First record of Trichophyton benhamiae isolated from domestic cats in Russia

Abstract

We report a first case of Trichophyton benhamiae isolation from domestic cats in Russia. Genetically affiliated to European strains T. benhamiae were deposited in NCBI. T. benhamiae strains formed zonal cream-colored colonies, with reversum pigmentation ranging from intensive yellow to orange-brown in one and orange-brown to chocolate in the second strain. Mycelium is colorless, hyphae are septated, rapidly aging with the formation of arthrospores and microconidia. The formation of macroconidia was recorded after 48 hours. A favorable outcome of treatment was recorded after two weeks.

1. Introduction

Feline dermatophytosis is a superficial fungal skin disease. The most commonly isolated pathogen is Microsporum canis. Infection with Microsporum persicolor, Microsporum gypseum and Trichophyton species can also occur, though pathogens other than M. canis are rarely associated with outbreaks of dermatophytosis in multi-cat situations [1].

Over 82–90% of feline dermatophytosis cases worldwide are caused by Microsporum canis [2]. Others are caused by M. gypseum, T. mentagrophytes, T. quinckeanum, T. verrucosum or other agents [3]. In addition, Nannizzia gypsea and Trichophyton mentagrophytes were described as the causative agent of ringworm [4], and Microsporum distortum as zoophilic dermatophyte in dogs and cats [5]. Microsporum gypseum, Trichophyton terrestre and Trichophyton ajelloi are geophilic dermatophytes mainly isolated from dogs and cats without lesions [3].

Recently, there have been reports of the isolation of a new causative agent of dermatophytosis, T. benhamiae, from animals. More often, the pathogen is recorded in humans, which, apparently, is associated with a higher-level diagnostics in medicine.

Dermatomycete Trichophyton benhamiae was first isolated by Ajello L. and Cheng S. (1967) as a result of human and dog infections in North America and was described under the name Arthroderma benhamiae [11]. The most common host of T. benhamiae is the guinea pig (Cavia porcellus); human infection is often associated with contact with these animals. Trichophyton benhamiae can cause severe inflammatory ringworm of the trunk and scalp in humans [10].

Species of the Trichophyton benhamiae complex are predominantly zoophilic pathogens distributed throughout the world [6]. According to Baert F., et al. (2021), the Trichophyton benhamiae complex includes six closely related species: the zoophilic species T. benhamiae, T. erinacei, T. eriotrephon, T. verrucosum, and T. bullosum and the anthropophilic species T. concentricum [11]. According to Čmokova, A. et al. (2020), these pathogens have recently gained importance due to their epidemic spread among pets and their owners [6].

Cases of T. benhamiae isolation from human and animals are continuously increasing. An analysis of publications shows that isolation of T. benhamiae has recently been recorded in the Czech Republic, Poland, Germany, Italy, Finland, Switzerland, Iran, Egypt, Taiwan, Japan, and the USA [6]. The fungus Trichophyton of the species A. benhamiae is most often found in children and adolescents who become infected from guinea pigs or other animals (Cavia porcellus form. domestica), causing pronounced inflammatory dermatomycosis of the face and scalp [7]. Also, foxes [8], rabbits, dogs [6], stray cats [9], and pets (guinea pigs, cats and dogs) [10] can serve as source of T. benhamiae infection in humans.

A case of isolation of T. benhamiae in Russia was described in the Moscow region, from a guinea pig [15]. T. benhamiae has not been previously recorded on the territory of Western Siberia of Russia.

For the first time, we have established a case of feline ringworm caused by the pathogen T. benhamiae in the Siberian region of Russia. The causative agent was isolated from pathological material obtained from British and Maine Coon cats from two breeders in Novosibirsk.

The main biological properties of the new causative agent of ringworm in cats T. benhamiae have been studied and its species has been established.

2. Case presentation

Two cats were admitted to the veterinary clinic with suspected carnivore microsporia.

