Granulomatous rhinitis by Neoconidiobolus lamprauges in a mule

ABSTRACT: Conidiobolomycosis has a wide distribution, predominantly in humid tropical regions, affecting several species with significant mortality rates. The genus Conidiobolus is now divided into four genera: Capillidium, Conidiobolus, Microconidiobolus, and Neoconidiobolus. There are no confirmed reports of infection by these fungi in Equidae in Brazil. We present a rhinofacial rhinitis caused by Neoconidiobolus lamprauges in a mule from Rio de Janeiro, Brazil. The mule presented bilateral semi-occlusion of the nostrils, difficulty breathing, and weight loss. The histological examination of the nostril biopsied mass revealed multifocal necrotizing areas with nonstained images of fungal hyphae in the Splendore-Hoeppli reaction and surrounded by macrophages, eosinophils, neutrophils, and multinucleated giant cells. The Grocott methenamine silver staining revealed thin-walled, rarely septated, irregular branching hyphae, with a varying diameter of 12 μm (± 3.63 μm), and terminal ballooning dilations. The determining etiology of this rhinitis was based on the hyphae staining by immunohistochemistry and by amplifying the DNA fragment of N. lamprauges by polymerase chain reaction. Conidiobolomycosis should be included in the differential diagnosis of the causes of rhinitis in equids, mainly in tropical regions.

We described the clinic-pathological findings of a confirmed case of nasal conidiobolomycosis in a mule in Brazil, intending to alert veterinarians about this respiratory disease.
In October 2018, a nodule fragment from an incisional biopsy of a bilateral intranasal lesion of a 13-year-old mule from the municipality of Mangaratiba, coastal region of Rio de Janeiro, Brazil (S 22 57 35, W 44 2 28), was sent to the Setor de Anatomia Patológica (SAP) of the Federal Rural University of Rio de Janeiro (UFRuralRJ) for diagnosis.
Clinical and epidemiological data were obtained from the veterinarian who sent the lesion biopsied for diagnosis.The sedation protocol was administered by application of xylazine.After the mule reached the desired sedation plan, an incision on the lesion was done, and the obtained tissue was fixed in 10% buffered formalin.Finally, silver sulfadiazine and cypermethrin repellent were applied to the lesion site.Nasal tissues were routinely processed for histology and stained with hematoxylin and eosin and Grocott methenamine silver (GMS) and evaluated microscopically.The hyphae's diameter was determined from the GMS stained sections (MILLER & CAMPBELL, 1984).
The 3µm sections of the nodule were subjected to immunohistochemistry (IhC) using polyclonal antibodies anti-C.lamprauges and anti-Pythium insidiosum.The antibodies were raised by immunizing rabbits with C. lamprauges exoantigens from purified cultures; or P. insidiosum after subcutaneous inoculation of zoospores (UBIALI et al., 2013).The isolates used to develop the antigens for the production of the anti-C.lamprauges antibodies were recorded by SILVEIRA et al. ( 2013) (GenBank GQ478281.1).Antigen retrieval was performed with citrate buffer (10 mM, pH 6.0) at 96 ºC for 20 min.Both primary antibodies were used at a concentration of 1:1000, incubated at 38 ºC for two hours.The secondary antibody used was streptavidin-biotin-peroxidase (LSAB + System hRP, Agilent Technologies, Santa Clara, CA, USA).Substrate development occurred due to the addition of 3,3-diaminobenzidine chromogen (DAB + Substrate Chromogen System, DakoCytomation, Carpinteria, California).The sections were contrasted with Mayer's hematoxylin.During the test, tissues previously cultured and PCR positive (conidiobolomycosis from sheep) and negative controls (replacement of primary antibody by phosphate buffer saline and normal nasal tissue) were used simultaneously.
Nasal lesion tissue, formalin-fixed paraffin-embedded, was sent to the Molecular Biology Laboratory.The DNA was extracted according to ShI et al. (2004), and it was subjected to the PCR technique with oligonucleotides based on the 18S ribosomal gene, which amplified 222 bp, specific for the identification of C. lamprauges (SILVEIRA et al., 2013).Subsequently, the DNA was subjected to the PCR technique with the oligonucleotide pair encoding the sequence of the ITS1 rDNA gene of P. insidiosum, which amplifies 105 bp, specific for the identification of P. insidiosum (AZEVEDO et al., 2012).Positive controls for conidiobolomycosis and pythiosis were nasal and cutaneous lesions from sheep and horse, respectively, previously diagnosed by culture and PCR.
