Development of superficial lung lesions monitored on farm by serial ultrasonographic examination in sheep with lesions confirmed as ovine pulmonary adenocarcinoma at necropsy.

BACKGROUND: This ultrasonographic study monitored lesions involving the lung surface suspected to be the early stages of ovine pulmonary adenocarcinoma (OPA) tumours over 4 months in commercially farmed sheep. The enlargement of these lesions defined ultrasonographically, which likely represent the development of OPA tumours, have important implications for ultrasound screening schedules in veterinary management plans attempting to eliminate OPA by test-and-cull. RESULTS: The lungs of 58 adult Scottish Blackface sheep with ultrasonographic changes at the lung surface consistent with early OPA tumours were examined two to six times over 40 to 290 days. Lesion development, represented in early video recordings by 2-3 mm lesions involving the visceral pleural and comet tails, then a decreasing length of the hyperechoic line representing the normal visceral pleura and increasing depth of the sharply-demarcated and largely uniform hypoechoic areas into the lung parenchyma, was found in 26 of the 58 sheep. The rate at which the sonographic lesions progressed varied considerably and in 10 of 17 Group 1 sheep developed quickly from an estimated depth of 2-30 mm up to 70 mm between 60 and 120 days later. These sonographic lesions were confirmed as OPA at necropsy; histological changes of concurrent bacterial infection were detected in one of these 10 Group 1 sheep. Thirty-one sheep had sonographic changes ≤30 mm consistent with very early OPA at the first examination which had reduced or were not observed at subsequent examination. Five of these 31 sheep were necropsied, 3 had small OPA lesions while 2 had no significant pathology. CONCLUSION: Lesions involving the visceral pleura, with sonographic changes consistent with previous published findings of early OPA, developed over 40-120 days to large masses in 10 of 17 Group 1 sheep with the provisional sonographic diagnosis confirmed histologically at necropsy. While it is possible that atalectic lung could have caused some of the minor sonographic changes there was no microscopic evidence of pathologies other than OPA in nine of 10 Group 1 sheep. We conclude that some small tumours progress to large tumours within 3 months questioning the assumption that OPA is a slow growing tumour in adult sheep taking several years to cause clinical disease. The findings that a proportion of small ultrasonographic lesions are not found again at subsequent scanning illustrates the challenges of interpreting small (< 1-2 cm) lesions during rapid whole flock ultrasonographic examination and we continue to recommend re-scanning suspicious sonographic changes 2 months later.

Complex Gill Disease: an Emerging Syndrome in Farmed Atlantic Salmon (Salmo salar L.).

Gill disorders have become a significant problem during the marine phase of farming Atlantic salmon (Salmo salar L.). The term complex gill disease (CGD) includes a wide range of clinical gill disease presentations generally occurring from the end of summer to early winter on marine Atlantic salmon farms. The gross and histological lesions observed are the resultant culmination of exposure to a mixture of environmental insults, pathogenic organisms and farm management practices. None of the three principal agents purportedly associated with CGD (Desmozoon lepeophtherii, salmon gill poxvirus or Candidatus Branchiomonas cysticola) have been cultured successfully in-vitro, so individual in-vivo challenge studies to identify their pathogenesis have not been possible. Studies of cohabitation of single pathogen-infected fish with naïve fish, and epidemiological investigations are required urgently to elucidate the roles of these pathogens and other factors in CGD.

Differences in Virulence Between Bovine-Derived Clinical Isolates of Pasteurella multocida Serotype A from the UK and the USA in a Model of Bovine Pneumonic Pasteurellosis.

