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.     

Brucellosis in rabbits induced by an experimental infection with Brucella suis germs

Hares were experimentally infected with Brucella suis germs, biotype 2, to study the development of morphological changes in the organs. It was demonstrated that conjunctival and intranasal infection caused morphological brucellosis changes in liver. After conjunctival infection affections were observed in 16 days, and in 27 days after intranasal infection. The morphology of changes did not differ from those occurring in spontaneous brucellosis. The results of morphological examination corresponded to the serological results. It was proved by the results of the experiment that the course of brucellosis could be chronic and that in the process of the natural infection of hares with Brucella suis germs the conjunctival and intranasal modes of infection played an important role. Hares were highly sensitive and responsive to the conditions of the environment where experimental infection was performed. In laboratory conditions the health condition and functions of the organs of hares were impaired by a complex of negative, mainly psychogenic stress factors, which resulted in the death of hares and the experiment had to be finished prematurely on the 41st day after infection.     

A Disease Resembling Paratuberculosis (Johne’S Disease) in Roe Deer (Capreolus Capreolus L.) an Aetiological and Patho-Anatomical Study

A disease in wild living roe deer (Capreolus capreolus L.) caused by acid-fast bacteria is described.The morphological and cultural properties of these bacteria agree closely with corresponding properties of Mycobact. johnei.Enlarged lymph nodes, especially mesenteric lymph nodes with greyish-yellow necroses, were the most prominent macroscopic lesions. No intestinal lesions were present.The histopathological picture of the lymph nodes resembled mostly lesions which can occur in paratuberculous sheep and goats. The epithelioid cells contained masses of acid-fast bacteria. Such rods were also demonstrated in the intestinal villi.The acid-fast bacteria could be isolated from the mesenteric lymph nodes, spleens, bone marrow, mammary gland and also from the lymph nodes of other organs.The organism did not produce tuberculosis in guinea pigs. Intravenous injections into hens and rabbits resulted in miliary nodules resembling those of tuberculosis in the livers and spleens of the hens, and in joint and tendon sheath lesions in the rabbits. Microscopically the lesions mostly resembled those in paratuberculosis.One of the strains which was inoculated intravenously into two calves caused no lesions. The results of the allergy tests and serological blood tests in one animal indicate that infection with Mycobact. johnei cannot be excluded.A goat which was inoculated in the same manner and with the same strain as the calves died after six weeks. Miliary epithelioid cell granulomas in the liver, spleen, kidneys, bone marrow of long bones and in the submucosa of the ileum, as well as patchy infiltration of epithelioid cells in, among others, the mesenteric lymph nodes were observed on microscopic examinations.By intravenous infection the disease could be reproduced in a roe deer (fawn) (Gapreolus capreolus L.). The animal died nine weeks after the infection, and during the last two days before death it had a profuse diarrhoea. Masses of short acid-fast bacteria in clumps were present in the faeces. Nor in the experimental animal did macroscopic intestinal lesions occur. Enlarged villi infiltrated with epithelioid cells containing acid-fast bacteria were demonstrated by histological examination.The acid-fast bacteria could be recovered, but only on the Taylor/ Finlayson medium, from all experimental animals except the guinea pigs. Concerning the experimental calves, acid-fast bacteria were recovered from only one of them and then nearly three years after the infection.The acid-fast bacteria did not reduce nitrate. They showed positive neutral red reactions and were sensitive to isoniazid in a concentration of 2.5 µg per ml medium after three weeks’ incubation.The possibility that the isolated acid-fast bacteria and the lesions caused by them might be avian tubercle bacilli and avian tuberculosis has been discussed by the author. He does not, however, find any relevant reason for such an assumption. The author considers that the bacteria and the lesions exhibit the greatest similarity to Mycobact. johnei and paratuberculosis (Johne’s disease).On account of the organism’s pathogenicity for hens and rabbits, and necroses in the course of the disease, the author suggests that these bacteria possibly constitute a variety of the classic bovine Mycobact. johnei, different from the pigmented and the Icelandic varieties.     

