Analytical cell adhesion chromatography reveals impaired persistence of metastatic cell rolling adhesion to P-selectin.

Selectins facilitate the recruitment of circulating cells from the bloodstream by mediating rolling adhesion, which initiates the cell-cell signaling that directs extravasation into surrounding tissues. To measure the relative efficiency of cell adhesion in shear flow for in vitro drug screening, we designed and implemented a microfluidic-based analytical cell adhesion chromatography system. The juxtaposition of instantaneous rolling velocities with elution times revealed that human metastatic cancer cells, but not human leukocytes, had a reduced capacity to sustain rolling adhesion with P-selectin. We define a new parameter, termed adhesion persistence, which is conceptually similar to migration persistence in the context of chemotaxis, but instead describes the capacity of cells to resist the influence of shear flow and sustain rolling interactions with an adhesive substrate that might modulate the probability of extravasation. Among cell types assayed, adhesion persistence to P-selectin was specifically reduced in metastatic but not leukocyte-like cells in response to a low dose of heparin. In conclusion, we demonstrate this as an effective methodology to identify selectin adhesion antagonist doses that modulate homing cell adhesion and engraftment in a cell-subtype-selective manner.

Implications of Lymphatic Transport to Lymph Nodes in Immunity and Immunotherapy.

Adaptive immune response consists of many highly regulated, multistep cascades that protect against infection while preserving the health of autologous tissue. The proper initiation, maintenance, and resolution of such responses require the precise coordination of molecular and cellular signaling over multiple time and length scales orchestrated by lymphatic transport. In order to investigate these functions and manipulate them for therapy, a comprehensive understanding of how lymphatics influence immune physiology is needed. This review presents the current mechanistic understanding of the role of the lymphatic vasculature in regulating biomolecule and cellular transport from the interstitium, peripheral tissue immune surveillance, the lymph node stroma and microvasculature, and circulating lymphocyte homing to lymph nodes. This review also discusses the ramifications of lymphatic transport in immunity as well as tolerance and concludes with examples of how lymphatic-mediated targeting of lymph nodes has been exploited for immunotherapy applications.

Panfungal Polymerase Chain Reaction for Identification of Fungal Pathogens in Formalin-Fixed Animal Tissues.

Identification of fungal organisms often poses a problem for pathologists because the histomorphology of some fungal organisms is not specific, fresh tissues may not be available, and isolation and identification in culture may take a long time. The purpose of this study was to validate the use of panfungal polymerase chain reaction (PCR) to identify fungal organisms from formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed paraffin-embedded curls were tested from 128 blocks containing canine, feline, equine, and bovine tissues with cutaneous, nasal, pulmonary, and systemic fungal infections, identified by the presence of fungi in histologic sections. Quantitative scoring of histologic sections identified rare (11.9%), occasional (17.5%), moderate (17.5%), or abundant (53.1%) fungal organisms. DNA was isolated from FFPE tissues and PCR was performed targeting the internal transcribed spacer 2 (ITS-2) region, a segment of noncoding DNA found in all eukaryotes. Polymerase chain reaction products were sequenced and identified at ≥97% identity match using the Basic Local Alignment Search Tool and the NCBI database of ITS sequences. Of the 128 blocks, 117 (91.4%) yielded PCR products and high-quality sequences were derived from 89 (69.5%). Sequence and histologic identifications matched in 79 blocks (61.7%). This assay was capable of providing genus- and species-level identification when histopathology could not and, thus, is a beneficial complementary tool for diagnosis of fungal diseases.

GRANULOMATOUS FILARIAL ENCEPHALOMYELITIS CAUSED BY CHANDLERELLA QUISCALI IN A NORTHERN CRESTED CARACARA (CARACARA CHERIWAY).

A northern crested caracara (Caracara cheriway) was presented after being found nonambulatory in a field. On physical examination, the bird had severe hind-limb paresis. The bird did not improve after 10 days of hospitalization and was euthanized. Histologic examination of the cerebrum and spinal cord revealed multiple adult filarial nematodes surrounded by granulomatous inflammation with several multinucleated giant cells. These parasites were confirmed to be Chandlerella quiscali with polymerase chain reaction. This is the first report of C. quiscali in a bird of prey.

