Pathogenesis of porcine Actinobacillus pleuropneumonia, part II: roles of proinflammatory cytokines.

The in vitro production of proinflammatory cytokines after stimulation with Actinobacillus pleuropneumoniae and the relation of these cytokines in vivo with the disease caused by A. pleuropneumoniae were investigated. Within 24 h, in vitro stimulation by A. pleuropneumoniae (serotype 1) preparations, including killed bacteria, bacterial culture supernatant, lipopolysaccharide, and bacterial extracts, porcine pulmonary alveolar macrophages (PAM) produced significant (P < 0.05) amounts of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) as measured by bioassays. The supernatants containing interleukin-8 from PAM after stimulation by bacterial preparations showed significant neutrophil chemotaxis, while bacterial preparations alone did not. After in vivo infection with A. pleuropneumoniae, the mean levels of TNF-alpha and IL-1 in serum, as measured by bioassays, were elevated 37- to 27836-fold for TNF-alpha and 11- to 5941-fold higher for IL-1 within 4 d post-infection, depending on the treatments, and remained elevated up to Day 7. Both cytokines were also detected in porcine lungs by bioassays and immunocytochemistry. The results indicated that both secreted and surface components of A. pleuropneumoniae can stimulate PAM to produce proinflammatory mediators. Neutrophil chemoattractants rather than bacterial components are the major factor causing acute lung inflammation. The elevation of TNF-alpha and IL-1 in pigs occurred coincident with the onset of acute clinical disease.

Depletion of penicillin G residues in tissues, plasma and injection sites of market pigs injected intramuscularly with procaine penicillin G.

Procaine penicillin G was administered by intramuscular (i.m.) injection to groups of healthy 100 kg market pigs at the approved label dose (15,000 IU/kg body weight), once daily for three consecutive days; or an extra-label dose (66,000 IU/kg body weight), once daily for five consecutive days. Penicillin G residue depletion was followed in plasma, tissue and injection sites using a liquid chromatographic method. Groups of pigs were killed 1, 2, 3, 4, 5 and 8 days after the last injection with the label dose. Penicillin G was not detected in liver after 1 day of withdrawal, in muscle and fat after 2 days of withdrawal, in plasma after 4 days of withdrawal, in skin after 5 days of withdrawal, or in kidney and the injection sites after 8 days of withdrawal. Other groups of pigs were killed 1, 2, 3, 5 and 7 days after injection with the extra-label dose. In these pigs penicillin G was not found in liver after 2 days of withdrawal, in fat after 3 days of withdrawal, or in the muscle, skin, plasma and injection sites after 7 days of withdrawal. Penicillin G was found at all times in the kidneys of the groups of pigs that received the high dose. The technique used for neck injections was critical to obtain intramuscular rather than intermuscular injections. The Bureau of Veterinary Drugs, Health Protection Branch, Health Canada calculated that the appropriate withdrawal period for pigs was 8 days for a dose of 15,000 IU procaine penicillin G/kg body weight and 15 days for a dose of 66,000 IU/kg.

Pathogenesis of porcine Actinobacillus pleuropneumonia: Part I. Effects of surface components of Actinobacillus pleuropneumoniae in vitro and in vivo.

To understand the role of non-secreted components of Actinobacillus pleuropneumoniae in virulence, we investigated in vitro cytotoxicity and in vivo pulmonary changes in pigs due to various A. pleuropneumoniae (serotype 1) fractions. Following 1.5 h incubation, lipopolysaccharide (LPS), 2 crude extracts and bacterial culture supernatant (BCS) at high concentrations were cytotoxic to porcine pulmonary alveolar macrophages (PAM), peripheral blood mononuclear leucocytes, neutrophils and a cultured porcine bone marrow cell line. Heat-killed bacteria were cytotoxic to PAM after 24 h incubation. The 2 crude extracts were prepared by shaking either intact bacteria after removing culture supernatants (crude surface extract, CSE), or whole bacterial culture (crude surface plus culture supernatant extract, CSSE) with glass beads in saline at 60 degrees C. Further experiments showed that proteins from the bacterial membrane were partially involved in cytotoxicities of these 2 extracts. Both BCS and CSSE caused multivocal hemorrhage and neutrophil infiltration when inoculated into porcine lungs, but CSE did not. The lung:whole body weight ratios of the pigs treated with CSSE were significantly higher (P < 0.05) than those of pigs treated with BCS, CSE, or control solution. It is concluded that beside the secreted proteins, bacterial surface components including LPS and non-secreted proteins were cytotoxic in vitro; and secreted and non-secreted components act synergistically to cause lung lesions.

