This summer 2011 I have visited the School of Veterinary Medicine at the Washington State to University at Pullman . Professor William Davis kindly sent me the invitation to discuss the possible joint research on the Paratuberculosis . 

Early diagnosis of MAP infection is a pressing need to enable efficient intervention with the spread of MAP infection in herds. Hence, study of lymphocyte subsets and their expressed adhesion molecules could contribute in defining a distinct diagnostic marker (or markers) at the subclinical period of the infection that could in turn facilitate the development of effective diagnostic approach. In accordance with this objective, milk and blood samples were collected from two groups of cattle naturally infected with MAP and their corresponding negative controls. Group (C) comprised 3–4 year-old ELISA negative/ PCR positive-cattle that were considered as subclinical seronegative low shedder group (early stage). Group (A) included 6–8 year-old ELISA positive-cattle, which were considered as a clinical seropositive group (late stage). Flow cytometry of B cells, CD8+, CD4+ and cd cells and the adhesion molecules CD44+, CD62L, LFA-1 and LPAM-1 indicated increase in CD4+ and B cells levels, with higher levels in blood than milk of group A, and significant expression of CD44+ in blood and milk and LPAM-1 in blood only. The CD8+ cells count in milk was higher than blood in the late stage. The peculiar feature of the early stage (group C) was the high level of cd cells in the blood and milk, with tendency to express high level of CD62L. Compelling evidence could support the assumption that the dominant cd cells at early stage of MAP infection could be of CD8CDWC+1+ phenotype. cd cells appear as promising markers in defining early changes of MAP infection due to their important role in priming innate and cell mediated immunity. Possible utilization of these peculiar changes in the cd cells level in the early diagnosis of MAP infection should be the subject of further research.

The etiology of Crohn’s disease (CD) has drawn heated controversy in the literature. Compelling evidence in the literature has accumulated lately that could incriminate the Mycobacterium avium subspecies paratuberculosis (MAP), the well-known agent of John’s disease in cattle. The evidence is isolation of the organism or its DNA and RNA, detection of the anti-MAP antibodies in Crohn’s patients, increasing incidents of CD in areas close to the cattle pastures, and the possibility of treating the disease with the antibiotics. The group that favors the immune dysregulation theory considered this evidence circumstantial due to the variations in these reports. The treatment of CD with humanized anti-tumor necrosis factor-alpha antibodies is considered great endorsement to the immune dysregulation theory. The endless debate could jeopardize public health rather than bring a final solution. Reconciliation between the 2 theories appears inevitable in view of possible classification of this disease as a zoonotic.

The lack of efficacy of conventional strategies for the maintenance of healthy udders in domestic cattle has prompted studies on the use of cytokines for this purpose. The adjuvant use of recombinant bovine cytokines, such as IL-2, IFN-c and TNF-a, in normal mammary gland, mobilizes innate and acquired immunity. However, stimulated immunity does not prevent or eradicate infection, particularly in the case of Staphylococcus aureus mastitis. Cytokines do, however, improve the bactericidal efficiency of certain antibiotics. The subtle and sensitive changes in the cytokine network of normal and mastitic bovine mammary gland may encourage the use of cytokines in the diagnosis and prognosis of udder health. Numerous studies support this hypothesis, and detection and monitoring of cytokines could become an important alternative management for udder health. The use of cytokines in the im- munotherapy, diagnosis and prognosis of mastitis will grow with knowledge of the cytokine network in bovine mammary glands and the development of efficient cytokine diagnostic techniques.

Here we present a novel methodology to quantitate bovine cytokines and growth factors contributing to immunity against bacterial infections of the mammary gland in cattle. Real-time TaqManI PCR systems were developed to overcome limitations of conventional quantitative PCR methods. The TaqManI method is based on the cleavage of ̄uorescent dye-labeled probes by the 50 ±30 exonuclease activity of the Taq DNA polymerase during PCR and measurement of ̄uorescence intensity by an automated spectrophotometer integrated in a sequence detection system (Applied Biosystems, Foster City, CA). The bovine-speci®c TaqManI probes were designed to encompass an intron, thus allowing differentiation between complementary DNA (cDNA) and genomic DNA (gDNA) ampli®cation products. Quantitative analysis of cytokine cDNA was performed in comparison to bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an ampli®cation control and allowed for correction of variations in different numbers of cells in the starting material, in the ef®ciencies of RNA extraction and reverse transcription. With this method, high-throughput analysis of large numbers of samples was possible within a short time. In addition, decreasing the numbers of working steps shortened the time for analysis and increased accuracy. Pro®les of cytokines (interleukin (IL)-2, IL-6, IL-8, IL-12 p40, TNF-a, IFN-g) and granulocyte±macrophage colony stimulating factor (GM-CSF) were established in normal lactating cattle. Differences ofcytokine pro®les obtained with the real-time TaqManI PCR system and conventional methods are discussed.

The expression of bovine interferon-gamma (IFN-γ) and interleukin-2 (IL-2) in the late stage of the lactation period in milk cells was detected with reverse transcriptase-polymerase chain reaction (RT-PCR). IFN-γ was detected with all primer concentrations, 0·2, 0·5 and 1 μM. IL-2 however, was faintly detected only with 0·5 μM primer concentration. It was shown that IFN-γ mRNA expression can be demonstrated clearly with RT-PCR at the late stage of the lactation period. In contrast, IL-2 was weakly expressed at this stage of the lactation period. The work is important for substantiating the therapeutic use of recombinant bovine IFN-γ and IL-2 and for help- ing to reveal the activity of cytokine in the mammary gland at the late stage of the lactation period.