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The avidity of IgG antibodies directed to Neospora caninum was measured using an IgG avidity enzyme-linked immunosorbent assay (ELISA) employing N. caninum proteins incorporated into immunostimulating complexes as antigen. In this ELISA, low-affinity antibodies were eluted by adding an incubation step with urea after the serum incubation. The antibody titers obtained with and without incubation with urea were then used to calculate the IgG avidity values. Analysis of sequential sera collected from experimentally infected calves revealed that the avidity increased during the course of infection. Three weeks after infection, the IgG avidity was 9-18%, and 24 weeks later it had increased to 58-76%. Cattle naturally infected for more than 6 months all had avidities >50%. The results in this study, however preliminary, indicate that the IgG avidity ELISA can be used to discriminate between recent and chronic N. caninum infections and may therefore be a valuable complement to IgG assays in epidemiologic studies of N. caninum infection in cattle.

Neospora caninum is a cyst-forming coccidian parasite which in cattle can cause abortion and birth of feeble calves. For the serological diagnosis of neosporosis, antibodies directed to the parasite can be demonstrated by ELISA, utilizing N. caninum proteins incorporated into iscoms (immunostimulating complexes) as antigen. Electrophoretic and immunoblotting studies had earlier revealed that N. caninum iscoms contained a restricted number of proteins compared with soluble parasite extracts, but the cellular origin of the incorporated proteins has not yet been determined. In the present study, monoclonal antibodies were raised against N. caninum iscoms. Six of these, named Ncmab-4, 7, 10, 13, 17 and 24, were used to characterize the N. caninum iscom antigen. The nature of the reactive epitopes and their localization within N. caninum tachyzoites were determined by means of immunological methods, including immunoblot, IFAT and immunogold electron microscopy. The apparent molecular weights of the dominant iscom antigens were found to be 18, 30, 32, 41 and 61 kDa. While the 61 kDa antigen was located intracellularly, the others were found on the parasite surface as well as within distinct intracellular compartments.

Immunostimulating complexes (iscoms) are cage-like structures of about 40 nm composed of Quil A, cholesterol, phospholipids and antigen. Their main area of use has been as adjuvants and carriers of immunogens in vaccines. Iscoms can also be used for selection of surface membrane proteins of micro-organisms for use in immunoassays, thus decreasing the number of internal proteins that might cause problems with non-specific binding and cross-reactivity. Enzyme-linked immunoassays (ELISAs) utilising parasite antigens incorporated into iscoms have been developed for demonstration of antibodies directed to the intracellular coccidian parasites Toxoplasma gondii and Neospora caninum. These iscom ELISAs have proved very reliable, with high sensitivity and specificity. The preparation of T. gondii and N. caninum iscoms is described, and ELISAs based on iscom antigen preparations that have so far been used for diagnosis of protozoal infections are reviewed.

In order to obtain data on the occurrence of the protozoan parasite Neospora caninum as a potential factor in the aetiology of reproduction problems in cattle, 97 postabortion sera were examined by ISCOM ELISA and IFAT for the presence of antibodies in N. caninum. The results showed 10% and 9% seropositivity by the ELISA and IFAT, respectively. In 2 of the 10 seropositive cases no other fetopathogenic agents (such as Chlamydia sp., Leptospira spp. or bovine viral diarrhoea virus) were detected. These data confirm the presence of N. caninum in cattle in Hungary.

Serum samples from 70 (33 aborting and 37 non-aborting) dairy cows from a herd in California were analyzed for Neospora caninum antibodies in different laboratories by various serologic assays including enzyme-linked immunosorbent assay (ELISA) with recombinant antigens (Nc4.1 and Nc14.1), kinetic ELISA, whole tachyzoite lysate ELISA, immunostimulating complex (iscom) ELISA, antigen capture competitive inhibition ELISA, and by the indirect fluorescent antibody test. Eighteen percent of pregnant cows in this herd had aborted within 2 mo of the index case. All 70 cows had antibodies to N. caninum by at least 1 of the tests. Antibody levels to N. caninum in aborting cows as a group were higher than in nonaborting cows. However, it was concluded that no serological test could be used to establish definitively that N. caninum caused the abortion in an individual cow.

An indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Neospora caninum in serum from cattle is described. Extracted tachyzoite proteins incorporated into immunostimulating complexes (iscoms) were used as coating antigen and a mouse monoclonal antibody to bovine immunoglobulin G1 as conjugate. Western blot analysis of the iscom preparation revealed a restricted number of antigens compared with whole parasite homogenates. When probed with a serum from an experimentally infected calf, heavily stained antigens with apparent molecular masses of 28, 35, 45 and 78 kDa were seen. The sensitivity and specificity of the ELISA was 100% and 96%, respectively, against an indirect fluorescent antibody test as indicator of true status. The applicability of the ELISA for demonstration of antibodies in milk was evaluated and the agreement between serum and milk ELISA was 95%.

The brain of a stillborn calf, seropositive to Neospora caninum and born to a seropositive cow, was homogenized and cultured on Vero cells, where growth of Neospora-like tachyzoites was detected after 8 weeks. The ultrastructural features of the new isolate (Nc-SweB1) corresponded to those of previously published Neospora isolates. In indirect immunofluorescence tests, antigens on Nc-SweB1 tachyzoites were recognized by antibodies raised to a canine N. caninum isolate (Nc-1) but not by antibodies to Toxoplasma gondii, Sarcocystis cruzi, S. tenella, Eimeria alabamensis, Babesia divergens, or B. motasi. Immunoblot analyses revealed no major antigenic difference between Nc-SweB1 and Nc-1, whereas several differences were seen between Nc-SweB1 and protozoa related to N. caninum. The sequences of 16S-like rRNA and the internal transcribed spacer 1 of Nc-SweB1 revealed complete homology with corresponding sequences of two canine N. caninum isolates. Thus, no dissimilarity between Nc-SweB1 and the canine isolates was found, confirming that Nc-SweB1 is N. caninum and suggesting that Neospora-like organisms isolated from cattle are indeed N. caninum.

Objective: To investigate the route of transmission of Neospora sp in a herd of dairy cattle in which sporadic abortions had been observed since the establishment of the farm in 1980., Design: Serum samples were screened for antibodies to Neospora sp, and records from an artificial insemination program were analyzed., Animals: 58 female cattle., Procedure: An ELISA was used to screen serum samples of antibodies to Neospora sp. Fertility, calf mortality, and relationships between specific cattle were investigated. Statistical analysis was performed on the fertility data., Results: Antibodies were detected in 17 of 58 (29%) tested cattle. All seropositive cattle were descendants of 2 cows purchased in 1980. Cattle that were descendants of those 2 cows were compared with their herdmates, but significant differences were not detected in the number of inseminations per confirmed pregnancy or in the number of cattle that required more than 1 insemination/ pregnancy. Since 1980, there were 323 confirmed pregnancies in the herd, and calf mortality (prenatal and perinatal mortality) was 24 of 323 (7%)., Clinical Implications: Congenital transmission of Neospora organisms together with the apparent lack of horizontal transmission observed in the herd reported here indicated that Neospora sp has the ability to be transmitted from dam to offspring for several generations. This mode of transmission would explain the maintenance of infection in a population of cattle despite the lack of a definitive host for the parasite.

The concentrations of sodium, potassium, calcium, magnesium, phosphorus, sulphur and chlorine, in the skeletal muscle fibres of cows with parturient paresis, downer cows, normally calving cows and unmated heifers were determined by means of an X-ray microanalysis technique. The most consistent finding was an increase in the concentration of chlorine in the muscle samples from the downer cows. There were no other significant differences between the concentrations of these elements in samples from the different groups of cows, but the calcium concentration was higher in the muscle samples collected one month after parturition than in the samples collected close to parturition.

Several experiments were conducted to determine whether the ingestion of diterpenoids (resin acids) by pine sawfly larvae influences the survival of postlarval stages. Larvae of two diprionid sawfly species were reared on shoots of two Scots pine clones, one with a low (1.5% dry wt) concentration of resin acids and the other with a high (5.2% dry wt) concentration. No significant treatment-related differences were found in any of the experiments with respect to (1) resistance against parasitoids, (2) preference of predatory shrews and carabids, and (3) apparency of cocoons in the field to predators. A preference of sawfly prepupae to spin cocoon in feces from larvae reared on high resin acid needles was found. Possible explanations for these results are discussed. Detection of an unknown compound, possibly a breakdown product of the major resin acid in pine needles (pinifolic acid), in prepupae indicate that resin acids may be metabolized by the sawflies.

