**7. Laboratory diagnosis of Marek's disease**

Generally, the diagnostic methods for Marek's disease can be divided into the following methods: serological, histopathological and virological. In the case of the latter, they include methods based on molecular biology.

Live birds (5 to 10 birds per flock) showing symptoms of the disease and an additional 20 to 25 blood or preferably serum samples should be provided for virological examination. We can also examined feathers collected from birds from the shoulder glass or the inner thigh surface. These feathers should be protected in such a way that does not dry out during the transport of the samples to the laboratory. Sick birds delivered to the diagnostic examinations should be euthanized using methods compliant with applicable legislation. Then, a thorough anatomopathological changes should be performed, describing the visible changes. During the anatomopathological examination, samples of the internal organs should be collected (in sterile way) for laboratory diagnostic tests, (classical virological and molecular). Most often samples of the liver and spleen have been collected, but also samples of other internal organs with pathological changes were examined to better understand the virus virulency. In turn, for histopathological examinations, apart from liver and spleen sections, also bursa of Fabricius, glandular stomach and peripheral nerves (sciatic and brachial plexus) have been examined. The possible presence of lesions in peripheral nerves is also diagnostic tool for differentiating between Marek's disease and avian leukemia infections, but also other neoplastic diseases. Homogenates are prepared from the collected internal organs, which are used to infect cell cultures: SPF (Specific Pathogen Free) chicken embryo fibroblast cultures (CEF) or Chicken embryo kidney cultures (CEK) or chicken embryos. The infected cell cultures are incubated at 37.5°C and the possibly cytopathic effect (CPE) in the form of clustered fine light refracting cells is observed on a daily basis. The formation of CPE in infected cell cultures indicates the presence of Marek's disease virus [67]. Sometimes, plaque formation is found, i.e. places where the connectivity of the cell layer has been interrupted by a proliferating Marek's disease virus strains.The cytopathic effect and plaques created by, for example, the vaccine strain HVT FC 126 (serotype 3) usually begins to appear as early as 72 hours after infection of the cell culture. In turn, field strains form CPE only in the 2nd or even 3rd passage. Under the microscope, starting from the 5th day after infection of the culture, a cytopathic effect is visible in the form of clustered small cells, strongly refracting light, and it most often appears between 7 and 9 days after infection. The final results of incubation are read under the microscope after about 12–14 days culture incubation. Clusters of small cells may also be visible, often overlapping each other and forming so-called focuses [68].

Marek's disease virus can also be isolated directly in a sterile culture prepared from kidney cells collected from diseased birds in a similar manner as described above. Isolation of Marek's disease virus strains in chicken embryos is used very

rarely, mainly due to the time cost consuming nature of this method. Commercial Marek's disease virus antigen and commercial anti-MDV serum are used in serological method. Blood is taken from the examined birds in a sterile manner, from which the serum is obtained after centrifugation. Feathers, are collected from places where there is an intensive replication of complete virus particles (wings, thighs and shoulder pathway). The presence of the specific anti-MDV antibodies in the serum samples collected from infected birds is determined by the agar gel immunodiffusion method (AGID), while the agar gel radial immunodiffusion test (RID) is used to detect the presence of the specific antigen of Marek's disease virus in the feather follicles of sick birds.

Among the molecular methods, the most frequently used technique is the polymerase Chain Reaction (PCR) is the amplification method with other variations. The amplification reaction allows the detection of the genetic material of Marek's disease virus strains and the differentiation of field and vaccine strains.Organ samples, blood, feather follicles, as well as dust collected from poultry houses serve as a matrix for DNA isolation. Traditional PCR consists in amplifying a fragment of a gene specific for MDV. Primers whose nucleotide sequence is complementary to the amplified fragment of the MDV genome are most often used for this purpose. The primers used are usually complementary to the sequence of genes such as: meq gene, 132 bp repeat sequence, pp38 gene or fragment of the SORF1 gene specific for turkey herpesvirus FC 126 strain (serotype 3) [69]. The advantage of the PCR method is its high sensitivity and specificity. The high specificity of the PCR allows the differentiation between field strains belonging to serotype 1 and the vaccine strain HVT FC 126 belonging to serotype 3 and the vaccine strain Rispens CVI 988 belonging to serotype 1.The results of PCR methods have been used to answer the question whether the birds have been vaccinated correctly or not, and whether the birds were infected with a virulent, field strains of Marek's disease virus.

Frequently in birds vaccinated with the bivalent vaccine (Rispens CVI 988 and HVT FC 126), in the case of infection with the field strain, DNA of the Rispens CVI 988 strain is absent. This is most often the case in birds over 6 weeks of age. On the other hand, these birds have DNA of the vaccine strain HVT FC 126 in practically every case. Such a PCR result clearly proves that the examined birds were vaccinated against Marek's disease. It should be remembered that vaccination does not protect birds against infection with a field strain, but only against the manifestation of the clinical symptoms and the occurrence of pathological changes.
