A 2-year-old, castrated male, mixed-breed dog was presented to the University of Florida Veterinary Medical Center with swelling, edema, ulceration, and draining tracts in the region surrounding the left hock. The dog had mild monocytosis and moderate hyperglobulinemia. Fine-needle aspirate specimens of the left popliteal lymph node revealed pyogranulomatous lymphadenitis with hyphal organisms. The diameters of the hyphae were variable, ranging from 11 to 22 mum. The organism was considered as most consistent with Lagenidium caninum; although Pythium insidiosum or Lagenidium karlingii were not conclusively excluded, hyphal diameter in these organisms is typically smaller (6.6-8.8 and 2.5-11 mum, respectively). A positive Western blot confirmed the presence of serum antibodies reactive against Lagenidium sp. and the absence of antibodies to P. insidoisum, Basidiobolus, and Conidiobolus antibodies. Careful assessment of hyphal diameter in cytologic specimens may be useful in differentiating L. caninum from P. insidiosum or L. karlingii.

Objective-To evaluate the radial growth assay for use in in vitro susceptibility testing of Pythium insidiosum and a Lagenidium sp and to assess susceptibility of representative isolates to itraconazole, posaconazole, voriconazole, terbinafine, caspofungin, and mefenoxam. Sample Population-6 isolates each of P insidiosum and Lagenidium sp. Procedures-Isolates were plated in triplicate onto agar supplemented with antifungal compounds at concentrations of 0.025 to 8 mug/mL. Isolates on dimethyl sulfoxide- and water-supplemented agar served as control samples. Effect of antifungal concentration on colony diameter was assessed with a mixed linear model. Assay variability was assessed with the coefficient of variation. Results-Colony growth was uniform (mean intra-assay and interassay coefficients of variation were < 5%). Minimal inhibition was evident with voriconazole and posaconazole at 8 mug/mL. Terbinafine at 8 mug/mL significantly reduced growth of P insidiosum and at >/= 1 mug/mL significantly reduced growth of the Lagenidium sp. Caspofungin and mefenoxam (concentrations >/= 1 mug/mL and >/= 0.025 mug/mL, respectively) significantly reduced growth of both pathogens. Mefenoxam at 0.1 mug/mL caused > 50% growth inhibition in 11 of 12 isolates and at 1 mug/mL caused > 90% inhibition in all isolates. Conclusions and Clinical Relevance-Results suggested that the radial growth assay was a simple, reproducible technique for susceptibility testing of P insidiosum and a Lagenidium sp. Azoles had limited activity, whereas terbinafine and caspofungin caused significant but minimal to moderate inhibition. Only mefenoxam had a profound effect on both pathogens at concentrations likely to be achievable in tissues.

We describe the ecological niche of the human and animal pathogen Pythium insidiosum within endemic agricultural areas of Thailand. Samples were collected from irrigation water, including rice paddy fields, irrigation channels and reservoirs. Zoospores of P. insidiosum were captured from water by the use of a sterile human hair baiting technique. Pythium isolates were identified based on phenotypic characteristics and by using a specific PCR assay for P. insidiosum. In addition, internal transcribed spacer (ITS) regions of P. insidiosum rDNA were sequenced and used in the phylogenetic analysis of 20 other known P. insidiosum DNA sequences available in the database and 11 related DNA sequences of other Pythium species including Lagenidium giganteum. The sequences of 59 environmental isolates of Pythium spp. recovered from Thailand confirmed 99% identity to P. insidiosum. Three well supported phylogenetic groups within P. insidiosum were found. The protein profiles of P. insidiosum environmental strains were determined and compared with reference strains. A typical 45-30 kDa band was consistently found in all isolates of P. insidiosum but not in closely related Pythium species. This study provides the first evidence for the natural occurrence of P. insidiosum in endemic aquatic environments. The highest recovery rate of this hydrophilic pathogen was found to be from water reservoirs and our data show that irrigation water may be an important source of P. insidiosum infection for individuals working in endemic agricultural areas.

A case of eosinophilic granulomatous gastroenterocolitis and hepatitis in a 1-year-old male Siberian Husky is described. The dog presented with a history of diarrhea, weakness, lethargy, and anorexia of several months' duration. Hematologic and biochemical examinations, abdominal ultrasonography, computer tomography, and exploratory laparotomy were performed. Histopathologic examination of full-thickness biopsies from the gastrointestinal tract and liver revealed the presence of eosinophilic granulomatous lesions in the submucosa and tunica muscularis of stomach, jejunum, ileum, colon, and liver. Infectious agents were not detected by light microscopic and electron microscopic examination or by immunohistochemistry. On the basis of the findings, it is concluded that the disease in this dog represents an unusual manifestation of chronic idiopathic inflammatory bowel disease.

Two young adult male Domestic Shorthair cats living in the southeastern United States were evaluated for signs attributable to partial intestinal obstruction. Physical examination indicated a palpable abdominal mass in each animal. Exploratory laparotomy revealed a large extraluminal mass involving the ileum and mesentery with adjacent mesenteric lymphadenopathy in cat No. 1 and an abscessed mass in the distal duodenum in cat No. 2. Mass resection and intestinal anastomosis were performed in both cats. Histologic evaluation indicated that the intestinal lesions involved primarily the outer smooth muscle layer and serosa and consisted of eosinophilic granulomatous inflammation with multifocal areas of necrosis. In Gomori methenamine silver-stained sections, broad (2.5-7.5 microm), occasionally branching, infrequently septate hyphae were observed within areas of necrosis. A diagnosis of Pythium insidiosum infection was confirmed in both cats by immunoblot serology and by immunoperoxidase staining of tissue sections using a P. insidiosum-specific polyclonal antibody. Cat No. 1 was clinically normal for 4 months after surgery but then died unexpectedly from an unknown cause. Cat No. 2 has been clinically normal for at least 9 months after surgery and appears to be cured on the basis of follow-up enzyme-linked immunosorbent assay serology.

An 11-months-old mixed Terrier male originally from Venezuela, was referred to a Veterinary Hospital with signs of depression, anorexia, vomiting and diarrhea. The illness had begun 1 month earlier. Despite antibiotic chemotherapy and vitamins, the disease progressed. Radiological exams showed involvement of the small intestine. Histopathological studies of tissue samples taken during surgical intervention revealed eosinophilic areas in the center of which, abundant eosinophils, histiocytes and giant cells were observed. Silver stained cross-sections of the small intestine showed slender sparsely septate hyphae within the necrotic areas. Attempts to isolate the etiologic agent in pure culture were fruitless. The dog died without a definitive diagnosis. Fixed tissue samples of the small intestine were later investigated using specific fluorescent antibodies for pythiosis and molecular tools. These exams indicated that the hyphae in the infected tissues belong to the straminipilan pathogen Pythium insidiosum. This is the first confirmed case of dog pythiosis in Venezuela.

A 7-month-old, male jaguar presented with dyspnea and leukocytosis unresponsive to antibiotic therapy. Radiographs revealed unilateral pulmonary consolidation. An exploratory thoracotomy was performed, and the left lung, which contained a large multilobular mass with extensive fibrosis and numerous caseonecrotic foci, was removed. Microscopically, eosinophilic granulomatous inflammation surrounded broad (4.4-8.3 microm) rarely septate hyphae. A diagnosis of Pythium insidiosum infection was confirmed by immunohistochemistry, immunoblot serology, culture, and polymerase chain reaction. Dyspnea recurred despite treatment, and the animal succumbed 3 weeks after surgery. Necropsy findings indicated that death resulted from occlusion of the right main stem bronchus by a fungal granuloma. The oomycete P. insidiosum typically causes granulomatous disease of the skin or gastrointestinal tract in animals and arteritis, keratitis, or cellulitis in humans. Infection is uncommon in felines, and pulmonary involvement is rare. This report details the first case of P. insidiosum infection in an exotic felid and provides the first description of primary pulmonary pythiosis in any species.

Pythium insidiosum is a pathogen that causes disease in both animals and humans. Human infection is rare; however, when it does occur, most patients, especially those having underlying hemoglobinopathy syndromes, such as thalassemia, exhibit a severe form. We identified four isolates of P. insidiosum. Two were recovered from tissue biopsy specimens from thalassemic and leukemic patients, one was derived from brain tissue from a thalassemic patient, and another was isolated from a corneal ulcer from a fourth patient. Western blotting and an enzyme-linked immunosorbent assay (ELISA) were performed with a serum sample derived from one thalassemic patient. The methods used to identify the P. insidiosum isolates were based on morphology, nucleic acid sequencing, and a PCR assay. To confirm the identification, portions of the 18S rRNA genes of these four isolates were sequenced. The sequences were shown to be homologous to previously described P. insidiosum DNA sequences. In addition, PCR amplification of the internal transcribed spacer region specific for P. insidiosum was positive for all four isolates. The ELISA with the serum sample from the thalassemic patient gave a positive result from a serum dilution of 1:800. Finally, Western immunoblotting with this serum sample showed positive immunoglobulin G recognition for proteins of 110, 73, 56, 42 to 35, 30 to 28, 26, and 23 kDa. The results of this study show that both molecularly based diagnostic and serodiagnostic techniques are useful for the rapid identification of human pythiosis. The predominant antigens recognized by Western blotting may be useful in the development of a more sensitive and specific diagnostic tool for this disease.

