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.

Structural Development, Cellular Differentiation and Proliferation of the Respiratory Epithelium in the Bovine Fetal Lung.

Fetal bovine lung samples of 11 different gestational ages were assigned to a classical developmental stage based on histological morphology. Immunohistochemistry was used to characterize the morphology of forming airways, proliferation rate of airway epithelium and the presence of epithelial cell types (i.e. ciliated cells, club cells, neuroepithelial cells (NECs) and type II pneumocytes). Typical structural organization of pseudoglandular (84-98 days gestational age [DGA]), canalicular (154-168 DGA) and alveolar (224-266 DGA) stages was recognized. In addition, transitional pseudoglandular-canalicular (112-126 DGA) and canalicular-saccular (182 DGA) morphologies were present. The embryonic stage was not observed. A significantly (P <0.05) higher proliferation rate of pulmonary epithelium, on average 5.5% and 4.4% in bronchi and bronchioles, respectively, was present in the transitional pseudoglandular-canalicular phase (112-126 DGA) compared with all other phases, while from 8 weeks before term (224-266 DGA) proliferation had almost ceased. The first epithelial cells identified by specific marker proteins in the earliest samples available for study (84 DGA) were ciliated cells and NECs. Club cells were present initially at 112 DGA and type II pneumocytes at 224 DGA. At the latest time points (224-226 DGA) these latter cell types were still present at a much lower percentage compared with adult cattle. This study characterized bovine fetal lung development by histological morphology and cellular composition of the respiratory epithelium and suggests that the apparent structural anatomical maturity of the bovine lung at term is not matched by functional maturity of the respiratory epithelium.

Assessment of transthoracic ultrasound diagnosis of ovine pulmonary adenocarcinoma in adult sheep.

Ovine pulmonary adenocarcinoma (OPA), caused by Jaagsiekte sheep retrovirus (JSRV), is a disease of increasing concern in the sheep industry. There is no commercial antemortem test for OPA; therefore, an early evaluation phase study was undertaken to examine the accuracy of transthoracic ultrasound examination using a 5-6.5 MHz sector ultrasound machine widely available in veterinary practice in the UK to diagnose OPA. Restraint, preparation and examination time was restricted to five minutes per sheep to represent the cost limitations of commercial sheep farming. One hundred sheep were examined. All 41 cases identified with suspect OPA lesions during transthoracic ultrasound examination had the diagnosis confirmed at postmortem examination, while sheep without ultrasonographic changes characteristic of OPA had no gross lesions of OPA at postmortem examination. This demonstrates the specificity of transthoracic ultrasound for diagnosis of OPA. The authors propose that, in the absence of any other reliable preclinical diagnostic test, the use of transthoracic ultrasound examination should be considered for a second opinion on an initial diagnosis of OPA, for screening purchased adult flock replacements for OPA, or for screening sheep in a known OPA-affected flock. However, the authors emphasise that a negative scan cannot provide a guarantee that the animal is free of JSRV infection nor early OPA.

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.

Diagnostic accuracy of PCR for Jaagsiekte sheep retrovirus using field data from 125 Scottish sheep flocks.

Using a representative sample of Scottish sheep comprising 125 flocks, the sensitivity and specificity of PCR for Jaagsiekte sheep retrovirus (JSRV) was estimated. By combining and adapting existing methods, the characteristics of the diagnostic test were estimated (in the absence of a gold standard reference) using repeated laboratory replicates. As the results of replicates within the same animal cannot be considered to be independent, the performance of the PCR was calculated at individual replicate level. The median diagnostic specificity of the PCR when applied to individual animals drawn from the Scottish flock was estimated to be 0.997 (95% confidence interval [CI] 0.996-0.999), whereas the median sensitivity was 0.107 (95% CI 0.077-0.152). Considering the diagnostic test as three replicates where a positive result on any one or more replicates results in a positive test, the median sensitivity increased to 0.279. Reasons for the low observed sensitivity were explored by comparing the performance of the test as a function of the concentration of target DNA using spiked positive controls with known concentrations of target DNA. The median sensitivity of the test when used with positive samples with a mean concentration of 1.0 target DNA sequence per 25µL was estimated to be 0.160, which suggests that the PCR had a high true (analytical) sensitivity and that the low observed (diagnostic) sensitivity in individual samples was due to low concentrations of target DNA in the blood of clinically healthy animals.

