Agreement between Clinical Assessment and Laboratory Diagnosis of Ringworm in Calves at Auction Markets

Simple Summary Cattle ringworm is a mycotic infection of the bovine skin caused by dermatophytes that are also transmissible to humans. To limit the spread of bovine ringworm, calves at auction markets in Styria, Austria, are visually inspected and excluded from auction if they display skin lesions that are typical of ringworm. To investigate whether these clinical assessments correspond to laboratory diagnoses, samples from skin lesions were examined through microscopy, culture, and nested PCR approaches; the relatedness of the isolated dermatophytes was determined using multi-locus sequence typing (MLST). Overall, the clinical assessments were largely supported through the results of the nested PCR laboratory diagnosis, possessing an analytical sensitivity superior to that of the culture approach. Thus, this represents a fast and sensitive diagnostic test for the detection and identification of dermatophytes. Most of the isolated dermatophytes were assigned to a unique Trichophyton (T.) verrucosum MLST genotype, indicating that ringworm in calves at auction was predominantly caused by a single T. verrucosum strain. Abstract To limit the spread of bovine ringworm, control measures such as movement restrictions are highly recommended. In this context, calves at auction markets in Styria, Austria, displaying skin lesions characteristic for bovine ringworm, are excluded from the auctions. To investigate whether these clinical assessments correspond to laboratory diagnosis, a total of 166 samples taken from skin lesions assigned to the three clinical categories ‘ringworm very likely (v), likely (l) or unlikely (u)’ were mycologically examined using microscopy, culture, and nested PCR followed by amplicon sequencing. Further, the relationships of isolated dermatophytes were determined through multi-locus sequence typing (MLST). Overall, a high agreement between clinical assessment and laboratory results were observed with microscopy and nested PCR, providing more consistent results and molecular detection possessing an analytical sensitivity superior to that of cultural isolation (culture 21.7% vs. nested PCR 48.2%). Phylogenetic analyses revealed that most of the isolated dermatophytes belong to a unique Trichophyton verrucosum MLST genotype. In conclusion, clinical assessments were largely confirmed through laboratory diagnosis with nested PCR and sequencing, providing rapid, sensitive, and species-specific detection of dermatophytes in calves at auction markets displaying skin lesions typical for ringworm; this seems to be predominantly caused by a single Trichophyton verrucosum strain.


Introduction
Cattle dermatophytosis, also known as bovine ringworm, is a contagious mycotic infection of the hair and the keratinized layers of the bovine skin, most frequently caused Animals 2024, 14, 390 2 of 11 by the dermatophyte Trichophyton (T.) verrucosum [1][2][3][4].This zoophilic dermatophyte is also transmissible to humans with farmers and their families, farm workers and veterinarians being at risk of infection, sometimes developing severe inflammatory skin lesions [4][5][6].Bovine ringworm is enzootic in cattle herds in many countries all over the world [4,[7][8][9][10][11] and usually occurs in temperate areas during the autumn, winter, and spring months when animals are kept predominantly in-house.Over-crowding, high humidity, low-intensity illumination, and poor stable hygiene promote direct transmission between animals and spreading of the dermatophyte within the herd.Moreover, dermatophytes contaminating the farm environment are a major maintaining factor for cattle dermatophytosis [12].
Clinical symptoms of bovine ringworm vary from mild to severe depending on the virulence of the dermatophyte strain, the host's immune status, and other factors such as husbandry conditions, management, and production systems [1,2,13].Ringworm is more prevalent in calves which may be explained with reference to stressors (e.g., weaning, inappropriate diet, parasitic burden, transport, crowding) which are frequently present in this age group [2,9,11].In general, ringworm is considered a self-limiting disease with symptoms lasting between 4 and 12 weeks.Treatment or vaccination may shorten the duration of clinical symptoms and reduce the risk of spread of infection [12][13][14].In addition, strict hygienic measures are advised to eliminate dermatophytes from the environment and movement restrictions are recommended to limit the spread of the disease between herds [12].
Conventional methods for the detection and identification of dermatophytes including microscopy and fungal culture are time-consuming, labor-intensive, not always discriminating (microscopy), and require profound expertise in the diagnostician [15].To circumvent these drawbacks of conventional diagnostics, rapid and sensitive molecular assays such as PCR or real-time PCR are increasingly used for the detection and identification of bovine ringworm dermatophytes in clinical samples [11,13,16,17].Nevertheless, in most countries, bovine ringworm is considered a relatively harmless disease with little impact on farm economy and animal welfare.Hence, veterinarians usually refrain from performing mycological tests in affected animals and control measures are rarely implemented among veterinary authorities.
In Austria, however, calves gathered for auction in calf markets are visually inspected by official veterinarians at arrival and those displaying skin lesions characteristic for bovine ringworm are excluded from auction.Since this control measure in preventing the spread of bovine ringworm is solely based on clinical assessment, the present study aimed (i) to investigate whether the clinical assessments correspond to laboratory diagnostic results, (ii) to identify the congruence between results of the laboratory methods applied, and further (iii) to gain insight into the epidemiology of bovine ringworm in Austria through determining the phylogenetic relatedness of the dermatophyte strains isolated.

