In order to specifically monitor Crithidia mellificae, additional oligonucleotides complementary to the C. mellificae rRNA and kinetoplast sequence were designed and included on the APM beginning in October 2009. These additional oligonucleotides Figure 6 ). In addition, we screened samples throughout the time-course (April 2009-Jan. 2010) by PCR and qPCR specific to the C. mellificae rRNA gene. C. mellificae infection was detected by PCR at every time-point and in turn from every geographic location sampled in our study (MS, SD and CA). Likewise, C. mellificae was readily detected in pooled monthly RNA samples by qPCR throughout the year ( Figure 6C ). In contrast to BQCV, SBV, ABPV and Nosema ceranae, which exhibited peak levels in late summer and early fall, peak trypanosomatid levels occurred in January 2010. Despite this, C. mellificae infections statistically associated with N. ceranae infections (Chi Square p = 0.004, OR = 3.1). C. mellificae was also detected in numerous hobbyist and study hives in the San Francisco Bay Area (CA), as well as samples from a CCD-affected apiary in Oklahoma, indicating wide geographic distribution (Table S1) .