Figure

Figure. Agarose gel electrophoresis of Coxiella burnetii Trans-polymerase chain reaction products amplified from total DNA of bulk tank milk samples. Lanes 1 to 20, bulk tank milk samples; N, water negative control;...

The samples in this study were somatic cells extracted from bulk tank milk aliquots submitted to the New York State Animal Health Diagnostic Laboratory to detect bovine viral diarrhea that persistently infected lactating dairy cattle. The samples tested do not represent a random sampling, as tests were done only on samples available. The samples are heavily weighted to the Northeast, but some are from the Midwest and West. We tested 316 bulk tank milk samples from dairy herds in the United States during a 3-year period from January 2001 to December 2003 by using trans-PCR (Figure). Positive results were confirmed by nested PCR and DNA sequencing. The sequencing results of the 687-bp PCR product were consistent with the published sequence of IS1111 with 100% homology. A summary of the PCR test results of the bulk tank samples is shown in Table 1. The overall prevalence of C. burnetii in the tested samples was 94.3% with little variation (93.2% to 94.7%) from year to year. Samples from New York State did not show significant variation from other states, which indicates that C. burnetii infection in the dairy herds was persistent or steady, with little temporal or regional variations. Milk samples were collected 6 times from 2002 to 2004 from specific cattle in a small, C. burnetii–positive dairy to monitor the infection in specific cattle. A summary of the results of tests on milk samples from specific cattle is presented in Table 2. While 28 (52.8%) of 53 cattle were C. burnetii positive in 2002, 23.5% and 31.3% of the cattle were positive in 2003 and 2004, respectively. Daily and weekly shedding levels of 5 C. burnetii– positive cattle were assayed by real-time PCR. Real-time PCR was conducted by using the primers and probe designed by the Primer Express program (Applied Biosystems, Foster City, CA). The primer set consisted of primers trans-f (5′-GGGTAAAACGGTGGAACA ACA-3′) and trans-r (5′-ACAACCCCCGAATCTCATTG-3′). The internal probe trans-p (5′-AACGATCGCGTATCTTTAACAGCGCTTG-3′) was labeled with the reporter dye 5-carboxyfluoroscein (FAM) on the 5′ end and the quencher dye N′, N′, N′, N′-tetramethyl-6-carboxyrhodamine (TAMRA) at the 3′ end. The reactions and assay conditions were according to the manufacturer’s instructions (Applied Biosystems). Each cattle shed a similar amount of C. burnetii daily over 7 days; weekly shedding over 4 weeks was also similar. Shedding levels of positive cattle were estimated to be 10¹–10⁴ cells/mL each. The bulk tank milk samples of the herd stayed at a level of 10² cells/mL over 3 years.

Bulk tank milk has been used for surveillance samples in dairy herds for several bovine diseases including bovine viral diarrhea. More than 90% of U.S. dairy herds sampled were infected with C. burnetii based on bulk tank milk testing over a 3-year period. This high prevalence did not show temporal or regional variation, suggesting that C. burnetii infections in dairy herds are common throughout the United States. Our report of C. burnetii in dairy herds is not surprising if earlier reports regarding an increase of bovine infection in North America are considered. An early investigator concluded that C. burnetii was endemic throughout the United States in the 1950s and predicted that a high bovine infection rate could occur in other parts of the country, as it did in southern California where a 98% infection rate was reported (11). A California survey reported that 20 (100%) of 20 herds in 17 counties throughout the state contained seropositive cattle, and 82% of 1,052 specific cattle from the herds were seropositive (12). An increase in the prevalence of Q fever from 2.3% in 1964 to 66.8% in 1984 was reported in Ontario dairy herds (13). Our study found a notable decrease in shedding of C. burnetii in milk by specific cattle from 52.8% to 23.5% over 3 years (Table 2). This decrease was not because animals stopped shedding the organism but because uninfected animals replaced shedding animals with an average annual replacement rate of 30%. With the exception of the first year of our study, the shedding rate appeared to be steady at 20% to 30% over 2 years. A similar shedding rate was found in a California study; 23% of 840 cattle were shedding C. burnetii in their milk (13). Continual daily and weekly shedding in the milk by the infected cattle suggests chronic infection by C. burnetii. Chronic C. burnetii infection of dairy cattle could be the most important source of human infection simply based on sheer numbers (1). Extrapolation of our data to the national dairy herd suggests that nearly 3 million lactating cattle are shedding C. burnetti daily. Though the mode and extent of transmission from bovine to human has not been determined, epidemiologic studies indicate that Q fever develops in farmers, veterinarians, and slaughterhouse workers who are in contact with domestic animals (14). While infection from commercial milk is unlikely because of the pasteurization process, ingestion of raw milk has been linked to higher seroprevalence rates.

In the aftermath of September 11, 2001, and the anthrax incidents, the use of biologic warfare is no longer a distant possibility. C. burnetii is considered a potential bioterrorism agent because of its high infectivity (a single organism may cause disease in human), its ease of dispersion in aerosols (because of its small, sporelike structure), and its resistance to extreme environmental conditions and chemicals (15). Therefore, further investigations are needed to determine the implications of the high prevalence of C. burnetii in dairy herds, to address the potential risk to public health, and to be prepared for outbreaks and bioterrorism events. Currently, no commercial vaccines are available for cattle, and no effective treatment protocol exists for infected animals.