Evaluation of diagnostic techniques for the detection of Bovine Viral Diarrhoea Virus (BVDV) in infected and persistently infected cattle

Bovine Viral Diarrhoea Virus (BVDV) is a significant pathogen associated with gastrointestinal, respiratory, and reproductive diseases of cattle worldwide. The ability of BVDV to cross the placenta during early pregnancy can result in the birth of persistently infected (PI) calves, which are capable of shedding large quantities of virus throughout their lives and are considered the primary reservoirs for BVDV. PI animals do not show antibody response, and diagnostic tests such as virus isolation, immunohistochemistry (IHC), Antigen-Capture ELISA (ACE), and RT-PCR are used in their detection. Control efforts based on the identification and elimination of PI animals have been successful in a number of countries around the world. In Italy, little research has been done concerning BVDV prevalence and at this time no compulsory/voluntary control program exists. In order to investigate the seroprevalence and to identify PI animals in Central Italy, a serological survey on 1,929 unvaccinated Holstein Friesian animals from 21 farms from the Marche, Umbria and Lazio regions was carried out. Sera samples were tested to detect BVDV antibodies using a commercial ELISA kit (Antibody BVDV IDEXX HerdCheck). In order to detect PI cattle, all young (2 years old) and seronegative animals were tested individually using a commercial ACE (Serum/ACE BVDV IDEXX HerdCheck) and a RT-PCR protocol using pooled sera samples. In order to compare two different epidemiological situations, a short survey of 2 months was carried out at the Veterinary Diagnostic Laboratory of the Colorado State University (Rocky Ford, Colorado, USA) where a BVD voluntary program based on the identification and elimination of PI animals has been implemented. The protocol was carried out testing surnatants of ear-notches individually by ACE (ACE BVDV IDEXX, USA) derived from positive pools previously tested by RT-PCR. A total number of 14,508 samples were received, conforming 360 pools with an average number of 40 samples each one. The results obtained in the serological survey of Central Italy demonstrated a total seroprevalence of 26.43%, meanwhile seroprevalences of 20.35%, 24.92% and 31.66% were observed in Umbria, Marche and Lazio regions respectively. These results showed the presence of BVDV infection with low prevalence. According to the age, 5.33% and 85.28% of animals younger and older than 2 years old respectively were positive. Only 10% (2/20) of farms showed recent infection of BVDV due to the presence of seropositive animals younger than 24 months old. All seronegative / young animals tested (No. 490) were negative with the Serum/ACE using both, the standard and the overnight incubation methods with the intent to improve the sensitivity of the test, as well as by pooled RT-PCR. As additional Serum/ACE kit evaluation, 10-fold serial dilutions of BVDV TMV-2 strain in BVDV negative serum were tested, and positive results were seen in a 10-3 dilution confirming its validity. Therefore, no PI animals were identified in our research. Several factors such as the low PI prevalence, intermittence of BVDV excretion, the presence of a low percentage of PI animals that do not show detectable viral titers, along with the changing levels of viremia in PI animals must be considered. Our results demonstrate the presence of BVDV infection even in the absence of PI animals, illustrating the possible important role of transiently infected (TI) animals in the long term circulation of BVDV. Probably risk of indirect or external avenues of transmission must be considered due to the presence of on-going infections. In addition, the current herd management practices and low cattle density in Central Italy may be contributors to the low presence of PI animals. On the basis of these results the BVDV spread in Central Italy does not appear critical. The application of other diagnostic techniques such as IHC, RT-PCR and ACE using buffy coat and/or ear notches samples to detect the presence of PI animals is strongly recommended. In other hand and following the protocol of the Colorado State University, 15 Hereford of 4 months old animals were detected as PI animals, representing a total prevalence of 0.1%. This prevalence has been described everywhere, ranging from 0.5 to 2%. The methodology used offered a low-cost and highly sensitive system to detect BVDV infected animals. Important differences as the number of animals sampled, the absence/ presence of a Control Program, the different samples used in each survey (serum vs. ear notch), differences in size, density and management, as well as the role of the presence of wild animals in the Colorado State are factors that must be considered between the different results observed in both surveys.