Oral and Poster Abstracts

University of Warmia and Mazury in Olsztyn, Department of Animal
Genetics, Olsztyn, Poland
To determine the actual carrier frequency of the CVM (Compex
Vertebral Malformations) in a population of Polish Holstein-Friesian
Black-and-White cattle, a study was undertaken that examined 355
proven bulls (active within 2001 - 2007) used by 4 domestic artificial
insemination companies. A total of 69 T/G heterozygotes were
diagnosed (19,4%), being mostly sons of known carriers of the CVM
defect. Identification of a polymorphism in a bovine solute carrier
family 35 member 3 gene, named SLC35A3, was carried out with the
use of a new PCR-SSCP method (polymerase chain reaction - single
stranded conformation polymorphism) which, due to ease of use and
high reliability, can be applied in widespread screening programs.
All CVM carriers were diagnosed once more by licensed allelespecific
- PCR method. Complete accordance between the numbers
275 Phenotypic and Genotypic Identification of Methicillin
Resistance of Staphylococci Isolated from Mastitic Milk
Samples
B. Sareyyupoglu 1 , Z. Cantekin 2 , M. Akan 1
1 Faculty of Veterinary Medicine, Ankara University, Department of
Microbiology, Ankara, Turkey
1 Faculty of Veterinary Medicine, Mustafa Kemal University,
Department of Microbiology, Hatay, Turkey
In this study, phenotypic and genotypic identification of methicillin
resistance of staphylococci isolated from mastitic milk samples
collected from 3 different districts (Polatli, Çubuk and Haymana) of
Ankara were investigated. A total of 83 isolates, distribution of
which was, 22, 29, and 32 from Polatli, Çubuk and Haymana,
respectively, were used in the study. Isolates were identified with
conventional identification tests. Kirby-Bauer disc diffusion method
using oxacillin (1 µg) discs were used for the phenotypic
identification of methicillin resistance. Furthermore, susceptibilities
of isolates to amoxycillin+clavulanic acid (30 µg), amoxycillin (10
µg), ampicillin (10 µg), and penicillin (10 µg) were also investigated
in the study. For the genotypic determination of methicillin
resistance, a multiplex-PCR technique co-amplifying the specific
fragment of 16S rDNA in staphylococci, mecA gene encoding the
methicillin resistance, and femA gene discriminating S. aureus from
other staphylococci were used. Moreover, for the determination of
beta lactamase activity, nitrocefin sticks and a PCR technique
detecting blaZ gene were used. Following the tests, distribution of
oxacillin resistance were detected 31.8%, 58.6% and 0%,
respectively in isolates from Polatli, Çubuk and Haymana, while,
mecA gene was detected 31.8%, 31% and 3.1% in these districts.
BlaZ gene was detected 59.1% in Polatli isolates, 41.4% in Çubuk
isolates and 59.4% in Haymana isolates. MecA gene was detected in
17 (20.5%) out of 83 isolates, while 10 (58.8%) of these were
determined to be methicillin-resistant S. aureus (MRSA). As a
conclusion, multiplex-PCR technique was found to be a fast, specific
and reliable technique for the simultaneous detection of methicillin
resistant isolates and the discrimination of S. aureus isolates from
other staphylococci.
Key words: mastitis, methicillin resistance, multiplex-PCR,
staphylococci
276 Extended Biofilm Susceptibility Assay for Staphylococcus
aureus Bovine Mastitis Isolates: Evidence for Association
between Agr-type and Biofilm Susceptibility.
M. Melchior 1,4 , M. Van Osch 1 , T. Lam 2 , W. Gaastra 3 ,
J. Fink-Gremmels 1
1
Utrecht University, Department of Pharmacology and Toxicology,
Utrecht, Netherlands
2
GD Animal Health Service, Udder Health Service, Deventer,
Netherlands
252 XXV. Jubilee World Buiatrics Congress 2008
of CVM carriers genotyped by both methods was observed. Due to
high breeding value of selected CVM carrier bulls’ - breeders
hesitate whether to inseminate cows with them or not. In “milk
productive farms” the risk to produce potential CVM homozygotes
is very low if mating will take into account CVM status of their
ancestors, already available in breeding documents. Special attention
should be paid in “breeding herds” producing elite bulls and dams.
Mating these animals should be assisted with continuous diagnostics
to remove gaps in CVM status of pedigree and active education
carried out by inseminators and A.I. dealers. Only CVM-free young
bulls are allowed to enter progeny testing program in A.I. station.
This strategy should reduce the incidence of CVM carriers in very
short time. Closer international integration of national associations
of Holstein cattle breeders and coordination of genetic defects
discovery programs is necessary to decrease the number of carriers
and affected animals at a faster rate.
Good Veterinary Practice (Antibiotic Resistance)
3
Utrecht University, Infectious Diseases and Immunology, Utrecht,
Netherlands
4
Central Veterinary Institute, Bacteriology and TSE's, Lelystad,
Netherlands
Staphylococcus aureus is one of the most prevalent causes of bovine
mastitis. The antimicrobial treatment of this disease is currently
based on antimicrobial susceptibility tests according to CLSI
standards. However, various studies have shown that there is a
discrepancy between the results of this standard susceptibility test
and the actual cure rate of the applied antimicrobial treatment.
Increasing evidence suggests that biofilm formation by S. aureus is
associated with this problem. Previous data obtained with a limited
number of strains revealed that the extended biofilm antimicrobial
susceptibility assay allows differentiation between strains, which
cannot be derived from a standard susceptibility test or from a 24hour
biofilm susceptibility test (Melchior, et al., 2007). The
objective of this study was to test a representative collection of S.
aureus bovine mastitis strains in the extended biofilm susceptibility
assay. Based on the results from a previous study with the same
collection of strains the effects of Agr-type (Accessory Gene
Regulator gene) and the presence of IS257 (Insertional Sequence
257) in Agr-type II strains on the biofilm susceptibility were also
studied. The Agr locus of S. aureus controls the expression of most
of the virulence factors it represses the transcription of a number of
cell wall-associated proteins, and activates several exoproteins
during the post exponential phase (Bronner, et al., 2004). The IS257
gene has been related to biofilm formation in vitro (Cramton, et al.,
1999) and was found earlier in 50% of the Agr-type II strains.The
results of this study revealed differences in biofilm susceptibility
between Agr-type I, III and IV strains and Agr-type II strains which
were also dependent on the antimicrobial used. The presence of
IS257 in Agr-type II strains has a marked effect on the in vitro
biofilm density and on antimicrobial susceptibility of these strains
growing in biofilm. The decreasing antimicrobial efficacy with older
biofilms and the increasing efficacy with longer duration of
antimicrobial challenge were shown. These data explain the better
therapy results for penicillin susceptible strains in vivo obtained in
several epidemiological studies (Sol, et al., 1997, Taponen, et al.,
2003). The data presented here also offer an explanation for the
higher efficacy of early antimicrobial treatment and for treatments of
longer duration in bovine mastitis S. aureus infections described in
the literature (Sol, et al., 2000).
Key words: mastitis, antimicrobial susceptibility, bacterial biofilm,
Staphylococcus aureus, Agr- typing
277 Longitudinal Study on Antimicrobial Resistance Patterns of
Salmonella spp. and E. coli obtained from Dairy Cattle in
Colorado, USA
A. Villarroel 1 , D. Dargatz 2 , M. Salman 3 , S. Ladely 4 ,
P. Fedorka-Cray 4