High producing multiparous dairy cows were fed either diets differing in energy content or diets with identical energy and protein content but differing in roughage content at the end of the dry period and beginning of lactation. Basal insulin and ketone bodies were analysed every week from 3 weeks before to 7 weeks after calving. Pancreatic glucagon was estimated 3 weeks before, 1-3 days after, and 3 weeks after calving. Before calving the feeding regimen had a very strong influence on the basal insulin level. High amounts of concentrate increased basal insulin levels until one week before calving and caused an interruption in the physiological decreasing course. After calving the insulin levels were low in all groups of cows. Before calving there were small variations in the glucagon levels, and no influence of feeding was observed. After calving there was a strong increase, especially in the cows fed the highest amounts of concentrate. Feeding high amounts of concentrate resulted in varying and in many cases increased levels of ketone bodies in plasma. Hyperketonemic cows had lower insulin and higher glucagon levels than normal cows. The influence of non-structural carbohydrates in the feed on pancreatic hormones is a cause of ketogenesis is discussed.

Blood samples were collected from the coccygeal artery and a jugular vein two weeks before expected calving, one to five days after calving and three weeks after calving of cows which were fed either a high roughage or a low roughage diet from four weeks before to 14 weeks after calving. The mean venous acetate concentration ranged from about half to three-quarters of the arterial concentration. No differences were observed at any time between concentrations of acetate in either the arterial or venous blood of the cows on the different diets.

The concentration of acetate in blood plasma was measured by both a gas-liquid chromatographic method and an enzymatic method using acetyl-CoA synthetase. When acetate was measured enzymatically without previous protein precipitation, the apparent concentration was lower than when the concentration measured by either a gas-chromatographic method or by the enzymatic method after protein precipitation with perchloric acid and neutralization.

Changes in needle nitrogen and resin acid concentrations in young Scots pine trees fertilized with ammonium nitrate were followed over 3 years. Sawfly larvae (Neodiprion sertifer) were reared on fertilized and control trees the year after fertilization. Both nitrogen and resin acid concentrations increased in fertilized trees. The fact that resin acid concentrations increased contradicts predictions of the carbon/nutrient balance hypothesis. We suggest that needle resin-acid concentrations are limited more by the size of the resin ducts than by the availability of substrate for resin acid synthesis, and that the formation of resin ducts is limited by the availability of nitrogen. A modification of the carbon/nutrient balance hypothesis, relating compartment formation to allelochemical synthesis, is discussed. Performance of sawfly larvae was not affected by fertilization treatment, probably because concentrations of nitrogen (positively affecting performance) and resin acids (adversely affecting performance) increased simultaneously in fertilized trees. Thus, the results of this study do not support the notion that fertilization increases the resistance of trees to needle-eating insects.

The idea of trade-offs among antiherbivore defences in plants is examined using data from a South American blackberry (Rubus bogotensis). Two distinct morphs of R. bogotensis, one with glandular trichomes and one without, were compared with respect to leaf toughness, number of prickles and prickle length. The two morphs were sympatric and grew under similar environmental conditions. The morph lacking trichomes had significantly tougher leaves and also tended to have more and longer prickles. Bioassay showed that Ithomiid larvae fed to a lesser extent on tough leaves than on more tender ones. Correlations between antiherbivore defences within each phenotype revealed three significant or almost significant negative relationships. The comparisons support the hypothesis that trade-offs exist among antiherbivore defences.

1. Comparative data are presented of the activities of pyruvate dehydrogenase complex and acetyl-CoA synthetase and of the acetate content in homogenates from ventral grey matter in spinal cord from cows and two non-ruminant species, pigs and horses. The methods used in the study are evaluated and discussed. 2. The total pyruvate dehydrogenase complex activity was 24.9-29.9 mU/mg protein and did not differ between the species. The part of the complex that was in active form at the sampling occasion was 60, 85 and 95% in cows, pigs and horses, respectively. 3. Acetyl-CoA synthetase activity differed significantly between the species and was 0.93, 1.28 and 2.61 mU/mg protein in pigs, cows and horses, respectively. The highest cytosolic activity was found in the horses. Acetate concentration at half maximal reaction velocity (at saturating CoA and ATP levels) was found to be 0.15-0.70 mM and did not differ between the species. 4. Acetate content was 63, 83 and 96 micrograms/g wet wt in cows, horses and pigs, respectively. 5. It is concluded that there seems to be no striking difference in acetyl-CoA synthesis in peripheral nerves between ruminants and non-ruminant species.

