Vaccination Against Dictyocaulus Filaria with Irradiated Larvae*

Brit. vet. J. (1965),

IlU, 212

VACCINATION AGAINST DICTYOCAULUS FILARIA WITH IRRADIATED LARVAE* By A. SOKOLIC, M. JOVANOVIC, K. CUPERLOVIC and M. MOVSESIJAN Institute for the Application of Nuclear Energy in Agriculture, Veterinary Medicine and Forestry, Zemun, Yugoslavia

SUMMARY Active anthelmintic immunization, by means of vaccination with irradiated larvae, represents the most recent approach to solution of the problem of control of parasitic diseases. An irradiated vaccine against Dictyocaulus filaria was tested in nearly 2,000 animals in both laboratory and field experiments. In laboratory experiments, doubly-vaccinated lambs were highly resistant to challenge, in comparison with non-vaccinated controls. When challenged on the 15th day after vaccination, 8 1·8 per cent of vaccinated animals were resistant, and on the 60th day after vaccination 100 per cent were resistant. Field experiments on the infected lambs and sheep established that a postvaccination decrease took place in both the number of infected animals and the degree of infection. The average percentage for both decreased by about 50 to 60 per cent of the values in the controls at the end of the first pasture season. The epidemiological aspect of dictyocauliasis is discussed as well as the need for procedures for evaluating anthelmintic vaccines.

INTRODUCTION The outward manifestations of parasitic and .infectious diseases appear to be somewhat similar; both may have the character of zoonoses and be potentially dangerous to people and animals, and parasitoses may be even more costly because of the heavy losses caused to the livestock industry. Often as difficult to control and eradicate as infectious diseases, the parasitoses now lend themselves to control by active immunization. Extensive studies on parasitic immunity in general (Soulsby, 1959, 1960a, b, c, 196w, b, 1962, 1963; Stoll, 1961; Tromba, 1962) and the more detailed work on helminth immunity (Soulsby, 1962) have strongly supported the view that active immunization might be used against parasites, and Soulsby (1962) reported that there is no evidence that the immune response associated with the host-parasite system differs markedly in its physical or chemical characteristics from that stimulated by other infectious agents. The possibility of actively immunizing animals against certain helminths --Some data were presented to the Panel on the Production and Utilization of Radiation Vaccines, IAEA, Vienna, D ecember 16-18, 1963.

VACCINATION AGAINST D . FILARIA

21 3

has been demonstrated recently in the laboratory and the field with irradiated pathogen parasitic forms (Jarrett et al., 1958a, b, 1959a, b, 1961 ; Jones & Nelson, 1960; Poynter et al., 1960; Dow et al., 1961, 1962; Engelbrecht, 1961; Jovanovic et al., 1961, 1963; Sokolic et al., 1961, 1963a, Edds et al., 1962; Erickson & Caldwell, 1962; Smithers, 1962; Thorpe & Broome, 1962; Villella et al., 1962; Miller) 1963; Movsesijan et al., 1963; Urquhart, 1963; Urquhart et al., 1963). In this report it is intended to present the results oflaboratory and field work with a radiation vaccine against the sheep lungworm, Dictyocaulus filaria. The epidemiological aspect of sheep dictyocauliasis will be discussed as well as that of vaccine assessment. MATERIALS AND METHODS

Animals

One thousand nine hundred and fifty-eight sheep of the Tsigaya and Pramenka breeds and crossbred lambs of these with Merino, Shropshire or Hampshire were used. Except for 240 yearling lambs in two field flocks, animals were two to four months old at the beginning of the experiment. A group of 112 lambs, used in the laboratory studies, was raised parasite-free as described by Pribicevic et al. (1962). The field experiments were carried out in the districts with a previous history of losses caused by sheep dictyocauliasis. Irradiated larvae

