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1.  Comparative assessment of two commonly used commercial ELISA tests for the serological diagnosis of contagious agalactia of small ruminants caused by Mycoplasma agalactiae 
Contagious agalactia (CA) of sheep and goats caused by Mycoplasma agalactiae is a widely occurring economically important disease that is difficult to control. The ELISA is commonly used for the serological detection of CA but it has some limitations and the performance of the available tests have not been properly evaluated.
Two commercial ELISA kits are widely used, one involving a fusion protein as target antigen and the other a total antigen. The objectives were to compare these tests by evaluating:
i. Their diagnostic sensitivity and specificity, the relevance of the recommended cut-off points, the correlation between the two tests, and, the correlation between serology data and the milk shedding of M. agalatiae;
ii. The influence of extrinsic factors such as the targeted animal species, geographical origin of the samples, intra-specific variability of M. agalactiae and concurrent mycoplasma infections.
A sample of 5900 animals from 211 farms with continuous CA monitoring for 20 years and no prior vaccination history was used. The infection status was known from prior bacteriological, epidemiological and serological monitoring with a complementary immunoblotting test.
The average diagnostic sensitivity was 56% [51.8–59.8] for the fusion protein ELISA and 84% [81.3–87.2] for the total antigen ELISA, with noteworthy flock-related variations. The average diagnostic specificity for the fusion protein ELISA was 100% [99.9–100], and for the total antigen ELISA differed significantly between goats and sheep: 99.3% [97.4–99.9] and 95.7% [93.8–97.2] respectively.
Experimental inoculations with different M. agalactiae strains revealed that the ELISA kits poorly detected the antibody response to certain strains. Furthermore, test performances varied according to the host species or geographical origin of the samples.
Finally, the correlation between milk shedding of M. agalactiae and the presence of detectable antibodies in the blood was poor.
These serological tests are not interchangeable. The choice of a test will depend on the objectives (early detection of infection or disease control program), on the prevalence of infection and the control protocol used. Given the variety of factors that may influence performance, a preliminary assessment of the test in a given situation is recommended prior to widespread use.
PMCID: PMC3439703  PMID: 22776779
2.  Serological Diagnosis of Ovine Enzootic Abortion by Enzyme-Linked Immunosorbent Assay with a Recombinant Protein Fragment of the Polymorphic Outer Membrane Protein POMP90 of Chlamydophila abortus 
Journal of Clinical Microbiology  2002;40(11):4235-4243.
Ovine enzootic abortion (OEA) resulting from infection of sheep and goats with Chlamydophila abortus is of major economic importance worldwide. Over the last 50 years the serological diagnosis of infection has been based mainly on the complement fixation test (CFT), which lacks both sensitivity and specificity because of cross-reactive antibodies to other gram-negative bacteria, including another common chlamydial pathogen of sheep, Chlamydophila pecorum. In the present study, a series of overlapping recombinant antigens representing the polymorphic outer membrane protein POMP90 of C. abortus was assessed by enzyme-linked immunosorbent assay (ELISA) with a panel of 143 serum samples from sheep experimentally infected with C. abortus, from sheep clinically free of OEA, and from specific-pathogen-free lambs experimentally infected with different subtypes of C. pecorum. The results were compared to those obtained by CFT and another recently described test, an indirect ELISA (iELISA) with the recombinant OMP91B (rOMP91B) fragment (rOMP91B iELISA) (D. Longbottom, E. Psarrou, M. Livingstone, and E. Vretou, FEMS Microbiol. Lett. 195:157-161, 2001). The rOMP90-3 and rOMP90-4 ELISAs were identified as being more sensitive and specific than CFT. Assays with both fragments were evaluated further with a panel of 294 field serum samples from flocks with documented histories of abortion, from flocks with no clinical histories of abortion but which had a high proportion of samples seropositive by CFT, and from animals with no histories of abortion but from which various C. pecorum subtypes had been isolated. ELISAs with both POMP90 fragments outperformed CFT with serum samples from C. pecorum-infected animals, producing no false-positive results. However, the ELISA with the rOMP90-4 fragment appeared to be more sensitive than the one with rOMP90-3, as it identified more of the OEA-positive samples. The ELISA with the rOMP90-4 fragment was also able to identify apparently healthy animals that were infected with an enteric strain of C. abortus in flocks that were probably infected with both enteric C. abortus and C. pecorum strains. The identification of animals infected with enteric C. abortus is extremely important in controlling the spread of OEA. Overall, the new rOMP90-4 ELISA was found to be a more sensitive and specific test than CFT for differentiating animals infected with C. abortus from those infected with C. pecorum.
PMCID: PMC139646  PMID: 12409404
3.  Experimental infection of lambs with C and S-type strains of Mycobacterium avium subspecies paratuberculosis: immunological and pathological findings 
Veterinary Research  2014;45(1):5.
The two main genotypes of recognized isolates of Mycobacterium avium subsp. paratuberculosis (Map) are cattle (C) and sheep (S) strains. An experimental infection was conducted to establish the effect of Map strain on the pathogenesis of ovine paratuberculosis. Twenty-four out of thirty 1.5-month-old Assaf lambs were divided into 4 groups of 6 and infected orally with three low passage field isolates, two of S- (22G and the pigmented Ovicap49) and one of C– (764) type, and the reference K-10 strain (C type). The remaining six animals were unchallenged controls. Animals were euthanized at 150 and 390 days post-infection (dpi). Throughout the experiment, the peripheral immune response was assessed and histological and molecular (PCR) studies were conducted on samples of intestine and related lymphoid tissue. Specific antibody and IFN-γ production was significantly higher in animals infected with the C strains, while no consistent IFN- γ responses were observed in the S-type strain infected groups. A positive intradermal skin test response was detected in all infected groups. Lambs infected with S-type strains had granulomatous lesions restricted to the lymphoid tissue with no differences in the lesion intensity over time. In both C–type strain groups, lesions were more severe at 150 dpi while at 390 dpi lesions, characterized by well-demarcated granulomas with fibrosis, decreased in severity. Only infected lambs were positive to PCR. These results suggest that the strain of Map has a strong influence over the immune and pathological responses developed by the host. Lesions induced by C–type strains in lambs show a regressive character and tend to decrease as the infection progresses.
