Maturation of Paramphistomum cervi in sheep in India

Veterinary Parasitology, 15(1984) 239--245 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

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MATURATION OF PARAMPHISTOMUM CERVI IN SHEEP IN INDIA

B.C. G U P T A , V.R. P A R S H A D

and S.S. G U R A Y A

Department o f Zoology, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana-141004 (India) (Accepted for publication 22 November 1983)

ABSTRACT Gupta, B.C., Parshad, V.R. and Guraya, S.S., 1984. Maturation of Paramphistomurn cervi in sheep in India. Vet. Parasitol., 15: 239--245. Histological studies of the trematode Paramphistomum cervi collected throughout the year from the rumen of sheep, revealed the occurrence of gravid worms from April to August and immature worms from September to March. The maturation of the gonads and the accessory sex glands i.e., the prostate-, Mehli's- and vitelline-gland began during the period March--April and showed functional peaks during the period July--August, which coincided with the availability of the intermediate host, Indoplanorbis exustus in nature. Maturation as well as the incidence of infection of P. cervi have also been discussed in relation to various ecological factors, i.e., rainfall and temperature.

INTRODUCTION

The frequent outbreaks of clinical paramphistomiasis due to Paramphist o m u m cervi in sheep during the early winter months, September-October (Dutt, 1980) relate to the immature phase of intestinal development of worms (Sharma and Katiyar, 1967; Dutt, 1980). The life cycle of P. cervi involves the snarl Indoplanorbis exustus as the intermediate host which hibernates during winter (Dutt and Bali, 1981). Therefore, taking into consideration the survival factor of the eggs and metacercariae under natural climatic conditions (Dutt, 1980), some synchrony is to be expected between the release of eggs by the ruminal worms and the availability of I. exustus in nature, and also between the release of metacercariae and their availability to grazing sheep. Therefore, in order to determine the ;annual maturation period and reproductive events related to it, the reproductive tissues of P. cervi were studied throughout the year. MATERIALS AND METHODS

The specimens of P. cervi were collected at fortnightly intervals from May 1980 to April 1981 from the rumen of naturally infected sheep, slaugh-

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TABLE I Seasonal changes in the reproductive organs ofParamphistomum eervi

Months

Mean temp.

Total Status rainfall of worms

Ovary

Testis

Vitenine glands

Prostate Mehli's Inseminated gland gland

(ram) January (50)" 4b February (75) 4 March (65) 4 April (86) 6 May (165) 6 June (360) 6 July (400) 6 August (600) 4 September (50) 5 October (350) 4 November (350) 4 December (200) 3

12.4°C

75.0

Immature

OG,IO

PSG

IM

NS

NS

15.3°C

16.0

Immature

OG,IO

PSG,SSG,TSG,IM PSC

IM

NS

NS

18.3 ° C

59.0

Immature

OG,IO

PSG,SSG,TSG,PSC

IM

NS

NS

26.1°C

0.00

Mature

OG,IO,MO

PSG,SSG,TSG,PSC, SSC,ST,SP

IM,MA,M

S

S

+

32.0°C

11.4

Mature

OG,IO,MO

PSG,SSG,TSG,PSC, SSC,ST,SP

IM,MA,M

S

S

+

32.9°C

41.9

Mature

OG,IO,MO

PSG,SSG,TSG,PSC, SSC,ST,SP

IM,MA,M

S

S

+

29.8°C

554.7

Mature

OG,IO,MO

PSG,SSG,TSG,PSC, SSC,ST,SP

IM,MA,M

S

S

+

29.3°C

132.2

Mature

OG,IO,MO

PSG,SSG,TSG,PSC,

IM,MA,M

S

S

+

SSC,ST,SP 28.1°C

30.4

Immature

OG,IO

PSG

IM

NS

NS

24.7°C

26.4

Immature

OG

PS~

IM

NS

NS

18.5°C

17.4

Immature

OG

PSG

IM

NS

NS

Immature OG

PSG

IM

NS

NS

13.0°C

7.9

aTotal number of worms collected;bSample size; key; ÷, present; -, absent; IM, immature; IO, immature oocytes; M. mature; M A , maturing; M O , mature oocytes; NS, non-secretory;OG, oogonia; PSC, primary spermatocyte; PSG, primary spermatogonia; S, secretory; SP, spermatozoa; SSC, secondary spermatocytes; SSG, secondary spermatogonia; ST, spermatids; TSG, tertiaryspermatogonia.

