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23CLINICAL, LABORATORY FEATURES AND TREATMENT OF SPONTANEOUSLY INFECTED DOGS AND CATS WITH MICROSPORUM CANIS, IN BAKU, GANJA, SHAMKIR AND GOYGOL, AZERBAIJAN, DURING 2021-2022
NARMIN ALASGAROVA1, ASAF M.OMAROV2
Azerbaijan State Agrarian University, Ganja, Azerbaijan
ORCID NO: 0000-0003-2072-6186 2ADA University, associate professor, Baku, Azerbaijan https://orcid.org/0000-0002-2286-5510
Resume This article focuses on performing clinical, laboratory examination, isolation, and identification of M. canis and the successful treatment of M. canis infection in cats and dogs in Baku, Ganja, Shamkir and Goygol, Azerbaijan, during 2021-2022. The source of infection (SOI) was demonstrated to be cats and dogs in veterinary clinic operating under the Veterinary Scientific Research Institute, Baku, Azerbaijan and in small animal hospitals, Ganja, Azerbaijan. Samples were collected from a total of 27 animals. 11 of the sampled animals are dogs and 16 are cats. 20 of the 27 suspected animals we examined had the disease, in 2021. Twenty of the 27 samples (74%) were identified as M. canis macroscopically and microscopically. During 2022, 69 dogs and 53 cats were examined in Baku. Out of 69 dogs examined, 2 stray dogs were found to have the disease. Disease was detected in 16 cats among 53 cats examined. It was determined that 3 of the sick cats were domestic cats, 5 were shelter animals, and 8 were stray cats. The skin lesions observed in the M. canis infected cats and dogs were erythema, alopecia, scaly, and crusty distributed to the ear, body, neck, back and tail of cats, respectively. As a result of our study, 19 infected cats and 4 dogs were treated with itraconazole and one of two topical therapies including 2% chlorhexidine and 2% miconazole shampoo. The median time to clinical cure was six weeks and the median time to mycological cure was six weeks (range 7-21 weeks).
Keywords: dermatophytes; miconazole, itraconazole; zoonosis;
Abbreviation
FLZ - fluconazole
IT- Itraconazole
GRI- griseofulvin
SOI -The source of infection
TER - terbinafine
1. Introduction
Dermatophytosis is a disease caused by dermatophytes, a group of fungi that can cause disease both in humans and animals (Vena Chupia, Jirapat Ninsuwon et al., 2022). Infection withM. canis is usually associated with alopecia, and infection has been diagnosed by isolation of fungus, which has characteristic hyphae or arthroconidia, from the patients' hair lesions (Hsiao YH, Chen C, Han HS, et al., 2018). M. canis cause lesions to the glabrous skin (tinea corporis) and to the head (tinea capitis) (by Mario Pasquetti, Anna Rita Molinar Min et al., 2017). M. canis transmission occurs through direct contact with sick or subclinically infected animals, mainly cats, or with arthrospores, that remain viable in the environment for up to 18 months (A. H. Sparkes, G. Werrett et al., 1994). The pathogen can be found in the hair of cats with and without skin lesions, owners, keepers, veterinarians, and others who come into contact with these animals are at risk of infection if they are not aware or do not take precautions after contact with them (Vena Chupia, Jirapat Ninsuwon et al., 2022). The infected patients show hair loss with erythema and are diagnosed as having dermatophytosis, but the transmission routes of M. canis from animals to others are sometimes unclear, although they are critical to the treatment of patients and infection control (Maya Hariu, Yuji Watanabe et al., 2021). During our research, we did not find any literature information about the research of this disease in
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our republic during the last 30 years. It is known that the disease is detected in certain seasons of the year in Turkey, which is a neighboring country to Azerbaijan. The isolation rates of dermatophyte species from dogs and cats were 18.7% and 20.1%, respectively (Esra Seker, Nurhan Dogan, 2010). Microsporum canis (57.1%) was the most common species isolated from dogs and cats. The isolation rate of dermatophytes was relatively high in the spring and winter for dogs, and in the spring, summer and autumn for cats in western Turkey (Esra Seker, Nurhan Dogan, 2010). For the treatment of dermatophytosis, griseofulvin, ketoconazole, itraconazole and terbinafine are the drugs most commonly used in veterinary medicine (Boothe, 2012). According to our research since 2018, this disease is often found among both humans and animals. The disease is especially common in autumn, winter and spring for dogs and cats in Azerbaijan.
Aim of the research: to perform clinical, laboratory examination, isolation, identification and successful treatment of M. canis infection in cats and dogs in Baku, Ganja, Shamkir and Goygol, Azerbaijan.
