Studies on heterologous immunity in schistosomiasis*

Bull. Org. mond. Sant}1 1970, 429 745-749Bull. Wid Hlthi Org. f Studies on Heterologous Immunity in Schistosomiasis* 4. Heterologous Schistosome Immunity in Cattle M. F. HUSSEIN,' A. A. SAEED 2 & G. S. NELSON 2 Previous studies have shown that when mice and monkeys are infected with bovine schistosomes they develop a considerable degree of heterologous immunity against sub- sequent challenge with Schistosoma mansoni. The present report describes a study on the reverse effect in which calves were first exposed to cercariae of S. mansoni and then challenged with cercariae of S. mattheei. The calves developed patent infections with S. mansoni, excreting viable eggs in the faeces, and it is suggested that cattle may be a source of infection to man under natural conditions. The immunizing effect of exposure to S. mansoni was demonstrated by a reduction in the S. mattheei egg load and adult worm burden in the immunized, as compared with the control, animals. The results suggest that previous exposure ofcattle to S. mansoni may reduce the severity ofbovine schistosomiasis in endemic areas. MATERIALS AND METHODS Previous studies have shown that mice and monkeys can be partially protected against sub- sequent challenge with Schistosoma mansoni if they have previously been exposed to infection with the bovine schistosomes S. bovis and S. mattheei (Amin et al., 1968; Amin & Nelson, 1969; Nelson et al., 1968). The present report describes a study on the reverse effect to see whether calves could be pro- tected against S. mattheei by previously exposing them to infections of S. mansoni. A Puerto Rican strain of Schistosoma mansoni, maintained in Biomphalaria glabrata, and a South African strain of S. mattheei, maintained in Bulinus (Physopsis) globosus, were used in this experiment. Cercariae for the initial exposure were collected from 48 snails; those for the challenge from 60 snails. For each infection, cercarial counts were made using * From the Royal Veterinary College, London, and the London School of Hygiene and Tropical Medicine, London, England. This investigation was supported by the World Health Organization and the Tropical Medicine Research Board of the United Kingdom through the Ministry of Overseas Development. 1 Present address: Faculty of Veterinary Science, Uni- versity of Khartoum, P.O. Box 32, Khartoum North, Sudan. ' Present address: London School of Hygiene and Tropical Medicine. the ninhydrin staining technique of McClelland (1961). The calves were cross-bred Friesian- Ayrshire males, 10-12 weeks old. They were divided into 3 groups matched for age and body-weight. Group 1 consisted of 2 calves each exposed to 10000 cercarias of S. mansoni but not challenged. These animals were killed 9 weeks later in order to determine the pathogenic effects of the S. mansoni infection. Group 2 consisted of 3 calves each exposed to 10 000 cercariae of S. mansoni and challenged after 8 weeks with 6000 cercariae of S. mattheei; and group 3 consisted of 3 calves each exposed only to 6000 cercariae of S. mattheei and serving as controls for the calves of group 2. Insufficient numbers of S. mattheei cercariae were available to challenge all 6 calves on the same day. The calves were therefore challenged two at a time on successive days, one calf being taken from group 2 (the immunized group) and one from group 3 (the control group). Each calf was exposed to 3000 S. mattheei cercariae; this procedure was then repeated so that each calf was exposed to a total of 6000 cercariae of S. mattheei. Infections were made by partial tail immersion as described by Saeed et al. (1969). After 6-8 weeks, the hatching method of Standen (1949) was used to demonstrate viable eggs in the faeces. Autopsy of groups 2 and 3 was per- 2514 -745- M. F. HUSSEIN, A. A. SAEED & G. S. NELSON formed 8 weeks after challenge, using intravenous injections of sodium pentobarbital (Trinavin) to kill the