Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells.

Airway epithelial cells (AECs) play a key role in maintaining lung homeostasis, epithelium regeneration and the initiation of pulmonary immune responses. To isolate and study murine AECs investigators have classically used short and hot (1h 37°C) digestion protocols. Here, we present a workflow for efficient AECs isolation and culture, utilizing long and cold (20h 4°C) dispase II digestion of murine lungs. This protocol yields a greater number of viable AECs compared to an established 1h 37°C dispase II digestion. Using a combination of flow cytometry and immunofluorescent microscopy, we demonstrate that compared to the established method, the cold digestion allows for recovery of a 3-fold higher number of CD45-CD31-EpCAM+ cells from murine lungs. Their viability is increased compared to established protocols, they can be isolated in larger numbers by magnetic-activated cell sorting (MACS), and they result in greater numbers of distal airway stem cell (DASC) KRT5+p63+ colonies in vitro. Our findings demonstrate that temperature and duration of murine lung enzymatic digestion have a considerable impact on AEC yield, viability, and ability to form colonies in vitro. We believe this workflow will be helpful for studying lung AECs and their role in the biology of lung.

Cotransfer of antigen and contextual information harmonizes peripheral and lymph node conventional dendritic cell activation.

T cell responses against infections and cancer are directed by conventional dendritic cells (cDCs) in lymph nodes distant from the site of challenge. Migratory cDCs, which travel from the tissue to the lymph node, not only drive initial T cell activation but also transfer antigen to lymph node-resident cDCs. These resident cells have essential roles defining the character of the resulting T cell response; however, it is unknown how they can appropriately process and present antigens to suitably direct responses given their spatial separation. Here, using a novel strain of influenza A and a modified melanoma model, we show that tissue and lymph node cDC activation is harmonized and that this is driven by cotransfer of contextual cues. In the tumor, incomplete cDC activation in the tumor microenvironment is mirrored by lymph node-resident cDCs, whereas during influenza infection, pathogen-associated molecular patterns cotransferred with antigen drive TLR signaling in resident cDCs and their subsequent robust activation. This cotransfer mechanism explains how individual antigens can be handled distinctly by resident cDCs and how signals driving poor tumoral cDC activation further impact the lymph node. Our findings clarify how tissue context dictates antigenic and, consequently, T cell fate in the lymph node.

Immunohistochemical analysis of expression of VEGFR2, KIT, PDGFR-ß, and CDK4 in canine urothelial carcinoma.

Urothelial carcinomas (UCs), also known as transitional cell carcinomas, are the most common canine urinary tract neoplasms. Tyrosine kinases (TKs) are enzymes that tightly regulate cell growth and differentiation through phosphorylation. Receptor TK (RTK) inhibitors are currently used to treat UCs. Toceranib phosphate (Palladia; Pfizer) is an RTK inhibitor that blocks the activity of vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor-alpha and -beta (PDGFR-α, -ß), FMS-like tyrosine kinase 3, stem cell factor receptor (KIT, kinase inhibitor targeting), and colony stimulating factor receptor. To better understand UCs and validate treatment targets, we performed immunohistochemical staining for RTKs, as well as a novel target, cyclin-dependent kinase 4 (CDK4, a central regulator of the mammalian cell cycle), on formalin-fixed, paraffin-embedded tissues from bladder biopsies from 17 dogs with UCs, 17 dogs with cystitis (diseased controls), and 8 normal dogs (negative controls). Although immunohistochemical scores could not be extrapolated to prognostic value, response to treatment, and outcome of patients with UC, we demonstrated expression of PDGFR-ß and VEGFR2 in UCs; all UC samples staining positively for VEGFR2. Minimal positive staining for KIT was noted in the tumor samples. CDK4 staining intensity was significantly weaker in UCs compared with normal and cystitis bladder samples. The intense staining of VEGFR2 in UC cells suggested that VEGFR2 may be of prognostic and/or therapeutic value in dogs with UC. Overexpression of VEGFR2 in UC cells validates this receptor as a treatment target in UC.

A Scoping Review of the Global Distribution of Causes and Syndromes Associated with Mid- to Late-Term Pregnancy Loss in Horses between 1960 and 2020.

