RESUMO
The objective of this work was to comparatively study the tissue tropism and the associated pathology of 2 autochthonous small ruminant lentivirus (SRLV) field strains using an experimental infection in sheep through the bone marrow. Fifteen male, SRLV-free lambs of the Rasa Aragonesa breed were inoculated with strain 697 (nervous tissue origin, animals A1-A6), with strain 496 (articular origin, animals B1-B6), or with uninfected culture medium (C1-C3). Clinical, serologic, and polymerase chain reaction (PCR) evaluations were performed periodically. Two lambs from each infected group and a control animal were euthanized at 134, 273, and 319 days postinfection. Tissues were analyzed by gross and histopathologic evaluation; immunohistochemistry for CD3, CD4, CD8, CD68, and FoxP3 cell markers; lung morphometric evaluation; and tissue proviral quantification by PCR. All infected animals became positive either by enzyme-linked immunosorbent assay and/or PCR, with group B lambs showing the highest serologic values and more consistently positive PCR reactions. Group A lambs showed representative lung lesions but only mild histopathologic changes in the central nervous system (CNS) or in carpal joints. Contrarily, group B lambs demonstrated intense carpal arthritis and interstitial pneumonia but an absence of lesions in the CNS. Proviral copies in tissues were detected only in group B lambs. Experimental infection with these SRLV strains indicates that strain 496 is more virulent than strain 697 and more prone to induce arthritis, whereas strain 697 is more likely to reproduce encephalitis in Rasa Aragonesa lambs. Host factors as well as viral factors are responsible for the final clinicopathologic picture during SRLV infections.
Assuntos
Medula Óssea/virologia , Infecções por Lentivirus/veterinária , Lentivirus Ovinos-Caprinos/patogenicidade , Tropismo Viral , Animais , Medula Óssea/patologia , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Ensaio de Imunoadsorção Enzimática/veterinária , Articulações/patologia , Articulações/virologia , Infecções por Lentivirus/patologia , Infecções por Lentivirus/virologia , Pulmão/patologia , Pulmão/virologia , Masculino , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Ovinos/virologia , Tropismo Viral/fisiologiaRESUMO
We describe the clinicopathologic features of an arthritis outbreak in sheep induced by small ruminant lentivirus (SRLV), linked to the presence of a new SRLV isolate phylogenetically assigned to caprine arthritis encephalitis virus-like subgroup B2. Thirteen SRLV seropositive Rasa Aragonesa adult ewes were selected from 5 SRLV highly infected flocks (mean seroprevalence, 90.7%) for presenting uni- or bilateral chronic arthritis in the carpal joint. A complete study was performed, including symptomatology, histopathology, immunocytochemistry, immunohistochemistry, in situ hybridization, and microbiology. The carpus was the joint almost exclusively affected, with 10 sheep (76%) showing a moderate increase in carpal joint size (diameter range, 18-20 cm; normal range, 15-16 cm) without signs of locomotion problems and with 3 ewes (23%) showing severe inflammation with marked increase in diameter (21-24 cm), pain at palpation, and abnormal standing position. Grossly, chronic proliferative arthritis was observed in affected joints characterized by an increased thickness of the synovial capsule and synovial membrane proliferation. Microscopically, synovial membrane inflammation and proliferation and hyperplasia of synoviocytes were observed. More positive cases of SLRV infection were detected by immunocytochemistry of articular fluid than of bronchoalveolar lavage fluid. Immunohistochemistry and in situ hybridization also detected positive cells in the subsynovial connective tissue, lung, mediastinal lymph node, mammary gland, and mammary lymph node. All animals were negative for the presence of Mycoplasma or other bacteria in the articular space. The present outbreak likely represents an adaptation of a caprine virus to sheep. Our results underline the importance of the arthritis induced by SRLV in sheep, a clinical form that might be underestimated.
