Lipofuscin accumulates in ganglionic neurons in chronic equine dysautonomia.

Lipofuscin is a complex mixture of highly oxidized, cross-linked macromolecules that accumulates in neurons with age and some neurodegenerative diseases. Equine dysautonomia (ED) is a polyneuropathy that mainly affects autonomic and enteric nervous systems, resulting in alimentary tract dysfunction. Our main aim was to determine whether neuronal lipofuscin increased with increasing duration of ED. We investigated the prevalence of lipofuscin in cranial cervical ganglia of horses with acute (AED), subacute (SED), and chronic ED (CED), young controls (of similar age to ED cases), and aged controls (n = 8 per group). We used Schmorl stain for histologic detection of lipofuscin and assessed its accumulation in neurons using image analysis software. The percentage of neurons positive for lipofuscin increased with age in individual groups and all groups combined (p < 0.001). There were fewer positive neurons in AED and SED compared to aged controls (p < 0.001) and more in CED than AED cases (p = 0.042) and young controls (p = 0.012). We found a strong positive correlation between percentage positive neurons and percentage positive area of the neuron containing lipofuscin for combined groups (p < 0.001). Although neuronal lipofuscin increased in cranial cervical ganglion in CED cases, it remains to be determined whether this is a cause or consequence of neuronal degeneration.

Calf Management: Individual or Paired Housing Affects Dairy Calf Health and Welfare.

Previous research has indicated that preweaned dairy calves reared in pairs compared with individually have improved performance and indicators of animal welfare. One hundred and thirty Holstein female calves completed the trial, with eighty-five being allocated to paired housing and forty-five calves being allocated to individual housing. Daily live weight gain (DLWG), treatments and mortality were recorded throughout the preweaning period. Salivary cortisol, latency to feed and latency to approach a novel object were assessed at batching. There were no significant differences in DLWG, mortality and disease treatments between the average of the pair and the individually housed calves, although the pair-reared calves were quicker to approach the milk feed after batching and interacted more quickly with a novel object. The heaviest born calves within the pair had the highest DLWG from birth to weaning, with a higher percentage of calves approaching the novel object, compared with the lightest born calf within the pair. This study shows that calves within a pair may have significantly different performance and welfare during the preweaning period, with the heavier calf outperforming and displaying less fear and more exploratory behaviour than the lighter calf within a pair.

Cytological evaluation, culture and genomics to evaluate the microbiome in healthy rabbit external ear canals.

BACKGROUND: Lop-eared rabbits may be predisposed to otitis externa (OE) as a consequence of their ear conformation. Although otoscopy, otic cytological evaluation and culture are valuable tools in dogs and cats, published data on rabbits remain lacking. HYPOTHESIS/OBJECTIVES: This study aimed to assess the utility of otoscopy and cytological results in evaluating healthy rabbit external ear canals (EECs) and to characterise ear cytological and microbiological findings through culture techniques and metagenomic sequencing. ANIMALS: Sixty-three otitis-free client-owned rabbits. MATERIALS AND METHODS: All rabbits underwent otoscopy and ear cytological evaluation. In a subset of 12 rabbits, further bacterial and fungal culture, fungal DNA assessment and metagenomic sequencing were performed. RESULTS: Otic cytological results revealed yeast in 73%, cocci in 42.9% and rods in 28.6% of healthy rabbit EECs. Compared to upright-eared rabbits, lop-eared rabbits had more discharge and more bacteria per oil immersion field. Culture isolated eight different species yet metagenomic sequencing identified 36, belonging to the Bacillota (Firmicutes), Pseudomonadota and Actinomycetota phyla. Staphylococcus were the most commonly observed species with both methods. Ten of 12 rabbits were yeast-positive on cytological evaluation with only three yielding fungal growth identified as Yarrowia (Candida) lipolytica, Eurotium echinulatum and Cystofilobasidium infirmominiatum. CONCLUSIONS AND CLINICAL RELEVANCE: Healthy rabbit EECs lack inflammatory cells yet can host yeast and bacteria, emphasising the need to evaluate cytological results alongside the clinical signs. Lop-ear anatomy may predispose to bacterial overgrowth and OE. Notably, yeasts may be present despite a negative culture.