PMCID: PMC3897920  PMID: 24428881
4.  Mycoplasmoses of ruminants in France: recent data from the national surveillance network 
Ruminant mycoplasmoses are important diseases worldwide and several are listed by the World Organization for Animal Health to be of major economic significance. In France the distribution of mycoplasmal species isolated from clinical samples collected from diseased animals upon veterinary request, is monitored by a network known as VIGIMYC (for VIGIlance to MYCoplasmoses of ruminants). The veterinary diagnostic laboratories collaborating with VIGIMYC are responsible for isolating the mycoplasmas while identification of the isolates is centralized by the French Food Safety Agency (AFSSA) in Lyon. The VIGIMYC framework can also be used for specific surveys and one example, on the prevalence of M. bovis in bovine respiratory diseases, is presented here.
Between 2003 and 2008, 34 laboratories were involved in the network and 1904 mycoplasma isolates, originating from the main ruminant-breeding areas, were identified. For cattle, the high prevalence of M. bovis in bronchopneumonia, notably in young animals, was confirmed by VIGIMYC and an associated specific survey, whereas the non-emergence of species such as M. alkalescens and M. canis was also demonstrated. The etiological agent of bovine contagious pleuropneumonia was never isolated. The principal mycoplasmosis in goats was contagious agalactia with M. mycoides subsp. capri as main agent. Ovine mycoplasmoses, most of which were associated with pneumonia in lambs, were infrequently reported. One exception was ovine contagious agalactia (due to M. agalactiae) that has recently re-emerged in the Pyrénées where it had been endemic for years and was also reported in Corsica, which was previously considered free.
Although VIGIMYC is a passive network and somewhat biased as regards sample collection and processing, it has provided, in this study, an overview of the main mycoplasmoses of ruminants in France. The French epidemiological situation is compared to those existing elsewhere in the world.
PMCID: PMC2892444  PMID: 20525406
5.  A comparative study of clinical manifestations, haematological and serological responses after experimental infection with Anaplasma phagocytophilum in two Norwegian sheep breeds 
It has been questioned if the old native Norwegian sheep breed, Old Norse Sheep (also called Norwegian Feral Sheep), normally distributed on coastal areas where ticks are abundant, is more protected against tick-borne infections than other Norwegian breeds due to a continuously high selection pressure on pasture. The aim of the present study was to test this hypothesis in an experimental infection study.
Five-months-old lambs of two Norwegian sheep breeds, Norwegian White (NW) sheep and Old Norse (ON) sheep, were experimentally infected with a 16S rRNA genetic variant of Anaplasma phagocytophilum (similar to GenBank accession number M73220). The experiment was repeated for two subsequent years, 2008 and 2009, with the use of 16 lambs of each breed annually. Ten lambs of each breed were inoculated intravenously each year with 0.4 ml A. phagocytophilum-infected blood containing approximately 0.5 × 106 infected neutrophils/ml. Six lambs of each breed were used as uninfected controls. Half of the primary inoculated lambs in each breed were re-challenged with the same infectious dose at nine (2008) and twelve (2009) weeks after the first challenge. The clinical, haematological and serological responses to A. phagocytophilum infection were compared in the two sheep breeds.
The present study indicates a difference in fever response and infection rate between breeds of Norwegian sheep after experimental infection with A. phagocytophilum.
Although clinical response seems to be less in ON-lambs compared to NW-lambs, further studies including more animals are needed to evaluate if the ON-breed is more protected against tick-borne infections than other Norwegian breeds.
PMCID: PMC3042963  PMID: 21314927
6.  Unexpected genetic diversity of Mycoplasma agalactiae caprine isolates from an endemic geographically restricted area of Spain 
The genetic diversity of Mycoplasma agalactiae (MA) isolates collected in Spain from goats in an area with contagious agalactia (CA) was assessed using a set of validated and new molecular typing methods. Validated methods included pulsed field gel electrophoresis (PFGE), variable number of tandem repeats (VNTR) typing, and Southern blot hybridization using a set of MA DNA probes, including those for typing the vpma genes repertoire. New approaches were based on PCR and targeted genomic regions that diverged between strains as defined by in silico genomic comparisons of sequenced MA genomes.
Overall, the data showed that all typing tools yielded consistent results, with the VNTR analyses being the most rapid method to differentiate the MA isolates with a discriminatory ability comparable to that of PFGE and of a set of new PCR assays. All molecular typing approaches indicated that the Spanish isolates from the endemic area in Murcia were very diverse, with different clonal isolates probably restricted to separate, but geographically close, local areas.
The important genetic diversity of MA observed in infected goats from Spain contrasts with the overall homogeneity of the genomic background encountered in MA from sheep with CA in Southern France or Italy, suggesting that assessment of the disease status in endemic areas may require different approaches in sheep and in goats. A number of congruent sub-typing tools are now available for the differentiation of caprine isolates with comparable discriminatory powers.