Diagnosis of Elaphostrongylus cervi infection in New Zealand red deer (Cervus elaphus) quarantined in Canada, and experimental determination of a new extended prepatent period.

A modified Baermann assay was used to recover dorsal-spined, first stage larvae of Elaphostrongylus cervi from feces and lungs of red deer (Cervus elaphus elaphus) from three of four herds imported from New Zealand into Canadian quarantine facilities. Tests done on a series of fecal collections showed that larval output from infected red deer was low and sporadic, casting doubt on the efficacy of the Baermann assay to detect all infected individuals in the herds. The animals had passed repeated preembarkation Baermann tests for E. cervi in New Zealand. Seven larvae recovered from these red deer were used to establish a patent infection in a naive red deer. The prepatent period was 206 days and larval shedding was intermittent. Elaphostrongylus cervi is a foreign animal parasite in continental North America, which could become irrevocably established if it were introduced. The data reported indicates that there is currently no reliable method for the detection of E. cervi infection.

Ovine Haemophilus somnus: experimental intracisternal infection and antigenic comparison with bovine Haemophilus somnus.

Experimental infection was produced by two of four isolates of ovine Haemophilus somnus given by intracisternal inoculation into two to three-month-old lambs. Isolate 2041 (originally obtained from a septicemic lamb in Alberta) caused lethal infection in eight of nine lambs, isolate 67p from the prepuce of a normal lamb produced less acute disease in four of nine lambs, and the other two isolates (93p and 1190) caused no detectable disease. Significant lesions were limited to the brain and spinal cord. Purulent meningitis was characteristic but vasculitis or septicemia were not detected, perhaps due to the route of inoculation. Since a difference in virulence was noted among strains, we analyzed surface proteins thought to be virulence factors of bovine H. somnus. Protein profiles of bovine and ovine H. somnus done by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed similar patterns for virulent bovine isolates and ovine septicemic isolates. Preputial isolates showed a lower molecular mass major outer membrane protein than septicemic isolates. Antigenic analysis revealed that outer membrane proteins p270, p78, p76, p40, and p39 were detected in both ovine and bovine isolates except for 1190, which was probably not a true H. somnus isolate. Thus the preputial and septicemic isolates of ovine H. somnus were similar to bovine H. somnus in pathogenicity and in surface antigens.

Disposition of penicillin G after administration of benzathine penicillin G, or a combination of benzathine penicillin G and procaine penicillin G in cattle.

Plasma concentration of penicillin G was evaluated in beef steers after administration of either a combination of benzathine penicillin G and procaine penicillin G in a 1:1 mixture at a dosage of 9,000 U/kg of body weight, IM (n = 5), 24,000 U/kg, IM (n = 5), or 8,800 U/kg, SC (n = 5), or benzathine penicillin G alone at a dosage of 12,000 U/kg, IM (n = 7). Plasma concentration of penicillin G was measured by use of a high-performance liquid chromatography assay that had a limit of determination of 0.005 microgram/ml. At a dosage for this combination of 9,000 U/kg IM, and 8,800 U/kg, SC, which are approved label recommendations in Canada, and the United States, respectively, mean (+/- SEM) peak plasma concentration was 0.58 (+/- 0.15) and 0.44 (+/- 0.02) microgram/ml, respectively. Although plasma penicillin concentration was quantifiable for 7 days in the steers that received 9,000 U/kg, IM, and for 4 days in the steers that received 8,800 U/kg, SC, the concentration was < 0.1 microgram/ml in both groups after the first 12 hours. After administration of the combination at dosage of 24,000 U/kg, IM, there was an initial peak plasma concentration at approximately 2 hours; thereafter, plasma concentration decreased slowly, with half-life of 58 hours. Although plasma penicillin G concentration was quantifiable for 12 days at this dosage, concentration was < 0.1 microgram/ml after the first 48 hours. After the initial 48 hours, plasma concentration of penicillin was of similar magnitude and decreased at similar rate for the combination at dosage of 24,000 U/kg and for 12,000 U/kg of benzathine penicillin G alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Depletion of penicillin G residues in tissues and injection sites of yearling beef steers dosed with benzathine penicillin G alone or in combination with procaine penicillin G.

