3 1 Introduction
3.2 Materials and Methods
3.2.1 Isolation of Campylobacter spp. from sheep abortions
Aborted foetuses (1-5 per flock) were submitted for Campylobacter isolation from 50 commercial sheep flocks in the Hawke' s Bay region in which Campylobacter was clinically suspected to be the cause of the abortion. Information was also sought on whether the affected sheep had been vaccinated with the C. fetus subsp. fetus vaccine, Campylovexin@.
Campylobacter isolates were cultured from the foetal stomach contents by either Gribbles Veterinary Pathology Animal Health Laboratory, Palmerston North (then known as AgriQuality Farm Network Animal Health Laboratory) or Massey University Diagnostic Microbiology Laboratory, Palmerston North, according to the method detailed in Chapter 2. Briefly, foetal stomach contents were plated directly onto selective media and incubated for 48 hours at 37°C in a microaerobic atmosphere. Campylobacter spp. identification was confirmed by Gram stain or dark field microscopy and isolates were frozen in 15% glycerol for further analysis.
3.2.2 Speciation of Campylobacter isolates
Isolates were subsequently identified to species level by standard microbiological methods detailed in Chapter 2. Isolates were identified as C. fetus subsp. fetus by production of hydrogen sulphide in 0.02% w/v semi-solid cysteine medium (lead acetate strip detection) , lack of hydrogen sulphide production in triple sugar iron medium, catalase activity, susceptibility to cephalothin, resistance to nalidixic acid, and growth at 25°C. Isolates were identified as C. jejuni by production of hydrogen sulphide in 0.02% w/v semi-solid cysteine medium (lead-acetate strip detection), lack of hydrogen sulphide production in triple sugar iron medium, catalase activity, susceptibility to nalidixic acid, resistance to cephalothin, lack of growth at 25°C, and a positive hippurate hydrolysis test (Quinn et al. 1994).
Chapter 3: Typing of C. fetus subsp. fetus from sheep abortions using PFGE: a pilot study of Hawke 's Bay isolates
3.2.3 Pulsed-field gel electrophoresis of C. fetus sU
bsp.fetus isolates
Preparation and digestion of genomic DNA and PFGE of C. fetus subsp. fetus isolates was performed according to the method detailed in Chapter 2. In addition to digestion with the restriction enzyme SmaI, digestion with the restriction enzyme Sail was performed as follows. One third of each plug was equilibrated for 45 minutes on ice with 1 .2x Roche Applied Science Buffer H. The plug slices were then equilibrated with Ix Roche Buffer H and 30 units of Roche restriction enzyme Sail. Digestion reactions were incubated overnight at 37°C.3.2.4 Analysis of PFGE profiles
The PFGE profiles of the isolates were analysed using BioRad Diversity Database software according to the method detailed in Chapter 2. The PFGE types were named according to the method detailed in Chapter 2.
3.2.5 Campylobacter fetus subsp.f
etu
svaccine strain
The C. fetus subsp. fetus strain used to produce the Campylovexin® vaccine (strain 5915) was typed using PFGE in the same way as the abortion isolates from this study.
3.3 Results
3.3.1
Campy/obacter
isolationsCampylobacter isolates were cultured from abortion samples from 28 out of the 50 Hawke' s Bay farms. Three or more foetuses were submitted from 17 out of these 28 farms (60%). In total, 85 Campylobacter isolates were cultured, each isolate being cultured from a different aborted foetus. Of these, 81 were C. fetus subsp. fetus from 2 5 farms and four isolates were C. jejuni from the other three farms.
The results relating to the four C. jejuni isolates from three affected properties are presented in Chapter 5 : Pulsed-field gel electrophoresis of Campylobacter jejuni sheep abortion isolates.