Animals and Sampling Procedures
During the period from March 2020 to March 2021, a total number of 20,259 calves were auctioned at the calf markets of 'Rinderzucht Steiermark eG' in Greinbach and Traboch (Styria, Austria).As required, all calves were visually inspected by an official veterinarian on arrival.During these inspections, all calves with superficial skin lesions were assessed, given how likely it is that dermatophytosis (ringworm) is the underlying cause of the skin lesions.Based on the clinical signs, the skin lesions were categorized by the official veterinarian as category v, 'ringworm very likely' (Figure 1A), category l, 'ringworm likely' (Figure 1B), and category u, 'ringworm unlikely' (Figure 1C).Additionally, the skin areas were photographically documented with a ruler on the picture for later documentation and measurement of the area.After cleaning the skin lesion with 70% ethanol, a superficial skin scraping sample and a hair sample were taken from the margins of the affected area using disposable scalpels.The samples were packed in a paper envelope and sent the same day by messenger service (MedLog ® , St. Pölten, Austria) to the University of Veterinary Medicine, Vienna, where they arrived the next day for mycological examination.The study has been approved by the institutional animal use and protection committee (ETK-038/02/2020).Since the study did not include invasive or painful procedures, governmental approval was not required.Overall, 166 samples were obtained.The sampled calves were, on average, 95 days old (standard deviation 59.1 days) and mostly Fleckvieh (Simmental) calves (79.5%).Furthermore, crossbred calves (Fleckvieh × Limousin [5.6%], Fleckvieh × Belgian Blue [8.7%]) and calves of other breeds or crossbreeds (6.2%) were recorded.The photographs were assessed using the freeware image measurement software (IC Measure, version 2.0.0.245, https://www.theimagingsource.de/,downloaded on 19 May 2020).The scale was taken from the photographed ruler and the area of the lesion was marked, and from these settings, the software calculated the area of the lesion.
the skin areas were photographically documented with a ruler on the picture for later documentation and measurement of the area.After cleaning the skin lesion with 70% ethanol, a superficial skin scraping sample and a hair sample were taken from the margins of the affected area using disposable scalpels.The samples were packed in a paper envelope and sent the same day by messenger service (MedLog ® , St. Pölten, Austria) to the University of Veterinary Medicine, Vienna, where they arrived the next day for mycological examination.The study has been approved by the institutional animal use and protection committee (ETK-038/02/2020).Since the study did not include invasive or painful procedures, governmental approval was not required.Overall, 166 samples were obtained.The sampled calves were, on average, 95 days old (standard deviation 59.1 days) and mostly Fleckvieh (Simmental) calves (79.5%).Furthermore, crossbred calves (Fleckvieh × Limousin [5.6%], Fleckvieh × Belgian Blue [8.7%]) and calves of other breeds or crossbreeds (6.2%) were recorded.The photographs were assessed using the freeware image measurement software (IC Measure, version 2.0.0.245, https://www.theimagingsource.de/,downloaded on 19 May 2020).The scale was taken from the photographed ruler and the area of the lesion was marked, and from these settings, the software calculated the area of the lesion.