Screening wild and semi-free ranging great apes for putative sexually transmitted diseases: Evidence of Trichomonadidae infections.

Sexually transmitted diseases (STDs) can persist endemically, are known to cause sterility and infant mortality in humans, and could have similar impacts in wildlife populations. African apes (i.e., chimpanzees, bonobos, and to a lesser extent gorillas) show multi-male mating behavior that could offer opportunities for STD transmission, yet little is known about the prevalence and impact of STDs in this endangered primate group. We used serology and PCR-based detection methods to screen biological samples from wild and orphaned eastern chimpanzees and gorillas (N = 172 individuals, including adults, and juveniles) for four classes of pathogens that either commonly cause human STDs or were previously detected in captive apes: trichomonads, Chlamydia spp., Treponema pallidum (syphilis and yaws), and papillomaviruses. Based on results from prior modeling and comparative research, we expected STD prevalence to be highest in females versus males and in sexually mature versus immature individuals. All samples were negative for Chlamydia, Treponema pallidum, and papillomaviruses; however, a high percentage of wild chimpanzee urine and fecal samples showed evidence of trichomonads (protozoa). Analysis revealed that females were more likely than males to have positive urine-but not fecal-samples; however, there was no evidence of age (sexual maturity) differences in infection status. Sequence analysis of chimpanzee trichomonad samples revealed a close relationship to previously described trichomonads within the genus Tetratrichomonas. Phylogenetic comparisons to archived sequences from multiple vertebrate hosts suggests that many of the chimpanzee parasites from our study are likely transmitted via fecal-oral contact, but the transmission of some Tetratrichomonas sequence-types remains unknown and could include sexual contact. Our work emphasizes that only a fraction of infectious agents affecting wild apes are presently known to science, and that further work on great ape STDs could offer insights for the management of endangered great apes and for understanding human STD origins.

Reassessing Stephanofilaria stilesi dermatitis in cattle, with characterization of molecular markers for confirming diagnosis.

BACKGROUND: Stephanofilaria stilesi is a vector-borne filarioid nematode of cattle in North America that is transmitted via the hematophagous horn fly (Haematobia irritans) intermediate host. Despite being relatively common, little attention has been given to a thorough description of S. stilesi lesions and the potential integration of pathological and molecular diagnostic findings to confirm infection. METHODS: To characterize the cutaneous lesions caused by S. stilesi in cattle (Bos taurus taurus and Bos taurus indicus), skin of the ventral abdominal midline was collected from 22 animals during postmortem examination. Skin samples were processed for histology, transmission electron microscopy (TEM), DNA extraction, PCR, and Sanger sequencing targeting molecular markers cytochrome oxidase c subunit 1 (cox1), 12S, 18S rDNA, and 28S rDNA. RESULTS: Macroscopically, lesions ranged from 5 × 4 cm to 36 × 10 cm, consisting of one large single lesion, or two to four ovoid areas at the ventral abdominal midline, surrounding the umbilicus. Each lesion presented as ulcerative dermatitis with dry, serocellular crusts, or alopecic and lichenified areas. Histologically, eosinophilic, neutrophilic, and ulcerative dermatitis with furunculosis, folliculitis, and epidermal hyperplasia was observed. Cross sections of adult nematodes were identified in ~ 60% of the cases (n = 13) within intact follicles, sebaceous ducts, crusts, and areas of furunculosis. Stephanofilaria first-stage larvae (L1) were observed in five cases within "vitelline membranes" in the superficial dermis and crusts. Ultrastructurally, the L1 cross sections were compounded of smooth multilayered cuticle and somatic cells. The "vitelline membrane" is a tri-layered membrane where L1 are suspended in a matrix. Stephanofilaria stilesi DNA was found in 5 out of the 13 cases in which adults or L1 were histologically observed (38%) and in 1 out of the 9 cases without adults or L1 present (11%). Phylogenetic analyses suggest a closer relationship of the genus Stephanofilaria with Thelazioidea, instead of the family Filariidae (Filarioidea), in which it has been historically allocated. CONCLUSIONS: Our study improved the characterization of lesions and described ultrastructural findings of S. stilesi and highlights that molecular tools should be utilized in combination with histology for improved diagnostic resolution.