PMCID: PMC3514313  PMID: 22920649
Mycoplasma agalactiae; Molecular typing; Contagious agalactia; Goats
7.  Characterization and Analysis of a Stable Serotype-Associated Membrane Protein (P30) of Mycoplasma agalactiae 
Journal of Clinical Microbiology  2001;39(8):2814-2822.
The gene for a 30-kDa immunodominant antigen, P30, of Mycoplasma agalactiae was cloned from type strain PG2 and expressed in Escherichia coli. P30 is encoded on a monocistronic operon determined by two −10 boxes and a possible −35 region constituting the potential promoter, and a transcription termination site. The gene for the 266-amino-acid protein is preceded by a polypurine-rich region designed as the consensus sequence for a ribosome-binding site. Analysis of the amino acid sequence of P30 revealed the presence of a recognition site for a prokaryotic signal peptidase II at amino acid (aa) 24, indicating that P30 is a transmembrane protein. Moreover, Triton X-114 phase partitioning of M. agalactiae PG2 total antigen revealed that P30 is strongly hydrophobic and hence a possible membrane component. Immunoblot analysis using the monospecific polyclonal anti-P30-His serum indicated that P30 is specific to M. agalactiae. Furthermore, PCR amplification with specific primers for p30 and Southern blot analysis revealed the presence of the gene in all M. agalactiae strains tested and its absence in the other mycoplasma species. Among 27 strains of M. agalactiae studied, 20 strains belonging to the common serotypes A to D, including PG2, expressed P30 or part of it as detected by the monospecific polyclonal anti-P30 antibodies. The other seven strains belonging to the rarely isolated serotypes E to H were negative for P30. The p30 gene was sequenced in 15 strains of M. agalactiae, 10 of which expressed P30 or at least part of it and 5 of which did not express P30. The negative strains carried mutations in both −10 boxes of the promoters. These mutations seem to be responsible for the lack of P30 expression in these strains. Analysis of sera from sheep that were experimentally infected with M. agalactiae revealed that P30 induced a strong and persistent immune response which was still very high two months after infection. In contrast, currently used enzyme-linked immunosorbent assay serology gave only low titers.
PMCID: PMC88244  PMID: 11473997
8.  The indirect hemagglutination test for the detection of antibodies in cattle naturally infected mycoplasmas. 
Stable mycoplasma antigens for the indirect hemagglutination test (IHA) were prepared employing glutaraldehyde treated sheep erythrocytes sensitized with Mycoplasma agalactiae subsp. bovis and Mycoplasma bovigenitalium antigens. Employing these antigens mycoplasma antibodies were detected in sera from cattle which had mastitic symptoms due to natural infection with either M. agalactiae subsp. bovis or M. bovigenitalium. A total of 200 cows from four herds were examined at varying intervals for the presence of M. agalactiae subsp. bovis and for the detection of antibody using growth inhibition and IHA tests. Mycoplasmas were isolated from 37 animals. Growth inhibiting antibody was detected from 56 of the 200 animals. In the IHA tests, antibody titer greater than or equal to 1:80 were detected in 148 animals, 76 of these having antibody titers greater than or equal to 1:160, while sera of 116 normal control animals had no growth inhibiting antibody and none had IHA antibody titers greater than 1:40. M. bovigenitalium was isolated from the milk of three of 26 animals in a fifth herd during an outbreak of mastitis. Growth inhibiting antibodies were demonstrated in the sera of ten of the 26 animals. However, the IHA test detected antibody titers of greater than or equal to 1:160 in 13 animals and of 1:80 in one of the 26 animals. To determine the specificity of the IHA tests, M. agalactiae subsp. bovis and M. bovigenitalium antigens were reacted with rabbit hyperimmune typing sera produced against 12 species of bovine mycoplasmatales. Homologous antisera showed IHA antibody titers of 1:1280 and 1:2560 against M. agalactiae subsp. bovis and M. bovigenitalium respectively, whereas heterologous antisera showed IHA antibody titers of less than or equal to 1:20. Also eight type-specific bovine antisera were reacted with M agalactiae subsp. bovis and M. bovigenitalium antigens in homologous and heterologous tests. Homoogous reactions showed IHA antibody titers greater than or equal to 1:320, whereas heterologous reactions showed IHA titers of less than or equal to 1:20. This IHA test promises to be useful for the detection of bovine mycoplasma antibodies in sera from cattle infected with M. agalactiae subsp. bovis or M. bovigenitalium. Thes test is sensitive, reproducible and specific and the technique is relatively simple and rapid. The antigens were stable for at least seven months.
PMCID: PMC1277514  PMID: 1000374
9.  Development of a Sensitive and Specific Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigens for Rapid Detection of Antibodies against Mycoplasma agalactiae in Sheep▿ †  
Clinical and Vaccine Immunology  2007;14(4):420-425.
We developed a new recombinant enzyme-linked immunosorbent assay (rELISA) for serodiagnosis of contagious agalactia (CA), a disease caused by Mycoplasma agalactiae in sheep and goats. The assay is based on two M. agalactiae surface proteins, namely, P80 and P55. Identification of these immunodominant and common antigens was accomplished by examining the antibody response elicited in sheep during experimental infection and comparing it to the protein expression profiles of 75 M. agalactiae field strains. Our rELISA was tested with 343 sera, collected from sheep with a laboratory-confirmed diagnosis of CA (n = 223) and from healthy animals (n = 120). All sera had previously been tested by Western blotting (WB) for reactivity against M. agalactiae. In addition, our rELISA was compared with a commercial routine ELISA based on inactivated antigens (CHEKiT). Among the 223 samples that were WB positive for M. agalactiae, 209 (93.7%) tested positive for rP80-P55 with our ELISA, whereas only 164 (73.8%) tested positive with the CHEKiT ELISA. Among the 120 samples tested that were WB negative for M. agalactiae, 96.7% were confirmed as negative with our rELISA, while only 75.8% were confirmed as negative with the CHEKiT ELISA. A comparison of the results with receiver operating characteristic curves indicated that the differences observed between our rELISA and the CHEKiT ELISA are statistically significant. The use of recombinant peptides instead of inactivated antigens could significantly improve the discrimination of positive and negative animals, bringing significant advantages in controlling the import/export of live animals and helping in eradication of this economically detrimental disease.
