Bovine Herpes Mammillitis Virus

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BOVINE HERPES MAMMILLITIS VIRUS II. Standardization of an in-vitro serum neutralization test By M. M.

RWEYEMAMU AND R.

H.

JOHNSON

Department of Veterinary Medicine, University of Bristol, Langford House, Langford, Nr. Bristol '

SUMMARY

Criteria are presented for a standardized in-vitro serum-neutralization technique for bovine herpes mammillitis (BHM) virus. The test has been standardized to within a three-fold error margin under ordinary laboratory conditions, utilizing primary or low passage bovine kidney cells or an established line of feline lung cells. INTRODUCTION

Rweyemamu, johnson & Tutt (1966) have pointed out the difficulties involved in isolating virus from any other than very early lesions of bovine herpes mammillitis (BHM). Diagnosis and study of epidemiology must depend in considerable part on serological findings, and there is an obvious requirement for standardization of an in-vitro serum neutralization (SjN) test, particularly as detectable levels of antibody may be very low in resistant animals (R weyemamu, unpublished observation). Basic criteria for standardization ofin-vitro SjN tests have been outlined by Robson et at. (1961), Carmichael (1962), and Carmichael, Atkinson & Barnes (1963). The technique for a standardized quantitative serological test for BHM has been based on the criteria outlined by these authors. The initial part of the work involving assay of virus, growth curves, and thermal stability have already been reported (Rweyemamu & Johnson, 1967). The present report describes factors which must be considered to obtain sensitive and reproducible quantitative serological results for neutralizing antibody to BHM.

MATERIALS AND METHODS

Virus. The TV strain was used throughout this work. Aspects of production, assay and storage of virus, and the cell systems used have already been described (Rweyemamu & johnson, 1967). Serum. Bovine sera from either natural cases of the disease or from animals experimentally infected with TV-virus were used throughout this work. Unless otherwise stated all sera were heat-inactivated at 56°c for I hour before use. To provide continuity, basic techniques are incorporated with results.

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PROCEDURES AND RESULTS

Factors which a.ffect the rate of viral inactivation by immune serum (a) The neutralization slope. In order to study the relationship between virus dose and the amount of antibody required for neutralization, a checkerboard titration of ten-fold virus dilutions against doubling dilutions of serum BI6 (known positive) and serum EBl j l5 (known negative control) was carried out. Equal volumes (0'25 ml.) of virus dilutions and serum dilutions were mixed. The serum virus mixtures were shaken for 30 seconds and incubated at 37°C for I hour. Then 0'1 ml. of each serum virus mixture was inoculated and adsorbed to BK monolayers as previously described (Rweyemamu & Johnson, 1967). Cultures were examined daily, and the endpoint neutralizing level of serum dilutions at each virus challenge level was estimated from criteria outlined in section 2 (b). The neutralization slope pattern is summarized in Fig. I. It may be seen that there is an inverse relationship between the challenge concentration of virus used and the neutralizing titre obtained. Use of minimal virus concentrations, therefore, increases the sensitivity of the neutralization test. It was decided empirically that the lowest virus concentration which could be used to allow for technical variation and minimal observation times of monolayers, I.

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Neutralization slope for bovine herpes mammillitis virus in BK cells.