Dirofilaria immitis in an Asian small-clawed otter (Aonyx cinereus) from southeastern Louisiana, United States.

A single, male Dirofilaria immitis was found in the right ventricle of a captive, 10-month-old female Asian small-clawed otter (Aonyx cinereus) from East Feliciana parish in Louisiana, USA. Molecular analysis was performed for unequivocal species level identification using the cytochrome c oxidase subunit 1 (cox1) region of the mitochondrial DNA and comparing to known D. immitis cox1 sequences available on GenBank to which the specimen had a 99.54-100% pair wise identity. As there is no safe adulticidal treatment for D. immitis in mustelids, chemoprophylaxis using macrocyclic lactones is recommended to prevent infection. Further studies are needed to better characterize D. immitis infection and its propensity to cause disease in the Asian small-clawed otter.

Subcutaneous Implantation of Hepatocellular Carcinoma in a Dog.

A 13-year-old male neutered Cocker Spaniel mixed-breed dog developed a subcutaneous mass 2 years after undergoing surgery to remove a hepatocellular carcinoma. An approximately 4 × 3 cm subcutaneous mass was found on the ventral abdomen at the cranial end of the abdominal incision from the previous surgery. The subcutaneous mass was surgically removed and histopathological examination determined that it was an implantation of the previously excised hepatocellular carcinoma. The diagnosis was confirmed by immunohistochemical labelling with hepatocyte paraffin 1 antibody and pancytokeratin. Based on the location of the subcutaneous mass at the cranial end of the abdominal incision associated with the previous hepatocellular carcinoma resection, it is likely there was iatrogenic metastasis from the primary tumour excision. Subcutaneous iatrogenic metastasis of hepatocellular carcinoma is well recognized in humans but has apparently never been reported in dogs. Clinicians should be aware of this potential surgical complication.

Clostridium piliforme and canine distemper virus coinfection in 2 domestic dog littermates and a gray fox kit.

Concurrent Clostridium piliforme and canine distemper virus (CDV) infection was diagnosed in 2 canine littermates and 1 gray fox kit from Texas, USA. In all 3 animals, intracytoplasmic, filamentous bacteria, consistent with C. piliforme, were present along the margins of foci of hepatic necrosis. Additional histologic findings included intracytoplasmic and intranuclear inclusion bodies in bile duct and bronchial epithelial cells of the fox kit, and mild intestinal necrosis in 1 puppy. PCR assays confirmed the presence of C. piliforme in all 3 animals, CDV in both puppies, and canine parvovirus in 1 puppy. Fluorescent antibody testing confirmed the presence of CDV in the fox kit. Concurrent canine distemper and Tyzzer disease in canine littermates and the gray fox has not been reported previously, to our knowledge.

Autochthonous, zoonotic Onchocerca lupi in a South Texas dog, United States.

BACKGROUND: Onchocerca lupi is an emerging, zoonotic filarioid nematode associated with ocular disease in companion animals in North America and the Old World. The areas where this parasite is assumed to be endemic in the USA comprise southwestern states. Thus far, all cases reported outside of the southwest are associated with travel or animal movement. METHODS: An 11-year-old, castrated male Pitbull dog from McAllen, Hidalgo County, southern Texas, with no travel history, was diagnosed with a perforating corneal ulceration of the right eye. Enucleation was performed and tissues submitted for histopathology. RESULTS: Histologically, sections of two filarioid nematodes were observed. DNA was extracted from formalin-fixed paraffin-embedded tissue using a commercial kit. We performed PCR targeting the cox1 gene of the mitochondrial DNA, followed by sequencing and phylogenetic analysis. Altogether, these results confirmed the identification of the nematode specimens as O. lupi, phylogenetically belonging to haplotype 1. CONCLUSION: We report the first autochthonous case of O. lupi in a dog from Hidalgo County, southern Texas, USA. Our finding suggests Texas as an additional state where this zoonotic nematode is endemic. Further investigations are required to understand the epidemiology of this parasite along the USA/Mexico border.

Fatal cerebral cuterebrosis in a dog.