The time of onset and subsequent degree and progression of clinical signs, bacterial colonization and tissue pathology during experimental disease induced by intratracheal inoculation of either a UK or USA isolate of Pasteurella multocida serotype A recovered from clinical cases of bovine pneumonia were determined. Calves aged 8 weeks were challenged with 300 ml phosphate buffered saline (PBS) alone (group 1, n = 3, negative control) or containing 7.1 × 10(8) colony forming units (cfu) of UK isolate (group 2, n = 8) or 5.8 × 10(8) cfu of USA isolate (group 3, n = 8). Bronchoalveolar lavage (BAL) at 0, 1 and 4 days post challenge (dpc) and at the time of necropsy examination (7-8 dpc) showed no significant differences between groups 2 and 3 in bacterial numbers recovered. No P. multocida were recovered from group 1 animals. No clinical disease was present in group 1 calves and in group 3 was limited to scour in 1 calf at 1 dpc. All calves in group 2 had reduced food intake at 4-5 dpc, five had periods of dullness, three a mild nasal discharge at 1 dpc, four had mild to substantial respiratory stridor and one was killed at 6 dpc for humane reasons. Rectal temperatures remained about 39°C in group 1 calves, but increased in P. multocida-challenged calves to 40-41°C within 8-12 h of challenge. Significantly (P = 0.01) greater percentages of lung surface area were consolidated in group 2 (mean ± SD, 21 ± 10.1) compared with group 3 (7 ± 8.6) calves. Significantly more extensive and severe histological lesions were present in the lung lobes (P = 0.006) and lymph nodes (P = 0.02) of group 2 compared with group 3 calves. Pleurisy was present in group 2 calves only and no pathology was present in group 1. Pulsed-field gel electrophoresis (PFGE) produced 11 (group 2, UK isolate) or 10 (group 3, USA isolate) bands with differences in banding patterns. Results overall showed that two isolates, distinct geographically and genetically (by PFGE), caused pneumonic pasteurellosis in a single host with significantly different severity of pathology. This information is relevant to the development of novel vaccine control and interpretation of diagnostic results.

Primitive neuroectodermal tumour in a striped dolphin (Stenella coeruleoalba) with features of ependymoma and neural tube differentiation (Medulloepithelioma).

Primary brain tumours in cetaceans are rare with only four reported cases of intracranial tumours in the scientific literature. A juvenile female, striped dolphin live-stranded at Whitepark Bay, Co Antrim, Northern Ireland, UK, and died after an unsuccessful attempt at refloatation. Necropsy examination revealed a large, soft, non-encapsulated friable mass, which expanded and replaced the frontal lobes, corpus callosum and caudate nucleus of the brain and extended into the lateral ventricles, displacing the thalamus caudally. Microscopically, this comprised moderately pleomorphic neoplastic cells arranged variably in dense monotonous sheets, irregular streams, ependymal rosettes, 'ependymoblastomatous rosettes' and multilayered to pseudostratified tubules. Liquefactive necrosis, palisading glial cells, haemorrhage and mineralization were also observed. Immunohistochemically, the neoplastic cells expressed vimentin but not S100, glial fibrillary acidic protein, cytokeratin, neuron-specific enolase or synaptophysin. Based on these findings a diagnosis of primitive neuroectodermal tumour was made. Monitoring and recording such cases is crucial as neoplasia may be related to viral, carcinogenic or immunosuppressive chemical exposure and can ultimately contribute to assessing the ocean health.

Pathogenesis of scrapie in ARQ/ARQ sheep after subcutaneous infection: effect of lymphadenectomy and immune cell subset changes in relation to prion protein accumulation.