Larvae were separated from the faeces of artificially infected lambs which had been raised parasite-free. The larvae were suspended in tap-water at pH 7'5 to 8'5 and kept at 4 to 8°c in order to retard development. Third-stage larvae were obtained in approximately 24 days under these conditions. Cultures, not older than 30 days, were concentrated to about 25,000 larvae/ml. tap-water and exposed, at room temperature, to ionizing radiation in a layer 1 mm. deep. Following irradiation, larvae were resuspended into tap-water and stored at 4 to 8°c until used. Viability and safety tests on the vaccine were performed before administration to the lambs. Two types of radiation were used: X-rays of roentgen origin and y-rays of 60CO source. The dose rate from the X-ray source was 240 roentgens (r.) /min. , and 200 r. /min. from the y-ray source. In laboratory experiments vaccine consisted of larvae irradiated at levels of 20, 40, 60 kiloroentgens (kr. ) of X-rays, or at 40 and 60 kr. of y-rays. The vaccine used in the field consisted oflarvae irradiated at the level of 50 kr. of X-rays. Technical details concerning radiation sources are described elsewhere (Jovanovic et al., 1965). . The technique reported previously (Nevenic et al., 1962) was used in faecal examinations for larvae of D. filaria. Experimental procedure

In laboratory experiments all animals were examined clinically for respiratory disorders and faecal samples were examined for larvae. In field work the ewes,

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which constituted about 10 per cent of the total number of animals used, were examined similarly, while 10 per cent of the lambs and yearlings allotted for vaccination and all of the non-vaccinated controls were also examined. The vaccine was administered orally. In the laboratory studies this was given on the fifth and tenth day after irradiation, by means of a stomach tube; in the field, animals were drenched with the vaccine within IS days of its irradiation. Animals received either a single dose of vaccine (2,000 irradiated larvae of D. filaria) or a double dose, the first containing 1,000 and the second, one month later, containing 2,000 irradiated larvae. Resistance to infection was assessed, in the laboratory, by simultaneous challenge of the vaccinates and controls with non-irradiated larvae, the challenge dose depending upon the age and body weight of the animal. In most cases the challenge dose was divided into seven parts given over a period of two months, or t en parts each given at intervals of three days. In the field experiments, animals were challenged by being put to graze on infected pastures, which were checked periodically for the presence and number of D. filaria larvae. In the laboratory work, animals were examined after vaccination by faecal larval counts every five days and by clinical examination of the respiratory organs every seven days during the course of three to four months. Animals were then slaughtered for post-mortem examination. In the field experiments, similar examinations were conducted. The initial examinations coincided with the first and second vaccination and thereafter animals were examined at intervals not longer than six weeks until the end of the pasture season.

Criteria used for assessment if post-vaccination resistance The assessment of resistance was generally based on the following: (a) number of D. filaria larvae/g. of faeces as calculated from the total faecal counts; (b) total number of D. filaria isolated from the respiratory organs; (c) total number of the sexually mature females of D. filaria; and (d) number of deaths in the post-challenge period. Group averages were calculated for the vaccinates and were expressed as a proportion of the group averages for the controls (e.g. 1/8; see Table I). Where there were negative findings in the vaccinates the figure for the controls was the mean of the actual findings (e.g. 0/8). The percentage of resistant animals was calculated as follows: animals with values less than 50 per cent of the control group means were arbitrarily considered resistant while those with any measurement which was greater than 50 per cent of the controls were considered non-resistant; this was expressed as a percentage of the total number in the group (e.g. 50 per cent of eight sheep resistant indicates that four sheep in one or more measurements were in excess of 50 per cent of the control measurements). Post-vaccination resistance in the field flocks was based on the percentage of the infected animals and on the degree of infection, the latter being derived from the total counts of D. filaria larvae/g. offaeces. Group means were calculated from flock averages.

