Effects of long-term dietary lipids on mature bone mineral content, collagen, crosslinks, and prostaglandin E2 production in Japanese quail.

This study investigated the effects of long-term dietary lipids on mature bone mineral content, collagen concentration, crosslink levels, bone marrow and ex vivo prostaglandin E2 (PGE2) biosynthesis, as well as the relationship of PGE2 production to these bone formation parameters. One-month-old male Japanese quail were given a basal diet containing 1 of 4 lipid sources: soybean oil (SBO), hydrogenated soybean oil (HSBO), chicken fat (CF), or menhaden fish oil (FO) at 50 g/kg of the diet. At 8 mo of age, lipid treatments did not affect bone length, diameter, or weight in quail. Quail fed SBO or CF had significantly lower levels of mineral content in tibial bones compared with those given FO. Bone collagen level was significantly higher in quail consuming SBO than those given HSBO or CF. Collagen crosslink concentration was markedly increased in birds provided FO or HSBO compared with those fed SBO or CF. Prostaglandin E2 biosynthesis in bone organ culture and marrow were greatly increased in quail maintained on the SBO or CF diet compared with those given the FO or HSBO diet. Prostaglandin E2 production in the bone microenvironment was negatively correlated with tibial ash and collagen crosslinks but had a positive correlation with tibial collagen levels. These results support our previous findings that long-term exposure to diets high in SBO or CF impaired mature bone mechanical properties and histological characteristics. Further, the results suggest that long-term supplementation of SBO or CF in the diet had a significant adverse effect on mature bone metabolism, and that dietary lipids altered bone metabolism, perhaps partially by controlling the production of local regulatory factor in bone.

Long-term supplementation of various dietary lipids alters bone mineral content, mechanical properties and histological characteristics of Japanese quail.

The purpose of this study was to investigate the effects of long-term supplementation of fat in the diets on the fatty acid composition, chemical, mechanical, and histological properties of tibial bone. Month-old male Japanese quail were fed a basal diet containing either soybean oil (SBO), hydrogenated soybean oil (HSBO), chicken fat (CF), or menhaden fish oil (FO) at 50 g/kg of the diet and maintained on these diets for 7 mo. Lipid treatments did not affect body weight, food intake, tibial length, or diameter. The FO diet group had the highest percentage of tibial ash, and both the FO and HSBO significantly increased tibial mineral content compared to those given SBO or CF. The type and amount of fatty acids in the diets had a profound influence on fatty acid composition of lipids in tibial cortical bones. Quail fed FO had the highest concentration of (n-3) fatty acids, and those fed SBO were highest in (n-6) fatty acids. The HSBO diet, containing high level of trans-fatty acids, led to the accumulation of these fatty acids in bone. In quail, long-term supplementation of FO or HSBO increased tibial shear force and shear stress and improved histological cortical thickness and density when compared to those given SBO or CF. These results suggest that long-term exposure to a FO or HSBO diet have a significant beneficial effect on bone metabolism.

Maternal dietary lipids alter bone chemical composition, mechanical properties, and histological characteristics of progeny of Japanese quail.

This study evaluated the effects of maternal dietary lipids on chemical components and mechanical and histological properties of tibia in progeny of Japanese quail fed different dietary lipids. Laying hens were fed a basal diet containing either soybean oil (SBO), hydrogenated soybean oil (HSBO), chicken fat (CF), or menhaden fish oil (FO) at 50 g/kg of the diet. The various maternal dietary lipid treatments did not affect growth of progeny at any developmental stage. There were no differences in tibial length, diameter, or collagen content. Tibial percentage ash was significantly higher in newly hatched progeny from hens fed the FO and HSBO diets. The levels of tibial deoxypyridinoline and total pyridinium crosslinks were higher in the FO and HSBO groups at hatch. At 7 d of age, the tibial deoxypyridinoline links remained higher in the FO group compared to the CF and SBO groups. Likewise, progeny from hens consuming the FO or HSBO diet had higher tibial shear force and stiffness at 7 and 14 d of age. There were no pronounced differences in tibial fracture energy and deflection among treatments. Maternal FO or HSBO enlarged the cartilaginous proliferative and hypertrophic zones of the tibial proximal end in newly hatched quail, which was accompanied by a thicker cortical bone in the diaphysis. However, the width of the hypertrophic zones tended to be smaller in these two groups coupled with improvement in trabecular density and cortical thickness in the proximal end and cortical density in the diaphysis at 3 wk of age. These results suggest that maternal dietary lipids altered bone development by influencing organic matrix quality and mineralization in embryos.

