In vitro studies with lymphocytes from sheep orally inoculated with an aromatic-dependent mutant of Salmonella typhimurium.

It was previously shown that a live aroA-strain of Salmonella typhimurium of ovine origin was a safe and effective vaccine against salmonellosis in sheep. The protective effect was observed in the apparent absence of a detectable, systemic T cell response. In the present study, populations of B and T cells from the peripheral blood of sheep vaccinated with S25/1aroA were separated and their responsiveness in vitro to Salmonella was examined. The purified T cells proliferated very weakly in response to Salmonella in the absence of interferon-gamma and interleukin 2/4 production. However, whole peripheral blood mononuclear cells and purified B cells proliferated strongly in response to Salmonella, and Salmonella-specific IgM antibodies could be detected in cell supernatants. Furthermore, Salmonella-specific IgM-producing cells were detected at low frequency by enzyme linked immunospot techniques. These observations extend the earlier findings that oral vaccination with S25/1aroA primes predominantly antigen-specific B cells in the absence of strong Salmonella-specific T cell responses.

A native plasmid of Pasteurella haemolytica serotype A1: DNA sequence analysis and investigation of its potential as a vector.

The complete nucleotide sequence of a 4.3 kilobase pair plasmid, pAB2, isolated from a bovine strain of Pasteurella haemolytica serotype A1, was determined. It encodes a Rob-1 type beta-lactamase and a region with homology to the mobilisation (mob) region of the Escherichia coli plasmid, ColE1. An insertion mutant of pAB2 (pTC2/81) carrying a copy of Tn5 was transferred to E coli K12 by conjugation. Subsequently pTC2/81 could be transferred by transformation to E coli HB101, but not to P haemolytica serotypes A1 or A2. However, a derivative of this construct containing only a fragment of the Tn5 insertion sequence was able to transform P haemolytica. A further construct containing a fragment of the P haemolytica A1 leucotoxin A gene, was similarly restricted to transforming E coli. These results demonstrate that the pAB2 plasmid is capable of acting as an E coli/P haemolytica shuttle vector. However, the nature of the cloned DNA sequences are important to transformation.

Differences in the immune responses of mice and sheep to an aromatic-dependent mutant of Salmonella typhimurium.

A live mutant aroA Salmonella serotype Typhimurium ovine strain (S25/1) could be cultured from tissues of mice for up to 90 days after oral infection. Following vaccination, high levels of Salmonella-specific serum IgM, IgG and IgA were produced in addition to high levels of specific intestinal IgA. Moreover, there was also evidence of Salmonella-specific cell-mediated immunity in vaccinated mice in the form of strong delayed-type hypersensitivity and the production of interferon-gamma (IFN-tau) by spleen cells stimulated with Salmonella antigen. The aroA strain was also recovered from the mesenteric lymph nodes and most tissues examined from sheep vaccinated by the oral route. Salmonella-specific IgM was detected in the serum; however, specific IgG responses were very low and there was an absence of specific copro-antibody. Although strong Salmonella-specific lymphocyte proliferative responses were detected, they did not result in the production of IFN-tau and flow cytometric analysis revealed that the proliferating cells were predominantly B lymphocytes. Despite the absence of strong vaccine-specific immune responses in vaccinated sheep compared with those seen in mice, both mice and sheep were protected against challenge with virulent wild-type strain S25/1.

A 2359-base pair DNA fragment from Cryptosporidium parvum encoding a repetitive oocyst protein.

A Cryptosporidium parvum lambda gt11 expression library was constructed using EcoRI-digested genomic DNA extracted from in vitro-excysted oocysts. Screening of this library with rat anti-Cryptosporidium antiserum led to the isolation of a clone containing a 2359-bp EcoRI fragment. When this fragment was ligated into the EcoRI site of plasmid vector pMS1S, the resulting clone expressed a 200-kDa beta-galactosidase fusion protein. Western blot analysis using serum raised against this fusion protein indicated that the EcoRI fragment represented part of a gene encoding a 190-kDa oocyst wall protein of C. parvum. Sequencing of the fragment revealed a continuous open reading frame encoding 786 amino acids. The DNA sequence is relatively low in G+C (39.1%), and the third codon position contains only 17.9% G+C. The deduced peptide sequence has unusually high proportions of cysteine, proline, glutamine and histidine. Another striking feature of the amino acid sequence is the presence of distinctly repetitive regions based on conserved cysteine residues.

Occurrence and diversity of plasmids in ovine isolates of Pasteurella haemolytica.