Diagnostic accuracy of the Enferplex Bovine TB antibody test using individual milk samples from cattle.

Bovine tuberculosis is usually diagnosed using tuberculin skin tests or at post-mortem. Recently, we have developed a serological test for bovine tuberculosis in cattle which shows a high degree of accuracy using serum samples. Here, we have assessed the performance of the test using individual bovine milk samples. The diagnostic specificity estimate using the high sensitivity setting of the test was 99.7% (95% CI: 99.2-99.9). This estimate was not altered significantly by tuberculin boosting. The relative sensitivity estimates of the test using the high sensitivity setting in milk samples from comparative skin test positive animals was 90.8% (95% CI: 87.1-93.6) with boosting. In animals with lesions, the relative sensitivity was 96.0% (95% CI: 89.6-98.7). Analysis of paired serum and milk samples from skin test positive animals showed correlation coefficients ranging from 0.756-0.955 for individual antigens used in the test. Kappa analysis indicated almost perfect agreement between serum and milk results, while McNemar marginal homogeneity analysis showed no statistically significant differences between the two media. The positive and negative likelihood ratio were 347.8 (95% CI: 112.3-1077.5) and 0.092 (95% CI: 0.07-0.13) respectively for boosted samples from skin test positive animals. The results show that the test has high sensitivity and specificity in individual milk samples and thus milk samples could be used for the diagnosis of bovine tuberculosis.

Equine grass sickness is associated with major abnormalities in the ultrastructure of skeletal neuromuscular junctions.

BACKGROUND: Equine grass sickness (EGS) is a frequently fatal multisystem neuropathy of equids. The aetiology is unknown; proposed causes include toxicoinfection with Clostridium botulinum and a mycotoxicosis. The effect of EGS on the organisation and structural integrity of the skeletal neuromuscular junction (NMJ), the target of botulinum neurotoxins (BoNTs), is unknown. OBJECTIVES: To compare the organisation and structural integrity of skeletal NMJs from EGS horses, control horses and one horse with a presumptive diagnosis of botulism. STUDY DESIGN: Blinded, retrospective case control. METHODS: NMJs in samples of diaphragm or intercostal muscle from six EGS horses, three control horses and one equine botulism case were compared using electron microscopy, morphometry and confocal light microscopy. RESULTS: A significantly higher percentage of EGS NMJs had abnormal morphology (EGS 72.2%, 95% CI 55.6-84.4; Controls 6.9%, 1.7-23.8; OR 35.1, 8.47-244.8; p < 0.001). EGS NMJs had a significantly lower mean volume fraction occupied by synaptic vesicles (SVs) (EGS 18.7%, 12.6-28.0; Controls 36.3%, 20.8-63.4; p = 0.024). EGS NMJs had evidence of accelerated SV exocytosis and SV depletion, accumulation of neurofilament-like material in terminal boutons and/or bouton degeneration. NMJs from the botulism horse had dense packing of SVs towards the presynaptic membrane active zone, consistent with BoNT intoxication, but had absence of the abnormalities identified in EGS NMJs. MAIN LIMITATIONS: Group sizes were limited by difficulties obtaining suitably processed samples. Ages of control and EGS horses differed. Botulism was diagnosed based on clinical and post mortem findings. CONCLUSIONS: EGS is associated with major changes in skeletal NMJ ultrastructure that are inconsistent with the effects of BoNTs. SV depletion may reflect increased exocytosis coupled with reduced repopulation of SVs via anterograde axonal transport and endocytosis, consistent with the action of an excitatory presynaptic toxin and/or neurotransmitter reuptake inhibitor. Skeletal NMJs represent a previously unrecognised target for the toxin that causes EGS.