Pythium insidiosum, the only species in the genus that infects mammals, is the etiological agent of pythiosis, a granulomatous disease characterized by cutaneous and subcutaneous lesions and vascular diseases. Accurate diagnosis of pythiosis and identification of its causal agent are often inconsistent with current immunological diagnostic methods. A species-specific DNA probe was constructed by using a 530-bp HinfI fragment from the ribosomal DNA intergenic spacer of P. insidiosum. When the probe was incubated with dot blots of genomic DNA from 104 Pythium species, it hybridized only to the DNA of P. insidiosum and P. destruens-two species that have been considered conspecific. The probe also hybridized to DNA from 22 P. insidiosum isolates in this study, regardless of their geographic origin or animal host. When tested against genomic DNA from other pathogenic organisms (Aspergillus fumigatus, Basidiobolus ranarum, Conidiobolus coronatus, Lagenidium giganteum, Paracoccidioides brasiliensis, and Prototheca wickerhamii), no cross-hybridization of the probe was detected. The specificity of the probe to hybridize to genomic DNA from all isolates of P. insidiosum and not cross-react with DNA from other Pythium species or pathogens that cause symptoms similar to pythiosis in their hosts makes it a powerful tool for the accurate diagnosis of pythiosis. In addition, the probe has the potential for pathological and environmental diagnostic applications.

A 10-week-old, male pit bull dog presented to the referring veterinarian with hind limb paresis and epaxial muscle atrophy. No spinal lesions were identified at gross necropsy; however, histologically there was marked granulomatous myelitis in the spinal cord between T13 and L2 with occasional, intralesional nematode larvae. Based on morphologic characteristics, the nematode larvae were identified as Strongyloides spp., possibly Strongyloides stercoralis.

This report describes a pituitary acidophil macroadenoma in a goat. Antemortem clinical findings included hypothermia and rumen stasis. Clinicopathologic findings included refractory hypoglycemia, low total thyroxin and insulin concentrations, elevated bile acid concentration, and hyposthenuria. In addition to the pituitary macroadenoma, bilateral atrophy of the zona reticularis of the adrenal glands was observed histologically.

An adult female hyacinth macaw (Anodorhynchus hyacinthinus) was presented for sudden onset of severe weakness in the legs. Neurologic examination revealed bilateral paresis of the pelvic limbs and decreased proprioception. Results of radiographs and computed tomography (CT) revealed variably sized soft tissue nodules throughout the lungs and invading into the spine and vertebral canal. Soon after the CT scan, the bird went into cardiorespiratory arrest and died. At necropsy, several yellow, coalescing nodules that were firm with a caseous component were present in the lungs, and a focus of similar tissue was attached to the vertebrae and invaded the spinal canal. On histologic examination, the diagnosis was primary pulmonary bronchial adenocarcinoma with spinal invasion.

Objective-To evaluate the radial growth assay for use in in vitro susceptibility testing of Pythium insidiosum and a Lagenidium sp and to assess susceptibility of representative isolates to itraconazole, posaconazole, voriconazole, terbinafine, caspofungin, and mefenoxam. Sample Population-6 isolates each of P insidiosum and Lagenidium sp. Procedures-Isolates were plated in triplicate onto agar supplemented with antifungal compounds at concentrations of 0.025 to 8 mug/mL. Isolates on dimethyl sulfoxide- and water-supplemented agar served as control samples. Effect of antifungal concentration on colony diameter was assessed with a mixed linear model. Assay variability was assessed with the coefficient of variation. Results-Colony growth was uniform (mean intra-assay and interassay coefficients of variation were < 5%). Minimal inhibition was evident with voriconazole and posaconazole at 8 mug/mL. Terbinafine at 8 mug/mL significantly reduced growth of P insidiosum and at >/= 1 mug/mL significantly reduced growth of the Lagenidium sp. Caspofungin and mefenoxam (concentrations >/= 1 mug/mL and >/= 0.025 mug/mL, respectively) significantly reduced growth of both pathogens. Mefenoxam at 0.1 mug/mL caused > 50% growth inhibition in 11 of 12 isolates and at 1 mug/mL caused > 90% inhibition in all isolates. Conclusions and Clinical Relevance-Results suggested that the radial growth assay was a simple, reproducible technique for susceptibility testing of P insidiosum and a Lagenidium sp. Azoles had limited activity, whereas terbinafine and caspofungin caused significant but minimal to moderate inhibition. Only mefenoxam had a profound effect on both pathogens at concentrations likely to be achievable in tissues.

An 18-year-old Arabian mare was examined with a large mass on the left hind pastern and fetlock. The mare was located in the Central Valley of northern California, and had never been out of the state. Routine histopathological processing and examination of biopsy samples from the mass showed several hyphal organisms that were delineated with a silver stain. Using immunohistochemistry the organism was diagnosed as Pythium insidiosum. The owner declined debulking surgery, and despite treatment with an immunotherapeutic vaccine, the horse's condition deteriorated leading to euthanasia.

A 20-month-old castrated male Labrador Retriever with a 3-month history of anorexia, weight loss, and vomiting was evaluated. Plasma biochemical abnormalities included marked hyperglobulinemia and hypercalcemia. Serum levels of parathyroid hormone, parathyroid hormone-related protein, 25-hydroxyvitamin D, and 1,25-dihydroxyvitamin D were either low or within reference intervals. Gastric wall thickening and abdominal lymphadenomegaly were observed with abdominal ultrasonography. Cytologic evaluation of a sample obtained via fine-needle aspiration of the gastric wall revealed pyogranulomatous inflammation and numerous poorly stained hyphae. Partial gastrectomy was performed, and a diagnosis of gastric pythiosis was made by immunohistochemical staining of infected gastric tissue, as well as by immunoblot serology. This case demonstrates that diagnostic samples for cytologic evaluation can be obtained by fine-needle aspiration of Pythium insidiosum-infected tissues and that a presumptive diagnosis can be made by examination of a Romanowsky-stained smear. Furthermore, pythiosis should be considered as a differential diagnosis for hypercalcemia, especially in young dogs with inflammatory lesions that have a granulomatous component. The mechanism for the hypercalcemia in this dog was not determined; however, calcium concentrations normalized after surgical resection of the gastric lesion.

Tamoxifen is a non-steroidal anti-estrogen that prevents estrogen receptor-positive breast cancer in rodents and humans. Bexarotene, a selective agonist for retinoid X receptors, inhibits mammary carcinogenesis in rodents. The present study was conducted to support the preclinical development of tamoxifen citrate (TAM)+ bexarotene (BEX) for use in breast cancer chemoprevention, and to investigate the influence of these agents on hepatic gene expression. Female CD rats (20/group) received daily oral (gavage) exposure to TAM (0 or 60 mug/kg/day) and/or BEX (0, 5, 15 or 45 mg/kg/day) for a minimum of 90 days. BEX induced mild, dose-related anemia and dose-related increases in serum alkaline phosphatase, cholesterol, triglycerides, and calcium levels, and increased platelet counts. TAM had no biologically significant effect on any clinical pathology parameter and did not alter the effects of BEX on these endpoints. Microscopic alterations induced by BEX included epidermal hyperplasia, hyperkeratosis (stomach) and cytoplasmic clearing (liver). Microscopic changes in TAM-treated rats were limited to mucous cell hypertrophy in the cervix and vagina. The toxicity of administration of the combination of TAM + BEX can generally be predicted on the basis of the toxicity of each drug as a single agent. BEX induced dose-related alterations in the expression of several genes involved in steroid, drug and/or fatty acid metabolism; TAM did not alter these effects of BEX. Differential expression of genes involved in drug and lipid metabolism may underlie the observed effects of BEX on cholesterol and triglyceride levels and its effects on liver histology.

This study evaluated the relationship between blood iron parameters and hepatic iron concentrations, and correlation of histologic findings with hepatic iron concentrations in a captive population of Egyptian fruit bats (Rousettus aegyptiacus) and island flying foxes (Pteropus hypomelanus). Blood samples were collected for complete blood counts, plasma biochemical profiles, serum iron concentrations, total iron-binding capacity, whole-blood lead concentrations, and plasma ferritin assays. Liver samples obtained by laparotomy were divided, with one half processed for histologic examination and the other half frozen and submitted for tissue mineral analysis. The histologic sections were scored by two blinded observers for iron deposition, necrosis, and fibrosis. The Egyptian fruit bats had significantly higher liver iron (mean = 3,669 +/- 1,823 ppm) and lead (mean = 8.9 +/- 5.8 ppm) concentrations than the island flying foxes (mean [Fe]= 174 +/- 173 ppm, mean [Pb]= 1.9 +/- 0.5 ppm). Hepatic iron concentrations significantly correlated with tissue lead concentrations, histologic grading for iron and necrosis, serum iron, transferrin saturation, and plasma ferritin (P < 0.001). Blood lead concentrations negatively correlated with tissue lead concentrations (P < 0.001). When the product of transferrin saturation and serum iron was greater than 51, an individual animal had a high probability of having iron overload. When the product of these two variables was greater than 90, there was a high probability that the animal had hemochromatosis. On the basis of this study, it appears that evaluation of serum iron, transferrin saturation, and plasma ferritin are useful and noninvasive methods for diagnosis of hemochromatosis in Egyptian fruit bats.

