kurz nach der Geburt herriihren konnte, mit dadurch verursachtem friihem Vorhandensein exzessiven Parasitismus im Darmtrakt und iibergrossem oder ungleichformigem \Vachstum normaler saprophytischer Darmbakterien. Influencias del rnedio arnbiente sobre los niveles de inrnunoglobulinas sericas en el ganado vacuno (Curtain) Resumen. Se examinaron los sueros procedentes de 44 ternera~ Shorthorn-Hereford y 53 Brahman cruzadas y criadas bajo condiciones subtropicales (Rockhampton) por media de electroforesis en agar-gel a fin de determinar las concentraciones de los principales grupos proteicos del suero, y por ensayos radioinmunol6gicos para saber la cantidad de inmunoglobulinas IgGr e IgG2. Dichos analisis mostraron una cifra de albuminas sericas significativamente inferior y otra mas elevada de IgGr que las encontradas en los sueros procedentes de 58 terneras Hereford criadas bajo conditciones de climas mas frios (Tooradin). Los cruces-Brahman mostraron niveles significativamente mas bajos de IgGr y se observ6 una correlaci6n significativamente negativa entr el peso corporal y las cifras de IgGr tanto en las razas Brahman cruzadas como en los grupos de Shorthorn-Hereford. Se sugiri6 que los niveles mas elevados de lgGr en los animales Rockhampton, especialmente los referentes ala perdida de peso, podian deberse a una transferencia relativamente mas pobre de los anticuerpos del calostro derivado de la madre poco despues del parto, con el consiguiente establecimiento procoz de un parasitismo intestinal excesivo y un exceso o crecimiento desequilibrado de las bacterias normalmente saprofiticas del intestino.