The use of a mimic to detect polymerase chain reaction-inhibitory factors in feces examined for the presence of Lawsonia intracellularis.

Lawsonia intracellularis is an intracellular organism that causes proliferative enteritis in pigs. This bacterium is difficult to culture, and antemortem demonstration of the microbe is therefore often performed on fecal samples by polymerase chain reaction (PCR). Polymerase chain reaction is sensitive and specific, but inhibitory factors in feces might cause false-negative results. This article describes the construction and use of an internal standard, a mimic. The mimic is amplified by the same primers as those used for L. intracellularis DNA and thus could indicate false-negative results in clinical samples. The amplicon was clearly visible when as few as 10 mimic molecules were added per amplification reaction and when no inhibitors werepresent. When fecal samples were spiked with the mimic, the detection limit was 10(2) molecules per PCR. Sixty clinical samples, 20 from wild boars, 20 from growing pigs with diarrhea, and 20 from pigs without diarrhea, were prepared by a boiling procedure and subjected to PCR together with 10(3) mimic molecules. Nine samples were positive, of which 7 originated from pigs with diarrhea and 2 from pigs without diarrhea. In 14 samples from wild boars, in 8 samples from pigs without diarrhea, and in 3 samples from pigs with diarrhea, neither the mimic nor the target DNA was visible. This indicated the presence of inhibitors in these samples. It is concluded that the mimic can be used as an internal control in the diagnosis of L. intracellularis to indicate inhibition of PCR.     

Analysis of IgG response to experimental infection with RH Toxoplasma gondii in goats.

The IgG response of goats experimentally infected with RH Toxoplasma gondii has been analysed using an indirect ELISA and Western-blot analysis. Specific IgG antibodies were first detected at 14 days post-inoculation (p.i.), reaching a peak by day 35 p.i. and showing slight fluctuations until the end of the experiment (91 p.i.). Specific IgG showed a reactivity over a whole range of peptides (125-24 kDa approximately), but the highest reactivity was observed against a group of antigens with a molecular weight between 34 and 28 kDa, in particular against a 30 kDa fraction which is considered to represent the major surface protein of T. gondii named p30 or SAG-1.     

Serum antibody response and Cryptosporidium parvum oocyst antigens recognized by sera from naturally infected sheep.

The response of specific serum immunoglobulins (IgG, IgM and IgA) and the major antigens of Cryptosporidium parvum recognized by these isotypes were investigated by using enzyme-linked immunosorbent assay and immunoblot techniques in lambs and ewes naturally infected throughout an outbreak of cryptosporidiosis. Serum samples were collected from 20 lambs the first day they showed diarrhoea (D1), and Days 11 and 22, in addition to single serum samples from 17 of their dams. Serum anti-C. parvum IgG, IgM and/or IgA antibodies were detected in lambs as early as Day 1. Levels of IgM antibodies remained steady from D1 to D11 and increased at D22, whereas the IgG response decreased from D1 to D11 and subsequently increased. In contrast, IgA antibodies rapidly fell from D1 and all lambs were seronegative at D11 and D22. The highest levels of specific antibodies were detected in sera from ewes. In fact, all ewes were seropositives for IgM and IgA isotypes and most (16/17) showed positive levels of IgG. Four protein fractions (37-39, 42-48, 51-57 and 60-69 kDa) were the most frequently recognized by IgG and IgM from lamb sera. A low molecular weight fraction (12-14 kDa) reacting with IgG and IgA in most lamb sera was scarcely recognized by IgM and three broad bands were frequently recognized by IgA antibodies (23-25, 51-57 and 90-95 kDa). The recognition pattern of 23-25 kDa peptides by IgA from lamb sera clearly increased with the age. Peptides of 42-48, 51-57, 60-69 and 71-78 kDa were most frequently recognized by IgG and IgM from ewe sera. In relation to IgA antibodies from ewe sera, a frequent immunoreactivity was found with proteins in the intervals between 12 and 22 kDa as well as between 32 and 34 kDa and practically all sera reacted with fractions from 42 to 95 kDa.     