The contribution of benzathine penicillin G to residues in tissues and injection sites of yearling beef steers was assessed by treating seven groups of five to seven steers with either benzathine and procaine penicillin G together or benzathine penicillin G alone. Steers were injected with a commercial combination of benzathine and procaine penicillin G according to the Canadian (intramuscular) or United States (subcutaneous) label dosages of 8600 and 8800 IU penicillin G/kg body weight, respectively. They were killed 14 or 30 days after the intramuscular injections, and 30 days after the subcutaneous injections. At the label withdrawal times, Canadian 14 days and United States 30 days, the levels in the injection sites for all of the treatments were 30-60 times above the Canadian and United States' Maximum Residue Limit of 50 micrograms/kg, while liver, kidney and gluteal muscle levels were below the Maximum Residue Limit. Other steers were injected intramuscularly with 24,000 IU benzathine/procaine penicillin G/kg body weight and slaughtered 8, 14 or 50 days after injection. Fifty-day injection site residues were 24 times the Maximum Residue Limit. Another group of steers was injected intramuscularly with benzathine penicillin G alone at 12,000 IU/kg body weight and slaughtered 14 days later. Penicillin G levels in the injection sites were 156 times the Maximum Residue Limit. The persistence of penicillin G residues at the injection sites in all the treatment groups appears to be attributable primarily to benzathine penicillin G. Visual inspection of muscle surfaces did not reliably reveal all injection site lesions in the underlying musculature.

Depletion of intramuscularly and subcutaneously injected procaine penicillin G from tissues and plasma of yearling beef steers.

Withdrawal periods required when doses of 24,000 IU and 66,000 IU of procaine penicillin G/kg body weight were administered to yearling beef steers by intramuscular injection daily for five consecutive days were investigated. These dosages are in excess of product label recommendations, but are in the range of procaine penicillin G dosages that have been administered for the treatment of some feedlot bacterial diseases. The approved dose in Canada is 7,500 IU/kg body weight intramuscularly, once daily, with a withdrawal period of five days. Based on the tissue residue data from this study, the appropriate withdrawal period is ten days for the 24,000 IU/kg body weight dose and 21 days for the 66,000 IU/kg body weight dose when administered intramuscularly to yearling beef steers. In a related study, 18 yearling beef steers received 66,000 IU of procaine penicillin G/kg body weight administered by subcutaneous injection, an extra-label treatment in terms of both dose and route of administration, typical of current practice in some circumstances. Deposits of the drug were visible at subcutaneous injection sites up to ten days after injection, with more inflammation and hemorrhage observed than for intramuscular injections of the same dose. These results suggest that procaine penicillin G should not be administered subcutaneously at high doses; and therefore a withdrawal period was not established for subcutaneous injection.

A study of the disposition of procaine penicillin G in feedlot steers following intramuscular and subcutaneous injection.

The disposition of an aqueous suspension of procaine penicillin G (300,000 U/mL) was studied in feedlot steers. Four groups of three steers were used. Steers in groups 1 and 2 received procaine penicillin G once daily for 5 days intramuscularly (i.m.) at a dose of 24,000 U/kg (group 1) or of 66,000 U/kg (group 2). The injection on the last day was administered in the gluteal muscle. Steers in group 3 (i.m. neck injection) and group 4 [subcutaneous (s.c.) injection] each received a single dose of procaine penicillin G at a dose of 66,000 U/kg. From every animal, after the last injection in groups 1 and 2 and following the single injection in groups 3 and 4, a series of blood samples was taken at fixed time intervals. The plasma from these samples was analysed for penicillin G by a high performance liquid chromatography (HPLC) assay in order to determine the disposition of penicillin. The maximum plasma concentration (Cmax) and the area under the curve (AUC) were significantly different between groups 1 and 2, but we found no difference in the disappearance rate constant between these two groups. Group 4 single s.c. injections produced a lower mean Cmax (1.85 +/- 0.27 microgram/mL) than the mean Cmax (4.24 +/- 1.08 micrograms/mL) produced in group 3 by i.m. injections into the neck muscle or the mean Cmax (2.63 +/- 0.27 microgram/mL) produced in group 2 by i.m. injections into the gluteal muscle. However the mean Cmax produced by i.m. injections into the neck muscles (group 3) was higher than the mean Cmax produced by i.m. injections into the gluteal muscle (group 2). Additionally, the disappearance t1/2 was longer (18.08 h) in group 4 following the s.c. injection and shorter (8.85 h) in group 3 following the i.m. neck injection, than the t1/2 following administration of the same dose i.m. into the gluteal muscle (15.96 h) in group 2. In this study, when procaine penicillin G was injected into the gluteal muscle, doses of 66,000 U/kg were necessary to produce plasma concentrations that were above a minimum inhibitory concentration (MIC) for penicillin G of 1.0 microgram/mL as compared to doses of 24,000 U/kg.

Hyperparathyroidism in black patients: an update.

