The present study was undertaken to increase the yield of Eimeria tenella oocysts in primary chicken kidney cells (PCKCs) using a comparatively inexpensive cell-culture system. PCKCs growing on coverslips positioned on the bottoms of individual wells in 24-well tissue-culture plates were infected with sporozoites of E. tenella. The effects of changing the culture medium (RPMI 1640), medium pH, serum type, and serum concentration in the wells were determined by counting newly produced oocysts at 7 days postinoculation. There were significantly more (P < 0.01) oocysts produced when the medium was supplemented with 10% fetal bovine serum (FBS) and changed either daily or every other day compared with not changing the medium. When the same medium was changed daily, significantly more (P < 0.05) oocysts were produced at pH 7.4 than at pH 8.0 but not at pH 6.0. If the medium was changed daily, significantly more (P < 0.05) oocysts were produced when medium was supplemented with 10% FBS only rather than 5% and 10% chicken serum. The cell-culture system described in this study offers a convenient and efficient method for investigating the biological, biochemical, and immunological relationships between E. tenella and the host cell.
Bovine mastitis represents the most costly disease of the dairy industry. In an effort to better understand natural host defense mechanisms against mastitis causing pathogens we have begun to characterize the bovine chemokine CCL28 (bCCL28). CCL28 is a dual function chemokine which has been shown to mediate migration of antibody secreting cells to mucosal tissues; this chemokine has also been shown to function as an antimicrobial protein. We have found that expression of bCCL28 mRNA in bovine mucosal tissues is highly variable with highest expression in exocrine glands, including mammary gland and milk. Additionally, we demonstrate that recombinant bCCL28 has antimicrobial activity against mastitis causing microorganisms, including Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae. These results demonstrate that bCCL28 is expressed in mucosal tissues and exhibits potent antimicrobial activity in vitro, suggesting that this dual function chemokine may serve an important role in bovine immunity.
Chemokines play a vital role in mediating leukocyte homing and accumulation to tissues. The chemokine CCL28 also known as Mucosal Epithelial Chemokine (MEC) has been shown to be essential in mediating migration of IgA antibody secreting cells to the lactating mammary gland and gastrointestinal tract. This protein has also been shown to function as an antimicrobial peptide. Cows and other ruminants do not transfer antibody transplacentally as do humans and mice. Therefore efficient transfer of immunity to the suckling newborn in ruminants is highly dependant on efficient transfer of antibodies via colostrum. Neither the role of CCL28 in the transfer of immunity from mother to nursing neonate, nor the role of CCL28 as a potential antimicrobial compound in milk has yet been elucidated in the cow. To begin addressing these basic questions we are cloning and expressing bovine CCL28. Using a putative bovine sequence of CCL28 from GenBank and known sequences from mouse and human CCL28, appropriate primers with restriction sites were created for bovine CCL28. Salivary gland mRNA was collected and RT-PCR was employed to amplify the coding region of the gene. The amplified fragment was sequenced to establish concordance with GenBank records. The coding region was then inserted into a pMAL-c2G vector and is being used to generate recombinant bovine CCL28.
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