The detection of proviral DNA by semi-nested polymerase chain reaction and phylogenetic analysis of Czech Maedi-Visna isolates based on gag gene sequences

A semi-nested polymerase chain reaction (snPCR) for detecting proviral DNA of ovine lentivirus (OvLV) in peripheral blood mononuclear cells was developed. Primers for snPCR were situated within the gag gene of the Maedi-Visna virus (MVV) genome. A comparison between the snPCR and serological tests (agar gel immunodiffusion test, immunoblot) were performed using 98 ovine blood samples. Thirty (30.6%) of the 98 sheep examined had antibodies specific for the MVV. PCR showed 21 of them to be positive and nine seropositive animals to be PCR negative. Six of the 68 serologically negative sheep were found to be PCR positive, probably due to delayed seroconversion. The PCR amplification products of these six sheep were sequenced and subjected to phylogenetic analysis. The resulting phylogenetic tree of partial gag gene sequences confirmed that the ovine lentivirus genotype in the Czech Republic is more closely related to the prototype MVV isolates than to the caprine arthritis encephalitis viruses.     

Presence of Maedi Visna Virus (MVV)‐Proviral DNA in the Genital Tissues of Naturally Infected Ewes

Maedi Visna virus (MVV) causes progressive degenerative inflammatory disease in multiple organs including the lungs (pneumonia, ‘maedi’), mammary gland, joints and nervous system (meningoencephalomyelitis, ‘visna’) in sheep. Maedi Visna Virus has been detected in macrophages of several tissues and epithelial cells in vivo: bone marrow, cells of the central nervous system, lung and bronchial tissues, milk epithelial cells recovered from milk samples and epithelial cells of mammary tissue. However, the presence of MVV in the genital tracts of naturally infected ewes has not previously been studied. The aim of this study was to use nested‐PCR, targeting the gag gene, to determine whether genital tissues (ovaries, oviducts and uterus) from 83 ewes originating from various breeding herds in the South‐East of France were positive for MVV‐proviral DNA. Peripheral blood mononuclear cells (PBMC) tested positive for MVV‐proviral DNA, using nested‐PCR analysis, in 57.8% of ewes (48/83). The provirus was also identified in 47% (78/166) of the ovaries, 38.6% (64/166) of the oviducts and 45.8% (38/83) of the uteri sampled. These findings clearly demonstrate, for the first time, that tissue samples from the genital tract of ewes (ovary, oviduct and uterus) can be infected with MVV. This suggests that there is a risk of vertical and/or horizontal transmission of MVV during embryo transfer from embryos produced in vivo or in vitro.     

Comparison of a maedi-visna virus CA-TM fusion protein ELISA with other assays for detecting sheep infected with North American ovine lentivirus strains.

A maedi-visna virus CA-TM fusion protein ELISA (MVV ELISA) was evaluated for the detection of antibody in sheep infected with North American ovine lentivirus (OvLV). The results of the MVV ELISA were compared with other assays for OvLV antibody and with viral infection in an intensively studied group of 38 sheep with a high prevalence of OvLV infection and disease. The sensitivity, specificity, and concordance of assays for OvLV antibody (MVV ELISA, indirect ELISA, Western blot, and AGID), virus (virus isolation, PCR, antigen ELISA), and OvLV-induced disease in each animal were compared with OvLV infection status as defined by a positive result in two or more of the assays. Five sheep met the criteria for absence of OvLV infection. The sensitivity of the MVV ELISA in detecting OvLV infected sheep was 64%, whereas the sensitivity of the other three tests for antibody ranged from 85 to 94%. All the antibody assays were 100% specific in this group of animals. Of the assays for virus, the PCR test had the highest sensitivity and the best concordance with OvLV infection, but it also had the lowest specificity of any of the virus or antibody assays. Among the antibody tests, the concordance of the MVV ELISA compared most favorably with the AGID test for detecting OvLV-infected sheep. Analysis of serum samples from 28 lambs experimentally-infected with one of three North American strains of OvLV suggested that there were no significant strain differences detectable by antibody assay. Twenty virus-inoculated lambs were positive by both the MVV ELISA and the AGID test, five lambs were MVV ELISA negative and AGID test positive, and three lambs were MVV ELISA positive and AGID test negative. No pre-inoculation samples were positive by either assay. In a longitudinal study involving seven lambs, antibodies to OvLV were detected by AGID 3-5 weeks post-inoculation, but were not detected by MVV ELISA until 5-10 weeks post-inoculation. Among 128 naturally and experimentally-infected sheep that were seropositive in the AGID test, the overall sensitivity of the MVV ELISA was higher in the naturally infected sheep (84%) than in the experimentally infected sheep (69%). The data indicated that the MVV ELISA represents a less sensitive, but specific alternative for the detection of OvLV antibodies.     

