The rapid estimation of microbial contamination of raw meat by measurement of adenosine triphosphate (ATP)

Bacteria were separated from raw meat homogenate by a simple three-stage process. Centrifugation (10 s at 2000 g) removed coarse particles; stirring with the cation exchange resin Bio-Rex 70 removed smaller particles and filtration through 0.22 μm membranes removed soluble materials. By this process 70—80% of the microbial populations of meat homogenates were consistently isolated on the filters. A linear relationship was found between log₁₀ microbial ATP and log₁₀ colony count of meat over the range 10⁵—10⁹ cfu/g. The value of ATP/cfu for meat samples was within the range previously reported for pure cultures. These data indicated that ATP extracted from the filters originated from bacteria in the meat samples. Several samples can be analysed simultaneously in an elapsed time of 20—25 min. The variability associated with estimates of both colony counts and ATP levels has been determined.     

Blood Selenium Associated with Health and Fertility in Norwegian Dairy Herds

A survey of blood selenium (Se) concentrations in Norwegian Red heifers and dry period cows was conducted to reveal possible association to management, feeding, health and fertility. Selenium contents were determined in 254 herd blood samples consisting of pooled samples from individual non-lactating animals from herds in 5 counties. The Se concentrations showed a normal distribution with mean 0.09 μg Se/g blood, with a standard deviation (SD) of 0.05, and ranged from 0.02 to 0.23 μg/g, with 50 % of the samples being between 0.06 and 0.11 μg/g. The herds with Se concentrations below 0.06 μg/g were smaller (21.4 ± 8.7 cow-years) than those with Se levels above 0.11 μg/g (27.5 ± 14.1 cow-years) (P < 0.01), but there were no differences in milk yield, incidence of replacement, proportion of animal culling, amount of concentrate or grass silage as percentage of energy consumption between the groups. Treatment registration records showed a tendency that more animals in the low Se herds were treated for all the diseases included in this investigation (64.8 animals per 100 cow-years) than those in the high Se herds (57.5 per 100 cow-years), while no such differences were revealed for individual disorders. There was, however, a significant difference in bulk milk somatic cell counts (BMSCC) between low and high Se herds, their values being 137 000 and 155 000 cells/ml, respectively. This difference was significantly influenced by herd size. Furthermore, a total of 4 916 lactations were analyzed from individual health and fertility recordings, including 2 934 first lactations and 1 982 later lactations. The present study revealed a reduced incidence of disease treatment with increased Se concentrations from 0.02 to 0.23 μg Se/g blood. In this regard, there seemed to be an optimum of 0.10 to 0.15 μg Se/g for all types of mastitis treatments summarized, and for treatment of retained placenta. Thus, herd Se concentrations below and above these values was connected with increased probability for sum mastitis and retained placenta, reflecting the effect of the quadratic term of Se. The cow (composite) milk somatic cell count (SCC) was lower in lactations from low Se herds than in high Se herds with a marked SCC increase in the Se concentration interval from 0.11–0.13 μg/g blood. In conclusion, heifers and dry period cows in Norway are low in blood Se content and there seems to be a positive association between increased blood Se concentration pre partum and decreased incidence of mastitis, ovarian cysts and anoestrus/silent oestrus post partum.     

Biochemical identification of the bovine blood group M' antigen as a major histocompatibility complex class I-like molecule

Absorption and elution experiments showed that it was impossible to separate antibodies against blood group factor M' from antibodies against bovine lymphocyte antigen (BoLA) A16 in an antiserum showing haemolytic activity against M' as well as lymphocytotoxic activity against BoLA-A16. To elucidate the structural relationship between BoLA-A16 and blood group antigen M', immunoprecipitation experiments on red and white cell lysates isolated from M'-A16 positive and negative cattle were carried out. These results showed that M_r 44 000 and M_r 12000 polypeptides can be precipitated from both red and white cells isolated from M'-A16 positive animals, whereas no bands were seen in M'-A16 negative animals in precipitations with the same antibody. Precipitation with a crossreacting human β₂-microglobulin (β₂-m) specific antibody confirmed a class-I-like structure associated with β₂-m on M' positive red cells and the absence of such a structure on M' negative red cells. Sequential precipitations gave analogous results. Proteolytic degradation by papain and V8 protease did not reveal any substantial difference between red and white M'-A16 positive cells, but a slight difference in the pI of the immunoprecipitable components of red and white cells was observed. All together, this indicates that either the blood group antigen M' is the BoLA-A16 class I antigen or M' and BoLA-A16 are two different class I polypeptides with the same relative mass, sharing identical epitopes and both associated with β₂-m. Comparable results were obtained with M₁ and BoLA-A24.     

