Humoral immunity in disseminated Aspergillus terreus infection in the dog

Aspects of humoral immunity were studied in 17 dogs with disseminated aspergillosis (16 cases Aspergillus terreus, 1 case Aspergillus flavipes). All dogs had markedly raised serum IgG levels by single radial immunodiffusion (range 1500-6000 mg dl-1). Despite this, serum antibody to A. terreus was demonstrated in only 7/16 cases by agar gel diffusion, 9/16 cases by counter immunoelectrophoresis, 10/16 by ELISA and 11/16 by an indirect immunofluorescence assay. Serum antibody was also detected in 2/5 clinically normal relatives of 2 cases, indicating previous exposure or subclinical infection.     

Morphological changes in brucellosis in guinea pigs due to experimental infection with Brucella suis

Sixty-four guinea-pigs were infected with the germs of Brucella suis. biotype 2 via the vaginal, peroral, intranasal, conjunctival and subcutaneous routes. In the subcutaneous infection the length of the trial was 151 days and in the other methods of infection 56 days. As found, guinea-pigs are highly sensitive and brucellosis lesions were detected in all cases. Brucellosis was chronical and spread locally in the guinea-pigs. In the haematogenic spreading of the disease, lesions were constantly recorded in liver, spleen and lungs. In the cases of subcutaneous infection lesions were also observed in regional lymph nodes at the places of infection. The lesions had amorphous structure and regular borders. The lesions were characterized by pronounced exudation and the exudate was subject to caseous necrosis, followed by colliquation. Secondary central colliquation was regularly observed in older granulomas, so that colliquation is considered as a characteristic morphological trait of brucellosis process in guinea-pigs. Histiocytary granuloma constituted the microscopic basis of the lesions. In older granulomas the central necroses were wide and contained a large amount of nuclear detritus, owing to the karyorhexis of cells, mainly neutrophiles. In the necroses of caseous type lipoid droplets were found and calcification was observed on the 151st day. Big cells of Langhans type occurred sometimes in the histiocytary layer of granulomas; they were present most frequently in the lesions affecting lymph nodes. Interstitial pneumonia was a characteristic symptom in liver and big cells of Sternberg type were found in the granulomas. Follicular tumor was constantly observed in spleens. Morphologically detectable lesions first developed in liver after about 7 days from conjunctival infection and subcutaneous infection, after 21 days from vaginal and intranasal infection, and after 28 days from peroral infection. In the case of subcutaneous infection, granulomas were found in the regional lymph nodes on the 14th day. The most expressive lesions in organs, mainly liver, were found from the 49th to 56th day from infection.     

An immunohistochemical study of canine disseminated aspergillosis

An immunohistochemical study of 25 lesions from 7 dogs with disseminated aspergillosis (Aspergillus terreus) is presented. All had multiple fungal granulomas in many viscera, with centres of necrotic tissue and hyphal elements surrounded by a mixed infiltrate of predominantly mononuclear cells. Within these lesions, hyphae coated with immunoglobulin (IgG, IgM, IgA) and complement (C3, C4) were identified, together with peri-lesional mononuclear cells that reacted with antisera directed towards either IgG, IgM, IgA or a T lymphocyte marker (MUII). A conspicuous feature was the prominent hyphal fluorescence seen with IgA and C3 antisera. The IgA reagent also marked large numbers of mononuclear cells both around lesions and scattered throughout interstitial tissue, suggesting an abnormality of IgA production or regulation as a factor predisposing to this condition.     

Cecal and hepatic granulomas in chickens associated with Heterakis gallinarum infection

Heterakis gallinarum infection was detected in the ceca of chickens with cecal and hepatic granulomas found at slaughter. The chickens were diagnostic submissions from four small backyard flocks in Saskatchewan. Detailed dissection of some cecal granulomas in one flock and pepsin digestion of cecal granulomas in the three other flocks demonstrated the presence of the parasite within granulomas in three of the flocks. A possible causal relationship between Heterakis gallinarum and the cecal and hepatic granulomas is discussed.     