VACCINATION AGAINST D . FILARIA

21 5

RE SU LTS

It was consider ed to be a good approach, from the aspect of the possible use of th e vaccine for the control of sh eep dictyocauliasis, to examine the following: (a) safety of irradiated larvae, (b) effect of single a nd double vaccination, (c) time factor in post-vaccinal resistance, (d ) effect of single and r epeated infection, (e) effect of vaccination of alread y infected lambs, (f) effect ofvaccination on alread y infected yearling lambs, and (g) effect of vaccination of animals in a closed flo ck. Effect ~r various radiation doses Larvae were exposed to dosages of 20, 40, 60 kr. of X-rays and 40 and 60 kr. of y-rays and 2,000 larvae from each dose schedule were administered orally, on the fifth post-irradiation day, to each animal of one group consisting of two lambs. Four controls were treated with non-irradiated infective larvae from the same batch as the irradiated larvae. The animals were slaughtered on the 55th post-infection d ay and examin ed . A characteristic feature was the finding of a small number of sexually mature female larvae and a small total number of ma les in the respiratory organs. The total male to female ratio was I: I I .5, compared with I: 1·6 with non-irradiated larvae in control animals. The details of the clinical and pathomorphological changes in the respiratory organs have been reported elsewhere (Jovanovic et al. , Ig6I; Sofrenovic et al., 1961; Gligorijevic et al., Ig62 ). immunity test oj the vaccine This was carried out on 98 lambs, the design and the results being presented in Table 1. The results generally show that: (a) in the group of doubly vaccinated lambs, except in the group challenged on the third post-vaccinal day, a greater number of resistant animals occurred as compared with singly vaccinated animals; (b) the number of resistant animals increased with the post-vaccinal time. In the doubly vaccinated lambs 38 per cent r esist ed challenge on the third d ay aft er vaccination, 81·8 per cent on the 15th day, and 100 per cent on the 60th day. In singly vaccinated lambs the numb er of resistant animals incr eased from 50 to 62'5 per cent from the 60th to the goth d ay; (c) with the increase in time between vaccination and challenge it was generally found that there w as a decrease of all parameters selected for the assessment of the vaccine, thus indicating the increase of the d egree of resistance as compar ed with the controls; (d ) vaccination resulted in the inhibition of d evelopment and retardation of reproduction in the females. This led to a reduction in oogenesis which was evidenced by a marked decrease in larval output, sometimes amounting approximately to a factor of 500, as compared with the corresponding controls (see Table I, group IV). The results obtained by administering larvae irradiated with equal doses of X- or y-rays were almost identical. Field flocks were infected before vaccination. Twenty-five to 40 per cent of

.., 0">

TABLE I THE RES ULTS OBTAINED IN SHEEP BY SINGLE AND DOUBLE VACCINATION WITH IRRADIATED VACCINE AGAINST

Dictyocaulus filaria

Single

D ouble

with 2000 irradiated larvae

with 1000 and 2000 irradiated larvae

Vaccination

OJ

:;tI >-<

Group

I

Irradiation dose (kr )

40 controls

Day of post-vaccinal irifection

60th

Dose of infective D. filaria larvae No. in vaccinates set at I tlNo. in controls (group averages} : AlIaie female parasites in lungs Sexually mature female parasites in lungs Larvae in I g. faeces

+

(I

5000 x 5000)

/II

IV

V

40 controls

40 controls

40 controls

3 rd

15 th

60th

II 20

40

60

controls

9 0th (I X

5000

+

25,000 5 x 3000

+

I

2000t or 4000t 4000t or 8000t x 5000) (10 x 200) (10 x 400) ( 10 x 400) (10 X 800)

(I X

25,000 5000 + 5 x 3000 + I x 5000)

>-l

>-< CFJ

::r: <: t%j

>-l t%j

:;tI

..... Z

> :;tI 0--< t....;

1/2 1/5 1/8

1/ 18 0/3 0/8

1/4 1/ 14 1/42

1/3 1/ 14 1/ 104

% of resistant animals

50/ 0

50

62·5

62·5

% of lethality

0/0

0

0

No. of experimental animals

8/4

4

8

Duration of experiment, days

150

220

0 C

1/7 1/13 1/3 1

1/8 1/27 1/499

1/67 0/3 0/5

0

38 . 1/0

81·8/0

100/0

r>

0

25

0/ 12·5

0/ 12·5

0/25

;:; :-

8

4*

9t

+

13t/4t

+ 2t

9t

110-120

*-Controls common to groups II and V. t - Each dose was administered to the corresponding number of animals. t-Except in O-values (negative findings).

+

13t/2t

12 7- 140

+ 4t

6/4* 21 5

:;tI

Z

(.n

VACCINATION AGAI NST D. FILARIA

21

7

ewes were infect ed with D. filaria, or D. filaria and Protostrongylus rufescens, while in one flock up to 30 per cent oflambs excreted 0'4 larvae/g. of faeces. The results from the field experiments for the lambs studied in the same year have been summarized and are presented in Figs. I and '2, which show the course of the infection during the pasture season. The figures represent the group means for I , I 98 vaccinates and 1 24 controls, the latter grazing with the vaccinated flocks.

