THE USE OF INTRADERMAL SKIN TESTING AND HYPOSENSITIZATION INJECTIONS TO CONTROL SEASONAL DERMATITIS IN GREATER ONE-HORNED RHINOCEROSES (RHINOCEROS UNICORNIS).

Allergic dermatitis was diagnosed in a 25-yr-old female greater one-horned rhinoceros (Rhinoceros unicornis) and her 6-yr-old female offspring by skin biopsy, intradermal skin testing (IDST), and allergen-specific serum IgE testing. Dam and offspring presented with seasonal, erosive, and ulcerative dermatitis affecting the face, legs, and trunk starting at 6 and 2 yr of age, respectively. IDST was performed at the caudal pinnal base using 61 regionally specific allergens. Specific serum allergen responses were detected using Heska's Equine ALLERCEPT® Allergen Panel. Histopathology of the lesions was consistent with an allergic etiology. Injectable allergen-specific immunotherapy was initiated in both animals and within 6 to 18 mon after commencing hyposensitization clinical improvement was noted. This report documents a repeatable methodology for IDST and serological allergen testing for use in rhinoceroses. The hyposensitization protocol detailed here can help guide future treatment protocols.

Surgical stabilization of traumatic elbow joint luxation and proximal ulnar fracture in a silvery langur (Trachypithecus cristatus).

CASE DESCRIPTION: A 3-year-old 5-kg sexually intact female silvery langur housed in a single-species group at a zoological institution was presented because of acute trauma to the left forelimb. CLINICAL FINDINGS: Radiography of the left forelimb revealed a type II Monteggia fracture (proximal ulnar fracture with cranial displacement and caudal luxation of the radial head). During surgery, disruption of the annular ligament and rupture of the lateral collateral ligament were noted. TREATMENT AND OUTCOME: The langur underwent open reduction and internal fixation of the ulnar fracture and placement of a radioulnar positional screw, a prosthetic lateral collateral ligament, and a temporary hinged type 1A external skeletal fixator. The langur was returned to group housing, underwent behavioral training, and was periodically anesthetized for physical therapy sessions to improve range of motion of the left elbow joint. The external skeletal fixator was removed 4 weeks after surgery, and the radioulnar positional screw was removed 6 weeks after surgery. Three months after surgery, the range of motion of the langur's left elbow joint was considered normal, and the animal returned to normal activity. CLINICAL RELEVANCE: For the captive silvery langur of the present report, surgical stabilization and postoperative management of a type II Monteggia fracture of the left forelimb were successful with recovery of elbow joint function. These techniques may be applied to other captive nonhuman primates, including those that brachiate or are members of social species that must be housed with conspecifics in the postoperative period to maintain group dynamics.

ASSESSMENT OF MULTIANTIGEN PRINT IMMUNOASSAY AND RAPID LATERAL-FLOW TEST FOR THE DETECTION OF MYCOBACTERIUM BOVIS INFECTION IN MALAYAN TAPIR (TAPIRUS INDICUS).

A multiantigen print immunoassay (MAPIA) and rapid test (RT) developed and validated for detection of mycobacterial antibodies in elephants (Elephas maximus and Loxodonta africana) was assessed in Malayan tapir (Tapirus indicus). Retrospective analysis of banked serum from one Mycobacterium bovis infected and seven presumably uninfected tapir was performed by MAPIA and RT. A sample collected 2 mon prior to the death of a culture-confirmed M. bovis-infected tapir served as a positive control. Seroreactivity of this sample was demonstrated via both MAPIA and RT testing. Seven uninfected animals, including four without postmortem evidence of mycobacterial disease and three that remain healthy, were negative controls; none demonstrated seroreactivity to key antigens with either test. These results suggest that MAPIA and RT have potential utility for rapid detection of M. bovis infection in Malayan tapir.

Pseudomonas aeruginosa Interstrain Dynamics and Selection of Hyperbiofilm Mutants during a Chronic Infection.