Assuntos
Artrite/veterinária , Infecções por Lentivirus/veterinária , Lentivirus/fisiologia , Doenças dos Ovinos/patologia , Animais , Artrite/patologia , Artrite/virologia , Vírus da Artrite-Encefalite Caprina/genética , Vírus da Artrite-Encefalite Caprina/fisiologia , Genótipo , Lentivirus/genética , Infecções por Lentivirus/patologia , Infecções por Lentivirus/virologia , Filogenia , Estudos Soroepidemiológicos , Ovinos , Doenças dos Ovinos/virologia , Especificidade da Espécie , Membrana Sinovial/virologiaRESUMO
A single broadly reactive standard ELISA is commonly applied to control small ruminant lentivirus (SRLV) spread, but type specific ELISA strategies are gaining interest in areas with highly prevalent and heterogeneous SRLV infections. Short (15-residue) synthetic peptides (n=60) were designed in this study using deduced amino acid sequence profiles of SRLV circulating in sheep from North Central Spain and SRLV described previously. The corresponding ELISAs and two standard ELISAs were employed to analyze sera from sheep flocks either controlled or infected with different SRLV genotypes. Two outbreaks, showing SRLV-induced arthritis (genotype B2) and encephalitis (genotype A), were represented among the infected flocks. The ELISA results revealed that none of the assays detected all the infected animals in the global population analyzed, the assay performance varying according to the genetic type of the strain circulating in the area and the test antigen. Five of the six highly reactive (57-62%) single peptide ELISAs were further assessed, revealing that the ELISA based on peptide 98M (type A ENV-SU5, consensus from the neurological outbreak) detected positives in the majority of the type-A specific sera tested (Se: 86%; Sp: 98%) and not in the arthritic type B outbreak. ENV-TM ELISAs based on peptides 126M1 (Se: 82%; Sp: 95%) and 126M2 0,65 0.77 (Se: 68%; Sp: 88%) detected preferentially caprine arthritis encephalitis (CAEV, type B) and visna/maedi (VMV, type A) virus infections respectively, which may help to perform a preliminary CAEV vs. VMV-like typing of the flock. The use of particular peptide ELISAs and standard tests individually or combined may be useful in the different areas under study, to determine disease progression, diagnose/type infection and prevent its spread.
Assuntos
Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Lentivirus/veterinária , Doenças dos Ovinos/diagnóstico , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Vírus da Artrite-Encefalite Caprina/genética , Vírus da Artrite-Encefalite Caprina/imunologia , Surtos de Doenças/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Genes gag , Cabras , Infecções por Lentivirus/diagnóstico , Infecções por Lentivirus/epidemiologia , Dados de Sequência Molecular , Filogenia , Pneumonia Intersticial Progressiva dos Ovinos/diagnóstico , Pneumonia Intersticial Progressiva dos Ovinos/epidemiologia , Pneumonia Intersticial Progressiva dos Ovinos/imunologia , Ovinos , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/imunologia , Carneiro Doméstico , Espanha/epidemiologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Visna/diagnóstico , Visna/epidemiologia , Visna/imunologia , Vírus Visna-Maedi/genética , Vírus Visna-Maedi/imunologiaRESUMO
The restrictive properties of tripartite motif-containing 5 alpha (TRIM5α) from small ruminant species have not been explored. Here, we identify highly similar TRIM5α sequences in sheep and goats. Cells transduced with ovine TRIM5α effectively restricted the lentivirus visna/maedi virus DNA synthesis. Proteasome inhibition in cells transduced with ovine TRIM5α restored restricted viral DNA synthesis, suggesting a conserved mechanism of restriction. Identification of TRIM5α active molecular species may open new prophylactic strategies against lentiviral infections.