Objective-To evaluate a combination of 2 nonantibiotic microbicide compounds, sodium hypochlorite (NaOCl) and polyhexamethylene biguanide (PHMB), as a treatment to suppress or eliminate Salmonella spp from red-eared slider (RES) turtle (Trachemys scripta elegans) eggs and hatchlings. Sample Population-2,738 eggs from 8 turtle farms in Louisiana. Procedures-Eggs were randomly sorted into 3 or, when sufficient eggs were available, 4 treatment groups as follows: control, pressure-differential egg treatment with NaOCl and gentamicin, NaOCl and PHMB bath treatment, and pressure-differential egg treatment with NaOCl and PHMB. Bacterial cultures were performed from specimens of eggs and hatchlings and evaluated for Salmonella spp. Results-RES turtle eggs treated with NaOCl and PHMB as a bath (odds ratio [OR], 0.2 [95% confidence interval (CI), 0.1 to 0.3]) or as a pressure-differential dip (OR, 0.01 [95% CI, 0.001 to 0.07]) or with gentamicin as a pressure-differential dip (OR, 0.1 [95% CI, 0.06 to 0.2]) were significantly less likely to have Salmonella-positive culture results than control-group eggs. Conclusions and Clinical Relevance-Concern over reptile-associated salmonellosis in children in the United States is so great that federal regulations prohibit the sale of turtles that are < 10.2 cm in length. Currently, turtle farms treat eggs with gentamicin solution. Although this has reduced Salmonella shedding, it has also resulted in antimicrobial resistance. Results of our study indicate that a combination of NaOCl and PHMB may be used to suppress or eliminate Salmonella spp on RES turtle eggs and in hatchlings.

In the preceding pages we have presented evidence which we believe furnishes new light on the disease process in avian tuberculosis. From a well known strain of avian tubercle bacillus, A(1), four variants have been dissociated, each manifesting distinct colony topography and physical and chemical characteristics. From these studies we have learned that the variants are sometimes unstable, not only in vitro but also in vivo, and that this characteristic is one of the prominent factors influencing both the advancement and retrogression of the disease. The four variants remain fairly stable in vitro provided they are cultivated on proper culture media. About 80 per cent smooth S and flat smooth F.S. colonies will develop true to type on egg media; and rough R and the chromogenic Ch when cultivated on gycerine agar media, in about the same percentage. An early non-specific eosinophilia followed inoculation, no matter which variant was used. This usually subsided by the 18th hour. The early stages of tubercle formation produced by all the variants appeared similar. First there appeared an aggregation of eosinophiles and their ingested bacilli within the tissues and then followed replacement by large mononuclear cells which wandered in and phagocyted both eosinophiles and bacilli. After the formation of tubercles composed only of large mononuclear cells, certain differences between the virulent and avirulent variants became apparent. There was also a direct relationship between the dosage and the extent of the disease. In our experience from 0.16 to 0.25 mg. gave the most uniform results. If S variant was used, the early non-specific eosinophilia was followed by a second rise which continued to ascend, running parallel with the total leucocyte count. There was a slow increase of monocytes and a corresponding decrease of lymphocytes. The microscopic lesions, 2 weeks after inoculation, were composed of irregularly shaped clumps of necrosing large mononuclear cells. The margins were clear-cut and bordered by few or no lymphocytic cells. By the 3rd and 4th weeks eosinophiles began to migrate into the centers of the tubercles and abscess formation became evident. Death usually occurred after 5 or 6 weeks, although this varied with the dosage. The appearance of the lesions suggested an acute "toxic" nature, as manifested (1) by marked enlargement of the spleen and liver,(2) by the short fatal course of the disease, which never became very extensive,(3) by the presence of few organisms within the tubercles,(4) by destruction of reticulum and (5) by the marked response of the blood leucocytes. After inoculation with F.S. variant the early non-specific eosinophilia disappeared, returning again at the terminal stage, 5 to 6 weeks later. The number of eosinophiles then ascended sharply, accompanied by a marked leucocytosis which continued until death. During this eosinophilia a monocytosis occurred and lymphocytes fell away rapidly. During the 2nd week, clumps of closely packed large mononuclear cells containing masses of phagocyted bacilli were seen. In the 3rd and 4th weeks there appeared a prominent peripheral zone of hyalin, collagenous-like material associated with an increased reticulum formation which apparently walled off the lesion. The bacilli within the tubercle multiplied rapidly, it seemed as though they were more resistant to destruction than the S organisms, perhaps due to their higher lipin content. As a result of their rapid increase, progression of the tubercle continued, with necrosis and abscess formation, accompanied by the dissemination of the organisms both locally and distantly to new uninvolved tissue. Wide extension of the disease followed due to the increase in numbers of bacilli, terminating with death usually during the 5th and 6th weeks. The manifestations of "toxin" damage shown in S disease were not so marked until the approach of the end-stage. We interpret the type of lesion produced by the F.S. variant as a foreign body tubercle to distinguish it from the acute "toxic" nature of the S. A quantitative chemical determination showed that this organism contained 20.14 per cent lipin whereas in the S variant it was only 15.03 per cent. Following inoculation with R variant the initial eosinophilia returned to normal within 24 hours. There was then an increase of both lymphocytes and monocytes, the latter exceeding the former for a time. Later, usually after the 2nd month, the lymphocytes again became predominant over the monocytes, approaching the normal base line. Eosinophiles failed to react. This blood picture indicated a successful resistance against the infecting organisms, and was supported by the pathological findings. None of the chickens of this group died during the period of study (79 days) although infecting doses of 0.25 mg. were used. Necropsies on four chickens revealed no macroscopic evidence of disease approximately 11 weeks after inoculation. Microscopic lesions were present in the spleen and liver of some cases. In chickens inoculated 11 weeks previously these tubercles consisted only of very small, discrete clumps of degenerating mononuclear cells often surrounded by a border of lymphocytes. Necrosis, abscess formation or caseation was not found. Their small size and appearance was evidence of their retrogressive character. Isolated giant cells were rarely seen. It was impossible to find bacilli within these tubercles. In one instance it was possible to show complete innocuousness of this organism in the tissues, as far as producing recognizable tuberculous lesions. Reticulum played little part in the development of the lesion and it was neither increased or destroyed. Following inoculation with the chromogenic Ch variant the initial eosinophilia declined by the 24th or 42nd hour count and from then on remained low. The lymphocytes showed little decrease. A prominent monocytosis of prolonged character usually occurred during the 3rd to 6th weeks and was then displaced by a return of the lymphocytes. By the end of the period of observation the differential count had closely approached the normal values. Again the blood picture was truly representative of the relatively benign character of the lesions. The tubercle formation, even after 93 days' duration of the disease, remained limited to small clumps of two or three large mononuclear cells showing some degeneration. Lymphocytic cells were usually present within and about the lesion. There was never any evidence of abscess formation or actual necrosis. Bacilli could not be found within these tubercles. The reticulum in relation to the lesion showed neither increase nor destruction. None of the chickens died though a dose of 0.5 mg. was used. In three chickens infected with S variant, the blood picture did not follow the pattern set by the others similarly inoculated. Either a high monocytosis replaced the usual eosinophilia, or there was a marked recession of the total leucocyte count followed by a return of lymphocytes towards normal percentage. The blood picture indicated the conversion of an acute process into a subacute or chronic affair. These chickens survived for 78, 79 and 110 days, respectively. An explanation for this occurred to us later when cultures recovered from them, instead of consisting of S colonies, showed the topography of the intermediate type, closely resembling the F.S. type. Evidently this was due to a reversion; i.e., a loss of virulence of the organism occurring within the animal body. At necropsy the spleen and liver were somewhat enlarged and contained prominent, discrete yellow nodules resembling small shot in their size and shape, which, in one case could be lifted from the tissue by the point of the knife. Microscopically, they possessed small centers of caseation surrounded by numerous giant cells and large mononuclear cells with a thick border of lymphocytic cells. This tubercle formation could not be classified in any special group as it was too sharply circumscribed and walled off by lymphocytic cells to be considered as truly malignant in nature as the S type of lesion. Even the younger tubercles in these cases, which consisted of clumped epithelioid cells with prominent lymphocytic cell borders, could not be called acute or "toxic" in character. At present we make only limited deductions from our observations. In the bacterial dissociation phenomenon we have at least a new line of investigation, and different variants of tubercle bacilli must be taken into consideration when planning new tuberculosis studies. With the stabilization of the human type variants, experiments such as reported here may bring about the proper clinical interpretation, of the course of human tuberculosis.

