Small Ruminant Lentiviruses in Sheep: Pathology and Tropism of 2 Strains Using the Bone Marrow Route.

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.

Small ruminant lentivirus-induced arthritis: clinicopathologic findings in sheep infected by a highly replicative SRLV B2 genotype.

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.

Ovine TRIM5α can restrict visna/maedi virus.

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.

Serologically defined (SD) locus in cattle.

Using cytotoxic serums obtained from multiparous cows or by alloimmunization, we have detected 11 lymphocyte antigens controlled by codominant alleles at a serologically defined locus called BoLA-A (bovine lymphocyte antigens). This locus, along with the lymphocyte defined loci previously reported, establishes the existence of a major histocompatibility system of cattle.

Molecular characterization and phylogenetic study of Maedi Visna and Caprine Arthritis Encephalitis viral sequences in sheep and goats from Spain.

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.

Horizontal Maedi-Visna virus (MVV) infection in adult dairy-sheep raised under varying MVV-infection pressures investigated by ELISA and PCR.

A three year long experimental study was carried out to investigate horizontal MVV-infection by PCR and ELISA, in 191 one year-old latxa dairy-sheep raised in two separate groups under low and high MVV-infection pressure, respectively. Sheep originated from a previous MVV-transmission study in lambs and seroprevalence among one year-old sheep in both groups was 15% approximately. The high infection-pressure group (H-group) consisted of 147 replacement ewes that joined a milk-producing, housed dairy-flock with 42-66% MVV-seroprevalence and the low infection-pressure group (L-group) were castrated males raised in a separate shed. In contrast to results obtained when infection was investigated in lambs, the overall degree of agreement between ELISA and PCR results was very good and there was some indication that it increased further as sheep became older. MVV-prevalence did not change in the L-group and increased to 57% in three year-old sheep in the H-group (p<0.001). Random effects logistic regression confirmed seroconversion was significantly higher in the H-group compared to the L-group and was highest during the year after the sheep were introduced in the dairy flock and did not increase with age as in previous studies using less sensitive antibody assays. The evidence that horizontal transmission can be very low in spite of prolonged close contact between infected and non-infected sheep is valuable for MVV-control purposes. Furthermore it highlights the need to investigate virus excretion dynamics in infected animals and animal to animal transmission to improve our overall understanding of horizontal MVV transmission in MVV endemic populations.

PCR detection of colostrum-associated Maedi-Visna virus (MVV) infection and relationship with ELISA-antibody status in lambs.

A recent large-scale experimental study showed that bottle-feeding ovine colostrum from seropositive ewes results in high MVV-seroconversion in lambs. In contrast, relatively few lambs that naturally suckled colostrum from seropositive dams seroconverted as a result of it. Furthermore, lambs fed uninfected bovine colostrum readily seroconverted when mixed with ovine-colostrum lambs indicating that horizontal MVV transmission between lambs was efficient. MVV-infection was further investigated in the same samples using two PCR tests targeting sequences in the long-terminal repeats (LTR) and POL MVV genes. PCR-tests confirmed previous serological findings. However, the LTR-PCR was more sensitive and allowed detecting infection earlier than the other tests, including 5-8% of new-born lambs from seropositive dams, providing more evidence that prenatal MVV-infection may be more important than considered. The degree of agreement between PCR and antibody tests in individual samples was low up to 6 months of age and moderate at 10 months-old. Nine percent of lambs were always PCR-negative but seroconverted and 19% of lambs were PCR-positive at least once and did not seroconvert. However, seroconversion was associated with increasing number of times lambs were PCR-positive and ovine colostrum-fed lambs were more frequently PCR-positive than other lambs. The significance of these findings in terms of MVV-infection, epidemiology and control is discussed.

Relative contribution of colostrum from Maedi-Visna virus (MVV) infected ewes to MVV-seroprevalence in lambs.