Genetic characterization of maedi-visna virus (MVV) detected in Finland

The aim of the study was to characterize the small-ruminant lentiviruses (SRLVs) detected in Finland by defining their phylogenetic relationships and by studying the evolution of the virus based on a well-known epidemiology. The study material comprised lung tissue samples of 20 sheep from 5 different farms, a cell-cultured virus from one of the original sheep lung samples, and a blood sample of a goat. The sheep were identified as positive during seroepidemiologic screenings in 1994-1996 and the goat in 2001. Initial classification of a 251 nucleotide sequence within gag gene amplified from the uncultured samples as well as from the cell-cultured virus showed that the SRLVs were genetically close and that they were more closely related to the prototype ovine maedi-visna viruses (MVVs) than to the caprine arthritis-encephalitis virus (CAEV). The lentivirus detected from the goat aligned within the cluster of the Finnish ovine viruses, demonstrating a natural sheep-to-goat transmission. Further phylogenetic analysis of the proviral gag, pol and env sequences confirmed the initial classification and showed that they constituted a new subtype within the diverse MVV group. The sequence analyses also showed that the virus had remained genetically relatively stable, in spite of the time given for virus evolution, an estimated 20 years, and in spite of the virus crossing the host species barrier.     

Molecular characterization of lentiviruses from goats from Poland based on gag gene sequence analysis

Caprine arthritis-encephalitis virus (CAEV) infection in goats is worldwide but with higher prevalence in industrialized countries. While positive serology of CAEV in Polish goats was reported there was no genetic study of this virus. In this study, we described the molecular characterization of lentiviruses isolated from seropositive goats from Poland. We cloned and sequenced a fragment from the gag gene covering part of the coding sequences for the matrix (MA) p17 and for the capsid (CA) p25 proteins. Resulting nucleotide sequences were aligned with those from other ovine/caprine lentivirus isolates. We present data showing that the sequences of most goat lentivirus isolates are closer to the prototypic CAEV-Co isolate, nevertheless from one goat we isolated a virus that is closer to the sheep Maedi Visna virus (MVV) isolate. This might indicate a recent cross-species infection from sheep to goat.     

Impact of natural sheep-goat transmission on detection and control of small ruminant lentivirus group C infections

Dissemination of small ruminant lentivirus (SRLV) infections in Norway is affected by the different control strategies used for maedi-visna virus (MVV) infections in sheep and caprine arthritis-encephalitis virus (CAEV) infections in goats. Here we investigated SRLV phylogenetic group variants in sheep. CAEV-like isolates, belonging to phylogenetic group C, were found among both seropositive sheep and goats in mixed flocks, in which sheep and goats are kept together. Intra-herd clustering confirmed that mixed flock animals were infected by the same virus variant, suggesting ongoing interspecies transmission. Few sheep flocks were found to be infected with the MVV-like phylogenetic group A. The apparent absence of SRLV group A type in goats is probably due to the MVV control programme and animal management practices. SRLV group C targets lungs and mammary glands in sheep, and induces typical SRLV pathological lesions. SRLV group C isolated from the sheep mammary glands suggested a productive infection and potential for transmission to offspring. SRLV group C was most prevalent among goats. A lower PCR sensitivity in seropositive sheep suggested a lower load of SRLV group C provirus in sheep than in goats. Higher genetic divergence of group C than in other SRLV groups and extensive heterogeneity among group C isolates in the matrix C-terminal region demonstrate the need for identifying conserved target regions when developing PCR protocols for SRLV detection. As sheep and goats may serve as reservoirs for all SRLV genogroup types, successful control programmes require inclusion of both species.     