Pharmacokinetics of pipemidic acid in chickens after single intravenous and oral dosings

The pharmacokinetics of pipemidic acid after 2 single doses were studied in broiler chickens. Chickens were given single IV and oral doses of 10 and 30 mg of pipemidic acid/kg of body weight. Blood samples were collected over 8 hours after each dose administration. High-pressure liquid chromatography with UV detection was used to determine concentrations in plasma of pipemidic acid. The plasma concentration-time curves after IV administration followed 2-compartment characteristics, rapid initial distribution phase, and a terminal elimination phase. The pharmacokinetic variables differed significantly between single doses of 10 and 30 mg of pipemidic acid/kg. Mean disposition variables were a half-life at alpha phase of 0.06 hours or 0.33 hours, a half-life at beta phase of 1.18 hours or 1.72 hours, a volume of distribution in the central compartment of 0.12 L/kg or 0.31 L/kg, a volume of distribution during the elimination beta phase of 1.64 L/kg or 1.05 L/kg, and a total plasma clearance of 0.97 L/h.kg or 0.41 L/h.kg, for the 10 or 30 mg/kg dose, respectively. After oral administration, the pipemidic acid plasma profile could be adequately described by a 1-compartment model. After the single oral doses of 10 and 30 mg of pipemidic acid/kg, pipemidic acid was absorbed rapidly (time to maximal concentration of 0.31 hours or 0.71 hours) and eliminated with a mean half-life of 0.86 hours or 0.61 hours, respectively. The bioavailability was 39% at 10 mg of pipemidic acid/kg and 61% at 30 mg of pipemidic acid/kg.     

The effect of animal fat and vegetable oil supplementation of feeds of different energy concentration upon the digestibility of nutrients and some blood parameters in rabbits

The effect of mixed animal fat and sunflower oil supplementation (5%) of a feed of medium (12.02 MJ DE/kg) and low (8.54 MJ DE/kg) energy concentration upon the digestibility of nutrients and on some blood parameters was investigated. The ether-extractable content of feed and faecal samples was determined by diethyl ether extraction (after Soxhlet) and the total (true) fat level was measured by the method of Stoldt (1952), viz. petroleum ether extraction of samples pretreated with 4 N HCl. In the majority of cases the voluntary feed intake decreased after the addition of animal fat or vegetable oil. Mixed animal fat supplementation significantly (by 5 and 11 units) improved the digestibility of the ether extract if added to either of the basal diets. The digestibility of crude fibre and N-free extract increased only in the case of the basal diet of low energy concentration (by 4 and 7 units, respectively). Sunflower oil addition produced changes of the same tendency (9, 9, 28 and 5 units). The digestibility of the crude protein was practically not altered by either supplementation. The total fat content of feed and faecal samples proved to be higher than that of the ether extract, on an average by 27 and 100%, respectively. Consequently, the digestibility coefficients of the total fat are by 10 units lower than those of the ether extract. The DE concentrations, calculated by means of the digestible ether extract or the total fat content, differed significantly only between the basal diets. The addition of mixed animal fat to either of the basal diets increased the blood concentration of total lipids (by 18 and 32%, respectively), while that of sunflower oil did not cause such an alteration. Neither fat nor oil supplementation had an effect on the cholesterol level of the blood plasma. Both the animal fat and the vegetable oil surplus modified the fatty acid composition of blood lipids. It was characteristic of each treatment that the proportion of polyunsaturated fatty acids increased.     

Experimental control of the process of lead absorption and deposition in domestic ducks

A trial was conducted with 40 domestic ducks of the White Pekin breed at the age of 40 days: thirty pieces of lead shot were introduced as a single dose in the gizzard. The process of lead resorption and deposition was found not to be significantly influenced by the therapeutic treatments nor changes in the composition of feed. A week after the administration of shot a significant increase was recorded in the content of lead to 4.32 mg in liver, to 11.36 mg in kidney and to 1.15 mg in muscle (the respective values for the control animals were 0.07, 0.11 and 0.06 mg per kg). At the end of the trial after three weeks of treatment and administration of experimental diets, no significant differences were recorded in lead content in liver, kidney and muscle between the untreated and treated ducks nor ducks fed experimental diets.     