Comparison of phytase from genetically engineered Aspergillus and canola in weanling pig diets.

Ninety-six crossbred pigs with an average weight of 9.0 kg were used in a 5-wk trial to compare the efficacy of genetically engineered Aspergillus ficuum phytase, expressed in Aspergillus niger (Natuphos) or in canola seed (Phytaseed), for enhancing the utilization of phytate P in corn-soybean meal-based diets fed to young pigs and to evaluate the safety of Phytaseed phytase. Three levels of the two sources of phytase (250, 500, or 2,500 U/kg of diet) were added to a corn-soybean meal basal diet containing .35% total P, .09% available P, and .50% Ca. There were six pens per treatment (one barrow and one gilt/pen), except that the diet without added phytase was fed to 12 pens of pigs. Pen feed consumption and BW were recorded weekly. During wk 5, pen fecal samples were collected for determination of apparent digestibilities of DM, Ca, and P. At the end of wk 5, all barrows were killed, and the 10th rib on both sides was removed for determination of shear force and energy. Thirty pigs (six from the diet without added phytase and the diets with 500 and 2,500 U/kg phytase from both sources) were randomly selected for gross necropsy and histologic evaluation of liver, kidney, and bone tissues. Both sources of phytase were equally effective in increasing (P < .05) daily gain, gain:feed, apparent digestibilities of DM, P, and Ca, and 10th rib measurements. Fecal P excretion was reduced with phytase addition. Feed intake was increased by phytase levels during wk 4 to 5. No significant abnormalities were seen in any of the 30 pigs necropsied. The fit of a nonlinear function revealed that most measurements were reaching a plateau at 2,500 U/kg phytase. In summary, based on performance, bone measurements, and digestibilities of P, Ca, and DM of young pigs, the efficiency of Phytaseed was similar to that of Natuphos for enhancing the utilization of phytate P in corn-soybean meal-based diets. General necropsy and histologic examination of tissues indicated no toxic effect of phytase.

Tribological study of joint pathology.

Osteoarthritis (OA) is the most common among the numerous forms of arthritides, affecting millions of people worldwide. Low-grade synovitis is an inflammatory condition commonly seen in OA. In joint fluids aspirated from patients with low-grade synovitis, increased numbers of white blood cells have been detected. During periods of prolonged inflammation, these cells may lyse, releasing lysate into the synovial fluid. The effect of this lysate on cartilage wear and damage has not been investigated previously. A lysate of bovine white blood cells was added to normal bovine synovial fluid. Both lysate treated and normal synovial fluids were used in in vitro tribological wear tests to determine the effect of the white blood cell lysate on the wear and damage of articular cartilage. Cartilage wear increased by a factor of 2.6 when normal synovial fluid was treated with white blood cell lysate. Histology showed considerable damage and fibrillation of the lysate-treated cases, in addition to a loss of proteoglycans in the deep layer of the cartilage. The untreated control cases showed no significant damage or histological abnormalities. It is suspected that the wear and damage seen in the lysate-treated cases is partially due to enzymatic activity within the cartilage. The results of this study suggest that the products of joint inflammation, or synovitis, may have an adverse effect on cartilage wear and damage.

Comparison of genetically engineered microbial and plant phytase for young broilers.