Equine pregnancy loss is frustrating and costly for horse breeders. The reproductive efficiency of mares has significant implications for a breeding operation's economic success, and widespread losses can have a trickle-down effect on those communities that rely on equine breeding operations. Understanding the causes and risks of equine pregnancy loss is essential for developing prevention and management strategies to reduce the occurrence and impact on the horse breeding industry. This PRISMA-guided scoping review identified 514 records on equine pregnancy loss and described the global spatiotemporal distribution of reported causes and syndromes. The multiple correspondence analysis identified seven clusters that grouped causes, syndromes, locations and pathology. Reasons for clustering should be the focus of future research as they might indicate undescribed risk factors associated with equine pregnancy loss. People engaged in the equine breeding industry work closely with horses and encounter equine bodily fluids, placental membranes, aborted foetuses, and stillborn foals. This close contact increases the risk of zoonotic disease transmission. Based on this review, research is required on equine abortion caused by zoonotic bacteria, including Chlamydia psittaci, Coxiella burnetii and Leptospira spp., because of the severe illness that can occur in people who become infected.

Ichthyosis fetalis in Polled Hereford and Shorthorn calves.

Inherited forms of ichthyosis, or generalized scaling of the skin, have been reported in many animal species, including cattle, and are characterized by an autosomal recessive mode of inheritance. We investigated 2 calves affected with ichthyosis fetalis, a Polled Hereford and a Shorthorn. Both cases had hard white plaques on the skin consistent with excessive keratinization. This was confirmed by histopathology, which showed severe diffuse epidermal and follicular orthokeratotic hyperkeratosis. The known mutation (H1935R) in gene ABCA12, responsible for ichthyosis fetalis in Chianina cattle, was shown to be absent in both affected calves and their obligate heterozygous parents. These molecular findings indicate that allelic heterogeneity exists for this condition in cattle.

Clinical, diagnostic and pathologic features of presumptive cases of Chlamydia pecorum-associated arthritis in Australian sheep flocks.

BACKGROUND: Arthritis is an economically significant disease in lambs and is usually the result of a bacterial infection. One of the known agents of this disease is Chlamydia pecorum, a globally recognised livestock pathogen associated with several diseases in sheep, cattle and other hosts. Relatively little published information is available on the clinical, diagnostic and pathologic features of C. pecorum arthritis in sheep, hindering efforts to enhance our understanding of this economically significant disease. In this case series, a combination of standard diagnostic testing used routinely by veterinarians, such as the Chlamydia complement fixation text (CFT), veterinary clinical examinations, and additional screening via C. pecorum specific qPCR was used to describe putative chlamydial infections in five sheep flocks with suspected ovine arthritis. CASE PRESENTATION: Five separate cases involving multiple lambs (aged six to ten months) of different breeds with suspected C. pecorum arthritis are presented. In two of the five cases, arthritic lambs exhibited marked depression and lethargy. Arthritis with concurrent conjunctivitis was present in four out of five lamb flocks examined. Chlamydia CFT demonstrated medium to high positive antibody titres in all flocks examined. C. pecorum shedding was evident at multiple sites including the conjunctiva, rectum and vagina, as determined via qPCR. Two of the five flocks received antimicrobials and all flocks recovered uneventfully regardless of treatment. CONCLUSION: This case series highlights the features a field veterinarian may encounter in cases of suspected ovine chlamydial arthritis. Our analysis suggests a presumptive diagnosis of chlamydial arthritis in lambs can be made when there is evidence of joint stiffness with or without synovial effusion and elevated chlamydia antibody titres. C. pecorum-specific qPCR was found to be a useful ancillary diagnostic tool, detecting Chlamydia positivity in low or negative CFT titre animals. Variables such as symptom duration relative to sampling, sheep breed and farm management practices were all factors recorded that paint a complex epidemiological and diagnostic picture for this disease. These case studies serve to provide a platform for further research to improve diagnostic testing and new treatment and control strategies for C. pecorum infections in sheep.

Equine alopecia areata: a retrospective clinical descriptive study at the University of California, Davis (1980-2011).

BACKGROUND: Alopecia areata (AA) causes hair loss due to inflammatory changes within and around hair bulbs and lower portions of the hair follicles. Documentation of AA in horses is limited to a few case reports. HYPOTHESIS/OBJECTIVES: The aim of this retrospective study was to characterize equine AA by describing patterns in age, sex, breed and lesion distribution in a series of cases. An attempt was made to characterize the long-term course of the disease by surveying owners of affected horses. ANIMALS: Computerized records from 1 January 1980 to 1 July 2011 yielded 15 horses. METHODS: Descriptive statistics were calculated for age at presentation, breed, sex, dermatological signs, season when diagnosed and any recurrence of AA. The breed and sex distribution of horses with AA was compared with the equine hospital population during the study period. RESULTS: The prevalence of AA was 0.017%. Appaloosas and quarter horses were the most commonly recorded breeds. The median age was 9 years, with an age range from 3 to 15 years. Alopecia was the primary dermatological abnormality in all horses and commonly affected the mane, tail and face. More than half of the horses presented for other medical conditions. Five of seven (71.4%) owners who returned completed surveys reported a seasonal pattern to the disease, which usually worsened in spring and summer. CONCLUSIONS AND CLINICAL IMPORTANCE: Alopecia areata is a rare disease in horses, and is typically cosmetic in nature. To the authors' knowledge, this is the first study investigating the epidemiology of equine AA.

