Purification and serological analysis of tomato spotted wilt virus

A modified procedure for the purification of TSWV is described which consists of differential centrifugation, treatment with antiserum against sap from healthyN. rustica and density-gradient centrifugation in sucrose gradients. Centrifugation in the zonal-rotor proved efficient for processing large amounts of infected leaves.Antisera to purified TSWV were produced in rabbits. These sera reacted with healthyN. rustica sap. This reaction could be abolished by absorption with either healthyN. rustica sap or polysaccharides purified fromN. rustica. Unabsorbed sera reacted with the major TSWV structural proteins following their electrophoretic separation in polyacrylamide-gels. The positions of the precipitin lines corresponded with those of the structural proteins visualized by staining. The intensity of these lines was greatly diminished after absorption of the antisera with either healthyN. rustica sap or polysaccharides. It was concluded that the reaction of TSWV antiserum with the structural proteins is partly due to host-derived polysaccharides covalently bound to the virus proteins.Samenvatting Het bronsvlekkenvirus van de tomaat (TSWV) werd gezuiverd met een procedure die bestond uit een cyclus van differentiële centrifugering, behandeling van de virus-suspensie met antiserum tegen bestanddelen van gezonde planten (N. rustica) en dichtheidsgradiëntcentrifugering (Fig. 1 en Fig. 3). De zone rotor kon worden gebruikt wanneer grote hoeveelheden bladmateriaal werden gebruikt (Fig. 2).Twee antisera, die elk een verschillende activiteit tegen TSWV (Tabel 1, Fig. 4) hadden, werden bereid. Deze sera reageerden ook met sap en polysacchariden uitN. rustica (Fig. 6). Door verzadiging met polysacchariden werd de reactie tegen TSWV aanmerkelijk verzwakt. Analyse van de eiwitbandjes die na polyacrylamidegel-elektroforese waren gevormd, toonde aan dat de activiteit van de sera tegen de eiwitcomponenten van TSWV was gericht (Fig. 5) en dat deze activiteit na verzadiging met polysacchariden zwakker was. Uit deze reacties kan geconcludeerd worden dat polysacchariden van de waardplant aan de structurele eiwitten van TSWV zijn gebonden. Niet geabsorbeerd antiserum geeft met TSWV drie precipitatielijnen (Fig. 4), maar na absorptie met compleet virus trad nog slechts één precipitatielijntje op (Fig. 7). Mogelijk wordt dit lijntje gevormd door het nucleocapside-eiwit dat door de dissociatie van het virus vrij komt. Sera die tegen normale bestanddelen vanN. rustica zijn bereid, vormen geen zichtbare reactie met compleet virus; wel wordt er een lijntje gevormd indien het virus met guanidine-HCl wordt gedissociëerd (Fig. 8). Deze waarneming kan verklaren dat deze antisera kunnen worden gebruikt bij de zuivering van TSWV ondanks het feit dat dit virus polysacchariden uit de waardplant draagt.     

Effect of prostaglandin F2 alpha on hormone concentrations in dairy cows after parturition

This study was designed to evaluate the effects of exogenous prostaglandin F2 alpha (PGF2 alpha) on hormone secretion in cows without a corpus luteum. Blood samples were taken from 10 Friesian dairy cows at frequent intervals from a jugular vein and the caudal vena cava starting between nine and 20 days after parturition. PGF2 alpha (25 mg dinoprost) was injected intramuscularly into five cows after the first eight hours of sampling. Plasma concentrations of 13,14-dihydro 15-keto PGF2 alpha (PGFM) increased rapidly but had returned to baseline by 14 hours after injection. There was no significant effect of the treatment on the time taken by the cows to resume ovarian cycles, and it had no consistent effect on plasma luteinising hormone (LH) patterns; however the amplitude of pulses of LH was temporarily suppressed in two cows and the frequency of pulses of LH was immediately increased in one cow. Treatment with PGF2 alpha had no significant effect on the concentration of oestradiol in blood from the vena cava. It is concluded that any enhancement of the reproductive performance of cows treated with PGF2 alpha after parturition is not due to a direct effect on pituitary-ovarian function.     