Microscopic Examination and Cultivation
Samples from each individual calf were divided into three portions.The first portion was examined microscopically for fungal structures (e.g., arthroconidia) after incubation in 15% potassium hydroxide for 30 min at room temperature.The second portion was again divided and inoculated onto Sabouraud dextrose agar with chloramphenicol and gentamicin (BBL™, Becton Dickinson, Vienna, Austria) and Dermatophyte test medium agar (BBL™, Becton Dickinson) supplemented with thiamine (4 mg/L) and inositol (100 mg/L) (Sigma Aldrich, Merck KGaA, Vienna, Austria).Both plates were incubated at 30 °C for up to 4 weeks and checked for growth at 4-day intervals.Colonies of dermatophytes were identified according to their macro-and micromorphological characteristics, as previously described [18].In addition, DNA was extracted from cultured dermatophytes using a GenElute™ Plant Genomic DNA Miniprep Kit, according to the manufacturer's instruction (Sigma Aldrich, Merck KGaA).For definite species identification of presumptive dermatophyte isolates, amplification and sequencing of the internal transcribed spacer (ITS-1-5.8S-ITS-2cluster) were carried out as described below (phylogenetic analysis), and the resulting sequences were compared with those available in the NCBI database using

Microscopic Examination and Cultivation
Samples from each individual calf were divided into three portions.The first portion was examined microscopically for fungal structures (e.g., arthroconidia) after incubation in 15% potassium hydroxide for 30 min at room temperature.The second portion was again divided and inoculated onto Sabouraud dextrose agar with chloramphenicol and gentamicin (BBL™, Becton Dickinson, Vienna, Austria) and Dermatophyte test medium agar (BBL™, Becton Dickinson) supplemented with thiamine (4 mg/L) and inositol (100 mg/L) (Sigma Aldrich, Merck KGaA, Vienna, Austria).Both plates were incubated at 30 • C for up to 4 weeks and checked for growth at 4-day intervals.Colonies of dermatophytes were identified according to their macro-and micromorphological characteristics, as previously described [18].In addition, DNA was extracted from cultured dermatophytes using a GenElute™ Plant Genomic DNA Miniprep Kit, according to the manufacturer's instruction (Sigma Aldrich, Merck KGaA).For definite species identification of presumptive dermatophyte isolates, amplification and sequencing of the internal transcribed spacer (ITS-1-5.8S-ITS-2cluster) were carried out as described below (phylogenetic analysis), and the resulting sequences were compared with those available in the NCBI database using the BLAST algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi,accessed on 5 December 2023).

Phylogenetic Analysis of Dermatophyte Isolates
The phylogenetic relationship among T. verrucosum and T. mentagrophytes isolates were determined employing the multi-locus sequence typing (MLST) approach.For this purpose, the ITS rDNA region (ITS1-5.8S-ITS2cluster), partial gapdh gene (glyceraldehyde-3phosphate dehydrogenase), partial tubb gene (β-tubulin), and partial tef1α gene (translation elongation factor 1-α) were amplified using primer combinations and PCR protocols, as described previously [20,21].Reaction volumes of 25 µL were mixed as described for nested PCR.T. verrucosum CBS 365.53 (isolated from a cow; country and time of isolation unknown) from which sequences of the MLST scheme were publicly available (Genbank accession numbers: ITS-LR794143; gapdh-LR794254; tubb-KT155552; tef1α-LR792279), and T. mentagrophytes GP2015 isolated from a cow with dermatophytosis in 2015 in Austria; these were included in the MLST study as reference strains.Sequences of ITS, gapdh, tubb, and tef1α regions were aligned using ClustalW, trimmed, concatenated (total lengths for T. mentagrophytes 2342 bases, and for T. verrucosum 2337 bases), and a phylogenetic tree was constructed using the maximum likelihood method and Hasegawa-Kishino-Yano substitution model with bootstrapping (1000 replications) in MegaX [22].Sequence data were deposited at Genbank and are available under the accession numbers provided in Table S1.

Statistical Analysis
Animal-related data (age, gender, breed) were obtained from the calf market records and transferred into an Excel file.The categorical data from clinical assessment of the lesions and the results of microscopy, culture, and nested PCR were added.For assessing agreement among different diagnostic methods and between laboratory diagnosis and clinical assessment, Cohens Kappa coefficients (κ) and 95% confidence intervals were calculated.Results were interpreted as follows: κ values 0-0.20 as no agreement, 0.21-0.39 as minimal, 0.40-0.59as weak, 0.60-0.79as moderate, 0.80-0.90as strong, and >0.90 as almost perfect agreement [23].