tered at the local abattoir, Ludhiana (30°56'N, 75°52'E) India. They were fixed in alcoholic Bouin's fluid and 5--7 pm thick paraffin sections were stained with hematoxylin--eosin (Humason, 1979). Usually each collection consisted of a large number of worms (Table I), but a sample of 3--6 specimens from each batch was subjected to histological examination. RESULTS

Features o f the different reproductive organs of P. cervi collected throughout the year are summarized in Table I and illustrated in Figs. 1 and 2. Immature worms collected during September to March show very small irregular testes with shallow invaginations (Fig. 1A). The worms collected during the period September-January contain a small number of sperm-

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Fig. 1. (A) Testis of the worm, collected during March. Note the irregular shape and presence of spermatogonial cells. Hematoxylin--eosin (H. E.) X 100. (B) Ovary (O), ootype (OT) and uterus (U) of the worm collected during February. Note the presence of oogonia (OG) and immature oocytes (IO) in the ovary. H. E. X 100. (C) Vas deferens (VD) and uterus (U) of the worm collected during March. Note their thin walls and narrow lumens. H. E. X 100. (D) Mature testis of the worm collected during May. Note the presence o f various stages of spermatogenesis. H. E. X 400. (E) Ovary of the worm during July, completely filled with mature oocytes. H. E. × 100. (F) Vas deferens (VD) and uterus (U) of the worms, collected during July, is completely filled with spermatozoa (SP) and eggs (E) respectively. Note their wide lumens. H. E. x 100.

11.3 13.6 15.7 17.6 5.6 6.3 6.2 16.1 16.1 0 0 0

0 0 0 10.4 32.5 41.0 52.8 37.4 37.4 0 0 0

100 89.1 80.1 66.0 46.4 52.4 44.0 53.3 100 100 100 100

0 3.1 4.1 4.3 0 0 3.0 0 0 0 0 0

Secondary spermatogonia

Tertiary spermatogonia 0 3.9 4.2 5.2 3.1 1.9 7.3 0 0 0 0 0

Primary spermatogonia

Mature oocytes

Oogonia

Immature oocytes

Stages of spermatogenesis

Stages of oogenesis

January 88.6 February 86.3 March 84.2 April 72.3 May 61.8 June 52.9 July 40.8 August 46.4 September 73.1 October 100 November 100 December 100

Months

0 3.9 10.4 6.2 3.3 4.2 6.6 0 0 0 0 0

Primary spermatocytes

Seasonal changes in the percentage frequency of gametogenesis in Paramphistomum cervi

TABLE II

0 0 0 3.0 5.5 0 2.6 0 0 0 0 0

Secondary spermatocytes

0 0 0 2.1 11.8 9.9 33.0 9.3 0 0 0 0

Spermatids

0 0 0 7.9 26.7 31.6 35.1 30.6 0 0 0 0

Spermatozoa

bO

bO

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Fig. 2. (A) Prostate gland (PG) of the worms collected during October. Note the low heights and the absence o£ secretory material from them. H. E. x 100. (B) Worm collected during March shows immature vitelline cells (VC). H. E. X 400. (C) Mehli's gland (MG) of the worm during October. Note its smaller size and the absence of secretory material. H. E. X 100. (D) Fully developed prostate gland in the worm collected during July. Note the increased heights of prostate gland cells (PGC) and the egg (E) coming out the metraterm. H. E. X 100. (E) Mature vitelline cells (VC) in the worm during August. H. E. X 100. (F) Fully developed Mehli's gland (MG) in the worm during July. H. E. X 100.

