Efficacy of Corynebacterium pseudotuberculosis bacterin for the immunologic protection of sheep against development of caseous lymphadenitis

The efficacy of a Corynebacterium pseudotuberculosis bacterin to protect sheep immunologically against development of caseous lymphadenitis was evaluated in controlled challenge-exposure experiments. Sixty-three mixed-breed, white-faced lambs were used. The lambs were 10 to 12 weeks old and were randomly assigned to 3 groups (21 lambs/group). Group 1 was vaccinated once, using 2 ml of a C pseudotuberculosis bacterin (given subcutaneously) in the right axillary region at the beginning of the study. Group 2 was vaccinated twice; the 1st vaccination was given at the same time that lambs in group 1 were vaccinated and the 2nd vaccination was given 4 weeks later. Group 3 (nonvaccinated controls) was given physiologic saline solution (2 ml, subcutaneously). Each lamb was challenge exposed (ie, given 2 ml of live Corynebacterium pseudotuberculosis inoculum [6 X 10(6) colony-forming units/ml], subcutaneously at 4 different sites) during the 20th week of the study. All lambs were killed and necropsied during week 33. The mean number of abscesses per lamb was 7 for group 1, 4 for group 2, and 32 for group 3. Significant differences in the size of the abscesses were not found between the groups. Results of the study indicated that the vaccine provided immunologic protection of lambs against challenge exposure to Corynebacterium pseudotuberculosis.     

Corynebacterium pseudotuberculosis infection in adult ewes by inoculation in the external ear

Caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis, was studied in ewes to devise a reliable method of induced infection that would help to understand mechanisms by which abscesses develop in lymph nodes or internal organs, especially the lungs. Fifteen adult ewes, from a flock where the occurrence of caseous lymphadenitis was unknown, were inoculated SC in the auricle with various doses of a virulent strain of C pseudotuberculosis. Ewes inoculated with at least 10(8) viable corynebacteria develop a strong local reaction that was apparent 4 hours after challenge exposure and lasted 7 to 14 days. Of these ewes, all developed at least 1 abscess and 3 developed multiple abscesses in lymph nodes draining the inoculated auricle and in the lungs. All infected ewes developed a strong antibody response against C pseudotuberculosis exotoxin, which persisted for 3 months. Of 9 ewes infected with 1.2 X 10(8) corynebacteria, 3 developed lesions because of field strains of C pseudotuberculosis and had an increased capacity to limit the dissemination of challenge-inoculum bacteria by developing only one abscess in the draining lymph node (parotid or prescapular lymph node). Seemingly, inoculation in the external ear was a suitable method for obtaining a reproducible infection and indicated that a primary infection might protect against reinfection by limiting dissemination of C pseudotuberculosis.     

Comparison of protection induced in lambs by Corynebacterium pseudotuberculosis whole cell and cell wall vaccines

Colostrum-deprived lambs were vaccinated IM with 10 mg (dry weight) of Corynebacterium pseudotuberculosis whole cells (WC) or cell walls (CW) and their immunity was challenged by IV injection of 3.1 X 10(4) colony-forming units of C pseudotuberculosis. Before challenge exposure, the logarithmic mean antibody titers were 2.0837 in lambs that were vaccinated with WC, 2.6858 in lambs that were vaccinated with CW, and 1.4214 in control lambs. Significant protection was demonstrated by fewer abscesses and organisms in the lungs of lambs vaccinated with WC or CW (P less than 0.05) than in control lambs. By the same criteria, more protection was provided to lambs vaccinated with CW than to lambs vaccinated with WC.     

Experimental Corynebacterium pseudotuberculosis infection in lambs: kinetics of bacterial dissemination and inflammation

