Hyphae, pseudohyphae, yeasts, spherules, spores, and more: A review on the morphology and pathology of fungal and oomycete infections in the skin of domestic animals.

Fungi are among the most common infectious agents affecting the skin of animals. The skin can serve as a port of entry for fungal infections, which can eventually become disseminated. In some regions of the world, oomycetes, such as Pythium and Lagenidium, are also responsible for a significant number of severe cutaneous infections. Histologic evaluation of fungal morphology, including size, shape, septation, branching, and budding characteristics, combined with the distribution of inflammatory infiltrates within different skin layers can potentially identify etiologic agents, guiding selection of antifungals and additional diagnostics. Fungal infections of the skin surface are typically caused by Malassezia and rarely Candida, with opportunistic fungi also capable of colonizing the skin surface, especially when the barrier is broken. Folliculocentric infections, caused by dermatophytes, result in mild to severe inflammation and can occasionally penetrate deep into the skin. A wide range of fungi, including agents of hyalohyphomycosis, phaeohyphomycosis, and dimorphic fungal infections, as well as oomycetes, result in nodular cutaneous and subcutaneous lesions. With the occasional exception of dimorphic fungi, fungal speciation often requires cultures performed on fresh tissues. However, molecular techniques such as pan-fungal polymerase chain reaction on paraffin blocks is becoming an increasingly useful tool to distinguish between cutaneous fungal pathogens. This review focuses on describing the clinical and histologic features of the most common fungal and oomycete infections affecting the skin of animals, divided according to distribution patterns of lesions and fungal or oomycete morphology.

Intratracheal Inoculation with Brucella melitensis in the Pregnant Guinea Pig Is an Improved Model for Reproductive Pathogenesis and Vaccine Studies.

Reproductive failure is the hallmark of brucellosis in animals. An uncommon but important complication in pregnant women who become acutely infected with Brucella melitensis is spontaneous pregnancy loss or vertical transmission to the fetus. Unfortunately, the mechanism behind reproductive failure is still obscure, partially due to the lack of a proper study model. Recently, it was demonstrated that intratracheal (IT) inoculation of nonpregnant guinea pigs would replicate features of clinical disease in humans. To determine if IT inoculation would induce reproductive disease, guinea pigs were infected at mid-gestation and monitored daily for fever and abortions. Fever developed between day 14 to 18 postinoculation, and by 3 weeks postinoculation, 75% of pregnant guinea pigs experienced stillbirths or spontaneous abortions mimicking natural disease. Next, to investigate the guinea pig as a model for evaluating vaccine efficacy during pregnancy, nonpregnant guinea pigs were vaccinated with S19, 16MΔvjbR + Quil-A, or 100 µl PBS + Quil-A (as control). Guinea pigs were bred and vaccinated guinea pigs were challenged at mid-gestation with B. melitensis IT inoculation and monitored for fever and abortions. Vaccination with both vaccines prevented fever and protected against abortion. Together, this study indicates that pregnant guinea pigs are an appropriate animal model to study reproductive disease and offer an improved model to evaluate the ability of vaccine candidates to protect against a serious manifestation of disease.

Anaplastic Large T-Cell Lymphoma in the Intestine of Dogs.

Anaplastic large T-cell lymphoma (ALTCL) is a rare subtype of non-Hodgkin T-cell lymphoma that occasionally occurs in the gastrointestinal tract of humans. Enteropathy-associated T-cell lymphoma (EATL) type 1 is the most common type of intestinal lymphoma in dogs, and ALTCL has not previously been reported in the intestinal tract of dogs. Thirteen dogs with intestinal masses diagnosed as intestinal lymphoma with anaplastic morphology were reviewed. Clinical data, including treatment protocols, were available for 11 cases. Immunohistochemistry for CD3, CD20, and CD30 was performed for all cases in addition to PCR for Antigen Receptor Rearrangements (PARR) for assessment of clonality. Eight (62%) of the cases presented with intestinal perforation, and all cases had 1 or more masses arising from the small intestine. Histologically, all cases were characterized by transmural infiltrates of large, CD3-positive and frequently CD30-positive cells. Neoplastic T cells had marked anisocytosis and anisokaryosis, prominent nucleoli, and occasionally indented to reniform nuclei. There was abundant necrosis and inflammation with occasional vascular invasion within neoplastic masses. All cases had a monoclonal T-cell receptor γ gene rearrangement. The median survival time was 5 days, with 1 dog surviving 2 years after the initial diagnosis. ALTCL can occur as an aggressive transmural lymphoma in the gastrointestinal tract of dogs and commonly causes intestinal perforation. ALTCL can be differentiated from EATL type 1 and might have implications for accurate prognostication and selection of therapeutic options in the future.

