Eight lactating Holstein cows (cases 1 – 7 and 9) and one Holstein heifer (case no. 8) (age range 1 1/2 – 6 years, average: 4 years) of which three were pregnant were included in the study. The animals originated from four herds tested free for bovine virus diarrhoea virus and assumed to be free of several pathogens including bovine herpesvirus type 1, bovine leucosis virus, Brucella abortus, and Mycobacterium bovis due to the national disease status. The herds had naturally acquired infection with S. Dublin and participated in a S. Dublinintervention project. The animals were selected for the study based on antibody levels to S. Dublin LPS of at least 80 ODC % (background-corrected ratio of the optical density to a positive reference) during a period of at least 180 days measured by ELISA in three individual milk or blood samples collected every three months (Figure 1). While other cows in the herds had decreasing antibody levels these nine animals remained high in antibody levels for at least 180 days.
The animals were transported two at a time by truck for 4–6 hours from four herds of origin and were housed separately in isolation facilities at the research institution. The animals were separated by a wooden wall during transportation. The animals were examined clinically before they left the herd of origin and blood, milk and faeces were sampled (day 0). Additional blood, milk and faecal samples for serology, bacteriology or haematology were collected upon arrival at the isolation facility. Clinical examinations and sampling for bacteriological culture and serology were performed daily. The animals were treated with dexamethasone sodium phosphate (DSP) at a dose of 0.08 mg/kg intramuscularly (Dexadreson® Vet, Intervet International, Boxmeer, The Netherlands) on day 2, 3 and 4. The animals were euthanized by intravenous injection of a sodium pentobarbital solution seven to 14 days (average 7.8 days) after arrival and necropsied. Samples for bacteriology and histopathology were taken at necropsy. Case no. 9 was only included in a part of the study as it was euthanized on day one due to accidental injuries.
Housing and Management
The animals were housed separately in isolation facilities. Only two animals were allowed in the barn in the same period and they were housed alone in each of two fully closed isolation rooms. All daily routines were structured in a way to minimize the risk of cross contamination between two animals in adjacent isolation rooms. The animals were fed 4–6 kg concentrated feed twice daily and had free access to grass silage and water. Milking was done twice daily in lactating animals. The study was performed during a three-month-period with ambient outdoors temperature of 7 to 20°C.
Full clinical examination was performed daily, while rectal temperature, respiratory and pulse rates were recorded twice daily.
Milk samples (50 ml) of lactating animals were collected aseptically to avoid faecal contamination from all functional quarters twice a day and pooled, while faeces (50 g) was sampled rectally four times daily. The samples were stored at 4°C until analysis, which was started within 24 hours, except for samples taken on Fridays and Saturdays, which were stored for around 72 and 48 hours before analysis, respectively.
Tissue samples were taken at necropsy. Whenever possible, 25 g of tissue was sampled. The tissues included tonsils, lung, tracheobronchial lymph nodes, spleen, liver, liver lymph nodes, gall bladder, ileum, colon, colon lymph node, gut associated lymphoid tissue of colon (colon tonsil), cecum, cecal lymph node, uterus including placentomes if present, mammary gland and mammary lymph nodes. Lung, liver, gall bladder and abomasal content of an aborted foetus were also examined. Instruments were disinfected in 96% ethanol between each tissue sample.
The samples were cultured by conventional culturing as previously reported . In short, Buffered Peptone Water (BPW, Merck 1.07728) were added to 25 g sample to a dilution of 1:10 and incubated over night at 37°C. From the pre-enriched buffer, inoculation was performed on two enrichment medias: a) 100 ml onto Modified Semi-solid Rappaport Vassiliadis (MSRV, Oxoid CM910); b) 1 ml in 9 ml selenite cysteine broth (SC, Merck 1.07709). Both mediawere incubated at 41.5°C for 24 hours. Culture negative plates were incubated for further 24 hrs. After enrichment, 10 ml from the broths and material from swarming zones of probably positive MSRV-plant were inoculated in parallel onto Brilliant Green Agar (BGA, OxoidCM 329) and Xylose Lysine Deoxycholate agar (XLD, Oxoid CM469). These plates were incubated at 37°C for 24 hrs. Isolated strains were verified by serotyping according to the Kauffman-White scheme.
Daily blood samples were collected from the jugular or caudal vein in unstabilised vacutainer tubes. The samples were centrifuged at 2,000 g at 4°C for 10 minutes after coagulation, which occurred during five to 24 hours of storage at 4°C. Serum was aspirated and stored in cryotubes at -18°C until analysis.
The level of antibodies to S
. Dublin LPS was analysed at Steins Laboratory, Holstebro, Denmark as previously described [22
]. In short, analyses were performed by an indirect O-antigen based LPS serum- or milk-ELISA. Results were measured in optical density (OD) by an ELISA plate reader. The observed OD was corrected for background OD using negative test sera and expressed as ODC%:
ODC% = (ODsample - ODneg ref)/(ODpos ref - ODneg ref) × 100%
where ODsample = mean OD of the two test sample wells, ODneg ref = mean OD of the four negative reference sample wells, and ODpos ref = mean OD of the four positive reference test sample wells.
Daily blood samples were collected as for serology in 10 ml EDTA-stabilised vacutainers for haematological profiles. Analysis was performed within the same day except for samples taking during weekends, which were stored at 4°C until examination.
The samples were analysed by automatic flow cytometry using an automated analyser (ADVIA120, Bayer) with species-specific software. Automatic differential cell counts of leucocytes were carried out and histograms were visually inspected as were blood smears to evaluate if automatic counting was correct. If doubt arose, manual differential cell counts were performed on 100 leukocytes.
The animals were necropsied and evaluated for gross lesions. Lesions were sampled, fixed in 10% neutral buffered formalin, and processed for microscopy by routine histological methods. Tissue sections were stained by haematoxylin and eosin.