Maedi-visna virus (MVV) seroprevalence associated with consumption of colostrum from seropositive ewes was investigated in 276 housed lambs from birth to 300 days-old. At birth, lambs were allocated to five experimental groups according to the maternal MVV-serological status, source and mode of feeding colostrum (bovine or ovine and bottle fed or suckled from the dam) and type of horizontal MVV-exposure (raised with the dam or separately with other lambs). The risk of being seropositive at 300 days-old was associated with feeding ovine colostrum from seropositive ewes and increased with intake of bottle-fed ovine colostrum and was higher in lambs separated from their dams and raised with other experimental lambs compared to lambs raised with their dams. Approximately 75-87% of ELISA-positive results in lambs that had ovine colostrum was attributable to colostrum itself. However, approximately only 16% of naturally raised and 29-61% of bottle-fed ovine colostrum lambs were ELISA-positive as a result feeding ovine colostrum. These results confirm that ovine colostrum from seropositive ewes can be a major source of MVV but its overall contribution to seroprevalence in natural conditions is relatively low, and shows that horizontal MVV transmission can be an important source of infection in new-born lambs.

Small ruminant lentivirus infections and diseases.

Small ruminant lentiviruses include viruses with diverse genotypes that frequently cross the species barrier between sheep and goats and that display a great genetic variability. These characteristics stress the need to consider the whole host range and to perform local surveillance of the viruses to opt for optimum diagnostic tests, in order to establish control programmes. In the absence of effective vaccines, a comprehensive knowledge of the epidemiology of these infections is of major importance to limit their spread. This article intends to cover these aspects and to summarise information related to characteristics of the viruses, pathogenesis of the infection and description of the various syndromes produced, as well as the diagnostic tools available, the mechanisms involved in transmission of the pathogens and, finally, the control strategies that have been designed until now, with remarks on the drawbacks and the advantages of each one. We conclude that there are many variables influencing the expected cost and benefits of control programs that must be evaluated, in order to put into practice measures that might lead to control of these infections.

An efficient microtest to study adherence of bacteria to mammalian cells.

A simple, fast and highly reproducible microtest was developed for in vitro adherence studies. A rat epithelial cell line was investigated for the adherence of clinical and subclinical ovine and bovine Staphylococcus aureus strains isolated from mastitis. Staphylococcus aureus strains differed in their ability to adhere to epithelial cells, the degree of adherence being dependent on the concentration of bacteria used in the test.

A simple infection model using pre-colonized implants to reproduce rat chronic Staphylococcus aureus osteomyelitis and study antibiotic treatment.

Staphylococcus aureus biofilms formed on medical implants represent a serious problem, being difficult to eradicate with antibiotic therapy and leading to chronic infections. Simplified in vivo and in vitro antibiotic susceptibility assays using biofilm bacteria are needed. In this work, a novel chronic osteomyelitis infection model was developed in rats in the absence of bacterial suspension, requiring the use of only 10(6) bacteria in biofilms at the site of surgery, with a full success in reproducing infection. Stainless-steel implants pre-colonized for 12 h with a highly adherent S. aureaus isolate were introduced into the rat tibiae. In animals not submitted to antibiotic treatment, infection was found in the implants and spread to bone in all cases, indicating the high efficacy of the model to reproduce osteomyelitis. The effect of a 21-day treatment with cefuroxime, vancomycin, tobramycin or ciprofloxacin on infection was studied in this model 42 days after surgery. Bone colonization was inhibited by vancomycin and cefuroxime. Cefuroxime (the most efficient antibiotic, able to sterilize 1 out of 8 implants) reduced the number of bacteria in biofilms adhered to implants at a higher extent than vancomycin, trobramycin and ciprofloxacin. Analogous observations were made in this work in vivo and in vitro on the relative antibiotic efficacy against S. aureus biofilm bacteria. suggesting the usefulness of both tests as a potential tool to study antibiotic suceptibility, and the need for new antimicrobials against these bacteria.

Staphylococcus aureus capsule and slime as virulence factors in ruminant mastitis. A review.

Staphylococcus aureus is one of the most prevalent causes of ruminant mastitis. The interaction of this microorganism with the host is strongly dependent on its cell surface properties, specially concerning the presence of the exopolysaccharide-containing outer layers (glycocalyx), which appear to play an important role in virulence. In this article, the definition and recognition of the types of exopolysaccharide layers are described, together with their likely role in the pathogenesis of mastitis.

Factors influencing the degree of in vitro bacterial adhesion to ovine mammary gland epithelial cells.