Rhodococcus equi Infections in Goats: Characterization of Virulence Plasmids.

Rhodococcus equi is an uncommon cause of systemic pyogranulomatous infections in goats with macroscopic similarities to caseous lymphadenitis caused by Corynebacterium pseudotuberculosis. Caprine cases have previously been reported to be caused by avirulent R. equi strains. Six cases of R. equi infection in goats yielding 8 R. equi isolates were identified from 2000 to 2017. Lesions varied from bronchopneumonia, vertebral and humeral osteomyelitis, and subcutaneous abscesses, to disseminated infection involving the lungs, lymph nodes, and multiple visceral organs. Isolates of R. equi from infected goats were analyzed by polymerase chain reaction for R. equi virulence-associated plasmid ( vap) genes. Seven of 8 isolates carried the VapN plasmid, originally characterized in bovine isolates, while 1 isolate lacked virulence plasmids and was classified as avirulent. The VapN plasmid has not been described in isolates cultured from goats.

Adenocarcinoma of the distal esophagus and esophageal-proventricular junction in a yellow-naped parrot.

A 57-y-old male yellow-naped parrot (Amazona auropalliata) was presented because of lethargy, inappetence, and weight loss. Hematology and serum biochemistry were unremarkable, and imaging revealed a mass in the distal esophagus at the coelomic inlet. The luminal diameter of the esophagus was reduced in this area, and passage of ingesta was limited. Following gavage feeding, the patient died and was submitted for autopsy. At postmortem examination, the noted mass effect was a thickening of the distal esophagus with adherent, coalescing, soft, pale-tan plaques on the mucosal surface. Additional gross findings included pale-tan, opaque feed material oozing from the dorsum of the lungs and covering the cranial air sacs. Histology of the esophagus, esophageal-proventricular junction, and proximal proventriculus revealed an unencapsulated, infiltrative, transmural neoplasm that extended from the mucosal surface deep into the muscularis, almost to the adventitia. The neoplasm was composed of cuboidal cells arranged in islands and tubules, consistent with an adenocarcinoma, a rarely reported entity in the esophagus of psittacine birds and to our knowledge not reported previously at the esophageal-proventricular junction.

Fabrication and In Vivo Assessment of Oxidatively Responsive PolyHIPE Scaffolds for Use in Diabetic Orthopedic Applications.

Achieving surgical success in orthopedic patients with metabolic disease remains a substantial challenge. Diabetic patients exhibit a unique tissue microenvironment consisting of high levels of reactive oxygen species (ROS), which promotes osteoclastic activity and leads to decreased bone healing. Alternative solutions, such as synthetic grafts, incorporating progenitor cells or growth factors, can be costly and have processing constraints. Previously, the potential for thiol-methacrylate networks to sequester ROS while possessing tunable mechanical properties and degradation rates has been demonstrated. In this study, the ability to fabricate thiol-methacrylate interconnected porous scaffolds using emulsion templating to create monoliths with an average porosity of 97.0% is reported. The average pore sizes of the scaffolds range from 27 to 656 µm. The scaffolds can sequester pathologic levels of ROS via hydrogen peroxide consumption and are not impacted by sterilization. Subcutaneous implantation shows no signs of acute toxicity. Finally, in a 6-week bilateral calvarial defect model in Zucker diabetic fatty rats, ROS scaffolds increase new bone volume by 66% over sham defects. Histologic analysis identifies woven bone infiltration throughout the scaffold and neovascularization. Overall, this study suggests that porous thiol-methacrylate scaffolds may improve healing for bone grafting applications where high levels of ROS hinder bone growth.

Case report: Intraneural perineurioma in dogs: a case series and brief literature review.