Assuntos
Proteínas de Transporte/metabolismo , Doenças das Cabras/imunologia , Doenças dos Ovinos/imunologia , Vírus Visna-Maedi/fisiologia , Visna/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Doenças das Cabras/genética , Doenças das Cabras/metabolismo , Doenças das Cabras/virologia , Cabras , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Ovinos , Doenças dos Ovinos/genética , Doenças dos Ovinos/metabolismo , Doenças dos Ovinos/virologia , Visna/genética , Visna/virologiaRESUMO
This study investigates the nervous form of ovine maedi-visna by histological and immunohistochemical techniques. The aim was to study the lesion types and the local cellular immune response related to each lesion type, and the possible relationship between these parameters. Thirty-four Assaf ewes were studied, 29 of which had shown nervous signs. Microscopical lesion patterns were described according to location, extent and predominance of inflammatory cell type. Immunohistochemical labelling of T cells (CD3(+), CD4(+), CD8(+) and cells expressing the γδ form of the T-cell receptor), B cells and macrophages revealed clear differences between the lesion patterns. Two main lesion types were described. Lymphocytic lesions had areas of mild-moderate injury characterized by a predominance of infiltrating T cells. Histiocytic lesions were more severe and had extensive areas of malacia and dominant infiltration by macrophages and B cells. Each animal had a unique lesion pattern and these differences could be due to individual resistance to the progression of infection. The lymphocytic lesions appear to represent initial or latent phases of slow progression, in which the animal presents some natural resistance to the infection. The histiocytic pattern may reflect a poor immune response or a greater virulence of the viral strain.
Assuntos
Interações Hospedeiro-Patógeno , Imunidade Celular/imunologia , Pneumonia Intersticial Progressiva dos Ovinos/patologia , Vírus Visna-Maedi/imunologia , Animais , Antígenos CD/metabolismo , Linfócitos B/metabolismo , Linfócitos B/patologia , Biomarcadores/metabolismo , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Plexo Corióideo/imunologia , Plexo Corióideo/metabolismo , Plexo Corióideo/patologia , Progressão da Doença , Feminino , Histiócitos/metabolismo , Histiócitos/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Meninges/imunologia , Meninges/metabolismo , Meninges/patologia , Pneumonia Intersticial Progressiva dos Ovinos/imunologia , Pneumonia Intersticial Progressiva dos Ovinos/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Ovinos , Linfócitos T/metabolismo , Linfócitos T/patologia , Vírus Visna-Maedi/isolamento & purificação , Vírus Visna-Maedi/patogenicidadeRESUMO
An extensive outbreak characterized by the appearance of neurological symptoms in small ruminant lentivirus (SRLV) infected sheep has been identified in Spain, but the genetic characteristics of the strain involved and differential diagnostic tools for this outbreak remain unexplored. In this work, 23 Visna-affected naturally infected animals from the outbreak, 11 arthritic animals (both groups presenting anti-Visna/Maedi virus serum antibodies), and 100 seronegative animals were used. Eight of the Visna-affected animals were further studied post-mortem by immunohistochemistry. All had lesions in spinal cord, being the most affected part of the central nervous system in six of them. A representative strain of the outbreak was isolated. Together with other proviral sequences from the outbreak the virus was assigned to genotype A2/A3. In vitro culture of the isolate revealed that viral production was slow/low in fibroblast-like cells but it was high in blood monocyte-derived macrophages. The long terminal repeat (LTR) of the viral genome of this isolate lacked an U3-duplication, but its promoter activity in fibroblast-like cells was normal compared to other strains. Thus, viral production could not be inferred from the LTR promoter activity in this isolate. Analysis of the viral immunodominant epitopes among SRLV sequences of the outbreak and other known sequences allowed the design of a synthetic SU peptide ELISA that detected the Visna affected animals, representing a tool of epidemiological interest to control viral spread of this highly pathogenic strain.