Relationship of environmental pCO2 to selected blood parameters of Oncorhynchus kisutch (Walbaum)

Blood samples from sub-adult coho salmon ( Oncorhynchus kisutch ) were correlated with environmental conditions. Carbon dioxide tensions, pH and bicarbonate levels were determined in the blood for fish acclimated to environmental pCO₂ levels of 2.49 and 2.74 mmHg. Fish were removed from the high pCO₂ environment and placed in one with a low pCO₂. Their blood pH and pCO₂ were monitored during the course of acclimation. After 27 days of acclimation to a low pCO₂ environment, a group of fish was sampled and compared to a group of fish acclimated to a high pCO₂ environment. It was found that pCO₂ in the environment affects the rate of diffusion of carbon dioxide from the blood to the water. Under the environmental conditions prescribed in these experiments no significant change in bicarbonate was noticed. It is postulated that a bicarbonate chloride exchange is not a major pathway for the removal of carbon dioxide from the blood of O. kisutch .     

Diurnal variations in the kinetics of delayed luminescence from Scenedesmus obtusiusculus after exposure to various light and CO2 regimes

Delayed luminescence was measured from samples of a synchronously growing cell culture of the unicellular green alga, Scenedesmus obtusiusculus Chod., every second hour during the 24 h cell cycle under a 15/9 h lighi/dark regime. Both high (air + 2.5% CO₂) and low (0.03% CO₂) CO₂ conditions were used. Under high CO₂ conditions, while light excitation induces formation of a late (maximum reached after 10–60 s) transient peak in delayed luminescence in cells sampled after 10–16 h in the cell cycle. During most of the cell cycle low CO₂ conditions stimulate a late transient peak formation. Excitation with 700 nm light, but not with 680 nm light, induces a late transient peak in delayed luminescence under high CO₂ conditions. The transient peak is more or less pronounced depending on the cell stage. The variations might be due to a changing capacity for light-induced state I/stale II transitions during the cell cycle. It is assumed that the formation of a late transient peak in delayed luminescence is due to ATP hydrolyzation and is thus favoured by a high ATP/NADPH ratio. Hydrolyzation of ATP affects the transthylakoidal ΔpH, which regulates the reverse electron flow to the plastoquinone-pool and Q_A/Q_B, thus affecting the decay kinetics of the delayed luminescence.     

Immune modification of the pathogenesis of Streptococcus uberis mastitis in the dairy cow

Abstract Two groups of 4 cows were vaccinated subcutaneously with live Streptococcus uberis strain 0140J or a surface extract derived from the same strain, at 14 days prior to the cessation of lactation (drying off) and at calving. Both groups also received an intramammary administration of the surface extract 7 days after drying off. A third group of unvaccinated animals acted as controls. Following intramammary challenge of two quarters per cow with the vaccine strain, all quarters on control cows and those vaccinated only with surface extract developed clinical mastitis. However, only 12.5% of challenged quarters on cows which were vaccinated with live bacteria developed clinical mastitis. In addition, the numbers of bacteria in the milk following challenge were 10⁵ times higher from the control and extract vaccinated cows than those which received live vaccine. Serum levels of S. uberis specific IgG₂ were elevated in the animals vaccinated with the live organism when compared to that of either extract-vaccinates or controls, whilst S. uberis specific levels of IgG₁ and IgM were similar in all groups throughout the experiment. Specific antibody levels in milk were unaffected by vaccination. Despite increased levels of IgG₂, no increase in opsonic activity was detected in any serum or milk samples. Peripheral blood lymphocytes from animals vaccinated with live organisms showed a considerable increase in proliferative response to S. uberis antigen in vitro when compared with lymphocytes from control and extract-vaccinated animals. These results suggest that neutrophils and specific opsonising antibody may not form the major defence against infection with S. uberis .     