Intraneural perineurioma is an exceptionally rare neoplasm in animals. This case study comprises a series of three cases and a brief literature review focusing on canine intraneural perineurioma. The pathological and immunohistochemical findings are documented, revealing that canine intraneural perineurioma frequently affects adult dogs aged between 3 and 10 years old, with a male predominance. Clinical signs associated with intraneural perineurioma in dogs include spinal pain, lameness, and paresis, resulting from the involvement of spinal nerve roots of the pelvic limbs, brachial plexus, or distal part of the median nerve. Most neoplasms had characteristic pseudo-onion bulb patterns on histopathology. Neoplastic perineurial cells, in most cases, expressed laminin and claudin-1, and NF200 consistently highlighted the central axon. While the immunohistochemical (IHC) profile of intraneural perineurioma in veterinary medicine remains incompletely characterized, the available IHC data from all reported cases suggest that a combination of laminin and claudin-1 immunomarkers, along with distinctive histological features, can assist in establishing a definitive diagnosis of intraneural perineurioma.

Intratracheal inoculation results in Brucella-associated reproductive disease in male mouse and guinea pig models of infection.

Brucella species are considered a significant cause of reproductive pathology in male and female animals. Importantly, Brucella melitensis can induce reproductive disease in humans. Reproductive pathogenesis and evaluation of newly developed countermeasures against brucellosis studies have traditionally utilized female animal models. However, any potential, new intervention for use in humans would need to be evaluated in both sexes. Therefore, animal models for male reproductive brucellosis are desperately needed to understand disease progression. Accordingly, we evaluated guinea pigs and mice using B. melitensis 16 M in an intratracheal model of inoculation at different stages of infection (peracute, acute, and chronic) with an emphasis on determining the effect to the male reproductive organs. Aerosol inoculation resulted in colonization of the reproductive organs (testicle, epididymis, prostate) in both species. Infection peaked during the peracute (1-week post-infection [p.i.]) and acute (2-weeks p.i.) stages of infection in the mouse in spleen, epididymis, prostate, and testicle, but colonization was poorly associated with inflammation. In the guinea pig, peak infection was during the acute stage (4-weeks p.i.) and resulted in inflammation that disrupted spermatogenesis chronically. To determine if vaccine efficacy could be evaluated using these models, males were vaccinated using subcutaneous injection with vaccine candidate 16 MΔvjbR at 109 CFU/100 µl followed by intratracheal challenge with 16 M at 107. Interestingly, vaccination efficacy varied between species and reproductive organs demonstrating the value of evaluating vaccine candidates in multiple models and sexes. Vaccination resulted in a significant reduction in colonization in the mouse, but this could not be correlated with a decrease in inflammation. Due to the ability to evaluate for both colonization and inflammation, guinea pigs seemed the better model not only for assessing host-pathogen interactions but also for future vaccine development efforts.

Primary and memory immune responses against rough Brucella canis are less robust compared to smooth B. abortus and B. melitensis following intratracheal infection in mice.

Brucella canis is the cause of canine brucellosis, a globally distributed, zoonotic pathogen which primarily causes disease in dogs. B. canis is unique amongst the zoonotic Brucella spp. with its rough lipopolysaccharide, a trait typically associated with attenuation in gram-negative bacteria. Unfortunately, no vaccine is available against B. canis, and vaccine development is hampered by a limited understanding of the immune response required to combat it and the course of infection following a physiologically relevant, mucosal route of inoculation. To address these concerns and analyze the impact of the rough phenotype on the immune response, we infected mice intratracheally with rough B. canis or smooth B. melitensis or B. abortus. Bacterial colonization and histologic lesions were assessed in systemic target organs as well as locally in the lungs and draining mediastinal lymph node. Mice were also reinfected with Brucella following antibiotic treatment and cytokine production by T lymphocytes in the lung and spleen was assessed by flow cytometry to investigate the memory immune response. Despite its rough phenotype, B. canis established a persistent infection at the same level of colonization as the smooth strains. However, B. canis induced significantly less granulomatous inflammation in the spleen as well as a lack of bronchial-associated lymphoid tissue (BALT) hyperplasia in the lungs. These differences coincided with increased IL-10 and decreased IFN-γ in the spleen of B. canis-infected mice. Previous exposure to all Brucella strains provided protection against colonization following secondary challenge, although induction of IFN-γ by T lymphocytes was seen only in the lungs during B. canis infection while the smooth strains induced this cytokine in the spleen as well. Neither Brucella strain induced significant polyfunctional T lymphocytes, a potential immunomodulatory mechanism that appears to be independent of lipopolysaccharide phenotype.

Systemic proliferative arteriopathy and hypophysitis in a cow with chronic ovine herpesvirus 2-induced malignant catarrhal fever.