Actinobacillosis in bovine caesarean sections.

An infection with Actinobacillus lignieresii, which was spread by a veterinary surgeon, caused problems after caesarean sections in cows on several farms. The wounds became hard about six weeks after the operation, and a few weeks later small abscesses developed and later the wounds were covered with small and large granulomas. The general health of about 20 per cent of the affected cows was poor and in these cows multiple granulomas could be detected in the abdomen by rectal palpation.     

The development of morphologic changes and the antibody response in guinea pigs experimentally infected with Brucella suis, biotype 2 bacteria

Sixty-two guinea pigs were infected vaginally, perorally, intranasally, conjunctivally and subcutaneously with the bacteria B. suis, biotype 2. The inoculum contained 13.8.10(9) of brucellas in 1 ml of physiological solution. Subcutaneous infection lasted 151 days, the other types of infection 56 days. All types of infections resulted in brucellotic changes developing in relation to the type and duration of infection. The least marked changes were observed after peroral infection. Brucellosis was chronical and the changes in liver, spleen, lungs, and after subcutaneous infection also in regional lymphoglandulae, were found out incessantly. Microscopically these changes were histiocytic granulomas. The pathogenesis of brucellosis process in guinea pig organism is influenced primarily by haematogenous propagation. The highest serological reactivity was caused by subcutaneous infection, the lowest by peroral infection. In the course of different types of infection the titres of agglutination and complement-fixation antibodies varied and the highest values were recorded between the 21st and 56th day. The diagnostic effectiveness of the surface fixation test was verified and no decrease in its sensitivity, not even in the chronic phase of infection, was observed. An incomplete correlation between morphological changes and serological reactivity in guinea pigs was detected. The highest number of Brucella infections was identified by histopathological examination which helped to reveal the initial stages of granulomas at the time when specific antibodies were not yet determined by serological tests.     

Disseminated Rhodococcus equi infection in two goats.

Rhodococcus equi infection was diagnosed in two goats from the same herd. At necropsy, numerous caseating granulomas were disseminated throughout the liver, lungs, abdominal lymph nodes, medulla of right humerus, and the right fifth rib of goat No. 1, and the liver of goat No. 2. Histopathologic examination confirmed the presence of multiple caseating granulomas in these organs. Numerous gram-positive and Giemsa-positive coccobacilli were identified within the cytoplasm of macrophages. Aerobic bacterial cultures of the liver and lung from both goats yielded a pure growth of R. equi. R. equi antigens were immunohistochemically identified in caseating granulomas from both goats. However, the 15- to 17-kd virulence antigens of R. equi were not detected, suggesting possible infection by an avirulent strain of this organism.     

Characterization of tuberculous granulomas in different stages of progression and associated tertiary lymphoid tissue in goats experimentally infected with Mycobacterium avium subsp. hominissuis

Oral infection of goats with Mycobacterium avium subsp. hominissuis (MAH) resulted in a large variety of granulomas in organized gut-associated lymphatic tissues and intestinal lymph nodes. To characterize the cellular composition of granulomas, CD4⁺, CD8⁺, γδ, B lymphocytes and plasma, CD25⁺, CD68⁺, MHC-II⁺, Ki67⁺ and endothelial cells were labeled in consecutive frozen sections by immunohistochemistry and acid fast bacilli (AFB) by Kinyoun stain. Granulomas with extensive necrosis, little mineralization and variable numbers of AFB surrounded by many CD4⁺ T cells, but only few epitheloid macrophages were observed in severely sick goats at 2–3 mpi. They were interpreted as exuberant immune reaction. Organized granulomas with very few AFB were seen in clinically healthy goats at 13 mpi. The necrotic cores were surrounded by a zone of granulomatous infiltrate with many epitheloid macrophages and few lymphocytes. This zone was initially wide and highly vascularized and became progressively smaller. It was enclosed by an increasing layer of connective tissue. All organized granulomas were surrounded by compartimentalized tertiary lymphoid tissue. The granulomas in experimental infection of goats with MAH reflect the heterogeneity of lesions seen in mycobacterial infections of humans and ruminants and are therefore valuable for comparative research.     