PMCID: PMC1865618  PMID: 17287317
10.  Ovine Haemophilus somnus: experimental intracisternal infection and antigenic comparison with bovine Haemophilus somnus. 
Experimental infection was produced by two of four isolates of ovine Haemophilus somnus given by intracisternal inoculation into two to three-month-old lambs. Isolate 2041 (originally obtained from a septicemic lamb in Alberta) caused lethal infection in eight of nine lambs, isolate 67p from the prepuce of a normal lamb produced less acute disease in four of nine lambs, and the other two isolates (93p and 1190) caused no detectable disease. Significant lesions were limited to the brain and spinal cord. Purulent meningitis was characteristic but vasculitis or septicemia were not detected, perhaps due to the route of inoculation. Since a difference in virulence was noted among strains, we analyzed surface proteins thought to be virulence factors of bovine H. somnus. Protein profiles of bovine and ovine H. somnus done by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed similar patterns for virulent bovine isolates and ovine septicemic isolates. Preputial isolates showed a lower molecular mass major outer membrane protein than septicemic isolates. Antigenic analysis revealed that outer membrane proteins p270, p78, p76, p40, and p39 were detected in both ovine and bovine isolates except for 1190, which was probably not a true H. somnus isolate. Thus the preputial and septicemic isolates of ovine H. somnus were similar to bovine H. somnus in pathogenicity and in surface antigens.
PMCID: PMC1263697  PMID: 7954123
11.  Evidence for interference, coinfections, and intertypic virus enhancement of infection by ovine-caprine lentiviruses. 
Journal of Virology  1989;63(11):4682-4688.
The ovine-caprine lentiviruses share nucleotide homology and serological properties in their gag-pol genes and gene products but constitute two distinct biological groups represented by ovine visna virus of Icelandic origin and by caprine arthritis-encephalitis and ovine progressive pneumonia viruses of U.S. origin. Two members of each group, visna 1514 and its antigenic variant LV1-1 in the first group and CAEV/CO and S93, a field isolate virus from a local arthritic sheep, in the second group, were examined in the present study in competitive-binding studies in fibroblast and macrophage cell cultures. The cultures were preinoculated with each of the four viruses and then reinoculated with either 1514 virus or CAEV/CO, labeled with [35S]methionine. Both 1514 and CAEV/CO caused homologous interference. LV1-1 and S93 viruses shared the interference patterns of 1514 and CAEV/CO, respectively. 1514 and LV1-1 did not interfere with binding of CAEV/CO. Similarly, CAEV/CO and S93 did not interfere with binding of 1514. Remarkably, certain combinations, such as S93 plus 1514, resulted in enhanced binding of the second virus. Other experiments showed that the enhancement in binding extended to enhancement in replication of the second virus. These latter data suggested that individual cells supported replication of both viruses. Further testing of this phenomenon showed that goats could be doubly infected with two noninterfering viruses, 1514 and CAEV/CO. The ability of noninterfering related lentiviruses to infect the same cell and also the same host animal may be important in the natural history of these viruses in providing ideal conditions for the development of new recombinant viruses.
PMCID: PMC251103  PMID: 2552145
12.  Analysis of antibody responses to phenotypically distinct lentiviruses. 
Journal of Clinical Microbiology  1990;28(4):764-770.
To define the immune responses against phenotypically and pathogenically distinct lentiviruses, we used an immunoblotting assay to study antibodies to viral proteins of ovine lentivirus (OvLV) in 16 experimentally and 12 naturally infected sheep. Two distinct phenotypes of OvLV were used to experimentally infect lambs: strain 85/34, a "rapid/high" isolate which rapidly induced lysis in infected primary macrophage cultures and replicated to relatively high titers, and strains 84/28 and 85/14, "slow/low" isolates which induced slowly progressive syncytia with minimal lysis in vitro and replicated only to low titers in the same cell type. Serum antibodies against four major viral structural proteins, gp105, p25, p16, and p14, were detected. In a longitudinal study of experimentally infected lambs, the antibody to p25 (major gag protein) usually appeared first (average, about 3 weeks postinoculation [p.i.]) and was followed in about 2 weeks by p16, p14, and gp105 almost simultaneously. Six of 16 animals did not develop anti-p14 antibody by the time of necropsy at 9 to 29 weeks p.i. Two of 10 lambs which developed antibody to p14 had the antibody only transiently from 3 to 8 or 13 weeks p.i. and lost it by the time of necropsy at 21 or 22 weeks p.i. In contrast, antibodies to the other three structural proteins remained fairly constant until the time of necropsy. There were differences in the antibody responses of the experimentally infected lambs to the two phenotypes of OvLV. Seven of 10 (70%) lambs which were inoculated with the rapid/high strain developed antibody to p14, whereas only 17% of the lambs inoculated with the slow/low strains had antibody to this protein. In the longitudinal study, no decline was observed in the activity of any specific antibody such as that which occurs with anti-p24 antibody in human immunodeficiency virus infection, except in the case of anti-p14 antibody in two lambs. There were no significant differences in antibody titers against p25, p16, and p14 in final blood samples between rapid/high virus- and slow/low virus-infected groups. However, the rapid/high virus-infected group developed a fivefold-higher geometric mean titer of anti-env product (gp 105) antibody than did the slow/low virus-infected group (P
PMCID: PMC267790  PMID: 2159018
The United States control program for classical ovine scrapie is based in part on the finding that infection is typically spread through exposure to shed placentas from infected ewes. Transmission from goats to sheep is less well described. A suitable rodent model for examining the effect of caprine scrapie isolates in the ovine host will be useful in the ovine scrapie eradication effort. In this study, we describe the incubation time, brain lesion profile, glycoform pattern and PrPSc distribution patterns in a well characterized transgenic mouse line (Tg338) expressing the ovine VRQ prion allele, following inoculation with brain from scrapie infected goats.
