5.1 Tipo de estudio
6.1.7 Caracterización socioeconómica
Initially there are two technical points which require comment.
Firstly , some Wes tern blots of T. hydatigena onco sphere antigen showed predominant bands of antigen of approximately Itl, 20, 22, 26, 39, 50 kDa, which were recognized by serum samples from control animals. ELISA results of these pooled samples showed very low absorbances (Figure 2. 10) and, as mentioned in Section 2.2. 1 . 1 , only
1
animal out of all the sheep used in the experiments described in this thesis was found to harbour a T.hydatigena cyst from a natural infection. This indicates that these bands of antigen were recognized by cross-reacting antibodies in the control sera.S econdly, the cysts which resulted from the challenge infection of Experiment
1
numbered between 20 and 40. These resulted from an oral dose of T.hydatigena eggs estimated to con tain approx imately 50 ac tivatable onco spheres. I n Experiments 2 and 3 , the challenge dose was increased to contain an estimated 100 activatable oncospheres with the intention of increasing the numbers of larvae developing and enabling any subtle differences in the immunity induced in the recipients to be detected more readily. However, 28 days after the challenge, the control sheep in these experiments harboured approximately 200 metacestodes indicating that far more infective eggs, than indicated by the in vitro assessment, were present. It is clear from this that in vitro activation does not provide an accurate assessment of the number of metacestodes which can become established in vivo.The results presented in this chapter clearly establish that a considerable level of resistance can be transferred in the sera from hyperinfected or immunized sheep. It is interesting to note that, in Experiment
1,
the pooled sera from the donors, which had received a single oral infection of eggs estimated to contain only 50 activatable oncospheres, and which were completely resistant to a challenge infection, did not protect the recipients in the volumes administered (Figure 2. 12a). The most obvious explanation for this is that the level of antibody present was insufficient and this isFigure 2.12a 60 50 11 '" 40
0-
'0 30I
Z 20 1 0The numbers of cysts found in the recipients of saline, immune or non-immune serum in Experiment 1.
Recipients of: Saline � Control Serum
f0'l2
Immune Serum o Figure 2.12.h 300 200 1 00Live Dead Total
Cysts in the Peritoneal Cavity
The numbers of cysts found in the recipients of saline, immune or non-immune serum in Experiments 2 and 3.
Live Dead Total
Cysts in the Peritoneal Cavity
Recipients of: � Control Serum
f0'l2 =��eExPt
2 Immune . Serum,Expt 3
borne out by the Western blot and ELISA results which indicated substantially higher anti-T.hydatigena antibody levels in the sera of Experiments 2 and 3, which induced 70-80% protection in its recipients (Figure 2. 1 2b). Indeed, as judged by Western blots, the antibody level in the pooled serum u sed for the transfer in Experiment 1 was lower than in pooled sera collected from the serum donors only 24 hr after the last collection for transfer purposes. Sera for transfer were collected on 4 occasions and it is possible that earlier collections contained lower levels of antibody and so effectively diluted sera collected later.
The levels of protection achieved in Experiments 2 and 3 were substantial but total protection was not achieved. This leads one to question whether or not anti
T.hydatigena antibody alone, given in suf ficient quantities, would be capable of inducing 1 00% protection in recipients. One approach to establishing this, and to determine whether or not there is a quantifiable relationship between antibody titers and the level of protection, would be to carry out further experiments of the type described here but involving l arger numbers of animals and graded doses of antibody. This was not feasible in this study. Another approach to the question is through examination of the effects of passively transferred antibody in colostrum. This is described in Chapter 4.
Another matter of interest concerns the point at which the larvae are killed in an immune animal. Does it occur before grossly visible liver damage has been done, after this, or both? Clearly, if it were possible to distinguish reliably between liver lesions of different ages and to show a relationship between their nature and the numbers of live or dead larvae present in the peritoneal cavity, examination of the livers would shed some light on this. However, this did not prove practicable. The livers of the recipients were carefully examined but it was not possible to determine macroscopically which of the lesions in them were due to larvae which had emerged into the peritoneal cavity and which were caused by larvae that died before doing so.
Furthermore, the degree of liver damage was similar in the livers of all 3 recipient
groups of Experiment 1 and in the 4 recipient groups of Experiments 2 and 3 despite
two of these groups being significantly protected. Not only was it impossible to distinguish between the lesions caused by escaped or dead larvae, even following a single infection, but it was also impossible to distinguish with any certainty between the lesions re sulting from an initial immunizing infection and a subsequent challenge infection 28 days before necropsy. The livers of the infected donors which were challenged, therefore, also gave no indication of the point at which host resistance was effective. However, the livers of the FrS-immunized donors had not been subjected to an oral infection until the challenge infection was given. Not only
were no cysts found in the peritoneal cavity following this challenge but the livers of these animals showed only a few, barely visible, scars indicating that the larvae from the challenge infection had succumbed before they were able to cause gross liver damage (Figure 2. 1 3).
Whether or n ot thi s is the c ase in partially protected animals remains to be established. It could be i nvestigated by attempting to induce varying levels of immunity by using different antigen preparations and immunization regimes. Thus the livers would be free from any initial larval migration so that the liver damage due to the challenge infection alone could be assessed.
Figure 2.13
Control
The livers of the donors of the immune serum 28 days after challenge.
CHAPTER 3
T H E I N F L U E N C E O F I M M U N E S E R U M , C O M P L E M E N T A N D LEUKOCYTES ON ONCOSPHERE SURVIVAL IN VITRO.
3. 1 INTRODUCfION.
The importance .of factors in the serum of immune animals in protecting sheep against infection with T.hydatigena was described in the preceding chapter. It is clear that if enough antibody is present, highly significant protection against infection with T.hydatigena occurs in vivo. This chapter investigates the effect of immune serum and complement on the surv_val of T.hydatigena oncospheres in vitro and also examines the involvement of leukocytes from both immune and non immune sheep in the killing of oncospheres in vitro. The question as to whether the failure to obtain complete protection in the experiments described in the previous chapter could be attributable to a need for leukocytes from actively immunized animals, will be discussed.