Pathology and pathogenesis of ovine pulmonary adenocarcinoma.

Ovine pulmonary adenocarcinoma (OPA), also known as jaagsiekte, is a transmissible lung tumour of sheep caused by jaagsiekte sheep retrovirus (JSRV). JSRV induces neoplastic transformation of alveolar and bronchiolar secretory epithelial cells and the resulting tumours can grow to occupy a significant portion of the lung. Tumour growth is frequently accompanied by the overproduction of fluid in the lung, which further compromises normal respiration. The period between infection and the appearance of clinical signs may be several months or years and many JSRV-infected sheep do not exhibit clinical signs at all during their lifespan. This allows the spread of OPA into new flocks through contact with infected but apparently normal animals. OPA was first described in the early 19th century; however, it has still not been possible to devise effective methods for controlling its spread and it remains an important problem in most countries where sheep are farmed. This is due in part to the absence of an immunological response to JSRV in infected animals, which has hindered the development of serological diagnostic tests and vaccines. In addition to its veterinary importance, OPA is regarded as a potential large animal model for human lung adenocarcinoma and this has stimulated research into the pathogenesis of the ovine disease. This work has produced some significant results, including the finding that one of the JSRV structural proteins is directly involved in oncogenesis. The recent advances in understanding JSRV and the pathogenesis of OPA should lead to novel strategies for diagnosis and control of this disease and for its exploitation as a comparative model for human lung cancer.

SPECTCT cerebral perfusion scintigraphy; is the low-dose CT component of diagnostic value?

AIM: To assess the potential diagnostic value of the low-dose computed tomography (CT) component of dual-examination single photon emission CT and CT (SPECTCT) cerebral perfusion studies. METHOD AND MATERIALS: Two hundred and forty consecutive 99 mTc hexamethylpropylene amine oxime (HMPAO) SPECTCT studies were considered for inclusion. The images were acquired on a hybrid dual-head gamma camera/low-dose CT system. The CT component had a fixed tube current of 2.5 mA. The CT section thickness was 5mm and total acquisition time approximately 7.5 min. Studies in which no CT images were acquired, or those excessively degraded by movement artefact were excluded. The CT images of each of the remaining studies were retrospectively reviewed and categorized as normal or abnormal. Details of the abnormalities were recorded. RESULTS: Fifteen of the 240 studies were excluded as no CT images were obtained. A further 14 were excluded as they were considered excessively degraded by movement artefact. A single abnormality was demonstrated on 48 (23%), and two abnormalities on four (2%) of the remaining 211 studies. The most common abnormal findings were low attenuation in the deep cortical white matter (n=22), infarcts (n=12), cerebral atrophy (n=7), dilated ventricles (n=5), basal ganglia calcification (n=4), and post-surgical change (n=3). Other findings included a chronic subdural haematoma, a meningioma, and a posterior fossa cyst. Previous cerebral imaging was available for comparison in 31% of cases. There was 85% concordance between previous imaging and the low-dose CT images. CONCLUSION: Twenty-five percent of the low-dose CT images in this study demonstrated abnormalities. Therefore the CT component of cerebral perfusion SPECTCT investigations should be routinely reported in their own right.

PCR examination of bronchoalveolar lavage samples is a useful tool in pre-clinical diagnosis of ovine pulmonary adenocarcinoma (Jaagsiekte).

Ovine pulmonary adenocarcinoma (OPA) is a contagious lung tumour of sheep caused by Jaagsiekte sheep retrovirus (JSRV). The disease is a particular problem in flocks in many parts of the world. The aim of the study was to assess screening methods for individual animals as a prelude to future eradication trials. Results of histological examination were used as the standard to evaluate the relative sensitivity and specificity of an established heminested polymerase chain reaction (PCR) test for JSRV proviral DNA from blood and bronchoalveolar lavage (BAL) samples. PCR results from tissue samples are included as control data. PCR testing of blood samples was found to have an estimated sensitivity of only 10% (95% confidence interval (CI) 3-20) while the sensitivity of the PCR test on BAL samples was 89% (CI 79-96) in comparison to the results of histological examination. We conclude that PCR testing of BAL samples is an effective confirmatory test for sheep with suspected clinical OPA. It is also a useful tool for the pre-clinical identification of individual infected sheep within an infected flock and therefore may prove beneficial in future control or eradication programmes.