Determination of the More Adequate Modified Agglutination Test Cut‐off for Serodiagnosis of Toxoplasma gondii Infection in Sheep

Toxoplasmosis is one of the most common food borne disease world‐wide. Among food animals, sheep seems to having higher prevalence of Toxoplasma gondii infection. However, there is no consensus about the best cut‐off for serodiagnosis in sheep. To estimate the more adequate cut‐off value of Modified Agglutination Test (MAT) for serodiagnosis in sheep, a commercial ELISA kit was used as a golden standard. Evaluation of the optimal sensitivity and specificity was calculated using Youden’s J‐statistics. Values obtained were used to estimate the prevalence of sheep toxoplasmosis. One thousand four hundred and sixty seven blood samples were collected randomly from 160 farms from northern Portugal, representing approximately 10% of the ovine population from the region. All sera were tested for anti‐T. gondii antibodies using the MAT. One hundred nine sheep (7.4%) presented a MAT titer ≥1 : 80; 45 (3.0%) a MAT titer of 1 : 40; 97 (6.6%) a MAT titer of 1 : 20 and 1216 (83.0%) a MAT titer ≤1 : 20. The best Youden’s J‐statistic was obtained at 1 : 20 titer (0.752), with 86.15% of sensitivity and 89.09% of specificity with negative and positive predictive values of 90.32% and 84.48% respectively, suggesting that the 1 : 20 was the most appropriate cut‐off for serodiagnosis of toxoplasmosis in sheep. Assuming this cut‐off, the prevalence of toxoplasmosis in the studied population was 17.1% and 92 (57.5%) of the 160 studied flocks having one or more positive sheep. Those results indicate that toxoplasmosis in Portugal should be considered in the differential diagnosis of abortions in sheep and neurological signs in lambs. Furthermore, while Portugal produces ovine meat for internal consumption and for exportation, isolation of T. gondii from ovine meat and further characterization of the isolates will be needed to understand the risk that ovine toxoplasmosis may represent for human health.     

Human and ovine lentiviral infections compared

Maedi-visna virus (MVV) of sheep was the first lentivirus to be isolated. The genomic organization of MVV is very similar to that of human immunodeficiency virus (HIV) with several genes regulating the expression of the viral genome. Viral replication is severely restricted in the host and some cells apparently contain the genetic information in a DNA provirus form with little or no expression of viral antigens. This seems to be a major factor in causing the “slowness” of lentiviral infections and the persistence of the virus in the host since the immune system may not recognize the provirus-containing cells. The target cells for HIV and MVV are similar although T4 lymphocytes are not specifically destroyed in maedi-visna. There are also certain similarities in the pathological changes in both diseases, both in the central nervous system, the lungs and the lymphatic system. Although the severe final immunodeficiency state characteristic of AIDS has not been observed in maedi-visna, the basic biological features of the MVV and its interaction with host cells are so similar to HIV infection, that we consider ovine maedi-visna useful animal model for the human lentivirus infections.     

A 57‐bp deletion in the ovine KAP6‐1 gene affects wool fibre diameter

High glycine–tyrosine keratin‐associated proteins (HGT‐KAPs) are predominantly present in the orthocortex of wool fibres. They vary in abundance in different wools and have been implicated in regulating wool fibre properties, but little is known about the functional roles of these proteins in the fibre matrix. In this study, we used polymerase chain reaction – single‐strand conformational polymorphism (PCR‐SSCP) analysis to screen for variation in a gene encoding the ovine HGT‐KAP6‐1 protein. We identified three gene variants (A, B and C). Variants A and B were similar to each other, with only three nucleotide differences occurring downstream of the coding sequence. However, variant C had a 57‐bp deletion that would notionally result in a loss of 19 amino acids in the protein. The presence of C was found to be associated with an increase in mean fibre diameter (MFD), fibre diameter standard deviation (FDSD), coefficient of variation of fibre diameter (CVFD) and prickle factor (percentage of fibres over 30 microns; PF). Sheep of genotype BC produced wool of greater MFD, FDSD and PF than sheep of genotypes AA, AB and BB. The CVFD was greater in the BC sheep than the AB sheep. The results suggest that variation in ovine KRTAP6‐1 affects wool fibre diameter‐associated traits and that the 57‐bp deletion in this gene would lead to coarser wool with greater FDSD, CVFD and PF.     