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atogonial cens, which are at an advanced stage of spermatogenesis in the worms collected during the period February--April (Fig. 1A, Table II). At this stage, mature spermatozoa were neither seen in the testes nor in the vas deferens. The worms collected during the period April--August contain larger testes with deep testicular invaginations and show all the stages of spermatogenesis as well as mature spermatozoa (Fig. 1D). The percentage frequency of mature spermatozoa in the total spermatogenic cells shows a progressive increase from April and reaches a peak in July. After July the percentage frequency of mature spermatozoa decreases (Table II). The ovaries of the immature worms collected from September to March are very small and contain mainly oogonia and a few immature oocytes (Fig. 1B, Table II). A few mature oocytes together with an increased number of immature oocytes appear in worms during September (Table I). The ovaries of the worms collected during July contained the highest percentage of mature oocytes of the total (52.81%), indicating the peak during this period (Fig. 1E). The immature state of the genital ducts (Fig. 1C), small size of prostate(Fig. 2A), Mehli's- (Fig. 2C) and vitelline-glands (Fig. 2B) and the absence of secretory material within these glands, during the period S e p t e m b e r March, indicates that the secretory functions of accessory sex glands are not elaborated until the gonads begin to show active gametogenesis in the worms from July to August (Fig. 2D,E,F). However, during the maturation phase, these glands are well developed and their cells contain secretory materials. During May the vitelline follicles are small and contain mainly immature and maturing vitelline cells with only a small number of mature vitelline cells. The shell globules present in the vitelline cells are small; their size increases during the subsequent months. During June, the cells are mainly maturing and mature. The vitelline follicles increase in size (Fig. 2E) and fill the lateral sides of the worms. In comparison to the immature worms from September to March, the mature worms from April to August contain a well developed thick-walled uterus completely filled with eggs (Fig. 1F), and a vas deferens filled with spermatozoa (Fig. 1F). All the worms collected during these months show signs of insemination revealed by the presence of spermatozoa in the seminal receptacle and eggs in the uterus. DISCUSSION

In this study, the mature specimens of P. cervi were obtained, from the rumen of sheep, during the period April--August and the immature specimens during the period September--March, as was also observed in the moose Alcer alcer (Lankester et al., 1979). The pre-patent period of P. cervi in laboratory infection of sheep ranges from 103 to 115 days (see Dutt, 1980). If this period is added on to the time of occurrence o f / .

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exustus and metacercariae in nature (June--August), the mature flukes would be expected from September to November or December, but they begin to appear from April onwards. This suggests that either the sheep acquire infection during the late monsoon when the intermediate host begins to hibernate (Dutt and Balli, 1981), or maturation of the worms is delayed or slowed down in natural infections until the intermediate host is available in nature. The maturation of P. cervi from April to August coincides with the availability of the intermediate host in nature, as it hibernates during winter (Dutt and Bali, 1981). If the worms become fully mature and start producing eggs during the months of November or December, then during this period snails will not be available to pick up infection. Thus, the maturation of worms and release of eggs at a time when the intermediate host is available and all the meterological factors, e.g., rainfall and temperature (Table I) are favourable, provides a good example of biological adjustment leading to the successful completion of the life-cycle. In conclusion, it can be stated that there is some synchronization between the maturation of P. cervi, its egg-laying and emergence of I. exustus from hibernation. The possible factors controlling or delaying the maturation of P. cervi in naturally infected sheep are poorly understood. ACKNOWLEDGEMENT

Financial support from UGC is duly acknowledged.

REFERENCES Dutt, S.C., 1980. Paramphistomes and Paramphistomiasis o f Domestic Ruminants in India. Punjab Agricultural University Press, Ludhiana, 162 pp. Dutt, S.C. and Bali, H.S., 1981. Studies on the life history of the aquatic snails Lymnaea luteola Lamarck, L. aurieularia rufescens Grey and Indoplanorbis exustus Deshayes. Indian J. Anita. Sc~, 51: 215--220. Humason, G.L., 1979. Animal Tissue Techniques. W.H. Freeman, San Francisco, 681 pp. Lankester, M.W., Snider, J.B. and Jerrard, R.E., 1979. Annual maturation of Paramphistomum cervi (Trematoda: Paramphistomatidae) in moose Aicer alcer L. Can. J. Zool., 57: 2355--2357. Sharma, D . V ~ . and Katiyar, R.D., 1967. Studies on the _patho_genicity due to immature amphistomes among sheep and goats. Indian Vet. J., 44: 199---205.