animals. For worm recovery, the mesen- teric, portal and pancreatic veins were dissected, and the liver was sliced to collect any remaining parasites. Tissue egg counts were made according to the pepsin digestion technique of Nelson & Saoud (1966). In each case, the small and large intestines were opened and washed, and numerous small snips were taken from them to be digested. In addition, the liver was transected along a straight line 10 cm from its ventral extremity and the sepa- rated portion was homogenized and representative samples digested. This technique was adopted because it had previously been noticed that, in the bovine liver, most of the schistosome eggs accu- mulate in the left lobe (Hussein, 1969). RESULTS The results of this experiment are summarized in Tables 1 and 2. Calves of groups 1 and 2 devel- oped a mild infection of S. mansoni and some of them passed viable eggs in their faeces approximately 8 weeks after exposure. Eggs of this parasite were also found in digests of the liver and intestines, and adult worms were collected at autopsy. The pro- portions of worms recovered ranged from 0.2% to l.30.O; the worms were most numerous in the mesenteric veins draining the small intestine. Egg densities were highest in the small intestine, followed by% the liver and least in the large intestine. This pattern of distribution resembled that of S. mattheei but the accompanying pathological changes were considerably milder. Examination of the livers and intestines of calves 1 (a) and 1 (b), which had pure S. Zalnso0ni infections, revealed a few eggs each surrounded by a granulomatous reaction in which epithelioid cells, eosinophils, lymphocytes and fibroblasts were seen. The liver of calf 1 (a) also showed a degenerated S. mansoni worm, surrounded by a localized zone of inflammation. Traces of schistosome pigment were present in Kupffer cells, and some of the local portal vein branches showed a slight medial hypertrophy, subintimal eosinophilic infiltration and blockage by eosinophilic thrombi. These lesions were confined to a small part of the liver, about 2 cm from the lateral border of the left lobe, and the rest of the organ was normal. Other organs were also normal. As shown in Table 1, the reduction in numbers of S. mattheei adults in the three immunized calves compared with their challenge controls was found V_ -J co ~i: I- 1- LU (D z LU -J -J I 2 4C- Ii- LU N Z D Cl) W LUJ J° U Z Cl) Z D 0 C] 0 LJ 0 LU LI 0 0 U- 0L LU Ii~ C 'Z o ai (U 0 7CD o_M ._ Xto 'a 0 q) -C E I co M CC4 0 to 2) _o X1Ito Ei Irl, Ire-J 26 CI4 C) ~~ > co L-n COco 00 0 U E)_ o) 0 o E ) :2 U. u 2 Cl) C-)~ ~ co m CO r- COC 2 C'J ~~~~~~~~~~. L (U) 06 CD 01 >0U) ii% 0c' Q 0)01 0 0 01 CD ~ ~ C C O C M -0 _- _- (U) _ C_ .0 .0 C_) _ U CU 746 I- STUDIES ON HETEROLOGOUS IMMUNITY IN SCHISTOSOMIASIS. 4 TABLE 2 EFFECT ON TISSUE EGG COUNTS OF HETEROLOGOUS IMMUNITY IN CALVES IMMUNIZED WITH S. MANSONI CHALLENGED WITH S. MATTHEEI Tissue egg counts (eggs per g) Calf Liver Small intestine Large IntestineNo. S. mansoni S. mattheel S. mansoni S. mattheel S. mansoni S. maftheel 1 (a) 58 _ 290 _ 220 1 (b) 4 - 390 - 2 - 2 (a) 2 35 32 117 3 8 3 (a) - 132 - 225 - 135 Reduction of S. mattheei eggs per g of tissue in calf 2 (a) 73.5 - 48 - 94(%) 2 (b) 20 47 25 51 2 23 3 (b)a - - - - - - 2 (c) 37 208 67 310 20 110 3 (c) - 252 - 467 - 314 Reduction of S. mattheei eggs per g 17.5 - 33.6 _ 65 of tissue in calf 2 (c)(%) " No egg counts were made on calf 3 (b). to be 94.4%, 75.9% and 40.1%. There was also a fall in the tissue egg counts of calf 2 (a), showing a reduction of 73.5%, 48% and 94% in the liver, small intestine and large intestine, respectively, compared with the counts in control calf 3 (a). The corresponding reduction in tissue egg counts in calf 2 (c) were 17.5%, 33.6% and 65%, and although this animal had a relatively high adult worm count the worms were markedly small in size. The average length of 50 gravid female worms taken at random was 7.4 mm