It has been found that although there is some parallelism between the quantity of tubercle bacilli demonstrable histologically and the number of colonies that can be isolated from a given tissue, the culture method is far the more efficient in indicating quantitative relations. Tubercle bacilli were not perceived in the organs of rabbits 1 day after infection with the modified BCG although as many as 1,500 colonies were isolated from one of them. This may be solely because it is difficult to see widely dispersed single minute acid-fast rods in the diffuse infiltrations of mononuclears with their hyperchromatic nuclei and sparse cytoplasm. Later, with the formation of tubercle, the parallelism is much closer. The culture method gives evidence concerning the number of living tubercle bacilli in the tissue. The significance of the accumulation of acid-fast particles in the tissues has been discussed. It has been seen that from the beginning this accumulation is greater in the Kupffer cells of the liver, in the macrophages of the spleen and in the reticular cells of the bone marrow than within the mononuclears of the lung, the organ where the bacilli grow with the greatest rapidity and are destroyed with the greatest difficulty. Acid-fast particles are more prominent with the bovine than with the human bacillus or the BCG, the microorganism that is destroyed with the greatest difficulty thus leaving more incompletely digested bacillary debris at a given time within the cells. Thus it seems permissible to conclude from the presence of acid-fast material that some tubercle bacilli are undergoing destruction even 24 hours after infection. The initial accumulation of polynuclear leucocytes corresponds with the subsequent severity of the infection. Despite the greater primary localization of bacilli in the liver, this initial inflammatory reaction with all three infections is much greater in the lung than in the liver. In each organ it is more intense with the bovine than with the less virulent strains. The multiplication of the bacillus and its accumulation within large mononuclear and young epithelioid cells is accompanied by an intense formation of new mononuclears by mitosis. The more rapid the growth of the bacillus, the more conspicuous the regeneration of these cells. Thus with all strains mitosis is more intense in the more susceptible organ, as in the lung compared with the liver; with the most virulent strain the most extensive and diffuse accumulation of these new cells corresponds with the greater rise in the numbers of bovine bacilli after the lag of the 1st week. With the maturation of the epithelioid cells and the formation of tubercles the bacilli have already been greatly reduced numerically and the speed of this process diminishes with the virulence of the three strains used. The faster the development of tubercle the faster the destruction of the bacillus and the earlier the resorption of the tubercle. Tubercle bacilli never accumulate in such large numbers in the mononuclears of the liver as they do in the lung. Though at first the tubercles in the liver may be more numerous than those in the lung they never attain the same size. The formation of new mononuclears by mitosis is restricted and Langhans' giant cells appear very early (1st and 2nd weeks). In the lung, giant cells are not found until much later with the BCG and the human bacillus (4th week); they were not noted in the interstitial tubercles with the bovine type, but the extension of these tubercles was accompanied by an unabated mitosis of mononuclears until the death of the animal. The liver tubercles are resorbed early even with the bovine infection. Associated with these histological differences are the slow initial growth and the early and complete destruction of the tubercle bacilli even of bovine type in the liver, and the more rapid initial growth in the lung, with the later destruction of the BCG and the human bacillus and the unabated growth of the bovine bacillus. Similar differences were observed between the splenic pulp and corpuscle. In the former the accumulation of acid-fast particles was much greater and the tubercles developed earlier. Mitosis of mononuclears was less frequent and giant cells appeared earlier. Tubercle bacilli, always intracellular, disappeared from the tubercles in the pulp sooner than from those in the corpuscle, and the tubercles themselves first disappeared from the pulp. Consequently with the persistence of bacilli mitosis continued in the tubercles of the corpuscle and these attained a much larger size. Moreover individual resistance is linked with the ability to form mature tubercles early. In two animals simultaneously infected with the same strain and killed at the same time, the destruction or retardation of the bacillus is greater in that rabbit in which maturation of the tubercle and of epithelioid cells has proceeded further (Figs. 15 and 16). These observations indicate that the mononuclears of different organs or even of the same organ, as in the different parts of the spleen, have a different capacity to destroy the tubercle bacillus, and that the transformation of the mononuclear into the mature epithelioid cell follows its destruction of the tubercle bacilli. In the lung the more virulent types of bacillus are destroyed within the epithelioid cells of interstitial tubercles but persist in foci of tuberculous pneumonia. In this organ in rabbits infected with the human strain and to a lesser degree in rabbits infected with the bovine strain, the parasite largely disappears from the epithelioid cells of interstitial tubercles. But with both strains tubercle bacilli in large numbers may accumulate within epithelioid cells lying free in the alveoli. With the human type they are numerous within the cells and free in caseous material in the localized foci of caseous pneumonia. With the bovine infection, this caseous pneumonia is more often widespread and in the areas of caseous pneumonia the greater part of the vast accumulation of bovine bacilli in the lungs is found; as many as 200,000 colonies have been isolated from 10 mg. of tissue (Fig. 11). Flooding of the respiratory passages by the caseation of tuberculous lesions into the bronchi plays an important rôle in dissemination of tubercle bacilli through the lung. The process on the contrary is predominantly interstitial when the bovine bacillus is held in check (Fig. 12). Thus there is apparently some factor acting in the alveoli that favors the growth of the parasite. The accumulation of tubercle bacilli is seen especially in the peripheral epithelioid cells in immediate contact with the alveolar space. In the same lung the bacilli are much fewer in the interstitial tubercles. The accumulation in human tuberculosis of large numbers of tubercle bacilli in the tissues lining cavities is well known. Novy and Soule (20) have shown that within certain limits the growth of the bacillus in vitro is proportional to the oxygen tension of its environment. Corper, Lurie and Uyei (21) have confirmed these observations and have noted further that a difference in the gaseous environment of the bacilli equal to the difference between the conditions existing in the alveolar air and the venous blood is sufficient to cause a considerable increase in the growth of the microorganism in vitro. Loebel, Shorr and Richardson (22) by the use of Warburg's manometer have found that the oxygen consumption of tuberculous tissue is such that a tubercle 0.5 mm. thick would completely exhaust the oxygen of the air before it reached the center. These observations suggest that a factor responsible for the greater multiplication of the bacillus in the cells of the alveoli may be the greater oxygen tension of the alveolar air. In the liver, spleen and bone marrow even with the bovine infection many instances were found of the effective destruction of the parasite synchronously with the maturation of epithelioid cells and the formation of tubercle. On the other hand, in the spleen and bone marrow of some rabbits, living bacilli persisted within the epithelioid cells of isolated tubercles even 2 months after infection, a condition never found with the human type or BCG infection. Thus the epithelioid cell is the means of defense for the rabbit against the bovine type bacillus, and as such it is usually adequate in the liver, spleen and bone marrow though ineffective in the lung and kidney. In the latter, descending infection, and the occasional colony-like multiplication of bacilli in unorganized material, tubular casts, determine the long persistence of large numbers of bacilli in this organ. In differentiating the mononuclear phagocyte of the connective tissues into the monocyte and clasmatocyte Sabin and her coworkers (23) have maintained that the clasmatocyte can efficiently destroy the tubercle bacillus but that the monocyte and its derivatives, the epithelioid and Langhans' giant cells, cannot. With the progress of the disease they have noted that the monocytes accumulate in great numbers in the foci of infection and overflow into general circulation (4). White (24) and Sabin and her coworkers have concluded that tuberculosis is specifically a disease of the monocyte, and that this cell and its derivatives act as incubators for the tubercle bacillus. Doan and Sabin (25) have therefore sought, with indecisive results, to protect the body against tuberculosis by an antimonocytic serum. However it has been shown here that although an intense multiplication of mononuclears is associated with the growth of the tubercle bacillus, their transformation into mature epithelioid cells is constantly associated with its destruction, and the rapidity of the destruction varies with the rapidity of the maturation of tubercle. Even in the bovine infection the epithelioid cells destroy the bacilli in the liver, spleen and bone marrow as a rule, and even in the lung, keep them in check in the interstitial tubercles. The appearance of giant cells is associated with cessation or diminution of mononuclear regeneration by mitosis, and is coincident with cessation of multiplication or marked reduction in the number of living bacilli. They therefore appear earlier and in larger numbers in these organs or parts of organs that first destroy the bacillus (Figs. 16 and 17). They were not observed even 2 months after the bovine infection in the interstitial tubercles in the lung. Their absence and the continued mitosis of mononuclears, which accounts for the massive pneumonic and interstitial consolidation of the lung with this infection, were associated with the failure of the lung to destroy effectively the bovine parasite. The formation of giant cells in the pneumonic foci in the bovine infection would seem to be an exception to this rule. The Langhans giant cells have often been considered an indication of the chronicity of the pathological process. It would appear that they are formed from existing epithelioid cells when the multiplication of the bacillus has ceased and the stimulus for the formation of new cells has decreased or stopped. Giant cells were most conspicuous in the liver and splenic pulp where, with the BCG infection, no caseation ever developed, and in the liver before caseation was seen anywhere in the body. In the human and bovine infections, giant cells formed in the liver before caseation appeared. Hence caseation is not a necessary requirement for giant cell formation, as maintained by Medlar (26), though these cells frequently form about caseous material. Lymphocytes and granulation tissue do not cause the destruction of tubercle bacilli, these being destroyed in their absence. They usually appear about tubercles due to all strains and in all organs, after the greater part of the microorganisms have been destroyed (Fig. 18). The bacilli are not destroyed in the lung with bovine infection where the tubercles are usually little permeated by lymphocytes and granulation tissue. There is however, no constant relation between granulation tissue and destruction of tubercle bacilli, for in the lung after the human infection and even in other organs after the bovine infection isolated tubercles may be surrounded and penetrated by lymphocytes and granulation tissue at a time when considerable numbers of living bacilli are still histologically demonstrable within the epithelioid cells. Caseation is usually not caused by the local accumulation of tubercle bacilli. At first, when the BCG (after 1 week) and the human microorganism (after 2 weeks) are present in the cells in very large numbers as demonstrated both histologically and by culture (Figs. 4 and 13) there is no necrosis of these cells. An exception to this rule found in the lung with the bovine infection is considered below. Later, after the bacilli have been destroyed to a great extent and even though the number of bacilli is small, caseation appears (Fig. 14). After this preliminary destruction the extent of caseation apparently varies with the number of residual bacilli. With the least virulent microorganism, the BCG, few bacilli remained in the liver in the 4th week and no caseation was seen. In the tubercles of the splenic corpuscle at the same time bacilli were somewhat more numerous and there was scant caseation. On the other hand with the human bacillus after 4 weeks more bacilli survived and caseation was more extensive in both organs; with the bovine microorganism tubercle bacilli were much more numerous and caseation was far advanced. In the lung, however, caseation appeared with the first considerable accumulation of the bovine bacilli present 2 weeks after inoculation. That the bovine bacillus is primarily more injurious to the lung of rabbits than the BCG or the human bacillus is suggested by the greater intensity of the initial inflammation and by the more conspicuous accumulation of cells in the alveoli evident from the very beginning of infection. Maximow (27) showed that bovine bacilli even in small numbers cause the death of cells in tissue cultures of rabbit lymph nodes whereas the BCG or the human bacillus may accumulate within the cells in tremendous numbers without injuring them. Nevertheless in the liver, spleen and bone marrow of the living animal, caseation does not appear at the time when bovine bacilli are most abundant, but after they have been greatly reduced in numbers. Large numbers of the less virulent types of tubercle bacilli accumulated in different organs a short time after infection do not cause caseation, and with the bovine infection caseation under the same conditions occurs only in the lung. Later when the animal is sensitized caseation occurs in various organs in the presence of the small numbers of tubercle bacilli that remain in the tissues after most of them have been destroyed, and the extent of this caseation varies with the numbers of residual bacilli. These observations suggest that a large number of bacilli fail to cause necrosis soon after infection whereas a few bacilli produce caseation in the animal that is sensitized. Many investigators have held that caseation is due to sensitization. Krause (28), Huebschman (29) and Pagel (30) think that caseation is caused by the action of tuberculin-like substances on the sensitized tissues of the allergic animal. Rich and McCordock (31) view the process in essentially the same light. Recently Schleussing (32) has suggested that caseation is a coagulation necrosis in Weigert's sense of an allergically inflamed tissue, and is similar to the necrosis of the Arthus phenomenon.