BOVINE HERPES MAMMILLITIS VIRUS. II

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whilst still retaining high level sensitivity was to dilute virus stock to contain 10 3 •3 TCID so/mI., thereby incorporating an estimated 100 TCID 50 of virus into each o· I mi. inoculum of the serum virus mixture. Using the assay criteria previously outlined (Rweyemamu & Johnson, 1967), such a virus challenge was associated with CPE in controls which enabled the test to be read within 4 days without media change. (b) Serum virus incubation times. It has previously been shown that BHM virus is most stable on incubation in the presence of antibody-free calf serum (Rweyemamu & Johnson, 1967). Serum B16 was serially diluted by two-fold steps in HSLS containing 50 per cent of an antibody-free calf serum, and mixed in equal volumes with stock TV-virus diluted in HSLS to contain 10 3 • 3 TCID 50/mi. Incubation of the serum virus mixtures was allowed to proceed at 37°c for I and 2 hours, and then overnight at 4°c; samples were tested at each time interval, using four young BK monolayers per dilution. Controls were set up using antibody-free calf serum subjected to the same test criteria, and tubes were read daily for CPE, the final readings being made when control CPE was maximal as outlined in section 2 (b). Endpoint serum titres rose from I :20 after I hour at 3tc, to I :45 after. 2 hours at 37°c, to I :100 after overnight standing at 4°C. As virus titre is stable over this range of temperature/time (R weyemamu & Johnson, 1967) all subsequent neutralizations were made using an incubation regime of 2 hours at 37°c followed by standing overnight at 4°C, this being the same regime as adopted by Hull & Nash (1960) for B virus. (c) Heat inactivation oj serum and effect oj adding complement. Heat inactivation has been shown in some cases to lower the specific antibody titres of several viruses (Gordon, 1925; Sabin, 1950; Howitt, 1950; McCarthy & Germer, 1952), and in most of these cases a heat labile potentiating factor has been demonstrated. Recently Yoshino & Taniguchi (1964) have shown the presence of a complement requiring neutralizing antibody in early sera after infection with Herpesvirus hominis. Using techniques as outlined by these authors with negative, early and late bovine serum samples it was not possible to demonstrate any increase in sensitivity of the test system using either un-inactivated test sera or by addition of guinea-pig complement.

Factors affecting the accuracy qf the standardized technique (a) Dose-response patterns for the SIN test. To determine the dilution rate of test sera, and the number of tubes at each dilution level compatible with minimal variation, a dose-response graph was prepared (Robson et al., 1961). A known positive heat inactivated serum was serially diluted by two-fold steps in HSLS containing 50 per cent antibody-free calf serum, and equal quantities were mixed with TV-virus diluted to contain 103 •3 TCID 50/mi. Incubation was as described above, and infection of young BK monolayers was carried out by adsorbing 0·1 mI. of each serum virus mixture for I hour at room temperature to each of twenty-five monolayers. Similar control series were set up, using antibody-free calf serum as control and endpoint readings were made as outlined in section 2(b). 2.

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Figure 2 represents the sensitivity range graph (Robson et al., 1961) covering the range from 100 to 0 per cent neutralization. From this it may be calculated that the sensitivity log. range is 0·65, and by applying the formula given previously (Rweyemamu & Johnson, 1967), it may be calculated that, allowing for a three-fold error margin, and with a doubling dilution series, only one tube per dilution is strictly necessary. )00

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Bovine herpes mammillitis virus: dose-response pattern for SIN test in BK cells_

As FL cells are regularly used at these laboratories as alternative to BK cells for SIN work on BHM the sensitivity of these cells was tested by comparative neutralization tests for four bovine sera in both BK and FL cells, and by a similar dose-response pattern graph as described for BK cells. Table I represents the results of one set of comparative SIN tests in BK and FL cells, where it may be seen that the differences obtained are within the permissible two-fold error margin. From dose-response and other comparative experiments, it was evident that, provided strict criteria were followed to estimate endpoint neutralization, SIN titrations performed in FL cells were of comparable sensitivity to those made in BK cells. FL cells were more sensitive to residual virus than were BK cells, degrees of CPE being evident over a wider range of serum dilutions, and , if neutralization endpoints were read as complete absence of CPE, SIN tests in FL cells would be of lower sensitivity than those performed in BK cells. This observation led to the establishment of the following criteria for reading endpoint neutralization in BK and FL cells. (b) Endpoint criteria. In order to standardize the SIN test for both BK and