羊梅迪-维斯那病毒实时荧光定量PCR检测方法的建立

为了建立一种能快速检羊梅迪-维斯那病毒(MVV)的实时荧光定量PCR,根据GenBank中MVV gag基因的序列,设计了1对特异性引物及TaqMan探针,经过反应体系及条件优化,以定量的10倍系列稀释的阳性质粒为标准品进行实时荧光定量PCR扩增,建立了MVV实时荧光定量PCR。结果显示,该方法对MVV DNA最小检出量为10copies/μL,比普通PCR具有较高的检出率;组内及组间变异系数均低于2%,具有较好的重复性;该方法可以特异地检测到MVV,与其他病毒性样品无交叉反应。被检的92份临床样品中,实时荧光定量PCR和常规PCR的阳性检出率分别为4.3%和3.3%。为羊MVV快速检测和分子流行病学调查提供了一种有效的方法。     

Application of PCR technique in diagnosis of small ruminant lentivirus infection in sheep and goats

Detection of small ruminant lentiviruses (SRLVs) in sheep and goats usually relies on serological testing. In this study, we evaluated semi-nested PCR and nested PCR techniques applied as a diagnostic tool for detection of maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) in naturally infected sheep and goats, respectively. The examination of 193 ovine and 85 caprine serum samples by the ELISA revealed the presence of specific antibodies in 133 (69%) and 18 (21.2%) animals, respectively. Presence of proviral DNA was manifested in 103 (53.4%) sheep and 12 (14.2%) goats. Despite the relatively lower sensitivity of PCR, the fact of detection of proviral DNA in 19 out of 60 ovine samples and 7 out of 67 caprine samples collected from animals previously negative by ELISA was noteworthy. In conclusion, the data demonstrated that combinations of both ELISA and PCR might afford optimal detection of SRLVs infection.     

Molecular characterization of bovine foamy virus and its association with bovine leukemia virus infection in Vietnamese cattle

The detection of bovine foamy virus (BFV) in Vietnamese cattle was performed using conventional PCR targeting pol and gag genes. Out of 243 tested samples, ten (4.1%) and eight (3.3%) samples were positive for BFV gag and pol DNA, respectively. The prevalence of bovine leukemia virus (BLV) estimated by detection of proviral DNA using nested PCR targeting env gene was 26.7% (65/243). The results of nucleotide sequence alignment and the phylogenetic analysis suggested that Vietnamese BFV strains showed high homology to isolates belonging to either European or non-European clades. There was no significant correlation between BLV and BFV. This study provides information regarding BFV infection and confirms the existence of two BFV clades among Vietnamese cattle for the first time.     

Determination of prevalence,serological diversity,and virulence of Dichelobacter nodosus in ovine footrot with identification of its predominant serotype as a potential vaccine candidate in J&K,India

The aim of this study was to determine the prevalence, serological diversity, and virulence of Dichelobacter nodosus in footrot lesions of sheep and identification of its predominant serotype as a potential vaccine candidate. The overall prevalence of footrot in sheep was 16.19%, and ranged from 13.69 to 19.71%, respectively. A total of 759 flocks with 22,698 sheep were investigated for footrot and 2374 clinical samples were collected from naturally infected sheep exhibiting footrot lesions. Of the 2374 samples collected, 1446 (60.90%) were positive for D. nodosus by polymerase chain reaction (PCR). These positive samples when subjected to serogroup-specific multiplex PCR, 1337 (92.46%) samples carried serogroup B, 247 (17.08%) possessed serogroup E, 86 (5.94%) serogroup I, and one (0.069%) serogroup G of D. nodosus. While mixed infection of serogroups B and E was detected in 127 (8.78%), B and I in 46 (3.18%) and B, E, and I in 26 (1.79%) samples, respectively. The serogroup B of D. nodosus was the predominant (92.47%) serogroup affecting sheep population with footrot followed by serogroup E (19.91%) and serogroup I (4.57%), respectively. Virulent status of D. nodosus strains were confirmed by presence of virulence-specific integrase A (intA) gene and the production of thermostable proteases. The intA gene was detected in 709 (72.79%) samples while gelatin gel test carried out on 246 representative isolates all positive for intA gene produced thermostable proteases, confirming their virulence nature. The PCR-restriction fragment length polymorphism (PCR-RFLP) of whole fimA gene of serogroup B revealed the predominance of serotype B5 (82.97%) of serogroup B. This information suggests that serotype B5 is the predominant serotype of D. nodosus associated with severe footrot lesions in sheep in Jammu & Kashmir (J&K), India. Hence, this serotype can be a potential vaccine candidate for the effective control and treatment of ovine footrot.