Before proceeding to a discussion of the experiments upon cold-blooded animals, it is necessary to review briefly some of the work recently done with the bacillus of leprosy. The appearance of the bacillus in man and its behavior under artificial cultivation, and in the tissues of lower animals, should be considered in order that comparisons may be drawn. In their studies with the organism under cultivation, Duval and Gurd pointed out that the long, slender, and beaded appearance of the leprosy bacillus described by Hansen, in 1872, is lost when removed for several generations from the parent stem, and under artificial cultivation the organism becomes unbeaded, short, and coccoid. Duval also noted that these changes in morphology were always followed by rapid multiplication of the organism. Duval argues, a priori, that the bacillus is not in a favorable environment in the human tissues. If these deductions are correct, the morphology of the leprosy bacillus should vary according to the resistance offered by the tissues of different animals. The resistance of the human host to the leprosy bacillus becomes more evident in the light of the clinical aspect of the disease. The long period of incubation, the duration of the disease, and the disappearance of the bacilli preceding the healing of the infected foci show that the resistance offered to the bacillus by the human tissues is not to be overestimated. This opinion is confirmed when the behavior of the leprosy bacillus under cultivation and in the tissues of various mammals is compared. When cats, rabbits, bats, guinea pigs, and rats are inoculated either below the skin or into the peritoneal cavity with large quantities of Bacillus leprae, a slight local reaction follows within twenty-four to forty-eight hours, but no definite lesions are produced and the bacilli soon disappear. The resistance of some animals to Bacillus leprae is well illustrated by two cats which were inoculated subcutaneously and intraperitoneally with a heavy suspension of Bacillus leprae. These animals were killed and examined three days later, but the bacilli were not demonstrable from the regions about the sites of inoculation. Pigeons are likewise refractory. It is impossible to cause a local reaction in these birds, and the injected bacilli disappear rapidly. Hence, probably no multiplication takes place in them. Goats, young pigs, and white and dancing mice are in a degree susceptible to injections, and though undoubted lesions are produced, and multiplication of the bacilli occurs, the lesions and bacilli disappear after a limited time. Acid-fast bacilli which are recovered from the lesions are long, slim, and beaded, though the organisms used in the inoculations were short, unbeaded, and coccoid. Monkeys inoculated with cultures of the short unbeaded forms react promptly. The lesions resulting, though confined in most instances to the site of inoculation, occasionally appear at distant points. The number of bacilli present in the nodules and their arrangement within typical lepra cells show that multiplication has taken place. The organism has, however, changed from the short coccoid form to the long, slender, beaded form. Though the lesions induced and the bacilli present are in every way similar to those found in man, their tendency to disappear gradually after a quiescent stage clearly denotes that the tissues of the monkey, although less refractory than the tissues of the animals previously mentioned, still offer resistance to invasion. While mammals react but poorly to inoculations of the leprosy bacillus, this reaction manifests itself in various ways in different species. For example, while multiplication of the organism with the production of lesions occurs in some species, in others that are more refractory, the injected bacilli assume the involuted or beaded forms and do not multiply or produce lesions; in others, still more resistant to the action of the leprosy bacillus, the organisms quickly undergo granular metamorphosis and disappear. Furthermore, in some species the lesions are, in most instances, limited to the site of inoculation, and though presenting all the characteristics of the lesion in man, the nodules and the bacilli disappear after a variable time. This behavior of the leprosy bacillus can be accounted for only by the degree of resistance offered by the tissues of the individual host. Since the morphology of the organism invariably changes from the short coccoid to the large beaded form when placed in insusceptible animals, and conversely, from the long beaded forms to the short coccoid forms when placed in susceptible animals, the deduction can be drawn that the organism varies in morphology and rapidity of growth according to the susceptibility of the host. Examples of similar behavior of Bacillus leprae in the human subject are known to all investigators of leprosy. Ulcers and nodular areas often heal, and the bacilli disappear with little or no treatment. It is true that while older lesions are healing, new ones are constantly appearing, yet the duration of the disease and its undoubted tendency towards healing shows that conditions in the human subject are variable, and suggests that the organism has its natural habitat in some other host. The experiments presented here serve to show that the bacillus of leprosy meets but little or no resistance in the tissues of cold-blooded animals, multiplies in their tissues, and may be harbored by them without apparent discomfort or external evidence of the disease. That no appreciable resistance is offered to the multiplication of the leprosy bacillus by many species of cold-blooded animals is shown by the fact that aside from the trauma produced by the inoculation and the slight initial reaction of the tissues, the organism continues to grow profusely, and to invade the tissues without further reaction. Quite the opposite condition occurs in mammals: in some of these the leprosy bacillus degenerates into a granular mass shortly after inoculation; in others that are less refractory, typical lesions appear, but they seldom extend from the point of inoculation; and while the bacilli multiply slowly, they do not infiltrate the tissues, but disappear after a short time, the lesions healing. That multiplication of Bacillus leprae occurs in the tissues of cold-blooded animals is shown by the fact that while animals examined a few days after inoculation show but a few scattered organisms, those killed at longer intervals show a proportional increase in the number of bacilli. Furthermore, the few bacilli found at the early-period are extracellular and scattered, while after longer periods they tend to be massed and enclosed in large lepra cells. The supposition that these lepra cells are phagocytes has naturally arisen. Duval holds that they are not phagocytes in the true sense of the term, that the bacilli penetrate the cells rather than that the cells engulf them, after which, finding conditions for growth favorable, they multiply without causing serious injury to the cell. The size of the cell depends upon the size of the colony within. The experimental work bears out this view since the decrease in number of the organisms observed in animals killed shortly after inoculation depends not upon phagocytic action nor upon cells which appear later when active lesions are established. In early lesions, the lepra cells are smaller, barely measuring twenty to thirty microns in diameter, and contain but few bacilli; whereas in older ones, they attain a diameter of 100 microns or even more, and contain enormous numbers of bacilli. Were this increase in size due to phagocytic action, some cells would be found in which the limit of their capacity had been reached; and they would either contain a mass of dead and disintegrated bacteria or would themselves show evidence of disintegration. On the contrary, the bacilli, though they occupy most of the cell, show no signs of disintegration, and the nucleus and the cytoplasm of the cell retain normal staining properties. That the invasion and multiplication of the bacilli cause an irritation is evident by the amitotic divisions of the nucleus which occur in the larger cells. The absence of external evidence of invasion by Bacillus leprae in cold-blooded animals, and the apparent lack of discomfort caused by the presence of the organism within their tissues, are points which should be remembered in considering the sources from which leprosy may be transmitted. In not a single instance in the numerous experiments presented here would it have been possible, from any external sign, to suspect that the animals were harboring multitudes of leprosy bacilli. While the evidence in support of the opinion that leprosy may be transmitted from man to man appears sufficiently strong to warrant this belief, the number of cases in which infection can be actually traced to this source is small. Since leprosy is known to be prevalent where fish and sea-food are plentiful, and since the experiments here recorded prove that fish can be infected by being fed cultures of Bacillus leprae, or nodules from human lepers, or bits of fish previously infected with the leprosy organism, account should be taken of the possibility that leprosy, in certain localities, may arise from this source of infection. The question as to how and from what source leprosy bacilli enter the human body may be still regarded as an open one. Isolated examples of direct infection of healthy human beings from lepers have been reported by Arning and Nonne, by Manson, and others. The notion that the agency of infection is already infected human beings, that is lepers, is at the foundation of the modern practice of the isolation and segregation of lepers, which would seem to have brought about a definite decrease in the prevalence of the disease. It is an acknowledged fact, however, that the lepers confined in institutions practically never cause infection of nurses, etc. Some other factor than the human agency may therefore be considered as affecting this issue. It is well known that Jonathan Hutchinson has brought forward the idea that fish are the source of the infection, basing the view on the high prevalence of the disease along the coast countries of Norway and Sweden, and in the Pacific Islands, and in the countries bordering the Mediterranean and Black Seas, in all of which fish furnish the chief food material. No convincing proof was ever adduced in support of this contention. But now that it has been shown that the leprosy bacillus survives and multiplies in cold-blooded animals, at least at room temperature in a warm climate, and since methods have been devised for cultivating and identifying the leprosy bacillus, the question has been opened up to accurate investigation. Duval has shown that the leprosy bacillus in cultures grows better at room temperature than at 37 degrees C., so that growth in cold-blooded animals kept at room temperature is perhaps in some way connected with this phenomenon. What must now be ascertained, in order to test the Hutchinsonian theory more accurately is whether such growth takes place at a temperature corresponding with the average mean temperature of such a body of water as the North Sea and that of the fiords of Norway. Since cold-blooded animals possess the same temperature as their surroundings, they would be suitable media for the cultivation of leprosy bacilli at those temperatures. For the waters of the Mediterranean Sea and the tropical Pacific Ocean, this consideration would count less. But the theory will stand or fall according as it can account for the whole, and not only for a part, of the phenomena to be explained. As the length and shape of the bacilli and the number of chromatin masses are constant for a given species of cold- or warm-blooded animals, which features are governed by the resistance of the individual species, the following conclusions seem justified: that the morphology and rapid multiplication of the leprosy bacillus in cultures and in some species of cold-blooded animals indicate that Bacillus leprae under natural conditions is short, coccoid, and un-beaded, and that the long, slender, beaded variety which occurs in the mammalian species is atypical and the product of an unfavorable environment.