Assuntos
Surtos de Doenças/veterinária , Vírus Visna-Maedi/genética , Visna/diagnóstico , Visna/virologia , Animais , Anticorpos Antivirais/sangue , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Macrófagos/virologia , Masculino , Ovinos , Carneiro Doméstico , Espanha/epidemiologia , Sequências Repetidas Terminais , Visna/epidemiologia , Vírus Visna-Maedi/imunologia , Vírus Visna-Maedi/isolamento & purificaçãoRESUMO
Nucleotide sequences of small ruminant lentiviruses (SRLVs) were determined in sheep and goats, including progeny of imported animals, on a farm in Mexico. On the basis of gag-pol, pol, env and LTR sequences, SRLVs were assigned to the B1 subgroup, which comprises caprine arthritis-encephalitis virus (CAEV)-like prototype sequences mainly from goats. In comparison with CAEV-like env sequences of American and French origin, two putative recombination events were identified within the V3-V4 and V4-V5 regions of the env gene of a full length SRLV sequence (FESC-752) derived from a goat on the farm.
Assuntos
Vírus da Artrite-Encefalite Caprina/classificação , Vírus da Artrite-Encefalite Caprina/genética , Genes Virais , Doenças das Cabras/virologia , Infecções por Lentivirus/veterinária , Recombinação Genética , Doenças dos Ovinos/virologia , Animais , Sequência de Bases , Genes env , Genes gag , Genes pol , Cabras , Infecções por Lentivirus/virologia , México , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Ovinos , Carneiro Doméstico , Sequências Repetidas TerminaisRESUMO
Antibody-based diagnosis of small ruminant lentiviruses (SRLVs) has been efficiently achieved using serum and milk, but not semen, for which polymerase chain reaction (PCR) has been proposed as a confirmatory technique. This work, involving 296 ovine (Ovis aries) and caprine (Capra hircus) semen donors, investigates whether seminal fluid (SF) can be reliably used in antibody-based SRLV diagnosis. First, a gold standard was established to assess the infection status and determine the sensitivity and specificity of three commercial enzyme-linked immunosorbent assays (ELISAs) in serum testing using Western blot and PCR as confirmatory tests. For SF testing, both gold standard and serum testing results were used as reference. The performance of SF testing was affected not only by the ELISA assay sensitivity (related to antigen spectrum) compared with that of the gold standard (as it occurred in serum testing) but also by SF sample quality and SF working dilution. Nonturbid SF samples, commonly collected in artificial insemination centers (AICs), were required. Compared with serum, SF testing had a decreased sensitivity in two of the ELISA assays (with original serum working dilutions Assuntos
Ensaio de Imunoadsorção Enzimática/métodos
, Doenças das Cabras/diagnóstico
, Infecções por Lentivirus/veterinária
, Sêmen/virologia
, Doenças dos Ovinos/diagnóstico
, Animais
, Doenças das Cabras/virologia
, Cabras
, Infecções por Lentivirus/diagnóstico
, Lentivirus Ovinos-Caprinos/genética
, Lentivirus Ovinos-Caprinos/isolamento & purificação
, Estudos Longitudinais
, Masculino
, Sensibilidade e Especificidade
, Ovinos
, Doenças dos Ovinos/virologia
RESUMO
RNA transcripts of the B7 family molecule (CD80) are diminished in blood leukocytes from animals clinically affected with Visna/Maedi virus (VMV) infection. This work investigates whether the use of B7 genes enhances immune responses and protection in immunization-challenge approaches. Sheep were primed by particle-mediated epidermal bombardment with VMV gag and env gene recombinant plasmids together with plasmids encoding both CD80 and CD86 or CD80 alone, boosted with gag and env gene recombinant modified vaccinia Ankara virus and challenged intratracheally with VMV. Immunization in the presence of one or both of the B7 genes resulted in CD4+ T cell activation and antibody production (before and after challenge, respectively), but only immunization with CD80 and CD86 genes together, and not CD80 alone, resulted in a reduced number of infected animals and increased early transient cytotoxic T lymphocytes (CTL) responses. Post-mortem analysis showed an immune activation of lymphoid tissue in challenge-target organs in those animals that had received B7 genes compared to unvaccinated animals. Thus, the inclusion of B7 genes helped to enhance early cellular responses and protection (diminished proportion of infected animals) against VMV infection.