Borna disease virus 1 infection in alpacas: Comparison of pathological lesions and viral distribution to other dead-end hosts.

Borna disease is a progressive meningoencephalitis caused by spillover of the Borna disease virus 1 (BoDV-1) to horses and sheep and has gained attention due to its zoonotic potential. New World camelids are also highly susceptible to the disease; however, a comprehensive description of the pathological lesions and viral distribution is lacking for these hosts. Here, the authors describe the distribution and severity of inflammatory lesions in alpacas (n = 6) naturally affected by this disease in comparison to horses (n = 8) as known spillover hosts. In addition, the tissue and cellular distribution of the BoDV-1 was determined via immunohistochemistry and immunofluorescence. A predominant lymphocytic meningoencephalitis was diagnosed in all animals with differences regarding the severity of lesions. Alpacas and horses with a shorter disease duration showed more prominent lesions in the cerebrum and at the transition of the nervous to the glandular part of the pituitary gland, as compared to animals with longer disease progression. In both species, viral antigen was almost exclusively restricted to cells of the central and peripheral nervous systems, with the notable exception of virus-infected glandular cells of the Pars intermedia of the pituitary gland. Alpacas likely represent dead-end hosts similar to horses and other spillover hosts of BoDV-1.

Establishing and Maintaining an Etruscan Shrew Colony.

The Etruscan shrew (Suncus etruscus) is one of the smallest mammals on earth and is used in many fields of research, including physiology, behavioral science and neuroscience. However, establishing and maintaining a breeding colony of this species in the laboratory can be challenging, as it requires specific husbandry conditions that greatly differ from those of more common laboratory species such as mice or rats. Over the past 15 y, we have successfully established a long-term thriving colony of 150 to 200 animals originating from 36 founders. The colony shows longer life expectancy and larger litter sizes than wild conspecifics. Breeding occurs year-round, independent of seasons, and a breeding pair can regularly produce 2 to 6 offspring with an average life expectancy of more than 3 y. The shrews are housed in glass or plastic enclosures on a specific soil-sand-mixture bedding and are provided with hideouts and nesting material consisting of moss, wood, or bark. Due to their high basal metabolic rate, the shrews require food intake greater than their body weight per day, can hunt arthropods as large as themselves, and cannot survive more than a few hours without food. Live feed such as crickets or mealworms is crucial and must be provided daily or, at the very least, every 2 d. Although our husbandry practices have constantly been adapted and refined, shrew husbandry remains challenging, and great care is necessary to meet the specific needs of this species. Here, we describe the establishment of a long-term stable colony of Etruscan shrews in a research animal facility and the specific husbandry requirements for animal wellbeing.

Alternatives in Education-Evaluation of Rat Simulators in Laboratory Animal Training Courses from Participants' Perspective.

In laboratory animal science (LAS) education and training, five simulators are available for exercises on handling and routine procedures on the rat, which is-beside mice-the most commonly used species in LAS. Since these simulators may have high potential in protecting laboratory rats, the aim of this study is to investigate the simulators' impact on the 3R (replace, reduce, refine) principle in LAS education and training. Therefore, the simulators were evaluated by 332 course participants in 27 different LAS courses via a practical simulator training workshop and a paper-based two-part questionnaire-both integrated in the official LAS course schedule. The results showed a high positive resonance for simulator training and it was considered especially useful for the inexperienced. However, the current simulators may not completely replace exercises on live animals and improvements regarding more realistic simulators are demanded. In accordance with literature data on simulator-use also in other fields of education, more research on simulators and new developments are needed, particularly with the aim for a broad implementation in LAS education and training benefiting all 3Rs.

Alternatives in Education-Rat and Mouse Simulators Evaluated from Course Trainers' and Supervisors' Perspective.