Latent gammaherpesvirus infection enhances type I IFN response and reduces virus spread in an influenza A virus co-infection model.

Infections with persistent or latent viruses alter host immune homeostasis and have potential to affect the outcome of concomitant acute viral infections such as influenza A virus (IAV). Gammaherpesviruses establish life-long infections and require an on-going immune response to control reactivation. We have used a murine model of co-infection to investigate the response to IAV infection in mice latently infected with the gammaherpesvirus MHV-68. Over the course of infection, latently infected BALB/c mice showed less weight loss, clinical signs, pulmonary cellular infiltration and expression of inflammatory mediators than naïve mice infected with IAV and had significantly more activated CD8+ T cells in the lungs. Four days after IAV infection, virus spread in the lungs of latently infected animals was significantly lower than in naïve animals. By 7 days after IAV infection latently infected lungs express elevated levels of cytokines and chemokines indicating they are primed to respond to the secondary infection. Investigation at an early time point showed that 24 h after IAV infection co-infected animals had higher expression of IFNß and Ddx58 (RIG-I) and a range of ISGs than mice infected with IAV alone suggesting that the type I IFN response plays a role in the protective effect. This effect was mouse strain dependent and did not occur in 129/Sv/Ev mice. These results offer insight into innate immune mechanisms that could be utilized to protect against IAV infection and highlight on-going and persistent viral infections as a significant factor impacting the severity of acute respiratory infections.

Testing anatomy: Dissecting spatial and non-spatial knowledge in multiple-choice question assessment.

Limited research has been conducted on the spatial ability of veterinary students and how this is evaluated within anatomy assessments. This study describes the creation and evaluation of a split design multiple-choice question (MCQ) assessment (totaling 30 questions divided into 15 non-spatial MCQs and 15 spatial MCQs). Two cohorts were tested, one cohort received a 2D teaching method in the academic year 2014/15 (male = 15/108, female 93/108), and the second a 3D teaching method in the academic year 2015/16 (male 14/98, female 84/98). The evaluation of the MCQ demonstrated strong reliability (KR-20 = 0.71 2D and 0.63 3D) meaning the MCQ consistently tests the same construct. Factor analysis of the MCQ provides evidence of validity of the split design of the assessment (RR = 1.11, p = 0.013). Neither cohort outperformed on the non-spatial questions (p > 0.05), however, the 3D cohort performed statistically significantly higher on the spatial questions (p = 0.013). The results of this research support the design of a new anatomy assessment aimed at testing both anatomy knowledge and the problem-solving aspects of anatomical spatial ability. Furthermore, a 3D teaching method was shown to increase students' performance on anatomy questions testing spatial ability.

Response and survival of dogs with proteinuria (UPC > 2.0) treated with angiotensin converting enzyme inhibitors.

BACKGROUND: Angiotensin-converting enzyme inhibitors (ACEi) are a recommended treatment for glomerular proteinuria. Frequency of response to ACEi and the association of achieving proposed urine protein-to-creatinine ratio (UPC) targets on survival is unknown. OBJECTIVES: To determine response rates to ACEi therapy and whether a positive response is associated with improved survival. ANIMALS: Eighty-five dogs with proteinuria (UPC > 2.0). METHODS: Retrospective study including dogs (UPC > 2.0) prescribed an ACEi for treatment of proteinuria. Baseline creatinine, albumin, cholesterol, UPC, and systolic blood pressure were recorded, and cases reviewed to track UPC. Treatment response was defined as achieving a UPC of <0.5 or reduction of ≥50% from baseline within 3 months. Outcome data were collected to determine overall and 12-month survival. RESULTS: Thirty-five (41%) dogs responded to ACEi treatment. Treatment response was statistically associated with both median survival time (664 days [95% confidence interval (CI): 459-869] for responders compared to 177 [95% CI: 131-223] for non-responders) and 12-month survival (79% responders alive compared to 28% non-responders). Baseline azotemia or hypoalbuminemia were also associated with a worse prognosis, with odds ratios of death at 12 months of 5.34 (CI: 1.85-17.32) and 4.51 (CI: 1.66-13.14), respectively. In the 25 dogs with normal baseline creatinine and albumin, response to treatment was associated with 12-month survival (92% responders alive compared to 54% non-responders, P = .04). CONCLUSIONS AND CLINICAL IMPORTANCE: When the UPC is >2.0, achieving recommended UPC targets within 3 months appears to be associated with a significant survival benefit. Response to treatment is still associated with survival benefit in dogs with less severe disease (no azotemia or hypoalbuminemia).