Malignant catarrhal fever (MCF) is a severe, systemic, lymphoproliferative disease affecting domestic ruminants, caused by a group of MCF viruses in the genus Macavirus. Infection of cattle and bison with ovine herpesvirus 2 (OvHV2) is economically significant in North America. Sheep are the reservoir host of the virus, and only rarely manifest disease. Cattle and bison, however, frequently have lymphoproliferation, mucosal ulceration, and systemic vasculitis. OvHV2-induced MCF in cattle and bison is often fatal, with clinical recovery reported only rarely. Chronic cases are uncommon, but vascular changes of variable severity and ocular lesions have been described. Here we present a case of chronic MCF in a cow with proliferative arteriopathy, systemic vasculitis, and OvHV2-associated hypophysitis. We demonstrated OvHV2 nucleic acid in affected tissues with in situ hybridization.

When the Going Gets Rough: The Significance of Brucella Lipopolysaccharide Phenotype in Host-Pathogen Interactions.

Brucella is a facultatively intracellular bacterial pathogen and the cause of worldwide zoonotic infections, infamous for its ability to evade the immune system and persist chronically within host cells. Despite the frequent association with attenuation in other Gram-negative bacteria, a rough lipopolysaccharide phenotype is retained by Brucella canis and Brucella ovis, which remain fully virulent in their natural canine and ovine hosts, respectively. While these natural rough strains lack the O-polysaccharide they, like their smooth counterparts, are able to evade and manipulate the host immune system by exhibiting low endotoxic activity, resisting destruction by complement and antimicrobial peptides, entering and trafficking within host cells along a similar pathway, and interfering with MHC-II antigen presentation. B. canis and B. ovis appear to have compensated for their roughness by alterations to their outer membrane, especially in regards to outer membrane proteins. B. canis, in particular, also shows evidence of being less proinflammatory in vivo, suggesting that the rough phenotype may be associated with an enhanced level of stealth that could allow these pathogens to persist for longer periods of time undetected. Nevertheless, much additional work is required to understand the correlates of immune protection against the natural rough Brucella spp., a critical step toward development of much-needed vaccines. This review will highlight the significance of rough lipopolysaccharide in the context of both natural disease and host-pathogen interactions with an emphasis on natural rough Brucella spp. and the implications for vaccine development.

Characterization of epididymal and testicular histologic lesions and use of immunohistochemistry and PCR on formalin-fixed tissues to detect Brucella canis in male dogs.

In male dogs, Brucella canis frequently causes epididymitis, ultimately resulting in testicular atrophy and infertility. Although B. canis predominantly affects the epididymis, the misleading term "orchitis" is still commonly used by clinicians. Of additional concern, diagnosis in dogs remains challenging because of variable sensitivity and specificity of serologic assays and fluctuations in bacteremia levels in infected dogs, reducing the sensitivity of blood culture. We describe here the histologic lesions in the scrotal contents of 8 dogs suspected of being infected with B. canis and clinically diagnosed with orchitis. We explored the possibility of using immunohistochemistry (IHC) and real-time PCR (rtPCR) in formalin-fixed, paraffin-embedded (FFPE) tissues to detect the presence of B. canis. Epididymitis of variable chronicity was identified in all 8 dogs, with only 3 also exhibiting orchitis. Using rtPCR, the presence of B. canis was identified in 4 of 8 dogs, with 3 of these 4 dogs also positive by IHC. These results suggest that rtPCR and IHC are promising techniques that can be used in FFPE tissues to detect B. canis when other detection techniques are unavailable. Additionally, accurate recognition of epididymitis rather than orchitis in suspect cases could aid in accurate diagnosis.

Evaluation of the Efficacy of the Brucella canis RM6/66 ΔvjbR Vaccine Candidate for Protection against B. canis Infection in Mice.