2. Materials and methods
Samples were collected from spontaneously infected stray dogs, cats, shelter animals and domestic pets (dogs and cats) in veterinary clinic operating under the Veterinary Scientific Research Institute, Baku, Azerbaijan and in small animal hospitals Ganja, Azerbaijan, from 2021 to 2022. Totally 80 dogs, 69 cats were caught, 149 samples were collected. 38 cultures were isolated. For each animal, a complete clinical examination was performed. The animals were sampled using the toothbrush technique (Moriello K.A., 2001). We observed patchy hair loss and the infected areas were round, oval, or irregular and 1 to 4 cm in diameter, and multiple patches are common in sick animals. Skin scrapped from cats and dogs that clinically showed lesions of dermatitis i.e. combination of alopecia, erythema, papules, pustules, scaly, and crusty were used in this study (Table 1). Hair samples are handled in a manner to preserve any epithelial cells adhering to the hair shaft. Samples were collected into glassine evidence bags, dry-mount on microscope slides. Hair samples lifted with a conventional fingerprint tape was placed sticky side down on paper for shipping (https://vgl.ucdavis.edu/forensics/evidence-collection-sample-type). Infected animals were selected based on the clinical signs we observed. Examination of clinical lesions and screening tests using the Wood's lamps were performed prior to sampling (Figure 1). Wood's lamp examination is a useful technique.
Biosecurity and biosafety regulations
Collection, packaging, and transportation of samples were carried out in accordance with biosafety rules (Chapter 1.1.4 Biosafety and biosecurity: Standard for managing biological risk in the veterinary laboratory and animal facilities).
Ethical report: Samples from animals were collected in accordance with the bioethical and standard procedures of the "Bioethics Committee of the Azerbaijan National Academy of Sciences".
Out of 38 samples of 149 patients with culturally proven Microsporum canis infections, Wood's light examination was performed (Figure 2).
The growth medium used for the cultures was Sabouraud's dextrose agar (Sabouraud Dextrose Agar, Granulated-GM063-500Gmedium/ Sabouraud Dextrose Agar, Granulated is supplied by the company, HIMEDIA). Media was required final sterilization in an autoclave at 121°C for 20 minutes. Plates that were incubated at 25°C and examined daily for 2 weeks. Fungal colonies were identified at the species level based on their morphology and microscopic features. Fungal culture on Sabouraud dextrose agar showed flat, white and spread-out colonies with a cottony surface (Figure 3). Light microscopy was implemented for colony conformation.
Figure 1 Wood's lamps examination
Figure 2 Result of Wood's lamps examination
Figure 3 Colonies of M.canis on Sabouraud's dextrose agar
3. Conventional Therapy for Animals and Humans
The choice of proper treatment is determined by the site and extent of the infection, as well as by the efficacy, safety profile, and pharmacokinetics of the available drugs (Norris H.A., Elewski B.E., Ghannoum M.A., 1999). A vast range of antifungal classes, such as the first oral imidazole (e.g., ketoconazole-KTZ) and GRI have been used in human and veterinary medicine to treat dematophytoses (Elewski B.E., 1998). Later on, other azoles (i.e., FLZ, ITZ, efinaconazole, and luliconazole), allylamines (i.e., TER, butanafine, and naftifine) and amorolfine, and ciclopiroxolamine were employed (Matsuda Y., Sugiura K., Hashimoto T., Ueda A., Konno Y., Tatsumi Y. Efficacy, 2016). In animals, topical therapies (i.e., weekly application of lime sulphur, enilconazole, or a miconazole/chlorhexidine shampoo) are currently recommended (Chioma I. Aneke, Domenico Otranto, and Claudia Cafarchia, 2018).
4. Topical antifungal treatments
Transmission of dermatophytosis occurs via direct contact with infective material originating from the skin and hair coat of infected animals. Thus, the purpose of topical therapy is to decrease the infectious, contagious and zoonotic risks associated with this disease by disinfecting the hair coat and minimizing contamination of the environment (Karen A. Moriello, Kimberly Coyner, Susan Paterson, Bernard Mignon, 2017).
4.1 Miconazole/chlorhexidine formulations
After a clinical report showed efficacy of a combination miconazole/chlorhexidine shampoo in the treatment of dermatophytosis, two in vitro studies investigated the antifungal efficacy of stock solutions of miconazole, chlorhexidine, or a 1:1 combination of both against Microsporum spp. and Trichophyton spp (Karen A. Moriello, Kimberly Coyner, Susan Paterson, Bernard Mignon, 2017). the minimum inhibitory concentrations (MIC) of chlorhexidine, miconazole and chlorhexidine/miconazole ranged from 12.5 to 25 ^L/mL, 0.29 to 1.17 ^L/mL, and 0.14 to 0.39 ^L/mL, respectively. For nine of 10 of the isolates, the miconazole/chlorhexidine combination was more effective than either agent alone; there was either a synergistic (n = 5 isolates) or additive (n = 4 isolates) effect. This study protocol was repeated but this time evaluated these agents against T. mentagrophytes (n = 9), T. erinacei (n = 9) and M. persicolor (n = 5). The MIC of chlorhexidine, miconazole and miconazole/chlorhexidine ranged from 12.5 to 50 ^L/mL, 0.24 to 1.56 ^L/mL, and 0.11 to 1.66 ^L/mL, respectively. The mean MICs did not vary significantly between the three dermatophyte species tested, but the MICs of miconazole alone and in combination
with chlorhexidine for T. erinacei were significantly greater than
for T. mentagrophytes and M. persicolor. A synergistic or additive effect was seen in 15 of 23 isolates tested (Karen A. Moriello, Kimberly Coyner, Susan Paterson, Bernard Mignon, 2017).