Caseous Lymphadenitis in Goats: The Pathogenesis,Incubation Period and Serological Response after Experimental Infection

Twenty goats, in two groups of 10, were injected intradermally with Corynebacterium pseudotuberculosis. The doses of infection were 1×10⁵ and 5×10⁴ colony-forming units (cfu) for groups 1 and 2, respectively. Thereafter, a goat from each group was killed every 2–3 days and examined for gross and microscopic caseous lesions in the draining lymph nodes. Bands or zones of macrophages and polymorphonuclear granulocytes were observed microscopically on the second day of infection in both groups. Gross caseous lesions were observed from days 8 and 9 of infection, respectively. Positive bacterial agglutination test and haemolysis inhibition test titres were detected after 15–17 days and 20–25 days of infection, respectively. These results indicated that caseous lymphadenitis is a subacute disease with an incubation period of 8–9 days, but that it is not detectable serologically until after 15 days of infection.     

Mycobacterium avium abortion in a sow.

A 2-year-old sow aborted her entire near-term litter of 11. Gross and histologic examination of a fetus suggested a tuberculous infection, and a yellow-pigmented Mycobacterium avium serotype 1 was subsequently isolated from the fetal tissue. Efforts to rebreed the sow were unsuccessful. She was anergic to skin tests with purified protein derivative of M. avium on two occasions but had M. avium specific in vitro lymphocyte immunostimulation. Gross granulomatous lesions were found in the liver, kidneys, and endometrium when the sow was necropsied 5 months after the abortion. Histologic examination showed diffuse and focal non-encapsulated granulomas in lymph nodes, tonsils, kidney, liver, spleen, lung, and uterine and vaginal walls. There were a few encapsulated calcified foci in the endometrium. The centers of some granulomas in the tonsils, liver, kidneys, and some lymph nodes were caseated. The yellow-pigmented M. avium was isolated from the reproductive organs and from 11 of 12 other tissues cultured.     

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.     

Calcospherite-like bodies and caseous necrosis in tracheal mucus from a dog with tuberculosis

A 3-year-old Wirehaired Fox Terrier was presented to the University Veterinary Hospital, University College Dublin, for evaluation of chronic cough of 8-months duration. Bronchoscopy showed a severely dilated collapsed left principal bronchus filled with highly viscous white mucus. Cytologically, globular lipid-like material and round concentrically laminated crystalline structures were evident within the proteinaceous mucus. These findings resembled the calcospherites and granular caseous debris often observed in human tuberculous patients. A Ziehl-Neelsen-stained cytocentrifuged preparation of material obtained by bronchoalveolar lavage revealed a few acid-fast rods within macrophages, suggestive of tuberculosis. At necropsy, granulomas with caseous necrosis were present in the lung parenchyma, bronchial and mediastinal lymph nodes, liver, pancreas, and mesentery. Granulomas were adherent to both kidney capsules and to the diaphragm. Histologically, there was evidence of mild calcification within caseous granulomas, which was confirmed by von Kossa's stain. Using Ziehl-Neelsen stain, acid-fast rods were identified within granulomas; bacterial culture was positive for Mycobacterium bovis. The cytologic findings in this case have not been reported previously in dogs and demonstrate a possible correlation between tuberculosis and calcospherite-like bodies with caseous, globular material in bronchial mucus, similar to that described in human patients.     

Patterns of lesions of bovine tuberculosis in wild red deer and wild boar

The data obtained from a survey of Mycobacterium bovis infection in wild red deer (Cervus elaphus) and wild boar (Sus scrofa) conducted in France in the 2005/06 hunting season were used to describe and quantify the pathological findings in the two species. The red deer had caseous abscessed lesions in their organs and lymph nodes, whereas in the wild boar the lesions were predominantly caseocalcareous and occurred mainly in the lymph nodes. The severity of the gross tuberculosis-like lesions was estimated on the basis of a numerical score. The significant difference between the distribution of the scores in the two species indicated that the disease was more serious in the red deer than in the wild boar. Unlike the red deer, the wild boar did not show a generalised pattern of disease. Among the lymph nodes examined systematically, gross lesions were most frequently observed in the mesenteric lymph nodes in the red deer and in the retropharyngeal lymph nodes in the wild boar. In both species, the presence of gross lesions showed the closest agreement with the isolation of M bovis from the same lymph nodes. The different patterns of the lesions of tuberculosis in the two species suggest that red deer might play an important role in the intraspecies and interspecies dissemination of the infection, whereas in wild boar the spread of the infection would be more likely to be restricted to other wild boar.     