This study was conducted to compare the efficacy of genetically engineered microbial (Natuphos) and plant (Phytaseed) phytase for enhancing the utilization of phytate P in corn-soybean meal-based diets fed to young broilers and to evaluate the safety of Phytaseed phytase. Three levels of each of the two sources of phytase (250, 500, and 2,500 U/kg of diet) were added to a corn-soybean meal basal diet containing 0.46% total P, 0.21% nonphytate P, and 0.92% Ca. There were eight cages per treatment (eight birds per cage for Weeks 2 to 3 and seven birds for Weeks 4 to 5), except for the basal diet without added phytase that had 16 cages. Cage BW and feed consumption were recorded weekly. During Week 5, cage excreta samples were collected for determination of apparent retention coefficients of DM, Ca, and P. At the end of Week 5, all birds were killed, and the left and right toes were removed for determination of toe ash weight and percentage. Forty birds (one per cage from the diet without added phytase and diets with 500 or 2,500 U phytase/kg from both sources) were randomly selected for gross necropsy and histologic evaluation of liver, kidney, and bone tissues. Addition of both sources of phytase resulted in similar increases (P < 0.05) of BW gain; feed intake; gain:feed; apparent retention of DM, P and Ca; and toe measurements. Phosphorus excretion decreased as phytase addition increased. No significant abnormalities were seen in any of the 40 broilers necropsied. Further, the fit of a nonlinear function revealed that most measurements reached a plateau at 2,500 U/kg. Based on performance, bone characteristics, and retention of P, Ca, and DM of young broilers, the efficacy of Phytaseed phytase was similar to that of Natuphos phytase for enhancing the utilization of phytate P in corn-soybean meal-based diets. General necropsy and histologic examination of liver, kidney, and tibial tissues revealed no adverse effects of phytase source or level.

Evaluation of a method for experimental induction of osteoarthritis of the hip joints in dogs.

OBJECTIVE: To evaluate a method for experimental induction of osteoarthritis in the hip joints of dogs. ANIMALS: 12 mixed-breed dogs. PROCEDURE: A unilateral triple pelvic osteotomy was performed. In 6 dogs, the iliac osteotomy was repaired with 45 degrees of internal rotation, reducing coverage of the femoral head by the acetabulum. In the other 6 dogs, the fragments were repaired in anatomic alignment. Radiography, force plate evaluations, and subjective lameness evaluations were performed before and after surgery. Dogs were euthanatized 7 months after surgery, and samples of cartilage and joint capsule were examined histologically. RESULTS: Subjective lameness scores, radiographic appearance of the hip joints, and Norberg angles were not significantly different between groups; however, force plate evaluations did reveal significant differences in vertical ground reaction forces. Femoral head coverage was significantly decreased with rotation of the acetabulum. Mild inflammatory changes were discernible in the joint capsule and articular cartilage of some dogs in both groups. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that 45 degrees internal rotation of the acetabulum does not consistently induce biologically important osteoarthritic changes in the hip joints of dogs.

Toxin production by Pasteurella granulomatis.

Pasteurella granulomatis (Pg) is a recently identified bacterium associated with proliferative fibrogranulomatous panniculitis (also called "lechiguana") in Brazilian cattle. Recent attempts to experimentally reproduce this disease have only been partially successful. We hypothesized that Pg may produce hemolysin(s) and/or cytotoxin(s) which could contribute to its pathogenicity in susceptible cattle. The objective of this study was to determine the presence and degree of hemolytic and leukotoxic activity of selected isolates of Pg. Either ovine or bovine blood agar plates were streaked with 1 of 7 Pg isolates, incubated at 37 degrees C +/- 1 C for 48 hours, and examined for hemolysis. Two of seven isolates showed hemolytic activity on bovine plates, while all seven showed hemolytic activity on ovine plates. By use of the CAMP reaction, involving simultaneous intersecting cultures of Staphylococcus aureus and Pg, all seven Pg isolates showed enhanced (positive CAMP) hemolysis within 24 hours on bovine blood agar plates. Preliminary results using tetrazolium (MTT) dye reductions with bovine neutrophils showed leukotoxicity in 13 of 16 Pg cultures. Alamar blue tests indicate leukotoxic activity for all 7 Pg isolates. We conclude that some Pg isolates have variable hemolytic and/or leukotoxic properties and that this variability (presence and/or degree) of these 2 properties may affect the relative pathogenicity of Pg in susceptible cattle.

Cloning and mutagenesis of a serotype-specific DNA region involved in encapsulation and virulence of Actinobacillus pleuropneumoniae serotype 5a: concomitant expression of serotype 5a and 1 capsular polysaccharides in recombinant A. pleuropneumoniae serotype 1.