4.2 Vaccine Polivac-TM (Вакцина ПОЛИВАК-ТМ)
We contacted the veterinarians at the small animal clinics in Baku and Ganja and found out that the Polivac-TM vaccine is used for the treatment and prevention of infection with M. canis in our country. Veterinary doctor Taryel Mursalov in IDEA Animal Care Center was also informed that the mentioned vaccine is used in the treatment of all dermatophytosis. Polivac-TM prevents the infection of trichophytosis and microsporia during the year, therefore, annual double revaccination of animals with an interval of 10-14 days at the indicated doses is indicated.
For therapeutic purposes, cats are vaccinated two or three times:
at 1-5 months at a dose of 1.5 ml
from 6 months at a dose of 2 ml
If the animal is in the incubation period of fungal diseases, then immunization with Polivac can provoke the appearance of single or multiple lesion of disease. In this case, 10-14 days after the main course, the vaccine at a therapeutic dose is repeated. The main drawback of this method is that same vaccine is used in the treatment of Dermatophyte infections (Trichophyton, Microsporum, and Epidermophyton) and no means are used to eliminate the clinical signs directly on the skin during this treatment method.
5. Results
5.1 Case (February, 2021)
Microsporum canis is a fungal species that causes numerous forms of disease. It is part of a group of fungi known as Dermatophytes. Though mostly well known for ringworm in pets and other animals, it is also known to infect humans. This fact makes this pathogen both anthrophilic and zoophilic in nature. Microsporum canis is a contagious pathogen (https://www.viroxanimalhealth.com).
Table 1
Clinical manifestation of the disease in 2021
Animals erythema alopecia scaly crusty
Cat BA 01 + - - -
Cat BA 02 + + + +
Cat BA 03 + + + +
Cat BA 04 + + + +
Cat BA 05 + + - -
Cat BA 06 + + - -
Cat GA 07 - + + +
Cat GA 08 - + + +
Cat GA 09 - + + +
Cat GO 10 - + - -
Cat SH 11 + + - -
Cat SH 12 + + - -
Dog BA 13 - - + +
Dog BA 14 - - + +
Dog BA 15 + + - -
Dog BA 16 + + - -
Dog GA 17 - - + +
Dog GA 18 - - - -
Dog GO 19 + + - +
Dog SH 20 - - + +
The animals shown in the table are conventionally numbered. The mentioned first letters indicate the city, district and residential areas where the samples were taken (BA- Baku, GA- Ganja, GO- Goygol, SH- Shamkir). The sign "+" indicates the presence of a clinical sign, and "-" indicates its absence. During 2021, samples were collected from a total of 27 animals. 11 of the sampled animals are dogs and 16 are cats. 20 of the 27 suspected animals we examined had the disease. Clinical signs of the disease were detected in 20 infected animals. Erythema and crusty were observed in 11 animals (55%), alopecia was observed in 16 animals (80%), scaly was observed in 10 animals (50%).
Table 2
Clinical dissemination of the pathogen in the body areas of the spontaneously infected animals
Host animal head neck nose patchy hair loss
Cat BA 01 - - + -
Cat BA 02 + - + +
Cat BA 03 + + + +
Cat BA 04 + + - +
Cat BA 05 + - - +
Cat BA 06 + - + +
Cat GA 07 + + - +
Cat GA 08 + + - +
Cat GA 09 + + - +
Cat GO 10 + + - +
Cat SH 11 + + - +
Cat SH 12 + + - +
Dog BA 13 - - - -
Dog BA 14 - - - -
Dog BA 15 + + + +
Dog BA 16 + + + +
Dog GA 17 - - - -
Dog GA 18 - - - -
Dog GO 19 + + - +
Dog SH 20 - + - -
The animals shown in the table are conventionally numbered. The mentioned first letters indicate the city, district and residential areas where the samples were taken in 2021 (BA- Baku, GAGanja, GO- Goygol, SH- Shamkir). The sign "+" indicates the presence of a clinical sign, and "-" indicates its absence in the mentioned parts of the body (head, nose). The clinical signs of the disease were observed on the head of 14 animals (11 cats and 3 dogs), on the neck of 12 animals (8 cats and 4 dogs), and on the nose of 6 animals (4 cats and 2 dogs) and patchy hair loss was observed in 14 animals (11 cats and 3 dogs).