Effect of trenbolone and testosterone on the plasma elimination rates of sulfamethazine, trimethoprim, and antipyrine in female dwarf goats

Plasma elimination rates of sulfamethazine (100 mg/kg of body weight, IV), trimethoprim (20 mg/kg, IV), and antipyrine (35 mg/kg, IV) were studied in adult female dwarf goats (n = 5) before and after implantation with trenbolone acetate (5 mg/kg). Pretreatment with trenbolone caused a significant decrease in the elimination rate of the drugs tested: for sulfamethazine, 5 times; for antipyrine, 3 times; and for trimethoprim, 2 times. After treatment with testosterone (1 mg/kg, SC, twice weekly for 2.5 weeks), female goats (n = 5) had a similar decrease in the elimination rate of sulfamethazine. Other induced effects included a change in social behavior, a lower voice, and the development of a typical billy goat-like odor. Plasma creatinine concentrations after androgen administration were significantly higher than those before androgen administration; changes were not observed in plasma urea values. Because of the differences observed, we believe that more attention should be paid to the effects of androgenic agents on drug kinetic properties, with particular reference to studies on clinical efficacy, side effects, and drug residues in food products.     

Haptoglobin and serum amyloid A in milk from dairy cows with chronic sub-clinical mastitis

New tools are needed to detect chronic sub-clinical mastitis, especially in automatic milking systems. Haptoglobin and serum amyloid A (SAA) are the two most sensitive bovine acute phase proteins, and their concentrations increase in milk from cows with clinical mastitis and in milk from cows with experimentally induced chronic sub-clinical Staphylococcus aureus mastitis. The aim of this study was to further evaluate the potential for haptoglobin and SAA in milk as indicators of chronic sub-clinical mastitis. Quarter milk samples were collected from 41 cows with a mean composite milk somatic cell count (CSCC) above 300,000 cells/mL during at least two months prior to sampling. Quarter milk samples were also taken from eleven cows with a mean CSCC below 80,000 cells/mL during at least two previous months. These samples were analysed for haptoglobin, SAA, adenosine triphosphate (ATP) activity and bacterial growth. The samples were grouped according to their ATP, haptoglobin and SAA status. ATP+ samples had ATP > 2 x 10(-10) mol/mL, Hp+ and SAA+ samples had detectable levels of haptoglobin (> or = 0.3 mg/L) and SAA (> or = 0.9 mg/L), respectively. In udder quarter samples from healthy cows, 42 out of 44 samples belonged to the ATP-Hp-SAA- group. Among cows with chronic sub-clinical mastitis, the ATP+Hp+SAA+ group contained 66 out of 164 samples while 44 samples belonged to the ATP+Hp-SAA- group. Detectable levels of haptoglobin and SAA were found in 92 and 80 samples, respectively. Growth of udder pathogens was detected in 28 samples and Staphylococcus aureus was the most common bacteria. In conclusion, haptoglobin and SAA concentrations below the detection limit were considered as good indicators of healthy udder quarters. A substantial variation in haptoglobin and SAA concentrations in milk was observed in udder quarters with chronic sub-clinical mastitis.     

The diagnosis of listeria encephalitis in ruminants using cultural and immunohistologic methods. II. Immunohistologic studies in formalin-fixed paraffin sections]

The unlabelled peroxidase-antiperoxidase (PAP)-technique was compared with the avidin-biotin-peroxidase-complex (ABC)-technique in the identification of Listeria antigen in formalin-fixed paraffin sections of 58 ruminant brains, 44 of which showed histopathological lesions typical for listeric encephalitis. Rabbit hyperimmune serum, obtained by immunization with a Listeria monocytogenes strain of serotype 1/2 a, served as primary serum in the immunohistochemical investigations. The antiserum was tested for specificity using formalin-fixed smears of various bacterial species. Listeria antigen was demonstrated in 40 of the 44 brains with histopathological CNS alterations, as seen in listeriosis, using the ABC method, whereas their identification with the PAP method only succeeded in 34 of the brains. Despite the higher dilution of the primary antibody, the ABC method distinguished itself further, in comparison with the PAP method, through more intense immunohistochemical staining of Listeria antigen. Listeria spp, could be isolated from 46 of 52 brains, which were also examined bacteriologically. They could be isolated from 14 brains, which showed no histopathological lesions indicative of listeriosis. In contrast to this, Listeria antigen was only detected immunohistologically in brains with typical histological listeric CNS alterations. In three cases the presumptive histological diagnosis could not be confirmed immunohistologically.