Bernardino Moreno

MHC class II DRB1 gene polymorphism in the pathogenesis of Maedi–Visna and pulmonary adenocarcinoma viral diseases in sheep

Ovine pulmonary adenocarcinoma (OPA) and Maedi–Visna (Maedi) are two chronic respiratory diseases of retroviral origin which occur worldwide. It is known that different host genetic factors influence the outcome of viral infections. To determine if variation in the Mhc-DRB1 gene was associated with progression to these ovine diseases, sheep lungs with and without OPA and Maedi lesions were collected. A sequence-based method was applied and 40 different alleles were detected in the sample analysed. In the allele-by-allele association analysis, allele DRB1*0325 had a significant association with susceptibility to Maedi (P = 0.045). For OPA, DRB1*0143 and DRB1*0323 were significantly associated with susceptibility (P = 0.024 and P = 0.029), and allele DRB1*0702 was significantly associated with resistance (P = 0.012). Based on these results, the Mhc-DRB1 alleles were classified by effect in three categories—susceptible (S), resistant (R) and neutral (N)—and animals were reassigned the genotypes as S/S, S/R, S/N, R/R, R/N and N/N. In a second analysis, penalised logistic regression models including a flock effect were run. In Maedi, significant association was detected for the N/S heterozygote (P = 0.0007), but not for the S/S homozygote, probably as a result of the low number of S/S animals. In OPA, association was detected for both the S/S and R/R homozygotes (P = 0.005 and P = 0.047). This allele grouping method may be applied in association studies with highly variable genes. This is the first study demonstrating significant associations between sheep Mhc-DRB1 alleles and susceptibility to OPA and Maedi. Therefore, both diseases are suitable candidates for more comprehensive genetic studies.

Colostrum and milk can transmit jaagsiekte retrovirus to lambs

Ovine pulmonary adenocarcinoma (OPA) is a contagious disease caused by jaagsiekte sheep retrovirus (JSRV). In the three studies performed, we have obtained data of the importance of colostrum/milk (C/M) in the transmission of JSRV. In the first study, a group of sheep from a flock with a long history of OPA, samples from colostrum and peripheral blood leucocytes (PBLs) were collected. Two specific PCRs (U3-LTR and env of the JSRV) were carried out. Using U3PCR 8/34 sheep were positive in colostrum whereas with envPCR 7/34 were positive. From these animals only one was positive with U3PCR in the PBLs. Evidence of the transmission of JSRV infection by C/M was obtained in two more separate studies. In the second study, PBLs from five lambs from JSRV+ ewes and two from JSRV-ewes were tested by the U3PCR. They were fed C/M by their mothers during 3 months and slaughtered 7 months after birth. Three out of five lambs from the JSRV+ sheep become PBL positive at 3-4 months old and the other two were also positive at 4-6 months of age. One lamb of the JSRV-sheep became also PBL positive at an age of 3 months. In the third study, a group of lambs from JSRV negative mothers were fed with C/M from JSRV+ sheep and housed in separate unit. For comparison, another group of the same origin and maintained in another different unit, were fed with C/M containing a JSRV virus preparation. All lambs were blood sampled monthly and JSRV infection was detected as early as 15 days and several times onwards in both groups. Control groups fed with C/M from JSRV free flock and JSRV blood test negative sheep were always negative. Together these results indicate that suckling is an important natural transmission route for JSRV.

Pathological and Aetiological Studies in Sheep Exhibiting Extrathoracic Metastasis of Ovine Pulmonary Adenocarcinoma (Jaagsiekte)

Seven sheep with a histopathological diagnosis of pulmonary adenocarcinoma with extrathoracic metastases were included in this retrospective study aiming to describe the pathological findings and to establish their relationship with Jaagsiekte sheep retrovirus (JSRV). In order of frequency, extrathoracic metastases were found in the liver, kidneys, skeletal muscle, digestive tract, spleen, skin and adrenal glands. Intrathoracic metastases involved the chest wall, regional lymph nodes, diaphragm and heart. Immunohistochemistry and polymerase chain reaction allowed detection of JSRV-related protein and nucleic acid, respectively, in the extrathoracic tumours of all cases. It is concluded that extrathoracic metastases constitute a pathological event of ovine pulmonary adenocarcinoma and confirm the malignant character of this virus-induced neoplasia.

Transmission of sheep-bovine spongiform encephalopathy to pigs

Experimental transmission of the bovine spongiform encephalopathy (BSE) agent has been successfully reported in pigs inoculated via three simultaneous distinct routes (intracerebral, intraperitoneal and intravenous). Sheep derived BSE (Sh-BSE) is transmitted more efficiently than the original cattle-BSE isolate in a transgenic mouse model expressing porcine prion protein. However, the neuropathology and distribution of Sh-BSE in pigs as natural hosts, and susceptibility to this agent, is unknown. In the present study, seven pigs were intracerebrally inoculated with Sh-BSE prions. One pig was euthanized for analysis in the preclinical disease stage. The remaining six pigs developed neurological signs and histopathology revealed severe spongiform changes accompanied by astrogliosis and microgliosis throughout the central nervous system. Intracellular and neuropil-associated pathological prion protein (PrPSc) deposition was consistently observed in different brain sections and corroborated by Western blot. PrPSc was detected by immunohistochemistry and enzyme immunoassay in the following tissues in at least one animal: lymphoid tissues, peripheral nerves, gastrointestinal tract, skeletal muscle, adrenal gland and pancreas. PrPSc deposition was revealed by immunohistochemistry alone in the retina, optic nerve and kidney. These results demonstrate the efficient transmission of Sh-BSE in pigs and show for the first time that in this species propagation of bovine PrPSc in a wide range of peripheral tissues is possible. These results provide important insight into the distribution and detection of prions in non-ruminant animals.