Simulators allow the inexperienced to practice their skills prior to exercise on live animals. Therefore, they bear great potential in overcoming the dilemma between the present demand for high-quality practical training involving live animals whilst implementing the 3R principle according to the Directive 2010/63/EU. Currently, one mouse and six rat simulators are commercially available. As data on their impact are lacking, this project aimed at providing an overview of the awareness, implementation, and methodical and practical satisfaction provided by 35 course trainers and supervisors of laboratory animal training courses for mice and rats regarding the simulators available. Although simulators facilitate training of relevant techniques and relatively high awareness of them seemed to be present, their implementation is currently very low, possibly due to lack of meeting the respondents' demands. Thus, this study revealed the overall approval of simulator training and general demand for user-optimized, realistic, and financially affordable simulators and, hence, indicates a strong impulse for new developments strengthening the 3Rs as a benefit to all animals used in research.

Neoplasia and Tumor-Like Lesions in Pet Rabbits (Oryctolagus cuniculus): A Retrospective Analysis of Cases Between 1995 and 2019.

Prevalence and age distribution of tumors is largely unknown in pet rabbits. Currently available studies focused on specific organ systems or specific tumor types and never covered a comparative examination of all tumor types. Previous studies on laboratory rabbits suggested a low tumor prevalence but were mostly limited to young adult animals. In the present study, all tumor types and several tumor-like lesions of all organ systems were analyzed retrospectively in archived pet rabbit samples of all ages. Cases included necropsy cases (n = 2,014) or postmortem tissue samples (n = 102) as well as surgical biopsies (n = 854). All lesions suspicious of neoplasia were reevaluated by histopathology and, when indicated, by immunohistochemistry. Necropsy cases had a tumor prevalence of 14.4% in both sexes or 19.8% in female intact rabbits of all age groups, and up to 47.2% or 66.7%, respectively, in rabbits older than 6 years. Overall, the most common tumor types were uterine adenocarcinoma (prevalence in female intact animals: 13.1%), lymphoma (prevalence: 2.8%), and thymoma (prevalence: 2.1%). Lymphoma, the most common tumor of rabbits ≤24 months of age, were of B-cell immunophenotype in 96% of cases and most commonly located in the lymph nodes (57%), gastrointestinal tract (54%), kidneys (48%), spleen (42%), and liver (41%). Tumors accounted for 81.1% of surgical biopsies and mostly comprised cutaneous, mammary, and uterine tumors. In conclusion, tumor types and prevalence varied significantly with respect to age, revealing some differences from previous studies on laboratory rabbits.

Uterus duplex bicollis, Vagina simplex in laboratory Guinea pigs (Cavia porcellus), rats (Rattus norvegicus forma domestica) and mice (Mus musculus forma domestica).

The female genital tract originates from the Müllerian ducts during embryological development. Fusion of the ducts occurs in different segments depending on the animal species, resulting in a variational number of the respective organ. Current literature on genital tract morphology of laboratory rodents is controversial. Therefore, the present study aimed at determining an anatomically correct definition of the uterus in laboratory guinea pigs, mice and rats. In all three rodent species, we found two separate cervical canals that communicate with an individual uterus via discrete ostia uteri interna. The correct anatomical definition should therefore be uterus duplex bicollis, vagina simplex.

Effects of on-slide fixation on the cell quality of cytocentrifuged equine bronchioalveolar lavage fluid.

BACKGROUND: During bronchoalveolar lavage fluid (BALF) sample preparation in horses, several technical aspects can affect sample variability. To date, the effects of different fixatives on prepared equine BALF films have been insufficiently investigated. OBJECTIVES: This study aimed to determine the effect of various on-slide fixation methods on cell quality, including spray fixation of wet films, and acetone and methanol fixation of air-dried samples in comparison with unfixed, air-dried films. METHODS: Cytocentrifuged BALF samples from 5 horses were fixed in a wet state using a commercially available fixation spray. They were also fixed with acetone or methanol after air-drying using standard protocols or were air-dried with no fixation. After different postfixation storage durations and temperatures, the samples were stained with May-Grünwald Giemsa or immunocytochemistry stains. Subsequently, differential cell counts (DCCs) were performed, cell areas were measured, and cell morphologies and immunocytochemical staining intensities were assessed semiquantitatively. RESULTS: Optimal cell morphology results were achieved with the wet-spray fixation method. Acetone and methanol fixation, especially when performed at -20°C, caused reduced cell morphology quality, thereby significantly altering DCCs. For storage of unstained samples for 1 week at room temperature, no significant changes in cell morphology were observed for either fixation method. Wet-spray fixation resulted in enhanced preservation of macrophage, granulocyte, and mast cell sizes compared with air-drying techniques. Immunocytochemical staining of unfixed and acetone-fixed samples was the most intense. CONCLUSIONS: Wet-spray fixation resulted in the best preservation of cellular morphology and less cell shrinkage compared with unfixed specimens and is, therefore, recommended for BALF cytology.

