Applications in Veterinary Medicine
Applications of the TaqMan principle are extremely wide. There are three principle fields of interest for the real-time TaqMan PCR user: pathogen detection (i.e. viruses, bacteria, fungi, etc), gene expression (i.e. cytokines, growth factors, transcription factors, etc.) and allelic discrimination (detection of single nucleotide polymorphism, SNP). A few selected examples will illustrate the potential of the real-time TaqMan PCR technique.

Pathogen detection using TaqMan PCR
Feline immunodeficiency virus (FIV), a lentivirus isolated first in California (USA) [48], is similar morphologically and genetically to the human immunodeficiency virus (HIV) [22]. Because FIV is a naturally occurring pathogen which induces an AIDS-like disease in cats, it is considered an important animal model for the study of AIDS in human beings. Furthermore, the FIV model has proven to be useful for studying AIDS pathogenesis, for evaluating new anti-lentiviral drugs and for establishing criteria for the development of safe and efficacious vaccines against lentiviral infections [4,9]. To study the effect of candidate vaccines or therapeutics, highly sensitive and specific test systems were successfully established to quantify the FIV RNA and DNA load for vaccine and therapy studies [32,35]. Quantitative assays for both FIV provirus and viral RNA have a similar absolute sensitivity of 10 molecules.

The level of HIV-1 RNA in serum has the highest predictive value with regard to disease progression [20,42] and sensitive virus load assays have been critical in monitoring the status of HIV-1 infection [18]. Animal models provide great potential for research into such regimes; the most promising for studies of AIDS therapy is infection of rhesus macaques with simian immunodeficiency virus (SIV) [17,54] or with chimeras of SIV containing HIV-1 targets (SHIVs), such as reverse transcriptase (RT-SHIV) [64]. One considerable limitation of the SIV model in HAART-related research is the lack and/or expense of highly sensitive assays to measure viral burdens in plasma. The current test for detection of SIV has been the branched-chain DNA assay (Bayer, Emeryville, CA), which is expensive and not sensitive enough (1500 viral RNA copies/ml) to detect very low viral loads in the SIV system. Moreover, the assay is not adapted to all strains of SIV or to RT-SHIV [60]. Several assays with greater sensitivity than existing quantitative ones have been established [28,62]. In our laboratory, we have optimised a real-time TaqMan RT-PCR assay for SIV RNA which was more sensitive (50 vs 1,500 RNA copies/ml) and had fewer false positives and negatives than the current version of the SIV branched-chain DNA assay [36].

Feline coronavirus (FCoV) is known to be highly prevalent in the cat population, especially in catteries [1]. It is the most important fatal infectious disease in cats, with about 5 -12% of seropositive cats developing lethal FIP [2]. The pathogenicity of FCoV leading to the FIP syndrome may be linked to mutagenesis, due to increased viral replication. Successful management of FIP may eventually consist of better methods of disease prevention, as well as management of the disease after it occurs. Prevention of FIP can be accomplished by detection and separation of FCoV shedding from non shedding cats, resulting in the reduction of coronaviral load or even the elimination of FCoV from a cattery [21]. A commercial FIP vaccine is available and consists of a temperature-sensitive mutant form of FIP virus, delivered through the mucosa. The vaccine is supposed to undergo replication only in low temperature, outer oronasal cavities and thus triggers protective antibodies but not FIP. A few controversial studies have recorded a reduction in FIP as a result of vaccination, especially in FCoV naive cats at the time of vaccination [19,24,41,50,58]. It becomes evident that quantification of coronaviral load in FCoV positive shelter cats, or the development of strategies for the prevention or elimination of FCoV in catteries, will depend on PCR procedures that allow the reliable and fast analysis of large numbers of samples. A sensitive real-time TaqMan RT-PCR should enable this type of research [26]. The FCoV real-time TaqMan RT-PCR assay is based on the reverse transcription and amplification of a portion of the FCoV 7b gene, which is known to be highly conserved among coronavirus isolates. This assay, adapted from a previous study [27], has an analytical sensitivity between 10 and 100 times better than a nested RT-protocol. Real-time TaqMan RT-PCR detected most of the important laboratory and field strains of FCoV, including FIPV 204859, FIPV UCD1, UCD 5, FeCV UCD 1, FeCV RM but not the human coronavirus (HCV) strain 229E [26]. The assay allowed absolute quantification with high sensitivity, it was reliable, rapid, easy to use and enables a high sample throughput, making it an excellent tool for diagnostics and FCoV research.

Tick-borne zoonotic pathogens are well known in many areas of the world. Among the tick-borne diseases in Europe, Lyme disease (caused by Borrelia burgdorferi), ehrlichiosis (caused by various species of Ehrlichia) and tick-borne encephalitis (caused by the tick-borne encephalitis virus, TBEV) are the most important zoonotic diseases. Early diagnosis and treatment is necessary to prevent fatal infections and chronic damage to various tissues. Due to the variety of uncharacteristic clinical signs, tick-borne diseases are not easily recognised. Diagnosis is based on clinical findings, a history of exposure to ticks, and direct or indirect detection of the pathogen. The design and optimisation of real-time TaqMan PCR systems for a range of tick-borne pathogens has proved to be important for diagnosis and research and has initiated a series of exciting new projects in this field [33,38,51,52,53,69].

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