Differential gene expression and apoptosis markers in presymptomatic scrapie affected sheep

Neuronal loss is one of the characteristics of scrapie neuropathology. Previous analysis of brains from sheep naturally infected with scrapie that were in a terminal stage did not detect a clear induction of apoptosis, although molecular changes were evidenced. As neuronal death could be occurring early in scrapie, we developed a neuropathological and gene expression study of sheep infected with scrapie in a presymptomatic stage. The histopathology, immunolabelling of PrP(Sc), Bax and activated caspase-3, and the analysis of the expression of 7 genes involved in the regulation of the mitochondrial pathway of apoptosis were investigated in the following 4 central nervous system areas: medulla oblongata, diencephalon, frontal cortex and cerebellum. Moreover, TUNEL and NeuN immunolabelling was performed in the medulla oblongata. The PrP(Sc) immunolabelling in the four areas, as well as a neuropil spongiform change, were more evident in the terminal stage than in presymptomatic animals. Cytoplasmic Bax immunostaining was observed in the presymptomatic medulla oblongata. In contrast to symptomatic animals, the immunostaining was not extended to the hypothalamus, indicating the progression of Bax induction during the course of the disease. Although neither caspase-3 immunostaining nor the TUNEL technique detected neurons with apoptosis, NeuN-immunolabelled cell counting determined that presymptomatic animals have already suffered neuronal loss in a lower or equal degree than symptomatic animals. Finally, the gene expression profiles indicated that the mitochondrial pathway of apoptosis was activated with higher intensity in presymptomatic animals than in symptomatic sheep and confirmed the implication of genes such as BAX or AIF in the disease.

Isolation and phylogenetic characterization of Streptococcus halichoeri from a European badger (Meles meles) with pyogranulomatous pleuropneumonia.

Summary Clinical and pathological studies in European badgers (Meles meles) are limited. Badgers play a significant role in the epidemiology of bovine tuberculosis (TB) in some countries and an accurate diagnosis is needed for this infection. However, the lesions of bovine TB are similar to those associated with other pathogens, making pathological diagnosis difficult. In the present study, Streptococcus halichoeri was isolated from a European badger with pyogranulomatous pleuropneumonia and suspected of having tuberculosis. TB and other pathogens able to induce similar lesions were ruled out. Comparative 16S rRNA and rpoB gene sequencing studies showed an identity of 99.51% and 98.28%, respectively, with S. halichoeri. This report represents the third description of this bacterium and the first in an animal species other than the grey seal (Halichoerus grypus). It also shows that S. halichoeri can be associated with a pathological process characterized by granulomatous inflammation and resembling tuberculosis.

Amino acid signatures in the Ovar-DRB1 peptide-binding pockets are associated with Ovine Pulmonary Adenocarcinoma susceptibility/resistance

Potential relationships between amino acid motifs of various alleles of the ovine major histocompatibility complex DR (Ovar-DR) molecule and occurrence of clinical OPA caused by JSRV were investigated in a case-control study. Latxa sheep (n=132) screened for presence/absence of pulmonary OPA lesions were typed for their Ovar-DRB1 2nd exon alleles by PCR and sequence-based typing (PCR-SBT). The polymorphic amino acid residues derived from the obtained 34 DRB1 protein variants were subjected to a logistic regression-based association study. The amino acids at several positions showed significant associations with the presence/absence of pulmonary OPA lesions; some of the residues were located within the peptide binding cleft of the DRB molecule, including pockets P1, P4, P7 and P9.

Experimental transmission to a calf of an isolate of Spanish classical scrapie

Multiple theories exist regarding the origin of bovine spongiform encephalopathy (BSE). An early and prominent theory proposed that BSE was the result of the adaptation of sheep scrapie to cattle. The reports to date indicate that the distribution of the pathological prion protein (PrPSc) in experimental bovine scrapie is largely restricted to the central nervous system (CNS). Here, we describe pathological findings in a calf intracerebrally inoculated with a Spanish classical scrapie isolate. While clinical disease was observed 30 months after inoculation and PrPSc was detected in the CNS, the corresponding phenotype differed from that of BSE. Immunohistochemistry and PMCA also revealed the presence of PrPSc in the peripheral nerves, lymphoid tissues, skeletal muscle and gastrointestinal tract, suggesting centrifugal spread of the scrapie agent from the brain. To the best of our knowledge, this is the first report describing the detection of PrPSc in tissues other than the CNS after experimental transmission of scrapie to cattle.