Coexistence of enzootic nasal adenocarcinoma and jaagsiekte retrovirus infection in sheep.

Ten sheep naturally affected with enzootic nasal adenocarcinoma (ENA), a disease associated with ovine enzootic nasal tumour virus (ENTV-1), were found also to be infected with jaagsiekte sheep retrovirus (JSRV), the causal agent of ovine pulmonary adenocarcinoma (OPA). Only one of the sheep showed OPA lung lesions. The animals belonged to 10 flocks located in a geographical area in which OPA is frequently seen. ENTV-1 was found in all the ENA tumours but only occasionally in extra-tumoral sites, confirming the results of a previous study. In contrast, JSRV had a disseminated tissue distribution, similar to that previously reported for animals infected with JSRV. However, the occurrence of JSRV in lymphoid tissues was clearly greater than in sheep infected with JSRV but with no lesions of ENA. The data suggested a synergistic relationship between ENTV-1 and JSRV, resulting in increased proliferation of JSRV.

Enzootic nasal adenocarcinoma of sheep and goats.

Enzootic nasal adenocarcinoma is a contagious tumour of the mucosal nasal glands affecting young adult sheep or goats. The disease occurs naturally in all continents except Australia and New Zealand. Clinical signs include continuous nasal discharge, respiratory distress, exophthalmos and skull deformations. The tumour is classified histologically as a low-grade adenocarcinoma. Nasal glands of both respiratory and olfactory muosal glands seem to be the origin of the neoplasia. It has been experimentally transmitted in sheep and goats using either tumour extracts or concentrated nasal fluids. Two distinct retroviruses are implicated in the aetiology of the neoplasia one in sheep (ONAV) and one in goats (CNAV). We suggest that jaagsiekte sheep retrovirus (JSRV), ONAV, CNAV, and their endogenous counterparts represent a unique family of retroviruses. The similarities between these viruses suggests that any control strategies, including vaccination, may be appropriate to both diseases. The differences, however, represent a unique resource for delineating the function of individual regions of the virus. It is intriguing that whilst ONAV and CNAV appear to be as different to each other as they are to JSRV, that they have very similar disease pathologies, distinct from that of OPA. Additionally, all three exogenous viruses manage to avoid instigating any apparent immune response. Whether this is indeed a result of tolerance induced by the endogenous counterparts or whether the viruses themselves have unique immunosuppressive properties will be an important finding.

Jaagsiekte sheep retrovirus can be detected in the peripheral blood during the pre-clinical period of sheep pulmonary adenomatosis.

Peripheral blood leukocytes (PBLs) and tissue samples from 36 sheep were examined for jaagsiekte sheep retrovirus (JSRV) by hemi-nested PCR. Animals were classified according to the status of sheep pulmonary adenomatosis (SPA), which was confirmed by pathological examination, as follows: (i) sheep with classical SPA (cSPA, n=10), (ii) sheep with atypical SPA (aSPA, n=6), (iii) non-affected sheep from SPA-affected flocks (in-contact, n=10) and (iv) non-affected sheep from SPA-free flocks (control, n=10). JSRV proviral DNA was detected in the PBLs of 10/10 cSPA, 5/6 aSPA, 4/10 in-contact and 0/10 control sheep. Lung tumours and lymphoid organs were also found to be JSRV-positive. The number of positive PCR results was greater for sheep in the cSPA group than for those in the aSPA and in-contact groups. For the first time, it is concluded that JSRV can be detected in naturally infected sheep before the onset of clinical disease and even before the development of discernible tumours.

Sheep pulmonary adenomatosis: characterization of two pathological forms associated with jaagsiekte retrovirus.