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígeno B7-1/administração & dosagem , Pneumonia Intersticial Progressiva dos Ovinos/prevenção & controle , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Vírus Visna-Maedi/imunologia , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antivirais/sangue , Antígeno B7-1/genética , Antígeno B7-1/farmacologia , Antígeno B7-2/administração & dosagem , Antígeno B7-2/genética , Antígeno B7-2/farmacologia , Linfócitos T CD4-Positivos/imunologia , Produtos do Gene env/administração & dosagem , Produtos do Gene env/genética , Produtos do Gene gag/administração & dosagem , Produtos do Gene gag/genética , Vetores Genéticos , Imunização Secundária/métodos , Masculino , Ovinos , Linfócitos T Citotóxicos/imunologia , Vaccinia virus/genética , Vírus Visna-Maedi/genéticaRESUMO
Small ruminant lentiviruses (SRLVs) cause different clinical forms of disease in sheep and goats. So far in Spain, Maedi visna virus-like (MVV-like) sequences have been found in both species, and the arthritic SRLV disease has never been found in sheep until a recent outbreak. Knowing that arthritis is common in goats, it was of interest to determine if the genetic type of the virus involved in the sheep arthritis outbreak was caprine arthritis encephalitis virus-like (CAEV-like) rather than MVV-like. Alignment and phylogenetic analyses on nucleotide and deduced amino acid sequences from SRLV of this outbreak, allowed a B2 genetic subgroup assignment of these SRLV, compatible with a correspondence between the virus genetic type and the disease form. Furthermore, an isolate was obtained from the arthritic outbreak, its full genome was CAEV-like but the pol integrase region was MVV-like. Although its LTR lacked a U3 repeat sequence and had a deletion in the R region, which has been proposed to reduce viral replication rate, its phenotype in sheep skin fibroblast cultures was rapid/high, thus it appeared to have adapted to sheep cells. This outbreak study represents the first report on CAEV-like genetic findings and complete genome analysis among Spanish small ruminants.
Assuntos
Artrite Infecciosa/veterinária , Surtos de Doenças/veterinária , Infecções por Lentivirus/veterinária , Lentivirus Ovinos-Caprinos/genética , Doenças dos Ovinos/virologia , Animais , Artrite Infecciosa/genética , Artrite Infecciosa/virologia , Sequência de Bases , Plexo Corióideo/virologia , Clonagem Molecular , DNA Viral/genética , DNA Viral/isolamento & purificação , Genoma Viral , Infecções por Lentivirus/epidemiologia , Lentivirus Ovinos-Caprinos/classificação , Lentivirus Ovinos-Caprinos/isolamento & purificação , Filogenia , Reação em Cadeia da Polimerase/métodos , RNA Viral/genética , Ovinos , Espanha , Líquido Sinovial/virologia , Membrana Sinovial/virologia , Sequências Repetidas Terminais/genética , Vírus Visna-Maedi/classificação , Vírus Visna-Maedi/genética , Vírus Visna-Maedi/isolamento & purificaçãoRESUMO
To determine whether systemic immunization with plasmid DNA and virus vector against visna/maedi virus (VMV) would induce protective immune responses, sheep were immunized with VMV gag and/or env sequences using particle-mediated epidermal bombardment and injection of recombinant modified vaccinia Ankara. The results showed that immunization induced both humoral and cell-mediated responses prior to and after virus challenge. The vaccination protocol did not prevent infection, but immunization with the gag gene or a combination of gag and env genes resulted in significantly reduced provirus loads in blood and mediastinal lymph node, respectively. Provirus loads in lung and draining lymph node were unaffected, but p25 expression was undetectable in lungs of animals immunized with a combination of gag and env genes. Analysis of target tissues for lesions at post-mortem showed that immunization with the env gene caused a significant increase in lesion score, while the gag gene or a combination of gag and env genes had no effect. Inclusion of the ovine interferon-gamma gene in the initial priming mixture had minimal effect on immune responses, provirus load, or lesion development, although it resulted in a decreased p25 expression in the lung. The results thus show that systemic immunization with gag or a combination of gag and env genes reduces provirus load in blood and lymphoid tissue, respectively whereas env immunization has no effect on provirus load but increased lesion development.