A blocking ELISA for the detection of antibodies to psittacine beak and feather disease virus (BFDV).

Currently, the only diagnostic test available routinely for the sero-diagnosis of BFDV is the haemagglutination-inhibition (HI) assay. This test, whilst useful and applicable to samples from a wide range of psittacine birds, is not an ideal assay; it requires erythrocytes from live animals, virus purified from the feathers of infected birds and polyclonal antibody preparations in order to perform the assay. Variations in these reagents make consistency between tests difficult to achieve, underscoring the need for a new test with standardised reagents for the sero-diagnosis of BFDV infection which has led to the development of an antibody response. The methods used to develop a novel "blocking" (or "competitive") ELISA (bELISA) for the detection of anti-BFDV antibodies in psittacine sera are presented in this paper. The assay was developed using a baculovirus-expressed recombinant BFDV capsid protein and a newly developed monoclonal antibody raised against this protein. The assay was then validated with 160 samples from eastern long-billed corellas (Cacatua tenuiostris) vaccinated with the recombinant capsid protein and challenged with live virus and samples from 82 cockatiels known to be HI negative. The bELISA described in this study is a sensitive and specific diagnostic test and should have wide application for the sero-diagnosis of BFDV.

A quantitative, real-time polymerase chain reaction assay for beak and feather disease virus.

PCR-based assays for the detection of BFDV DNA are in widespread use throughout the world. Quantitative real-time PCR assays are extremely sensitive and have the advantages over standard PCR assays that they do not require post-reaction processing to visualise PCR products and can quantify the amount of DNA present in a sample. This study describes a quantitative real-time PCR assay for the detection of BFDV DNA, using primers designed to amplify a conserved 81 bp fragment of ORFV1 and SYTO9, a fluorescent intercalating dye. A synthetic oligonucleotide was used to make standard curves for the quantitation of viral load in blood and feather preparations. The assay was very sensitive, with a detection limit of 50 copies/microL. The assay was developed using DNA extracts from the feathers of 10 different species of birds which had tested BFDV-positive previously and was validated with blood and feather samples from corellas vaccinated with an experimental BFDV vaccine, then challenged with live virus. Viral DNA was detected consistently in the blood of all control (non-vaccinated) birds and in some vaccinated birds. Contamination of the environment with feather dander from BFDV-infected birds meant that feather preparations used for the haemagglutination assay were unreliable for the detection and quantitation of viral excretion. Nonetheless, the assay should prove to be a useful and sensitive test for the detection of viral DNA in a range of samples.

Assessment of recombinant beak and feather disease virus capsid protein as a vaccine for psittacine beak and feather disease.

Beak and feather disease virus (BFDV) is a significant pathogen of wild Australasian and African psittacine birds. We assessed the immunogenicity of recombinant BFDV capsid (recBFDVcap) to protect against the development of psittacine beak and feather disease (PBFD). Long-billed corellas (Cacatua tenuirostris) (n=13) received (by injection) 1 ml vaccine containing 10 microg recBFDVcap on day 0 and 0.4 ml vaccine containing 66.8 microg recBFDVcap on day 11. All vaccinated corellas and five non-vaccinated control corellas were given 0.4 ml BFDV suspension [titre=log(2) 12 haemagglutination units (HAU) 50 microl(-1)] intramuscularly and 0.1 ml orally 16 days after booster vaccination. Blood was collected during the vaccination period and blood and feathers were collected after BFDV administration. Testing of blood samples included BFDV DNA detection by PCR and quantitative PCR (qPCR) as well as antibody detection by haemagglutination inhibition (HI) and on feather samples, BFDV DNA and antigen was detected by haemagglutination (HA) and qPCR. Four of 97 blood samples collected from vaccinated birds after virus challenge tested positive by PCR, whereas 17 of 35 samples taken from non-vaccinated control corellas tested positive. Vaccinated birds did not develop feather lesions, had only transient PCR-detectable viraemia and had no evidence of persistent infection 270 days post-challenge using PCR, histopathology and immunohistochemistry. Non-vaccinated control corellas developed transient feather lesions and had PCR, HI and HA test results consistent with PBFD. They were BFDV PCR-positive for up to 41 days post-challenge and qPCR demonstrated reduced virus replication in vaccinated birds compared with non-vaccinated control birds.

