Mark R. Ackermann

Acute Fibrinopurulent Blepharitis and Conjunctivitis Associated with Staphylococcus hyicus, Escherichia coli, and Streptococcus sp. in Chickens and Turkeys

Multiple outbreaks of acute severe fibrinopurulent lesions of the eyelids occurred in chickens and turkeys. Lesions began as tiny foci of epidermal necrosis and ulceration and spread to involve the entire eyelid. Scabs overlying the epidermis contained large gram-positive cocci; lesser numbers of small cocci and gram-negative bacilli were in more superficial areas. Staphylococcus hyicus was isolated from birds in all stages of the disease. Escherichia coli and Streptococcus sp. were isolated only during severe stages; no anaerobic bacterial pathogens were isolated. Vasculitis and perivascular lymphocytic infiltrates in deep layers of the dermis suggested that a staphylococcal toxin may have been involved. The disease was not reproduced by scarifying S. hyicus onto the eyelids or by intravenous inoculation of retro virus-infected chickens.

Use of rats to compare atrophic rhinitis vaccines for protection against effects of heat-labile protein toxin produced by Pasteurella multocida serogroup D

Four bacterin-toxoid and three bacterin commercial vaccines against atrophic rhinitis were tested in rats for their capacity to immunize against the lethal and systemic effects of purified heat-labile protein toxin (D-toxin) produced by Pasteurella multocida serogroup D. Only one bacterin-toxoid vaccine stimulated sufficient immunity to prevent the death of all rats challenged with D-toxin. None of the vaccines prevented weight loss, leukocytosis or increases in serum complement titers in rats challenged with D-toxin. Rats provide an inexpensive animal model for testing the capacity of vaccines to generate antitoxic immunity against the lethal and systemic effects of D-toxin.

Low calcium diet and 1,25-dihydroxyvitamin D3 infusion modulate immune responses during Mycobacterium paratuberculosis infection in geige mice

A 12-month study was conducted to evaluate the effects of feeding a low calcium (Ca) diet or 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3) infusion on the persistence of Mycobacterium paratuberculosis infection using a mouse model. Male beige mice 6-8 weeks of age were assigned to one of the following treatments: (1) non-infected, (2) infected,(3) non-infected/1,25(OH)(2)D(3), (4) infected/1,25(OH)(2)D(3), and (5) infected/low Ca (0.15 percent) diet. Infected mice were inoculated intravenously with live M. paratuberculosis. At 1, 6 and 12 months postinfection, mice in Treatments 3 and 4 were implanted subcutaneously with mini-osmotic pumps to deliver 1,25(OH)(2)D(3). Infusion with 1,25(OH)(2)D(3) exacerbated M. paratuberculosis infection in most tissues at all time points. Mice infused with 1,25(OH)(2)D(3) had higher bacterial counts in spleen, liver, and ileum compared with control infected mice after 1 month of infection. In contrast, feeding a low Ca diet reduced the number of viable organisms cultured from the liver and ileum of infected mice. Plasma Ca and 1,25(OH)(2)D(3) were increased in mice infused with 1,25(OH)(2)D(3) at all time points but values for low Ca mice were not different than for non-infused mice. Splenocyte production of TNF, IL-1 and IL-6 was higher for mice fed the low Ca diet compared with control infected mice after 1 month of infection. Inducible IL-6 activity remained higher for this treatment at 6 months postinfection. These results suggest that feeding a low Ca diet to mice chronically infected with M. paratuberculosis appears to enhance their ability to clear the infection in a manner distinct from any effect of 1,25(OH)2D3.

Influence of chondroitinase on indirect hemagglutination titers and phagocytosis of Pasteurella multocida serogroups A, D and F

Capsules of Pasteurella multocida serogroups A, D and F contain mucopolysaccharides which block antigenic determinants and prevent phagocytosis. In this study, capsules of serogroup A, D and F strains of P. multocida were depolymerized by enzyme treatment. Capsule depolymerization of serogroup D and F strains with chondroitinase increased indirect hemagglutination (IHA) test titers and enhanced phagocytosis by swine neutrophils. Capsule depolymerization of serogroup A strains with hyaluronidase increased IHA titers, but depolymerization with chondroitinase did not. When serogroup A strains were treated with a combination of chondroitinase and hyaluronidase, IHA test titers were lower than titers of the same strains treated with hyaluronidase alone. Combined enzyme treatment of serogroup D strains resulted in IHA test titers similar to those of chondroitinase treatment alone.

Immunity induced in rats vaccinated with toxoid prepared from heat-labile toxin produced by Pasteurella multocida serogroup D.

Rats were vaccinated with a toxoid (D-toxoid) prepared from purified heat-labile toxin (D-toxin) produced by Pasteurella multocida serogroup D. Vaccination of rats with D-toxoid prevented death and other effects of D-toxin (hepatic necrosis, development of elevated leukocyte counts, lymphopenia, neutrophilia, and elevated complement titers) that occurred in phosphate buffered saline (PBS)-vaccinated control rats.

