Functional and Immunological Relevance of Anaplasma marginale Major Surface Protein 1a Sequence and Structural Analysis.

Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma marginale. The major surface protein 1a (MSP1a) has been used as a genetic marker for identifying A. marginale strains based on N-terminal tandem repeats and a 5'-UTR microsatellite located in the msp1a gene. The MSP1a tandem repeats contain immune relevant elements and functional domains that bind to bovine erythrocytes and tick cells, thus providing information about the evolution of host-pathogen and vector-pathogen interactions. Here we propose one nomenclature for A. marginale strain classification based on MSP1a. All tandem repeats among A. marginale strains were classified and the amino acid variability/frequency in each position was determined. The sequence variation at immunodominant B cell epitopes was determined and the secondary (2D) structure of the tandem repeats was modeled. A total of 224 different strains of A. marginale were classified, showing 11 genotypes based on the 5'-UTR microsatellite and 193 different tandem repeats with high amino acid variability per position. Our results showed phylogenetic correlation between MSP1a sequence, secondary structure, B-cell epitope composition and tick transmissibility of A. marginale strains. The analysis of MSP1a sequences provides relevant information about the biology of A. marginale to design vaccines with a cross-protective capacity based on MSP1a B-cell epitopes

Pathogenesis of reproductive failure induced by Trypanosoma vivax in experimentally infected pregnant ewes

The present study was aimed at investigating the effect of experimental infection by Trypanosoma vivax in different\ud stages of pregnancy, determining the pathogenesis of reproductive failure, and confirming transplacental\ud transmission. We used 12 pregnant ewes distributed into four experimental groups: G1, was formed by three ewes\ud infected with T. vivax in the first third of pregnancy (30 days); G2 comprised three infected ewes in the final third of\ud pregnancy (100 days); G3 and G4 were composed of three non-infected ewes with the same gestational period,\ud respectively. Each ewe of G1 and G2 was inoculated with 1.25 × 105 tripomastigotes. Clinical examination,\ud determination of parasitemia, serum biochemistry (albumin, total protein, glucose, cholesterol, and urea), packed\ud cell volume (PCV), serum progesterone, and pathological examination were performed. Placenta, amniotic fluid,\ud blood and tissues from the fetuses and stillbirths were submitted to PCR. Two ewes of G1 (Ewe 1 and 3) presented\ud severe infection and died in the 34th and 35th days post-infection (dpi), respectively; but both fetuses were\ud recovered during necropsy. In G2, Ewe 5 aborted two fetuses on the 130th day (30 dpi) of pregnancy; and Ewe 6\ud aborted one fetus in the 140th day (40 dpi) of gestation. Ewes 2 and 4 delivered two weak lambs that died five\ud days after birth. Factors possibly involved with the reproductive failure included high parasitemia, fever, low PCV,\ud body score, serum glucose, total protein, cholesterol, and progesterone. Hepatitis, pericarditis, and encephalitis were\ud observed in the aborted fetuses. The presence of T. vivax DNA in the placenta, amniotic fluid, blood, and tissues\ud from the fetuses confirms the transplacental transmission of the parasite. Histological lesion in the fetuses and\ud placenta also suggest the involvement of the parasite in the etiopathogenesis of reproductive failure in ewes

Ultrastructure of endogenous stages of Eimeria ninakohlyakimovae Yakimoff & Rastegaieff, 1930 Emend. Levine, 1961 in experimentally infected goat

The ultrastructure of endogenous stages of Eimeria ninakohlyakimovae was observed in epithelial cells of cecum and colon crypts from a goat experimentally infected with 2.0 x 10^5 oocysts/kg. The secondary meronts developed above the nucleus of the host cell. The nucleus first divides and merozoites then form on the surface of multinucleated meronts. Free merozoites in the parasitophorous vacuole present a conoid, double membrane, one pair of rhoptries, micronemes, micropore, anterior and posterior polar ring, a nucleus with a nucleolus and peripheral chromatin. The microgamonts are located below the nucleus of the host cell and contain several nuclei at the periphery of the parasite. The microgametes consist of a body, a nucleus, three flagella and mitochondria. The macrogamonts develop below the nucleus of the host cell and have a large nucleus with a prominent nucleolus. The macrogametes contain a nucleus, wall-forming bodies of type I and type II. The young oocysts present a wall containing two layers and a sporont

World A. marginale MSP1a molecular map.

