Serological Findings in Bovine Herpes Mammillitis

Br. vet. J. (1969),

125,

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SEROLOGICAL FINDINGS IN BOVINE HERPES MAMMILLITIS By M. M.

RWEYEMAMU*, R.

H. JOHNSON

AND ROSEMARY

E.

LAURILLARD

Department of Veterinary Medicine, University of Bristol SUMMARY

A serological investigation of BHM is described. It has been shown that natural exposure of cattle to BHM results in an antibody response. This response is so rapid that the first of paired serum samples for diagnosis ofBHM should be taken during the very early stages of development of lesions. Following recovery, antibody persists for at least 2 years. It is proposed that there are two epidemiological patterns of BHM, one a primary infection in a susceptible herd resulting in a high morbidity rate, and the second in previously exposed herds tending to be to newly calved heifers. The results of the serological survey have indicated modes of transmission of BHM infection other than by teat-cluster/milker's hands, and it is proposed that such transmission is by biting flies . It has also been shown that in the West of England approximately 19'5 per cent of cattle sera show evidence of previous exposure to BHM virus. INTRODUCTION

It has now been clearly established that bovine herpes mammillitis (BHM) is a distinct clinical entity (Martin, Martin, Hay & Lauder, 1966; Rweyemamu, I968a, b;Johnson, Gibbs & Rweyemamu, 1969). As part ofa long-term study on the epidemiology of BHM, serological investigations were carried out on farms with and without a history of clinical BHM, and on 400 random serum samples taken from lactating dairy cattle in the West of England. Serum neutralization techniques used were those previously standardized by Rweyemamu & Johnson (1968). The TV strain (R weyemamu & Johnson, 1967) was used as the BHM virus challenge in all the neutralization tests reported in this paper. METHODS AND RESULTS

Serological findings during outbreaks of BHM

During clinical investigations of outbreaks of BHM, serum samples were collected from clinically affected cows showing lesions of different duration. Fourteen such samples were collected from animals with lesions less than I week old. All but one animal demonstrated detectable antibody; this animal being one of two which were bled within 2 days of onset oflesions. The second animal had a titre of 1/4. Ten of the sera were from animals showing lesions 3-5 days old. These showed neutralizing antibody of titre between 1 / 22 and 1 / 100. • Present address: Central Veterinary Laboratory, Temeke, Dar-es-Salaam, Tanzania.

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Twenty serum samples taken from animals showing lesions 1- 6 weeks old all d emonstrated neutralizing antibody with titres ranging between 1/50 and 1/720 (Fig. 1). It will be noted from Fig. 1 that the majority of the twenty sera h ad titres between 1/200 and 1/500. In addition twenty p aired serum samples (Table I ) d emonstrated a sign i-

10 r

8 ~ <1) on

-

°6 z ci

4

r--

2r

Il 50

Fig.

I.

100 250 5 00 Reciprocal SIN titre

>500

Distribution of neutralizing BHM antibody in twenty sera taken from cows showing lesions 1-6 weeks old. TABLE I

THE R ESULTS OF NEUTRALIZATION TESTS FOR BHM ON TWENTY PAIRE D SERUM SAMPLES

R eciprocal SIN titre Serum riference Acute serum (lesions less than I week old)

Convalescent serum (4---6 weeks later)

B B B By By By By By By By By By By . By

23 24

22

25

22

360

4

200 250 5 00 25 0 100 25 0 50 50 100 00 5

By By By By By By

34 36 41 45 48

4 4 10 II

15 18

19 20

23 24 56

52

It> 50 22 22

-ve* -ve* -ve* -ve* -ve* 100

-ve (2 days) 22 (7 days) 6

-ve* -ve* 22

-ve* -ve*

* Collected before appearance of clinical lesions.

720 180

5 00 25 0 50 50 100 50 100

SEROLOGICAL FI NDINGS IN BHM

ficant rise in titre of BHM a ntibody during the course of the natural disease. It was quite apparent from these findings that during BHM infection there was an early antibody response so that for the purpose of diagnostic serology the first serum sample should be taken during the very early stages of the disease, i.e. within 2 days of onset of lesions, the second sample being taken at 3- 4 weeks. In all these outbreaks the disease was confirmed as BHM by isolation of virus in vitro, and/or d e monstratio~ of typical herpes virus p articles by electron mIcroscopy. Serological findings

1

year after clinical BHM

T en sera taken from previously affected cows were collected from two farms in Somerset I year after an outbreak ofBHM, and assayed for BHM antibody. The results of the neutralization tests on these sera are summarized in Table II, from which it is seen that BHM antibody is detectable for at least 1 year after r ecovery from natural infection. TABLE II R ESULTS OF NEUTRA LI ZATION TESTS ON I NDIVID UAL SERA CL I NICAL BHM