160 ovine isolates of Pasteurella haemolytica, representing each of the 16 serotypes and also untypable strains, were examined for plasmid content. Plasmid DNA was identified in, and prepared from, strains of serotypes A2, T3, A14 and A16 and also from an untypable strain. The relationship between the plasmids present in the different strains was examined both by restriction fragment profile analysis and by DNA/DNA hybridisation. Both methods gave broadly similar results and showed that each serotype tended to contain either a single plasmid species, or a limited range of species, and that structural similarities could traverse serotype boundaries. None of the plasmid-bearing strains showed any significant level of resistance to a range of antibiotics.

Identification and localization of an iron-regulated 35 kDa protein of Pasteurella haemolytica serotype A2.

Iodination of intact Pasteurella haemolytica serotype A2 cells labelled a sub-set of total cellular proteins. Comparison of the autoradiographic patterns obtained from iodinated cells grown on complete medium and on iron-depleted medium showed that expression of three proteins, of 100, 70 and 35 kDa, respectively, was increased by growth under iron-depleted conditions. Of these proteins, that of 35 kDa had not been reported previously. Like the 100 and 70 kDa proteins, the 35 kDa protein was expressed in natural infections, since it was recognized by antiserum from sheep that had recovered from an experimental infection with P. haemolytica A2. The 35 kDa protein was partially purified by reverse-phase HPLC and was found to be antigenic in both sheep and mice. A monoclonal antibody that was specific for the 35 kDa protein was used to identify the cellular location of the protein by immunoblotting of cell fractions enriched for particular cellular components. This demonstrated that the 35 kDa protein was located mainly in the periplasm.

The virulence plasmid of Salmonella dublin: detailed restriction map and analysis by transposon mutagenesis.

A detailed restriction map of the virulence plasmid of Salmonella dublin has been determined and used for comparison with the virulence plasmid from S. typhimurium. Two regions were identified which appeared to be similar based on blotting and restriction data. One, of about 22 kb, encompassed the virulence region; the other, of about 8 kb, was outside it. The locations of 259 transposon insertions on the S. dublin plasmid were determined and related to their effect on virulence. One gene involved in virulence but outside the essential virulence region was shown to affect citrate metabolism.

A common virulence region on plasmids from eleven serotypes of Salmonella.

Cured derivatives of Salmonella dublin and S. typhimurium showed reduced virulence following oral infection of mice (10(4)-10(5)-fold for S. dublin, 10(2)-fold for S. typhimurium). Large plasmids from S. dublin and S. typhimurium independently restored virulence to the cured S. dublin but truncated S. dublin plasmids with deletions in a previously identified virulence region did not. This common virulence region identified in plasmids from S. dublin and S. typhimurium was shown to be carried on plasmids from 11 other serotypes of Salmonella but was absent from 10 plasmid-containing serotypes. TnA and Tn10 were transduced from the virulence region of two TnA-insertion mutants of S. dublin and one Tn10-insertion mutant of S. typhimurium that showed diminished virulence to recipient wild-type strains of S. dublin, S. enteritidis and S. typhimurium. Each transductant showed a decrease in mouse virulence within the range 10(3)-10(5). It is therefore proposed that similar virulence determinants are expressed in different serotypes. It was also shown that integration that occurred during curing was Tn10 dependent.

The role of plasmid genes in the pathogenicity of Salmonella dublin.

The virulence (expressed as LD50 values) for mice of two mutant strains of Salmonella dublin, both containing TnA insertions in the resident plasmid, was reduced by 10(4)-10(5) when infection was by the oral or intravenous or intraperitoneal route. When the plasmid was lost from one of the mutants no further decrease in virulence was observed. Results also suggested that plasmid genes are not involved in the ability of S. dublin to cross the gut wall.

Evidence for related virulence sequences in plasmids of Salmonella dublin and Salmonella typhimurium.

Transposon-insertion mutants were prepared from virulent field isolates of Salmonella dublin and Salmonella typhimurium. Detailed restriction-enzyme mapping of the single sites of TnA insertion in two mutants (M51 and M173) of S. dublin that showed diminished virulence in a mouse assay indicated that these sites were about 5 kbp apart on the approximately 70 kbp plasmid harboured by the isolate. A Tn10-insertion mutant (M242) of S. typhimurium that showed diminished virulence was also identified. A single copy of Tn10 was inserted into the approximately 90 kbp plasmid harboured by this isolate. Hybridization studies indicated that homology existed between the region encompassing the sites of TnA insertion in M51 and M173 and that encompassing the site of Tn10 insertion in M242. Restriction mapping indicated that the two regions were very similar and could even be identical and, if so, the Tn10 insertion in M242 could be mapped to a point 1.5 kbp from the TnA insertion in M51 and 6.5 kbp from that in M173. It appeared that the maximal extent of the putative similarity/identity was between 13 and 23 kbp. It is proposed that this stretch of high homology could represent a virulence sequence that has been conserved during the evolutionary divergence of the two Salmonella serotypes.