Pathological and immunohistochemical studies were performed on the lungs of 10 sheep with lesions of "classical" sheep pulmonary adenomatosis (SPA) and six sheep with "atypical" lung tumours. Lung tumour samples and other tissues from the same 16 animals were tested for the presence of jaagsiekte retrovirus (JSRV) by a polymerase chain reaction (PCR) that amplified a portion of the U3 long terminal repeat. The differences in the gross appearance of the classical and atypical forms paralleled the histopathological differences. The latter mainly concerned the stroma of the tumours which in the atypical cases was more heavily infiltrated by inflammatory cells and connective tissue fibres. JSRV major capsid protein was detected immunohistochemically in the epithelial transformed cells of both classical and atypical tumours, but the immune reactivity was slightly milder in atypical SPA. Proviral U3 sequences of JSRV were detected by specific PCR in all the tumour samples. Furthermore, the sequences of amplimers obtained from the two different pathological forms of the tumour were very similar. However, the dissemination of JSRV to other organs was greater in sheep with classical SPA than in those with atypical SPA. The pathological and virological features of these two forms of tumour are compared in an attempt to clarify whether classical and atypical SPA are two separate diseases or different expressions of a single disease spectrum.

Complete sequence of enzootic nasal tumor virus, a retrovirus associated with transmissible intranasal tumors of sheep.

The sequence of the complete genome of ovine enzootic nasal tumor virus, an exogenous retrovirus associated exclusively with contagious intranasal tumors of sheep, was determined. The genome is 7,434 nucleotides long and exhibits a genetic organization characteristic of type B and D oncoviruses. Enzootic nasal tumor virus is closely related to the Jaagsiekte sheep retrovirus and to sheep endogenous retroviruses.

Jaagsiekte retrovirus establishes a disseminated infection of the lymphoid tissues of sheep affected by pulmonary adenomatosis.

Jaagsiekte retrovirus (JSRV) is an exogenous type D-related retrovirus specifically associated with a contagious lung cancer of sheep (sheep pulmonary adenomatosis; SPA). Recently, epithelial tumour cells in the lungs of SPA-affected sheep were identified as major sites of JSRV replication by immunological techniques and RT-PCR amplification of part of JSRV gag. JSRV was not detected outside the lungs and their draining lymph nodes. However, low levels of JSRV expression in non-respiratory tissues could have been masked by co-amplification of endogenous JSRV-related sequences, which were differentiated from JSRV by the lack of a Scal restriction site in the PCR product. To further investigate the pathogenesis of SPA, an exogenous virus-specific hemi-nested PCR was developed utilizing primers in the U3 region of JSRV LTR, where major differences between endogenous and exogenous sequences exist. This technique was shown to be > or = 10(5)-fold more sensitive than the previous gag PCR/ScaI digestion method. Using this new assay the tissue distribution of JSRV in sheep with natural and experimentally induced SPA was analysed. Proviral DNA and JSRV transcripts were found in all tumours and lung secretions of SPA-affected sheep (n = 22) and in several lymphoid tissues. The mediastinal lymph nodes draining the lungs were consistently demonstrated to be infected by JSRV (10/10). JSRV transcripts were also detected in spleen (7/9), thymus (2/4), bone marrow (4/8) and peripheral blood mononuclear cells (3/7). Proviral DNA was also detected in these tissues although in a much lower proportion of cases. JSRV was not detected in 27 samples from unaffected control animals (n = 15).

PCR-based detection and partial characterization of a retrovirus associated with contagious intranasal tumors of sheep and goats.

A type D-related retrovirus has been demonstrated in enzootic nasal tumors (ENTs) of sheep and goats. This retrovirus, ENT virus (ENTV), has antigenic cross-reactivity with the jaagsiekte sheep retrovirus (JSRV), which is associated with a contagious lung tumor of sheep (sheep pulmonary adenomatosis). Here, we present the first report of nucleic acid sequence from ENTV which confirms, at the nucleic acid level, that this retrovirus is related to JSRV yet apparently distinct from it. Reverse transcription-PCR followed by restriction enzyme digestion specifically identified ENTV. By this technique, ENTV was demonstrated exclusively in tumor tissues and exudates of animals with ENT. Thus, there is a unique and consistent association between ENT and the retrovirus, just as there is between JSRV and sheep pulmonary adenomatosis. This gives further weight to the hypothesis that these retroviruses are the etiologic agents of the tumors.

Unique long terminal repeat U3 sequences distinguish exogenous jaagsiekte sheep retroviruses associated with ovine pulmonary carcinoma from endogenous loci in the sheep genome.