Soluble mucus component CLCA1 modulates expression of leukotactic cytokines and BPIFA1 in murine alveolar macrophages but not in bone marrow-derived macrophages.

The secreted airway mucus cell protein chloride channel regulator, calcium-activated 1, CLCA1, plays a role in inflammatory respiratory diseases via as yet unidentified pathways. For example, deficiency of CLCA1 in a mouse model of acute pneumonia resulted in reduced cytokine expression with less leukocyte recruitment and the human CLCA1 was shown to be capable of activating macrophages in vitro. Translation of experimental data between human and mouse models has proven problematic due to several CLCA species-specific differences. We therefore characterized activation of macrophages by CLCA1 in detail in solely murine ex vivo and in vitro models. Only alveolar but not bone marrow-derived macrophages freshly isolated from C57BL6/J mice increased their expression levels of several pro-inflammatory and leukotactic cytokines upon CLCA1 stimulation. Among the most strongly regulated genes, we identified the host-protective and immunomodulatory airway mucus component BPIFA1, previously unknown to be expressed by airway macrophages. Furthermore, evidence from an in vivo Staphylococcus aureus pneumonia mouse model suggests that CLCA1 may also modify BPIFA1 expression in airway epithelial cells. Our data underscore and specify the role of mouse CLCA1 in inflammatory airway disease to activate airway macrophages. In addition to its ability to upregulate cytokine expression which explains previous observations in the Clca1-deficient S. aureus pneumonia mouse model, modulation of BPIFA1 expression expands the role of CLCA1 in airway disease to involvement in more complex downstream pathways, possibly including liquid homeostasis, airway protection, and antimicrobial defense.

Interspecies diversity of chloride channel regulators, calcium-activated 3 genes.

Members of the chloride channel regulators, calcium-activated (CLCA) family, have been implicated in diverse biomedical conditions, including chronic inflammatory airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, the activation of macrophages, and the growth and metastatic spread of tumor cells. Several observations, however, could not be repeated across species boundaries and increasing evidence suggests that select CLCA genes are particularly prone to dynamic species-specific evolvements. Here, we systematically characterized structural and expressional differences of the CLCA3 gene across mammalian species, revealing a spectrum of gene duplications, e.g., in mice and cows, and of gene silencing via diverse chromosomal modifications in pigs and many primates, including humans. In contrast, expression of a canonical CLCA3 protein from a single functional gene seems to be evolutionarily retained in carnivores, rabbits, guinea pigs, and horses. As an accepted asthma model, we chose the cat to establish the tissue and cellular expression pattern of the CLCA3 protein which was primarily found in mucin-producing cells of the respiratory tract and in stratified epithelia of the esophagus. Our results suggest that, among developmental differences in other CLCA genes, the CLCA3 gene possesses a particularly high dynamic evolutionary diversity with pivotal consequences for humans and other primates that seem to lack a CLCA3 protein. Our data also help to explain previous contradictory results on CLCA3 obtained from different species and warrant caution in extrapolating data from animal models in conditions where CLCA3 may be involved.

Role of goblet cell protein CLCA1 in murine DSS colitis.