We have studied how differences in needle resin acid concentrations between two clones of Scots pine influenced larval survival, larval developmental time, and cocoon weight in Neodiprion sertifer. Larvae were reared under controlled conditions in the laboratory on needles showing a three-fold difference, in resin acid concentration. Larval developmental time was significantly longer for larvae fed needles high in resin acids. No significant differences were found in cocoon weight. Larvae fed needles high in resin acids suffered significantly higher mortality, most of which occurred, in the first two instars. Data on relative growth rate for last-instar larvae, based on a traditional growth efficiency experiment, did not show any negative effects of resin acids, indicating that a possible induction takes place during early instars. Observations on the feeding behaviour of last-instar larvae, the high consumption of bark tissue (with high concentrations of resin acids) by larvae fed needles low in resin acids and preference for needle tissues high in resin acids, indicate that these larvae might actively search for tissues rich in resin acids. We conclude that the physiology and behaviour of N. sertifer has been shaped, in part, by the conflict between the negative effects of resin acids, primarily in early instars (longer developmental time with a corresponding increase in exposure to natural enemies and an increase in direct mortality), and the positive effect of resin acids in later instars (a need for resin acids for use in its own predator defence secretion).

Bird densities were estimated on 41 small islands and two mainland plots at a South Swedish lake both in 1976 and 1983. In the latter year, three additional plots were also censused. The ratio between combined densities of hole-nesting birds on the mainland and on islands was 3:1 both in plots without and with nest boxes. In plots with boxes combined densities of hole-nesting birds doubled compared with control plots. This increase was caused by a tenfold increase of pied flycatcher Ficedula hypoleuca. Territories of this species were on average established about a week later on the islands compared with the mainland. Furthermore, 50% of the males on the islands did not attract a female. Densities of great tit Parus major, marsh tit Parus palustris and nuthatch Sitta europaea were unaffected by increased nesthole availability. For P. major this result contrasts with those in other studies.The density of chaffinch Fringilla coelebs in habitats with similar height and vertical structure was two times higher on the islands compared to the mainland. On the islands the density was the same on islands with only one pair and on those with two or more pairs. In spring, there were no significant differences between islands and the mainland in the proportion of leaves with insect feeding traces. The proportion of Salix leaves with feeding traces increased with island size, but this was not so for Alnus and Betula leaves. In late summer, the proportion of leaves with feeding traces were halved inside a plot with nest boxes and hence increased bird densities compared to a nearby control plot. This result was the same along the lake shore and about 150 m away from the shore.The discussion centers on the effect of man on the food-and nest site-availability of hole-nesting birds, food limitation of insectivorous birds and density compensation on islands.

A gas chromatographic method was evaluated for the quantification of volatile fatty acids in blood plasma from ruminants. Amounts of .002 mg acetic acid and .001 mg of propionic and butyric acid could be quantified. Diurnal variation in acetate and propionate was found when blood samples were taken during 1 d from five cows. Jugular blood samples were taken from the five cows 8 wk before parturition to 10 wk after parturition and from three of their calves to 32 wk of age. Acetate decreased in cows at the weeks around parturition. Calves had very low acetate during the first 10 wk of life, but the concentration subsequently increased to that in adult ruminants. Propionate concentration was below .17 mM and butyrate concentration below .08 mM in all samples. It was concluded that this method was suitable for analyzing volatile fatty acids in blood plasma from ruminants, which is of importance in the study of metabolic diseases.

To examine if differences in egg predation rates could explain differences in bird community composition, egg predation was studied in two years on small islands in a South Swedish lake and on the nearby mainland using both natural and artificial nests.In plots with similar vegetation, the combined density of ground- and tree-nesting bird species did not differ between the islands and the mainland. Egg predation rates were similar on islands and the mainland for natural Turdus nests in two years, and for artificial Turdus and Phylloscopus nests. Unmarked and unvisited experimental nests suffered similar rate of egg predation as marked and visited nests. Egg predation rates were higher on natural nests when artificial nests were also put out, increasing the total nest density. Initial egg predation rates in artificial nests were also higher than later when nest density had decreased by 75%.The egg predators involved differed for artificial Phylloscopus nests between the islands and the mainland. Small mammals were apparently responsible for 29% of the predation on the mainland, but none on the islands. Artificial Turdus nests near crow nests suffered from a higher egg predation rate than nests further away from crow nests. Daily survival rates of Turdus nests increased from the laying to the incubation and further to the fledging state.Egg predation can not explain differences in bird community composition between islands and mainland in the present case.