A DNA region involved in Actinobacillus pleuropneumoniae serotype 5 capsular polysaccharide (CP) biosynthesis was identified and characterized by using a probe specific for the cpxD gene involved in CP export. The adjacent serotype 5-specific CP biosynthesis region was cloned from a 5.8-kb BamHI fragment and an 8.0-kb EcoRI fragment of strain J45 genomic DNA. DNA sequence analysis demonstrated that this region contained four complete open reading frames, cps5A, cps5B, cps5C, and cps5D. Cps5A, Cps5B, and Cps5C showed low homology with several bacterial glycosyltransferases involved in the biosynthesis of lipopolysaccharide or CP. However, Cps5D had high homology with KdsA proteins (3-deoxy-D-manno-2-octulosonic acid 8-phosphate synthetase) from other gram-negative bacteria. The G+C content of cps5ABC was substantially lower (28%) than that of cps5D and the rest of the A. pleuropneumoniae chromosome (42%). A 2.1-kb deletion spanning the cloned cps5ABC open reading frames was constructed and transferred into the J45 chromosome by homologous recombination with a kanamycin resistance cassette to produce mutant J45-100. Multiplex PCR confirmed the deletion in this region of J45-100 DNA. J45-100 did not produce intracellular or extracellular CP, indicating that cps5A, cps5B, and/or cps5C were involved in CP biosynthesis. However, biosynthesis of the Apx toxins, lipopolysaccharide, and membrane proteins was unaffected by the mutation. Besides lack of CP biosynthesis, and in contrast to J45, J45-100 grew faster, was sensitive to killing in precolostral calf serum, and was avirulent in pigs at an intratracheal challenge dose three times the 50% lethal dose (LD50) of strain J45. At six times the J45 LD50, J45-100 caused mild to moderate lung lesions but not death. Electroporation of cps5ABC into A. pleuropneumoniae serotype 1 strain 4074 generated strain 4074(pJMLCPS5), which expressed both serotype 1 and serotype 5 CP. However, serotype 1 capsule expression was diminished in 4074(pJMLCPS5) in comparison to 4074. The recombinant strain produced significantly less total CP (serotypes 1 and 5 CP combined) in log phase (P = 0.0012) but significantly more total CP in late stationary phase than 4074 (P < 0.0001). In addition, strain 4074(pJMLCPS5) caused less mortality and bacteremia in pigs and mice following respiratory challenge than strain 4074, indicating that virulence was affected by diminished capsule production. These results emphasize the importance of CP in the serum resistance and virulence of A. pleuropneumoniae.

Effects of supplemental phytase and phosphorus on histological, mechanical and chemical traits of tibia and performance of turkeys fed on soyabean-meal-based semi-purified diets high in phytate phosphorus.

Tibial traits were investigated for turkey poults fed on soyabean-meal-based semi-purified diets high in phytate P (2.2 g/kg) with added phytase and inorganic P. Dietary treatments were: (1) 2.7 g non-phytate P (nP)/kg; (2) diet 1 + 1000 U phytase/kg diet; (3) 3.6 g nP/kg; (4) diet 3 + 800 U phytase; (5) 4.5 g nP/kg; (6) diet 5 + 600 U phytase; (7) 6.0 g nP/kg. Added phytase and nP increased (P < 0.006) tibial dry matter, ash weight and content, body-weight gain, feed intake and gain:feed. The Mg and Zn concentrations in the tibial ash were also increased (P < 0.001 and P < 0.09 respectively) by added phytase or nP; tibial P and Ca concentrations tended to be increased. Hypertrophy zone width of the tibial proximal end decreased (P < 0.001), while proliferating zone width, tibial length, and widths at the long and short axes increased (P < 0.003) as phytase and nP were added. The addition of phytase also tended to enlarge the cartilaginous zone width, which was linearly increased (P < 0.05) by added nP. Disorganization scores of the hypertrophy zone and trabecular bone were low, approaching normal (P < 0.05), for turkey poults fed on diets with phytase supplementation, and tibial abnormality scores were linearly decreased (P < 0.001) as nP levels increased (zero score is considered normal). Adding phytase and nP improved the orderliness of development, mineralization and arrangement of cartilage and bone cells, and alleviated the effects of P deficiency on the histological and gross structure of the tibias. Tibial shear stress increased (P < 0.04) as phytase and nP were added. In summary, similar improvements in bone characteristics were achieved for turkey poults fed on a P-deficient diet supplemented with either phytase or nP.