Cervical exploration with removal of the pathologic gland or glands is effective treatment for parathyroid adenomas and hyperplasia. This article reports the results of a retrospective study of 41 patients who underwent elective cervical exploration for primary hyperparathyroidism at Howard University Hospital between 1974 and 1989. Preoperative localization studies for primary neck exploration consisted of an ultrasound of the neck. Removing the diseased gland/glands resulted in the resolution of the patients' symptoms and the return of calcium levels to normal. Complications included transient hypocalcemia, a wound hematoma, and a postoperative death.

Performance of Five Screening Tests for the Detection of Penicillin G Residues in Experimentally Injected Calves.

Three calves were each injected with a single intramuscular (IM) dose of penicillin G procaine at either 3750, 7500, or 15000 IU per kg of body weight and killed at 24 h postinjection, along with a control calf that had not received penicillin. Tissues were tested by the Swab Test on Premises (STOP), the Calf Antibiotic and Sulfa Test (CAST), the Brilliant Black Reduction Test (BBRT), the Charm Test II, thin layer chromatography - bioautography (TLC/BA), and high performance liquid chromatography (HPLC). Samples of muscle, liver, and kidney from all injected calves contained detectable penicillin residues when analyzed by HPLC. The BBRT and Charm Test II were the most sensitive test kits for penicillin G in muscle, while the Charm Test II also detected residues in livers and kidneys from all injected animals. The STOP and CAST were less sensitive, although improved performance was observed for the STOP using a modified growth medium. Penicillin residues were detected in all livers and kidneys from injected animals using TLC/BA. Urine collected from injected animals 12 and 24 h postinjection was positive by the Live Animal Swab Test (LAST). All urine and tissue samples from the control animal were negative. The BBRT and Charm Test II appear to offer greater sensitivity for penicillin G residues than such currently used procedures as STOP and CAST but should be confirmed by a suitable laboratory method, such as the HPLC procedure used in this study.

Bioassay techniques and high-performance liquid chromatography for detection of oxytetracycline residues in tissues from calves.

Tissue specimens from muscle, liver, kidney, and injection sites were collected, and serum was obtained from 3 calves euthanatized on each of posttreatment days 5 and 22. Calves were treated with 6.7, 13.4, or 20 mg of oxytetracycline (OTC)/kg of body weight, IM, once daily for 3 days; these dosages are 1, 2, and 3 times the label dose, respectively. One control calf was euthanatized on each of posttreatment days 5 and 22. In treated male calves killed 2 days after the last injection, OTC residues were detected in all tissues and serum, using high-performance liquid chromatography. Tissues from all injection sites also were considered positive for antimicrobial residues, using the swab test on premises (STOP), microbial inhibition test (MIT), and thin-layer chromatography-bioautography (TLCB) test. Kidney tissues from a calf given 13.4 mg of OTC/kg and kidney and liver tissues from a calf given 20 mg of OTC/kg also were considered positive, using the MIT and TLCB. Results of the STOP only were considered positive for the liver and kidney of a calf given 20 mg of OTC/kg, but substitution of Saskatoon antibiotic medium-3 for the original medium (antibiotic medium-5) allowed the STOP to detect residues in these tissues from all treated calves. In female calves killed 19 days after the last injection, the STOP, MIT, and TLCB procedures revealed positive results for tissues from some injection sites, but revealed negative results for other tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Persistence of chloramphenicol residues in calf tissues.

Twenty-one 5 to 18 day old calves were administered 11 mg chloramphenicol in propylene glycol per kg body weight intramuscularly twice daily for three days. Groups of calves were euthanized with a barbiturate overdose at 5, 21, 42 and 70 days after the last dose was administered. Serum, kidney, analyzed for the drug using a quantitative gas chromatographic method with a detection limit of five parts per billion. After five days of withdrawal, chloramphenicol was detected in all the injection sites and in 6 out of 16 of the other samples. After 21 days of withdrawal, chloramphenicol was detected in all the injection sites and in one each of the serum, liver and muscle samples. After 42 days of withdrawal, chloramphenicol was detected in the injection sites only, and after 70 days of withdrawal it was not detected in any of the samples.

Association of eosinophilic myositis with an unusual species of Sarcocystis in a beef cow.

The carcass of a mature cow had numerous, disseminated lesions typical of eosinophilic myositis. To elucidate the nature and possible cause of the lesions, histological sections were examined by light microscopy and selected areas were removed and processed for electron microscopy. The lesions were granulomatous in nature. Each granuloma contained at its centre an intact or ruptured sarcocyst associated with degenerate muscle fibers. Surrounding this was a layer of epithelioid cells and an intense accumulation of inflammatory cells, most of which were eosinophils. The primary cyst wall of the sarcocysts in these granulomas consisted of hair-like protrusions that featured many unusual electron-dense bodies. Sarcocysts with ultrastructures characteristic of Sarcocystis cruzi and Sarcocystis hirsuta were also present in muscle from the same animal, but these sarcocysts lacked any associated cellular responses. The eosinophilic myositis in this case appeared to be associated with sarcocystosis of an unknown species. Possibly, the inflammatory reaction was due to the host-parasite interaction in an unusual host.