PurifiedPasteurella multocida protein toxin reduces acid phosphatase-positive osteoclasts in the ventral nasal concha of gnotobiotic pigs

SummaryTo study thein vivo response of conchal (turbinate) osteoclasts toPasteurella multocida toxin, four gnotobiotic pigs (7 days of age) were inoculated subcutaneously with 0.2 μg/kg of purified toxin. One toxin-treated pig along with one control pig were necropsied at 2, 5, 9, and 14 days postinoculation. The entire length of nasal concha from the nasal planum to ethmoid region was removed, blocked by transverse cuts into five areas, decalcified, sectioned, and then stained with tartrate-resistant acid phosphatase (TRAP) to identify osteoclasts. In each section, total area of concha, total osteoclast cytoplasmic area, and number of osteoclasts were determined using an image analysis morphometric unit. Also collected from pigs were blood and serum for complete blood counts, electrolyte levels, liver enzymes, and TRAP levels. Conchal atrophy increased in severity with time after 2 days postinoculation. In general, the ventral conchae from toxin-treated pigs at 9 and 14 days postinoculation had decreased surface area, osteoclast cytoplasmic area, and numbers of osteoclasts. Serum levels of TRAP were mildly elevated when compared with age-matched controls. No other significant alterations in blood cells or chemistries occurred and no lesions were present histologically in tissues (liver, kidney, lung, heart, and spleen) other than concha. This study shows that theP. multocida toxin induces rapid bone resorption and increases serum levels of acid phosphatase but leads to diminished acid phosphatase expression and presumably, numbers of osteoclasts.

Identification of β2 Integrins in Bovine Neutrophils by Scanning Electron Microscopy in the Backscatter Mode and Transmission Electron Microscopy

Neutrophils (I x 105) were suspended in phosphate-buffered saline (PBS) and I% bovine serum albumin (BSA) for 10 minutes at 37 C to block nonspecific binding sites. Cells were centrifuged (900 rpm for 3 minutes) and rinsed twice with PBS (pH 7.2). Some of the cell suspensions were incubated with platelet activating factor (PAF; 100 ng/ml) for 5 minutes. Neutrophils were incubated with the optimal concentration of primary antibody (RI5.7; 5 f.Lg/ml final concentration) for I hour, rinsed, resuspended, and then exposed to the secondary antibody conjugated to 30-nm gold (I: 20 dilution final dilution) for 30 minutes. To assess possible nonspecific binding, control preparations used neutrophils with normal CD 18 expression but that either lacked the primary antibody or were exposed to a mouse antibody specific for unrelated antigens. Neutrophils were rinsed three times, fixed with I% glutaraldehyde (pH 7.4) for I hour, and rinsed again. A single layer of fixed neutrophils was applied to a round glass coverslip precoated with fresh poly-r.-Iysine and mounted with aluminum paint onto an aluminum stub; remaining neutrophils were processed for transmission electron microscopy. The preparation was dehydrated in graded alcohols, dried in a critical point dryer (Balzers CPO 020, Liechtenstein), coated with carbon (l0-20-nm layer), and examined on a JEOL scanning electron microscope (Tokyo, Japan) equipped with a backscatter detector. Gold sputter coating and osmium fixation could not be used because these heavy metals would cause haphazard and unorganized electron reflection and obliterate the colloidal gold particles. Carbon (atomic weight = 14), however, is light enough to coat the samples without interfering with the electroreflective properties of the gold particles and prevents undesirable charging effects. The optimal magnification settings were 18,000 x magnification and 15 kV. Gold particles were present on the surface of neutrophils from cows that expressed normal levels of CD 18. These foci were widely separated by 0.025-0.05 f.Lm (Fig. I) and were present on both the tips of surface pseudopodia and along the deeper recesses of the plasma membrane. The particles were easily visible in the backscatter (BE) mode but were undetectable in the conventional scanning mode. For quantitative work, five random cells per stub were photographed (18,000 x ) from three replicate tests, and gold particles were counted. By scanning electron microscopy, the number of gold particles averaged 62.0 and 51.4 particles per field of

Reduced physeal area and chondrocyte proliferation in Pasteurella multocida toxin-treated rats

Pasteurella multocida toxin depresses weight gain in rats and pigs. It also affects tissues with rapidly dividing cells. In the present study, we investigated the role of this protein toxin on chondrocyte growth in vivo. Rats were divided into a single- or multiple-dose group and were given, respectively, either a single injection (0.15 or 0.6 μg/kg toxin subcutaneously) or multiple injections (0.01-0.2 μg/kg subcutaneously) of toxin. Bone (humerus) and other selected tissues were stained for bromodeoxyuridine immunoreactivity (BrDU-IR) in order to gauge cell proliferation. Physeal area was measured in rats from the multiple-dose group. Serum from single-and multiple-dose groups were tested for tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) activity using a bioassay system. Decreased weight gain, feed intake, and feed efficiency were observed in single- and multiple-dose groups of rats. Decreased BrDU-IR indices were present in the resting and proliferative zone chondrocytes of the humeral physis in rats from the multiple-dose group, as was decreased physeal area. Increased serum IL-6 bioactivity was present in rats after 24 hours, and no changes in TNF-α bioactivity were seen in any group. No alterations in BrDU-IR were seen in rats fed restricted (80% of control) diets. These studies show that sublethal doses of toxin decrease weight gain and affect growth of long bones through suppression of chondrocyte proliferation. These effects may be mediated by direct binding of the toxin to target cells or IL-6 but are not associated with altered feed intake or TNF-induced cachexia.

Clinical and Immunological Features Associated with Bovine Leukocyte Adhesion Deficiency

Granulocytopathies in dogs (1), humans (2), and cattle (3,4) characterized by persistent progressive neutrophilia in patients affected with severe recurrent bacterial infections and failure to form pus were reported between 1975 and 1987. In all three species, these conditions were determined to be heritable deficiencies of leukocyte surface glycoproteins associated with diminished cell adherence between 1984 and 1990 (5–9). Although published reports of its diagnosis are few, the bovine granulocy-topathy syndrome has been diagnosed at veterinary schools throughout the world during the past 8 y. In vitro assessments have identified abnormalities of motile, phagocytic, and oxidative functions of neutrophils which appear to mediate inflammatory deficits in vivo (3,4,7,8). Factors contributing to low frequency of diagnosis of this syndrome may relate to the impracticality of intensive clinical laboratory studies in food-producing animal species.