Studies of Pasteurella haemolytica : (1) comparison of serotyping techniques, (2) prevalence of serotypes in New Zealand sheep : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
P.haemolytica, which is the aetiological agent of
pasteurellosis, is the major cause of mortalities in sheep in
Great Britain and is a problem in several other countries
including New Zealand. Furthermore, P.haemolytica, acting as a
secondary invader, exacerbates lesions of chronic
non-progressive pneumonia (CNP) initiated by Mycoplasma
ovipneumoniae and this disease causes considerable economic loss
to the New Zealand sheep farming industry.
P.haemolytica exists as 15 serotypes and immunity is
serotype specific. P.haemolytica vaccines are marketed overseas
and their use in New Zealand is under active consideration. It
is logical however, to establish which serotypes are present in
New Zealand before a vaccine is produced, but there is at
present, no information on this point. This is largely because
of technical difficulties in typing isolates. This situation
stimulated the present investigation which has 2 major aims: To
develop an improved method of typing P.haemolytica and to gather
some data on the prevalence of the various serotypes in Ne>..J
Hith respect to the first aim, several approaches \..Jere
taken to the problem of differentiating between serotypes.
(1) Indirect heamagglutination (IHA), the standard method by
which P.haemolytica is serotyped, was found to be laborious and
gave many cross-reactions.
(2) SDS-PAGE of total protein showed similar patterns for all
serotypes within a biotype, whereas the 2 biotypes had different
patterns. Thus, SDS-PAGE cannot be used to identify the
serotype, but could be useful for identifying the species and
(3) Latex beads, coated with antibody pre~ared against whole
cells, agglutinated homologous cells, but also gave many
cross-reactions. This test should, in principle, become
type-specific if purified capsule were used as the immunising
antigen, but further work is required to prepare capsular
polysaccharide free from endotoxin which stimulates the
production of cross-reacting antibody.
(4) Gel precipitation was simple to perform and was serotype
specific. However, serotype A2 required a concentrated antigen
preparation for the detection of a precipitation line. The
concentrated antigen could not be routinely used with the other
types because of cross-reactions between antigens.
(5) Counter immunoelectrophoresis (CIE) was not
specific due to endotoxin causing cross-reactions.
(6) Bacterial agglutination was a rapid and simple test to
perform and showed some limited cross-reactions.
Since IHA, gel precipitation and agglutination showed some
potential, they were then compared using 40 isolates from the
lungs of 60 sheep with CNP These findings reinforced the
conclusions drawn when prototype strains were used.
We initially proposed that the serotype of isolates should
be primarily determined by agglutination tests, but since this
is not 100% specific, the results must be confirmed by gel
precipitation. This approach was investigated using 110
isolates from the nasopharynx of sheep and it was satisfactory
for all serotypes except A2. Strains within the A2 serotype
showed some heterogeneity at least when examined by gel
precipitation and a hypothesis is presented to explain this.
We conclude that at present, the best method of serotyping
isolates is by gel precipitation, but that all isolates which
are not positively serotyped by this approach should be retested
Information concerning the prevalence of serotypes was
obtained during the above studies. 110 isolates were obtained
from the nasopharynx of 50 sheep from each of 4 farms. All nAn
serotypes, except A12 and A1 u, were isolated.
up 35% of these isolates. 60 lungs with
Serotype A2 made
CNP lesions were
obtained from 20 farms in the Manawatu region and 40 isolates of
P.haemolytica were obtained. All were serotyped and they were:
A1 (25%), A2 (55%), A6 (7.5%), A7 (7.5%), A8 (2.5%), and A9
(2.5%). No "Ttt types were found in either the nasopharynx or
the lungs. The implications of these findings for vaccine
production are discussed.