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Embryos and uterine flush fluids from cattle with bovine spongiform encephalopathy are not infective for mice
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Theriogenology
EMBRYOS AND UTERINE FLUSH FLUIDS FROM CATTLE WITH BOVINE SPGNGIFORM ENCEPHALOPATHY ARE NOT INFECTIVE FOR MICE A E Wrathall’, K F D Brown’, H Fraser*, A Chreez and C A Ferguson3 Qntral Veterinary Laboratory, Addlestone, Surrey, KT15 3NB, UK ZNeuropathogenesis Unit, Edinburgh, UK; 31nstitute for Animal Health, Compton, Berkshire, UK This work was part of an ongoing project to assess the risk of transmitting bovine spongiform encephalopathy (BSE) by embryo transfer (Wrathall et al Theriogenology 1994; 41: 337). All the donor cows, and some of the bulls used for artificial insemination (AI), were clinical BSE cases. Embryos and fluids were collected by non-surgical uterine flushing seven days after AI. Further collections were at monthly intervals until the donors had to be killed. Non-transferable embryos and ova with intact zonae pellucidae and free from adherent debris were washed ten times (IETS Manual, 1990) then frozen using glycerol cryoprotectant. After thawing they were sorted into groups of similar donor/bull status, sonicated, and injected iutracerebrally into weanling mice (20 embryos in 0.02ml suspension per mouse). Embryos of status +/+ were from BSE+ve cows given semen Tom BSE+ve bulls while those of status +/- were from +ve cows given semen from -ve bulls. Status of BSE+ve cows and bulls was conlirmed post-mortem. Flush IIuid samples, with the sediment, were frozen (-200C). After thawing, samples from 40 cows (successive flush fluids from a cow were pooled if of same donor/bull status) were injected into groups of mice by the combined intracerebral(O.02ml) and intraperitoneal (0.1Oml) routes. BSE-susceptible strains of mice (Sine s7 RIB and Sine s7 C57BL) were used, with groups of approx. 25 mice (same strain) for each individual inoculm In bioassays of BSE-infected brain samples these two mouse strains have incubation periods of 316-327 and 407-438 days respectively. Gur mice were kept for up to 700 days post-injection (d.p.i.), then killed. Their brains, including those of most mice culled for inter-current illness, were examined histologically for spongiform encephalopathy (SE). See Table 1. Table 1. Results for the embryos (E) and the flush fluids from donor cows (C). Donor Mice Nil Result Interval (d.p.i) to brain exam status +ve +ve -ve result <399 400-499 500-599 >599 used +/25 0 0 22 3 0 3 14 5 +I+ 26 0 0 26 0 8 1 3 14 +t578 0 1 535 42 49 53 104 329 +I+ 422 0 0 410 12 19 23 58 310
Type and No. E=500 E=520 C=23 c=17
All but one mouse (an RIII mouse given flush fluid of status +/-) were negative for SE. The survival time (447 d.p.i.) of this non-negative mouse was inconsistent with even a hypothetically very low infective dose of BSE which this would represent ifit was a single positive case in a batch of 25 injected mice. The spongiform changes in its brain may have been due to senility. We conclude that neither the embryos nor the flush fluids contained detectable BSE infectivity in the mouse bioassay. However, since it is possible that mice are less sensitive to BSE than cattle, viable embryos from our BSE+ve donors were transferred in 1991 - 1992 into heifers imported Born New Zealand. So fhr there has been no evidence of BSE in these heifers or in their embryo transfer offspring, but the project is not scheduled to fInish until the year 2000.
Theriogenology
EMBRYOS AND UTERINE FLUSH FLUIDS FROM CATTLE WITH BOVINE SPGNGIFORM ENCEPHALOPATHY ARE NOT INFECTIVE FOR MICE A E Wrathall’, K F D Brown’, H Fraser*, A Chreez and C A Ferguson3 Qntral Veterinary Laboratory, Addlestone, Surrey, KT15 3NB, UK ZNeuropathogenesis Unit, Edinburgh, UK; 31nstitute for Animal Health, Compton, Berkshire, UK This work was part of an ongoing project to assess the risk of transmitting bovine spongiform encephalopathy (BSE) by embryo transfer (Wrathall et al Theriogenology 1994; 41: 337). All the donor cows, and some of the bulls used for artificial insemination (AI), were clinical BSE cases. Embryos and fluids were collected by non-surgical uterine flushing seven days after AI. Further collections were at monthly intervals until the donors had to be killed. Non-transferable embryos and ova with intact zonae pellucidae and free from adherent debris were washed ten times (IETS Manual, 1990) then frozen using glycerol cryoprotectant. After thawing they were sorted into groups of similar donor/bull status, sonicated, and injected iutracerebrally into weanling mice (20 embryos in 0.02ml suspension per mouse). Embryos of status +/+ were from BSE+ve cows given semen Tom BSE+ve bulls while those of status +/- were from +ve cows given semen from -ve bulls. Status of BSE+ve cows and bulls was conlirmed post-mortem. Flush IIuid samples, with the sediment, were frozen (-200C). After thawing, samples from 40 cows (successive flush fluids from a cow were pooled if of same donor/bull status) were injected into groups of mice by the combined intracerebral(O.02ml) and intraperitoneal (0.1Oml) routes. BSE-susceptible strains of mice (Sine s7 RIB and Sine s7 C57BL) were used, with groups of approx. 25 mice (same strain) for each individual inoculm In bioassays of BSE-infected brain samples these two mouse strains have incubation periods of 316-327 and 407-438 days respectively. Gur mice were kept for up to 700 days post-injection (d.p.i.), then killed. Their brains, including those of most mice culled for inter-current illness, were examined histologically for spongiform encephalopathy (SE). See Table 1. Table 1. Results for the embryos (E) and the flush fluids from donor cows (C). Donor Mice Nil Result Interval (d.p.i) to brain exam status +ve +ve -ve result <399 400-499 500-599 >599 used +/25 0 0 22 3 0 3 14 5 +I+ 26 0 0 26 0 8 1 3 14 +t578 0 1 535 42 49 53 104 329 +I+ 422 0 0 410 12 19 23 58 310
Type and No. E=500 E=520 C=23 c=17
All but one mouse (an RIII mouse given flush fluid of status +/-) were negative for SE. The survival time (447 d.p.i.) of this non-negative mouse was inconsistent with even a hypothetically very low infective dose of BSE which this would represent ifit was a single positive case in a batch of 25 injected mice. The spongiform changes in its brain may have been due to senility. We conclude that neither the embryos nor the flush fluids contained detectable BSE infectivity in the mouse bioassay. However, since it is possible that mice are less sensitive to BSE than cattle, viable embryos from our BSE+ve donors were transferred in 1991 - 1992 into heifers imported Born New Zealand. So fhr there has been no evidence of BSE in these heifers or in their embryo transfer offspring, but the project is not scheduled to fInish until the year 2000.