Antibodies Against Staphylococcus Aureus Nuclease in Bulk Milk As An Indicator of Mastitis in Dairy Herds

Titres of staphylococcal antinucleases and cell counts in bulk milk samples were compared as indicative criteria of the mastitis situation in dairy herds. The correlation coefficients between the prevalence rate of mastitis and the antinuclease titre and cell count, respectively, were of the same order of magnitude. The incidence of clinical mastitis showed a better correlation to the antinuclease titre than to the cell count. When cell counts and antinuclease titres were combined a more reliable selection of herds with a possible mastitis problem was achieved. The antinuclease test is consequently recommended as a supplement to cell counting when screening bulk milk.     

Characterization of ATPase activities in rice root plasmalemma vesicles isolated by two-phase partitioning

Plasma membrane vesicles were isolated from the roots of 7-day-old rice plants ( Oryza sativa L. cv. Bahía) by utilizing an aqueous polymer two-phase system with 6.2%:6.2% (w/w) Dextran T500 and polyethylene glycol 3350 (PEG) at pH 7.6. Plasmalemma vesicles of high purity were obtained as indicated by the vanadate-sensitive K⁺, Mg²⁺-ATPase activity that was 18 times higher in the upper (PEG-rich) phase than in the lower (Dextran-rich) phase and by specific staining with sodium silicotungstate. Two peaks of ATPase activity were found. One showed a pH optimum at 6.0 in the presence of 150 m M KCl and 3 m M ATP with apparent K_m (ATP) and V_max of 0.75 m M and 79 μmol (mg protein)⁻¹ h⁻¹, respectively. With 50 m M KCl and 7 m M ATP a pH optimum of 6.5, an apparent K_m (ATP) of 6.3 m M and V_max of 159 μmol (mg protein)⁻¹ h⁻¹ were determined. Both activities were specific for ATP, unspecific for monovalent cations, sensitive to sodium vanadate and Ca²⁺ but insensitive to azide and nitrate.     

31P NMR Relaxation Does Not Affect the Quantitation of Changes in Phosphocreatine, Inorganic Phosphate, and ATP Measured In Vivo During Complete Ischemia in Swine Brain

Abstract: Ischemia-induced changes in ³¹P NMR relaxation were examined in 16 piglets. NMR spectra were acquired under control conditions and during complete cerebral ischemia induced via cardiac arrest. Changes in T ₁ were assessed directly in six animals during control conditions and after 30–45 min of complete ischemia when changes in brain P₁ levels had reached a plateau. The T ₁ for P₁ did not change, i.e., 2.3 ± 0.5 s during control conditions versus 2.4 ± 1.0 s during ischemia. To evaluate phosphocreatine and ATP, two types of spectra, with a long (25-s) or short (1-s) interpulse delay time, were collected during the first 10 min of ischemia (n = 10). Both types of spectra showed the same time course of changes in phosphocreatine and ATP levels, implying that the T ₁ relaxation times do not change during ischemia. There were no changes in the linewidths of phosphocreatine, ATP, or P₁ during ischemia, implying that the T *₂ values remain constant. Our results suggest that the ³¹P T ₁ and T *₂ for phosphocreatine, P_i, and ATP do not change during ischemia, and therefore changes in ³¹P NMR peak intensity accurately reflect changes in metabolite concentrations.     

Carbon dioxide anaesthesia in rainbow trout: effects of hypercapnic level and stress on induction and recovery from anaesthetic treatment