100 65

62

29

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17

./

/

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-0

./

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c:

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6 ,/

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--.....

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~'5

o

c:

controls (12q Vaccinates (1 '198) - "-

Q)

u

... Q)

0...

o Vaccine 30

75

I dose IT Fa e c a

120

165 days

exam i nations I

I Fig.

I .

I

R esults of field experiments.

The results show that the number of infected vaccinates decreased by 55 to 65 p er cent and the d egree of infection by about 40 to 60 per cent as comp ared with the controls. The effect of vaccination on an existing infection was studied in 140 yearling lambs, 80 per cent of which had an average of 3'3 larvae/g. of faeces. The infection d ecreased after vaccination so that at the end of the first p asture season only 2 I per cent of the vaccinates were infected and passing an average of

BRITISH VETERINARY JOURNAL,

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0·66 D. filaria larvae/g. of faeces, in contrast to a 60 per cent infection of the controls, which passed an average of I · 44 larvae/g. Studies of the effect of vaccination on the animals in a closed flock, with no import of other infected animals, extended over two successive pasture seasons and included 384 animals, IOO of which were yearling lambs. The results are presented in Table II. There was a general decrease in the number of infected animals and in the number oflarvae/g. of faeces at the end of the second pasture season, compared with the pre-vaccinal findings. A slight difference from the general trend of d ecrease of infection is observed in larval counts in the flock of vaccinated lambs. The lambs in the other flock were revaccinat ed the next year, before being put to graze. .

2·9

1·3

.......-.. -.. .

II>

'"u '"o

.. ".,.,., .. ,..- ,.

-

1·2 " -"--Q

'y "-1·0

' ·0

/

--

Infected controls Infected vacc inates

a

d

;z

0·1

o Voce ine 30

I dose n Foe e Q I

Fig.

e xa m I

2.~Results

165 doys

120

75

not i

0

n s

I

of fi eld experiments.

DISCUSSION

As in bacterial or viral vaccines, the potency of an anthelmintic vaccine is influenced by a variety of factors associated with both the parasite and host. Factors to be studied in the parasite are the a ntigenic materials, their "protective" quality, quantity, and their route and time of administration. Important factors to be studied in a host are the immunological competence