     

PCR detection of lentiviral GAG segment DNA in the white blood cells of sheep and goats

A PCR assay for the detection of small ruminant lentiviral gag DNA (provirus) in the white blood cells of sheep and goats was developed and compared with a serological test (AGIDT). A sample of the DNA prepared from the white blood cells in 3 ml of blood from 208 sheep and goats from 18 different flocks was subjected to PCR assay. One of 85 animals from flocks accredited under the Dutch national MVV/CAEV control programme was positive by PCR while none was positive by AGIDT. In infected flocks, the AGIDT appeared slightly more sensitive, but preliminary results show that the sensitivity of the PCR assay may be further improved by increasing the number of monocytes tested. The PCR assay, however, was clearly more sensitive in detecting animals in the early stages of infection. With the use of a set of mixed primers and probes, the assay was able to detect the variety of CAEV and MVV strains occurring in the field.     

In vivo depletion of CD8+ cells does not affect primary maedi visna virus infection in sheep

T-cells have been implicated both, in promoting and reducing viral replication during lentivirus infection. CD8+ lymphocytes are believed to be important in controlling viral load through direct killing of virus-infected cells and by secretion of inhibitory chemokines and cytokines. To evaluate the role of CD8+ T-cells in the induction and control of the primary phase of a lentivirus infection, we have used a non-T-cell tropic lentivirus, maedi-visna virus (MVV), to study the initial pathogenesis and subsequent immune responses in sheep depleted in vivo of CD8+ cells. Sheep were depleted of CD8+ cells in both blood and efferent lymph for up to 14 days. No difference in MVV replication was observed in either the draining efferent lymph or lymph node of these sheep. Surprisingly, these animals displayed a normal induction of pCTL whereas the virus-specific proliferative responses were reduced. This could reflect either that a proportion of functional CD8+ lymphocytes remained in these animals, as suggested by the appearance of pCTLs, or that CD8+ cells are not required for control of primary MVV infection.     

Detection of Chlamydophila abortus in Ovine Milk by Immunomagnetic Separation–Polymerase Chain Reaction

The present study was carried out to investigate the presence of Chlamydophila abortus, the causative agent of ovine enzootic abortion, in milk samples collected from sheep flocks with and without the history of abortion in Eastern Turkey by means of immunomagnetic separation (IMS) in conjunction with the polymerase chain reaction (PCR). A total number of 201 milk samples collected from 10 flocks with abortion and four flocks without abortion were tested. In the analysis of the milk samples by IMS–PCR, correct amplification was obtained with three (1.5%) samples originating from one flock with abortion. In the digestion of PCR positive products by AluI, restriction profiles observed in all three samples were determined to be the same as C. abortus S26/3.     

Ovine Lentivirus is Aetiologically Associated with Chronic Respiratory Disease of Sheep on the Laikipia Plateau in Kenya

A study was undertaken to investigate the occurrence of ovine lentivirus (OvLV) infection in sheep with chronic respiratory disease on the Laikipia Plateau, Kenya. All seven Merino crossbred sheep with chronic dyspnoea and emaciation examined for gross and microscopic lesions had lymphoid interstitial pneumonia (LIP), and one also had pulmonary abscesses. Two of the sheep with LIP also had lesions of ovine pulmonary carcinoma (OPC, jaagsiekte). Using in situ hybridization, OvLV DNA localized to a high proportion of pulmonary macrophages in lungs with lesions of LIP. Lung tissue samples from six of these sheep were positive for a syncytium-inducing virus in cultures of lamb testis cells. Thin-section electron microscopy of infected cells showed virions with morphogenesis typical of lentiviruses. In a western blotting assay, monoclonal antibodies to the OvLV capsid (CA, p27) and matrix (MA, p15) proteins of a North American OvLV isolate reacted with similar-sized bands of the virus, and serum from six of the sheep were reactive with CA from the Kenyan viral isolate. Using an OvLV agar gel immunodiffusion (AGID) test, all seven sheep were positive for serum antiviral antibody, as were 29% of 63 clinically normal sheep from Laikipia District. However, when sera from the healthy sheep were tested in a western blot assay, only 52% had IgG reactive to the OvLV CA, indicating a high rate of false negative reactions with the AGID test. Serum samples from 87 Red Maasai or Dorper crossbred sheep from two farms in other parts of Kenya were OvLV seronegative by both the AGID test and the western blot assay. These results document the first identification of OvLV as a cause of chronic respiratory disease in sheep in Kenya and show a high rate of infection in sheep flocks, with a high prevalence of chronic respiratory disease.     