The physiological and anaesthetic effects of three different levels of air-saturated and buffered CO₂ anaesthesia, P _w co ₂=31, 78, or 125 mmHg, were examined in cannulated rainbow trout Oncorhynchus mykiss . Complete anaesthesia (no opercular movements) was not achieved by these hypercapnic levels after 20 min of CO₂ exposure. Although increasing P _w co ₂ reduced the induction times to the early stages of anaesthesia, it also resulted in increasing hyperventilatory, hypoxaemic, and acid-base disturbances. After a 10-min recovery period, while the respiratory acidosis component of the acid-base disturbance was corrected, there was a significant metabolic acidosis. Recovery time was longest in the high P_w co₂ treatment where 33% of the fish died. Two additional groups ( P _w co ₂=37 and 78 mmHg) were exposed to an acute stress prior to the anaesthetic treatment. Stress reduced the hypoventilatory effects of the low P _w co₂ treatment, increased the recruitment of anaerobic metabolism, and prolonged recovery time. Although the increase in plasma catecholamines elicited by the stress was small relative to the response obtained with the anaesthetic, stress prior to CO₂ anaesthesia impaired the efficiency of the treatment. Overall, our results suggest that P _w co₂ levels above 37 mmHg and/or stress prior to the anaesthesia impair the efficiency of air-saturated and buffered CO₂ anaesthesia by exacerbating the hypoxaemic effects of the hypercapnic treatment.     

Vitamin B12 Production by the Gastro-intestinal Microflora of Baboons Fed either a Vitamin B12 Deficient Diet or a Diet Supplemented with Vitamin B12

The vitamin B₁₂-producing capacity of micro-organisms isolated from baboon faeces and gastric contents was measured using Lactobacillus leichmannii . The animals were fed either a diet deficient in or supplemented with vitamin B₁₂ (controls). Samples of gastric and small intestinal contents, obtained at laparotomy from two young vitamin B₁₂-deprived baboons, contained varying quantities of vitamin B₁₂. Many of the organisms isolated from these aspirates produced vitamin B₁₂ in vitro . The highest levels of vitamin B₁₂ were produced by anaerobic organisms. Gastric juice samples from vitamin B₁₂ deprived and control baboons contained similar types of organisms with like vitamin B₁₂-producing capacity. The vitamin B₁₂ content, pH and total bacterial counts of gastric juice samples aspirated after a 6 h fast from vitamin B₁₂ deprived baboons were not significantly different from those of the control animals. The pH values of gastric juice samples aspirated 18 h after feeding, however, were significantly lower than those of 6 h fasting samples in both groups. The mean vitamin B₁₂ levels in the total volumes of gastric juice aspirated after each fasting period were similar. The possible involvement of the gastrointestinal flora in the vitamin B₁₂ status of the baboon is discussed.     

Prostaglandin E2 production by rat peritoneal macrophages: role of cellular and humoral factors in vivo in transfusion-associated immunosuppression

Abstract The transfusion of blood is associated with long-term immunosuppression, which has been postulated to influence immunosurveillance and cancer cell killing. The mononuclear phagocyte synthesises large quantities of PGE₂, and PGE₂ has been shown to inhibit the activity of a range of immunocompetent cell types. The role of mononuclear phagocyte PGE₂ synthesis in transfusion-associated immunosuppression, and the elements of transfused blood which control this immunosuppression, were investigated using a transfused rat model. A significant increase in macrophage PGE₂ synthesis was detected 7 days after transfusion with blood and serum. The storage of blood for 24 h increased the stimulatory activity of transfused blood. The effects of storage and serum on macrophage PGE₂ synthesis were greater than effects due to genetic differences between blood donor and recipient, and the serum effects indicated that a major factor activating PGE₂-mediated immunosuppression in transfused subjects may be humoral in nature.     

ONTOGENETIC AND SEASONAL VARIATION IN BLOOD PARAMETERS IN SOUTHERN ELEPHANT SEALS

Blood samples were collected from 156 free-ranging southern elephant seals ( Mirounga leonina ) from Peninsula Valdes to study the variation in blood parameters related to ontogeny and the annual cycle. Samples ranged from newborn pups to adults. Interactions between age and sex showed different trends in ontogenetic changes of blood parameters (MANOVA, Wilkis' Lamb-da_12,243= 0.75, P < 0.01). The hematocrit (HCT) and hemoglobin concentration (HB) reached adult levels early in life. Weanlings had 14 % higher HCT than pups, and similar levels to juveniles and adults (HCT range = 46%-62 %). HB of males were below those of females from weaning (18.4 vs . 20.3 g/dl) to adulthood (19.8 vs . 22.3 g/dl). Red blood cell counts (RBC) did not change significantly from pups to juveniles (2.8–3.2 10⁶/μl), but varied for adults at different times of the annual cycle. Breeding females had higher RBC than molting females (3.1 vs. 2.4 10⁶/μl). Changes in blood parameters are related to the development of diving capabilities from pups to juveniles. Changes in adults were associated with different stages of the annual cycle, and these may be the result of the requirements imposed by pregnancy and fasting duration.     