5.2 The source of infection (SOI) was demonstrated to be cats and dogs in Baku and Ganja, Azerbaijan (Figure 4, 5).
Table 3 Datebase of sick cats and dogs examined in the veterinary clinic operating under the Veterinary Scientific Research Institute in Baku, during 2022
5.3 The skin lesions observed in the M.canis infected cats and dogs were erythema, alopecia, scaly, and crusty distributed to the ear, body, neck, back and tail of cats and dogs, respectively (Table 2). Twenty of the 27 samples (74%) were identified asM. canis macroscopically and microscopically, during 2021.
5.4 Case (April, 2022)
According to the database of sick dogs and cats examined in the veterinary clinic operating under the Veterinary Scientific Research Institute, infection by Microsporum canis is more common among cats than dogs in Baku, during 2022. Out of 69 dogs examined, 2 stray dogs were found to have the disease. Disease was detected in 16 cats among 53 cats examined. It was determined that 3 of the sick cats were domestic cats, 5 were shelter animals, and 8 were stray cats (Table 3). Clinical signs of the disease were also observed in the vast majority of animal owners who applied to the veterinary clinics in Baku. Therefore, dogs and cats play an exceptional role in the spread of the disease among people.
5.5 The cats and dogs were successfully treated using itraconazole (IT) with oral therapy and miconazole with topical therapy (Figure 6) and disinfecting of washable textiles via mechanical washing. Contamination was monitored over time, and cleaning procedures were stopped when fungal culture results were negative.
5.6 As a result of our study, twice weekly application of lime sulfur, a miconazole/chlorhexidine shampoo are currently recommended effective topical therapies in the treatment of generalized dermatophytosis in cats and dogs.
5.7 Miconazole shampoos (Davis Miconazole Pet Shampoo, 12 Oz) are effective in vitro but in vivo are most effective when combined with chlorhexidine.
5.8 In our study, 19 infected cats and 4 dogs were treated with itraconazole and one of two topical therapies including 2% chlorhexidine and 2% miconazole shampoo (Pet MD Micoseb-CX Medicated Shampoo for Dogs, Cats, & Horses with Miconazole, & Chlorhexidine - 12 oz). The median time to clinical cure was six weeks and the median time to mycological cure was six weeks
Figure 6 Infected dog after the treatment
6. Discussion
6.1 The natural reservoir of M. canis is dogs and cats, and secondary infection in humans causes tinea capitis and tinea corporis. These hair and skin lesions have been observed in severe cases when M. canis infects immunocompromised hosts (Mock M, Monod M, Baudraz-Rosselet F, et al, 1998). In this present case, the origin was directly identified, so the source of infection (SOI) was demonstrated to be cats and dogs in Baku, Ganja, Shamkir and Goygol, Azerbaijan. These cats were very likely the reservoir of the M. canis and the origin of transmission to the family. M. canis has a characteristic morphology and produces septate hyphae and macro-conidia that are spindle-shaped and have asymmetrical, apical knobs (Hubka V, Dobiasova, Dobias R, et al., 2014). According to our research, this disease is also observed in neighboring countries in certain seasons of the year and infected cats play an important role in the spread of the disease in other countries, as well as in Azerbaijan. The disease is especially common in autumn, winter and spring for dogs and cats in Azerbaijan but the isolation rate of dermatophytes was relatively high in the spring and winter for dogs, and in the spring, summer and autumn for cats in western Turkey (Esra Seker, Nurhan Dogan, 2010).
6.2 The main cons of this method are that, Vaccine Polivac-TM (Вакцина ПОЛИВАК-ТМ) is used in the treatment of Dermatophytes (Trichophyton, Microsporum, and Epidermophyton) and no means are used to eliminate the clinical signs directly on the skin during this treatment method. In contrast, the method of treatment (using itraconazole (IT) with oral therapy and miconazole with topical therapy) that we apply is designed only for the treatment of infection with M. canis. In addition, the application of a miconazole/chlorhexidine shampoo has an important role in faster elimination of problems caused by M. canis on the skin.
Outcomes
1. Fungal infections were spread among stray cats and dogs and stray animals are main reservoirs of infection in Baku, Ganja, Shamkir and Goygol
2. M.canis was one of the main infections among dermatophytes in Baku, Ganja, Shamkir and Goygol
3. Antifungal drugs were demonstrated differently in "in vitro" performance
4. "in vitro" concerts were excused on the spontaneously infected animals
5. Combination of antifungal prescriptions demonstrated more effective treatment of mycosis
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