Infection and pyogranulomas induced by Corynebacterium pseudotuberculosis were experimentally reproduced in lambs. In two separate experiments, bacterial multiplication and dissemination were studied in 30 male lambs inoculated subcutaneously into the right ear with 1.1 or 1.5 X 10(8) viable C. pseudotuberculosis strain 19R. Infected lambs were necropsied at various times until the 28th day following inoculation. After a transient hyperthermia and a strong local inflammatory reaction, an abscess developed in the right ear from postinoculation day (PID) 6; it enlarged until PID 14 and stabilized thereafter and was associated with adenopathy of lymph nodes draining the head. Three acute phase indicators of inflammation were followed in 14 out of 30 lambs; plasma levels of copper and haptoglobin increased rapidly following inoculation whereas zinc levels decreased. The peaks were reached from PID 1 to 5, and thereafter the values came back slowly to the baseline. Antibodies against C. pseudotuberculosis exotoxin increased from PID 5 and reached a plateau on PID 21. Bacterial dissemination, assessed by the number of infected organs per lamb, was maximal on PID 16 and then stabilized until the end of the experiment. Lungs were infected in seven out of 18 lambs necropsied on PID 28. These results demonstrate a significant relationship between the clinical score of superficial lymph nodes or inoculation site and the infection level of these organs, and an early localization of pyogranulomatous lesions in regional lymph nodes. The subsequent development of the disease was related to the enlargement of these lesions and, in some animals, to a bacterial dissemination from primary sites of infection in the right prescapular lymph node and in the lung.     

Protection in alpacas against Corynebacterium pseudotuberculosis using different bacterial components

Corynebacterium pseudotuberculosis is a Gram positive bacterium that produces caseous lymphadenitis in sheep and goats, and a granulomatous lymphadenitis in llamas and alpacas. To evaluate the immune potential of different doses of cell wall and toxin components of C. pseudotuberculosis from alpaca origin, 12 adult alpacas were allotted at random to four groups, and SC inoculated in the left flank with vaccines composed of low and high doses of bacterial crude antigens, cell wall: 250 and 500 microg/ml and toxin: 133 and 265 microg/ml, respectively. The vaccines were supplemented with 20 microg/ml of muramyl dipeptide as adjuvant. Three alpacas were sham inoculated with adjuvant as a control. After 3 weeks, immunized and naive alpacas were challenged intradermally in the right flank with 1 x 10(6) colony forming units (CFU) of C. pseudotuberculosis. The alpacas were sacrificed at days 28, 58 and 112 after inoculation, and the degree of protection induced by vaccines was demonstrated by the absence of abscesses and/or bacteria. The alpacas vaccinated with high dose of toxin, did not show abscesses. In contrast, the alpacas vaccinated with a low dose of toxin showed abscesses at the inoculation site, regional, and renal lymph nodes. The cell wall vaccinated alpacas showed a lesser degree of protection than the other groups with superficial and internal abscesses. The control alpacas had persistent fever and abscesses at the inoculation site, regional, and internal lymph nodes. In addition, a robust and early humoral response was observed in all vaccinated alpacas after challenge, lasting at least 3 months. The results suggest that the toxin of C. pseudotuberculosis is a very important antigen, inducing a dose dependant protective immunity against this bacterium in alpacas.     

Histopathology of the early phase during experimental Corynebacterium pseudotuberculosis infection in lambs.

Histological responses during experimental Corynebacterium pseudotuberculosis infection in lambs were investigated in parotid lymph nodes for ten days following inoculation. Lambs were infected by the subcutaneous route into the right eyelid with a virulent strain of C. pseudotuberculosis. Multiple microscopic acute abscesses, predominantly infiltrated with polymorphonuclear (PMN) leucocytes, were seen in the right parotid lymph node on the 1st day post-inoculation (PI). This massive PMN infiltration coincided with a peripheral blood granulocytosis. On day 3 PI, an influx of histiocytes was observed, while the microabscesses became confluent. From day 3 to day 10 PI, these lesions became enlarged and transformed into typical pyogranulomas with a central necrosis and a peripheral mantle of mononuclear cells composed of macrophages, epithelioid cells and lymphocytes; these histological changes were associated with a bacterial dissemination limited to the superficial lymph nodes. A lymphoid hyperplasia with prominent germinal centers was observed in the draining lymph nodes from day 3 PI. These results illustrate the dual role of granulomatous lesions in chronic bacterial infections: although they limit bacterial dissemination, the granulomas do not impair the persistence of infectious organisms in the host, leading to focal tissue damage.     