A 3-year-old British Shorthair cat presented with a round spot near the orbit of the left eye. The lesion had distinct boundaries, was accompanied by exudation and complete loss of hair. The skin area was almost naked and intensely red (Fig. 1). Desquamation of the skin wasobserved around the lesion, which was not immediately noticeable under the coat. Before contacting the veterinarian, clinical signs were observed for three days. The lesion was accompanied by itching, anxiety, constant scratching and washing of the affected area. The cat after ovariectomy was brought from the European part of the country (Moscow) and kept at home. The animal is alone in the apartment, did not take part in exhibitions, and did not walk on the street. During the collection of anamnesis, it was established that in the summer the cat was taken to the country house, where it freely moved around the territory of the country house village. The cat has been repeatedly seen catching mice and other rodents. Treatment for helminths was carried out once at the age of two months. Vaccinationagainst viral infections was carried out once two weeks after helminthization. A 1-year-old castrated Maine Coon cat presented with an indistinct red spot with branny desquamation on the ventral side of the tail, equidistant from both ends (Fig. 2). In the lesion, the hair was sparse and thinned; broken hair in the form of stipes, typical for dermatomycosis, was not detected. The cat was purchased in a cattery for breeding cats of this breed. As a kitten, at the age of three months, he suffered from infectious rhinotracheitis. Deworming was carried out regularly once every six months since the cat was kept in an apartment with two dogs of the Russian Greyhound breed, which were walking outside. Before contacting the veterinarian, clinical signs were observed for two days. In both animals, the presence of skin symptoms differed from the characteristic signs of microsporia. The animals were not previously vaccinated or treated against dermatomycosis, symptomatic treatment after the appearance of clinical signs was not subjected.

Fig. 1.

British Shorthair cat, from which the strain T. benhamiae №19 was isolated. With lesion in front of the head near the eye: a) before treatment, b) after treatment.

Fig. 2.

Maine Coon cat with dermatomycosis lesion on a tail, caused by T. benhamiae №20, а) external view b) lesion before treatment, c) after treatment.

The spot had a rounded shape and was located near the orbit of the left eye. The lesion had distinct boundaries, was accompanied by exudation and complete loss of hair. Hair loss in the affected area is heavy, the skin area is almost naked, intensely red. Desquamation of the skin was observed around the lesion, which was not immediately noticeable under the coat.

The growth of the lesion during the first weeks did not bother the cat. As the lesion area increased, the disease was accompanied by itching, anxiety, constant scratching and washing of the affected area.

In a one-year-old Maine Coon cat, the lesion was found on the ventral side of the tail, equidistant from both ends (Fig. 2).

An indistinct red spot with branny desquamation was found on the tail. In the lesion, the hair is sparse, thinned; broken hair in the form of stipes, typical for dermatomycosis, was not detected.

Sampling of biological material in the form of hairs from the border of the affected area and skin scrapings was performed on day 0 after proper sterilization of the affected area with 70% alcohol. Samples of the biomaterial were delivered to the diagnostic Institute of Experimental Veterinary Science of Siberia and Far East (SFSCA RAS) for further research by cultural and luminescent methods.

During the primary isolation of the pathogen, the surface cultivation of the fungus was carried out at a temperature of 28 °C for at least 10–14 days, until distinctive colonies were formed. Cultural diagnostics of the primary culture made it possible to establish the growth of a dermatomycete, remotely resembling the growth of M. canis. The appearance of the formed colony had a pronounced zonality: along the edge of the colony - the growth of fluffy mycelium, in the center - the formation of powdery foci of beige shades.

Luminescent diagnostics did not confirm the presence of Microsporum canis.

Further identification of pathogens was accomplished in the mycological laboratory at Research Platform agricultural biotechnology NJSC "S. Seifullin Kazakh Agrotechnical University”, Nur-Sultan, Kazakhstan.

Mycological diagnostics were carried out over +18–21 days. To study the cultural and morphological properties of the isolated dermatomycete strain, Sabouraud dextrose agar was used (Fig. 3).

Fig. 3.

Macroscopic appearance of cultured colony of T. benhamiae on Sabouraud agar: а) external view, b) reversum: 1 – strain №19, 2 – strain №20.

Colonies grew rapidly on Sabouraud agar, velvety at the beginning, powdery at the end of formation, and gave off various shades of beige. The reversum was intensely pigmented, with distinct zoning, 3.3 × 3.6 cm in diameter. The growth of the colony began with an uncharacteristic white mycelium.

Both strains of T. benhamiae produced cream-colored colonies of various shades with well-defined growth zones and active pigment formation on the underside of the colony. The reverse is bright orange to chocolate brown in one strain and intense yellow to orange brown in the second strain. The center of the colony was raised.