Farm reference

SM

I

SM 3

Serum reference

FROM

TWO

FA R MS

I

3 4

5 00 50 100 100

B B B B B B

18 20 16 19 15 17

50 50 100 100 25 0 5 00

I

AFTE R

Reciprocal SIN titre

B B B B

2

YEAR

A more detailed investigation was carried out on one farm on which clinical BHM, in the form of "skin gangrene of the bovine udder", had been investigated a year previously (Rweyem amu, Johnson & Tutt, 1966). The results of the neutralization tests on sera taken from all the lactating cattle and the r eplacement h eifers on this farm are presented in Table III. It will be noted that go per cent (70/77 animals) of all the lactating cattle had neutralizing antibody although only six a nimals had shown clinical disease a year previously. All fourteen animals that had no d etectable antibody were either replacement heifers or first calvers. It was further observed that all seven first calvers which showed no detect able a ntibody had calved subsequent to the disappearance of the clinical disease from the farm . Three a nimals that had shown no clinical BHM had neutralizing antibody titres greater than 1/500. These were a second, a fifth and a seventh calver. Their r espective titres were 1/ 1250, 1/1250 and 1/6250. The last animal was one of three cows which

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were on the farm in 1959 when a syndrome clinically identical to the one investigated in 1966 was experienced on this farm (Tutt, 1966, p ersonal communication). The titres of the other two animals were 1/250 and 1/500. TABLE III RESULTS OF NEUTRALIZATI ON TESTS ON ALL CATTLE ON FARM A, I YEAR AFTER AN OUTB REAK OF BOVI NE HERPES MAMMILLITIS I N WHICH SIX ANIMALS WERE CLINICALLY AFFECTED

Numbers ofpositive sera Reciprocal SIN titre

Replacement First heifers near calvers calving

0

7

2-22 50-100 250-5 00 > 500

3

Total

7

I

9 6

0 0

0 2

9

0

II

Serological findings

Second calvers

23

2

13

Third calvers

Fourth calvers

0

Fifth calvers

0

2

0 0 2

5 0

I

0

8

3

I

Sixth calvers

Older than sixth calvers 0

Total

I

0 2

2

9

4

5 0

3

33 3

8

16

6

88

14 II

27

years after clinical BHM

R epresentative animals, from three farms in Somerset (SM1, SM2 and SM3) which h ad experienced clinical BHM 2 years previously, were bled . On farm SM l six animals were bled ; these included two animals which had been clinically affected, two animals which were on the farm during the BHM outbreak b ut did not d evelop the clinical disease a nd two animals which had been bought-in since the time of outbreak. As farms SM2 and SM3 were selfcontained, it was decided to bleed eighteen animals from each herd to include six animals which had been clinically affected, six animals which were in the milking herd at the time of the outbreak but did not show clinical signs of BHM, and six animals which were either calves or yearlings during the outbreak, so that at the time of bleeding they were first or second calvers. In addition young stock from farm SM3 were bled. These included four calves up to 3 months old, four calves between 3 and 6 months old, six calves between 6 and 12 months old, and seven heifers between 1 and 2 years old, three of which had been born during the BHM outbreak. The r esults of the neutralization tests on these sera are summarized in Table IV, from which it will be appreciated that the animals which had developed clinical lesions of BHM still h ad antibody 2 years post infection. It also seems th at the majority of the animals that were on these farms at the time of the outbreak d eveloped antibody which app arently p ersisted for at least 2 years. The fact that the majority of calves up to 6 months old had detect able neutralizing antibody, and that young stock older than 6 months had no such antibody suggests that this could be due to passive immunity to BHM lasting up to 6 months. Such persistence of passive immunity in calves h as been demonstrated under experimental conditions (Rweyem amu, 1968b). From this observation

SEROLOGICAL FINDINGS IN BHM

321

it seems reasonable to assume that the antibody detected in heifers which were calves or yearlings during the BHM outbreak was due to direct exposure of these animals to the virus during the outbreak, apparently without any obvious clinical disease. TABLE IV RES U LTS OF NEUTRALIZATION TESTS ON THREE FARMS 2 YEARS AFTER AN OUTBREAK OF BOVINE HERPES MAMMILLITIS

Results are expressed as proportion of total number of animals bled in each group.