First passage incubation times of caprine tissue in Tg338 ovinized mice varied widely but second passage intervals were shorter and consistent. Vacuolation profiles, glycoform patterns and paraffin-embedded tissue blots from terminally ill second passage mice derived from sheep or goat inocula were similar. Proteinase K digestion products of murine tissue were slightly smaller than the original ruminant inocula, a finding consistent with passage of several ovine strains in previous reports.
These findings demonstrate that Tg338 mice propagate prions of caprine origin and provide a suitable baseline for examination of samples identified in the expanded US caprine scrapie surveillance program.
PMCID: PMC3489715  PMID: 22472560
Prion; Mouse; Transgenic; Caprine
The Journal of Hygiene  1980;85(2):247-256.
Nine caprine and ovine mycoplasma strains, said to be indistinguishable serologically from Mycoplasma mycoides subsp. mycoides (the causative organism of contagious bovine pleuropneumonia; CBPP) were examined in mice by (1) a mycoplasmaemia test, and (2) a cross-protection test. Of the nine strains, two from goats belonged to a small colony (SC) type; four caprine and three ovine strains belonged to a large colony (LC) type.
The two SC strains — like a single SC strain examined in an earlier study — were indistinguishable from genuine M. mycoides subsp. mycoides as isolated from CBPP. They produced mycoplasmaemia readily. In a cross-protection test, the two SC strains and a CBPP strain immunized completely against each other.
Of the seven LC strains, six — like six LC strains examined in an earlier study — were easily distinguished from genuine M. mycoides subsp. mycoides; except for one that was not tested, all were shown to lack the ability to produce mycoplasmaemia readily. In cross-protection tests all six strains immunized partially but not completely against a CBPP strain.
The seventh LC strain (Mankefår 2833) was exceptional: it produced mycoplasmaemia readily, resembling the SC strains in this respect. Like other LC strains, in cross-protection tests it protected only partially against a CBPP strain. Strain Mankefår 2833 was isolated in ca. 1965 by Brack from a Barbary sheep (Ammotragus lervia) in a German zoo.
The ability of Mankefår 2833 to produce mycoplasmaemia enabled it to be used as a challenge strain in cross-protection tests. For the purpose of such tests the collection of nine mycoplasma strains referred to above was augmented with six LC strains from an earlier study. Partial but not complete protection against Mankefår 2833 was produced by two caprine SC strains, one CBPP strain, and nine LC strains. Three further LC strains gave protection that may have been as strong as that produced by the homologous strain, but confirmatory experiments are needed. A strain of M. mycoides subsp. capri gave no protection against Mankefår 2833.
PMCID: PMC2133930  PMID: 7005327
Infection, Genetics and Evolution  2014;22(100):208-215.
•97 global ovine S. aureus isolates characterised using MLST and spa typing.•Majority of 261 global ovine isolates belong to one of three clonal complexes (CC).•One CC has spread across the New World; two are restricted to Europe and Africa.•Clonal complex spread matches the route and timing of sheep domestication.•Patterns of clonal diversification of sheep isolates differ from human isolates.
Staphylococcus aureus is an important pathogen of many species, including sheep, and impacts on both human and animal health, animal welfare, and farm productivity. Here we present the widest global diversity study of ovine-associated S. aureus to date. We analysed 97 S. aureus isolates from sheep and sheep products from the UK, Turkey, France, Norway, Australia, Canada and the USA using multilocus sequence typing (MLST) and spa typing. These were compared with 196 sheep isolates from Europe (n = 153), Africa (n = 28), South America (n = 14) and Australia (n = 1); 172 bovine, 68 caprine and 433 human S. aureus profiles. Overall there were 59 STs and 87 spa types in the 293 ovine isolates; in the 97 new ovine isolates there were 22 STs and 37 spa types, including three novel MLST alleles, four novel STs and eight novel spa types. Three main CCs (CC133, CC522 and CC700) were detected in sheep and these contained 61% of all isolates. Four spa types (t002, t1534, t2678 and t3576) contained 31% of all isolates and were associated with CC5, CC522, CC133 and CC522 respectively. spa types were consistent with MLST CCs, only one spa type (t1403) was present in multiple CCs. The three main ovine CCs have different but overlapping patterns of geographical dissemination that appear to match the location and timing of sheep domestication and selection for meat and wool production. CC133, CC522 and CC700 remained ovine-associated following the inclusion of additional host species. Ovine isolates clustered separately from human and bovine isolates and those from sheep cheeses, but closely with caprine isolates. As with cattle isolates, patterns of clonal diversification of sheep isolates differ from humans, indicative of their relatively recent host-jump.
PMCID: PMC3969713  PMID: 24035790
S. aureus; Ovine; Caprine; MLST; spa typing; Global diversity
Journal of Clinical Microbiology  2008;47(2):445-450.