Assuntos
Biolística , Genes env/genética , Genes gag/genética , Pneumonia Intersticial Progressiva dos Ovinos/prevenção & controle , Vacinas de DNA , Vaccinia virus/genética , Animais , Anticorpos Antivirais/sangue , Epiderme/virologia , Feminino , Genes env/imunologia , Genes gag/imunologia , Imunização , Masculino , Pneumonia Intersticial Progressiva dos Ovinos/virologia , Provírus/isolamento & purificação , Ovinos , Linfócitos T/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vaccinia virus/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/imunologia , Vírion/genética , Vírion/imunologia , Vírus Visna-MaediRESUMO
Sheep were immunized against Visna/Maedi virus (VMV) gag and/or env genes via the nasopharynx-associated lymphoid tissue (NALT) and lung using polyethylenimine (PEI)-DNA complexes and modified vaccinia Ankara, and challenged with live virus via the lung. env immunization enhanced humoral responses prior to but not after VMV challenge. Systemic T cell proliferative and cytotoxic responses were generally low, with the responses following single gag gene immunization being significantly depressed after challenge. A transient reduction in provirus load in the blood early after challenge was observed following env immunization, whilst the gag gene either alone or in combination with env resulted in significantly elevated provirus loads in lung. However, despite this, a significant reduction in lesion score was observed in animals immunized with the single gag gene at post-mortem. Inclusion of IFN-gamma in the immunization mixture in general had no significant effects. The results thus showed that protective effects against VMV-induced lesions can be induced following respiratory immunization with the single gag gene, though this was accompanied by an increased pulmonary provirus load.
Assuntos
Produtos do Gene env/imunologia , Produtos do Gene pol/imunologia , Pneumonia Intersticial Progressiva dos Ovinos/prevenção & controle , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Vírus Visna-Maedi/imunologia , Animais , Anticorpos Antivirais/sangue , Proliferação de Células , Testes Imunológicos de Citotoxicidade , Feminino , Produtos do Gene env/genética , Produtos do Gene pol/genética , Vetores Genéticos , Leucócitos Mononucleares/imunologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Masculino , Nasofaringe/imunologia , Provírus/isolamento & purificação , Índice de Gravidade de Doença , Ovinos , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/administração & dosagem , Vaccinia virus/genética , Carga Viral , Vacinas Virais/administração & dosagemRESUMO
Small ruminant lentiviruses (SRLV) are widely spread in many countries, including Spain. However, little is known about the genetic characteristics of Spanish goat and sheep SRLV. In this study, segments from three genomic regions (pol, gag-p25 and LTR) were amplified using DNA isolated from three Spanish autochthonous sheep (one) and goats (two). Animals (one per flock) belonged to distantly located, single-species flocks (goat or sheep). Sequence analysis showed conservation of regions that are putatively relevant to viral survival. Sequences of Spanish goat and sheep SRLV were allocated into phylogenetic trees (phylograms) with known SRLV groups. The phylograms corresponding to the pol, gag-p25 and LTR regions analyzed presented a compatible topology. This showed that Spanish caprine and ovine SRLV sequences belonged to the A or D phylogenetic groups and were closer to sheep SRLV prototypes (A1 group) than to goat SRLV prototypes (B or C groups), according to the current classification [Shah, C., Boni, J., Huder, J.B., Vogt, H.R., Muhlherr, J., Zanoni, R., Miserez, R., Lutz, H., Schupbach, J., 2004a. Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: evidence for regular sheep-to-goat transmission and worldwide propagation through livestock trade. Virology 319 (1), 12-26]. It was not possible to amplify in the three genetic regions the expected fragment in additional Spanish caprine and ovine SRLV proviral DNA sequences with the PCR primers used. This suggests that there is heterogeneity at the primer binding site among Spanish SRLV sequences. It also illustrates the need to develop diagnostic tests that are sensitive in local breeds.