Detection of foot-and-mouth disease virus in milk samples by real-time reverse transcription polymerase chain reaction: Optimisation and evaluation of a high-throughput screening method with potential for disease surveillance

Abstract

This study aimed to evaluate the utility of milk as a non-invasive sample type for the surveillance of foot-and-mouth disease (FMD), a highly contagious viral disease of cloven-hooved animals. Four milking Jersey cows were infected via direct-contact with two non-milking Jersey cows that had been previously inoculated with FMD virus (FMDV: isolate O/UKG/34/2001). Milk and blood were collected throughout the course of infection to compare two high-throughput real-time reverse transcription polymerase chain reaction (rRT-PCR) protocols with different RT-PCR chemistries. Using both methods, FMDV was detected in milk by rRT-PCR one to two days before the presentation of characteristic foot lesions, similar to detection by virus isolation. Furthermore, rRT-PCR detection from milk was extended, up to 28 days post contact (dpc), compared to detection by virus isolation (up to 14 dpc). Additionally, the detection of FMDV in milk by rRT-PCR was possible for 18 days longer than detection by the same method in serum samples. FMDV was also detected with both rRT-PCR methods in milk samples collected during the UK 2007 outbreak. Dilution studies were undertaken using milk from the field and experimentally-infected animals, where for one sample it was possible to detect FMDV at 10^-7. Based on the peak C_T values detected in this study, these findings indicate that it could be possible to identify one acutely-infected milking cow in a typical-sized dairy herd (100-1000 individuals) using milk from bulk tanks or milk tankers. These results motivate further studies using milk in FMD-endemic countries for FMD surveillance.

Keywords: Foot-and-mouth disease virus; Milk; Real-time RT-PCR; Surveillance.

Fig. 1

Comparison of the analytical sensitivity for Methods A (used the TaqMan® Fast Virus 1-Step Master Mix (Applied Biosystems®)) and B (used the Superscript III Platinum® One-Step qRT-PCR Kit (Invitrogen^™)). C_T values are the average of three replicates, and bars represent standard deviation. : Method A, : Method B.

Fig. 2

Comparison of both methods tested with experimental whole milk samples. Each square represents the average C_T value of the whole milk sample at each day post contact. White squares represent a ‘No C_T’ value – no detection. Black squares represent any C_T value including or below 45 (Method A) or 50 (Method B) in all replicate wells – FMDV positive. Grey squares represent instances where a ‘No C_T’ value was observed in one or two wells, but a positive result was observed in the other replicates. N/A represents where there was not sufficient sample available for testing.

Fig. 3

FMDV detection in samples collected at regular intervals from all cows. Virus titrations in BTY cells (A) and rRT-PCR using Method B (B) for skimmed and whole milk fractions and serum (B only). Average C_T is derived from the mean of 2 replicates. The development of lesions in at least one foot indicates the onset of clinical signs. : Onset of clinical signs, : whole milk, : skimmed milk, : serum.

Fig. 4

Detection of FMDV by rRT-PCR using Method B on ten-fold dilutions in Jersey whole milk of two milk samples: animal 867 (4.5 days post contact infection) and c27, a field sample from the UK 2007 outbreak (Table 2). C_T values are the average of three replicates with standard deviation error bars. : 867 (4.5 dpc), : c27.