Phylogenetic analysis reveals a high prevalence of Sporothrix brasiliensis in feline sporotrichosis outbreaks

Abstract

Sporothrix schenckii, previously assumed to be the sole agent of human and animal sporotrichosis, is in fact a species complex. Recently recognized taxa include S. brasiliensis, S. globosa, S. mexicana, and S. luriei, in addition to S. schenckii sensu stricto. Over the last decades, large epidemics of sporotrichosis occurred in Brazil due to zoonotic transmission, and cats were pointed out as key susceptible hosts. In order to understand the eco-epidemiology of feline sporotrichosis and its role in human sporotrichosis a survey was conducted among symptomatic cats. Prevalence and phylogenetic relationships among feline Sporothrix species were investigated by reconstructing their phylogenetic origin using the calmodulin (CAL) and the translation elongation factor-1 alpha (EF1α) loci in strains originated from Rio de Janeiro (RJ, n = 15), Rio Grande do Sul (RS, n = 10), Paraná (PR, n = 4), São Paulo (SP, n =3) and Minas Gerais (MG, n = 1). Our results showed that S. brasiliensis is highly prevalent among cats (96.9%) with sporotrichosis, while S. schenckii was identified only once. The genotype of Sporothrix from cats was found identical to S. brasiliensis from human sources confirming that the disease is transmitted by cats. Sporothrix brasiliensis presented low genetic diversity compared to its sister taxon S. schenckii. No evidence of recombination in S. brasiliensis was found by split decomposition or PHI-test analysis, suggesting that S. brasiliensis is a clonal species. Strains recovered in states SP, MG and PR share the genotype of the RJ outbreak, different from the RS clone. The occurrence of separate genotypes among strains indicated that the Brazilian S. brasiliensis epidemic has at least two distinct sources. We suggest that cats represent a major host and the main source of cat and human S. brasiliensis infections in Brazil.

Figure 1. South America map showing sampling localities in Brazil and total number of animals (n = 33) and humans Sporothrix spp.

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

Figure 2. Clinical aspects of feline sporotrichosis in Brazil.

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

Figure 3. 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).

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 Table 1.

Figure 4. Median-joining haplotype network of Sporothrix schenckii complex isolates based on partial nucleotide sequences of the calmodulin-encoding gene (A) and the translation elongation factor-1 alpha (EF1α) loci regions (B).

The EF1α haplotype showed a clear intraspecific separation resultant from a nucleotide transition from A to G, between S. brasiliensis 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 Table S2.

Figure 5. Split decomposition analysis of the Sporothrix brasiliensis isolates from zoonotic epidemic outbreaks in different geographic regions in Brazil according to sequences of the calmodulin-encoding gene (A) and the translation elongation factor-1 alpha (EF1α) locus region (B).

The inset Box represents the S. brasiliensis species alone, showing the absence of recombination possibilities within this species. The absence of reticulated phylogenetic structure in the S. brasiliensis haplotypes suggests a clonality spread of this species among human, cats and dogs in Brazil for both loci.

Figure 6. In vitro temperature fitness in the Sporothrix species.

Growth inhibition at 37°C compared to 30°C incubation. S. brasiliensis from feline (n = 30) or human source (n = 27) are more resistant to heat incubation and differ statistically when compared to S. schenckii (n = 25), S. globosa (n = 7) and S. mexicana (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 S. globosa and S. mexicana. No isolate were able to growth at 40°C.