It is well established that the infectious agent of scrapie can replicate in the lymphoreticular system (LRS). However, the effects of removal of LRS target tissues on the pathogenesis of the infection and the accumulation of disease-associated prion protein (PrP(d)) in LRS tissues on specific immune cell subsets are poorly understood aspects. To address these questions 16 ARQ/ARQ sheep were subcutaneously inoculated in the drainage area of the prefemoral lymph node with brain homogenate derived from Suffolk sheep naturally infected with scrapie. Fourteen sheep were then subjected to either early (14-17 days post-inoculation [dpi]) or late (175-201 dpi) lymphadenectomy and culled at preclinical or clinical stages of infection. Neither late nor even early lymphadenectomy prevented infection or had any effect on the accumulation of PrP(d) in the LRS or CNS suggesting a rapid organic dissemination of the infectious agent after inoculation. Lymph nodes from eight scrapie inoculated sheep selected on the basis of the amount of PrP(d) in their LRS tissues (negative, low or high) were examined for six different immune cell markers. The PrP(d) negative lymph nodes from two sheep with no evidence of scrapie infection showed lower numbers of cluster of determination (CD) 21 positive cells than PrP(d) positive nodes, irrespective of their location (hind leg or head). However, quantitative differences in the expression of this marker were not detected when comparing lymph nodes with low and high levels of PrP(d) accumulation, suggesting that proliferation of CD21 positive cells is related to scrapie infection, but not directly linked to the magnitude of PrP(d) accumulation. An additional observation of the study was that sheep that were methionin-threonine at codon 112 of the prion protein gene showed lower attack rates than methionine homozygotes (67% and 100%, respectively) and also generally lower levels of PrP(d) accumulation in the LRS and brain and increased survival times, suggesting an influence of such polymorphism in the susceptibility to scrapie.

Cellular differentiation and proliferation in the ovine lung during gestation and early postnatal development.

This study investigates epithelial cell differentiation and proliferation in specific anatomical regions of the ovine lung during prenatal and postnatal development. Immunohistochemistry was used to identify ciliated epithelial cells, Clara cells, neuroepithelial bodies and type II pneumocytes in the lungs of preterm (67, 127 and 140 days of gestation), full-term (147 days) and postnatal (9, 16 and 91 days old) lambs. Differentiation of ciliated epithelial cells was seen at 67 days of gestation and at term for Clara cells. Neuroepithelial bodies were first detected at 127 days of gestation. From 16 to 91 days of age there was a significant (P <0.05) increase in beta-tubulin (present in ciliated epithelial cells) and Clara cell protein (present in Clara cells) in multiple regions of the lung. Detection of Ki67, a marker of proliferation, in preterm lambs showed a reduction in proliferation index in multiple anatomical regions of the lung between 70 days of gestation and term. Cell proliferation increased following parturition, and then decreased between 16 and 91 days of age, with the largest reduction occurring in the alveolar compartment. Knowledge of which cells are present at specific times of lung development provides valuable information on the anatomy of the ovine lung, improving its use as a model for ovine and human neonatal disease. In addition, the antibodies used here will be valuable for future studies requiring the identification and quantification of respiratory epithelial cell phenotypes in the sheep lung.

The first report of otarine herpesvirus-1-associated urogenital carcinoma in a South American fur seal (Arctocephalus australis).

Otarine herpesvirus (OtHV)-1-associated urogenital carcinoma has been well documented in the California sea lion (Zalophus californianus, CSL), but this is the first report of this tumour in a captive South American fur seal (Arctocephalus australis, SAFS). The gross and microscopical morphology of the tumour in the SAFS was identical to that described previously in CSLs and the tumour in the present case had metastasized within the urogenital tract and draining lymph nodes and to the lungs and one kidney. Immunohistochemistry revealed intra- and extracytoplasmic labelling of herpesvirus antigen in the cells of the tumour tissue and transitional epithelium of the urethra. OtHV-1 nucleic acids were detected within tumour tissue and from a urogenital swab by polymerase chain reaction. The ranges of these two species of pinniped do not overlap normally in the wild, suggesting that transmission of OtHV-1 probably occurred in captivity. This confirmed susceptibility of the SAFS to the development of OtHV-1-associated urogenital carcinoma suggests that all species of Otariidae should be screened for OtHV-1 infection prior to movement within and between zoological collections.

Mycobacterium avium subsp. hominissuis Infection in a captive-bred kiang (Equus kiang).