The susceptibility of Lymnaeid snails to Paramphistomum cervi infections in Mexico

The occurrence of Paramphistomum cervi has been reported in Mexico, but its intermediate hosts have not been identified. Five species of snails of the genus Lymnaea, L. bulimoides, L. columella, L. cubensis, L. humilis and L. palustris, were collected in Mexico. Only three of the exposed species, L. palustris, L. cubensis and L. humilis, became infected with P. cervi. The first of these snail species was highly susceptible to infection and a higher number of the exposed snails shed cercariae. It is suggested that L, palustris may act as an important vector of P. cervi in Mexico.     

The effect of ionizing radiation on structural changes in the energy nutrients in animal feed

For the purpose of testing the effect of ionizing radiation, feeds for dogs (meat feed mixture VETACAN and loose feed mixture VETAVIT) irradiated by 60Co radioisotope at the dose of 25 kGy/kg were studied for 60 days. It has been found out that the total volume of energetic and non-energetic nutrients is not changed. Qualitative structure, however, displays a significant, on the average 35% disintegration of essential amino acids, decrease of proteins and increase of free ammonic bases. A significant oxidation effect of radiation is exerted on the decomposition of fats with a release of free fatty acids from glycerol bond in a process similar to rancidification (from 13.3-37.10 mg/g in meat mixture, from 103.1-103.04-135.04 mg/g in loose mixture). A certain disintegration of nutrients, only within the limits of significance, occurred also in the saccharide proportion of the loose feed mixture (acidity of water extract 348.8-403.99-436.60 mg/100 g). It has been proved that radiosterilization reliably secures microbiological and mycological sanitation of feeds and causes no sensory changes noticeable by human senses. It follows from the results that ionizing radiation has a pronounced antimicrobial and antimycotic effectiveness. However, it causes significant structural changes of energetic nutrients in the feeds of animal as well as of vegetable origin.     

The effect of feeds treated with ionizing irradiation on biochemical indicators of the nutritional value of energy nutrients

The influence of feedstuffs treated with ionizing radiation on the nutrition of dogs was tested in four groups of animals. Two groups were administered for 90 days a ration, the main part of which (VETACAN meat feed mixture and VETAVIT loose feed mixture) was irradiated with radioisotope Co 60 of the intensity of 25 kGy/kg, in other two groups of dogs the nonirradiated ration was used for the same time period. The control groups of dogs were put together for these two diets. The laboratory examination of irradiated feedstuffs confirmed their complete microbiological and mycological intactness. However, the irradiation brought about a significant 35% degradation of essential amino acids with an increase of ammonia nitrogen, destructive changes in the lipid component of feedstuffs and a partial decomposition of the saccharide part of the VETAVIT feed mixture, expressed by the acidity of water extract. The sensory evaluation of irradiated feedstuffs did not show any perceptible alterations. The haematological examination of the blood of animals, which had been administered irradiated feed rations, demonstrated a significant negative influence on the blood picture. The biochemical examination of the blood serum and plasma revealed that total proteins of experimental dogs dropped and the creatinine level was also significantly decreased. Neither was the level of carbohydrate nutrition nor the energy saturation affected by irradiation. The glucose levels in the blood serum of dogs fluctuated within the range of physiological reference values. The growth of free ammoniacal bases of feedstuffs, evoked by ionizing radiation, conditioned obviously the level of actual pH of blood in dogs as determined in this study. The destruction of lipoid fraction in the feedstuffs induced a decrease in the activity of lipophile retinol and thus the biological value of feeds was impaired. The biochemical examination of ALT, AST and ALP enzyme activity did not show any increased activity of parenchyma, in particular of liver cell. A decisive role of the biological quality of feed ration for utilization of some minerals was demonstrated by a significant decrease of the magnesium level in animals administered irradiated feed rations without any biological supplementation. On the contrary, the potassium, calcium and phosphorus levels did not reflect this dietary difference between the groups.