Use of a toxoid vaccine to protect goats against intradermal challenge exposure to Corynebacterium pseudotuberculosis

Two groups of male, 9-week-old goats (5 goats/group) were vaccinated subcutaneously with formalized exotoxin of Corynebacterium pseudotuberculosis, with Freund's incomplete adjuvant. Each goat was given 2 vaccinations, 2 weeks apart. At each vaccination, each group 1 goat was given 0.5 ml of toxoid, and each group 2 goat was given 1 ml of toxoid. Twenty days after the 2nd vaccination, vaccinated goats and 5 nonvaccinated 12-week-old goats (controls) were inoculated intradermally (challenge exposed) with live C pseudotuberculosis, monitored for 13 weeks, and euthanatized. At necropsy, 5 of the 10 vaccinated goats did not have C pseudotuberculosis lesions, 3 had abscesses limited to the inoculation site and draining lymph node, and 2 had disseminated bacterial lesions. Of the 5 nonvaccinated controls, 4 had disseminated abscesses and 1 had a single abscess in an internal node. Serologically, 9 of the 10 vaccinated goats developed positive (greater than or equal to 1:8) antibody titers against the exotoxin within 1 week after inoculation; the 10th goat seroconverted 2 weeks after inoculation, whereas control goats required 3 weeks to develop a positive antibody response. Therefore, early during an infection with C pseudotuberculosis, antibodies against the exotoxin may protect a goat against spread of the organism. All goats were injected intradermally before challenge exposure, 10 days after challenge exposure, and at 4, 8, and 12 weeks after challenge exposure with a skin-test reagent composed of fragmented bacterial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Abscess disease, caseous lymphadenitis, and pulmonary adenomatosis in imported sheep

The occurrence of abscess disease, caseous lymphadenitis, and pulmonary adenomatosis in sheep in Denmark is reported for the first time. Subcutaneous abscesses were observed in imported 4- to 5-month-old lambs of the Lacaune breed 10 days after arrival in Denmark. Abscesses were mostly located in the head, neck and shoulder regions close to the regional lymph nodes. Bacteriological examinations revealed growth of Staphylococcus aureus ssp. anaerobius in all animals with subcutaneously located abscesses containing a viscous white-yellow odourless mass. In addition, Corynebacterium pseudotuberculosis was isolated from abscesses in one animal and lesions consistent with pulmonary adenomatosis were found in four animals.     

Serologic response and lesions in goats experimentally infected with Corynebacterium pseudotuberculosis of caprine and equine origins

Fifteen goat kids were experimentally inoculated with Corynebacterium pseudotuberculosis. Five were given a strain of caprine origin (nitrate-negative biotype) intradermally, 5 were given a strain of equine origin (nitrate-positive biotype) intradermally, and 5 were inoculated intranasally with the caprine-origin strain. Animals were monitored for 127 days. The goats given the inocula intradermally developed abscesses; those given caprine-origin strain had multiple lesions both peripherally and in visceral locations (primarily endothoracic abscesses), whereas those given the equine-origin strain had abscesses only at injection sites and draining nodes. The difference in extent of lesions could be due to biotypic bacterial differences or to the individual strains used. Intranasally inoculated goats did not develop abscesses and were essentially no different from controls. The cranial part of the respiratory tract may not be an important portal of entry for C pseudotuberculosis. Serum samples obtained monthly from all animals were subjected to the synergistic hemolysis-inhibition test, which measures antibodies to the exotoxin of C pseudotuberculosis. Animals with abscesses developed titers within 1 month of inoculation. Animals without abscesses remained seronegative. The synergistic hemolysis-inhibition test may be a reliable diagnostic assay for caseous lymphadenitis in goats.     

The incidence of caseous lymphadenitis in Alberta sheep and assessment of impact by vaccination with commercial and experimental vaccines.

In Alberta, caseous lymphadenitis (CLA) is one of the leading causes of lamb and mutton carcass condemnation. In this study, serologic results confirmed a high (50-94%) incidence of exposure to Corynebacterium pseudotuberculosis, the causative agent of CLA, in mature, unvaccinated sheep in southern Alberta. To assess the efficacy and impact of vaccination with 2 commercial (Glanvac-6 and Case-Vac) and 1 experimental (WC+ MDP-GDP) CLA vaccines, a series of 3 field trials in 3249 ewes and lambs was conducted in affected flocks from 1992-1996. Efficacy was assessed from the serological response to vaccination, prevalence and size of injection site reactions by treatment, and the incidence of CLA abscesses. Overall, agglutinating antibody titres to C. pseudotuberculosis in lambs vaccinated with WC+MDP-GDP and Case-Vac remained significantly elevated above nonvaccinated control lambs for the 12 mo period after the initial vaccination. Lambs vaccinated with the WC/MDP-GDP maintained higher titres (P < 0.06) than those vaccinated with Case-Vac for the period from 6 to 12 mo after vaccination. Agglutinating antibody titres for lambs vaccinated with Glanvac did not differ from those of controls at any point during the 12 mo period after vaccination. The number of injection site reactions was elevated in lambs vaccinated with Glanvac as compared to those vaccinated with WC+MDP-GDP but the size of injection site reactions did not significantly differ. Sheep vaccinated with WC+ MDP-GDP also had a reduced incidence of putative CLA abscesses, although confirmation of the presence of C. pseudotuberculosis was only successful in a small number of instances.     