We evaluated the capacity of the Mycoplasma agalactiae p40 gene as a diagnostic marker for contagious agalactia in sheep by quantitative real-time PCR. The p40 gene encodes an immunodominant adhesin that plays a key role in cytoadhesion of M. agalactiae. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent (GE), a quantification that is highly linear (R2 > 0.992) and efficient (PCR efficiency, >0.992) over a 6-log dynamic range, down to 10 GE. We evaluated the capacity of the assay to detect Mycoplasma agalactiae in 797 milk samples (373 raw sheep milk samples from refrigerated tanks of different farms and 424 milk samples from individual sheep of a flock positive for M. agalactiae). In parallel, we also tested the samples by using microbiological isolation coupled with microscopy identification and by a PCR method recommended by the World Organization for Animal Health. While our assay was able to detect 57 (15.28%) positive samples of the 373 milk samples from different farms, identification by microbiological isolation coupled with microscopy detected only 36 (9.65%) samples, and the conventional PCR detected 31 (8.31%) samples. These findings showed that our assay based on the p40 gene is more specific and sensitive for the detection of M. agalactiae in actual natural samples and, thus, can be a promising alternative tool for diagnosis and epidemiological studies of M. agalactiae infection.
PMCID: PMC2643663  PMID: 19020058
PLoS Pathogens  2011;7(3):e1001319.
A new variant of Creutzfeldt Jacob Disease (vCJD) was identified in humans and linked to the consumption of Bovine Spongiform Encephalopathy (BSE)-infected meat products. Recycling of ruminant tissue in meat and bone meal (MBM) has been proposed as origin of the BSE epidemic. During this epidemic, sheep and goats have been exposed to BSE-contaminated MBM. It is well known that sheep can be experimentally infected with BSE and two field BSE-like cases have been reported in goats. In this work we evaluated the human susceptibility to small ruminants-passaged BSE prions by inoculating two different transgenic mouse lines expressing the methionine (Met) allele of human PrP at codon 129 (tg650 and tg340) with several sheep and goat BSE isolates and compared their transmission characteristics with those of cattle BSE. While the molecular and neuropathological transmission features were undistinguishable and similar to those obtained after transmission of vCJD in both transgenic mouse lines, sheep and goat BSE isolates showed higher transmission efficiency on serial passaging compared to cattle BSE. We found that this higher transmission efficiency was strongly influenced by the ovine PrP sequence, rather than by other host species-specific factors. Although extrapolation of results from prion transmission studies by using transgenic mice has to be done very carefully, especially when human susceptibility to prions is analyzed, our results clearly indicate that Met129 homozygous individuals might be susceptible to a sheep or goat BSE agent at a higher degree than to cattle BSE, and that these agents might transmit with molecular and neuropathological properties indistinguishable from those of vCJD. Our results suggest that the possibility of a small ruminant BSE prion as vCJD causal agent could not be ruled out, and that the risk for humans of a potential goat and/or sheep BSE agent should not be underestimated.
Author Summary
Prion diseases, also referred as transmissible spongiform encephalopathies, are fatal neurodegenerative diseases caused by proteinaceous infectious particles denominated “prions.” Prion diseases acquired their first real public relevance with the outbreak of bovine spongiform encephalopathy (BSE) (“mad cow disease”) in the United Kingdom in the 80s and its link with the appearance of a new, variant form of Creutzfeldt-Jakob disease in humans. Recycling of ruminant tissues in meat and bone meal has been proposed as origin of the BSE epidemic. During this episode, sheep and goats have also been exposed to BSE-contaminated meal, so transmission to this species may have occurred. We analyzed the human susceptibility to sheep and goat passaged-BSE prions by using transgenic mice expressing human prion protein (PrP). When different sheep and goat BSE isolates were inoculated in these transgenic mice, higher susceptibility than that observed for cattle BSE was detected and the disease manifestation was similar to that observed in mice inoculated with the new variant of Creutzfeldt-Jakob disease. Our findings suggest that humans are at least equally, and might be even more, susceptible to a sheep or goat BSE agent compared to a cattle BSE one.
PMCID: PMC3060172  PMID: 21445238
Acta Veterinaria Scandinavica  2001;42(4):453-458.
Tick-borne fever (TBF) is caused by the rickettsiae Ehrlichia phagocytophila and is a common disease in sheep in tick (Ixodes ricinus) infested areas in Norway. Earlier investigations have shown that some sheep could remain infected for several months after the primary infection. In this study, the persistence of E. phagocytophila after experimental infection was investigated in 2 age groups of lambs. Six lambs (1–2 weeks old) and 14 lambs (6–8 months old) were inoculated intravenously with an ovine strain of E. phagocytophila and thereafter examined clinically (including daily body temperature recording) and by haematological and serological (E. equi antibodies) methods for the next 4 months. At the end of this period, the lambs were examined for a TBF infection by blood smear investigation and blood inoculation studies. The infection was demonstrated in 19 (95%) of the 20 lambs.
PMCID: PMC2203224  PMID: 11957373
granulocytic Ehrlichia; tick-borne fever; sheep; age
Veterinary Research  2014;45(1):58.