From the foregoing description of the histological changes in the leptomeninx it is quite evident that we are dealing with a chronic, stationary, healing form of tuberculous inflammation. This statement is substantiated, in the first place, by the clinical history. The only reasonable interpretation of the symptoms would establish the duration of the process as four months. The imaginable contingency that there existed first a meningeal syphilitic lesion that was dispersed by the iodide of potassium only to be followed by a tuberculous infection is so remote and unlikely that it need not be discussed. At all events the tuberculous leptomeningitis, which presented a typical distribution, began insidiously, existed at times in a latent condition, and pursued a very anomalous course, marked by a relative mildness of all the symptoms, and thus it came about that when an apparent or real improvement followed the administration of iodide of potassium able observers were induced to make an erroneous diagnosis. Death occurred as a result of an intercurrent infection. The long duration of the process is also shown, anatomically, by the thick layer of firm, translucent and gelatinous material that matted together the structures at the base, and also by the evident adhesions between the pia and the brain. The histological examination furnishes proof positive of the correctness of the conclusion in regard to the peculiar character of this process because it shows:(1) That the tuberculous proliferation is uniform in development and has reached nearly the same stage of evolution throughout the entire extent of the leptomeninx involved; it is not a process that has advanced by exacerbations and irregular extensions; the lesions are, generally speaking, of nearly the same age everywhere and must have begun at about the same time.(2) That only a very limited degree of caseous degeneration is present, pointing to an early arrest of the activity of the tubercle bacillus or to a very decided diminution or attenuation of its virulence.(3) That the subendothelial intimal proliferations of epithelioid cells, so generally found in acute tuberculous leptomeningitis,* have in this case become more or less completely changed into distinct fibrous tissue in which but very slight, if any, direct evidence of its tuberculous origin can be found. It is only by recognizing that the chronic endarteritis is most marked in correspondence with the most advanced adventitial tuberculous changes, and by finding an imperfect, much altered giant cell in one district of intimal thickening, that we were able to establish the direct kinship of the endovascular changes with those of the pia in general.(4) That acute inflammatory changes, in the form of emigration of polymorphonuclear leucocytes and of fibrinous exudation, are entirely absent in all parts of the district involved. The presence of a turbid serous fluid is of course not at all inconsistent with the view that the anatomical changes are of long duration.(5) That the granulation tissue present is, in general, undergoing fibrillation and contains a rich supply of enabryonal capillary vessels as well as of larger blood-vessels of evidently new formation. The absence of any considerable extent of polymorphonuclear leucocytic infiltration in this tissue has already been referred to. The cells in the granulation tissue correspond to the cells of embryonal or formative connective tissue. Vacuolation is rarely present.(6) That the unusually large number of giant cells present are remarkably free from evidences of necrosis and degeneration of the character ordinarily observed in tuberculous proliferations, that they do not contain in demonstrable form tubercle bacilli, and that the majority of the giant cells seem to be separating into individual cells and smaller masses often with, but sometimes also without, evidences of nuclear disintegration. The possibility that these phenomena may signify fusion instead of the sundering of cells will be discussed below. For these reasons there can be no doubt that the general claim that we are dealing with an instance of chronic, healing tuberculous meningitis must be regarded as established beyond dispute. The growth of tubercle bacilli in the glycerine-agar tubes, inoculated with the fluid from the pial meshes, and the demonstration of tubercle bacilli, though in very small numbers, between the cells of the embryonal tissue, furnish the positive evidence that we are actually dealing with a tuberculous process due to living and not to dead bacilli. The degree of virulence of the cultures of tubercle bacilli was, unfortunately perhaps, not studied. The presence of living tubercle bacilli in a tissue free from active and acute changes characteristic of tuberculosis demonstrates that, whatever the actual degree of virulence of the bacilli may have been, the tissue in which they were found was at this time relatively immune from their action. The manner in which this immunity was produced, and in which the process of healing was initiated, need not be discussed at this time any further than to again direct attention to the fact that the bacilli lost their virulency as regards the cells in this leptomeninx before these cells underwent any marked degree of degeneration. The cells of the tuberculous proliferations survived the further action of the bacilli whose original effect it was to initiate cell accumulation or proliferation; the cells also retained sufficient vitality to develop, in some instances at any rate, into formative cells according as their origin would dictate, e. g. into fibroblasts. That fibroblasts are formed only by embryonal connective tissue cells, and not by wandering cells, such as the large mononuclear leucocytes, we are well aware, is possibly still a disputable assumption, and we do not consider it pertinent to discuss the question any further in connection with this study, but would only emphasize the point that some of the cells of tuberculous proliferations may, under favorable circumstances, become formative cells, and, furthermore, that the amount of formative tissue produced may be far in excess of what is actually needed for purposes of repair only. Surely the appearances here noted indicate that the bacillus of tuberculosis has the power to stimulate fixed cells to multiply, unless one assumes that all, or almost all, the formative cells here seen are derived from wandering cells attracted by the presence of the bacillus and its products. As to the ultimate fate of the formative and other cells in this healing tuberculous tissue no final statements can be made. It must be remembered that it is only one stage in the process of healing that is dealt with. The well marked evidences of fibrillation, the quite extensive formation of new vessels, the absence of evidences of degenerative changes in the uninuclear cells, all point to the production of new fibrous tissue as sure to occur, but it seems quite probable that occasional epithelioid cells may undergo or have undergone dropsical or other forms of degeneration, although it is certainly apparent that so far as the small cells are concerned the involution of the tuberculous tissue is not occurring through disintegration. Perhaps the most interesting feature in this case is the opportunity it affords to study the changes in the giant cells of healing, non-degenerated tuberculous tissue. In the first place, the large number of giant cells is quite remarkable. The general characters of the tissue in which they are found recall the fact that giant cells are regarded as quite constant elements in chronic mild tuberculosis; often the giant cells are the only cells that contain bacilli (Koch). In this instance the giant cells do not contain bacilli that are demonstrable by the usual methods; neither do they contain bodies that can be definitely interpreted as degenerate forms of bacilli such as those found by Metchnikoff, Stchastny, Weicker, and others, in the giant cells of Spermophilus guttatus, in avian and in human tuberculosis. Metchnikoff states, however, that he knows of the occurrence of such degenerate forms only in the Spermophilus guttatus under the circumstances mentioned, and in the rabbit and guinea-pig in mammalian tuberculosis, but not in man; consequently, the manner in which the giant cells rid themselves of the bacilli undoubtedly present in their interior at some time during their existence, must as yet remain without any explanation. In the description of the histological changes the various appearances presented by the giant cells are described somewhat minutely. The essential observations made concern, in my opinion, the further fate of giant cells which are still found to persist in healing nondegenerated tuberculous tissue. It was, I believe, quite conclusively shown that the consecutive changes appear to consist in the breaking up of the nuclei, the removal of the detritus by phagocytes, and the formation of a few apparently viable uninuclear cells in the case of more degenerated, exhausted giant cells, while other, and, as it would seem, better preserved or younger giant cells, separate into a number of individual, uninuclear cells with but little or no nuclear disintegration. Objection might be raised to this interpretation of the appearances in the giant cells. While no one could very well dispute the view that part of the giant cells are undergoing retrogressive and absorptive changes with the production of some viable cells, a question might well be raised concerning the nature of the process taking place in those giant cells that have been spoken of as splitting up or dividing into uninuclear cells and smaller multinucleated masses without much evidence of nuclear disintegration. It might be claimed that the process is one of fusion of many cells to form giant cells, and not one of division of fully formed giant cells into small cells. But a broad view of the processes described speaks against fusion. In the first place we are not dealing with a stage of tuberculous proliferation (Baumgarten), or cell accumulation (Metchnikoff), in which one would look for the production of giant cells, no matter which view concerning the histogenesis of tubercle be assumed as the correct one, because it has been demonstrated that, from whichever point of view the lesions are examined, the same positive conclusion that they are in the process of healing is reached; there is, therefore, no occasion for the formation of new giant cells in such wide-spread degree throughout the district involved. It might he claimed that the cells became arrested and, as it were, fixed in the act of fusion which was taking place in the early stage of the meningitis, but it would be difficult to understand the nature of the stimulus that could hold the cells together in such a peculiar manner for such a long time. It must be remembered that bacilli or bacillary detritus could not be found among the incomplete or in the complete giant cells. In the second place the difference between the cells that are undergoing disintegration and those regarded as dividing is essentially, to a certain extent at any rate, one of degree, because in the first instance there is not much, if any, doubt but that viable smaller cells are also formed, and in the second instance some, though often very slight, evidence of nuclear fragmentation is nearly always present; it would also be correct to infer that in advanced subdivision of a giant cell much, and perhaps all, of the nuclear detritus produced might have been removed up to the last trace; finally, the two extremes of these changes in the giant cells are connected by transition stages passing by gradation from the one to the other. Hence it is justifiable to conclude, for the time being, that in healing non-degenerated tuberculous tissue, the multinucleated giant cells may in part disintegrate and undergo absorption, in part form viable small cells; that both these changes may, and usually do, affect the same cell, but that in one class of cells-presumably the older or the more exhausted-the retrogressive process is predominant, while in a second class of cells-presumably the young and vigorous-the progressive changes are the more marked. In this connection it may be pointed out that while there cannot very well be any question but that we are dealing only with dividing and not coalescing cells, yet if this conclusion should be disputed and found incorrect, then the only remaining alternative would be to infer that this tissue furnished a unique and striking example of the formation of plasmodial masses by fusion in human tuberculosis, a conclusion to which many pathologists would refuse to subscribe, if for no other reason than because it is not in accordance with the almost universally accepted teachings of Baumgarten and Weigert in regard to the mode of formation of the giant cells in tuberculosis. Believing as I do that the giant cells under consideration are in the act of division and not at all of fusion, there remain to be discussed some of the histological and other features presented by the dividing cells. Many of the giant cells, perhaps the majority, contain larger and smaller vacuoles in the protoplasm. The exact significance of this vacuolation is not always clear. When the vacuolation accompanies an evident solution of the nucleus (karyolysis), there cannot be any doubt but that we are in the presence of a distinctly retrogressive process. Vacuoles are also most numerous in the giant cells that present other evidences of degeneration, such as coarseness of the granules in the protoplasm and extensive nuclear disintegration, but they occur as well around nuclei that stain deeply, around cells that seem to be separating from the giant cell, and even about nuclei that present mitoses. The formation of vacuoles seems to be responsible, to a certain extent at any rate, for the diminution in the volume of disintegrating and dividing giant cells, as shown by the clear spaces that form about them; these spaces are too large and occur too uniformly to be attributed solely to artificial shrinking produced by the hardening in alcohol. Further undoubted evidence of retrogression in certain giant cells is the occurrence of nuclear disintegration, or karyorhexis, which sets free larger and smaller chromatin masses that are recognized in the giant cell as well as in the interior of the phagocytes usually found around such cells. Almost all the polymorphonuclear leucocytes found in this tissue are met with around giant cells with broken-up nuclei. In many nuclei of disintegrating giant cells can be noted appearances that correspond well to certain stages in the complicated karyorhexis observed in anaemic necrosis by Schmaus and Albrecht; some of the nuclei with budding processes correspond particularly well with those in certain of their drawings; the interior of giant cells of tuberculous tissue may, it would seem, present conditions favorable to the development of this series of postnecrotic nuclear change. Vacuolation, karyolysis and karyorhexis are the essential steps that lead to destruction of the whole or parts of some of the giant cells; associated with these processes there is usually observed a splitting up of the body of the giant cell into irregular fragments with as well as without nuclei; and, as described, more or less phagocytosis of the resulting remnants of various kinds is seen. But evident degenerative and necrotic processes in a giant cell may be associated with progressive changes. While some nuclei undergo vacuolation or break up, others seem to become richer in chromatin and to stain more deeply at the same time that they seem to acquire cell bodies quite distinct from the protoplasm of the giant cells: this hyperchromatosis does not, therefore, seem to be a stage in karyorhexis. A very few but undoubted karyokinetic figures were found, together with evidences of division of the cell body formed in the giant cell protoplasm. Precisely similar changes are described by Klebs in healing pulmonary tuberculosis of the guinea-pig; the nuclei of the giant cells became rich in chromatin and karyokinetic figures occurred. Krückmann among others has found occasional mitoses in giant cells around foreign bodies, as well as elsewhere, but it would seem that such mitoses have always been interpreted as indicating the probable mode of formation of the giant cells rather than of their involution. The question of mitosis in existing multinucleated cells has recently been studied by Krompecher, who concludes that the individual nuclei of such cells may undoubtedly divide by mitosis, either simultaneously or at separate times. Division by amitosis can also occur, but mitosis is the only progressive form of division, amitosis being a retrogressive, disintegrating process that must be looked upon as an evidence of degeneration of the nucleus. Ziegler states that in division of giant cells whose nuclei have multiplied by mitosis it may happen that the separating cell remains enclosed in the protoplasm of the mother cell. A singular phase in the involution of the giant cells in this pia is to be found in the existence of progressive changes side by side with nuclear necrosis and with degeneration; this finding indicates that giant cells may contain many independent elements which, though apparently fused into one large cell, may preserve their individuality so that while some nuclei die, others proliferate and perhaps feed on the remnants of their dead brethren and form new, viable small cells. The nuclei in giant cells may be looked upon as representing independent centres, capable at times of existing even though the cell protoplasm is disintegrated. Many of the giant cells separate into individual cells, unaccompanied or unassociated with much evidence of necrosis. These cells may be regarded as the more vigorous forms. Here also are observed occasional mitoses-but on the whole extremely few-and very constantly an evident increase in the amount of chromatin in the nuclei of the new cells as compared with the amount ordinarily found in the nuclei of giant cells. These deductions concerning the persistence of the vitality of some of the nuclei, even in the presence of molecular and morphological changes in the cytoplasm and in other nuclei of the giant cell that lead to disintegration, are not entirely without the support of previous observations on cells, which, although made under different conditions, are nevertheless, it would seem, applicable to cells in general. Thus the brilliant investigations of Loeb upon the effects of various unfavorable surroundings, such as absence of oxygen or reduction of the amount of water, upon the cleavage of eggs of many kinds, show that the conditions which arrest development are qualitatively alike for nucleus and protoplasm, but quantitatively less for the protoplasm; when the irritability of the protoplasm is suspended the nucleus may segment without segmentation of the protoplasm, but upon re-establishment of favorable conditions the protoplasm may divide into about as many spheres as there are nuclei preformed-the nucleus persists, preserves the irritability of the cell and stimulates the protoplasm to segmentation. From the appearances of the giant cells here described it would seem, then, that some nuclei are able to maintain their vitality longer than others in the same cell, and under certain conditions to stimulate parts of the protoplasm to segment; in other cells all the nuclei have, as a rule, preserved their irritability. The groups of cells formed by the dividing of the giant cells can be traced by studying the process at the different stages in the different parts of the tissue. They assume an oval or spindle-shaped form, becoming more and more like the formative and endothelioid cells of young connective tissue, but their ultimate fate cannot be determined because it concerns essentially only one limited period in the involution of the tissue. It may be said with reasonable certainty, however, that the new cells do not form blood-vessels, but as regards their forming lymph-vessels nothing definite can be concluded. It would not be safe to draw any definite conclusions, from the appearances described, with regard to the origin and the mode of formation of the giant cells. The resulting small cells in general resemble very much endothelial and formative cells, but some of them are, at certain stages at any rate, not unlike large mononuclear leucocytes; their final fully developed or mature condition being unknown, no positive inference can be drawn as to their pre-giant-cell origin. The evidence points to the fact that the most probable origin of the giant cells, as indicated by their form and the apparent future career of their descendants, would be the fixed mesoblastic cells of the pia. In regard to the mode of formation of the giant cells it is quite clear that it must involve some process which is not incompatible with the viability of the small cells which may spring from the giant cells. Whether this would speak more in favor of formation by fusion than by karyokinesis of a single cell without division of the cell body cannot be well determined, and as long as authors are not agreed upon the question of the production of living, procreative cells by amitosis (direct segmentation, direct and indirect fragmentation) it would not be profitable to discuss the compatibility or incompatibility of the views of those investigators who trace the origin of giant cells to amitotic division, with the progressive changes that giant cells have been shown to be capable of. The fact that giant cells in tuberculous tissue, under certain conditions, undergo progressive changes and separate into small, living cells proves that they are not, as claimed by Baumgarten, Weigert and others, necrobiotic elements that are doomed to destruction from their very inception. On the other hand it lends more strength, if that were necessary, to the teleological view urged by Metchnikoff that they are living, defensive cells (whatever their origin may be), formed for the distinct purpose, like plasmodial masses in general, of isolating and removing foreign, harmful bodies, in this case the tubercle bacillus, and, having accomplished their object without being destroyed or exhausted, or the cause of their formation being removed or neutralized in some way, they, or their nuclei, may retain enough irritability to form a larger or smaller number of living, small, uninuclear cells.