Beak and feather disease virus infection in cockatiels (Nymphicus hollandicus).

Psittacine beak and feather disease is known to occur in a wide range of psittacine species; however, there are no scientific or credible anecdotal reports of psittacine beak and feather disease occurring in the cockatiel (Nymphicus hollandicus) despite it being one of the world's most commonly kept companion bird species. Consequently, this has resulted in speculation that the species may have some innate resistance to beak and feather disease virus (BFDV) infection. To investigate this hypothesis we conducted a survey of cockatiels (n=88) at commercial aviaries to investigate whether BFDV infection occurs in cockatiels, and found that all birds were virus-free by polymerase chain reaction and haemagglutination assay and had no detectable antibody titre by haemagglutination-inhibition assay. In addition to this, we sequenced the genome of two BFDV isolates obtained from diseased cockatiel feathers and performed cross-reactivity assays using virus eluted from these feathers and sera from naturally immune psittacine birds. Serological cross-reactivity results and phylogenetic analysis of the nucleotide sequences indicated that the cockatiel virus isolates were serologically and genetically different to other BFDV isolates. This is the first paper to report evidence of an antigenically distinct BFDV in psittacine birds.

Elimination of false-positive polymerase chain reaction results resulting from hole punch carryover contamination.

The collection of biological material (e.g., blood) directly onto filter paper for subsequent use in laboratory assays such as polymerase chain reaction (PCR), has become a common practice. Dried cells or fluid on the paper can be readily rehydrated and retrieved into a standard volume of an appropriate elution buffer but introduces a dilution factor to the sample. The use of a common cutting instrument for excising a standard-sized piece of paper that contains the material also introduces the potential for transferring biological material from one sample to subsequent samples, causing false-positive results by PCR. In the present study, filter-paper-collected blood that contained beak and feather disease virus was used to determine if viral DNA could be transferred between samples by a hole punch used to excise sequential filter papers. It was determined that false-positive results could be obtained at least 13 times after a positive sample. Subsequently, the efficacy of 4 methods of hole punch disinfection, flaming, VirkonS, bleach, and a bleach-ethanol combination, was assessed. The only effective and practical method to destroy DNA was a method where the hole punch was agitated in commercial bleach, rinsed in water, the water was displaced with 100% ethanol and air-dried. This method was simple, cheap, and relatively rapid, and allowed for the use of a single hole punch for a series of samples, without carryover contamination and consequent false-positive results.

Development and applications of a monoclonal antibody to a recombinant beak and feather disease virus (BFDV) capsid protein.

The development of diagnostic assays for detecting beak and feather disease virus (BFDV) has traditionally been hampered by the difficulty associated with producing suitable reagents, namely purified virus and polyclonal antibodies. In an effort to develop a consistent and standardised source of antibody, a monoclonal antibody to a recombinant BFDV capsid protein has been developed and its use in western blotting, immunohistochemistry (IHC), ELISA and haemagglutination-inhibition (HI) assays characterised. The antibody was specific for both the recombinant BFDV capsid protein and the whole virus and had similar optimal titres when used in western blotting and IHC. The antibody also had HI activity and detected BFDV virus from three genera of psittacine birds, including the recently described cockatiel BFDV isolate. The monoclonal antibody should have widespread application in both research and the development of diagnostic assays for BFDV.

Baculovirus expression of beak and feather disease virus (BFDV) capsid protein capable of self-assembly and haemagglutination.

Beak and feather disease virus (BFDV) is a common avian circovirus infection of wild Psittaciformes and is a recognised threat to endangered psittacine species. Currently, there is a requirement to develop BFDV antigen for diagnostic purposes and since efforts to propagate BFDV in vitro have so far been unsuccessful the entire coding region of BFDV ORF C1 was expressed in Sf9 insect cells using a baculovirus expression system. The entire coding region of BFDV ORF C1, the presumptive capsid, was expressed in Sf9 insect cells using baculovirus expression system. Electron microscopic examination of negatively stained material demonstrated that the recombinant protein self-assembled to produce virus-like particles (VLPs) thus confirming that ORF C1 is likely to be the sole determinant for capsid construction in vivo. BFDV VLPs also possessed haemagglutinating activity which provides further evidence that self-assembled BFDV VLPs retain receptor mediated biological activity and that the determinants for BFDV haemagglutination activity rely solely on the capsid protein. The recombinant protein reacted with anti-BFDV sera from naturally immune parrots and cockatoo and from chickens experimentally inoculated with native BFDV in both Western blots and haemagglutination inhibition (HI) assay. BFDV VLPs were also a suitable replacement antigen for serological detection of BFDV antibody by HI.