Lewerin et al. Acta Veterinaria Scandinavica 2010, 52:7 http://www.actavetscand.com/content/52/1/7 Subtyping The three animal isolates showed the same MLVA pro- file. No perfect match to other published profiles was found. However, this study’s profile clustered with iso- lates in the A lineage that, unlike other major lineages, is known be present throughout the world [21]. Discussion and Conclusions This case illustrates the difficulties in detecting a disease that has been absent for a long period of time. The absence of typical signs such as dark blood failing to clot, or lack of rigor mortis, in combination with a non- typical history of animals kept indoors fed only rough- age, caused a delay in the diagnosis that led to a number of potential human exposures and consequent antibiotic treatments. Most cases reported from other countries are in grazing animals and “barn anthrax” is rarely reported now when meat-and-bone meal is no longer fed to ruminants [4]. However, one similar case has been described [4] where heifers indoors on a strict hay diet contracted anthrax via contamination of the hay. In that case, spores could be detected in the hay. In the current case, no samples of dust from either hay or straw were positive, in spite of great efforts to obtain representative samples. The culture method that was used before PCR on these samples had not been evalu- ated earlier and the detection limit of the method is unknown. It is most likely that low concentrations of contamination would not have been detected. Any such contamination is believed to have been of a low concen- tration possibly originating from fields close to the river Viskan. In these fields, flooding occurred the previous year and the next year there was a draught with a very low water level in the river allowing for the harvesting on soil not usually exposed. According to the farmer, the feed in question was mixed with soil and dust and this was also obvious at the time of sampling. In the early 1900s, animal carcasses are said to have been dumped in this river during anthrax outbreaks and it is most likely that some anthrax spores could remain in the area. The history of flooding followed by drought is typical for areas where old anthrax spores surface and cause outbreaks [4,22]. A low initial dose may be one reason for the less typi- cal appearance of the first carcasses. Bleeding from ori- fices [22] or failure of the blood to clot is reported to be the most reliable sign of anthrax carcasses [23], but this was not seen in the field and was only obvious after opening the carcass of animal 8. The lack of a laboratory diagnosis in animals investi- gated before anthrax was confirmed is, with hindsight, not surprising. Animal 2 had been transported for 4 days and was badly decomposed when the spleen swab was taken and thus the only remaining viable bacteria, Page 6 of 8 that appeared on culture, were from the post mortem contamination flora. Later, when the swab was re-ana- lysed by specific PCR, it was positive, demonstrating the need for this method when samples are from older car- casses. Animal 7 was culled by the owner and did not die from terminal bacteraemia. This was, however, not known by SVA at the time of investigation and thus the lack of bacterial growth on culture was at the time taken as contradicting the suspicion of anthrax. The his- tological findings were unclear and only indicated some type of infectious origin. In contrast, the necropsy of animal 8 revealed typical signs and immediate direct smears performed in the regional laboratory had a more typical appearance than when smears were performed on blood sent to SVA. The absence of encapsulated B. anthracis in the latter smears could be due to the fact that the blood samples were not fresh, and the capsules present in the blood of diseased animals became subsequently decomposed. Direct smears stained with “old methylene blue” have been widely used in the field and provide a quick preli- minary diagnosis provided the carcass is fresh, a good microscope is available and the person performing the microscopic examination has some experience. It would not be practical in Swedish field circumstances today and even the regional laboratories rarely have adequate experience in microscopic examinations. However, rapid detection is important and a robust field test to replace direct smears would be of great benefit. Both PCR and culture were used for the diagnosis and both methods are needed if a quick diagnosis on any sample, regardless of state of decomposition, is to be made while securing material for subtyping and antibiograms. The positive PCR results from DNA extracted from the soil samples showed that it was possible to detect genetic material from B. anthracis in a well-known com- plex environmental matrix such as soil [24]. Our results demonstrate the potential of using PCR as a tool for mapping B. anthracis-contaminated areas and possibly elucidate the coordinates of the source. A careful sam- pling strategy is a prerequisite for such a study, and was beyond the scope of this reported work. The MLVA results were not surprising. As the histori- cal anthrax cases in Sweden were mainly associated with the feeding of imported meat-and-bone meal, it is to be expected that the spores remaining in Swedish soil are of the A lineage. Detailed subtyping of old anthrax strains has been performed in some parts of the world [25-27], revealing different pictures of genetic linkage between strains as well as possible clues to the origin of some strains. Unfortunately, the old anthrax strains that were formerly stored at SVA have been destroyed so no further studies can be made on old historical material