Opportunistic pathogens establishing new infections experience strong selection to adapt, often favoring mutants that persist. Capturing this initial dynamic is critical for identifying the first adaptations that drive pathogenesis. Here we used a porcine full-thickness burn wound model of chronic infection to study the evolutionary dynamics of diverse Pseudomonas aeruginosa infections. Wounds were infected with a mixed community of six P. aeruginosa strains, including the model PA14 strain (PA14-1), and biopsies taken at 3, 14, and 28 days postinfection. Hyperbiofilm-forming rugose small-colony variants (RSCVs) were the earliest and predominant phenotypic variant. These variants were detected on day 3 and persisted, with the majority evolved from PA14-1. Whole-genome sequencing of PA14-1 RSCV isolates revealed driver mutations exclusively in the wsp pathway, conferring hyperbiofilm phenotypes. Several of the wsp mutant RSCVs also acquired CRISPR-Cas adaptive immunity to prophages isolated from the P. aeruginosa wound isolate (B23-2) that was also present in the inoculum. These observations emphasize the importance of interstrain dynamics and the role of lysogenic phages in the survival of an invading pathogen. Rather than being a side effect of chronicity, the rapid rise of RSCVs in wounds is evidence of positive selection on the Wsp chemosensory system to produce mutants with elevated biofilm formation capacity. We predict that RSCVs provide a level of phenotypic diversity to the infecting bacterial community and are common, early adaptations during infections. This would likely have significant consequences for clinical outcomes.IMPORTANCE Bacteria adapt to infections by evolving variants that are more fit and persistent. These recalcitrant variants are typically observed in chronic infections. However, it is unclear when and why these variants evolve. To address these questions, we used a porcine chronic wound model to study the evolutionary dynamics of Pseudomonas aeruginosa in a mixed-strain infection. We isolated hyperbiofilm variants that persisted early in the infection. Interstrain interactions were also observed, where adapted variants acquired CRISPR-mediated immunity to phages. We show that when initiating infection, P. aeruginosa experiences strong positive selection for hyperbiofilm phenotypes produced by mutants of a single chemosensory system, the Wsp pathway. We predict that hyperbiofilm variants are early adaptations to infection and that interstrain interactions may influence bacterial burden and infection outcomes.

Pseudomonas aeruginosa rugose small-colony variants evade host clearance, are hyper-inflammatory, and persist in multiple host environments.

Pseudomonas aeruginosa causes devastating infections in immunocompromised individuals. Once established, P. aeruginosa infections become incredibly difficult to treat due to the development of antibiotic tolerant, aggregated communities known as biofilms. A hyper-biofilm forming clinical variant of P. aeruginosa, known as a rugose small-colony variant (RSCV), is frequently isolated from chronic infections and is correlated with poor clinical outcome. The development of these mutants during infection suggests a selective advantage for this phenotype, but it remains unclear how this phenotype promotes persistence. While prior studies suggest RSCVs could survive by evading the host immune response, our study reveals infection with the RSCV, PAO1ΔwspF, stimulated an extensive inflammatory response that caused significant damage to the surrounding host tissue. In both a chronic wound model and acute pulmonary model of infection, we observed increased bacterial burden, host tissue damage, and a robust neutrophil response during RSCV infection. Given the essential role of neutrophils in P. aeruginosa-mediated disease, we investigated the impact of the RSCV phenotype on neutrophil function. The RSCV phenotype promoted phagocytic evasion and stimulated neutrophil reactive oxygen species (ROS) production. We also demonstrate that bacterial aggregation and TLR-mediated pro-inflammatory cytokine production contribute to the immune response to RSCVs. Additionally, RSCVs exhibited enhanced tolerance to neutrophil-produced antimicrobials including H2O2 and the antimicrobial peptide LL-37. Collectively, these data indicate RSCVs elicit a robust but ineffective neutrophil response that causes significant host tissue damage. This study provides new insight on RSCV persistence, and indicates this variant may have a critical role in the recurring tissue damage often associated with chronic infections.

Small sarcocysts can be a feature of experimental infections with Sarcocystis neurona merozoites.

Several reports indicate the presence of small tissue cysts associated with Sarcocystis neurona infections. Several failed attempts to develop tissue cysts in potential intermediate host using in vitro derived parasites originally isolated from horses with equine protozoal myeloencephalitis suggest that the experimental methods to achieve bradyzoites with those isolates was not possible. Those prior studies reported the lack of detectable sarcocysts based on histology and in vivo feeding trials. A recent report of successful production and detection of small sarcocysts triggered us to review archived tissues from earlier experimental infection studies. The retrospective review sought to determine if small sized sarcocysts were not detected due to their relatively smaller size and infrequency as compared to larger sized sarcocysts produced with other isolates in these experimental inoculation trials. Tissues from two prior in vivo inoculation studies, involving in vitro-produced parasites inoculated into laboratory-reared cats and raccoons, were re-examined by immunohistochemistry staining to more easily detect the tissue cysts. In the experimental cat study no small tissue cysts were seen, consistent with the original publication results. However, in the experimental raccoon study, one raccoon inoculated with an EPM-derived isolate, SN-UCD1, had small sarcocysts not reported in the original publication. This retrospective study suggests that much closer scrutiny of tissues, including the use of immunohistochemistry on tissue sections is required to detect the smaller S. neurona sarcocysts associated with the experimental inoculations of the isolates originally derived from horses with EPM.