Lesion development and immunohistochemical changes in granulomas from cattle experimentally infected with Mycobacterium bovis

Mycobacterium bovis, the causative agent of bovine tuberculosis, persists within granulomas. Formation of granulomas involves a complex array of immune activation and cellular migration. To examine temporal changes in granuloma development, we inoculated 32 cattle with M. bovis of deer origin. Tissues from 4 calves each were examined at 15, 28, 42, 60, 90, 180, 270, and 370 days after inoculation. Granulomas in the medial retropharyngeal lymph node were staged (I-IV) on the basis of cellular composition and the presence or absence of necrosis and peripheral fibrosis. Immunohistochemistry for inducible nitric oxide synthase (iNOS), CD68, CD4, CD8, and gamma/delta T cells was performed. Fifteen days after inoculation only stage I granulomas were seen, while between 28 and 60 days, there was a steady progression through granuloma stages such that by day 60, granulomas of all 4 stages were seen. Acid-fast bacilli were present in moderate-to-large numbers in stage I granulomas 15-60 days after inoculation. Stage IV granulomas contained large numbers of acid-fast bacteria. Abundant iNOS immunoreactivity was associated with granulomas from day 15 through day 60 but was minimal from day 90 to the termination of the experiment. The relative number of CD4+ and CD68+ cells remained constant throughout the study. In contrast, at time points >60 days, numbers of CD8+ and gamma/delta T cells diminished. Tuberculous granulomas are dynamic lesions that follow an orderly progression through disease stages. Diminished expression of iNOS and reduced numbers of CD8+ and gamma/delta T cells late in the progression of tuberculous granulomas may represent a failure of the host response to control infection.     

Disseminated aspergillosis in a dog

A dog with disseminated Aspergillus terreus infection died after a protracted course of hospitalization. Treatment with amphotericin B methyl ester was without effect. The causative organism was found in bone, myocardium, spleen, kidneys, liver, thymus, lymph nodes, and both eyes. Treatment with antimicrobials and corticosteroids prior to hospitalization may have contributed to dissemination of the fungus.     

Natural Heterobilharzia americana infection in horses in Texas

The schistosome Heterobilharzia americana infects dogs, raccoons, and other mammals in the southeastern United States. Migration of eggs into the liver results in parasitic granulomas with varying degrees of fibrosis and inflammation. Recently, hepatic parasitic granulomas in horses were shown to be caused by H. americana infection. In the present study, samples of liver from 11 of 12 horses with hepatic granulomas identified at necropsy (n = 11) or surgical biopsy (n = 1) were used for DNA extraction, polymerase chain reaction amplification and sequencing using primers specific for a portion of the H. americana small subunit ribosomal RNA gene. A polymerase chain reaction amplicon of the correct size was produced from the extracted DNA in 8 of the 11 horses. Amplicons from 5 of the 8 positive horses were sequenced and had 100% identity with H. americana. In all but 2 of the 12 horses, Heterobilharzia was not responsible for the primary clinical disease, and the hepatic granulomas were considered an incidental finding.     

Treatment of Mycotic Rhinitis With Itraconazole in Three Horses

Itraconazole, a third-generation azole, was evaluated for treatment of resistant nasal mycotic infections in horses. Two horses with Aspergillus spp nasal granulomas and 1 horse with Conidiobolus coronatus nasal infection were treated with itraconazole (3 mg/kg PO bid). One of the horses with nasal aspergillosis was also treated by surgical resection of the nasal septum. The treatment time for the horses ranged from 3 to 4.5 months. No adverse effects were noted in any of the horses during the treatment period. Peak and trough serum itraconazole concentrations were < 0.5 μg/mL in all 3 horses. Itraconazole (3 mg/kg PO bid) appears to be effective in the treatment of nasal Aspergillus spp infections in horses because the fungal infection was eliminated in both horses. One horse still had excessive nasal sounds during exercise and was retired from training, whereas the other horse returned to normal. The nasal C. coronatus infection appeared resistant to itraconazole treatment in the affected horse because the granulomas were still present after 4.5 months of treatment.     