Lesion development in white-tailed deer (Odocoileus virginianus) experimentally infected with Mycobacterium bovis

The recent discovery of tuberculosis in free-living white-tailed deer in northeastern Michigan underscores the need for increased understanding of the pathogenesis of tuberculosis in wildlife species. To investigate lesion development in white-tailed deer, 32 deer were experimentally infected by intratonsilar instillation of 300 colony-forming units of Mycobacterium bovis. Three deer each were euthanatized and examined at days 15, 28, 42, and 56 after inoculation, and five deer each were euthanatized and examined at days 89, 180, 262, and 328 after inoculation. Microscopic lesions first were seen in the medial retropharyngeal lymph node and lung 28 and 42 days after inoculation, respectively. Lung lesions were present in 12 (38%) of 32 deer, involving 23 lung lobes. Left caudal and right middle and caudal lobes were involved in 17 (74%) of the 23 affected lung lobes. Lesions in the medial retropharyngeal lymph node first appeared as granulomas composed of aggregates of macrophages and Langhans-type giant cells. Some early granulomas contained centrally located neutrophils. As granulomas developed, neutrophils were replaced with a central zone of caseous necrosis that first showed signs of mineralization 42 days after inoculation. Granulomas increased in size as the zone of caseous necrosis expanded. Peripheral fibrosis, first seen at 56 days after inoculation, progressed to only a thin fibrous capsule by 328 days after inoculation. By the termination of the study, the central necrotic core of the granuloma contained abundant liquefied necrotic material and grossly resembled an abscess. Although tuberculous lesions in white-tailed deer follow a developmental pattern similar to that in cattle, fibrosis is less pronounced and the advanced lesions may liquefy, a change seldom reported in cattle. An understanding of lesion development will aid in the identification of the spectrum of disease that may be seen in this important wildlife reservoir of tuberculosis.     

Experimental induction of caseous lymphadenitis in sheep by intralymphatic inoculation of Corynebacterium ovis

Lesions histologically similar to those of natural caseous lymphadenitis developed in sheep popliteal lymph nodes following injection of viable Corynebacterium ovis cells into afferent lymphatic ducts. Histopathological examination of affected lymph nodes suggested that exotoxin might be involved in the establishment, progressive development and persistence of caseous lymphadenitis lesions through localised cytotoxicity.     

A serological investigation of caseous lymphadenitis in four flocks of sheep

A double antibody sandwich ELISA developed by ID-DLO, Lelystad to detect Corynebacterium pseudotuberculosis infection was used on 329 sheep from four pedigree Suffolk flocks in which clinical cases of caseous lymphadenitis (CLA) had occurred. At subsequent necropsy, typical CLA lesions were seen in 133 sheep, and the diagnosis was confirmed on culture. Lesions were most commonly seen in lungs (n = 46), parotid lymph nodes (n = 44), prescapular lymph nodes (n = 38) and mediastinal lymph nodes (n = 31). The sensitivity of the ELISA test for detecting culture-positive sheep was 0.88, while the specificity of the test was 0.55. The antibody ELISA detected 87.5 per cent of sheep that had CLA lesions restricted to internal organs only. It was concluded that the ELISA test has a valuable role in detecting sheep with both clinical and subclinical CLA.     

Pathogenesis of caseous lymphadenitis in sheep and goats

The progress of caseous lymphadenitis in sheep and goats is described as involving primary wound infection, lymphatic and haematogenous dissemination, and secondary infection of lymph nodes and various visceral organs. This is followed by elimination or containment of infection, the latter being represented by the characteristic caseous lesions. Resistance to infection is complex involving components of both the specific and non-specific host response. The role of these in pathogenesis is discussed. Bacterial factors including the phospholipase exotoxin and cytotoxic lipid contribute to pathogenesis at a local level with little likelihood of systemic effect in the natural disease. In a disease such as caseous lymphadenitis, lesion site, size or number do not indicate severity of disease but rather, reflect the ability of the host to mount a cellular response to infection.     