<p>The worldwide molecular characterization of A. marginale MSP1a sequences is shown. The number of A. marginale strains (S), tandem repeats (TR), tandem repeat 2D structures (TR-2D), functional tandem repeats (FTR) containing D and E at position 20 and B cell epitope types (BCE) and microsatellites (MS) are represented for each country. Primary data is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065243#pone-0065243-g001" target="_blank">figures 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065243#pone-0065243-g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065243#pone-0065243-g006" target="_blank">6</a>. The information on 5′ UTR microsatellites is not available (NA) for some sequences.</p

Number of tandem repeats among A. marginale strains.

<p>The total number of strains classified in our study were organized by the number of MSP1a tandem repeats. The percent of A. marginale strains (external numbers) containing different number of tandem repeats (internal numbers) is shown. The most common numbers of MSP1a tandem repeats among strains were 3 (yellow), 4 (light blue) and 5 (violet).</p

B-cell epitope analysis in A. marginale MSP1a tandem repeats.

<p>The B-cell epitopes were predicted using BCPRED server. The type 1 B-cell epitope was used as reference (Model) for comparisons. (A) Clustalw alignment and amino acid changes in the 5 more represented MSP1a tandem repeat B cell epitopes. B-cell epitope types model (light violet), 1 (blue), 10 (yellow), 11 (dark violet) and 17 (red) are shown. (B) Percent of tandem repeats containing each type of B cell epitopes. (C) Neighbor joining phylogenetic tree based on B cell epitope amino acid sequences showing the two clusters formed by the 5 more represented B cell epitopes. Cluster-1: Types 1 and 11 and Cluster-2: Types Model, 10 and 17. Correlations between VaxiJen/Blastp (D), BCPRED/Blastp scores (E) and VaxiJen/BCPRED (F) scores are shown. These correlations suggest that the epitopes with higher homology (Blastp score) share in common the immunogenic properties represented by VaxiJen/BCPRED.</p

Association of <i>A. marginale</i> MSP1a R1 sequences with world ecoregions.

<p>World ecoregions were built upon temporal series of NDVI values.</p>(a)<p>R1 sequences recorded in one ecoregion only.</p>(b)<p>R1 sequences that have been reported in other ecoregions.</p

Phylogenetic tree based on MSP1a tandem repeat amino acid sequences.

<p>The MSP1a sequences from tick-transmissible and non-transmissible strains (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065243#pone-0065243-t002" target="_blank">Table 2</a>) were included in the phylogenetic analysis. The phylogenetic tree was reconstructed using the neighbor joining and maximum likelihood methods. Reliability for internal branch was assessed using the bootstrapping method with 1000 bootstrap replicates. Bootstrap values are shown as % in the internal branch. The tree shows four phylogenetic clusters containing different patterns of MSP1a tandem repeat 2D structures. Cluster β-α-c (blue), cluster α-1 and cluster α-2 (beige) contain tick-transmissible A. marginale strains while in cluster β (red) fall the non-tick-transmissible strains.</p

Geographical occurrence of the most common <i>A. marginale</i> strains.

<p>The most frequent A. marginale strains and their geographical occurrence are shown. The most common tandem repeats found among all the A. marginale strains are underlined and there were found more than 60 (<b><u>M</u></b>), 80 (<b><u>β</u></b>) and 90 (<b><u>B</u></b>) times.</p

Changes in putative 2D structure and disorder analysis of A. marginale MSP1a tandem repeats.

<p>The PSIPRED web server was used to predict the 2D structure. The tandem repeats were grouped into fourteen 2D structure models. Tandem repeats shown represent prototypes of corresponding tandem repeat 2D structures. The second column shows (model presented) the ID of the tandem repeat presented as prototype. Models ID in red represent tandem repeats in R1 position (first tandem in the MSP1a sequence).</p