BACKGROUND: The secreted goblet cell protein CLCA1 (chloride channel regulator, calcium-activated-1) is, in addition to its established role in epithelial chloride conductance regulation, thought to act as a multifunctional signaling protein, including cellular differentiation pathways and induction of mucus production. Specifically, CLCA1 has recently been shown to modulate early immune responses by regulation of cytokines. Here, we analyze the role of CLCA1, which is highly expressed and secreted by colon goblet cells, in the course of murine dextran sodium sulfate-induced colitis. FINDINGS: We compared Clca1-deficient and wild type mice under unchallenged and DSS-challenged conditions at various time points, including weight loss, colon weight-length-ratio and histological characterization of inflammation and regeneration. Expression levels of relevant cytokines, trefoil factor 3 and E-cadherin were assessed via quantitative PCR and cytometric bead arrays. Lack of CLCA1 was associated with a more than two-fold increased expression of Cxcl-1- and Il-17-mRNA during DSS colitis. However, no differences were found between Clca1-deficient and wild type mice under unchallenged or DSS-challenged conditions in terms of clinical findings, disease progression, colitis outcome, epithelial defects or regeneration. CONCLUSIONS: CLCA1 is involved in the modulation of cytokine responses in the colon, albeit differently than what had been observed in the lungs. Obviously, the pathways involved depend on the type of challenge, time point or tissue environment.

The Goblet Cell Protein Clca1 (Alias mClca3 or Gob-5) Is Not Required for Intestinal Mucus Synthesis, Structure and Barrier Function in Naive or DSS-Challenged Mice.

The secreted, goblet cell-derived protein Clca1 (chloride channel regulator, calcium-activated-1) has been linked to diseases with mucus overproduction, including asthma and cystic fibrosis. In the intestine Clca1 is found in the mucus with an abundance and expression pattern similar to Muc2, the major structural mucus component. We hypothesized that Clca1 is required for the synthesis, structure or barrier function of intestinal mucus and therefore compared wild type and Clca1-deficient mice under naive and at various time points of DSS (dextran sodium sulfate)-challenged conditions. The mucus phenotype in Clca1-deficient compared to wild type mice was systematically characterized by assessment of the mucus protein composition using proteomics, immunofluorescence and expression analysis of selected mucin genes on mRNA level. Mucus barrier integrity was assessed in-vivo by analysis of bacterial penetration into the mucus and translocation into sentinel organs combined analysis of the fecal microbiota and ex-vivo by assessment of mucus penetrability using beads. All of these assays revealed no relevant differences between wild type and Clca1-deficient mice under steady state or DSS-challenged conditions in mouse colon. Clca1 is not required for mucus synthesis, structure and barrier function in the murine colon.

Murine CLCA5 is uniquely expressed in distinct niches of airway epithelial cells.

The murine mCLCA5 protein is a member of the chloride channel regulators, calcium-activated (CLCA) family and is suspected to play a role in airway mucus cell differentiation. Although mCLCA5 mRNA was previously found in total lung extracts, the expressing cells and functions in the naive murine respiratory tract are unknown. Therefore, mCLCA5 protein expression was identified by immunohistochemistry and confocal laser scanning microscopy using entire lung sections of naive mice. Moreover, we determined mRNA levels of functionally related genes (mClca3, mClca5, Muc5ac and Muc5b) and quantified mCLCA5-, mCLCA3- and CC10-positive cells and periodic acid-Schiff-positive mucus cells in naive, PBS-treated or Staphylococcus aureus-infected mice. We also investigated mCLCA5 protein expression in Streptococcus pneumoniae and influenza virus lung infection models. Finally, we determined species-specific differences in the expression patterns of the murine mCLCA5 and its human and porcine orthologs, hCLCA2 and pCLCA2. The mCLCA5 protein is uniquely expressed in highly select bronchial epithelial cells and submucosal glands in naive mice, consistent with anatomical locations of progenitor cell niches. Under conditions of challenge (PBS, S. aureus, S. pneumoniae, influenza virus), mRNA and protein expression strongly declined with protein recovery only in models retaining intact epithelial cells. In contrast to mice, human and porcine bronchial epithelial cells do not express their respective mCLCA5 orthologs and submucosal glands had fewer expressing cells, indicative of fundamental differences in mice versus humans and pigs.