Whole-body metabolism of glucose and lactate in productive sheep and cows.

Constant infusions of D-[U-14C]glucose, D-[6-3H]glucose and L-[U-14C]lactate were used to determine rates of apparent turnover, de novo production, disposal and interconversions of glucose and lactate, together with total recycling of glucose-C, in ewes and dairy cows during late pregnancy and early lactation. The cows were also examined while being fasted. In the fed animals, infusions were made within 5 h after the morning meal when steady-state conditions appeared to exist. In the ewes, circulating concentrations of glucose and lactate, and magnitudes of apparent turnovers of glucose and lactate, tended to be higher during lactation than during pregnancy, while the extent of interconversions of glucose and lactate tended to be lower. Although the metabolic pattern seen in the cows appeared to be similar to that of the ewes during pregnancy, there were clear differences during lactation. Thus, in the lactating cows, as compared with the lactating ewes, circulating concentrations of glucose and lactate were lower, as was apparent turnover related to metabolic body-weight. Furthermore, the percentage of lactate turnover converted to glucose was higher. In the cows, fasting was characterized by low rates of apparent turnover of glucose and lactate and relatively high rates of interconversion of the two compounds. The results indicated that, under the conditions used in this study and when feeding is to recommended levels, carbohydrate metabolism in ewes is more precarious during late pregnancy than during early lactation, while in dairy cows it is more or less equally precarious in both physiological states. A further conclusion is that the extent of glucose-lactate interconversions, and thus Cori cycle activity, seems to be lower in ruminants than in other species.

Tissue glucose and lactate metabolism and interconversions in pregnant and lactating sheep.

Continuous infusions of [14C]glucose and [14C]lactate on separate days, and measurements of blood flow-rate, were used to obtain values for rates of unidirectional metabolism and of interconversion of glucose and lactate in the portal-drained viscera, liver and hind-quarters of ewes during late pregnancy and early lactation. All infusions were made within 5 h after the morning meal, when steady-state conditions appeared to exist. Use was made of ewes that had been appropriately catheterized during pregnancy, and whose catheters remained patent through into lactation. The liver was the main source of glucose production (67-70%) during both pregnancy and lactation. Other sources were the portal-drained viscera (absorbed glucose) and, presumably, the kidneys. Over 80% of the glucose was utilized by the peripheral tissues with approximately 35-40% of utilization being attributable to the hind-quarters. Of the total lactate production, 76% occurred in the peripheral tissues during pregnancy but only 36% during lactation. While the liver utilized 73% of lactate during pregnancy, this value fell to only 42% during lactation, at which time the portal-drained viscera utilized 26% of the lactate. During pregnancy, approximately 80% of the lactate arose from glucose, chiefly in peripheral tissues, while at least 12% of the glucose arose from lactate, chiefly in the liver. During lactation the extent of these interconversions was decreased. Despite the interconversions, whole-body turnover rates for glucose and lactate were under- or overestimated by only 4-10% and 2-5% respectively. Furthermore, a comparison of turnover rates obtained with [U-14C]- and [6-3H]glucose indicated that there was only 6 and 2% recycling of glucose-C during pregnancy and lactation respectively. Under the conditions employed in this study, lactate does not appear to be a major precursor of glucose in the ruminant, and most of the lactate taken up by the liver must be used for purposes other than gluconeogenesis, such as oxidation or alternative anabolic pathways.

Blood flow and nutrient exchange across the liver and gut of the dairy cow. Effects of lactation and fasting.

The rate of blood flow in the portal and hepatic veins, and the net exchange across the gut and liver of volatile fatty acids (VFA), glucose, lactate, pyruvate, amino acids, ketone bodies, glycerol, non-esterified fatty acids (NEFA) and oxygen, were measured in lactating and non-lactating cows (a) in the normal, fed state and (b) before, during and after 6 d of fasting. Blood flow rate through the liver was 52% higher in normal, fed, lactating cows as compared with non-lactating cows, and was decreased by fasting in both groups of cows. Portal blood flow rate increased with an increase in metabolizable energy (ME) intake. Lactating, as compared with non-lactating, cows exhibited lower arterial concentrations of glucose and lactate, higher net portal outputs of VFA and ketone bodies, a higher net hepatic output of glucose, and higher net hepatic uptake of propionate and lactate. The splanchnic outputs of acetate, glucose and hydroxybutyrate were all apparently greater in the lactating cows. Fasting caused a rapid decrease in the blood concentrations of the VFA and an increase in those of glycerol and NEFA. The portal, i.e. gut, outputs of VFA, lactate, ketone bodies, alanine and (serine + threonine), and the portal uptake of O2, were all decreased by fasting. Fasting for 6 h also decreased the hepatic output of glucose and acetate by 77 and 95% respectively, increased the hepatic uptake of pyruvate, glycerol and NEFA, and doubled hepatic ketone-body output. The splanchnic output of acetate and glucose and the splanchnic uptake of O2 were also decreased by fasting. The net portal outputs of VFA, lactate and hydroxybutyrate, and the net hepatic output of glucose, were all correlated with ME intake in fed and fasted cows. Hepatic glucose output was also correlated with milk yield. The net hepatic uptake of gluconeogenic precursors measured in this study could account for net hepatic glucose output in the fasted cows, but not in the fed cows. The net hepatic uptake of the ketogenic precursors butyrate and NEFA was sufficient to account for the hepatic output of ketone bodies in both fed and fasted cows, but it is unlikely that the hepatic uptake of ketogenic precursors could also account for the observed hepatic output of acetate.