Proportion Reciprocal titre showing - -- -- - - - - - - - - - - - -antibody o 2-22 50-100 250-500 > 500 o

Were clinically affected F

arm

5M 1

Farm 5M2

{

Were not clinically affected Bought in since outbreak

1/ 2

o

1/ 2

1/ 2

o

o

o

o

o

o

o

Were clinically affected

6/ 6

o

o

5/ 6

1/6

o

Were not clinically affected

3/ 6

3/ 6

2/6

1/6

o

o

l

C alves/yearlings during outbreak

1/6

5/ 6

1/6

o

o

o

Were clinically affected

6/6

o

1/6

4/6

1/6

o

6/6

o

2/6

2/6

2/6

o

6 / 10

o

o

3/10

o

l

Were not clinically affected Were calves/yearlings during outbreak Calves up to 6 months old at time of bleeding Calves 6- 12 months old at time of bleeding Heifers 1-2 years old at time of bleeding; born after BHM outbreak

{

Farm SM3

7/ 8

1/8

6/8

1/8

o

o

0/6

6/6

o

o

o

o

0/4

4/4

o

o

o

o

Other serological investigations In addition to the a bove investigations sera were collected from the University milking cows (thirty-nine sera), from six other farms that had been visited in connection with other types of teat lesions (seventy sera), plus 400 random bovine sera that had been submitted to the Veterinary Investigation Centre, Langford, in connection with diagnosis of diseases other than teat lesions. None of the sera from the University farm stock demonstrated detectable neutralizing antibody. Of the seventy sera from farms with teat problems other -than BHM, only four sera (5·7 per cent) had detectable antibody to BHM. These four sera included two from a farm with a high proportion of bought-in stock. Of the 400 random sera from Veterinary Investigation Centre, seventyeight (19·5 p er cent) had detectable neutralizing BHM antibody, suggesting a possible exposure to BHM virus in the West of England of 19·5 per cent.

322

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~ISCUSSION

This paper has d emonstrated that natural exposure to BHM infection results in an antibody response which apparently persists for more than 2 years. This finding should have an important bearing on any immunization procedures to be d eveloped. It may also explain why no recurrence of clinical disease has been observed on any of the farms investigated over the 2 year period (Rweyemamu, 1968b), although this could be fortuitous because of lack of re-exposure to the virus. In contrast, paravaccinia infection (pseudocowpox), another common cause of teat lesions in cattle in Britain, apparently does not elicit any persisting neutralizing antibody (Plowright & Ferris, 1959; Liebermann, 1967). One difference in the epidemiology of this disease and BHM is that periodic recurrence of lesions, which is not observed for BHM, appears to be characteristic (Cheville & Shey, 1967; Gibbs & Rweyem amu, 1968, unpublished). In contradiction, Martin et at. (1966) reported a recurrence of BHM in one cow after 13 months. This diagnosis is, however, questionable as it was made on clinical grounds without laboratory confirmation. It should be pointed out that pseudocowpox can be confused with some stages of BHM lesions and that the two infections can occur on the same farm or on the same animals simultaneously (Huck, 1968; Rweyemamu, 1968a, b; Johnson, Gibbs & Rweyemamu, 19 69) . It may be postulated that some recovered animals become latent carriers as in the case of h erpes simplex (Scott & Tokumaru, 1965) and that such carriers would "break down" under physiological stress to show mild sores or teat chapping, thereby disseminating virus to in-contact susceptible heifers. It may also be postulated that, alternatively, some recovered animals which become carriers excrete virus continually over a long period as in the case of infectious laryngotracheitis of chickens, another herpes virus infection (Hofstad, 1965; Jordan, 1966). The r esults of this serological study are not adequate to support or refute these hypotheses. On farm A it was noted that old cows which had been on the farm at the time of a previous outbreak of a disease clinically identical to BHM in 1959 d emonstrated high levels of antibody. Although it is possible that some of these animals with very high antibody titres could have been asymptomless carriers, it is also likely that such levels represented a sharp rise in titre due to a secondary antibody response. The results of a 2-year serological investigation on recovered herds show that young stock which were born after evidence of clinical BHM had disappeared, d emonstrated passively acquired neutralizing antibody. In one herd (SM I ) animals which had been bought-in after clinical BHM, showed no detectable antibody when tested a year later. These findings seem to suggest that none of the recovered animals in these herds h ad become carriers excreting virus to incontact susceptible cattle. From the data accumulated during these investigations and that published by other workers (Deas & Johnston, 1966; Martin et at., 1966; Pepper , Stafford,