BOVINE HERPES MAMMILLITIS VIRUS. II TABLE I COMPARATIVE ANTIBODY ASSAY OF FOUR SERA IN BK AND FL CELLS

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* Endpoints expressed as -IOglO of the final serum dilution associated with 50 per cent neutralization against 100 TCID 50/0"1 ml. FL cells, and to allow for maximal sensitivity, endpoint neutralization was read as significant retardation of CPE as seen in control cultures, the following scores being given for CPE: = No CPE + = Five or less established CPE foci + + = Five to fifteen CPE foci + + + = Twenty foci to 3/4 monolayer involvement + + + + = 3/4 monolayer involvement to complete sheet destruction. When CPE in control tubes had reached + + + to + + + + all tubes were read, and tubes showing no CPE or +CPE were read as positive neutralization. On these standards the test is readable in both FL and BK cells within 4 days of incubation" Neutralization titre 50 per cent endpoints were calculated according to the method of Reed & Muench (1938)" DISCUSSION

This work has established standards for performance and evaluation of an in-vitro neutralization test for bovine herpes mammillitis" In standardizing the technique the constant virus, varying serum technique has been adopted in preference to the constant serum, varying virus technique because this method appears the more satisfactory for measuring differences in serum antibody content (Rose, 1952; Tyrell & Horsfall, 1953). A system of scoring CPE and reading endpoint neutralization as significant retardation of CPE has been adopted, in a similar manner to that used by other investigators (Schmidt & Lennette, 1961). Such endpoint standards eliminate the necessity for recognizing terminal virus "breakthrough", and in consequence are associated with a reduced incubation time (obviating media change), with a minimal scanning time for infected cultures, and, in the present instance, allow comparable and reproducible results to be obtained in BK or FL cells. Using this standardized SIN technique considerable numbers of sera have been examined, and the results obtained have been shown to be reproducible,

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whilst the standardized test is of sufficient sensitivity to detect antibody in sera which are apparently negative when tested by non-standardized procedures.

Conclusions Based on this study the following protocol for the serum neutralization test for bovine herpes mammillitis is proposed: 1. Stock virus of a titre not less than 10 5 TCID 50/ml., is stored at 4°c or -50°C for not more than I month, and is diluted when required in tissue culture medium containing 50 per cent antibody-free, heat-inactivated calf serum, to contain 10 3 •3 TCID 50/ml. 2. Test sera are heat-inactivated at 56°c for I hour, and serially diluted by two-fold steps in tissue culture serum-free medium. 3. Equal volumes of serum dilutions are mixed with diluted virus suspension, and mixtures shaken for 30 seconds to ensure adequate mixing. Incubation of serum virus mixtures is made at 37°C for 2 hours and then overnight at 4°c (18-24 hours). 4. Serum virus mixtures are warmed to 37°C and 0'1 ml. of the mixtures inoculated onto young BK or FL monolayers using two tubes per dilution. Adsorption is allowed to proceed for I hour at room temperature and maintenance medium containing 5 per cent antibody-free, heat-inactivated, calf serum is then added. 5. Virus controls are set up containing a known BHM antibody-free calf serum at a ! final dilution, incubation and inoculation proceeding as with test serum/virus. In addition, control virus at the end of incubation is titrated to check the final titre used in the test, and a standard bovine serum with a known titre to BHM virus is incorporated in the series. 6. Provided that CPE progresses normally in virus controls, that the amount of virus in the challenge dosage lies between 50 and 200 TCID 50, and that the standard positive serum does not vary from known titre by more than twofold, results are read when controls are showing maximal involvement of the monolayer, endpoint being read as significant retardation of CPE. 7. For screening tests serum is used at a final dilution of 1:2. ACKNOWLEDGMENTS

We are grateful to Mr G. Fowler, Miss R. Laurillard and Mrs W. Glew for technical assistance, and to Professor C. S. Grunsell for his constant interest and encouragement. Finally we are grateful to the Agricultural Research Council for financial assistance and to the Ministry of O verseas Development for supporting one author (M.M.R.). REFERENCES

CARMICHAEL, L. E. (I962). 66th Ann. Proc. U.S. Livestock San. Ass., p. 59. CARMICHAEL, L. E., ATKINSON, G. F. & BARNES, F. D. (1963). Cornell Vet., 53, 369. GORDON, M. H. (1925). Special Report Series: Medical Research Council, London, No. 98, given by Schmidt & Lennette (I96I).