The role of Corynebacterium pseudotuberculosis lung lesions in the transmission of this bacterium to other sheep

SUMMARY: :Five groups of 5 shorn and 5 unshorn caseous lymphadenitis (CLA)-free Merino wether weaners were each placed in feedlot pens with 6 Merino ewes, 2 or more of which had CLA lung lesions but no discharging superficial lesions. The sheep were kept together for 5 months.
Twenty-eight per cent of the shorn weaners and 20% of the unshorn weaners developed antibodies to Corynebacterium pseudotuberculosis. At slaughter, 8% of the shorn weaners and 12% of the unshorn weaners had CLA lesions in either lungs, lymph nodes or both. In the absence of contact with CLA-infected ewes, a control group of 5 shorn and 5 unshorn weaners failed to develop antibodies to C. pseudotuberculosis or CLA lesions in the same period. This showed that sheep with CLA abscesses in the lungs but no discharging superficial abscesses were a source of C. pseudotuberculosis infection to other sheep. Aust Vet J. 64: 261–263     

Efficacy of a flock-specific pseudotuberculosis vaccine in goats

In a flock with 33 adult goats repeated abscesses due to Pseudotuberculosis occurred. For the owners refused a sanitation program based on testing and removing, the goats were vaccinated with a flock specific, whole-cells lysate vaccine of Corynebacterium pseudotuberculosis. Partly simultaneously or 14 days later vaccinations against Clostridiosis (Covexin8) and against Erysipelas (Erysorb plus) were performed at different application sites. After a twofold basal vaccination in a 4 week interval the goats were revaccinated in half year intervals. The local (swelling at the injection site) and systemic reactions (temperature elevation, antibody activity) after vaccination and the clinical status of the flock (core body temperature, adspection, abscesses, lymph nodes) were investigated over a period of two years. Due to the vaccination program the rate of new cases of clinical pseudotuberculosis were reduced significantly. In the last 3 months of the investigation no clinical cases occurred. The vaccination with the C. pseudotuberculosis-vaccine induced a significant increase in antibody activities detected by ELISA. Due to interaction of maternal antibodies with the antigen of the vaccine lambs should be vaccinated at the earliest at an age of 9 weeks. Vaccinating the C. pseudotuberculosis-vaccine separately in time to Covexin8 and Erysorb plus induced with increasing numbers of revaccinations higher antibody activities against C. pseudotuberculosis than simultaneous vaccinations with all the 3 vaccines. But this difference does not justify the additional expense of separate vaccinations in time. Despite considerable local and systemic reaction the C. pseudotuberculosis-vaccine is recommended to support sanitation programs.     

Immunisation against ovine caseous lymphadenitis: comparison of Corynebacterium pseudotuberculosis vaccines with and without bacterial cells

Sheep were immunised with Corynebacterium pseudotuberculosis vaccines prepared from cell-free toxoid or from toxoid with formalin-killed cells of C pseudotuberculosis added. Resistance of sheep to infection was tested 6 months after immunisation by inoculation with caseous lymphadenitis pus. The outcome was assessed 3 months later by slaughter and inspection of the sheep for lesions of caseous lymphadenitis. immunised sheep were adequately protected against infection as shown by a significant reduction in the number of sheep exhibiting lesions compared with control sheep, and by fewer abscesses in affected vaccinated sheep than in affected control sheep. The protective potency of the vaccines was not improved by the inclusion of cells of C pseudotuberculosis.     