Diseases of wapiti utilizing cattle range in southwestern Alberta.

Specimens from 28 wapiti (Cervus elaphus canadensis) were collected by hunters in southwestern Alberta in 1984. Various tests were performed to detect infections and conditions that could affect cattle sharing the range or cause disease in wapiti. Serum antibodies were present against leptospiral serovars autumnalis (25%), bratislava (4%), and icterohaemorrhagiae (8%), and the viruses of bovine virus diarrhea (52%), infectious bovine rhinotracheitis (45%), and parainfluenza type 3 (13%). No serological evidence of bovine respiratory syncytial virus, Brucella, Anaplasma, bluetongue virus, or epizootic hemorrhagic disease virus was found, nor were any lesions of vesicular diseases, necrotic stomatitis or nutritional myopathy evident. Focal interstitial nephritis and sarcocystosis were diagnosed histologically in 40% and 75%, respectively, of the wapiti tested. The prevalence of giant liver flukes (Fascioloides magna) was 50% and of lungworms (Dictyocaulus viviparus) 32%. Leptospiral serology on cattle in the area did not indicate that wapiti or cattle were a serious source of infection to each other. The giant liver fluke was the parasite most likely to be amplified by wapiti for cattle. Within the limits of this study, the results indicated that wapiti in the Waterton area do not pose a disease threat to the cattle with which they range, but periodic observational studies in these wapiti would be a useful means of early detection of any changes in the interspecies relationship.

Rabies in Western Canada 1978-1984.

The results of laboratory examination of 22,503 specimens for the diagnosis of rabies by the standard fluorescent antibody and mouse inoculation tests over a seven year period are presented. Specimens were received from British Columbia, Alberta, Saskatchewan, the Yukon and Northwest Territories. Of the 1,445 positive cases, 10.50% involved human contact. The main reservoirs of rabies were skunks, bats and foxes. The reliability of the fluorescent antibody test for the diagnosis of rabies was reaffirmed by agreement with the mouse inoculation test in over 99% of cases.

Serum complement-fixing antibody to Pasteurella haemolytica A1 and its effect on experimentally induced pneumonic pasteurellosis in calves.

Ninety-three calves comprising 16 experimental groups were exposed to viral (bovine herpesvirus-1 or parainfluenza-3 virus) and Pasteurella haemolytica aerosols. Serum samples from these calves were tested before and after exposure for antibodies to P haemolytica by a modified direct complement-fixation test. At slaughter of the calves, the extent of pneumonia produced was estimated for each calf and compared with the results of the modified direct complement-fixation tests. The extent of pneumonia was not related (P greater than 0.05) to the amount of anti-P haemolytica antibody produced by either naturally occurring or experimentally induced infection.

Anticytotoxin activity of bovine sera and body fluids against Pasteurella haemolytica A1 cytotoxin.

Toxin neutralizing activity of bovine sera and body fluids against Pasteurella haemolytica type A1 cytotoxin was evaluated by 51Cr release assay using bovine peripheral blood mononuclear leukocytes as the target cells. Sera collected from precolostral calves did not exert anticytotoxin activity at 10(-1) or higher dilutions, whereas randomly selected complement fixing antibody-negative sera neutralized on average over 90% of cytotoxin activity at the 10(-1) dilution and less than 50% of the toxin activity at 10(-2) or higher serum dilutions. Nasal secretions and lung washings of some of the cattle tested also contained cytotoxin neutralizing activity. The antibody nature of the cytotoxin neutralizing activity was demonstrated by its neutralization with bovine immunoglobulin G2 purified from pooled seropositive sera. Sera from a group of cattle which were vaccinated with a potassium thiocyanate extract of P. haemolytica, but which subsequently developed fibrinous pneumonia after aerosol challenge with bovine herpesvirus 1 and P. haemolytica, had significantly lower anticytotoxin activity than sera from another group of cattle which did not develop the disease after similar vaccination and challenge. Cattle which survived a natural outbreak of shipping fever had higher anticytotoxin activity than those having fibrinous pneumonia in the aforementioned experimental group, although there was no statistical difference between them and a randomly selected CF seronegative group. It is probable that this cytotoxin neutralizing antibody exerts a beneficial effect in protection of cattle against pneumonic pasteurellosis.