The F1FO ATP synthase genes in Methanosarcina acetivorans are dispensable for growth and ATP synthesis

There is a long-standing discussion in the literature, based on biochemical and genomic data, whether some archaeal species may have two structurally and functionally distinct ATP synthases in one cell: the archaeal A₁A_O together with the bacterial F₁F_O ATP synthase. To address a potential role of the bacterial F₁F_O ATP synthase, we have exchanged the F₁F_O ATPase gene cluster in Methanosarcina acetivorans against a puromycin resistance cassette. Interestingly, the mutant was able to grow with no difference in growth kinetics to the wild type, and cellular ATP contents were identical in the wild type and the mutant. These data demonstrate that the F₁F_O ATP synthase is dispensable for the growth of M. acetivorans .     

Effects of graded hypoxia on Atlantic salmon infected with amoebic gill disease

Atlantic salmon Salmo salar with amoebic gill disease (AGD) were exposed to a graded hypoxia (135–40 mmHg water P O₂) and blood samples analysed for respiratory gases and pH at 119, 79·5 and 40 mmHg water P O₂. There were no differences in the rate of oxygen uptake between infected and control fish. However, arterial P O₂, and pH were significantly lower in the infected fish whereas P CO₂ was significantly higher in infected fish compared with controls prior to hypoxia and at 119 mmHg water P O₂. At 79·5 and 40 mmHg water P O₂ saturation, there were no significant differences in blood P O₂ or pH although blood P CO₂ was elevated in AGD affected fish at 50% hypoxia (79·5 mmHg water P O₂). The elevated levels of P CO₂ in fish affected by AGD resulted in a persistent respiratory acidosis even during hypoxic challenge. These data suggest that even though the fish were severely affected by AGD, the presence of AGD while impairing gas transfer under normoxic conditions, did not contribute to respiratory failure during hypoxia.     

Heritabilities and quantitative trait loci for blood gases and blood pH in swine

Maintaining pH and blood gases in a narrow range is essential to sustain normal biochemical reactions. Decreased oxygenation, poor tissue perfusion, disturbance to CO₂ expiration, and shortage of HCO₃⁻ can lead to metabolic acidosis. This is a common situation in swine, and originates from a broad range of medical conditions. pH and blood gases appear to be under genetic control, and populations with physiological traits closer to the pathological thresholds may be more susceptible to developing pathological conditions. However, little is known about the genetic basis of such traits. We have therefore estimated phenotypic and genetic variability and identified quantitative trait loci (QTL) for pH and blood gases in blood samples from 139 F₂ pigs from the Meishan/Pietrain family. Samples were taken before and after challenge with Sarcocystis miescheriana , a protozoan parasite of muscle. Twenty-seven QTL influencing pH and blood gases were identified on nine chromosomes. Five of the QTL were significant on a genome-wide level; 22 QTL were significant on a chromosome-wide level. QTL for pH-associated traits have been mapped to SSC3, 18 and X. QTL associated with CO₂ have been detected on SSC6, 7, 8 and 9, and QTL associated with O₂ on SSC2 and SSC8. QTL showed specific health/disease patterns that were related to the physiological state of the pigs from day 0, to acute disease (day 14), convalescence (day 28) and chronic disease (day 42). The results demonstrate that pH and blood gases are influenced by multiple chromosomal areas, each with relatively small effects.     