Aspergillus terreus as a cause of mycotic abortion in cows

The fungus Aspergillus terreus Thom et Church was subjected to microscopic and cultivation study in the organs of an aborted foetus and was demonstrated to be the causative agent of abortion in cattle. The culture of the isolated organism is described and brief data reporting on its ecology are presented. Attention is drawn to the probable source of infection and to the insufficiently known aspects of the pathogenesis of this disease.     

Susceptibility to tuberculosis: composition of tuberculous granulomas in Thorbecke and outbred New Zealand White rabbits

We sought to characterize the lung cellular immune responses to inhaled Mycobacterium tuberculosis (Mtb) of the susceptible inbred Thorbecke rabbit (the genomically sequenced strain, now unavailable) and compare it to outbred, Mtb-resistant, New Zealand White rabbits. Using Mtb CDC1551, we confirmed that the inbred rabbits allowed establishment of infection with this low virulence strain, compared to poor establishment in outbred rabbits. With a more virulent strain, Mtb Erdman, that establishes infection well in both rabbit strains, we analyzed granulomas from rabbit lungs 5 weeks after aerosol infection. The lung granulomas of inbred rabbits had significantly higher frequencies of cells expressing MHC Class II and CD11b, and lower frequencies of CD8+ T cells than the outbred controls. Macrophage-sized cells expressing MHC Class II in inbred rabbit granulomas showed significantly decreased intensity of expression, suggesting impaired maturation. Although the inbred dermal tuberculin reactions were decreased, the in vitro IFN-gamma mRNA responses of hilar node lymphocytes to tuberculin were higher than those of outbred rabbits. Further delineation of the outbred rabbit's resistant immune response to Mtb infection is warranted.     

Prophylactic effect of monensin sodium against experimentally induced paratuberculosis in mice.

Monensin sodium (0, 15, or 30 mg/kg of complete feed) was fed ad libitum for 1 week to female mice (strain C57BL6/J) that were genetically susceptible to infection with Mycobacterium paratuberculosis. Ten mice in each of the 3 groups were inoculated intraperitoneally with M paratuberculosis (10(9) organisms). Sterile saline solution was injected intraperitoneally into 10 other mice in each group. Rations were continued for 50 days, then mice were euthanatized, and body weight, splenic weight, and hepatic weight for each mouse were recorded. Ratios of body weight to splenic weight and of body weight to hepatic weight were calculated for each mouse. Hepatic granulomas in 50 light microscopic fields were counted, and presence of acid-fast organisms in those granulomas was recorded. Infected mice given monensin had higher body weight and fewer hepatic granulomas than did mice not given monensin. Although hepatic granulomas were fewer in these mice, they contained acid-fast organisms. Effects of 15 mg of monensin and those of 30 mg of monensin/kg of complete feed were not different.     

T-lymphocyte predominance in lesions of canine coccidioidomycosis

Coccidioidomycosis is a systemic fungal infection endemic to the southwestern United States. Although cell-mediated immunity is considered critical in control of the infection, little is known of the cellular population in naturally occurring lesions. To characterize the lymphocytic infiltration, archived formalin-fixed, paraffin-embedded tissues (subcutis, pericardium/heart, lung, bone, and synovium) from 18 dogs with coccidioidomycosis were studied with immunohistochemistry for CD3 and CD79a. In nearly all lesions, T lymphocytes were more numerous than B lymphocytes and were distributed throughout the lesion with concentration in the periphery of granulomas, whereas B lymphocytes were mostly confined to the periphery of granulomas. The predominance of T lymphocytes in lesions of canine coccidioidomycosis was independent of the tissue evaluated, the number of intralesional organisms, and the nature or severity of the inflammatory response.     