compared with 9.8 mm in the control calf 3 (c). Unfortunately, no egg counts were made on calf 3 (b) so it was not possible to estimate the effect of immunization on the egg count in calf 2 (b), which had shown a 75.9% reduction in adult worm counts. Gross and histopathological examinations of the tissues of both immunized and non-immunized animals revealed a much more severe pathology in the latter group. In these calves, the intestinal tract, particularly the small intestine, was coated with mucus and blood, and its mucosa studded with numerous tiny granulomas or "sandy patches". The liver was also involved, and the left lobe showed characteristic lymphoid nodules and granulomas. In sections, the branches of the portal vein exhibited various changes including marked medial hyper- trophy and endothelial proliferation, and many of them were blocked by eggs. Eggs, adult worms and associated pathological lesions were also noted in the pancreas and in other organs. DISCUSSION Two interesting observations came out of this study. The first is that calves can develop patent infections with S. mansoni in the laboratory, sug- gesting that they could be involved in the transmis- sion of this parasite to man under natural conditions. The epidemiological significance of this finding has been discussed in more detail in a previous com- munication (Saeed et al., 1969). The second obser- 747 M. F. HUSSEIN, A. A. SAEED & G. S. NELSON vation is that although S. mansoni has little patho- genicity in calves, it confers on them some degree of heterologous immunity against subsequent infec- tions with S. mattheei. Heterologous immunity is thus reciprocal since the experiments with monkeys, S. bovis being challenged with S. haematobium (Hsu et al., 1966) and S. mattheei being challenged with S. mansoni (Amin et al., 1968), have demon- strated a considerable protective effect. The immu- nity in the calves was shown by the marked reduc- tions in worm loads and tissue egg counts of the challenge. In one calf it was also manifested by the stunting of adult worms. Several other workers have also accepted the stunting of worms as a sign of immunity in schistosomiasis (Kagan & Lee, 1952; Meleney & Moore, 1954; Purnell, 1966). On the other hand, Nelson et al. (1968) reported that stunting of schistosomes could also be produced by factors other than the immune state of the host, such as the crowding of worms and a predominance of females. A further interesting finding in the calves is that, in the immunized group, the S. mattheei females were often seen in copula with S. mansoni males. A similar phenomenon was observed by Taylor et al. (1969) in mice and they suggested that cross- specific pairings could affect the tissue egg counts in experiments on heterologous immunity. The mating of the different species, whereby the females of the challenge infection are taken up by males of the immunizing infection, may result in the females producing infertile eggs that become fixed in the tissues because of a lack of enzyme activity. These eggs may be deposited in abnormal sites due to the migratory activities of the male worms. The immunology of schistosomiasis of cattle, like many other aspects of the bovine disease, has been neglected in the past, even though the first evidence of schistosome immunity was based on observations made nearly 60 years ago on calves infected with S. japonicum (Fujinami, 1916). It is therefore needless to emphasize that cattle, as well as other domestic stock, should be used extensively for assessing immunity against their own schisto- somes. It is also important to determine the longevity of such immunity and the various mechanisms involved in its production. There is no immediate prospect of using the heterologous system for immunizing man, but if further studies confirm the present observations there should be no difficulty in setting up a field experiment to test the efficiency of heterologous immunity in protecting