Effect of muramyl dipeptide on immunogenicity of Corynebacterium pseudotuberculosis whole-cell vaccines in mice and lambs

Colostrum-deprived lambs and CF1 mice were vaccinated with water-in-oil emulsion vaccines containing nonviable whole cells (WC) of Corynebacterium pseudotuberculosis with and without muramyl dipeptide (MDP). Efficacy of vaccines was determined from the survival of mice and lesions in lambs after IV injection of 10(4) colony-forming units of C pseudotuberculosis. In mice, protection was related to the concentration of WC in the vaccine. At 50, 100, or 150 micrograms of WC, protection was good (78.8%). At 10 or 25 micrograms of WC, protection was considerably less (54.7%). At high WC concentrations, protection could only be moderately increased to 82.3% with high (50 and 100 micrograms) concentrations of MDP or increased to 90% protection with low (5 and 10 micrograms) concentrations of MDP. At low WC concentrations, protection significantly decreased to 32% (P less than 0.025) with high concentrations of MDP, but significantly increased to 72.5% (P less than 0.025) with low concentrations of MDP. Therefore, the amount of protection with lower concentrations of WC and MDP was comparable with the amount of protection with higher concentrations of WC without MDP. In lambs, high prechallenge antibody titers (geometric mean titers from 5.1 to 5.4 by day 35) were observed after vaccination with WC. Protection and vaccination site abscesses in lambs were related to the concentration of WC and MDP. Pulmonary or vaccination site abscesses were not observed in 4 of 4 lambs vaccinated with 1 mg of WC + 50 micrograms of MDP.     

The effect of parainfluenza virus type 3 and Pasteurella haemolytica on oxygen-dependent and oxygen-independent bactericidal mechanisms of ovine pulmonary phagocytic cells

Groups of caesarean-derived, colostrum-deprived lambs were inoculated by the intratracheal route with Pasteurella haemolytica, either alone or 4 or 6 days after the inoculation of parainfluenza virus type 3 (PI3). Other groups were inoculated with PI3 followed by veal infusion broth, or with uninfected cell culture fluid followed by veal infusion broth (controls). All lambs were killed 24 h after the second inoculation. Pulmonary phagocytic cells were recovered by lavage and separated into alveolar macrophage (AM) and neutrophil fractions by density gradient centrifugation. Bacterial proliferation was detected in the lungs of all five lambs inoculated with P. haemolytica 6 days after PI3 but in only one of five inoculated with P. haemolytica 4 days after PI3 and one of five inoculated with P. haemolytica alone. The number of phagocytic cells recovered from the lungs was highest in animals inoculated with P. haemolytica 6 days after PI3 and, overall, a greater number of both AM and neutrophils was recovered from the lungs of animals where bacterial proliferation occurred (greater than 10(5.0) P. haemolytica 100 g-1 lung) than from those that controlled the bacterial infection. Oxygen-dependent bactericidal activity of AM and neutrophils was measured by chemiluminescence. Infection with PI3 and P. haemolytica increased the chemiluminescence responses. The highest responses were recorded from lambs inoculated with P. haemolytica 6 days after PI3, the group where pulmonary clearance was poorest. Overall, responses were higher in lambs in which bacterial proliferation occurred than in those that controlled the infection. On the other hand, oxygen-independent bactericidal activity, measured by the direct effects of neutrophil lysates on Escherichia coli, was lowest in lambs inoculated with P. haemolytica 6 days after PI3 and was lower in lambs where bacterial proliferation occurred.     

利福平在脓肿病灶内的残效期及其耐受菌株的分离鉴定

将伪结核棒状杆菌人工接种５０只实验小鼠,４周后用利福平对其中存活的２７只进行治疗试验,疗程为１周。然后每周解剖３只实验小鼠,检测药物在其脓肿病灶内有效杀菌作用的持续时间即残效期;同时对耐受该药物的菌株进行分离鉴定。结果发现：停药后第８周病灶内不仅仍有药物残存,而且仍然具有强大的杀菌作用;同时由４只小鼠的脓肿病灶分离到接种菌,经鉴定确认均为利福平耐受菌。表明伪结核棒状杆菌对利福平极易产生耐受性。     

