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.     

Clinical experience with kennel-specific E. coli oral vaccines in dogs

Kennel-specific oral E. coli vaccines were tested for efficacy and side effects at breeding and boarding kennels with severe diarrhoea problems. Oral vaccines contained heat inactivated E. coli bacteria from specific kennels and were given once daily for 14 days. Oral vaccines were administered directly orally to puppies while for adult dogs vaccines were mixed with daily food rations. In breeding kennels with 4 to 10 week old puppies suffering from non-fatal diarrhoea, the oral immunization led to a decrease in morbidity from 86.5% to 0%. In kennels with some cases of fatal diarrhoea the rate of morbidity decreased from 45% to 21% and the mortality rate from 25% to 10.3% after using the vaccination. By vaccinating adult dogs in boarding kennels the morbidity rate dropped from 83.5% to 6.5% and the mortality rate from 4.1% to 0.5%. The kennel specific oral E. coli vaccine was found to be free of side effects. No adverse effects were observed in either puppies or adult dogs.     

On the structure of the adrenal gland of the common seal (Phoca vitulina vitulina)

The adrenal gland is a vitally important endocrine gland that occupies a central role in the regulatory mechanisms of the body metabolism. Environmental stress factors lead to permanent strain and overload of the body resulting in structural alterations of the adrenals that in turn are followed by hormonal imbalances. This leads to an increased susceptibility to bacterial and viral diseases. The recurrence of numerous fatalities in the different seal populations of the North Sea (during the years 1988, 1989 and 2002), of the Baikal Lake and Caspian Sea (during the years 2000 and 2001) were the motive for a morphological investigation of the species-specific structure of the adrenal gland of the common seal in order to differentiate environmental stress-induced pathological alterations from the physiological structure of this organ. The study was based on adrenals of 112 common seals (Phoca vitulina vitulina) using light microscopic and transmission and scanning electron microscopic methods. The phocine adrenal gland displays several structural characteristics. Originating from the connective tissue organ capsule, narrow and broad septa intersperse the adrenal cortex. These septa contain blastemata as a reserve for the regeneration of hormone-producing cortical cells. Such blastemata are also occurring in the form of an intermediate zone in between the zona glomerulosa and zona fasciculata in the phocine adrenal cortex. Another species-specific characteristic is an inverse part of the adrenal cortex encircling the central vein of the organ. These structural features have to be considered in assessment and definition of pathological alterations of the adrenals as observed in the form of exhausted blastema cell pools in the adrenocortex of seals perished in the mentioned phocine mass mortalities.     

Evidence of shedding of porcine circovirus type 2 in milk from experimentally infected sows

Detection of porcine circovirus type 2 (PCV2) was to evaluate the milk from experimentally infected sows using polymerase chain reaction (PCR) and virus isolation. Six pregnant sows were inoculated intranasally with PCV2 at 93 days of gestation, and milk samples were collected from all sows at 1, 3, 6, 9, 12, 15, 18, 21, 24, and 27 days of lactation. PCV2 was detected in milk as early as day 1 of lactation in all six sows. Thereafter, all infected sows remained positive by PCR for PCV2 in milk until 27 days of lactation. In addition, PCV2 itself was isolated from milk collected from a virus-infected sows. These results suggest that PCV2 may be shed in milk following infection of pregnant sows by the virus.     

Comparison of a new synthetic, peptide-derived, polyclonal antibody-based, immunohistochemical test with in situ hybridisation for the detection of swine hepatitis E virus in formalin-fixed, paraffin-embedded tissues

A synthetic, peptide-derived, polyclonal antibody-based, immunohistochemical test was developed to detect swine hepatitis E virus (HEV) and was compared with in situ hybridisation for the detection of HEV in formalin-fixed, paraffin-embedded tissues from experimentally infected pigs. Solid-phase peptide synthesis was used to generate peptides from swine HEV open reading frame 2, and the purified peptides were injected into rabbits to produce polyclonal antibodies. The specificity and sensitivity of the test were both 100%. Liver was most consistently positive for swine HEV antigen and RNA by immunohistochemistry and in situ hybridisation, respectively, but both were detected much less frequently in extrahepatic tissues such as lymph node, tonsil, spleen, and intestine. Swine HEV antigen and RNA showed a similar distribution in virus-infected hepatocytes in serial sections. The novel test developed in this study is suitable for consistently detecting swine HEV antigen in formalin-fixed, paraffin-embedded tissues.     

Membrane-coating granules and the intercellular cementing substance (membrane-coating material) in the epidermis in different regions of the equine hoof

This study aimed to carry out a comprehensive morphological examination of the structure and ultrastructure of the membrane-coating granules (MCG) and membrane-coating material (MCM) common to all hoof segments as well as segment-specific features in the MCG and MCM regarding their synthesis, structure and biochemical composition. Tissue samples from the hooves of the left front and right hind limb of 20 adult riding horses were used. Samples were preserved and embedded for histological routine methods as well as for histochemical techniques and tracer techniques for light and electron microscopy. The MCM shows a segment-specific structure, biochemical composition and function, which were demonstrated for the different parts of the hoof. The MCM and its properties are related to the segment-specific horn quality. Besides intercellular structural factors the MCM is of major importance for the structural integrity and the biomechanical properties of the hoof horn. The physiological structure and composition of MCM provides explanation for the existence of clinically well-known sites of predilection for hoof disorders such as the White Line Disease. Alterations of the MCM play a role in dysfunction and diseases of the hoof epidermis; they are particularly involved in the pathogenesis of infectious diseases of the hoof.     

The regional distribution and relative frequency of gastrointestinal endocrine cells in SHK-1 hairless mice: an immunohistochemical study

The regional distributions and relative frequencies of some gastrointestinal endocrine cells in the eight portions (fundus, pylorus, duodenum, jejunum, ileum, caecum, colon and rectum) of the gastrointestinal tract of SKH-1 hairless mice were investigated using immunohistochemical methods and seven types of specific antisera against somatostatin, serotonin, glucagon, cholecystokinin (CCK)-8, secretin, pancreatic polypeptide (PP) and gastrin. In this study, somatostatin-, serotonin-, glucagon-, CCK-8-, secretin- and gastrin-immunoreactive (IR) cells were identified. Most of these IR cells in the intestinal portion were generally spherical or spindle-shaped (open-type cell) while cells that were round in shape (close-type cell) were occasionally found in the stomach regions. Their relative frequencies were varied according to each portion of gastrointestinal tract. Somatostatin-IR cells were found throughout the gastrointestinal tract except for the large intestine. Serotonin-IR cells were detected throughout the whole gastrointestinal tract and were the most predominant endocrine cell types in this species of mouse. Glucagon-IR cells were restricted to the fundus, occurring rarely. CCK-8-IR cells were observed in the pylorus, duodenum and jejunum with frequencies that were numerous, moderate and few, respectively. Peculiarly, secretin-IR cells were demonstrated in the whole intestinal tract with either few or rare frequencies. Gastrin-IR cells were restricted to the pylorus and were numerous. However, no PP-IR cells were found in this study. In conclusion, some peculiar distributional patterns of gastrointestinal endocrine cells were found in SKH-1 hairless mouse.