Histopathological comparisons of Staphylococcus aureus and Pseudomonas aeruginosa experimental infected porcine burn wounds.

Chronic skin wounds are a significant human health concern and are often complicated by infection with Pseudomonas aeruginosa and Staphylococcus aureus, particularly methicillin resistant S. aureus (MRSA). Translating the knowledge gained from extensive study of virulence mechanisms and pathogenesis of these bacterial species to new treatment modalities has been lacking in part due to a paucity of animal models able to recapitulate human disease. Our groups recently described a novel porcine chronic burn wound model for the study of bacterial infection; however, the histopathology of infection has yet to be described. The objective of this study is to define the histopathology of this model using important human chronic wound bacterial isolates. Porcine full-thickness burn wounds topically inoculated with P. aeruginosa strain PAO1, MRSA S. aureus strain USA300 or both bacteria were used to define and quantify histopathologic lesions. The development of a systemic, well-defined rubric for analysis allowed for evaluation of differences between infection groups. These differences, which included epithelial migration and proliferation, stromal necrosis, fluid accumulation and intensity and character of the innate and adaptive inflammatory cell responses, were identified temporally between infection groups. Mono-species infected wounds developed a hyper-proliferative wound edge. Coinfected wounds at day 35 had the largest wound sizes, increased amounts of neutrophilic inflammation, immaturity of the wound bed, and retention of necrotic tissue. Infection, regardless of species, inhibited wound contracture at all time points evaluated. Most importantly, this model recapitulated key features of chronic human wounds. Thus, this model will allow researchers to study novel treatment modalities in a biologically relevant animal model while monitoring both host and bacterial responses.

Sarcocystis neurona manipulation using culture-derived merozoites for bradyzoite and sporocyst production.

Equine protozoal myeloencephalitis (EPM) remains a significant central nervous system disease of horses in the American continents. Sarcocystis neurona is considered the primary causative agent and its intermediate life stages are carried by a wide host-range including raccoons (Procyon lotor) in North America. S. neurona sarcocysts mature in raccoon skeletal muscle and can produce central nervous system disease in raccoons, mirroring the clinical presentation in horses. The study aimed to develop laboratory tools whereby the life cycle and various life stages of S. neurona could be better studied and manipulated using in vitro and in vivo systems and compare the biology of two independent isolates. This study utilized culture-derived parasites from S. neurona strains derived from a raccoon or from a horse to initiate raccoon infections. Raccoon tissues, including fresh and cryopreserved tissues, were used to establish opossum (Didelphis virginiana) infections, which then shed sporocyts with retained biological activity to cause encephalitis in mice. These results demonstrate that sarcocysts can be generated using in vitro-derived S. neurona merozoites, including an isolate originally derived from a naturally infected horse with clinical EPM. This study indicates the life cycle can be significantly manipulated in the laboratory without affecting subsequent stage development, allowing further purification of strains and artificial maintenance of the life cycle.

Histone H1 phosphorylation in breast cancer.

Breast cancer is the second leading cause of cancer-related deaths in women. The need for new clinical biomarkers in breast cancer is necessary to further predict prognosis and therapeutic response. In this article, the LC-MS histone H1 phosphorylation profiles were established for three distinct breast cancer cell lines. The results show that the extent of H1 phosphorylation can distinguish between the different cell lines. The histone H1 from the metastatic cell line, MDA-MB-231, was subjected to chemical derivitization and LC-MS/MS analysis. The results suggest that the phosphorylation at threonine 146 is found on both histone H1.2 and histone H1.4. Cell lines were then treated with an extracellular stimulus, estradiol or kinase inhibitor LY294002, to monitor changes in histone H1 phosphorylation. The data show that histone H1 phosphorylation can increase and decrease in response to extracellular stimuli. Finally, primary breast tissues were stained for the histone H1 phosphorylation at threonine 146. Variable staining patterns across tumor grades and subtypes were observed with pT146 labeling correlating with tumor grade. These results establish the potential for histone H1 phosphorylation at threonine 146 as a clinical biomarker in breast cancer.