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Howrrr, B. F. (1950). J. Immunol., 64. 73· HULL, R . N. & NASH, j. C. (1960). Am. J. Hyg., 7I, 15. MCCARTHY, K. & GERMER, W. D. (1952). Br. J. expo Path., 33, 529. REED, L. j. & MUENCH, H. (1938). Am. J. Hyg., 27, 493. ROBSON, D. S., HILDRETH, B. P., ATKINSON, G. F., CARMICHAEL, L. E., BARNES, F. D ., PAKKALA, B. & BAKER, j. A (1961). 65th Ann. Proc. U.S. Livestock San. Ass., p. 74. ROSE, H. M. (1952) . J. Immunol. , 68, 687. RWEYEMAMU, M. M.,jOHNSON, R. H. & TUTT,j. B. (1966) . Vet. Rec., 79, 810. RWEYEMAMU, M. M. & JOHNSON, R. H. (1967). Br. vet. J., I23,482. SABIN, A. B. (1950). Bact. Rev., I4. 225. SCHMIDT, N. j. & LENNETTE, E. H. (196 1). Prog. med. Virol.,3, I. TYRELL, D. Aj. & HORSFALL, F. L. (1953).J. expo Med., 97, 845. YOSHINO, K. & TANIGUCHI, S. (1964). Virology, 22, 193. (Acceptedfor publication 12th July 1967). Le virus de la m.arnilite herpetique bovine. n. Standardisation d'une epreuve de sero-neutralisation in-vitro (Rweyemamu et Johnson) Resume. Les criteres pour une technique standardisee de sero-neutralisation du virus de la mamilite herpetique bovine (MHB) in-vitro sont presentes. L'epreuve a ete standardisee de farron a se tenir dans les marges d'un triple ecart, dans les conditions courantes de laboratoire, par l'emploi de cellules renales bovines de premier passage ou d'un faible nombre de passages, ou par l'emploi d'une lignee etablie de cellules pulmonaires felines. Herpes Mamillitis Virus des Rindes. n. Eine standardisierte SerUD1 Neutralisationsprobe in Zellkulturen (Rweyemamu und Johnson) Zusammenfassung. Das standardisierte Verfahren zeigte unter gewohnlichen Laboratoriurnsbedingungen eine dreifache Fehlergrenze. Es wurden primare Kulturen, die ersten Subkulturen von KaIbernierenzellen odeI' ein bestatigter Stamm von Katzenlungenzellen benutzt. El virus de la mamilitis herpetica bovina. n. Estandardizaci6n de una prueba de seroneutralizaci6n in-vitro (Rweyemamu y Johnson) Resumen. Se presentan los criterios para una tecnica estandardizada de seroneutralizacion in-vitro para el virus de la mamilitis herpetica bovina (MHB). La prueba ha sido estandardizada para estar entre los limites de un triple error en las condiciones de laboratorio, empleando celulas renales bovinas de primer pasaje 0 de un bajo numero de pasajes, 0 sea empleando celulas pulmonares felinas.

Control and Prevention of Inherited Disorders Causing Infertility-ContinuedJrom page 8 Bekampfungsprogrammen nahm das Auftreten von erblich bedingten Fruchtbarkeitstorungen stark abo Control y profilaxis de des6rdenes heredados que ocasionan esterilidad (Bane) ResUD1en. Se pasa revista a la literatura sobre la esterilidad producida por desordenes heredados. Entre los defectos morfologicos, se estudian la hipoplasia gonadal, anormalidades del desarrollo de los conductos de Muller y de Wolff, y anormalidades de la espermiogenesis. Entre los desordenes funcionales se incluyen los ovarios quisticos, la impotencia de monta y las perturbaciones de la espermiogenesis. Se discute el control y la profilaxis de los desordenes heredados, y se pasa revista a los procedimientos usados en Suecia. Despues de introducirse los programas de control, se ha manifestado una disminucion marcada en la frecuencia de los desordenes heredados que producen esterilidad.