Staphylococcus aureus and bovine mastitis: recent findings

Bovine mastitis is a very common disease in dairy herds worldwide. In Switzerland, the incidence density is 32.7 cases per 100 cow years (Stärk et al., 1997), resulting in total costs of about CHF 130 Mio. per year (Heiniger et al., 2014). Udder diseases are the most common causes for slaughtering primiparous cows in Germany (Brade and Brade, 2007). Many of these losses are directly related to intramammary infections caused by Staphylococcus aureus (Staph. aureus). This pathogen normally causes subclinical chronic mastitis in several to many cows within one herd (Sears and McCarthy, 2003). Although the clinical changes are normally mild, the cure rates (30%) for antimicrobial treatment are low (Gruet et al., 2001). Diagnosis of Staph. aureus by bacteriological testing of quarter milk samples is not satisfactory. The diagnostic sensitivity under the routine conditions reaches an overall diagnostic sensitivity of only 75% for single sampling (Sears et al., 1990; Studer et al., 2008). In some cases, this sensitivity may be as low as 21.4% (Studer et al., 2008). Based on these results, a satisfying diagnostic sensitivity is only achieved, if at least 3 consecutive milk samples are analysed. As triple sampling is normally too expensive and too laborious, routine testing is accomplished with single sampling. This fact, however, is a major reason why sanitation of Staph. aureus-infected herds frequently fails.

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A highly sensitive assay for detecting Staph. aureus in clinical milk samples

From a clinical point of view, the limited diagnostic sensitivity of bacteriological culturing methods for Staph. aureus is not sufficient, as too many infected quarters and cows remain undetected (Sears et al., 1990; Studer et al., 2008). As a consequence, the control and eradication of Staph. aureus mastitis in a herd is difficult and often impossible. A novel assay for Staph. aureus was, therefore, developed (Graber et al., 2007). It includes a fast and reliable preparation procedure for bacteria from bovine milk and a specific detection by real-time quantitative PCR (qPCR). This method is quantitative, highly specific for Staph. aureus (Graber et al., 2007; Studer et al., 2008) and is 500 to 2000 times more sensitive than standard bacteriology (Graber et al., 2007; Studer et al., 2008). Using this assay, quarters infected with Staph. aureus are detected very reliably and single sampling is now possible, whereby the actual shedding quantity is no more of concern. Additionally, it was shown that shedding of Staph. aureus in milk of infected quarters was normally sinusoidal with a cycling period of about 13 days, but log-linear shedding was observed as well (Studer et al., 2008).

 

Figure 1. The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for Staphylococcus aureus. It allows a high throughput at moderate costs and is suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with the in-house developed software (freely available) assignment of the pattern of bands to a genotype is standardized and straight forward. Full details including a complete laboratory protocol and a video for RS-PCR are given in Graber 2016 (J. Vis. Exp. (117), e54623, doi:10.3791/54623 (2016)).

Genotypes of bovine Staph. aureus in Switzerland

Using ribosomal spacer PCR (RS-PCR), more than 100 genotypes and variants of bovine Staph. aureus have now been observed (Figure 1). In the initial study by Fournier et al. (2008), 17 subtypes were found whereby genotype B (GTB) and

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