Equids are considered highly resistant to mycobacterial infections and clinical cases have been described in domestic horses only. Mycobacterium bovis is the most common species reported, although a single report exists of disease due to definitively diagnosed infection with Mycobacterium avium subsp. hominissuis in two domestic horses. This is the first report of a mycobacterial infection in a kiang (Equus kiang), or indeed any wild equid. The animal had chronic loss of condition and serum biochemical changes suggestive of liver disease and chronic infection. Further investigation showed a chronic granulomatous enteritis, lymphadenitis and hepatitis with focal granulomatous pneumonia due to systemic infection with M. avium subsp. hominissuis. The distribution and severity of the lesions suggested that the route of infection was alimentary.

Immunophenotype of cells within cervine rectoanal mucosa-associated lymphoid tissue and mesenteric lymph nodes.

Rectoanal mucosa-associated lymphoid tissue (RAMALT) is a part of the lymphoid system that can be sampled easily in live animals, especially ruminants. RAMALT biopsy is useful for the diagnosis of transmissible spongiform encephalopathies, including scrapie in sheep and goats and chronic wasting disease (CWD) in cervids. Diagnosis is reliant on detection of abnormal prion protein (PrP(d)), which is associated with lymphoid follicles. For enzyme linked immunosorbent assays (ELISAs) detecting PrP(d) it is necessary to ensure that lymphoid follicles are present in biopsy samples to avoid false-negative results. Monoclonal antibodies known to recognize specific immune cell subsets present in lymphoid tissues of sheep were tested for cross-reactivity with cervine RAMALT and mesenteric lymph nodes (MLNs) preserved in zinc salts fixative. The distribution of cells expressing CD3, CD4, CD79, CD21 and class II molecules of the major histocompatibility complex was determined in these tissues. Cells of each immunophenotype had similar distributions in RAMALT and MLNs and these distributions were similar to those reported previously for sheep and cattle. The identification and validation of cervine lymphoid follicle cell markers (CD79 and CD21) may allow reduction in false-negative results during diagnosis of CWD by ELISA.

The first report of disease in a basking shark (Cetorhinus maximus).

Few diseases have been reported in any species of shark and none in the basking shark (Cetorhinus maximus) despite the latter being the subject of targeted hunting for over two centuries. This is the first report to describe the clinical signs and gross and microscopical pathology in a diseased basking shark that was live-stranded on the east coast of Scotland. Pyogranulomatous meningoencephalitis was present together with multifocal, predominantly non-suppurative, myocarditis with myocyte necrosis, oedema and haemorrhage. Additionally, there was full thickness ulcerative and fibrinonecrotizing dermatitis with underlying granulomatous inflammation. The aetiology could not be determined, but the lesions were suggestive of an infectious process, possibly bacterial.

Characterization and time course of pulmonary lesions in calves after intratracheal infection with Pasteurella multocida A:3.

Pasteurella multocida A:3 is a common cause of suppurative bronchopneumonia in calves and results in significant production losses and mortality. Here we describe the lesions in three calves at each of four time points (1 day and 4, 7 and 10 days) after experimental intratracheal infection with approximately 1x10(9) colony-forming units of P. multocida A:3 Moredun Research Institute (MRI isolate 671/90). Equivalent age- and time-matched sham-dosed negative control animals were also studied. Infected calves developed significantly elevated mean rectal temperatures (P<0.001) and respiratory rates (P<0.001) compared with negative control animals. Extensive consolidation of multiple lung lobes was present on each of the day/s post-infection (dpi). Histologically, large numbers of alveoli contained either or both polymorphonuclear neutrophils (PMNs) and oedema fluid (1 dpi). At 4 dpi a severe fibrinosuppurative bronchopneumonia had developed. At this time, PMNs and macrophages formed focal lesions containing central necrotic and mineralized debris, while the interlobular septa were severely distended by oedema. Early abscess formation was present in the lung parenchyma at 7 dpi and many of the interlobular septa were thrombosed. At 10 dpi abscesses within the lung parenchyma were mature and comprised of central necrosis with surrounding layers of PMN, macrophages and fibrous tissue. This study describes, for the first time, the commencement, nature and progression of lesions in bovine pneumonic pasteurellosis caused by P. multocida A:3 and provides the foundations for further investigation of the pathogenesis of this disease in cattle.