A 4-year-old intact female Yorkshire Terrier presented an acute onset of hypersalivation and depressed mentation. The owner reported that the dog displayed tremors, right-sided torticollis, right-sided head tilt, and lifting of the left forelimb. The dog appeared restless and confused, and the condition progressed to lethargy and death on the way to an emergency hospital. At necropsy, a single 10 mm long x 1 mm wide, pale gray, botfly larva with black spines was found along the cerebral meninges. Areas of hemorrhage were noted in the right cerebral hemisphere. Microscopically, these areas also had evidence of necrosis and inflammation. Morphology of the larva allowed confirmation of subfamily-level identification as Cuterebrinae, and presumed genus-level identification of Cuterebra. Species-level identification of the larva as C. abdominalis was achieved through DNA extraction, PCR and sequencing at the cytochrome oxidase subunits 1 and 2 (COI and COII), followed by phylogenetic analysis. Aberrant cuterebrosis is a poorly documented condition in dogs that may cause neurologic signs and lead to death.

Fatal Chagas myocarditis in government working dogs in the southern United States: Cross-reactivity and differential diagnoses in five cases across six months.

We describe the diagnostics surrounding the deaths of five working dogs over six-months to provide an enhanced clinical and diagnostic understanding of canine Chagas disease. Cases were five dogs with antibodies to Trypanosoma cruzi. Medical records were reviewed for diagnostic history. Testing was performed from samples collected before or immediately after death, including measurement of cardiac troponin I, histology, PCR and serology for Leishmania spp. and T. cruzi. Four dogs had a 2 to 7-year history of T. cruzi antibodies, while one positive dog had an unknown duration of exposure. Age at death ranged from 2 to 11 years and four dogs were actively working. The cardiac troponin I was elevated in all four dogs for which it was measured, although postmortem reference ranges are not established. Histopathologic diagnoses included mild to severe, chronic, lymphoplasmacytic to histiocytic myocarditis with variable fibrosis. Notably, only one dog had T. cruzi amastigotes observed in the heart histologically. T. cruzi DNA was detected in three other hearts. Although all five dogs tested antibody-positive for T. cruzi using three independent tests, all were also indirect fluorescent antibody-positive for Leishmania spp., interpreted as cross-reaction. Chagas disease in dogs is a diagnostic challenge owing to cross-reactions and variable clinical, histologic and molecular presentations. The use and interpretation of multiple diagnostic strategies is useful in diagnosis. This study demonstrates techniques used to diagnose and characterize Chagas disease in an at-risk dog population.

Retrospective Survey of Amphibian Pathology Cases at Texas A&M University System (2016-2020).

Dramatic declines in amphibians worldwide highlight the need for a better understanding of diseases affecting these species. To delineate the health issues of amphibians submitted to Texas A&M University System (2016-2020), the databases were queried on the basis of defined selection criteria. A total of 502 anurans (157 frogs [44 species] and 345 toads [10 species]) and 30 caudatans (23 salamanders [6 species] and 7 newts [4 species]) were reviewed. A most likely cause of death or major pathological finding (CD-MPF) leading to euthanasia was identified in 295 (55%) Anura cases and 15 (50%) Caudata cases. Of the 532 records reviewed, anurans included 492 captive, seven free-ranging and three undetermined specimens. All caudatans were captive. The most common CD-MPF in anurans was infectious/inflammatory (228/295; 77%), involving mycobacteriosis (73/228; 32%), chlamydiosis (44/228; 19%) and mycosis (32/228; 14%). Neoplasia was less common (28/295; 9%). Infectious/inflammatory lesions (14/15; 93%) were the main CD-MPFs in caudatans. Infectious diseases are a significant threat to captive amphibians in Texas and these results may aid personnel involved in amphibian conservation programmes, veterinarians and diagnosticians.

Angiostrongylus cantonensis central nervous system infection in captive callitrichids in Texas.