Bacterial adhesion to mammary gland epithelial cells (EC) may play a role in the pathogenesis of mastitis. In vitro adherence systems have been developed to study mastitis in cattle but little has been done in sheep. In this work, a method is described for obtaining mammary gland cell preparations containing greater than or equal to 65% EC from live or dead ewes, using a Ficoll-Hypaque flotation method (cell viability = 70-90%). An in vitro adhesion assay procedure was also developed to study the interaction between EC and ovine mastitis bacterial strains. It was observed that, under the test conditions, adherence increased as the incubation time was prolonged from 30 to 120 min (P less than 0.05). Adhesion was greater at incubation temperature of 37 degrees C than at 22 degrees C (P less than 0.001). An acidic pH (5.9) was associated with an increase in adhesion, when compared with a higher pH (7.2; P less than 0.05). Tween 20, Tween 80 and bovine serum albumin helped to eliminate a background of unbound bacteria from the test slides, but they also inhibited adhesion to some strains. Strain differences in adhesion and in ability to form a background were also observed. Some of these findings may have in vivo implications.

Effect of slime on adherence of Staphylococcus aureus isolated from bovine and ovine mastitis.

The interactions between slime, Staphylococcus aureus and ovine mammary gland epithelial cells (MGEC) were studied in vitro. Suspensions of radiolabelled bacteria incubated with slime significantly increased the ability of S. aureus strains to adhere to a filter. When suspensions of radiolabelled bacteria were incubated with MGEC treated with trypsin, the ability of slime to improve S. aureus adherence was also shown, indicating that it was not dependent on cell membrane proteins. The interaction of radiolabelled bacteria with slime prior to the adherence test with MGEC demonstrated that the adherence process requires the interaction between slime and bacteria. This interaction is inhibited by anti-slime antibodies. This study provides evidence that a specific interaction between bacteria coated with slime and MGEC could be a critical part of mammary gland infection.

Adherence of ruminant mastitis Staphylococcus aureus strains to epithelial cells from ovine mammary gland primary cultures and from a rat intestinal cell line.

Staphylococcus aureus isolated from mastitis (14 bovine and 11 ovine strains) exhibited an ability to adhere to epithelial primary cultures from ovine mammary gland and to a rat epithelial cell line, RIE-1. Strain differences in the degree of adherence were observed in both cases. These differences were maintained when comparing different epithelial sources (rat vs. ovine). RIE-1 cells can thus be used as a model for studying staphylococcal adherence to epithelial cells. Changes in bacterial adherence were observed according to the bacterial growth phase. The magnitude of these changes differed among strains. Bacterial cell surface hydrophobicity was not related to the degree of adherence to mammalian epithelial cells.

Effect of in vitro maedi-visna virus infection on adherence and phagocytosis of staphylococci by ovine cells.

This work was aimed at studying the effect of maedi-visna virus (MVV) infection in vitro on the ability of sheep cells to adhere to staphylococci (Staphylococcus aureus and Staphylococcus epidermidis), and phagocytose these bacteria. Adherence was studied in sheep choroid plexus cells (SCPC) using an ELISA test and phagocytosis was studied in pulmonary alveolar macrophages (PAM) by chemiluminescence. A 5- and 7-day of in vitro MVV infection resulted in syncytium formation and a significant increased adherence (P < 0.01) of SCPC to bacteria. SCPC endogenous fibronectin was significantly higher (P < 0.01) on days 5 and 7 than on day 0 of MVV infection. A significantly decreased phagocytosis (P < 0.05) was also observed on days 5 and 7 of MVV infection in PAM when compared to MVV-free controls. Comparatively, phagocytosis was highest for S. aureus non-slime producing strains, followed by S. epidermidis, and S. aureus slime producing strains, in that order. Finally, increased expression of both, class I and class II major histocompatibility antigens was also observed in MVV-infected PAM on days 5 and 7, whereas SCPC only demonstrated upregulation of MHC class I. These results, indicative of an alteration of some cell functions in MVV-infected cells, may help to understand interactions between MVV-infected cells and bacteria in simultaneous infections and may provide clues to the possible in vivo interactions of both pathogens.

Early detection of maedi-visna (ovine progressive pneumonia) virus seroconversion in field sheep samples.