Protective Efficacy of BCG Vaccination in Calves Vaccinated at Different Ages.

Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), is a globally prevalent pathogen with significant animal welfare, economic and public health impacts. In the UK, the control of bTB relies on detection via tuberculin skin tests with ancillary interferon gamma (IFN-γ) release assays, followed by culling infected animals. Vaccination with Bacille Calmette-Guérin (BCG) could be an important element of bTB control, and a number of studies have demonstrated its protective efficacy, particularly when young calves are vaccinated. Here, we compared immune responses and the protective efficacy of BCG in calves vaccinated within the first day of life and at three weeks of age. Significant protection from M. bovis infection was observed in BCG-vaccinated calves compared to non-vaccinated, age-matched controls. No significant differences were shown between calves vaccinated at one day and at three weeks of age when assessing the protective efficacy of BCG (measured as a reduction in lesions and bacterial burden). Antigen-specific IFN-γ levels were similar between the BCG-vaccinated groups, but significantly different from the non-vaccinated control animals. Antigen-specific IFN-γ expression post-BCG vaccination was correlated significantly with protection from M. bovis infection, whereas IFN-γ levels post-challenge correlated with pathology and bacterial burden. These results indicate that early-life vaccination with BCG could have a significant impact on M. bovis infection and, therefore, bTB incidence, and they demonstrate that age, at least within the first month of life, does not significantly impact the protective effect of vaccination.

Diagnostic accuracy of the Enferplex Bovine Tuberculosis antibody test in cattle sera.

Bovine tuberculosis is a contagious bacterial disease of worldwide economic, zoonotic and welfare importance caused mainly by Mycobacterium bovis infection. Current regulatory diagnostic methods lack sensitivity and require improvement. We have developed a multiplex serological test for bovine tuberculosis and here we provide an estimate of the diagnostic accuracy of the test in cattle. Positive and negative reference serum samples were obtained from animals from Europe and the United States of America. The diagnostic specificity estimate was 98.4% and 99.7% using high sensitivity and high specificity settings of the test respectively. Tuberculin boosting did not affect the overall specificity estimate. The diagnostic sensitivity in samples from Mycobacterium bovis culture positive animals following tuberculin boosting was 93.9%.The relative sensitivity following boosting in tuberculin test positive, lesion positive animals and interferon gamma test positive, lesion positive animals was 97.2% and 96.9% respectively. In tuberculin test negative, lesion positive animals and in interferon gamma test negative, lesion positive animals, the relative sensitivity following tuberculin boosting was 88.2% and 83.6% respectively. The results show that the test has high diagnostic sensitivity and specificity and can detect infected animals that are missed by tuberculin and interferon gamma testing.

Exploration of cultural norms and behavioural beliefs about zoo animal behaviour, welfare, ethics and husbandry practices in a sample of the international zoo community.

