Feline sporotrichosis cases around the world, 1952–2016.

<p>(A) Since the mid-20th century, feline sporotrichosis has typically occurred in isolated cases and small outbreaks, and only a few reports of zoonotic transmission have been described in the literature. The Southeast Brazil region has the largest absolute number of cases with an overwhelming prevalence of <i>S</i>. <i>brasiliensis</i> during epizootic outbreaks. Outside Brazil, most feline cases are due to the classical agent <i>S</i>. <i>schenckii</i>. (B) Spatiotemporal evolution of feline sporotrichosis cases in Brazil. Over the last two decades (1998–2016), Brazil has experienced a long-lasting outbreak of cat-transmitted sporotrichosis in Rio de Janeiro, with 4,669 cases reported. Cat-borne sporotrichosis due to <i>S</i>. <i>brasiliensis</i> often appears in the form of outbreaks or epidemics within a short period of time. Remarkably, before the 1990s, Rio de Janeiro reported a low number of cases, nearly always unrelated to feline transmission types.</p

Phylogenetic trees generated by Neighbor-joining, Maximum Likelihood and Bayesian analysis using partial nucleotide sequences of the calmodulin-encoding gene (A) and the translation elongation factor-1 alpha (EF1α) locus region (B).

<p>Bootstrap and posterior probabilities values were added to respective branches (NJ/ML/BI). Each species are indicated at each respective position at the phylogenetic tree. Calmodulin and EF1α accessions number are indicated in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002281#pntd-0002281-t001" target="_blank">Table 1</a>.</p

<i>In vitro</i> temperature fitness in the <i>Sporothrix</i> species.

<p><b>Growth inhibition at 37°C compared to 30°C incubation.</b><i>S. brasiliensis</i> from feline (n = 30) or human source (n = 27) are more resistant to heat incubation and differ statistically when compared to <i>S. schenckii</i> (n = 25), <i>S. globosa</i> (n = 7) and <i>S. mexicana</i> (n = 4). Statistical significance in one-way ANOVAs followed by Tukey's tests: * p<0.05, *** p<0.0001. The line in the boxes and upper and lower bars show the median, maximum and minimum values, respectively. Isolates were not compared at superior temperature (38–40°C) due to low growth observed to <i>S. globosa</i> and <i>S. mexicana</i>. No isolate were able to growth at 40°C.</p

Median-joining haplotype network of <i>Sporothrix schenckii</i> complex isolates based on partial nucleotide sequences of the calmodulin-encoding gene (A) and the translation elongation factor-1 alpha (EF1α) loci regions (B).

<p>The EF1α haplotype showed a clear intraspecific separation resultant from a nucleotide transition from A to G, between <i>S. brasiliensis</i> isolates recovered from Rio de Janeiro (H9) and Rio Grande do Sul (H11 and H12) feline epidemics. The size of the circumference is proportional to the haplotype frequency. Black dots (median vectors) are hypothetical missing intermediates. Calmodulin and EF1α haplotypes are detailed in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002281#pntd.0002281.s002" target="_blank">Table S2</a>.</p

Strains, species, origin, and GenBank accession numbers of <i>Sporothrix</i> spp. isolates used in this study.

1<p>Calmodulin literature reference. IPEC, Instituto de Pesquisa Clínica Evandro Chagas, Fiocruz, Brazil; FMR, Facultat de Medicina i Ciències de la Salut, Reus, Spain; CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; KMU, Kanazawa Medical University, Ishikawa, Japan; CMW, Culture Collection of the Forestry and Agricultural Biotechnology Institute (FABI); AFTOL, Assembling the Fungal Tree of Life project; NK, not known;</p>T<p>, type strain. All “Ss” strains belong to the culture collection of Federal University of São Paulo (UNIFESP). MG, Minas Gerais; RS, Rio Grande do Sul; PR, Paraná; SP, São Paulo; RJ, Rio de Janeiro; ES, Espírito Santo; PA, Pará; CE, Ceará; GO, Goiás, PE, Pernambuco.</p

Clinical aspects of feline sporotrichosis in Brazil.