<: ~

o

o

TABLE II

~

THE RESULTS OF THE VACCINATION OF INFECTED S HE E P IN A CLOSED F L O C K

vcu:cinated

vcu:cinated percentage of infected animals

>-<

Lambs

Yearlings

larvae/g. of faeces

percentage of infected animals

oz

vaccinated

larvae/g. of faeces

Pre-vaccination irifection

44

2 '1

30

0' 4

Infection at the end of the first pasture season

30

1'5

22

0 '5

lnfection at the end of the second pasture season

12

0 '5

10

1'7

percentage of irifected animals 79

~

larvae/ g. of f aeces 2' 9

60 4.6 revaccina ted before grazing 6 1'5

Q

~

Z

en >-l ~

~

~

::;, ~

IV
220

BRITISH VETERINARY JOURNAL,

121 ,5

associated with age and health status, especially with other parasite infections. The epidemiological aspects of immunoprophylaxis of helminth disease must take note of important factors such as the st ability and duration of artificially acquired resistance, effect of revaccination, d esirability of multiple vaccination of immature animals as well as vaccination of adults, and the significance of the natural infection (Taylor, 1960). Certainly time will be n eeded before many of the basic problems are elucidated, but a number of experimental variables were included in the laboratory and field work on the radiation vaccine against D. filaria. The initial results presented in this paper, although obtained on almost 2,000 animals, should be considered as only preliminary. It is intended, instead of going into detail, to emphasize the general features of the work, e.g. the fact that an increasing resistance to infection with D. filaria can be produced by the vaccination of animals. In the laboratory experiments a satisfactory degree of resistance to the challenge was established in doubly vaccinated lambs from the 15th post-vaccinal day onwards. Inhibition and retardation of oogenesis still existed 3t months after the first challenge dose, as compared with the controls, and larval output decreased by a factor of about 500 (see Table I, group IV). This resulted from the decreased number of reproductive females (Sokolic et at., 1963 ). Vaccination of previously infected animals in the field resulted, in almost all of the experimental flocks, in a decrease in larval output and in the number of infected animals. The post-vaccinal decrease of larval numbers in the faeces and the number of infected animals is a significant factor from the epidemiological point of view. It gives perspective for eradication of the disease if vaccination is carried on over a period of several years. When the results are considered in terms of "infection potentials" the difference between vaccinates and controls might be even greater. "Infection potential" is defined as the average number of larvae/g. of faeces calculated from all faecal counts of all infected animals of one group. Fig. 3 shows that the infection potential for the pasture is an important factor in the epidemiology of dictyocauliasis. It is derived from the results presented in Fig. 1 and 2 by multiplying the percentage of the infected animals with the degree of infection and dividing the product by 100. Fig. 3 shows that possible pasture infection might be four to six times lower in the vaccinates (1,198 animals) than in the controls (124 animals). However it should be noted that the controls formed part of a flock in which about 90 per cent of the animals were vaccinated. The decrease of larval output in the controls might be explained partly by the . decrease of the infection in the vaccinates and hence to a reduced infection potential. The lack of a suitable method for testing the potency of an anthelmintic vaccine has to be considered . Criteria such as the number of eggs or larvae per g. of faeces, or the number of parasites or reproductive females per animal, may reflect the immune response in the host in a general way, but more specific methods for assessing the immune response of the host are urgently needed.

22 1

1·9

1·0

0·8

0·3

II>

<1.1

u

0·3

0·1

<1.1

o

.....

.....o 0;

:z:

ControLs Vaccinates - ,, -

0.02

0'.01

o Vaccine I

30 dose n Fa e c a I

75 e x a m I

10 n a t i

0

165 days n s

I

Fig. 3. Derived from Fig. I and 2 , by multiplying the percentage of infected animals by degree of infection and dividing by 100. ACKNOWLEDGEMENTS

The authors greatly appreciate the valuable contributions of Professor C. L. Comar, from Cornell University, who provided initiative and encouragement for this work, and of Professor E. J. L. Soulsby, of the University of Pennsylvania, who criticized and discussed the manuscript. REFERENCES

Dow, C.,JARRETI', W. F.j.,JENNINGS, F. W., McINTYRE,W.j. M. & MULLIGAN, W. (1961 ). Amer. J. vet. Res., 22, 352. Dow, C.,JARRETI', W. F . j.,JENNINGS, F. W ., McINTYRE, W.J. M. & MULLIGAN, W. (1962). Amer. J. vet. Res., 23, 146.

