Chapter 4: Typing of C. fetus subsp. fetus sheep abortion isolates using PFGE: a New Zealand study
Pulsed-field gel electrophoresis typing of C. fetus subsp. fetus isolates from sheep abortions in the Hawke's Bay region in 1 999 showed that there was a variety of types, with the predominance of a single type, R I (Chapter 3). This investigation of the strain variation of C. fetus subsp. fetus implicated in sheep abortion was expanded in 2000 to
include isolates from throughout New Zealand.
In addition, seven freeze-dried C. fetus subsp. fetus isolates from the New Zealand Reference Culture Collection representing restriction types a-g found amongst New Zealand sheep abortion isolates in a 1 987 study (de Lisle et al. 1 987), were typed using PFGE. This chapter describes the results of PFGE typing of C. fetus subsp. fetus isolates from sheep abortions throughout New Zealand in 2000, and the comparison of these results with those of the earlier studies.
4.2 Materials and Methods
4.2.1 Collection of Campylobacter isolates from sheep abortions in
2000
Campylobacter isolates cultured from sheep abortions by the principal veterinary diagnostic laboratories in New Zealand during the 2000 season were collected. The laboratories were:
• LabNet Invermay Ltd., Mosgiel • Lab Works Animal Health, Lincoln
• Gribbles Veterinary Pathology Animal Health Laboratory, Palmerston North • Alpha-Scientific Ltd., Hamilton
The approximate geographical location of the farms from which isolates were cultured was supplied by the laboratories. Using this information, the farms were categorised as belonging to the following districts of New Zealand: Waikato, Gisbome, Hawke's Bay, Manawatu, Wairarapa, Marlborough, Canterbury, Otago and Southland (Figure 4. 1 ) .
Campylobacter was cultured according to the methods of the individual laboratories. In general, foetal stomach contents were examined microscopically for the presence of Campylobacter-like organisms, and Campylobacter spp. were cultured on selective media (Skirrows media from Fort Dodge Animal Health or Fort Richard Laboratories Ltd.) under microaerobic conditions (CampyGen sachets, Oxoid Ltd. or BBL ® CampyPak Plus, Becton Dickinson Microbiology Systems, Cockeysville, MD, USA). Isolates were identified to species level by the laboratories using standard microbiological methods.
Subcuituring of the isolates was kept to a minimum and a single colony on selective media was sub-cultured onto a Blood Agar plate (Fort Dodge Animal Health or Fort Richard Laboratories Ltd.) for purity. In the case of Gribbles Veterinary Pathology Animal Health Laboratory (Palmerston North) , the selective media and Blood Agar plates were brought to Massey University from the laboratory. A maximum of two sub cultures from the original growth was performed on Blood Agar before growth from a pure culture was harvested and stored in 1 5% glycerol at -70°C. In the case of the other
Chapter 4: Typing ofe. fetus subsp. fetus sheep abortion isolates using PFGE: a New Zealand study diagnostic laboratories, growth from the purity plate was harvested onto Amies medium (Cultureswab™ Transport Systems, Difco or Copan Diagnostics Inc., Corona, CA, USA) and couriered to Massey University, where fresh Blood Agar plates were inoculated from the swabs, and the growth harvested and stored in 1 5% glycerol at - 70°C.
Southland
Manawatu
Hawke's Bay
West Coast
Figure 4. 1 Map of New Zealand showing the districts the farms of the 2000 national study were categorised as belonging to. No
4.2.2
Campylobacter fetus sUbsp.fetus sheep abortion isolates from the
New Zealand Reference Culture Collection
Several C. fetus subsp. fetus isolates were acquired from the New Zealand Reference Culture Collection, Medical Section (NZRM) . The C. fetus subsp. fetus Type strain (NZRM 2398; National Collection of Type Cultures (NCTC) , Central Public Health Laboratory, Colindale, London, England 1 0842) was originally isolated from the brain of an aborted sheep foetus in France in 1 952 (Veron and Chatelain 1973) .
In addition, seven strains (NZRM 2939-2945) were lodged with the culture collection as representatives of the restriction types a-g, after a New Zealand study in which 70 C. fetus subsp. fetus sheep abortion isolates from 67 farms were examined using restriction
endonuclease analysis (REA) (de Lisle et al. 1 987) .
• Campy!obacter fetus sUbsp.fetus Type strain • REA type a • REA type b • REA type c • REA type d • REA type e • REA type f • REA type g NZRM 2398 NZRM 2939 NZRM 2940 NZRM 2941 NZRM 2942 NZRM 2943 NZRM 2944 NZRM 2945
The above freeze-dried C. fetus subsp. fetus isolates from the NZRM were revived according to accompanying instructions and growth on Blood Agar was harvested and stored in 15% glycerol at -70°C.
4.2.3 Pulsed-field gel electrophoresis of C. fetus subsp.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 2.4.2.4 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.
Chapter 4: Typing of C. fetus subsp. fetus sheep abortion isolates using PFGE: a New Zealand study
4.3
Results
·
4.3.1
Campyiobacter sheep abortion isolates
In total, 320 Campylobacter isolates were cultured from aborted sheep foetuses from
221 farms. The majority of these were C. fetus subsp. fetus: 289 isolates from 198
farms. Twenty-three C. jejuni isolates were cultured from 20 farms and one C. coli
isolate was cultured from one farm. From a further two farms, both C. fetus subsp. fetus
and C. jejuni isolates were cultured from separate foetuses, which added a further four
C. fetus subsp. fetus and three C. jejuni isolates to the collection. This resulted in a total
of 293 C. fetus subsp. fetus isolates from 200 farms, 26 C. jejuni isolates from 22 farms
and one C. coli isolate from one farm for analysis.
The results relating to the C. jejuni and C. coli isolates are presented in Chapter 5:
Pulsed-field gel electrophoresis of Campylobacter jejuni sheep abortion isolates.