sheep or cattle against severe and lethal schistosome infections such as have been reported by Le Roux (1929) in Rhodesia and Hussein (1969) in the Sudan. ACKNOWLEDGEMENTS We are grateful to Professor E. Cotchin of the Royal Veterinary College, London, for his advice and encourage- ment, and to Dr R. Smithers of the National Institute for Medical Research, London, and Dr C. A. Wright of the British Museum (Natural History), London, for supplying infective material. We are also indebted to Mr J. Rodney of the Royal Veterinary College for his care of the animals. RItSUMIt ETUDES SUR L'IMMUNITt HtTtROLOGUE DANS LA SCHISTOSOMIASE: 4. IMMUNITt HITtROLOGUE CHEZ LES BOVINS On a montr6 pr6c6demment qu'il est possible de conf6rer i la souris et au singe unetprotection partielle contre une infection d'epreuve par Schistosoma mansoni en les exposant au pr6alable a une infection par les schistosomes bovins, S. bovis et S. mattheei. La pr6sente 6tude rapporte les resultats d'une exp6rience inverse destin6e a v6rifier si le betail acquiert un certain degre d'immunit6 envers S. mattheei apres avoir 6t6 en contact avec S. mansoni. Chez le veau soumis a une infection immunisante unique par 10 000 cercaires de S. mansoni puis expose A une infection d'epreuve par 6000 cercaires de S. mattheei, on constate une nette r6duction de la charge en parasites bovins et du nombre de leurs ceufs contenus dans les tissus par rapport aux animaux temoins. Certaines cer- caires de S. mansoni poursuivent leur evolution, parvien- nent A l'etat adulte et produisent des aeufs viables qui sont elimin6s dans les selles. Cette observation devrait inciter 748 STUDIES ON HETEROLOGOUS IMMUNITY IN SCHISTOSOMIASIS. 4 749 a se pr6occuper davantage du r6le possible du betail dans la transmission de la schistosomiase humaine A S. mansoni. L'immunite heterologue a pour r6sultat d'attenuer dans une tres large mesure la gravit6 de l'infection par les schistosomes bovins. II n'est pas impossible qu'un pheno- mene du meme ordre intervienne dans les conditions naturelles, ce qui expliquerait que la morbidit6 par schisto- somiase bovine soit en general moins eleve-e dans les regions oiu la schistosomiase intestinale est particulire- ment fr6quente chez l'homme. Si ces premiers resultats sont confirm6s, on pourrait envisager des essais pratiques d'un vaccin h6t6rologue destin6 a proteger le betail contre les infections graves a Schistosoma que l'on rencontre en grand nombre dans certaines regions. REFERENCES Amin, M. B. A. & Nelson, G. S. (1969) Bull. Wld Hlth Org., 41, 225-232 Amin, M. B. A., Nelson, G. S. & Saoud, M. F. A. (1968) Bull. Wld Hith Org., 38, 19-27 Fujinami, A. (1916) Kyoto Igatu Zasshi, 22, 176-185 (Abstracted in China Med. J., 1917, 31, 81) Hsu, H. F., Hsu, S. Y. Li., Chu, K. Y., Tsai, C. T. & Eveland, L. K. (1966) Z. Tropenmed. Parasit., 17, 264-278 Hussein, M. F. (1969) The pathology of spontaneous and experimental bovine schistosomiasis. A study ofthe spon- taneous disease and of experimental infestations of calves and mice with Schistosorma bovis and Schistosoma mattheei. Ph.D. thesis, University of London Kagan, 1. G. & Lee, G. L. (1952) J. infect. Dis., 91, 147-158 Le Roux, P. L. (1929) Rep. vet. Res. S. Afri, 15, 347-407 McClelland, W. F. J. (1961) Laboratory investigations. In: East African Institute for Medical Research Annual Report, 1960-61, Nairobi, Government Printer, p. 12 Meleney, H. E. & Moore, D. V. (1954) Exp. Parasit., 3, 128-139 Nelson, G. S., Amin, M. B. A., Saoud, M. F. A. & Teesdale, C. (1968) Bull. Wid Hith Org., 38. 9-17 Nelson, G. S. & Saoud, M. F. A. (1966) Trans. rov. Soc. trop. Med. Hyg., 60, 429-430 Purnell, R. E. (1966) Trans. roy. Soc. trop. Med. Hvg., 60, 463465 Saeed, A. A., Hussein, M. F. & Nelson, G. S. (1969) Trans. roy. Soc. trop. Med. Hyg., 63, 456458 Standen, 0. D. (1949) Ann. trop. Med. Parasit.. 43, 13-22 Taylor, M. G., Amin, M. B. A. & Nelson, G. S. (1969) J. 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