Mycobacterium bovis infection in the brush-tailed possum (Trichosurus vulpecula): I. Preliminary observations on experimental infection

An Australian cattle strain of Mycobacterium bovis was injected intramuscularly into 10 brush-tailed possums (Trichosurus vulpecula) which were shown to be highly sensitive to experimental infection with this organism. The possums were killed and examined throughout the course of infection. At necropsy, gross and microscopic lesions were recorded and several tissues cultured for recovery of M. bovis. Infection spread rapidly via the lymphatic system from the injection site to the lumbar lymph nodes, then to the spleen. There was a bacteraemia after 2 week and by 6 weeks lesions were present in spleen, lymph nodes, lungs and kidneys; M. bovis proliferated rapidly and host response was minimal. Few organisms were detected in the liver where miliary lesions were found. M. bovis was excreted in large numbers in urine, faeces and discharging sinuses from subcutaneous abscesses. In two possums that died early in the infection, stress had rendered then more susceptible; infection spread more rapidly than in other possums and liver involvement was more severe. Although aerosol transmission was considered to be a possible means of spreading M. bovis, three in-contact possums did not acquire infection.     

Corynebacterium pseudotuberculosis infection in goats. VIII. The effect of vaccination against experimental infection

The effect of an inactivated vaccine against C. pseudotuberculosis infection was tested on castrated male kids from a herd free from caseous lymphadenitis. The animals were divided into 3 groups with 8 animals in each. Group 1 was immunized with crude filtrated C. pseudotuberculosis toxoid and whole killed organisms, while Group 2 in addition was given levamisole. The kids were vaccinated twice at an interval of 4 weeks. Group 3 consisted of unvaccinated animals. All groups were challenged subcutaneously with live bacteria 4 weeks after the last vaccination. Unvaccinated animals showed the most severe course of illness after challenge. Development of abscesses in the regional lymph nodes (Inn. subiliaci) was significantly more common in unvaccinated than in vaccinated kids at necropsy 2 months after challenge. There was, however, no such difference between the vaccinated groups, and there was no difference between any of the groups as regards abscess formation at the inoculation site. In each of the 2 vaccinated groups, there was a titre rise following vaccination in the hemolysis inhibition test, whereas no such rise was seen in the bacterial agglutination test. The titre values in both tests increased significantly after challenge in all the groups, the increase being most rapid in the vaccinated animals. The present investigation indicates that development of caseous lesions in lymph nodes in goats, following subcutaneous inoculation with C. pseudotuberculosis, can be reduced by an inactivated vaccine containing whole organisms and crude toxin.     

An abattoir study of tuberculosis in a herd of farmed elk.

The purpose of this study was to examine the prevalence and distribution of grossly visible lesions of tuberculosis in a herd of 344 North American elk (Cervus elaphus) depopulated during a three-month period in 1991. Abattoir inspection detected mycobacterial lesions in 134 (39.8%) of the 337 animals received for slaughter. The prevalence of lesions increased with the age of the animals. Lesions were predominantly suppurative rather than caseous, and mineralization was less evident than in tuberculous lesions in cattle. Lesions occurred predominantly in lymph nodes, and lungs were the only organs in which mycobacterial lesions were found. The distribution of lesions suggested that aerosol transmission was the most significant means of spread of the disease within the herd. Giant liver flukes (Fascioloides magna) were observed in approximately 80% of the adult elk.     

Prevalence and spectrum of Johne’s disease lesions in cattle slaughtered at two abattoirs in Kampala, Uganda

This study was conducted to determine the prevalence and characteristics of Johne’s disease (JD) lesions in Ugandan cattle slaughtered at two of the main abattoirs in Kampala. Ileocaecal junction and the associated lymph nodes of 1,022 cattle were examined for gross and microscopic lesions, followed by Ziehl Neelsen staining of the tissues bearing lesions. Confirmation of Mycobacterium avium subsp. paratuberculosis infection was done in some of the tissues using culture and IS900 PCR. The lesions were then described, characterised and tabulated. Characteristic Johne’s disease granulomas were found in 4.7% of the samples examined, derived from Zebu, Ankole longhorn, Friesian breeds of cattle and their crosses. Lesions were found both in the lymph nodes and ileocaecal junction mucosa. The lesions tended to be more severe in the lymph node than in the mucosa. There were also some unique and atypical lesions found in association with Johne’s disease granulomas. The diagnostic values of various gross lesions and criteria of lesion classifications and diagnosis are revisited and discussed based on the findings of this study. The prevalence of Johne’s disease lesions among slaughtered cattle in Kampala’s two abattoirs indicates that the disease is well established in the cattle population in the country. The diverse manifestations in lesions of JD need to be considered when making histological diagnosis in tissues where the disease is suspected.     