<p><b>Cats presenting ulcerated cutaneous lesions in the cephalic region.</b> (A) and (B) felines from Rio de Janeiro; (C) and (D) felines from Paraná.</p

Proteomics-Based Characterization of the Humoral Immune Response in Sporotrichosis: Toward Discovery of Potential Diagnostic and Vaccine Antigens

<div><p>Background</p><p><i>Sporothrix schenckii</i> and associated species are agents of human and animal sporotrichosis that cause large sapronoses and zoonoses worldwide. Epidemiological surveillance has highlighted an overwhelming occurrence of the highly pathogenic fungus <i>Sporothrix brasiliensis</i> during feline outbreaks, leading to massive transmissions to humans. Early diagnosis of feline sporotrichosis by demonstrating the presence of a surrogate marker of infection can have a key role for selecting appropriate disease control measures and minimizing zoonotic transmission to humans.</p><p>Methodology</p><p>We explored the presence and diversity of serum antibodies (IgG) specific against <i>Sporothrix</i> antigens in cats with sporotrichosis and evaluated the utility of these antibodies for serodiagnosis. Antigen profiling included protein extracts from the closest known relatives <i>S</i>. <i>brasiliensis</i> and <i>S</i>. <i>schenckii</i>. Enzyme-linked immunosorbent assays and immunoblotting enabled us to characterize the major antigens of feline sporotrichosis from sera from cats with sporotrichosis (n = 49), healthy cats (n = 19), and cats with other diseases (n = 20).</p><p>Principal Findings</p><p>Enzyme-linked immunosorbent assay-based quantitation of anti-<i>Sporothrix</i> IgG exhibited high sensitivity and specificity in cats with sporotrichosis (area under the curve, 1.0; 95% confidence interval, 0.94–1; <i>P</i><0.0001) versus controls. The two sets of <i>Sporothrix</i> antigens were remarkably cross-reactive, supporting the hypothesis that antigenic epitopes may be conserved among closely related agents. One-dimensional immunoblotting indicated that 3-carboxymuconate cyclase (a 60-kDa protein in <i>S</i>. <i>brasiliensis</i> and a 70-kDa protein in <i>S</i>. <i>schenckii</i>) is the immunodominant antigen in feline sporotrichosis. Two-dimensional immunoblotting revealed six IgG-reactive isoforms of gp60 in the <i>S</i>. <i>brasiliensis</i> proteome, similar to the humoral response found in human sporotrichosis.</p><p>Conclusions</p><p>A convergent IgG-response in various hosts (mice, cats, and humans) has important implications for our understanding of the coevolution of <i>Sporothrix</i> and its warm-blooded hosts. We propose that 3-carboxymuconate cyclase has potential for the serological diagnosis of sporotrichosis and as target for the development of an effective multi-species vaccine against sporotrichosis in animals and humans.</p></div

South America map showing sampling localities in Brazil and total number of animals (n = 33) and humans <i>Sporothrix</i> spp.

<p><b>(n = 49) isolates evaluated in Rio de Janeiro, Minas Gerais, São Paulo, Paraná and Rio Grande do Sul.</b> = 17) outside the gray area and used as control are not shown in the picture.</p

ROC analysis of assay sensitivity and specificity.

<p>Samples consisted of sera from 49 infected cats and 19 uninfected cats. Sb, <i>S</i>. <i>brasiliensis</i>; Ss, <i>S</i>. <i>schenckii</i>; PPV, positive predictive value; NPV, negative predictive value.</p

Frequency and diversity of serum-derived antibodies (IgG isotype) against <i>S</i>. <i>brasiliensis</i> and <i>S</i>. <i>schenckii</i> antigens in naturally infected cats.

<p>3-carboxymuconate cyclase (gp60 in the <i>S</i>. <i>brasiliensis</i> proteome (A); gp70 in the <i>S</i>. <i>schenckii</i> proteome (B)) is the immunodominant molecule in feline sporotrichosis; it was recognized by 100% of the cat sera tested here irrespective of disease form and severity. (C) Diversity of recognition of <i>S</i>. <i>brasiliensis</i> antigens (outer ring, CBS 132990; inner ring, CBS 132021). (D) Diversity of recognition of <i>S</i>. <i>schenckii</i> antigens (outer ring, CBS 132974; inner ring, CBS 132984). Charts are proportional. Molecular weights are colored as indicated. Further information about the frequency of antigen recognition appears in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004016#pntd.0004016.s003" target="_blank">S2 Table</a>.</p