SEROLOGICAL FINDINGS IN BHM

Johnson & Osborne, 1966 ; White, D erbyshire, Pattinson & Wilson, 1959) it appears that there are two epidemiological patterns for BHM. In some outbreaks, the disease may spread through the majority of the herd (up to 90 per cent) either r apidly, or slowly over a period of up to 2 months. In other outbreaks the disease may be confined to newly calved heifers. It seems that primary infection results in a high morbidity rate in a susceptible herd. Following BHM infection, the majority of animals in such recovered herds show persisting neutralizing antibody. If subsequently, BHM infection should get into such a recovered herd, only young stock or newly purchased susceptible animals would be expected to d evelop lesions. This may explain why in outbreaks of "skin gangrene" (White et at., 1959), the clinical disease has been noticed to be almost solely confined to newly calves heifers. It appears that BHM infection is probably endemic in some areas of Britain; White et at. (1959) had experienced "skin gangrene" in south Wiltshire for 20 years, and R weyemamu et at. (1966) have indicated that probably the lesions described by Hare (1925, 1926, 1927) under the designation of "impetigo" may have been due to BHM virus. The transmission pattern of BHM has yet to be fully elucidated. Clinical observations suggest that transmission within an infected herd may be by the milking machine and/or milker's hands (Martin et at., 1966; Deas & Johnston, 1966; Pepper et at., 1966; Rweyemamu et at., 1966). The fact that in the present investigation neutralizing antibody has been demonstrated in young stock which at the time of the outbreak were not in the milking herd, suggests another mode of transmission. Such an alternative method of transmission could be by biting flies. Flies might transmit the virus mechanically from infected animals to the young stock in the field thereby, presumably, introducing the virus into the skin on sites other than the teats without causing any adverse reaction in the animal. Such animals would show an antibody response. On this basis, it should be feasible to immunize animals by introducing the virulent virus intradermally in a site other than the teat or udder. Indeed such an approach has been part of a study on the d evelopmen t of a vaccine for BHM (R weyemamu, Johnson & Osborne, 1968; Rweyemamu & Johnson, 1969). A long-term study on the transmission of the disease between herds and into new primary centres is obviously required. ACKNOWLEDGMENTS

The kind cooperation of many veterinary surgeons in the West of England is gratefully acknowledged. We are also grateful to Mr A. D. Osborne for assistance in field studies and to Professor C. S. Grunsell for constant encouragement. The work reported in this paper represents part of a project financed by the Agricultural Research Council. REFERENCES

CHEVILLE, N. F. & SHEY, D. J. (1967) . J. Am. vet. med. Ass., 150, 855. DEAS, D. W. & JOHNSTON, W. S. (1966). Vet. Rec., 78, 828. HARE, T. (1925) . Vet. Rec., 5, 943· HARE, T. ( 1926). Vet. Rec., 6, 1083.

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HARE, T. (1927). Vet. Rec., 7, 885. HOFSTAD, M. S. (1965). In Diseases ofPoultry, fifthedn (ed. byH. E. Biesterand L. H. Schwarte), p. 621. Ames: Iowa State University Press. HUCK, R. A. (1968). Vet. Rec., 82 (clinical supplement No. 14, p. I) . JOHNSON, R. H., GIBBS, E. P. j. & RWEYEMAMU, M. M. (1969). In The Blue Book. Hoechst Pharmaceuticals Ltd. In preparation. JORDAN, F . T. W. (1966) . Avian. Dis., 10, I. LIEBERMANN, H. (1967). Z. Arst!. Fortbild., 61, 447. MARTIN, W. B., MARTIN, B., HAY, D. & LAUDER, I. M. (1966) . Vet. Rec., 78, 494. PEPPER, T. A., STAFFORD, L. P.,jOHNSON, R. H. & OSBORNE, A. D. (1966) . Vet. Rec., 78, 569. PLOWRIGHT, W. & FERRIS, R. D . (1959). Vet. R ec., 71, 718. RWEYEMAMU, M. M. (1968a). Int. Vet. Students Union Mag., 3, 15. RWEYEMAMU, M. M. (1968b). Studies on Bovine H erpes Mammillitis. Ph.D. Thesis, submitted University of Bristol. RWEYEMAMU, M . M. & JOHNSON, R. H. (1967). Br. vet.]., 123, 482. RWEYEMAMU, M. M. & JOHNSON, R. H. (1968). Br. vet. ]., 124, 9. RWEYEMAMU, M. M. & JOHNSON, R. H . (1969). Res. vet. Sci. In press. RWEYEMAMU, M. M., JOHNSON, R. H. & OSBORNE, A. D. (1968). Vet. Rec., 82, 85. RWEYEMAMU, M. M.,jOHNSON, R. H. & TUTT,j. B. (1966). Vet. R ec., 79, 8ro. SCOTT, T. F. McN. & TOKUMARU, T. (1965). In Viral and RickettsialInfections of Man, fourth edn (ed. by F. L. Horsfall and I. Tamm), p. 892. London: Pitman Medical Publishers. WHITE, j. B., DERBYSHIRE, j. B., PATTISON, I. H . & WILSON, C. D . (1959) . 71 , 764. (Acceptedfor publication 18 December 1968)