Plasma membrane damage to Candida albicans caused by chlorine dioxide (ClO2)

Aim:  To investigate the plasma membrane damage of chlorine dioxide (ClO₂) to Candida albicans ATCC10231 at or below the minimal fungicidal concentration (MFC).
Methods and Results:  ClO₂ at MFC or below was adopted to treat the cell suspensions of C. albicans ATCC10231. Using transmission electron microscopy, no visible physiological alteration of cell shape and plasma membrane occurred. Potassium (K⁺) leakages were significant; likewise, it showed time- and dose-dependent increases. However, adenosine triphosphate (ATP) leakages were very slight. Research shows that when 99% of the cells were inactivated, the leakage was measured at 0·04% of total ATP. Compared with the mortality-specific fluorescent dye of DiBAC₄(3), majority of the inactivated cells were poorly stained by propidium iodide, another mortality-specific fluorescent dye which can be traced by flow cytometry.
Conclusion:  At or below MFC, ClO₂ damages the plasma membranes of C. albicans mainly by permeabilization, rather than by the disruption of their integrity. K⁺ leakage and the concomitant depolarization of the cell membrane are some of the critical events.
Significance and Impact of the Study:  These insights into membrane damages are helpful in understanding the action mode of ClO₂.     

Carbohydrate partitioning, photosynthesis and growth in Lemna gibba G3. II. Effects of phosphorus limitation

The relationship between CO₂ assimilation rate, growth and partitioning of carbon among starch, sucrose, glucose and fructose were studied in phosphorus (P_i)-limited Lemna gibba L. G3. Two experimental models were used: 1) Cultures were grown at various stable, suboptimal rates regulated by the supply of P_i; 2) cultures growing at optimal rates were transferred to P_i-free medium. The response to a P_i deficiency can be divided into two phases. Phase I is characterized by hyperactivity of the sucrose synthesis pathway, leading to high levels of glucose and fructose. Phase II is characterized by starch accumulation associated with a decrease in the cytoplasmic pools of soluble sugars owing to inhibition of carbon export from the chloroplast. A strong negative correlation was found between the CO₂ assimilation rate and starch levels. No significant correlation was found between assimilation and ATP levels and decrease in relative growth rate did not significantly affect the adenylate energy charge (EC). The regulatory aspects of the partitioning of carbon among soluble sugars and starch as well as the negative correlation between carbohydrate levels and CO₂ assimilation at P_i-limited growth are discussed.     

Association between BoLA-DRB3 and somatic cell count in Holstein cattle from Argentina

Different studies have proved that the resistance/susceptibility to mastitis is genetically determined. The major histocompatibility complex in cows is known as bovine lymphocyte antigen (BoLA). Genes from the BoLA have been associated with the occurrence of infectious diseases such as mastitis and leukosis, especially the BoLA-DRB gene. The object of the present study was to detect associations between BoLA-DRB3 alleles and somatic cell count (SCC), as an indicator of resistance/susceptibility to mastitis in Holstein cattle (N = 123) from La Pampa, Argentina. Fisher's exact test and Woolf-Haldane odds ratio were applied to study the association between SCC and BoLA-DRB3 allele frequencies. Significant association was noted between BoLA-DRB3.2*23 and *27 alleles (p < 0.05) and protective or susceptibility effects, respectively. In addition, alleles BoLA-DRB3.2*20 and *25 exhibit suggestive association with high SCC (p < 0.1). These results were partially in agreement with data reported from Japanese Holstein cattle, but differed from those published by other authors. A possible explanation for the contrasting results could be that the mastitis is a multifactor disease caused by different pathogens. Moreover, most of the studies were carried out using PCR-RFLP method, which has less resolution than PCR-SBT because PCR-RFLP defined alleles included more than one sequenced alleles.     

Chromosome numbers of C3 and C4 variants within the species Alloteropsis sernialata (R.Br.) Hitchc. (Poaceae)

FREAN, M. L. & MARKS, E., 1988. Chromosome numbers of C₃ and C₄ variants within the species Alloteropais semialata (R.Br.) Hitch. (Poaceae). In a study of mid-lamina leaf sections, different variants of A. semialata were found to have C₃ or C₄ anatomy. The C₄ leaf showed a specialized photosynthetic vascular bundle sheath not present in the C₃ form. Chromosome counts made from pollen mother cell squashes showed that the C₃ form of A. semialata is a fertile diploid 2n = 2x = 18 and the C₄ form, a fertile allohexaploid 2n = 6x = 54. The cytological evidence suggests that the two forms should be considered as separate species.