The purpura accompanying the two foregoing cases of sarcoimatosis would seem to find its explanation in the coexistence of several factors, the main feature being an involvement of the vascular system by the sarcomatous elements. There existed in Case I a direct lesion of the vessel wall whereby the sarcoma cells invaded directly the various coats, and were found mainly between the intima and the adventitia, dissecting their way, as it were, along these tracts in the vessel walls. There was further an extensive involvement of the perivascular lymphatics, from which point, indeed, it would seem that the sarcoma cells had invaded the walls of the vessels themselves. In Case II, moreover, not only was there a definite invasion of the lymph spaces near the vessels, but, furthermore, there was undoubted evidence of the existence of emboli of sarcoma cells in the lumina of the blood vessels; and in the immediate vicinity of such conditions haemorrhages were invariably found. While some vessels, and indeed a great many, were quite free from such emboli, in others the lumina were completely occluded by spindle cells, so as to preclude the possibility that these were merely a collection of desquamated endothelial cells, such as is frequently found as the result of post-mortem changes. That such an embolic condition can exist is by no means an unreasonable supposition, and, while it is generally recognised that multiple sarcomata are usually made up of small round cells, in this case we have an undoubted example of sarcomatosis of the spindle-celled variety. There are numerous instances of this " embolic purpura," as it may be called, especially in French and German literature, the condition being associated with rheumatism, valvular lesions of the heart, and other diseases which induce directly or indirectly the formation of emboli. Krauss, Gimard, Leloir, and others have insisted with considerable emphasis on the embolic origin of many purpuric conditions, and in some instances they have verified their observations by histological examination. Leloir assumes that, in addition to the presence of the ordinary emboli and the changes in the vessel walls with desquamative endarteritis, the blood itself may be much altered chemically, and that in the cachectic conditions clots may be thrown down from the circulating blood and be carried onward to form capillary emboli, with resulting haemorrhagic infarctions. Krogerer, some ten years ago, in examining the skin removed from patients with symptomatic purpura, found definite thromboses in the smaller veins, and even in the arteries. According to his view, the alterations in the vessel walls gave rise to slowed circulation and tendency to thrombosis, bringing about a liability to haemorrhages. His plates bear out his theories regarding the thrombi, many of which show considerable organization. But a careful examination of the purpuric areas shows further that a mere invasion of the vascular system by sarcoma cells can not explain all the various blood effusions present. On examining the skin, for instance, in those areas where large irregular haemorrhages had occurred, there was but little evidence of vascular invasion, while the emboli, on the other hand, seemed to exist mainly in the localized smaller and more circumscribed patches. One must therefore conclude that in such instances a combination of factors will alone afford a rational explanation of the purpura, and that in the general condition of the patient we shall find another cause for the enormous effusions of blood. In both of our cases there were high fever, cachexia, and a rapid progressive asthenia, all being the results of a sarcomatosis, and implying also grave alterations in the composition of the blood. From this we may infer an altered condition of the vessel walls, and hence probably a combination of circumstances sufficient to explain the incidence of haemorrhage. The raised cutaneous nodules in our second case, some of which were haemorrhagic, can not be regarded as pure sarcomatous metastases, for on microscopic examination they merely revealed haemorrhage or necrosis, or both, and sometimes plugging of the vessels. There was nowhere in these nodules evidence of new growths. Such elevations, then, must have been produced rather by a temporary serous or cellular exudation coincident with or following upon the haemorrhage-a probability which is emphasized by the fact that during the last days of the patient's illness many of the nodules diminished in size. Whether the oedema and infiltration were secondary to the embolic process in the subcutaneous vessels or whether they were merely coincident with the haemorrhage would be difficult to decide. The ringlike spots, however, are of special interest, inasmuch as it has been shown that they have been present in more than one case of sarcoma. It is not impossible that such spots may be definitely related either to the embolic processes or to a direct invasion of the cutaneous vessels, though, so far as we know, there do not exist any experimental proofs to bear out such a theory. From what has been said, however, it is evident that the cutaneous vessels were plugged during the last few days of the illness, at a time when the walls of the smaller vessels and capillaries were already greatly enfeebled. The result of the embolic formation may therefore mean a decided deficiency in the supply of nutriment to the involved area, the collateral circulation naturally being poor under the circumstances. As soon, then, as the vessels had become plugged, the surrounding blood supply would be poured in to a limited extent, and, on meeting the enfeebled vessels, might possibly break through their thin walls, thus producing a zone of haemorrhage around the area deprived of its normal nutrition. In other words, the condition may be regarded as in many respects analogous to that presented in embolic infarcts in regions with end arteries, central necrosis with peripheral congestion and haemorrhage being induced, the latter being chiefly limited to the outer zone of the necrotic area. The cutaneous vessels under such circumstances may be regarded as end arteries in a functional sense, since the collateral circulation would be so diminished under the altered conditions that no complete nourishment could be afforded to the area supplied normally by the plugged vessel. Von Recklinghausen has directed especial attention to the occurrence of cutaneous haemorrhages following embolic or thrombotic occlusion of peripheral arteries. The possibility of some toxic condition as a factor in the production of the purpura in our cases may also be suggested; but while we would not exclude this possibility, we are unable to find any positive evidence in its favour. Focal necroses, which are often associated with toxic and infectious processes, were present only in direct association with the haemorrhages, and were not distributed in the liver, spleen, and kidneys in the manner characteristic of toxic infections. Nevertheless the absence of these necroses does not exclude the possibility of the existence of some form of toxaemia. Infection demonstrable by bacteriological examination was absent, and there is no reason to regard our cases as allied to the infectious purpuras. The thermic theory suggested by Fagge at all events finds no place in the production of the multiple tumours in our cases, inasmuch as in each instance extensive visceral growths had given rise to the metastases.