L'etude de serunJ. dans la nJ.anJ.ellite herpetique des bovins (RweyenJ.anJ.u et at.) ResUDle. Description d'une etude serologique de la mamellite herpetique des bovins. 11 a ete prouve que les bovins exposes naturallement a la mamellite herpetique produisent des anticorps. Cette reaction est si rapide que Ie premier des deux echantillons de serum d estines au diagnostic de l'affection doit etre preleve au tout premier stade de l'apparition d es lesions. Apres la guerison, les anticorps subsistent pendant encore au moins 2 ans. Les chercheurs suggerent qu'il existe deux formes epidemiologiques de la maladie: une infection primaire qui atteint des troupeaux sensibles a la maladie et qui a un taux eleve de morbidite et une infection qui tend a etre limitee aux genisses recemment nees dans les troupeaux affectes anterieurement. Les resultats de l'etude serologique indiquent la presence de modes de transmission autres que Ie contact des pis et des mains du trayeur et il est suggere que cette transmission se ferait par des mouches piqueuses. L'etude revele aussi que, dans l'ouest de l'Angleterre, environ 19,5 pour cent des bovins presentent les signes d'une infection anterieure par Ie virus de la mamellite herpetique. Serologische Befunde bei RinderherpesnJ.anJ.illitis ('RHM) (RweyeDlaDlu et at.) ZusaDlDlenfassung. Eine serologische Untersuchung der RHM wird beschrieben. Es wurde gezeigt, daB nach natiirlichem RHM-Befall eine Antikorperreaktion bei den Tieren erfolgt. Diese Reaktion erfolgt so rasch, daB die ersten der paarweisen Serumproben zur RHM-Diagnose wiihrend der friihen Anfangsstadien der Bildung von Liisionen entnommen werden miissen. Nach erfolgter Genesung bleibt der Antikqrper fUr mindestens 2 jahre erhalten. Es wird darauf hingewiesen, daB es zwei epidemiologische RHM-Arten gibt, die erste ist ein Primiiraffekt und tritt bei einer fiir diese Krankheit anfalligen H erde auf, was zu einer hohen Sterblichkeitsziffer fiihrt, die zweite tritt bei friiher befallen Herden auf und neigt dazu, sich bei Fiirsen zu zeigen, die gerade gekalbt haben. Die Ergebnisse des serologischen Erfassungsberichts haben auf andere Ubertragungsweisen, als durch die VerschIl!-utzung der ZitzenJHiinde des Schweizers hingewiesen, und es wird angenommen, daB diese Ubertragung durch Stechftiegen erfolgt. Es wurde ebenfalls herausgearbeitet, daB in Westengland nahezu 19,5 Prozent der Rinderseren einen Beweis hinsichtlich eines bereits durchgemachten Befalls durch das RHM-Virus liefem.

SEROLOGICAL FINDINGS IN BHM DescubriInientos serol6gicos en la D1anillitis herpetica bovine (MHB) (RweyenmD1u et al.) Res1UDen. Se describe una investigacion serologica de la marnilitis herpetica bovina. Se ha demostrado que la exposicion natural del ganado a la MHB resulta en una reaccion de anticuerpos. Esta reaccion es tan rapid a que la primera de las muestras de suero apareadas para la diagnosis de la MHB debe tomarse durante las primeras etapas de desarrollo de las lesiones. D espues de la recuperacion , el anticuerpo persiste por 10 menos durante 2 anos. Se propone que hay dos tipos de MHB epidemiologicos, uno que consiste en una infeccion primaria en un rebano susceptible que resulta en una alta razon de morbilidad, y el segundo en rebanos previamente expuestos que tienden a confinarse a vaquillas recien paridas. Los resultados del examen serologico han indicado modos de transmision de la infeccion de la MHB que no se deben a las manos del ordenador 0 a la agrupacion de tetillas, y se propone que dicha transmision se debe a las moscas mordaces. Se ha demostrado tabrnien que en el Oeste de Inglaterra aproximadamente el 19,5 por ciento de los sueros de ganado muestran evidencia de previa exposicion al virus de la MHB. .