The growth characteristics of both strains were studied by the agar block method. Phenotypic identification was carried out using a review of scientific publications (Čmoková, A., Kolařík, M., Dobiáš, R. et al., 2020) [6].

Swelling and appearance of growth tubes from spores in strains occurred within 18 and 24 hours, the growth and branching of the mycelium after 18 and 36 hours, and the appearance of microconidia and macroconidia after 48 hours, respectively. By the end of the first day, active deep growth of mycelium began in the thickness of the agar.

The microscopic structure of the T. benhamiae strain is characterized by the development of mycelium and characteristic morphological structures (Fig. 4).

Fig. 4.

Microscopic structures of T. benhamiae observed by agar block method. Mycelium is septate, branching and colorless а) third-day b) seventh day:1 - convoluted mycelium, 2 - septa, 3 - arthrospores, 4 - chlamydospores, 5 microconidia, 6 - macroconidia. Scale bar = 50 μm.

The mycelium is colorless, spiral (Fig. 4.1), or straight, with partitions (Fig. 4.2), rapidly aging with the formation of arthrospores (Fig. 4.3) and chlamydospores (Fig. 4.4) by the end of the seventh day. On the third day, the formation of microconidia (Fig. 4.5) and characteristic macroconidia (Fig. 4.6) began on a short mycelium stalk, more pronounced in strain No. 20.

Genomic DNA was extracted from fungal strain using liquid nitrogen and standard phenol-chloroform method with proteinase K followed by ethanol precipitation. The amount and purity of the isolated DNA were determined by measuring the absorbance at 260 and 280 nm using a NanoDrop 2000 instrument (Thermo Scientific, USA). DNA was dissolved in ddH2O and stored at −20 °C. The ITS region on rDNA was amplified by using specific primers ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) (Integrated DNA Technologies, Inc., USA) and received the PCR product with size of 526 bp. The PCR reaction was done in a SimpliAmp thermal cycler (Applied biosystems) under the following conditions: an initial denaturation set up at 94 °C for 5 min was followed by 35 cycles of denaturation at 95 °C for 30 sec, annealing at 52 °C for 40 sec and extension at 72 °C for 50 sec, with a final extension step of 72 °C for 7 min. The sequencing was done by using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).

The GenBank/EMBL/DDBJ accession number for the sequence of the Т. benhamiae strain OVB_T.b-19 and OVB_T.b-20 a-domain-encoding gene determined in this study is ON479483/ON479484. (ITS). These sequences were compared with other sequences in the GenBank by using the BLAST analysis. The phylogenetic analysis was carried out with MEGA X software.

Thus, the use of molecular methods to identify the pathogen in our case made it possible to identify the pathogen in a short time.

The British Shorthair cat was treated with Terbinafine 10 mg. The cream was applied externally 2 times a day during the first week and, with a positive dynamics of recovery, 1 time per day - in the second week. The Maine Coon was treated with Ketoconazole in the form of 2% for external use according to the same scheme. In both cases, drugs licensed for humans were used due to the lack of available veterinary drugs containing terbinafine and ketoconazole for the treatment of animals. Treatment effectiveness was monitored by mycological examination of biomaterial samples taken from these animals. Apparent clinical recovery of the animals occurred on the 15th day, mycological recovery (negative results of the study) after another week, no relapses were observed. The full course of treatment lasted two weeks.

As the results of studies have shown, the use of ointments based on fungicidal preparations in non-toxic doses was effective and did not lead to the formation of mycocarriage in domestic cats.

3. Discussion

In the present study, we isolated and characterized 2 strains of fungi from pathological material for the first time isolated from two cats in Russia, which were identified as Trichophyton benhamiae (accession nos. ON479483 and ON479484) using DNA sequence analysis.

Search for BLAST nucleotides from 636 bp showed maximum homology of 99% with Trichophyton benhamiae (MF152781) of Polish origin, isolated from a fox, 98% with Arthroderma benhamiae strain from Finland (AB458165), isolated from a rabbit, 97.9% with T. benhamiae strain from the Moscow region of Russia (OK376997), isolated from a guinea pig. The results of genetic identification by ITS sequencing of T. benhamiae isolates made it possible to reveal their genetic affiliation to European strains in a comparative aspect with Asian and African strains (Fig. 5).

Fig. 5.

Phylogenetic analysis (tree) generated with using ITS primer.