Diagnosis of preclinical scrapie in live sheep by the immunohistochemical examination of rectal biopsies.

In most sheep infected with a transmissible spongiform encephalopathy (tse) the disease-associated prion protein (PrP(d)) accumulates in tissues of the lymphoreticular system, suggesting that it might be detected in biopsy specimens. A procedure has been developed to obtain biopsy specimens of rectal mucosa in which PrP(d) has been detected by immunohistochemistry in preclinically infected sheep of all susceptible PrP genotypes. It is probable that PrP(d) increases with the age of sheep or period of incubation. PrP(d) was detectable approximately halfway through the incubation period, with sheep of some PrP genotypes showing positive results earlier than others. For a preclinical diagnosis, the risk of a false negative result was approximately 9 per cent for samples containing 10 follicles, a figure that was reached in 87 per cent of the biopsies. The rectal biopsies had the same sensitivity and time of onset of PrP(d) accumulation as biopsies of the palatine tonsil, but provided larger numbers of follicles. The procedure is simple and quick, does not require dedicated specific instruments, sedation or general anaesthesia, and can be performed repeatedly on the same sheep without detrimental effects to either the animal or the number of follicles obtained.

Kinetics of pulmonary neutrophil recruitment and clearance in a natural and spontaneously resolving model of airway inflammation.

BACKGROUND: Neutrophil apoptosis and phagocytic clearance have been proposed as key determinants affecting the resolution of airway inflammation. Objective To determine the kinetics of neutrophil priming, recruitment, activation and subsequent clearance in a naturally occurring equine disease model of neutrophilic pulmonary inflammation. METHODS AND RESULTS: A 5 h mouldy hay/straw challenge in hypersensitive horses induced transient pulmonary dysfunction lasting 4 days. At 24 h circulating neutrophils were primed and displayed delayed rates of spontaneous apoptosis in vitro. Neutrophil numbers in the airspaces peaked at 5 h and then fell abruptly, returning to pre-challenge levels by 4 days. Airspace neutrophils demonstrated increased respiratory burst activity compared with circulating cells and equine neutrophil elastase 2A concentrations increased in parallel with neutrophil numbers indicating in vivo priming and degranulation. The number of apoptotic neutrophils and proportion of alveolar macrophages containing phagocytosed apoptotic neutrophils increased significantly at 24 h and 4 days post-challenge corresponding to the period of most rapid neutrophil clearance. CONCLUSION: This is the first demonstration of spontaneous neutrophil apoptosis and phagocytic removal in a natural disease model of airway inflammation and provides critical kinetic data to support the hypothesis that this clearance pathway plays a central role in the resolution of neutrophilic inflammation.

Chemoattractant properties of conditioned medium from equine corpora lutea collected at various stages of the oestrous cycle.

This study investigated the chemotactic activity of equine CL at different stages of the oestrous cycle. The purpose of this was to ascertain whether luteal tissue itself contributes to the massive influx of leucocytes around the time of natural and induced luteal regression. Corpora lutea were collected at different stages of dioestrus and after treatment with PGF2alpha. Culture medium harvested after incubation of luteal tissue for 20 h was chemotactic for both polymorphonuclear and mononuclear cells in late dioestrus (before functional regression) as well as after natural and induced luteal regression. By contrast, midluteal tissue showed no chemotactic activity. This is the first report of the ability of equine luteal tissue actively to recruit inflammatory cells in vitro and supports our earlier findings that this infiltration starts prior to functional luteolysis. We hypothesise that this early influx of inflammatory cells may play an active role in luteal regression. Further research is needed to identify the specific chemotactic factor(s).