Identification of Cytokeratins in Bovine Sperm Outer Dense Fibre Fractions

Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)‐insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll^® density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB‐insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS‐insoluble material was collected and quantitatively dissolved in 8 M urea. SDS‐gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti‐cytokeratin antibodies detected two urea‐soluble, SDS‐insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45‐kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1–18 and CK 20), KL1, was the only antibody that reacted with the 66‐kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope‐like structures, we suggest that these intermediate filaments play an important structural or tension‐bearing role in sperm flagella.     

A specific DNA probe which identifies Babesia bovis in whole blood.

A genomic library of Babesia bovis DNA from the Mexican strain M was constructed in plasmid pUN121 and cloned in Escherichia coli. Several recombinants which hybridized strongly to radioactively labeled B. bovis genomic DNA in an in situ screening were selected and further analyzed for those which specifically hybridized to B. bovis DNA. It was found that pMU-B1 had the highest sensitivity, detecting 25 pg of purified B. bovis DNA, and 300 parasites in 10 microliters of whole infected blood, or 0.00025% parasitemia. pMU-B1 contained a 6.0 kb B. bovis DNA insert which did not cross-hybridize to Babesia bigemina, Trypanosoma evansi, Plasmodium falciparum, Anaplasma marginale, Boophilus microplus and cow DNA. In the Southern blot analysis of genomic DNA, pMU-B1 could differentiate between two B. bovis geographic isolates, Mexican strain M and Thai isolate TS4. Thus, the pMU-B1 probe will be useful in the diagnosis of Babesia infection in cattle and ticks, and in the differentiation of B. bovis strains.     

Two cases of colitis cystica profunda in dogs

Colitis cystica profunda in dogs has been diagnosed in one case only. The two own cases were characterized by repeated, partly bloody diarrhea, vomitus, and painful defecation. The disease was diagnosed by clinical examination and colonoscopy with the ensuing histological examination of biopsy specimens. The disease could be managed by administration of a diet, sulfasalazine and corticosteroids.