Detection of Theileria ovis in naturally infected sheep by nested PCR

A nested polymerase chain reaction (PCR) for the detection of Theileria ovis in sheep using oligonucleotide primers designed from the small subunit ribosomal RNA (SSU rRNA) gene sequence of T. ovis from sheep in eastern Turkey is described. A 398-bp DNA fragment was specifically amplified from blood samples from sheep, naturally infected with T. ovis. No PCR products resulted from T. lestoquardi, T. annulata, T. parva, T. buffeli and Babesia spp. DNA using these specific primers. The sensitivity of the nested PCR for T. ovis, which was assessed showed that one infected cell in 10(7) sheep erythrocytes, equivalent to a blood parasitemia of 0.00001%, could be detected. This is more sensitive than examining 200 fields under light microscopy. In addition, of the 124 field samples obtained from sheep in eastern Turkey tested, 19.35% (24/124) were positive for the presence of Theileria spp. by microscopic examination compared to 54.03% (67/124) positive for T. ovis by nested PCR. The primer pairs described in this study will be useful for epidemiological studies on ovine theileriosis and for discrimination between T. lestoquardi and T. ovis infections in sheep.     

Genetic variation in the 5′UTR of the KRT2.13 gene of sheep

KRT2.13 is a type II keratin wool intermediate filament (IF) protein. Extensive variation was revealed in the 5′ untranslated region (UTR) of the ovine KRT2.13 gene (KRT2.13) using polymerase chain reaction – single strand conformational polymorphism (PCR‐SSCP) analysis. Nine unique PCR‐SSCP patterns were obtained with individual sheep having either one (homozygous), or a combination of two (heterozygous) of these patterns. Seven of the amplicons that produced the apparently homozygous patterns were successfully sequenced (GenBank FJ217670 – FJ217676), revealing eight single nucleotide insertions, 10 single nucleotide substitutions, a nucleotide deletion and a 16 nucleotide insertion that occurred in only one of the sequences. The seven sequences showed between 85% and 95% homology to the previously identified KRT2.13 sequence (GenBank X72379). This study emphasizes the power of PCR‐SSCP analysis in genotyping, as this extensive variation was found in only 100 sheep, of a variety of breeds. Since variation in the 5′UTR of genes may affect their expression, this genetic variation needs to be further studied to establish its role if any, in influencing gene expression and consequently wool traits.     

Transmission of ovine herpesvirus 2 among adult sheep

Previous studies from this laboratory have defined the pattern of acquisition of ovine herpesvirus 2 (OHV-2) in lambs under natural flock conditions. This study examined the question of whether OHV-2 could be transmitted between adult sheep. Two potential routes of transmission were examined: (1) direct inoculation of either viable leukocytes or whole blood from OHV-2 positive sheep, and (2) horizontal transmission through natural contact with OHV-2 positive sheep. Two groups of OHV-2 negative adult sheep were inoculated with material from infected sheep, one with 5x10(8) viable peripheral blood leukocytes (PBL), and the other with 100 ml of whole peripheral blood. No PCR signals were detected in any of the three sheep inoculated with the PBL during the 20 weeks following inoculation. In the group of five sheep inoculated with whole blood, two became PCR-positive at 7 and 8 weeks post-inoculation, respectively, and the remaining three sheep maintained their negative status until termination of the experiment at 20 weeks post-inoculation. In two experiments conducted in different flocks, a total of 20 adult sheep were used to examine horizontal transmission by contact; all animals became PCR-positive within 12 months of mixing the uninfected and infected animals. The results of these experiments support two conclusions. First, the susceptibility to OHV-2 is not limited to young lambs; adult sheep remain fully susceptible. Second, the fact that whole blood, but not PBL, from infected sheep was able to transmit the infection to only two of five inoculated sheep suggests that the infection in peripheral blood cells may be largely non-productive.