Beliefs influence the intentions of people to behave in certain ways towards animals. This study presents survey responses from 237 people working in zoos in China and Europe and describes their demographic characteristics. It explores their beliefs about zoo animal behaviour, welfare and ethical issues, and zoo practices, using a survey methodology. These beliefs may be influenced by individual demographic or cultural factors such as age, gender and region of employment, as well as experiential or situative 'norms' within the work environment. Beliefs were significantly influenced by the region of employment with Chinese respondents beliefs being significantly different to beliefs from respondents in the United Kingdom or the rest of Europe. Hierarchical cluster analysis of the survey generated clusters of people from both regions who indicated positive beliefs about zoo animal welfare as well as clu sters indicating a lack of understanding of some zoo animal welfare issues. In addition, a cluster suggesting cognitive dissonance between beliefs about animals welfare and zoo practices was generated from Chinese responses. Factor analysis identified that prioritisation of in-situ conservation within good animal welfare was a key feature in Chinese respondents, whereas European beliefs prioritising in-situ conservation were distinct from those on supporting good animal welfare. This paper identifies similarities and differences in beliefs about zoo animal welfare and zoo husbandry practices between Europe and China, and discusses the underlying norms and values that these beliefs may reflect.

Zookeepers - The most important animal in the zoo?

Education to improve knowledge of animal welfare is not a universal component of training for zoo staff, and little is reported about the perspectives of zoo staff on the need for such education. This paper reports results from structured telephone interviews of a diverse sample of eight Chinese and eight European zoo staff about aspects of zoological animal welfare, education and zoological practices. These qualitative data were thematically analyzed and key themes generated. Similar themes emerged across regions: Zoo staff consider professional attributes including motivation and enthusiasm to be important alongside formal training, zoo staff value learning opportunities but don't always feel supported, and contextual information including wild animal ecology was considered important content in zoo animal welfare education.

Animal welfare requirements in publishing guidelines.

Descriptions of measures taken to optimize animal welfare are often absent from scientific reports of animal experiments. One reason may be that journal guidelines inadequately compel authors to provide such information. In this study, online English language versions of the 'Guidelines to authors' (GTAs) from 54 national biomedical journals were examined for neutral (unrelated to welfare) and non-neutral keywords referring to: animal welfare; the '3Rs'; the ARRIVE (2010) guidelines, and regulations pertaining to animal experimentation. Journals were selected from nine countries (UK, US, China, Canada, India, Brazil, Germany, Japan and Australia) and seven biomedical specialties (oncology, rheumatology, surgery, pharmacology, medicine, anaesthesia and veterinary medicine). Total GTA word counts varied from 1137 to 31,609. The keyword count identified per category were expressed per myriad (10,000) of total word count. One-way analyses of variance followed by post hoc Tukey pairwise comparisons revealed greater non-neutral per myriad word counts for (a) veterinary GTAs compared with medicine, oncology, rheumatology or surgery; (b) British, compared with Australian, Canadian, German and Japanese GTAs; and (c) no differences between non-neutral categories. The English language versions of GTAs of British and veterinary medical journals contain more words associated with animal welfare, the 3Rs and the ARRIVE guidelines than those from eight other countries and six other medical specialities. The exclusion of 'national' language versions from analysis precludes attempts to identify national differences in attitudes to laboratory animal welfare.

Real-time three-dimensional echocardiography for left atrial volume assessment in Thoroughbred racehorses: Observer variability and comparison with two-dimensional echocardiography.