Our research was expanded the list of pathogens of domestic cats in Russia and proposed a treatment regimen.

Trichophyton benhamiae was not registered as a causative agent of ringworm in domestic cats in the Russian Federation, although there is a case of T. benhamiae isolated from guinea pigs in the European part of Russia [15]. According to M. Sabou et al. (2017) Trichophyton, benhamiae is a zoophilic dermatophyte transmitted to humans mostly from guinea pigs and occasionally other animals. It presents two distinct phenotypes: yellow and white. Identification of T. benhamiae has been difficult, as it was described under more than three names, two phenotypes, and in several different possible host species [13].

We were unable to trace the epidemiological chain of infection of cats with T. benhamiae in the cattery. A possible reason was the popularity of guinea pigs as pets for human infections. Therefore, the cause may be the emergence of a new, virulent, and easily transmitted T. benhamiae genotype from the guinea pig, as was previously suggested [19].

The unique case of the isolation of T. benhamiae from purebred cats in the Novosibirsk cattery explains difficulties in making a diagnosis.

Sick cats had slight, not characteristic, signs of dermatomycosis. This was also discussed by other authors who reported that the animals had alopecia and crusting or no clinical signs at all [18].

Identification was carried out by morphological signs in vitro: the color of colonies and reversum, the size and shape of macroconidia, arthrosporous and chlamydospores, the rate and nature of the formation of structures on agar media. These signs must be taken into account during differentiation from other pathogens.

If the culture of strain T. benhamiae No. 19 differed in the presence of the characteristic properties described earlier for Microsporum canis, then in the culture of strain T. benhamiae No.20, cultural characteristics were more typical for Trichophyton mentagrophytes. The formation of T. benhamiae colonies, which were later classified as the yellow variant, complicated the diagnosis.

Difficulties in morphological diagnosis of T. benhamiae and misidentification of strains in 20% of cases were reported by Nenoff et al. [eleven]. At the same time, it should be noted that the strains we isolated were similar to other T. benhamiae isolates described earlier. When performing cultural and morphological identification of T. benhamiae, we revealed signs of similarity of strain 19 with T. europaeum, T. japonicum and, T. mentagrophytes [6], and strain 20 with T. erinacei [11].

The fact that morphological differentiation between M. canis and T. benhamiae is difficult since both show colonies with a yellow phenotype T. benhamiae can also produce rough and spindle-shaped macroconidia similar to those observed in M. canis, wrote [ 20].

At the same time, obtaining data on the morphological characteristics of T. benhamiae makes it possible to see their difference from classical pathogens. Microscopic differentiation showed the presence of similar morphological structures in these two strains, especially macroconidia of a specific shape, which are absent in both M. canis and, T. mentagrophytes. Despite the differences in the growth rate and formation of morphological structures in both strains up to 18–24 hours, the appearance of micro- and macroconidia was recorded at the same time, after 48 hours. These signs must be taken into account when differentiating from other pathogens.

It was the results of microscopic identification that allowed us to suggest that a new pathogen was identified from sick cats.

The difficulties of morphological diagnostics in our case can be explained by the absence of cases of detection of T. benhamiae in the Siberian region of Russia and the lack of experience in identifying dermatomycetes by veterinarians.

Since morphological differentiation between M. canis and, T. benhamiae was difficult, this required genetic identification of isolates by ITS sequencing.

We were unable to trace the epidemiological chain of infection of cats with T. benhamiae in the cattery. A possible reason was the popularity of guinea pigs as pets for human infections. Therefore, the emergence of a new, virulent, and easily transmitted T. benhamiae genotype from the guinea pig, as previously suggested [19], may be the cause.

Given the high probability of missed diagnosis due to misidentification of morphology, it is possible that the true incidence of T. benhamiae in Russia is underestimated.

Therefore, for successful diagnosis of T. benhamiae, microbiological differential diagnosis in animals should be recommended in regions where it has not yet been described. For accurate identification of species, we consider it necessary to use the methods of molecular diagnostics.

In conclusion, we report that the correctly chosen tactics and strategy of therapeutic measures made it possible to completely cure cats of the disease.

Funding source

Research project 0118RK10321 for 2018–2022.

Consent

Written informed consent was obtained from the patient or legal guardian(s) for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.

Conflict of interest

There are none.

Declaration of competing interest

There are none.

Handling Editor: Dr Adilia Warris