Virulence-associated protein characterisation of Rhodococcus equi isolated from bovine lymph nodes

Rhodococcus equi has a low pathogenicity in cattle, but it occasionally causes lymph node granulomas, which are detected at abattoir post mortem inspection, and must be distinguished from tuberculous granulomas. Lymph node lesions were detected in 6719 cattle, from a total of 3,263,622 cattle examined post mortem in abattoirs, in the Republic of Ireland, during 1997 and 1998. Histological examination was performed on all lesions, principally for the purpose of identifying animals with tuberculosis. A total of 1122 of the lesions were cultured on blood agar and on Stonebrinks and Lowenstein-Jensen medium containing pyruvate, because the histological findings were difficult to interpret or were suggestive of R. equi infection. R. equi was isolated from 264 lesions. Almost all of the R. equi granulomas were confined to a single lymph node, and were present predominantly in the retropharyngeal, bronchial and mediastinal lymph nodes. R. equi granulomas were present in a significantly higher proportion of the lesions detected in steers and heifers compared to cows. The prevalence in the total population of 3.3 million cattle examined post mortem was 0.008%. The 15-17kDa antigens, associated with virulence in this organism, and the 20kDa antigen, associated with intermediate virulence, were not detected in isolates from 146 cattle, analysed by immunoblot assays. A PCR assay to detect the plasmid gene encoding the 15-17kDa antigens was also negative for isolates from these 146 animals. Plasmids were not detected in 30 isolates which were examined.     

In vivo pulmonary response to Aspergillus terreus spores

The fate of intratracheally instilled Aspergillus terreus spores was followed in both rabbits and rats. Phagocytosis of the spores by the pulmonary macrophage was rapid in that approx. 42% of the observed spores were associated with the macrophages immediately after instillation. Direct penetration of the lung architecture by the spores was not observed but spores were seen in the alveolar interstitium at 3 hr after instillation and in the tracheobroncheal lymph nodes at 24 hr. Granulomas formed between 48 hr and 1 week after exposure. In the absence of apparent spore or spore extract toxicity and precipitating antibodies against Aspergillus terreus, the observed reactions preclude the possibility that the lesions were the consequence of hypersensitivity. This model of pulmonary response to fungal spores may be of future value for characterizing further the pathology associated with certain occupational exposures to moldy materials.     

Larval toxocarosis in sheep: the immunohistochemical characterization of lesions in some affected organs

Morphological and immunohistochemical responses of lambs following oral infection with 10,000 infective Toxocara canis (T. canis) eggs were studied up to 28 days post-infection (pi). The small intestine, liver, lungs and brain of both infected and control lambs were examined using the routine histological methods for paraffin sections and immunohistochemical techniques for frozen tissue sections. Eosinophil-rich hepatic granulomas and diffuse T. canis-induced pulmonary inflammation were the most prominent pathological features. CD2+, CD4+, CD8+, IgM bearing cells, and macrophages were demonstrated in the liver granulomas. The observed histomorphologic changes were similar to other paratenic hosts. It was concluded that larval toxocarosis followed the classical migratory path in the infected lambs.     

Immunologic study of systemic aspergillosis in German shepherd dogs

Data are presented from a series of eight cases of disseminated canine aspergillosis (A. terreus) in German Shepherd dogs referred to Murdoch University Veterinary Hospital. Immunoglobulin determination revealed depression of serum IgA (cases 1 and 5) and IgM (case 2) levels and elevated levels of IgG in all cases. Total complement activity (CH50) and complement components tests, (C3, C4) were present in normal amounts in all cases. Using agar gel diffusion, serum antibody to A. terreus was found in only one case and aspergillus antigenaemia in two of the remainder. Lectin transformation of lymphocytes in two dogs was found to be depressed relative to normal controls in case 1 and initially in case 2. Two dogs failed to respond to the intradermal injection of A. terreus antigen.