Effects of supplemental phytase and phosphorus on histological and other tibial bone characteristics and performances of broilers fed semi-purified diets.

Two trials with day-old chicks were conducted to investigate the effects of supplemental phytase (Natuphos) on histological, mechanical, and chemical properties of tibia, and performances of broilers fed semi-purified diets containing soybean meal as the only organic P source [0.11% nonphytate P (nP)]. Dietary treatments in Trial 1 were: 1) 0.20% nP, 2) Diet 1 + 800 U of phytase/kg of diet, 3) 0.27% nP, 4) Diet 3 + 600 U of phytase, 5) 0.34% nP, 6) Diet 5 + 400 U of phytase. Supplemental phytase and inorganic P increased tibial length (P < 0.01), shear force (P < 0.001), shear stress (P < 0.05), ash content (P < 0.001), and BW gain and feed intake (P < 0.001) during Trial 1. The hypertrophic zone width at the proximal end of the tibia was decreased (P < 0.05), and the tibial width (P < 0.05) of the long axis of the tibia was increased by the phytase and P supplementation. Supplemental phytase enlarged the cartilaginous and proliferative zones of the tibial proximal end (P < 0.05), and an increase in nP levels produced similar effects. Supplementation of phytase and P also tended to improve the orderliness of development and arrangement of cartilage and bone cells. Dietary treatments in Trial 2 were: 1) 0.27% nP, 2) Diet 1 + 350 U of phytase, 3) Diet 1 + 1,050 U of phytase, 4) 0.45% nP, 5) 0.54% nP, 6) Diet 5 + 1,050 U of phytase. Broilers fed diets containing relatively high levels of nP and phytase supplementation in Trial 2 gave results similar to those observed in Trial 1. Marked improvements (P < 0.05) in the ash content, shear force, shear stress, length of tibia, BW gain, and feed intake, and reduced hypertrophic zone width were achieved for broilers fed the P-deficient diet supplemented with phytase. Also, supplemental phytase tended to increase the width of cartilaginous and proliferative zones, to increase trabecular bone density, and to improve the orderliness of development and mineralization of cartilage and bone cells. In summary, supplementing a low-nP diet with inorganic P or phytase resulted in similar beneficial effects on bone development.

Comparison of hematologic reference intervals between culture system and type of hybrid striped bass.

OBJECTIVE: To determine and compare hematologic reference intervals for hybrid striped bass from different culture systems and for 2 types of hybrid. DESIGN: Observational comparison study. ANIMALS: Clinically normal sunshine bass (reciprocal hybrid striped bass, female Morone chrysops X male M saxatilis) raised in high-density recirculating systems, sunshine bass raised in tanks with biofilters, and palmetto bass (original hybrid striped bass, female M saxatilis X male M chrysops) raised in tanks with biofilters. PROCEDURES: Hematologic reference intervals were determined for fish from the different production systems, following the National Committee for Clinical Laboratory Standards guidelines. Reference intervals from the 3 groups were compared. RESULTS: Reference intervals were significantly different between sunshine bass in the 2 culture systems and between sunshine and palmetto bass. Many of the differences were slight, but notable differences were observed. Sunshine bass in recirculating systems had higher total leukocyte, lymphocyte, neutrophil, and monocyte counts than did either hybrid in tanks. Palmetto bass had a greater number of neutrophils than monocytes, whereas sunshine bass had more monocytes than neutrophils. Additionally, palmetto bass had a lower PCV and hemoglobin value than did either group of sunshine bass. CONCLUSIONS: Separate reference intervals should be developed for hybrid striped bass in different culture systems and for different types of hybrids. CLINICAL RELEVANCE: Determining reference intervals for hybrid striped bass provides a tool to assess the health status of these fish.

Blood biochemical reference intervals for sunshine bass (Morone chrysops x Morone saxatilis) in three culture systems.

