Hasle et al. Acta Veterinaria Scandinavica 2010, 52:55 http://www.actavetscand.com/content/52/1/55 included one inland farm approximately 30 kilometres from the sea (Farm 7) that was included because human Lyme borreliosis had been reported in this municipality, thus indicating the presence of ticks, according to the Norwegian Surveillance System for Communicable Dis- eases (MSIS) [30]. Blood samples were collected in May 2004 on farms 20 and 21, and samples were collected from all other sites in October and November 2005. The blood samples were stored at 4°C within a few hours after collection, and the serum portion of the samples was separated and frozen within 72 hours. All of the sera were tested using an indirect immuno- fluorescent antibody test (IFAT) [28] for IgG as described by Christensson [31,32], and Christensson and Moren [33] with the following modifications: Antigen was prepared in 2002 from blood of a calf infected with Babesia divergens with approx. 10% infected erythro- cytes as described by Christensson [32]. The antiserum used was was FITC conjugated rabbit anti bovine IgG, produced by ICN Cappel, code 55280, lot 03683, diluted at 1/200 to give comparable readings with control sera used by Christensson and Morén [33]. Control sera were obtained from calves used for vaccine production in the year 2001 drawn before infection and four weeks after having showed acute parsitaemia. Negative control serum showed no or uncertain reaction at a dilution of 1/20 or higher. The positive control sera had an end- point titre of 1/1280-1/2560. For each day of reading IFAT-slides a negative control at 1/40 and a positive control at 1/40, 1/160 and 1/1280 were included. As the purpose of the test was to identify seropositive/serone- gative animals sera were read at dilutions at1/40 and 1/ 160. Slides were read blindly and scored by Christensson as having uncertain (+), positive (++) or strongly posi- tive immunofluorescence (+++), at dilutions of 1:40 and 1:160. To minimise the risk of false positives, only sera with a minimum +++ score at a dilution of 1:40 were counted as positive. Farmers were interviewed to determine if there had been cases of redwater on their farms and if they had experienced redwater in cows that were imported to the farm. Data on the cases of babesiosis in the included farms were obtained from the NDHRS. To test the suitability of using PCR on full blood, we chose samples for a pilot study from four farms where redwater was common, according to the local farmers, and DNA from 100 μl from 20 samples of frozen EDTA-blood, and 25 samples of 100 μl blood clot, fro- zen after spinning and removal of the serum, were iso- lated in a spin-column, using DNeasy Blood & Tissue Kit (Qiagen), and eluated to 200 μl, according to the manufacturer’s protocol. The isolation of DNA con- tained a lysis step and washing. Five μl of the eluate was run in B. divergens real-time PCR for 40 cycles with Page 4 of 9 primers BdiF, BdiR and BdiT. The PCR was performed by Telelab (Skien, Norway), using an in-house method, as described by Lundsett [26]. The laboratory used a synthetic amplicon with the sequence of B. divergens, serially diluted in human DNA as a positive control. The reaction mix and human DNA was used as a nega- tive control. The observed cutoff for detection was 30 B. divergens DNA copies, i.e. 15 to 30 individual cells, depending of whether they are asexual, diploid cells or sexual, haploid cells. Exact confidence intervals for binomial proportions were calculated using the principles introduced by Clop- per and Pearson [34] and implemented in R (R Develop- ment Core Team, 2008). Results Of the 306 sera that we tested, 84 (27%) had positive IFAT results. A high percentage of these positive results were found in the western and eastern ranges of the study area, and a much lower rate of positive test results was found in the middle range of the study area (Table 1; Figure 2). Farm 23 had 3 positive test results among Table 1 Municipality of the test localities in Figure 1 and test results of indirect immunofluorescence antibody tests (IFAT) for Babesia divergens. Farm 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Total Municipality Farsund Farsund Lyngdal Mandal Mandal Søgne Songdalen Søgne Kristiansand Kristiansand Lillesand Lillesand Grimstad Grimstad Arendal Arendal Arendal Risør Kragerø Kragerø Kragerø Bamble Porsgrunn Larvik Neg 6 6 6 0 5 2 13 3 9 3 16 8 29 19 10 14 6 4 11 6 2 12 13 19 222 Pos1 5 9 2 9 7 7 1 1 0 0 2 0 2 0 0 1 4 13 1 12 5 0 3 0 84 N 11 15 8 9 12 9 14 4 9 3 18 8 31 19 10 15 10 17 12 18 7 12 16 19 306 % pos 45 60 25 100 58 78 7 25 0 0 11 0 6 0 0 7 40 76 8 67 71 0 19 0 27 1. IFAT IgG titres scored as 1:40 (+++) or higher are defined as positive.