Evaluation of mono- and polyclonal antibody-based antigen detection immunoassays for diagnosis of Trypanosoma evansi infection in the dromedary camel.

Two enzyme-linked immunosorbent assays (ELISA), one based on a mouse anti-Trypanosoma brucei group-specific monoclonal antibody and the other on rabbit anti-Trypanosoma evansi polyclonal antibodies, have been evaluated for their ability to detect circulating trypanosome antigens in camel sera as a means for the diagnosis of T. evansi infections. All 91 sera from a negative control camel herd from Kenya gave negative antigen-ELISA results in the monoclonal antibody-based ELISA and only 2 of them (2.2%) gave false positive results in the polyclonal antibody-based ELISA. In subsequent analyses of sera from infected camels (as determined by mouse inoculation), the monoclonal antibody-based ELISA detected antigens in 90 (83.3%) out of the 108 sera tested. This percentage was lower for the polyclonal antibody-based ELISA which was able to detect antigens in 67 (60.9%) out of the 110 sera tested. The two tests detected probably different antigens and when the results were combined, 99 out of 107 (92.5%) sera were shown to be ELISA positive. In a survey involving 316 camels from the Gao and Nara areas, in Mali, a high proportion of animals tested were antigen positive (43.5 and 42.9%, respectively for the mono- and polyclonal antibody-based ELISA) compared to only 22 (7.0%) diagnosed by the parasite detection techniques. Thus, these immunoassays were at least six times more sensitive than the haematocrit centrifugation technique. As a large proportion of cases may be antigen positive but parasite negative, these two of "surra" immunoassays should be used in routine diagnosis in addition to the parasite detection techniques in the dromedary camel.     

The effect of aging of milk samples on the accuracy of infrared analysis of milk composition]

The effect of a decrease (and/or fermentation) in the lactose content during milk storage under different conditions was investigated on the accuracy of the results obtained on a Milko-Scan apparatus to contribute to the present knowledge of this problem. The results were in agreement with some results cited in the literature. These wavelengths are used for infrared spectrophotometry on the above apparatus: for fat 3.48 microns, for proteins 6.46 microns and for lactose 9.60 microns. Bulk milk samples used for the tests were untreated or treated with potassium dichromate, bronopol, sodium azide and Milkofix at the temperatures of storage in darkness 20 degrees C and 4 degrees C. The differences against the reference values (measured on the first day) were determined and evaluated in milk composition and characteristics as arising during milk storage. These differences were used in form of either cumulative means of differences (Figs. 1 to 5) or individual differences (Fig. 8). In the first part significant correlation coefficients (P less than 0.001) were calculated for the relationship between the variations of lactose content and the fat and protein contents: r = -0.59 and/or -0.73 (Figs. 6 and 7). This suggests that the decrease in the lactose content by 0.10% recorded by the infrared analysis and caused by lactose decomposition is accompanied by a "seeming" increase in the fat and protein content by about 0.04%. In the second part the correlation coefficients for the fat and protein contents r = -0.96 and -0.96 (P less than 0.001; Figs. 9 and 10; Tab. II) were calculated on the basis of an observation of the lactose decrease in an untreated milk sample (20 degrees C for 28 hours). These coefficients are somewhat different from the preceding ones; this is due to the lower homogeneity of the first set where the milk samples were treated in a different way, but the coefficients confirm the same conclusions. The values of the correlation coefficients for the dependence between the development of the acquired titratable acidity (SH) and the variations of fat (F), protein (P) and lactose (L) contents were as follows: r = 0.95; 0.95; -0.99 (P less than 0.001; Figs. 12, 13; Tab. II). Thus the above-mentioned "seeming" increase in the F and P contents can be explained to the extent of 92.2% from the decrease in the L content, which also causes the increase in titratable acidity to the extent of 98.0%.(ABSTRACT TRUNCATED AT 400 WORDS)     

‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3′-ends, but also the 5′-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

Changes in the plasma concentrations of prolactin,luteinising hormone,progesterone and d‐(β)‐hydroxybutyrate in turkey hens (meleagris gallopavo), during treatment of broodiness under commercial conditions

1. An experiment was done under commercial conditions to investigate the physiological effects of isolating broody turkey hens, for 72 h, in sand and wire floored pens on the third, 10th and 16th weeks of production.