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EDDS, G. T., SWANSON, L. E. & ENGELBRECHT, E. (1963) . Amer. J. vet. Res., 24, 139. ENGELBRECHT, H. j. (1961), J. Parasit., 47 (supp!.), 21. ERICKSON, D. G. & CALDWELL, W. L. (1962). J. Parasit., 48 (supp!. ), 19. GLIGORIjEVIC, j ., JOVANOVIC, M., SOKOLIC, A., CUPERLOVIC, K. & MOVSESIjAN, M. (1962) . Vet. Glasn., 16, 1027. JARRETT, W. F. H., JENNINGS, F. W., McINTYRE, W. j. M., MULLIGAN, W. & URQUHART, G. M. (I958a). Proc. R. Soc. Med., 51, 743. JARRETT, W. F. H., JENNINGS, F. W., MARTIN, B., McINTYRE, W. j. M., MULLIGAN, W., SHARP, N. C. C. & URQUHART, G. M. (I958b). Vet. Rec., 70, 451. J ARRETT, W. F. H., JENNINGS, F. W., McINTYRE, W. j. M., MULLIGAN, W., SHARP, N. C. C. & URQUHART, G. M. (I959a) . Amer. J. vet. R es., 20, 522. JARRETT, W. F. H., J ENNINGS, F. W., MciNTYRE, W.j. M., MULLIGAN, W. & SHARP, N. C. C. ( I959b) . Amer. J. vet. Res., 20, 527 . JARRETT, W. F. H., J ENNINGS, F. W., MciNTYRE, W.j. M., MULLIGAN, W. & SHARP, N. C. C. (1961) . Amer. J. vet. Res., 22, 186. JONES, B. V. & NELSON, AM. R. (1960). Vet. Rec., 72, 395. JOVANOVIC, M., NEVENIC, V., SOKOLIC, A, SOFRENOVIC, D. j., GLIGORIJEVIC, j., CUPERLOVIC, K . & MOVSESIJAN, M. (1961 ) . Vet. Glasn., 15,455. JOVANOVIC, M., SOKOLIC, A., MOVSESIJAN, M. & CUPERLOVIC, K. (I965a). IAEA, Vienna, 1963 Technical Report Series, in press. JOVANOVIC, M., SOKOLIC, A, MOVSESIJAN, M. & CUPERLOVIC, K. (1965). Brit. vet. J., 121, 119. MILLER, T. A (1963) . Proc. XVI! WId. vet. Gongr., Hanover, I, 829. MOVSESIjAN, M., MLADENOVIC, Z., CUPERLOVIC, K., SOKOLIC, A & JOVANOVIC, M. (1963). Vet. Glasn, 17, 837. NEVENIC, V., JOVANOVIC, M., SOKOLIC, A., CUPERLOVIC, K. & MOVSESIJAN, M. (1962). Vet. Glasn., 16, 203. POYNTER, D., JONES, B. V., NELSON, A. M. R., PEACOCK, R., ROBINSON, j., SILVERMAN, P. H. & TERRY, R. j. (1960). Vet. Rec., 72, 1078. PRIBICEVIC, S.,jOVANOVIC, M., SOKOLIC, A., CUPERLOVIC, K., MOVSESIJAN, M. & STOJANOVIC, N. (1962). Vet. Glasn. , 16, 737. SMITHERS, S. R. (1962). Nature, Lond., 194> II 46. SOFRENOVIC, D. j., JOVANOVIC, M., SOKOLIC, A., NEVENIC, V., CUPERLOVIC, K. & MOVSESIJAN, M. (1961). Vet. Giasn., 15,871. SOKOLIC, A., JOVANOVIC, M., NEVENIC, V., SOFRENOVIC, D.j., CUPERLOVIC, K. & MOVSESIJAN, M. (1961). Vet. Glasn., 15,6.35. SOKOLIC, A., JOVANOVIC, M ., CUPERLOVIC, K. & MOVSESIJAN, M. (1963). J. Parasit., 49, 612. SOKOLIC, A,jOVANOVIC, M., CUPERLOVIC, K . & MOVSESIJAN, M. (1965). IAEA, Vienna, 1963 Technical Report Series, in press. SOULSBY, E. j. L. (1959). Proc. XVI Int. vet. Congr., Madrid, 2,571. SOULSBY, E. j. L. (I960a). Vet. Rec., 72, 322. SOULSBY, E. j . L. (196ob). Brit. vet. J., 116,302. SOULSBY, E. j. L. (1960c). Brit. vet. J. , 116, 315. SOULS BY, E. j. L. (I96xa). J. Amer. vet. med. Ass., 138, 355. SOULS BY, E. j. L. (196Ib). Vet. Rec., 73, 1053. SOULSBY, E. j. L. (1962). Advanc. Immunol., 2,265. SOULSBY, E. j. L. (I963b). Amer. J. Hyg., Monograph. Ser., No. 22,47. STOLL, N. R. (1961). Amer. J. trap. Med. Hyg., 10, 293. TAYLOR, E. H. (1960). Vet. Rec. 72, 684. THORPE, E. & BROOME, A W. j. (1962). Vet. Rec., 74> 775. TROMBA, G. F. (1962). J. Parasit., 48, 839. URQUHART, G. M. (1965). IAEA, Vienna, 1963 Technical Report Series, in press. URQUHART, G. M., McINTYRE, W. J. M., MULLIGAN, W., JARRETT, W. F. H. & SHARP, N. C. C. (1963). Proc. XVll Wid. vet. Congr., Hanover, I, 769. VILLELLA, j. B., GOMBERG, H. j. & GOULD, S. E. (1962). J. Parasit., 48, (supp!.) 19.

(Receivedfor publication 30 January, 1965)