Oral and Poster Abstracts

we measured sensitivity of the entire assay, including the DNA
extraction procedure. For this experiment, ten mastitis milk samples
shown to be negative for the tested targets were randomly chosen, spiked
with known quantities of the target bacterial colony forming units (CFU)
and analyzed using the PCR assay. When sensitivity was measured based
on the dilution series of purified DNA, the analytical detection limit of
the test was as low as 5.6 bacterial genomes per l. When using mastitis
milk samples spiked with the bacterial targets, the detection limit was as
low as 100 CFUs per one millilitre of milk. Both experiments showed
variation in the sensitivity of the test between the different bacterial
targets. In conclusion, the results of this study demonstrate that the
PathoProof Mastitis PCR Assay can detect low quantities of bacterial
DNA. This presentation also discusses some of the challenges involved
in sensitivity validation of a PCR test in a controlled manner when using
raw bovine milk as a sample matrix. Sensitivity of the PathoProof
Mastitis PCR Assay should next be validated against the golden standard
method, i.e. bacterial culture.
43 Investigating the Use of Viscosity Measurements for the
Quantification of the California Mastitis Test (CMT) in Dairy
Cattle
J. Roberts
University of Cambridge, Department for Veterinary Medicine,
Cambridge, United Kingdom
The California Mastitis Test (CMT) is widely used as a qualitative
cow side test for the detection of subclinical mastitis in dairy cattle.
The viscosity of the milk-CMT gel reaction has been characterised
but at present no attempt made to determine whether the reaction can
assess quantitatively the somatic cell count (SCC) in milk samples.
This initial investigation looks at the milk and milk-CMT gel reaction
viscosity for various SCC quarters and characterises the sample
viscosity for these SCCs. 57 quarter samples were collected
aseptically from 16 Holstein-Friesian cows in a commercial dairy
herd within the UK. Viscosity measurements were made using a
Brookfield R/S Rheometer at constant shear rate for milk alone and
milk with CMT reagent. Each quarter SCC was measured at a
commercial approved laboratory. The correlations between average
viscosity, initial viscosity, final viscosity, and change in viscosity for
both milk and milk-plus-CMT reagent against SCC are demonstrated.
The removal of the milk viscosity from the milk-plus-CMT reagent
viscosity may allow quantification of the SCC between animals. The
milk-CMT gel reaction used qualitatively to demonstrate subclinical
infection may be able to be measured quantitatively if the process of
analysing viscosity is carefully controlled as shown by this initial
analysis. Further work will investigate the CMT reaction at various
constant shear rates and using controlled shear rates. It is anticipated
that quantification of the viscosity of the CMT test may allow the
development of a portable device for more accurate determination of
SCC cow side and be developed for an in-line device in automated
milking systems.
44 Prevalence of Intramammary Infections in Heifers around
Calving in 40 Dairy Herds in the West of France.
L. Durel 1 ,C. Guidarini 2 , P. Moroni 3 , C. Locatelli 3 , L. Scaccabarozzi 3 ,
E. Schmitt-Van de Leemput 4
1
Clinique Vétérinaire de Marigny, Marigny, France
2
Boehringer Ingelheim France, Technical & Development
Department, Reims, France
3
Facolty di Medicina Veterinaria di Milano, Patologia animale,
Igiene e Sanit’Pubblica Veterinaria, Milano, Italy
4
Clinique Vétérinaire de Villaines-la-Juhel, Villaines-la-Juhel,
France
Intramammary infections (IMI) in dairy heifers around calving are
responsible for important economical losses. A better understanding
of the prevalence, the responsible germs and the risk factors of such
infections is needed to improve heifers udder health status in France.
To investigate the prevalence and the risk factors of IMI in heifers,
an epidemiological study has been carried out in two veterinary
practices in the west of France from June to December 2007. In this
abstract, the bacteriological data are reported. Sterile milk samples
of the 4 individual quarters (n=1.496) were collected of dairy heifers
(n=378) between 3 and 7 days post partum from 35 dairy herds and
frozen until analyses. For each sample, somatic cell count was
46 XXV. Jubilee World Buiatrics Congress 2008
determinate and bacteriological analyses were performed according
to the recommendations of the NMC and the IDF. 1% of the quarters
(n=16) were not functional at calving time. In total, 22.3% (n=333)
of the milk samples was positive in bacteriological analyses, 77.3%
(n=1.156) was negative; 2.1% of the samples was contaminated. In
general there was no clear relationship between elevated cell count
(> 100.000 cells/ml) and positive result in bacteriological analyses.
However, when the data were analyzed per type of bacteria, it
showed that the presence of Streptococcus spp. (Streptococcus
uberis) coincided in 77% (85%) of the cases with an elevated cell
count, whereas the presence of Staphylococcus spp in 48% (S.
aureus) and 64% (CNS). Among infected heifers (n=214), the
number of animals with 1, 2, 3 or 4 quarters infected, were
respectively, 63.0%, 22.4%, 10.3% and 4.2%. 43.4% (n=164) of the
heifers didn’t have any quarter infected. The frequency of presence
of CNS, Staphylococcus aureus, Streptococcus spp (Streptococcus
uberis) and Gram negative spp. were respectively 64.2%, 8.4%,
16.2% (9.3%) and 4%. On the herd level, the lowest prevalence of
infected animals was 20% and the highest 100%, but for most of the
farms (n=26) the infection rate varied from 45 to 75 %. In
conclusion, IMI in heifers has a high prevalence in France and most
of the herds have numerous infected animals at calving. Further
analyses of the data are needed to provide more information on the
economical impact and the risk factors of IMI infections of heifers.
45 Bovine Mastitis: Genotypes of Staphylococcus aureus Differ in
Their Clinical Outcome after Intramammary Infection
HU. Graber
Clinic for Ruminants, Vetsuisse Faculty, University of Bern, Berne,
Switzerland
Objectives of study: Based on our clinical experience on bovine
mastitis, we hypothesized that there are subtypes of Staphylococcus
aureus (S. aureus) which differ in their contagious and pathogenic
properties after intramammary infection (IMI).
Materials and Methods: 210 strains of S. aureus were isolated from
bovine milk samples collected during a representative epidemiological
study on S. aureus in cow herds of Switzerland. In each of the 26 herds
investigated, all lactating cows (n=449) were evaluated clinically for
udder health. Milk of each quarter was then aseptically collected for
bacteriological testing and analysis of somatic cell counts (SCC). The
genotypes of S. aureus were determined by PCR amplification of the
16S-23S rRNA intergenic spacer (RS-PCR).
Results: In total, 17 genotypes were obtained. Two of them (type B
and C) were predominant (81%). Each of the remaining types (OG)
occurred at a very low frequency. Considering genotype B, IMI was
always present in many cows of a herd. Frequently more than 1
quarter per cow was infected. In the case of genotypes C and OG,
IMI was restricted to 1 or very few cows per herd. Constantly, not
more than 1 quarter per cow was infected. Cow prevalence of S.
aureus was highly associated with the genotypes (p