2. Hens identified as broody and removed from the flock had higher plasma prolactin concentrations than the laying hens at each of the three experimental stages.

3. Confinement in sand and wire floored pens, induced a decline in plasma prolactin concentrations. This decline probably impeded immediate development of broody behaviour. Alternately, levels of prolactin higher than those of laying hens were again measured 7 and 14 days after treatment during third week but not after the 10 th and 16th week of production.

4. Confinement did not induce consistent changes in luteinising hormone (LH) and progesterone concentrations from one period to an other.

5. An increase in the plasma concentration of D‐(/?)‐hydroxybutyrate was observed in the hens which had an egg present in the oviduct on day 2, 3 and/or 4 of the treatment. Subsequently, a decrease in ovulation rate was observed in the hens with higher concentrations of D‐(β)‐hydroxybutyrate while under treatment, during the 10th week of production.

6. These data confirm that the effectiveness of the traditional methods for broodiness prevention under commercial conditions is related to the induction of a decrease of prolactin.     

Calving patterns in seasonal dairy herds

Two field studies examined the calving patterns of cows in seasonal dairy herds in the Waikato (Field Study 1) and South Taranaki regions (Field Study 2). The first study examined patterns for cows commencing their second or subsequent lactation in herds which had used an inseminating service during the previous season. The second study included first lactation heifers only in 15 herds where animals had been naturally mated, and in 15 herds in which they had been synchronised and then artificially inseminated at the synchronised oestrus. The parameters describing calving patterns were based on the date for each herd's planned start of calving (PSC), which was 282 days from the date on which breeding commenced in the preceding season. The average interval from PSC to mean calving date for the 35 herds in Field Study 1 was 22 days, with individual herds ranging from 15 to 30 days. In herds with heifers which had been naturally mated (Field Study 2), it was 17.6 days compared to 11.0 days for previously synchronised animals. Calculating the intervals from PSC to median calving date and separately for the last two quartiles more effectively described a herd's calving pattern. The duration for the last quartile of the calving pattern was influenced by the extent and timing of induced calving. In Field Study 1, 88.6% of the 35 herd owners induced premature parturition in at least one cow. In these herds, 11.3% of cows were treated and calved prematurely. Only 61.7% of heifers which had previously been naturally mated calved by 3 weeks after PSC. Their calving dates were not evenly distributed over this 3-week period, with 9.8% in the first week and 25.6% in the third week. The calving pattern for heifers which had been previously synchronised showed several distinct peaks. Calvings to the synchronised mating were completed 15 days after PSC, by which time 64.7% of animals had calved. By 3 weeks after PSC, 72.9% of these heifers had calved. The results showed that there was considerable variation in calving patterns in seasonal dairy herds. This variation would have been due to differences in conception pattern, and the way induced calving had been applied. The calving pattern in heifers which had been naturally mated was less concentrated than had been expected. Synchronisation can significantly concentrate the calving pattern of these first lactation animals. The parameters used to describe calving patterns may be less applicable in herds in which a high proportion of animals is induced to calve prematurely, or where a whole herd is synchronised. Nonetheless, they do serve as an illustrative example of the variation in calving patterns among herds.     

Efficacy of a topical formulation of ivermectin against naturally acquired gastro-intestinal nematodes in weaner cattle

The efficacy of a topical formulation of ivermectin against naturally acquired gastro-intestinal nematodes in weaner cattle was evaluated. At slaughter, 14-15 days after treatment, burdens of Ostertagia spp, Trichostrongylus axei and Oesophagostomum radiatum were significantly lower in the treated calves than in the untreated controls (p<0.01). Efficacies (based on geometric mean worm burdens of treated and control groups) were 99.6%, 95.1% and 100% respectively. The sizes of the Cooperia spp and Trichuris ovis burdens in the treated group did not differ significantly at the 5% level of confidence from those in the control group.