The mule presented bilateral semi-occlusion of the nostrils, difficulty breathing, and weight loss.These clinical signs were due to inflammatory masses that invaded the alar fold and dorsal meatus and partially occluded both nostrils.The mass was firm and measured approximately 5.3 x 4.2 x 3.0 cm, with an irregular surface, with multifocal brown and yellow areas.A moderate bilateral increase of submandibular lymph nodes was also observed.
The biopsied nostril mass was 2.5 x 1.2 x 1.2 cm, in its largest axes, with an irregular surface, and the cut surface was firm, compact with multifocal necrotic brown-yellow areas (Figure 1A).The histological evaluation demonstrated the mucocutaneous nasal mass with ulceration, multifocal areas of necrosis containing eosinophilic clots (Splendore-hoeppli reaction) and non-stained images of fungal hyphae, a significant amount of macrophages, a moderate number of multiple giant cells (Figure 1B), eosinophils, and neutrophils, fibrosis, and neovascularization.The histochemistry with GMS showed round tubuliform hyphae, with a varying degree of parallelism and diameter average of 12 μm (± 3.63 μm), with thin walls and terminal balloon dilations (Figure 1C).
The IhC and the PCR for C. lamprauges revealed intense immunolabelling of intralesional hyphae (Figure 1D) and the amplicon of 222 bp, respectively.The primers are putatively specific for N. lamprauges.After a clinical evolution of five months in the field, the mule died, and the necropsy was not undergone.
The diagnosis of conidiobolomycosis was based on clinical respiratory signs due to bilateral  nostrils semi-occlusion by an inflammatory mass, histological pattern, and confirmed by IHC and PCR.The testing of P. insidiosum by IhC and PCR was negative.We emphasized the importance of auxiliary methods such as IhC (UBIALI et al., 2013) and PCR (VILELA et al., 2010) as valuable techniques in providing a quick and accurate diagnosis compared to mycological cultures, which generally require more time and experience from the mycologist.The genus Conidiobolus was reclassified into four genera (Capillidium, Conidiobolus, Microconidiobolus, and Neoconidiobolus) (NIE et al., 2021).Based on the author's experience, it is complicated to distinguish among them without molecular testing.
Only one case of conidiobolomycosis in a mule was reported in the literature (CARVALhO et al., 1976).however, those authors based only on histomorphological patterns and did not present diagnostic confirmation through etiological exams.The current case confirmed the susceptibility of mules to infection by Neoconidiobolus lamprauges and expands the range of host species known for conidiobolomycosis.There are no confirmed reports of Conidiobolus spp.infection in Equidae in Brazil.
An important differential diagnosis of nasal conidiobolomycosis in horses is nasal pythiosis (SOUTO et al., 2016).When comparing both diseases, in a 35-year-study, the author's findings revealed that 195 horses, six mules, and one donkey were diagnosed with pythiosis (SOUTO et al., 2021).A research in the Brazilian Pantanal showed that horses were more affected (97.4%) by pythiosis than domestic equine hybrids (2.6%) (SANTOS et al., 2014).A resistance of equid hybrids by oomycete or fungi infection compared to equine is a hypothesis.Conversely, Brazil has different regions with heterogeneous populations of horses, mules and donkeys.Therefore, we encouraged other groups to compare host-pathogen interaction in horses and their hybrids.
In a survey conducted in Florida, in the United States, with horses that presented mycotic rhinitis and sinusitis, the diagnostic rate for conidiobolomycosis was 62% (32/51), which corresponds to the largest group of lesions (MORE et al., 2019).This high frequency represents the involvement of the nasal mucosa by saprophytic fungal spores (MANNING et al., 2007).The hypotheses on animal and human contamination are dust inhalation, nasal mucosa contact with contaminated water, traumatic injuries by plants with thorns and exposure to insects that transport different conidia species (KETTERER et al., 1992;VILELA et al., 2010, VILELA & MENDONZA, 2018, CARMO et al., 2021).It was not possible to determine the field infection source in this mule with conidiobolomycosis.
Conidiobolomycosis should be included in the differential diagnosis of the causes of rhinitis in equids, mainly in tropical regions.We emphasized the diagnostic importance through histology, immunohistochemistry, and molecular identification.