Brucella canis is a Gram-negative, facultative intracellular bacterium and the causative agent of canine brucellosis, a highly contagious disease of dogs that can be transmitted to humans. Unfortunately, no vaccine is available to prevent infection. We recently characterized the kinetics of B. canis infection in the mouse model, establishing the required dose necessary to achieve systemic infection. The objective of this study was to investigate the utility of the mouse model in assessing canine brucellosis vaccine candidates and to subsequently investigate the safety and efficacy of a live attenuated vaccine, the B. canis RM6/66 ΔvjbR strain. Mice vaccinated with a dose of 109 CFU of the vaccine strain by both intraperitoneal and subcutaneous routes were afforded significant protection against organ colonization and development of histopathologic lesions following intraperitoneal challenge. Addition of an adjuvant or a booster dose 2 weeks following initial vaccination did not alter protection levels. Vaccination also resulted in a robust humoral immune response in mice, and B. canis RM6/66 ΔvjbR was capable of activating canine dendritic cells in vitro These data demonstrate that the B. canis RM6/66 ΔvjbR strain shows promise as a vaccine for canine brucellosis and validates the mouse model for future vaccine efficacy studies.IMPORTANCE Canine brucellosis, caused by Brucella canis, is the primary cause of reproductive failure in dogs and represents a public health concern due to its zoonotic nature. Cases in dogs in the United States have been increasing due to the persistent nature of the bacterium, deficiencies in current diagnostic testing, and, most importantly, the lack of a protective vaccine. Current estimates place the seroprevalence of B. canis in the southern United States at 7% to 8%, but with the unprecedented rates of animals moving across state and international borders and the lack of federal regulations in regard to testing, the true seroprevalence of B. canis in the United States may very well be higher. Vaccination represents the most effective method of brucellosis control and, in response to the demand for a vaccine against B. canis, we have developed the live attenuated B. canis RM6/66 ΔvjbR vaccine strain capable of protecting mice against challenge.

Pathologic and flow cytometric features of a case of canine ventral cervical lymphangiosarcoma.

An 11-y-old spayed female German Shepherd was presented for a second opinion of ventral cervical swelling of 3-mo duration. On examination, the dog had significant dependent ventral cervical swelling. Enlarged lymph nodes with cystic changes and severe edematous facial swelling were noted on computed tomography. Fine-needle aspiration of the ventral cervical swelling revealed yellow-tinged fluid, with a predominance of lymphoid cells noted on cytologic examination. On cervical exploratory surgery, the left mandibular lymph node was surrounded by a large fluid pocket; biopsies of the lymph node were obtained. Impression smear cytology, flow cytometry, PCR for antigen receptor gene rearrangements, and histopathology were performed on samples from the left mandibular lymph node. Impression smear cytology revealed a population of atypical discrete cells. Flow cytometry identified a population of CD34+/CD45- large cells. A tumor of endothelial origin within the medulla of the lymph node was identified by histopathology, and lymphangiosarcoma was confirmed based on prospero-related homeobox gene 1 (PROX1) immunoreactivity. Our study describes the challenges in the diagnosis of a rarely reported entity and highlights that neoplastic endothelial cells should be considered as a differential when high proportions of CD34+/CD45- cells are present in flow cytometry.

Characterization of Brucella canis infection in mice.

Canine brucellosis, caused by Brucella canis, is a disease of dogs and represents a public health concern as it can be transmitted to humans. Canine brucellosis is on the rise in the United States and there is currently no vaccine for use in dogs. Mice have been extensively utilized to investigate host-pathogen interactions and vaccine candidates for smooth Brucella species and could serve a similar role for studying B. canis. However, comparatively little is known about B. canis infection in mice. The objective of this study was to characterize the kinetics of colonization and pathogenicity of B. canis in mice in order to evaluate the mouse as a model for studying this pathogen. C57BL/6 mice were inoculated intraperitoneally with 105, 107, or 109 CFU of Brucella canis RM6/66 and euthanized 1-, 2-, 4-, 6-, 9-, and 12-weeks post-inoculation. B. canis induced splenomegaly in mice infected with 109 CFU at 1- and 2 weeks post-inoculation while no gross lesions were observed in other dose groups. Infection at the two higher doses resulted in dose-dependent granulomatous hepatitis and histiocytic infiltration of the spleen and mesenteric lymph nodes by 1-2 weeks. B. canis was cultured from the liver, spleen, uterus, bone marrow, lung, and kidney in all groups with colonization declining at a slow but steady rate throughout the experiment. Clearance was achieved by 9 weeks 105 CFU group and by 12 weeks in the 107 CFU group, while B. canis persisted in the spleen until 12 weeks in the highest dose group. Although B. canis does not demonstrate significant replication in C57BL/6 mice, it has the ability to establish an infection, induce splenomegaly, and persist for several weeks in multiple organs. Moreover, 1 x 107 CFU appears to be a suitable challenge dose for investigating vaccine safety.