The quantitative role of sheep in the transmission of foot-and-mouth disease virus (FMDV) is not well known. To estimate the role of sheep in the transmission of FMDV, a direct contact transmission experiment with 10 groups of animals each consisting of 2 infected lambs and 1 contact calf was performed. Secretions and excretions (oral swabs, blood, urine, faeces and probang samples) from all animals were tested for the presence of FMDV by virus isolation (VI) and/or RT-PCR. Serum was tested for the presence of antibodies against FMDV. To estimate FMDV transmission, the VI, RT-PCR and serology results were used. The partial reproduction ratio R0p i.e. the average number of new infections caused by one infected sheep introduced into a population of susceptible cattle, was estimated using either data of the whole infection chain of the experimental epidemics (the transient state method) or the final sizes of the experimental epidemics (the final size method). Using the transient state method, R0p was estimated as 1.0 (95% CI 0.2 - 6.0) using virus isolation results and 1.4 (95% CI 0.3 - 8.0) using RT-PCR results. Using the final size method, R0p was estimated as 0.9 (95% CI 0.2 - 3.0). Finally, R0p was compared to the R0’s obtained in previous transmission studies with sheep or cattle only. This comparison showed that the infectivity of sheep is lower than that of cattle and that sheep and cattle are similarly susceptible to FMD. These results indicate that in a mixed population of sheep and cattle, sheep play a more limited role in the transmission of FMDV than cattle.
PMCID: PMC4058432  PMID: 24886222
Virology Journal  2014;11:65.
Small ruminant lentiviruses escaping efficient serological detection are still circulating in Swiss goats in spite of a long eradication campaign that essentially eliminated clinical cases of caprine arthritis encephalitis in the country. This strongly suggests that the circulating viruses are avirulent for goats.
To test this hypothesis, we isolated circulating viruses from naturally infected animals and tested the in vitro and in vivo characteristics of these field isolates.
Viruses were isolated from primary macrophage cultures. The presence of lentiviruses in the culture supernatants was monitored by reverse transcriptase assay. Isolates were passaged in different cells and their cytopathogenic effects monitored by microscopy. Proviral load was quantified by real-time PCR using customized primer and probes. Statistical analysis comprised Analysis of Variance and Bonferroni Multiple Comparison Test.
The isolated viruses belonged to the small ruminant lentiviruses A4 subtype that appears to be prominent in Switzerland. The 4 isolates replicated very efficiently in macrophages, displaying heterogeneous phenotypes, with two isolates showing a pronounced cytopathogenicity for these cells. By contrast, all 4 isolates had a poor replication capacity in goat and sheep fibroblasts. The proviral loads in the peripheral blood and, in particular, in the mammary gland were surprisingly high compared to previous observations. Nevertheless, these viruses appear to be of low virulence for goats except for the mammary gland were histopathological changes were observed.
Small ruminant lentiviruses continue to circulate in Switzerland despite a long and expensive caprine arthritis encephalitis virus eradication campaign. We isolated 4 of these lentiviruses and confirmed their phylogenetic association with the prominent A4 subtype. The pathological and histopathological analysis of the infected animals supported the hypothesis that these A4 viruses are of low pathogenicity for goats, with, however, a caveat about the potentially detrimental effects on the mammary gland. Moreover, the high proviral load detected indicates that the immune system of the animals cannot control the infection and this, combined with the phenotypic plasticity observed in vitro, strongly argues in favour of a continuous and precise monitoring of these SRLV to avoid the risk of jeopardizing a long eradication campaign.
PMCID: PMC3995504  PMID: 24708706
CAEV; VMV; MVV; Visna; Maedi; SRLV; Virulence; Pathogenicity; Attenuation; Eradication
Journal of Virology  1984;52(2):672-679.
Ovine and caprine lentiviruses share the capacity to induce slowly progressive and inflammatory diseases of the central nervous system (leukoencephalitis or visna), lungs (progressive pneumonia or maedi), and joints (arthritis) in their natural hosts. Studies on their replication indicated that ovine lentiviruses and caprine arthritis-encephalitis virus (CAEV) recently isolated in the United States establish persistent infection in ovine and caprine fibroblasts, whereas older prototype ovine lentiviruses such as Icelandic visna virus or American progressive pneumonia virus irreversibly lyse fibroblast cultures. Since all of the recent isolates were found to be persistent, Narayan et al. (J. Gen. Virol. 59:345-356, 1982) concluded that the highly lytic viruses were only tissue-culture-adapted strains. In the present report, we isolated new ovine lentiviruses from French sheep with naturally occurring progressive pneumonia which are either highly lytic (five isolates), as are the Icelandic strains of visna virus, or persistent (one isolate), as are CAEV or American persistent ovine lentiviruses. Protein and nucleic acid content analyses of these new highly lytic (type I) and persistent (type II) isolates indicated that type I and type II ovine lentiviruses were genetically distinct, type I and type II viruses being closely related to the Icelandic strains of visna virus and to CAEV, respectively. We conclude that (i) highly lytic ovine lentiviruses, such as the Icelandic prototype strains of visna virus and persistent lentiviruses more related to CAEV, are naturally present in the ovine species, and (ii) irreversible cell lysis induced by highly lytic viruses does not result from a tissue culture adaptation of field isolates that were originally persistent but is instead the consequence of a genetic content distinct from that of persistent viruses.
PMCID: PMC254572  PMID: 6092689
Infection and Immunity  2002;70(10):5612-5621.
An immunodominant protein, P40, of Mycoplasma agalactiae was analyzed genetically and functionally. The gene encoding P40 was cloned from type strain PG2, sequenced, submitted to point mutagenesis in order to convert mycoplasma-specific TGATrp codon to the universal TGGTrp codon, and subsequently expressed in Escherichia coli. Nucleotide sequence-derived amino acid sequence comparisons revealed a similarity of P40 to the adhesin P50 of Mycoplasma hominis and to protein P89 of Spiroplasma citri, which is expected to be involved in adhesion. The amino acid sequence of P40 revealed a recognition site for a signal peptidase and strong antigenic and hydrophilic motifs in the C-terminal domain. Triton X-114 phase partitioning confirmed that P40 is a membrane protein. Fab fragments of antibodies directed against recombinant purified P40 significantly inhibited adherence of M. agalactiae strains PG2 to lamb joint synovial cells LSM 192. Sera taken sequentially from sheep infected with PG2 revealed that P40 induced a strong and persistent immune response that gave strong signals on immunoblots containing recombinant P40 even 3 months after infection. The gene encoding P40 was present in a single copy in all of the 26 field strains of M. agalactiae analyzed and was not detected in closely related mycoplasma species. P40 was expressed as a protein with an apparent molecular mass of 37 kDa on sodium dodecyl sulfate-acrylamide gels by all M. agalactiae strains except for serotype C strains, which showed nonsense mutations in their p40 genes.