Angiostrongylus cantonensis is a tropical lungworm of rats known for central nervous system migration in aberrant primate hosts. Here, we describe A. cantonensis infection in three captive callitrichids from a Texas zoo. The affected animals included a Goeldi's monkey (Callimico goeldii), a cotton-top tamarin (Saguinus oedipus), and a pied tamarin (Saguinus bicolor) that ranged from 8 to 18 years old. Clinical signs included lethargy, ataxia, and seizures. Histologically, these animals had an eosinophilic meningoencephalitis to myelitis and some areas had abundant macrophages and parasite migration tracts. All cases had intralesional metastrongyle parasites, and nematodes were extracted from the formalin-fixed brain specimen in one case. This extracted parasite was identified as Angiostrongylus cantonensis based on morphologic features and diagnosis was confirmed with PCR. These cases represent the first report of this parasite in non-human primates in Texas, highlighting the western spread of A. cantonensis in the continental United States.

Photoconversion and chromatographic microfluidic system reveals differential cellular phenotypes of adhesion velocity versus persistence in shear flow.

An integrated photoconversion and cell sorting parallel-plate chromatography channel enabling the measurement of instantaneous and average velocities of cells mediating adhesion in flow fields was engineered to study the mechanisms underlying adhesion to selectins by metastatic cancer cells. Through the facile enrichment of cells into subfractions of differing adhesive behaviors and a fluorescent velocity probe amenable to off-chip analysis, underlying, causal molecular profiles implicated in differing adhesive phenotypes of metastatic cancer cells could be interrogated. This analytical method revealed selectin-mediated rolling adhesion to be strongly associated with expression of selectin ligands, correlations that vary with ligand type and rolling velocity magnitude. Discrete selectin ligand expression profiles were also found to underlie persistent versus non-persistent adhesion on selectins, suggestive of divergent regulatory mechanisms. This integrated cell sorting and photoconversion microfluidic platform thus enables in vitro analysis and comparisons of adhesive phenotypes as they relate to mechanisms of cancer cell metastasis in the context of selectin mediated adhesion, revealing new insights into potential cancer dissemination pathways.

PATHOLOGY AND DISCRETE TYPING UNIT ASSOCIATIONS OF TRYPANOSOMA CRUZI INFECTION IN COYOTES (CANIS LATRANS) AND RACCOONS (PROCYON LOTOR) OF TEXAS, USA.

Trypanosoma cruzi is a vector-borne, protozoal parasite of mammals. Infected humans, dogs (Canis lupus familiaris), and nonhuman primates may remain asymptomatic or may develop Chagas disease, most commonly characterized by lymphoplasmacytic myocarditis with myocardial degeneration and fibrosis, ultimately resulting in heart failure. Although wildlife species have important roles as sylvatic reservoirs, investigations into the pathology of T. cruzi in wildlife are limited to a few studies documenting histologic lesions in opossums (Didelphis spp.) and raccoons (Procyon lotor). Pathology in coyotes (Canis latrans) has not, to our knowledge, been described, despite their recognition as a reservoir and close genetic relationship to domestic dogs. Our objectives were to perform a detailed, comparative cardiac pathology study of sympatric, naturally infected coyotes and raccoons, to characterize the overall T. cruzi infection prevalence in the heart and blood of each species via PCR, and to identify infecting discrete typing units (DTUs). We sampled hunter-harvested coyotes (n=120) and raccoons (n=24) in a 28 county region of central and south Texas, US. Raccoons were significantly more likely to have positive PCR results (P<0.001) with a prevalence of 62% (15/24), comprising DTU TcIV exclusively, with mild to no evidence of cardiac pathology. In contrast, coyotes had a lower infection prevalence (8%, 10/120), comprising DTU TcI exclusively, with lymphoplasmacytic myocarditis observed in four of the six PCR-positive animals. Many raccoons had PCR-positive blood and heart tissue simultaneously, supporting previous reports that raccoons maintain parasitemia into chronic stages of infection; in contrast, none of the PCR-positive coyotes were positive in both heart and blood. Our findings demonstrate marked differences in T. cruzi infection dynamics between coyotes and raccoons, with important implications for reservoir potential and their role in transmission cycles.

Follicular choristoma in the third eyelid of an eclectus parrot (Eclectus roratus).