OBJECTIVE: To determine and compare biochemical reference intervals for sunshine bass (hybrid striped bass) from 3 culture systems. DESIGN: Observational comparison study. ANIMALS: Clinically normal sunshine bass (reciprocal hybrid striped bass, female Morone chrysops x male M saxatilis raised in high-density recirculating systems (80 g/L), low-density tanks (5 g/L) with biofilters, and cages (70 g/L) in a fresh water pond. PROCEDURE: Biochemical reference intervals were determined for fish from the different production systems following the National Committee for Clinical Laboratory Standards guidelines. Reference intervals from the 3 groups were compared. RESULTS: Reference intervals were significantly different between sunshine bass in the various culture systems. Though most of the differences in reference intervals were minor, fish in the high-density recirculating system had higher concentrations of total protein, albumin, globulin, creatinine, and phosphorus, and lower chloride values. There were no significant differences in glucose concentrations among the 3 groups of fish, and no differences in cortisol concentrations between fish in tanks and cages. CONCLUSIONS: Separate reference intervals should be developed for hybrid striped bass in different culture systems. CLINICAL RELEVANCE: Determining biochemical reference intervals for hybrid striped bass provides a tool to assess the health status of these fish.

Molecular investigation of the role of ApxI and ApxII in the virulence of Actinobacillus pleuropneumoniae serotype 5.

The extracellular hemolytic toxins (ApxI and ApxII) of Actinobacillus pleuropneumoniae are thought to be important factors in this microorganism's virulence and the pathogenesis of swine pleuropneumonia. Using the polymerase chain reaction, the apxI locus of a non-hemolytic, avirulent mutant of A. pleuropneumoniae serotype 5 (mIT4-H) generated by chemical mutagenesis (Inzana T. J., Todd J., Veit H. P. Microb Pathog 1991; 10: 281-96) was found to contain deletions that affected major parts of the entire apxICABD operon, thus inactivating each gene in the operon. The apxII locus was not affected. Monoclonal antibodies to ApxI and ApxII were used to confirm that ApxI was not synthesized, and that ApxII was synthesized but not secreted from the cell. The apxICABD genes and apxIBD genes were cloned into a broad host range vector to obtain plasmids pJFF800 and pJFF801, respectively. Each recombinant plasmid was electroporated into strain mIT4-H to obtain strain mIT4-H/pJFF800 and strain mIT4-H/pJFF801, respectively. Strain mIT4-H/pJFF800 exported ApxI and ApxII, and produced hemolytic activity comparable to or exceeding that of wild type strain J45. Strain mIT4-H/pJFF801 exported only ApxII and produced weak hemolytic activity. Strain mIT4-H/pJFF800 was virulent in mice, and had an LD50 of about 2 x 10(6) colony forming units. In contrast, mIT4-H/pJFF801 and mIT4-H were essentially avirulent in mice, and LD50s for these strains could not be calculated. Strain mIT4-H/pJFF800 was virulent in pigs and caused lethal pleuropneumonia, whereas parent strain mIT4-H was avirulent. Strain mIT4-H/pJFF801 was also able to induce pleuropneumonia in pigs, although a higher dose was required to induce lesions similar to those caused by mIT4-H/pJFF800. Thus, A. pleuropneumoniae strains that produce ApxI and ApxII require ApxI for full virulence and toxic activity in pigs. However, other factors including ApxII contribute to the virulence of A. pleuropneumoniae in pigs.

Muskrat populations in Virginia's Elizabeth River: physiological condition and accumulation of environmental contaminants.

This study evaluated the physiological condition and environmental contaminant concentrations in muskrats inhabiting the contaminated lower region of the Elizabeth River, Virginia, (USA). Muskrats trapped in the lower region of the Elizabeth River weighed less, had lower mean fat indexes, lower relative spleen weights, greater relative adrenal weights, and an increased incidence of disease and parasitism compared to muskrats trapped in the less contaminated upper region of the Elizabeth River or Nansemond River. Concentrations of the metals aluminum, cadmium, copper, nickel, and zinc and the number of polyaromatic hydrocarbon compounds were greater in lower Elizabeth River muskrats compared to upper region Elizabeth River or Nansemond River muskrats. These physiological data indicated that muskrats in the lower region of the Elizabeth River were in poor health relative to muskrats from the other regions studied. However, no significant difference in fecundity or density was observed among regions studied, suggesting that the level of environmental contaminant exposure was not sufficient to elicit a population level response. Greater population declines following severe natural environmental stresses (large storms, extreme cold, etc.) would be anticipated in lower region Elizabeth River muskrats; however, the muskrat population in this region does not now appear to be at risk of extirpation.