Tuberculosis in a New Zealand (Hooker's) sea lion

Extract

Tuberculosis has been described in seven species of pinnipeds. Based on host preference, phenotype and genetic and antigenic testing, the causal bacterium has been classified as a distinct species, Mycobacterium pinnipedii, within the M. tuberculosis complex. An adult male New Zealand sea lion (Phocarctos hookeri) was found dead on the Otago coastline in mid 2005. Gross necropsy revealed multiple caseous foci throughout the lungs, subcutaneous and thoracic lymph nodes, and mesenteric lymph nodes. Histopathological examination showed granulomatous lesions containing numerous intra-lesional acid fast organisms. The organism was confirmed as being a member of the M. tuberculosis complex, based on restriction enzyme analysis. Spoligotyping is pending. This is the first confirmed case of mycobacterial disease in a New Zealand sea lion.     

Granuloma development in cattle after intratonsilar inoculation with Mycobacterium bovis

OBJECTIVE: To examine the temporal development of tuberculous lesions in cattle inoculated with Mycobacterium bovis. ANIMALS: 15 mature crossbred cows obtained from a herd with no history of M bovis infection. PROCEDURE: Inoculation of cattle was done by intratonsilar instillation of 1.48 X 10(5) to 5.4 X 10(7) colony-forming units of M bovis strain 2045T. At 3 to 4 hours, 4 weeks, 6 weeks, and 8 weeks after inoculation, tissues were examined for gross and microscopic lesions and processed for isolation of M bovis. RESULTS: Retropharyngeal lymph nodes from cattle examined 4 weeks after inoculation contained microgranulomas consisting of aggregates of macrophages with few neutrophils. Retropharyngeal lymph nodes from all cattle examined 6 and 8 weeks after inoculation contained multiple, large, coalescing granulomas consisting of central areas of necrosis with mild fibrosis, numerous macrophages, lymphocytes, plasma cells, multinucleated giant cells, and neutrophils. Three of 8 cattle examined 6 or 8 weeks after inoculation had lesions in nonretropharyngeal sites with morphologic characteristics similar to that seen in retropharyngeal lymph node granulomas from cattle examined 4 weeks after inoculation. CONCLUSION: Granulomas can develop in draining lymph nodes of cattle in as little as 4 weeks after inoculation via intratonsilar instillation of M bovis. Intralesional morphologic changes between 4 and 6 weeks after inoculation indicate an increase in cellular chemotaxis and differentiation. Dissemination of bacteria to distant sites most likely was by lymphatic and hematogenous routes after establishment of the primary infection in retropharyngeal lymph nodes.     

Histoplasmosis in the lung of a race horse with yersiniosis.

A 4-year-old female thoroughbred race horse died of acute peritonitis caused by necrotizing granulomatous duodenitis. Yersinia enterocolitica was immunohistochemically demonstrated in macrophages in granulomas developed in the duodenum, lung, liver and abdominal lymph nodes. The yeast-like fungi were found in the cytoplasmic vacuoles of macrophages in the lung that infiltrated into the granulomas and surrounding alveoli with congestive edema. The yeast-like fungi were positively stained by Gomori-Grocott chromic acid methenamine silver stain and immuno-histochemically stained with anti-histoplasma antibody. In this case, it was considered that granulomas formed in the duodenum, lung, liver and abdominal lymph nodes were primarily caused by Yersinia enterocolitica due to idiopathic weakening of the immune system. Yeast-like fungi immunohistochemically identified as histoplasmas secondarily infected the lung. This is the first case regarded as equine histoplasmosis capsulati in Japan.     

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.