PMCID: PMC128363  PMID: 12228289
Croatian Medical Journal  2010;51(4):320-326.
To present the surveillance data on Brucella melitensis, B. suis, and B. ovis infection in cattle, sheep, goats, and swine in Croatia obtained in 2008 by serological, bacteriological, and molecular methods for diagnostics of brucellosis in domestic animals.
We serologically tested 42 785 cattle serums, 22 686 sheep and goat serums, and 28 520 swine serums using the Rose Bengal test, complement fixation test, and various immunosorbent assays. We also tested 10 173 ram blood samples for B. ovis infection using the complement fixation test. Bacteriological examination was conducted on 214 samples collected from 34 serologically positive animals. Different molecular methods were employed in the identification and typing of 20 isolates from the samples.
B. melitensis biovar (bv.) 3 was confirmed with different identification methods in 2 flocks in 2 Croatian counties and B. suis bv. 2 in 3 herds in 3 counties. B. melitensis in cows was confirmed for the first time in Croatia. Infection with B. ovis was serologically confirmed in 202 rams in 12 counties.
In 2008, the size of the brucellosis-affected area in Croatia and the efficiency of detection and prevention of brucellosis in sheep, goats, and swine were satisfactory. Infection with B. melitensis in cattle was confirmed for the first time and possible links for infection in humans were detected. More efficient measures for suppression and control of ovine epididymitis are required and a new strategy may be necessary for complete eradication of this disease.
PMCID: PMC2931437  PMID: 20718085
The Journal of Hygiene  1980;85(1):1-33.
A three-part epidemiological investigation was made on flaviviruses:
1. As a preliminary to tests on cattle sera from the field, the antigenic cross-reactivity of Wesselsbron, Spondweni, Usutu, Banzi, West Nile and yellow fever flaviviruses was studied in antisera prepared in guinea pigs. As described earlier for flaviviruses, sera were found to be highly cross-reactive in haemagglutination-inhibition (HAI) tests, less cross-reactive in complement-fixation (CF) tests and were virtually monospecific in microneutralization (NT) tests in Vero cell cultures.
2. Infection with Wesselsbron (WSL) virus produced mild febrile illness and viraemia in 5 out of 6 newborn calves, 3 out of 4 pregnant heifers and 3 out of 4 ewes. One heifer produced a weak calf which died soon after birth with WSL antibodies in its serum, indicating that infection had occurred in utero. The 3 other heifers produced healthy calves which lacked antibody in pre-colostral serum. Pathological changes occurred in the foetus in 2 out of 3 pregnant ewes and the ewe produced a healthy lamb which had antibodies to WSL virus in pre-colostral serum.
Unlike the situation in guinea pigs, cattle sera were monospecific for WSL virus in CF tests, but sheep sera cross-reacted with Banzi and yellow fever viruses. Re-infection of the cattle with Banzi, West Nile, Spondweni and Usutu viruses failed to induce marked antibody responses. The results suggest that antibodies to WSL virus in cattle sera from the field can be distinguished from those induced by other flaviviruses by quantitative serological tests.
3. HAI antibodies to WSL virus were detected in 2648/14395 cattle sera tested over 11 years from 1967 to 1978 in the course of investigation of abortion, infertility and other diseases. Results of quantitative HAI, CF and NT tests with six flaviviruses on 409 selected sera confirmed that infection was due to WSL virus. Serological evidence failed to implicate WSL virus as a cause of abortion in cattle. In a prospective study, abortion occurred in only one out of 21 heifers observed to gain WSL infection during pregnancy in the field, but abortion also occurred in five out of 207 heifers which did not become infected with WSL. No histopathological lesions diagnostic of WSL disease were observed in 1998 specimens from cattle, sheep and goats examined over 44 months prior to October 1972, and WSL virus was isolated once, from the organs of a cow, out of 2106 specimens from cattle sheep and goats tested virologically over six years from October 1972 to September 1978. HAI antibodies to WSL virus were detected in one out of 374 sera from aborted cattle foetuses. It was concluded that WSL virus is not an important cause of disease in cattle, despite widespread occurrence of infection.
PMCID: PMC2134009  PMID: 6300226
PLoS ONE  2014;9(4):e93970.
Mechanisms underlying pathogenic processes in mycoplasma infections are poorly understood, mainly because of limited sequence similarities with classical, bacterial virulence factors. Recently, large-scale transposon mutagenesis in the ruminant pathogen Mycoplasma agalactiae identified the NIF locus, including nifS and nifU, as essential for mycoplasma growth in cell culture, while dispensable in axenic media. To evaluate the importance of this locus in vivo, the infectivity of two knock-out mutants was tested upon experimental infection in the natural host. In this model, the parental PG2 strain was able to establish a systemic infection in lactating ewes, colonizing various body sites such as lymph nodes and the mammary gland, even when inoculated at low doses. In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal. In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host. The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals. The exact role of the NIF locus remains to be elucidated but these data demonstrate that it plays a key role in the infectious process of M. agalactiae and most likely of other pathogenic mycoplasma species as many carry closely related homologs.
PMCID: PMC3974822  PMID: 24699671

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