Research Results - (PDF, 101 mb) - USAID

vitro propagation of its causative retrovirus in
order to develop serological tests to detect
subclinically infected sheep and eventually a
safe, reliable vaccine.
Project Progress
Initial attempts to obtain homologous
OPC capsid (major core gag polypeptide) were
based on affinity chromatography using a
Sepharose CL 4B column with anti-SMRV
serum. This column was shown to bind and
concentrate culture supernatant SMRV, but
when OPC lung homogenate was passed
through the column, the eluted proteins were
found to be sheep serum protein rather than
OPC capsid protein. This was found to be
related to antibody to bovine albumin in the
anti-SMRV serum crossreacting with sheep
serum proteins in the sample. Therefore, this
approach was temporarily abandoned.
In attempts to rescue the OPC virus, we
previously cocultured infectious material with a
variety of cell lines and attempted transfection
of Cf2th cells with high molecular weight DNA
from OPC-affected lungs; this was not successful.
Additionally, using virus pelleted from
lung fluid, we generated a cDNA library in the
expression vector lambda gtl1 in order to
identify and isolate OPC molecular clones.
Preliminary screening of the library showed that
about 30% of the plaques contained insert.
However, the positive reaction of most of these
p!aques with antiserum to MPMV p27 was
found to be due to antibody of E.coli present in
the serum. We will therefore attempt to absorb
out these non-specific antibodies and rescreen
the library.
Finally, we decided to attempt to amplify
OPC DNA sequences using the polymerase
chain reaction (PCR). For screening the PCR
producs, we obtained a plasmid containing the
full length MPMV genome and produced four
subclones in riboprobe vectors (p72, p73). These
were mapped and found tc be useful in detection
of LTR, gag, and pol-env regions of MPMV.
Primers were designed for OPC DNA in the gag,
po/, and 3env taking advantage of short consensus
sequences among several type D or type B
74
virus. These were found to amplify MPMV and
SMRV sequences in the homologous regions,
but the pol primers also amplified ovine
lentivirus DNA. Attempts to amplify OPC
DNA using the gag and env primers yielded
multiple bands, some of which hybridized in
Southern blots with the MPMV probes. However,
the gagbands were found by sequencing
analysis to represent MPMV plasmid insert
contamination, a common problem using the
extremely sensitive PCR technique. The env
bands did not have open reading frames and
probably represented non-specifically amplified
sheep DNA rather than viral DNA. We have
now dealt with these problems and are preparing
to attempt this approach again.
Project Achievements
We have obtained evidence of the relationship
between the OPC retrovirus and other
type D retroviruses. This has permitted generation
of probes and reagents that can be used to
isolate and identify the cause of OPC. We are
thus well-poised to successfully isolate and
propagate the OPC retrovirus or its relevant
genes for use in preparation of diagnostic or
immunoprophylactic products. This work will
continue during the present year.
Changes in 1990 Workplans
There were no significant changes in the
workplans for this subproject.
SUBPROJECT 2. Enterotoxemia and
colibacillosis in alpacas and lambs.
Objectives
1. Evaluation of protection of neonatal alpaca
1. luain f protofena alaca
and lambs against enterotoxemia in by vaccination
of their dams with C. perfringens enterotoxin.
2. Identification of fimbriae (colonization
antigens) on E. coli isolated from cases of neonatal
alpaca diarrhea.