3.3.2 Pulsed-field gel electrophoresis analysis of C. fetus subsp.
fetus
abortion isolatesTen distinct PFGE profiles were identified amongst the 81 C. fetus subsp.fetus isolates (Figure 3 . 1). The PFGE profiles consisted of 1 3-16 DNA bands, which ranged in size between 3 1 .2 and 4 1 7.7 kb. Seven of these bands were invariable between all the PFGE profiles and the profiles were all at least 60% similar when a similarity matrix was calculated using the Dice coefficient (Appendix 1). A dendrogram showing similarity of the PFGE profiles of the isolates was produced using cluster analysis (Figure 3 .2). The PFGE profiles :::::84% similar by cluster analysis were considered to belong to the same PFGE group, as these profiles differed by the position of only one or two bands. Conversely, the PFGE profiles less than 84% similar by cluster analysis were considered to belong to different PFGE groups. The profiles fonned six PFGE groups, which were named A, B, C, D, E, and F.
Distinct PFGE profiles within the same PFGE group were further organised into types. Four of the PFGE groups (B, D, E, and F) each contained two PFGE types, which were named B 1 and B2, D1 and D2, El and E2, and F1 and F2, respectively (Figure 3 . 1). There were at least 12 invariable bands between the two PFGE types within each group. The similarity between the PFGE profiles of B l and B2, D 1 and D2, and E l and E2 was each 93%, and that of F 1 and F2 was 89% (Figure 3.2). Isolates of the same PFGE type
Chapter 3: Typing ofC. fetus subsp. fetusfrom sheep abortions using PFGE: a pilot study of Hawke 's Bay isolates
had indistinguishable PFGE profiles, for example the PFGE profiles of all the isolates
of PFGE type B 1 were indist inguishable.
Pulsed-field gel electrophoresis using the restriction enzyme San confirmed the strain
typing of the isolates as above, except that PFGE types 8 1 and 82 were not
differenti ated by San. No additional differentiation of types was generated by the use of
San. 436.5 339.5 291 .0 242.5 1 94.0 1 45.5 97.0 48.5 23.1 M A1 A2 B1 B2 C1 D1 D2 E1 E2 F1 F2 M
Figure 3. 1 P ulsed-field gel electrophoresis (PFGE) using the restriction enzyme SmaI of the ten PFGE types isolated from sheep abortions i n Hawke's Bay i n 1 999, and the Campylovexin vaccine strai n (AI). Lanes of C. fetus subsp.fetus are labelled with
the assigned name of the PFGE type. M = molecular size marker
0.67 0.67 0.71 0.75 0.74 0.80 0.82 0.85 0.83 0.90 0.89 0.89 0.95 0.93 0.93 0.93 1 .00 C1 82 81 A1 A2 F2 F1 E2 E1 D2 D1
Figure 3.2 Similarity of the PFGE proiIles of the C. fetus subsp. fetus isolates from the Hawke's Bay in 1999 and the Campylovexin@ strain (PFGE type Al). This dendrogram was produced using the unweighted pair group method using arithmetic averages (UPGMA) cluster analysis, with PFGE type Bl as the reference. Clusters of PFGE types that were �84% similar were classified as belonging to the same PFGE group.
3.3.3
Campylobacter fetus subsp
.f
etu
svaccine strain
The C. fetus subsp. fetus Campylovexin@ vaccine strain, although not isolated from aborted animals in this study, was most similar to PFGE type A2 at 89% and was named PFGE type Al (Figures 3.1 and 3.2).
Chapter 3: Typing of C. fetus subsp. fetus from sheep abortions using PFGE: a pilot study of Hawke 's Bay isolates
3.3.4 Incidence of PFGE types
The majority of the C. fetus sUbsp.fetus isolates (62 out of 81 , or 77%) were PFGE type B1 (Table 3. 1 , Appendix 2). Isolates of this PFGE type were cultured from aborted foetuses from 1 9 of the 25 farms from which C. fetus subsp. fetus was isolated. In contrast, types A2, Cl, D2, El , E2, F1 and F2 were each recovered from only one farm. The other types, B2 and D 1 , were each cultured from foetuses from two farms.
Table 3.1 Number of C. fetus subsp.fetus isolates of each PFGE type and number of farms from which each of the PFGE types was isolated.