CASE DESCRIPTION: A 1-year-old male eclectus parrot (Eclectus roratus) with a 3- to 4-month history of blepharospasm in the right eye was referred to a veterinary medical teaching hospital for further evaluation. Conventional medical treatments had been ineffective. The referring avian specialist had plucked a suspected ectopic feather from the right eye 6 weeks prior to the referral evaluation. CLINICAL FINDINGS: The parrot was sedated, and ophthalmic examination of the right eye with slit-lamp biomicroscopy revealed a 3 × 2 × 2-mm raised vascular mass with a focally pigmented center associated with the temporal aspect of the leading edge of the third eyelid. No abnormalities were detected in the left eye. TREATMENT AND OUTCOME: The parrot was anesthetized, and the right eye mass was excised and submitted for histologic examination. Histologically, there was a single pigmented feather follicle bulb surrounded by multiple discrete lymphoid follicles and moderate lymphoplasmacytic inflammation within the substantia propria of the third eyelid conjunctiva. The histologically normal feather follicle in an abnormal location classified the lesion as a choristoma. Nine months after surgery, the parrot had no signs of ocular discomfort and no overt regrowth of the feather follicle. CLINICAL RELEVANCE: For the eclectus parrot of this report, a lesion caused by normal differentiation of an ectopic feather follicle in the right third eyelid was successfully treated. A third eyelid choristoma appears to be a hitherto unreported pathological finding in avian species. Although rare, the presence of a choristoma should be considered as a differential diagnosis for birds with blepharospasm.

Analyzing Mechanisms of Metastatic Cancer Cell Adhesive Phenotype Leveraging Preparative Adhesion Chromatography Microfluidic.

An integrated, parallel-plate microfluidic device is engineered to interrogate and fractionate cells based on their adhesivity to a substrate surface functionalized with adhesive ligand in a tightly controlled flow environment to elucidate associated cell-intrinsic pathways. Wall shear stress levels and endothelial presentation of E-selectin are modeled after the inflamed vasculature microenvironment in order to simulate in vitro conditions under which in vivo hematogenous metastasis occurs. Based on elution time from the flow channel, the collection of separate fractions of cells-noninteracting and interacting-at high yields and viabilities enables multiple postperfusion analyses, including flow cytometry, in vivo metastasis modeling, and transcriptomic analysis. This platform enables the interrogation of flow-regulated cell molecular profiles, such as (co)expression levels of natively expressed selectin ligands sLex , CD44, and carcinoembryonic antigen, and cancer stem cell marker CD24. This additionally reveals E-selectin adhesivity exhibited by metastatic human colon carcinoma cells to be a transient phenotype. Facile and rapid, this methodology for unbiased, label free sorting of large populations of cells based on their adhesion in flow represents a method of studying flow-regulated adhesion in vitro for the identification of molecular drug targets for development as antimetastatic cancer therapeutics.

Blackleg in cattle: A case report of fetal infection and a literature review.

Clostridium chauvoei causes blackleg in cattle. The disease has been reported worldwide, and although it can be prevented by vaccination, sporadic cases and occasional outbreaks still occur. We describe a case of blackleg in a 2-y-old, pregnant Gyr cow with in utero transmission to the fetus. The cow had characteristic gross and microscopic lesions of blackleg including widespread necrohemorrhagic and emphysematous skeletal and myocardial myositis, and fibrinous pericarditis. Her uterus contained a near-term, markedly emphysematous fetus with skeletal muscle and myocardial lesions similar to those seen in the dam. Histopathology of dam and fetal tissues revealed numerous gram-positive bacilli, many of them with sub-terminal spores, in multiple tissues. These bacilli were identified as C. chauvoei by immunohistochemistry. Anaerobic culture and fluorescent antibody tests performed on skeletal muscle from both the dam and fetus were positive for C. chauvoei, confirming a diagnosis of blackleg. Blackleg is a so-called endogenous infection, and the currently accepted pathogenesis involves ingestion of spores that are transported to muscle tissues where they lie dormant until anaerobiosis prompts germination. Germinating bacteria are histotoxic, producing severe, local necrosis and ultimately lethal toxemia. This model, however, has not been confirmed experimentally and also fails to explain some cases of the disease. A presumptive diagnosis of blackleg is based on clinical, gross, and histologic findings. Diagnostic confirmation necessitates the detection of C. chauvoei by culture, PCR, or immunodetection methods.