Primary ketosis in the high-producing dairy cow: clinical and subclinical disorders, treatment, prevention, and outlook.

Bovine ketosis typically occurs in early lactation. Clinical signs include diminished appetite, decreased milk production, loss of weight, hypoglycemia, and hyperketonemia. Susceptibility to ketosis is probably due to the combination of appetite limitation and a high degree of precedence given to the demand of the mammary gland for nutrients, in particular glucose. The precipitating cause is likely to be development of a marked imbalance between glucose supply and glucose requirement. This imbalance then leads to decreased carbohydrate status, decreased insulin secretion, increased fat mobilization, and increased hepatic ketogenesis. Hepatic ketogenesis may be augmented by the diminished carbohydrate status. The role of hormones other than insulin in the etiology of ketosis, although probably important, has not yet been elucidated satisfactorily. Treatment of ketosis involves increasing glucose supply relative to glucose demand. Incidence of clinical ketosis can be minimized by correct nutrition and management as outlined in recommended guidelines. Besides decreasing milk field, clinical ketosis may affect productivity adversely in other ways, for example, by impairing fertility. Subclinical ketosis is important because it may remain undetected and yet have effects on productivity which parallel those elicited by clinical ketosis. Future research should be directed toward understanding mechanisms conferring priority on milk production and regulating appetite.

Metabolic modes indicative of carbohydrate status in the dairy cow.

Three modes of metabolism, indicative of differing levels of carbohydrate sufficiency, can be identified in nonpregnant dairy cows. The metabolic parameters defining these modes include: the hepatic content of glycogen and glucogenic metabolites; the in vivo net exchange of glucose and propionate across the liver, and of lactate and pyruvate across the liver and gut; and the concentration of insulin in the blood, and the secretion rate of insulin. In descending order of carbohydrate sufficiency, the three modes are; mode I, seen in fed nonlactating cows; mode 2, seen in fed lactating cows; and mode 3, seen in fasted or ketotic cows. The modes are interconvertible, because fasting will transform modes 1 or 2 into mode 3, while administration of either glucose, propionate, or glucocorticoid will, on the basis of at least one index, transform modes 2 or 3 into mode 1. It is concluded that a) carbohydrate sufficiency is jeopardized in lactating cows, and b) one reason for the therapeutic efficacy of antiketogenic agents used in the treatment of bovine ketosis is the ability of these compounds to increase carbohydrate sufficiency.

Net hepatic and splanchnic metabolism of lactate, pyruvate and propionate in dairy cows in vivo in relation to lactation and nutrient supply.

1. Circulating concentrations of glucose, propionate, lactate and pyruvate, and net exchange of these compounds across the liver and gut, were measured in lactating and non-lactating dairy cows (a) in the normal fed state, (b) before, during and after intravenous infusion of an aqueous solution of glucose, propionate or lactate (lactating cows only) in fed animals, and (c) before and during 6 days of food deprivation. 2. In the normal fed state, gut output of propionate, hepatic output of glucose and hepatic uptake of lactate were all higher in the lactating group. There was a net uptake of pyruvate across the liver in the lactating cows and a net output in the non-lactating cows. In the lactating cows there was a net uptake of lactate and pyruvate by the splanchnic bed (i.e. gut and liver combined). 3. In the lactating cows, the glucose and propionate infusions had the following effects: decrease in net hepatic uptake of lactate; a switch in pyruvate exchange across the liver from uptake to output; suppression of uptake of lactate and pyruvate by the splanchnic bed; increase in the magnitude of the liver (propionate uptake)/(glucose output) ratio. Lactate infusion did not affect hepatic propionate uptake. 4. Food deprivation increased hepatic extraction of lactate and pyruvate and decreased the liver (propionate uptake)/(glucose output) ratio in both groups. 5. It is concluded that mechanisms exist to ensure an inverse relationship between the availability to the cow of glucose or propionate and utilization by the splanchnic bed of endogenously derived lactate and pyruvate.