Innate and adaptive immune responses in the intestine of camel (Camelus dromedarius) naturally infected with Mycobacterium avium subspecies paratuberculosis.

The spread of John's disease in camel herds (Camelus dromedarius) has been worldwide reported. Despite extensive studies on Mycobacterium avium subspecies paratuberculosis (MAP) infection in camels, the complete pathogenesis and epidemiology of this infection have not been fully exploited. The objective of the study is focusing on the nature of the immune responses, and the types of the recruited cells were studied in the intestine of naturally infected camels employing immunohistochemistry to analyze the expression of CD335, CD103, CD11b, and CD38 markers. Marked expression of some or all of the markers was observed in the ileum, mesenteric, and supramammary lymph nodes of the old infected camels. The expression of CD335, a well-known natural killer (NK) cell marker, was detected in the mesenteric lymph node, while the dendritic cell (DCs) marker, CD103, was markedly expressed in the villi and propria submucosa (PS) of the ileum in old infected camels. CD103 + and CD11b + DCs were detected in the mesenteric lymph nodes of young infected camels. The expression of CD38, a crucial proinflammatory marker, was more noticeable in the peripheral region of the mesenteric lymph node. The expression of these markers in the infected camel intestine was peculiar and is reported for the first time. In summary, the unique expression patterns of CD335, CD103, CD11b, and CD38 markers in naturally infected camel intestines revealed through immunohistochemistry new insights into the immune responses associated with MAP infection. These first-time observations suggest potential roles of innate and adaptive immunity, highlighting specific aspects of MAP immunopathology. Further studies with targeted tools are crucial for a precise understanding of these markers' roles in the infected intestines.

The efficiency of ELISA and PCR in detecting subclinical paratuberculosis in the Saudi dairy herds.

This study was aimed to reveal the presence of John's disease in the Saudi dairy herds using the newly developed diagnostic tests, ELISA and PCR. A total of 687 serum and fecal samples were collected from dairy cattle of four different ages, one, two, four and six years age cattle of three geographically different dairy farms. IDEXX ELISA revealed 15 (2%) positive samples and 17 (2.5%) samples were inconclusive. PCR test were used only to test 62 ELISA-negative samples that their OD readings were the highest and all the inconclusive samples. The PCR disclosed more positive samples (22/62 = 32%), interestingly among the samples of the two-year old cattle. The study has conclusively confirmed the presence of the disease in the Saudi dairy herds. It has also approved the effectiveness of ELISA and PCR tests in revealing the MAP infection at the subclinical stage.

The cytokine markers in Staphylococcus aureus mastitis of bovine mammary gland.

TaqMan real time PCR was used to study the transcriptional activity of the bovine IL-2, IL-6, IL-12p40, IFN-gamma, TNF-alpha and granulocyte-monocyte colony stimulating factor of whole milk cells in bovine mammary gland experimentally infected with Staphylococcus aureus. Cytokine transcriptional activity was monitored at 7, 24 and 32 h Post-infection (Pi). IL-12 and TNF-alpha levels were significantly elevated at 24 h Pi followed by sharp decrease at 32 h pi. IL-2 level was decreased at 32 h pi. IL-12 and IFN-gamma showed a significant interaction at 24 h pi. The significant elevations of the IL-12 and TNF-alpha transcriptional level most likely indicate their important role in regulation of the immune responses of bovine mammary gland in S. aureus infection. Depression of IL-2 could reflect the suppressive nature of the S. aureus mastitis.

Cytokines gene expression patterns of bovine milk during middle and late stages of lactation.

The cytokine mRNA profiles of the bovine mammary gland were investigated using newly developed TaqMan real-time polymerase chain reaction systems (Applied Biosystems, Foster City, CA, USA). Transcriptional activity of six cytokines, interleukin (IL)-2, IL-6, IL-12, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and granulocyte-macrophage colony stimulating factor (GM-CSF) was studied during the mid- and late-lactation stages. Transcripts for TNF-alpha, GM-CSF, and IFN-gamma were detected in all samples of both stages. However, IL-12 was only detected in 80 and 58 % of late- and mid-lactation samples, respectively. IL-12 expression was up-regulated in late lactation in comparison with the corresponding level in mid-lactation. The cytokines interaction in late lactation was more co-ordinated and their transcriptional levels were significantly correlated among each other, whereas, in mid-lactation significant correlation of the cytokines transcription was only seen with the TNF-alpha, GM-CSF, and IFN-gamma. Cytokine mRNA profiles between mid- and late lactation showed significant differences, which can be attributed to the dramatic changes that the mammary gland is subjected to during late lactation. The significant elevation of IL-12 transcriptional activity in late lactation and its relevance to the mammary gland immunity is discussed.

Detection of IL-2 and IFN-gamma m RNA expression in bovine milk cells at the late stage of the lactation period with RT-PCR.

The expression of bovine interferon-gamma (IFN-gamma) 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-gamma was detected with all primer concentrations, 0.2, 0.5 and 1 microM. IL-2 however, was faintly detected only with 0.5 microM primer concentration. It was shown that IFN-gamma m RNA 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-gamma and IL-2 and for helping to reveal the activity of cytokine in the mammary gland at the late stage of the lactation period.

Quantitation of bovine cytokine mRNA in milk cells of healthy cattle by real-time TaqMan polymerase chain reaction.

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 TaqMan PCR systems were developed to overcome limitations of conventional quantitative PCR methods. The TaqMan method is based on the cleavage of fluorescent dye-labeled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by an automated spectrophotometer integrated in a sequence detection system (Applied Biosystems, Foster City, CA). The bovine-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation between complementary DNA (cDNA) and genomic DNA (gDNA) amplification 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 amplification control and allowed for correction of variations in different numbers of cells in the starting material, in the efficiencies 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. Profiles of cytokines (interleukin (IL)-2, IL-6, IL-8, IL-12 p40, TNF-alpha, IFN-gamma) and granulocyte-macrophage colony stimulating factor (GM-CSF) were established in normal lactating cattle. Differences of cytokine profiles obtained with the real-time TaqMan PCR system and conventional methods are discussed.

Role of surface glycoproteins in influenza virus pyrogenicity.

Eleven H3N2, seven H1N1 and three H3N1 influenza virus reassortants of the pyrogenic A/Puerto Rico/8/34-A/England/939/69 clone 7a (H3N2) (A/7A) and poorly pyrogenic A/Fiji/15899/83 (H1N1) (A/Fiji) parents were analysed genetically for the parental origin of their genes and for their pyrogenicity in ferrets. All H3N2 reassortants were pyrogenic and produced significantly more fever than A/Fiji but differences in pyrogenicity between them could not be correlated with either single or constellations of genes. All H1N1 reassortants were poorly pyrogenic compared with A/7A but one (Am29) produced significantly more fever than A/Fiji. No correlation of the increased fever with inserted A/7A genes was evident in Am29 and, while mutations were detected in the H1 haemagglutinin of this reassortant using monoclonal antibodies, similar mutations were present in the other H1N1 reassortants which showed no increase in fever production. The three H3N1 reassortants were intermediate in their pyrogenicity, being more pyrogenic than A/Fiji and less pyrogenic than A/7A. Overall, these results support previous conclusions that the haemagglutinin and/or neuraminidase play a major role in the pyrogenicity of influenza virus.

Caprine arthritis-encephalitis antibodies in indigenous sheep in Saudi Arabia.

A serological survey was conducted in indigenous sheep from Saudi Arabia for detection of antibodies against the caprine arthritis-encephalitis virus. Only 0.8% of the sera examined were positive. Epizootiological considerations of the disease in the country are discussed.