Abstract An adult male intact boxer was presented because of diffuse cutaneous nodules. Fine-needle aspirate revealed transmissible venereal tumour (TVT) cells. Neoplastic cells were also observed in the peripheral blood. Associated simultaneous diseases included leishmaniosis, demodicosis, papillomatosis and coccidiosis. Immunosuppression may have aggravated disease and triggered widespread metastases. The dog was hospitalized and administered oral amoxicillin/clavulanate, subcutaneous meglumine antimonite to treat leishmaniosis and oral chlortetracycline to treat coccidiosis. Intravenous injection of vincristine at weekly interval was used to treat TVT. A rapid regression of cutaneous nodules was noted; however, intractable diarrhoea developed, eventually leading to death after 18 days. This is the first report describing an unusual case of extragenital TVT associated with circulating neoplastic cells in an immunosuppressed dog presenting with multiple cutaneous nodules.

Abstract This report describes a case of cutaneous pythiosis in a 6-year-old female mixed breed dog, from the central west region of São Paulo State, Brazil. The cytological and histopathological analyses showed an intense inflammatory infiltrate with presence of numerous hyphal elements, suggesting infection due to Pythium insidiosum. The diagnosis was confirmed by nested-PCR, which was carried out with specific primers derived from the ribosomal DNA region. The pathogen occurs in Brazil and veterinarians should be aware of the importance of correctly diagnosing this disease and differentiating it from other fungal diseases.

Pythium insidiosum is a fungus that causes disease in both animals and humans. Human pythiosis is an emerging disease in the tropical, subtropical, and temperate regions of the world, occurring in localized and systemic or vascular forms. Most patients with arterial pythiosis have an underlying hemoglobinopathy, such as thalassemia. A case is presented of a thalassemic horse stable worker who developed an ulcerative cutaneous lesion on the lower left leg followed by progressive ascending involvement of the arteries of that extremity with a necrotizing arteritis with aneurysm formation. P. insidiosum was not isolated from the ulcer by culture or wet potassium hydroxide preparations but was diagnosed by histopathologic study of a biopsy. P. insidiosum infection was quickly confirmed by immunoblot method, aiding in preoperative decision making. Many systemic antibiotics or antimycotics have not been effective in the treatment of systemic pythiosis, and radical surgical removal of all infected tissue is the only method to ensure patient survival. An orally administered saturated solution of potassium iodide, amphotericin B-oral solution, and terbinafine has succeeded only in the cutaneous form but had no favorable effect on vascular pythiosis. It is likely that immunotherapy, successfully used in animal pythiosis, may be beneficial in the treatment of human vascular pythiosis.

During the 12 months of 2006, zygomycotic lymphadenitis was diagnosed in 194 of 198 feedlot steers (0.04% of cattle slaughtered during that period) in a California slaughterhouse as part of bovine tuberculosis surveillance. Mesenteric lymph nodes were involved in 190 cases. Affected lymph nodes were enlarged (2 to 42 cm in greatest dimension), firm, and mottled gray-white to yellow with multiple granular or caseocalcareous foci. Histologically, nodal architecture was effaced by necrosis, granulomatous inflammation and fibrosis. In approximately 20% of the cases, granulomas were mainly restricted to subcapsular sinuses and afferent lymphatic vessels, causing granulomatous lymphangitis. Non-septate, irregularly branching hyphae with non-parallel walls and bulbous enlargements were common in necrotic areas and within the cytoplasm of multinucleated giant cells. Fungal cultures were performed on 124 affected lymph nodes using 7 different media, but no zygomycetes were cultured. Fungal DNA was amplified from 20 lymph nodes. Amplicons from 16 nodes had nearly 100% homology with sequences for Rhizomucor pusillus; 4 amplicons had (>98%) homology with Absidia corymbifera sequences. Zygomycosis should be considered in the differential diagnosis for granulomatous lymphadenitis in feedlot steers.

Ectopic infection with Paragonimus miyazakii (P. miyazakii) was determined to be the cause of a subcutaneous inguinal mass in a 15-month-old, male, boar-hunting dog. Histologically, the mass comprised granulomatous panniculitis, intralesional adult trematodes and eggs, and lymphadenitis. Extrapulmonary paragonimosis in animals is rare; this appears to be the first report in a dog of ectopic P. miyazakii infection with mature trematodes and eggs involving the inguinofemoral lymphocenter and surrounding subcutis.