Ovine pulmonary carcinoma (OPC) is a contagious lung cancer of sheep that is presumed to be caused by an exogenous retrovirus of sheep, jaagsiekte sheep retrovirus (JSRV). The sheep genome carries 15 to 20 copies of endogenous sheep retrovirus (ESRV) loci that hybridize to JSRV DNA probes. In order to clarity the etiologic roles of ESRV and an exogenous JSRV-like retrovirus (exJSRV) in OPC, we assessed sequence differences between ESRV and JSRV. Molecular characterization of six ESRV loci revealed restriction sites specific for JSRV. Nucleotide sequences of ESRVs from sheep of different breeds were similar to those of JSRV in structural genes but divergent in U3. Therefore, primers specific for the U3 sequences of exJSRV were designed for use in the PCR. Of 13 tumor DNAs tested by PCR with these exogenous-virus U3 primers, 8 produced DNA fragments that hybridized with the JSRV gag probe, but neither lung DNAs from healthy sheep nor DNAs from nontumor tissues of diseased sheep produced similar DNA fragments. exJSRV PCR products from tumor DNAs of sheep with OPC from three continents had restriction profiles similar to each other but different from those of ESRVs upon digestion with EcoRI, HindIII, NdeI, KpnI, and ScaI. These exjSRVs could be classified into two genotypes according to U3 sequences and restriction profiles. U3 sequences of exJSRV proviruses in tumors strongly resembled those of JSRV but differed from those of ESRVs, suggesting that exJSRVs, rather than ESRVs, are primarily associated with oncogenesis in OPC.

The exogenous form of Jaagsiekte retrovirus is specifically associated with a contagious lung cancer of sheep.

Sheep pulmonary adenomatosis ([SPA] ovine pulmonary carcinoma) is a transmissible lung cancer of sheep that has been associated etiologically with a type D- and B-related retrovirus (jaagsiekte retrovirus (JSRV]). To date it has been impossible to cultivate JSRV in vitro and therefore to demonstrate the etiology of SPA by a classical approach. In addition, the presence of 15 to 20 copies of endogenous JSRV-related sequences (enJSRV) has hampered studies at the molecular level. The aim of this study was to investigate whether the expression of exogenous JSRV was specifically associated with neoplasia in SPA-affected animals. Initially, we found that enJSRVs were transcribed in a wide variety of normal sheep tissues. Then, by sequencing part of the gag gene of enJSRV we established a ScaI restriction site in gag as a molecular marker for the exogenous form of JSRV. Restriction enzyme digestion of PCR products obtained from the amplification of cDNA from a total of 65 tissues collected from SPA-affected and unaffected control sheep revealed that the exogenous form of JSRV was exclusively and consistently present in tumor tissues and lung secretions of the affected animals. In addition, exogenous JSRV provirus was detected only in DNA from SPA tumors and not from nontumor tissues of the same animals. This study has demonstrated clearly that the exogenous form of JSRV is specifically associated with SPA tumors.

Use of PCR-based methods for selection of integrated transgenes in preimplantation embryos.

The production of transgenic animals from ungulate species is an inefficient and expensive procedure. The development of selection methods to identify the small number of transgenic preimplantation embryos produced following DNA microinjection of one-cell embryos would greatly reduce both the cost and effort of these procedures. This study has examined the fate of the ovine beta-lactoglobulin-human alpha 1-antitrypsin (AATB) minigene construct or a subfragment of this following microinjection into one-cell mouse embryos. It has examined two PCR-based methods that were designed to identify a biochemical difference between microinjected DNA constructs to select preimplantation stage embryos in which chromosomal integration of exogenous DNA has occurred. The two methods involved the modification of the AATB DNA construct either by dam methylation or the substitution of dTTP by dUTP. The dam-sensitive DNA endonuclease DpnI, that was used to digest nonintegrated AATB sequences at sites located between PCR oligonucleotide sequences, was found to interfere with the activity of the subsequent PCR reaction. Analyses of the fate of dUTP-DNA indicated that either repair or replication of microinjected DNA interfered with the ability to distinguish between integrated and nonintegrated DNA constructs in the mid-preimplantation stage embryo. The distribution of microinjected AATB DNA between the blastomeres of individual four and eight-cell stage embryos was also examined by the PCR reaction. Microinjected DNA was not found to be evenly distributed between all the blastomeres of individual embryos.