BACKGROUND: Left atrial size predicts cardiac morbidity and mortality in humans and dogs. Real-time three-dimensional echocardiography (3DE) may be reliable for assessing left atrial volume (LAV) in horses. OBJECTIVES: To determine intra- and interobserver variability estimates of 3DE-LAV and compare it to that of 2DE-LAV estimates. STUDY DESIGN: Method comparison. METHODS: 3DE datasets were obtained from 40 horses, then graded for quality, creating a final study population of 22 horses. The 3DE and 2DE maximum LAV (LAVmax ) and minimum LAV (LAVmin ) were measured, and left atrial emptying volume (LA EV) and left atrial ejection fraction (LA EF) were calculated, from the same 3D dataset on four occasions using (a) a semi-automatic surface recognition algorithm and (b) a modified Simpson's method of discs. 3DE LAV measurements were repeated by a second observer. RESULTS: For 3DE, median LAVmax was 596cm3 for observer one, and 852 cm3 for observer two, LAVmin was 373 cm3 for observer one and 533 cm3 for observer two. Low intraobserver measurement variation was observed for LAVmax and LAVmin , with horse-level intraclass correlation coefficients (ICChorse ) for both observers between 76% and 85% (horse added as random effect). The interobserver ICC was 58% for LAVmax and 50% for LAVmin on averaged measurements (with observer added as random effect), indicating consistent differences between observers. While intraobserver variation was similar for 2DE LAVmax measurements, it was greater for LAVmin (ICChorse  = 67%). The intermethod ICC for 3DE vs 2DE was low at 14% for LAVmax and ~0% for LAVmin , indicating less-consistent differences with method. MAIN LIMITATIONS: Small study population, low observer number, use of different imaging modalities (fundamental frequency and octave harmonics). CONCLUSIONS: 3DE assessment of LAV was reliable, suggesting suitability for longitudinal evaluation of clinical cases. Clinicians should be aware of differences in LAV measurements between observers. More defined measurement guidelines may improve repeatability.

Is Animal Welfare an Internationally Understood Concept in the Zoo World? Thematic Analysis of Two Regional Groups of Zoo Staff.

Universal frameworks for zoo animal welfare have been suggested. However, there is little evidence of a cross-cultural understanding of zoo animal welfare. This paper reports themes emerging from a qualitative study of international (European and Chinese) zoo professionals on zoo animal welfare issues. Structured telephone interviews were conducted with eight Chinese and eight European zoo staff, covering aspects of zoological animal welfare, conservation and zoological husbandry practices. These qualitative data were thematically analysed, and key themes generated. This paper describes three themes relating to 'What is animal welfare' 'Animal welfare frameworks' and 'The human element in animal welfare'. This analysis indicates that the concept of animal welfare has cultural equivalence across Europe and between Europe and China, and that zoo staff are familiar with welfare frameworks. In China, a need for senior leadership and motivating staff to improve animal welfare emerged.

CLINICAL PROGRESSION OF LEPROSY IN EURASIAN RED SQUIRRELS (SCIURUS VULGARIS) IN A NATURALLY INFECTED WILD POPULATION.

Leprosy has been described in Eurasian red squirrel (Sciurus vulgaris; ERS) carcasses since 2014. Studies of ERS carcasses have not provided information about incubation or disease progression in this host but have provided important insights into pathogen presence and distribution throughout the United Kingdom. Here we present field study data on 31 live ERS from an island population naturally infected with Mycobacterium leprae that were assessed longitudinally over a 2-yr time period. Clinical assessment, serologic (anti-phenolic glycolipid-I antibody [αPGL-I] detection) and molecular methods (polymerase chain reaction) were used to diagnose and categorize ERS at each assessment as a leprosy case, a leprosy suspect, colonized by M. leprae, or a contact ERS. Eight ERS (25.8%) were identified as leprosy cases: four at initial assessment, two at 6 mon and two at 24 mon after initial assessment. One ERS was categorized a leprosy suspect when it developed typical lesions 12 mon after initial assessment, despite negative serologic and molecular test results at this time, though M. leprae DNA had been isolated during the initial assessment. Seven ERS (22.6%) were categorized as colonized and of these, six were reassessed but did not develop clinical signs of leprosy within 6 (n = 2), 12 (n = 3), and 18 (n = 1) mon. Most (48.4%, n = 15) were categorized as contact ERS. Progression of leprosy lesions varied between ERS, but always increased in severity over time and was paralleled with increased antibody response. Based on our dataset, we propose the hypotheses: 1) leprosy in ERS is a chronic, slowly progressing disease in this species, similar to that described for other hosts; 2) lesions can undergo repeated ulceration-healing cycles; and 3) in some instances M. leprae DNA and αPGL-I antibodies are detectable before the onset of clinical signs of disease. Future studies addressing the progression of leprosy in ERS should follow affected animals over a longer time period and include tissue samples to pair molecular diagnostics with serologic results.