Microorganisms associated with abscesses of sheep and goats in the south of Iran

A total of 86 abscesses (45 and 41) abscesses of sheep and goats, respectively) were examined for their causal agents; 44 of these abscesses were located subcutaneously, and the remaining 42 were in lungs, livers, intestines, and udders. A total of 23 different types of microorganisms were isolated from 78 abscesses; bacteria were not detected in the remaining eight abscesses. Microorganisms isolated were: species of the genera Corynebacterium, Staphylococcus, and Streptococcus and Pasteurella, Escherichia coli, and other gram-negative rods. Peptostreptococcus anaerobius and Eubacterium tortuosum were isolated in pure culture from one abscess each in goats. Two to four different microorganisms were associated with 25 of the 86 abscesses. It was determined that 11 isolates of Corynebacterium pyogenes, 2 isolates of Pasteurella haemolytica, 1 of P multocida, and 1 of C pseudotuberculosis were lethal to mice. Two of the C pyogenes isolates from subcutaneous spreading abscesses of goats were pathogenic for rabbits; these two isolates produced similar suppurative inflammation in goats experimentally, but did not cause death. As determined by experimental inoculation, goats were more susceptible than sheep to the two isolates. In nature, subcutaneous abscesses of goats associated with C pyogenes were of a more diffusive type which resulted in generalization of the infection and death of the animals. Postmortem examination of ten goats dying of field infection showed the presence of larvae of the warble-fly Przhevalskiana silenus at the site of infection.     

Immunisation against ovine caseous lymphadenitis: correlation between Corynebacterium pseudotuberculosis toxoid content and protective efficacy in combined clostridial-corynebacterial vaccines

Groups of sheep were dosed with vaccines containing Corynebacterium pseudotuberculosis toxoid combined in varying amounts with 5 clostridial antigens. Resistance of the sheep to infection with C pseudotuberculosis was tested at 1, 6 and 12 months after vaccination by infection with pus from ovine lymph glands actively infected with C pseudotuberculosis. The outcome was assessed 3 months after challenge by slaughter and inspection of the sheep for caseous lymphadenitis lesions. Protection was demonstrated by a significant reduction in the proportion of immunised sheep exhibiting lesions compared with control sheep, and by fewer abscesses in affected immunised sheep than in affected control sheep. A positive correlation was found between amount of C pseudotuberculosis toxoid administered and degree of protection obtained. Chromatographically-purified toxoid induced essentially the same protection, suggesting that anti-toxic immunity is the major factor in protection.     

Experimental colonization of the bovine teat duct with Corynebacterium bovis and the effect on milk somatic cell counts.

Colonization with Corynebacterium bovis was established in 59 of 64 (92%), 58 of 59 (98%) and 19 of 34 (56%) of uninfected bovine mammary quarters following inoculation of 83.3 X 10(4) colony-forming units (CFU) of the organism into the teat cistern, 4.7 X 10(3) CFU 5 mm into the teat duct or by exposure of the teat orifice to a milk culture containing 1.6 X 10(7) CFU/mL respectively. Mean somatic cell counts for foremilk samples from 122 quarters were significantly higher after colonization with C. bovis (145,900/mL) compared to before exposure (130,900/mL).     

Bacteriologic and histologic features in mice after intranasal inoculation of Brucella melitensis

OBJECTIVE: To characterize effects of intranasal inoculation of virulent Brucella melitensis strain 16M in mice. ANIMALS: Female Balb/c mice, 6 to 8 weeks old. PROCEDURE: Studies were designed to elucidate gross morphologic lesions, bacterial burden in target organs, and histologic changes in tissues following experimental intranasal inoculation of mice with B melitensis 16M, which could be used to characterize a model for testing vaccine efficacy. RESULTS: Measurable splenomegaly was evident at 3 and 7 weeks after inoculation. A demonstrable increase in splenic colony-forming units (CFU) from infected mice increased over time with increasing dose when comparing inocula of 10(3), 10(4), and 10(5) CFU. Recovery of brucellae from the lungs was possible early in infection with 10(1), 10(3), and 10(5) CFU, but only the group inoculated with 10(5) CFU consistently yielded quantifiable bacteria. At a dose of 10 CFU, few organisms were located in the spleen. Bacteria were recovered up to 140 days after inoculation in mice given 10(3) CFU. At an inoculum of 10(5) CFU, bacterial counts were highest early in infection. Histologic examination of tissues revealed an increase in white pulp and marginal zone in the spleen and lymphohistiocytic hepatitis. CONCLUSION AND CLINICAL RELEVANCE: Changes in the spleen and liver increased with increases in dose and with increased time following intranasal inoculation with B melitensis 16M. Surprisingly, histologic changes were not observed in the lungs of inoculated mice.