The aim of this work was to investigate whether an enzyme-linked immunosorbent assay (ELISA) was useful for early detection of maedi-visna virus (MVV) infection in sheep under field conditions. An ELISA based on p25 recombinant protein and a gp46 synthetic peptide was used. Sequentially obtained serum samples (n = 1,941) were studied for 4 years. ELISA results were compared with those of the agar gel immunodiffusion (AGID) test, and results of both tests were compared with a reference result established using consensus scores for at least 2 of 3 serologic techniques (AGID, ELISA, and western blotting, which was used to resolve result discrepancies between the other 2 techniques). A total of 247 discrepancies were observed between ELISA and AGID. Of these, 131 were due to an earlier detection of 120 sera by the ELISA and 11 sera by AGID. The remaining discrepancies (116) were due to the presence of false reactions in both tests. Fewer false-negative results were found by ELISA than with AGID (6 vs. 69 sera, respectively), whereas the number of false-positive results was virtually the same for ELISA and AGID (21 vs. 20, respectively). In relation to the reference result, ELISA sensitivity and specificity were 97.8% and 98.2%, respectively, whereas values for AGID were 76.3% and 98.3%, respectively. The agreement between ELISA and the reference result was higher than that between AGID and the reference result (K value: 0.96 and 0.77, respectively). A variation in the ELISA signal (based on optical density) was observed during the study period, suggesting different antibody levels throughout the animal's life. The ELISA was useful for detecting MVV-infected sheep in field conditions and has potential for use in control and eradication programs.

Infection of rabbit mammary glands with ovine mastitis bacterial strains.

An experimental model was developed in rabbits to study ovine mastitis. A total of 19 ovine mastitis bacterial strains (seven Staphylococcus aureus, four Staph. chromogenes, four Staph. hyicus and four Escherichia coli) were used for mammary gland infections. The histopathological results showed that the ovine mastitis types corresponded to experimental infections produced in the rabbit with the ovine strains. These results helped the grading of the bacterial species tested according to the severity of their effects on the mammary gland. The most pathogenic species was Staph. aureus, followed by E. coli, Staph. hyicus and Staph. chromogenes, in that order. There was, however, variation among strains within a given species (e.g. one out of seven Staph. aureus strains gave rise to a mild infection in sheep and rabbits). The procedure was simple and consisted of introducing bacterial suspensions through alternate teat ducts of does with the help of a cannula. It helped minimize the number of animals required in the experiments.

Role of an intramammary device in protection against experimentally induced staphylococcal mastitis in ewes.

An intramammary device (IMD) was adapted for use in ewes; this device was made of abraded polyethylene material (1.7 mm in diameter, 47 mm long) and formed a 15-mm-diameter loop in the gland cistern. The IMD was inserted in 1 gland in each of 43 ewes. A significant (P < 0.0001) increase in milk somatic cell count (SCC) was observed in glands provided with an IMD. This increase was attributable to an increase in neutrophil numbers and was observed during the first 12 weeks after insertion. The IMD had a protective effect against experimentally induced staphylococcal mastitis (Staphylococcus aureus and S epidermidis), although different milk SCC were required for protection from each bacterial species in most ewes (10(6) and 2 x 10(5) cells/ml, respectively). Histologic studies revealed that the IMD induced local squamous metaplasia in the glandular part of the lactiferous sinus. Erythrocytes were found in milk from glands provided with an IMD throughout the studied period (35 days of the 45-day lactation) and, in some cases, blood clots were observed during the first 2 weeks of lactation. Glands with IMD also had lower milk production and quality at 30 and 32 days of lactation. Eight ewes with IMD were studied throughout a subsequent lactation. Milk from the IMD-containing glands had an increase in SCC, as in the previous lactation period; did not contain blood clots or erythrocytes; and had normal composition (similar to that in glands without the IMD).

Computer methodology for simulation and prediction of alloimmune responses: expected antibody specificities.

The methodology presented here was developed for simulating planned alloimmunization results, with regard to the type and number of expected antibody specificities. The computer program designed for this purpose was adapted to an immunogenetic model using Boolean algebra. It was written to help immunogeneticists avoid handling routine data preceding selection of donor-recipient pairs, specially concerning blood type alloimmunizations in animals: all of the donor-recipient combinations and the expected antibody specificities (their limit number being specified in each particular immunization program) are provided in the print-out.