MRI of the optic nerve sheath and globe in cats with and without presumed intracranial hypertension.

OBJECTIVES: The study aimed to: (1) test MRI repeatability of measurements of optic nerve sheath diameter (ONSD), optic nerve diameter (OND) and eye globe transverse diameter (ETD); (2) investigate the associations between the OND, ONSD and ETD; (3) assess whether these measurements are affected by age or body weight; and (4) test the association between ONSD, OND, ETD and ONSD:ETD ratio with presumed intracranial pressure (ICP) status. METHODS: This was a retrospective and blinded study where patients were allocated to presumed normal or intracranial hypertension groups based on MRI findings. The ONSD and ETD were measured and recorded. Interclass correlation coefficient (ICC) was calculated to investigate interobserver agreement. Data were analysed using the Pearson correlation coefficient, two-sample t-test and general linear model ANOVA. RESULTS: Seventy-seven cats were included, 62 with presumed normal ICP and 15 with presumed intracranial hypertension. The ICC showed moderate-to-good reliability for all measurements. Positive correlations were identified for: (1) ETD and weight; (2) ONSD and age; (3) OND and age; (4) ONSD and ETD; (5) ONSD:ETD ratio and presumed ICP status; and (6) ONSD and presumed ICP status. No difference was detected between the presumed normal and intracranial hypertension groups and ONSD, as well as ONSD:ETD ratio and presumed ICP status when patient age was considered. CONCLUSIONS AND RELEVANCE: The measurement of the ONSD and the ONSD:ETD ratio on T2-weighted MRI might not be reliable as non-invasive tests for diagnosing intracranial hypertension in cats.

The interaction of Escherichia coli O157 :H7 and Salmonella Typhimurium flagella with host cell membranes and cytoskeletal components.

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of the ligands and mechanisms of binding are still debated. In this study, the molecular basis of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. Scanning electron microscopy revealed flagella disappearing into cellular surfaces and transmission electron microscopy of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6) and S. Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion for E. coli O157:H7 and S. Typhimurium, respectively.

Anatomical distribution of respiratory tract leukocyte cell subsets in neonatal calves.

Neonatal calves are highly susceptible to a number of diseases including those that infect via the mucosal surfaces of the respiratory and gastrointestinal tracts. In order to determine appropriate vaccine design and delivery systems, or to identify suitable immunostimulatory methods to combat these infections, a detailed understanding of the immune cell populations present at clinically relevant sites is key. Few studies have assessed the immune cell composition of the neonatal calf lung and comparisons with circulating immune cells in the blood are lacking. We describe immune cell populations present in the peripheral blood, bronchoalveolar lavage (BAL) fluid and lung tissue of young disease-free calves. Flow cytometric analysis revealed significant differences in cell subset distribution between the peripheral blood and respiratory tract, and between compartments within the respiratory tract. Notably, whereas WC1+ γδ TCR + T lymphocytes dominate the peripheral blood, both the BAL fluid and lung tissue contained a high proportion of myeloid cells which expressed CD14 and CD172a (SIRPα). Very low numbers of tissue myeloid cells expressed MHC Class II in comparison to circulating myeloid cells in the blood. Respiratory tract tissues had low frequencies of CD4+ and CD8 + T lymphocytes, which were significantly lower than in the blood. Differences in the proportion of NKp46+ natural killer cells were also observed between tissue compartments. In order to target vaccines or immunostimulatory therapeutics appropriately, these differences in immune cell populations in tissue compartments should be taken into consideration.