Vaccine-derived Pasteurella haemolytica alter the responses of ovine pulmonary artery and vein to drugs acting on adenylate cyclase.

Sheep were vaccinated with a live, vaccine-derived strain of Pasteurella haemolytica. The ex vivo response of isolated pulmonary artery and vein to isoproterenol, cholera toxin, sodium fluoride and calcium were examined three days after vaccination. In the pulmonary artery (endothelium intact), vaccination did not alter the response to isoproterenol, or sodium fluoride whereas the relaxation response to cholera toxin was impaired. In the pulmonary artery (endothelium removed), the maximum relaxation attained in response to isoproterenol was reduced and the response to exogenous calcium, sodium fluoride and cholera toxin not altered. In the pulmonary vein (endothelium intact), the response to isoproterenol and sodium fluoride was unchanged whereas the response to cholera toxin was impaired. In the pulmonary vein (endothelium removed), the response to isoproterenol and sodium fluoride was not altered following P haemolytica vaccination whereas the relaxation response to cholera toxin was enhanced and the response to exogenous calcium slightly impaired. These experiments suggest that vaccination with live strains of P haemolytica cause subclinical disturbances in the pulmonary circulation and may potentially alter the animals' response to pathogens.

Safety, stability, and efficacy of noncapsulated mutants of Actinobacillus pleuropneumoniae for use in live vaccines.

Clonal, noniridescent mutants of Actinobacillus pleuropneumoniae serotypes 1 and 5 were isolated following chemical mutagenesis with ethyl methanesulfonate. The absence of any detectable capsule was confirmed by inhibition radioimmunoassay. There were no differences between the parent and mutant strains in lipopolysaccharide or protein electrophoretic profiles or in hemolytic activity. There was no detectable reversion to the encapsulated phenotype in vitro after passage in mice or pigs or in microporous capsules that were implanted subcutaneously in pigs for 6 weeks. The mutants were able to survive for more than 1 week in pigs following subcutaneous inoculation, which resulted in a strong immune response to whole cells and Apx toxins I and II. Intratracheal challenge of pigs with the serotype 5 mutant at a dose 1 log greater than the 50% lethal dose for the parent resulted in no clinical disease or lesions except in one pig that had slight pneumonia and pleuritis. Twenty-four hours after challenge, A. pleuropneumoniae could not be recovered from the respiratory tracts of any of the challenged pigs except for the one infected pig; this isolate remained noncapsulated. Immunization of pigs with one or both serotypes of noncapsulated mutants protected all pigs against clinical disease following intratracheal challenge with the virulent homologous or heterologous serotype. Nonimmunized control pigs and pigs immunized with a commercial bacterin died or had to be euthanized within 24 h of challenge. Thus, live noncapsulated mutants of A. pleuropneumoniae may provide safe and cost-effective protection against swine pleuropneumonia. These observations support the possibility that noncapsulated mutants of other encapsulated, toxin-producing bacteria may also prove to be efficacious live-vaccine candidates.

Morphological and functional disturbances in pulmonary vascular endothelium following exposure of sheep to an "avirulent" strain of Pasteurella haemolytica.

Sheep were vaccinated with a live attenuated strain of Pasteurella haemolytica and killed 3 days later. Segments of main intrapulmonic artery and vein were removed for biophysical and scanning electron microscopic studies. In the pulmonary artery, vaccination with Pasteurella haemolytica caused an increase in the number of endothelial cell surface blebs and, in some cases, those blebs appeared to be splitting open, suggesting cell damage or irritation. There was a surprising lack of platelet adherence to the lesions, suggesting that an antiplatelet factor is released by the damaged endothelium. The endothelial-dependent relaxant response to bradykinin was enhanced following vaccination. In the pulmonary vein, ultrastructural lesions similar to those in the artery were present in vaccinated animals. Bradykinin caused a contraction, an effect that was reduced following vaccination with Pasteurella haemolytica. These experiments demonstrate that a live, vaccine-derived strain of Pasteurella haemolytica causes both morphological and functional changes in the pulmonary vascular endothelium.