Clinical Assessment
During the study period, samples were obtained from alopecic skin lesions of 166 calves (total number visually inspected 20,259), resembling a skin lesion frequency of 0.79%.Most of the lesions were found on the head and neck (65.5%) followed by the perianal/tail root area (25.7%).There were no differences between the prevalence of the breeds or gender in relation to the distribution of the 20,259 calves marketed during the period.The official veterinarians scored these 166 lesions into the three categories and collected skin scraping and hair samples: 47 cases were assigned to category v, 55 cases to category l, and 64 cases to category u.

Laboratory Diagnostic Results and Concordance between Laboratory Methods
From a total of 166 samples, 90 (54.2%) were found positive for fungal structures (arthroconidia) through microscopy.Dermatophytes were successfully cultured from 36 (21.7%) samples with 27 (16.3%)being identified as T. verrucosum, 8 (4.8%) as T. mentagrophytes, and 1 (0.6%) as Microsporum (M.) canis.Nested PCR detected dermatophyte DNA in 80 (48.2%) samples, producing 400 bp amplicons identified through sequencing as T. verrucosum (n = 59, 35.5%) or T. mentagrophytes (n = 20, 12.1%), and 1 420 bp amplicon identified as M. canis (n = 1, 0.6%) (Figure 2).From 28 (63.6%)out of 44 nested-PCR-positive samples that were missed in the culture, non-dermatophyte molds (e.g., Aspergillus spp., Alternaria sp., Cladosporium sp., Penicillium sp.) were abundantly isolated.None of the 76 (45.8%) samples found to be negative through microscopic examination were found to be positive in the culture or through nested PCR.The agreement between the microscopy and the culture examinations was moderate (κ: 0.6, CI 0.49-0.71)and the agreement with the PCR was strong (κ: 0.89, CI 0.85-0.93).None of the sample which were diagnosed as negative through nested PCR were found to be positive in the culture, and the agreement between culture and the PCR was weak (κ: 0.45, CI 0.33-0.57).Species identification was identical between the two methods (Table 1).No mixed infections of dermatophyte species were observed, neither through the culture nor through the nested PCR and amplicon sequencing.

Agreement between Laboratory Results and Clinical Assessment
Considering the individual clinical categories, 95.7% of samples formed category v, 72.7% formed category l, but only 7.8% of category u were microscopically positive for fungal elements.Similar results were obtained through nested PCR with 95.7% of samples from category v, 60% of samples from category l, and 3.1% of samples from category u yielding positive results.Of the 36 dermatophyte isolates, a majority (n = 33) were recovered from samples of category v and the remaining 3 were cultured from samples of category l (Table 2).

Agreement between Laboratory Results and Clinical Assessment
Considering the individual clinical categories, 95.7% of samples formed category v, 72.7% formed category l, but only 7.8% of category u were microscopically positive for fungal elements.Similar results were obtained through nested PCR with 95.7% of samples from category v, 60% of samples from category l, and 3.1% of samples from category u yielding positive results.Of the 36 dermatophyte isolates, a majority (n = 33) were recovered from samples of category v and the remaining 3 were cultured from samples of category l (Table 2).
With one exception (category l versus culture-no agreement), the clinical assessment showed moderate, strong, or almost perfect agreement with the laboratory results (Table 3).Taken together, the kappa coefficients for categories v and l (n = 102) under microscopy (85 positive samples) is 0.84 (CI 0.76-0.90)(strong agreement); for culture (36 positive samples), this is 0.35 (CI 0.26-0.45)(minimal agreement), and for the nested PCR (78 positive samples), this is 0.89 (CI 0.83-0.95)(strong agreement).Table 3. Cohens kappa coefficients (κ, 95% confidence interval) characterizing the agreements between the clinical assessment of skin lesion and laboratory methods (microscopy, culture, nested PCR).

Phylogenetic Relatedness among Dermatophyte Isolates
Trichophyton verrucosum showed a low level of intraspecific genetic variability.Overall, the 27 T. verrucosum isolates were separated into two unique MLST sequence types (TVa, TVb) represented by two substitutions in the ITS rDNA region and a single substitution in the gapdh gene fragment.All T. verrucosum isolates from the samples of category v (n = 25, of symptomatic ringworm in the monitored study population; this included 20,259 calves at auction.However, when considering cases with dermatological lesions (n = 166) or clinically suspected dermatophytosis (n = 102) only, the prevalence rates of ringworm confirmed using nested PCR were 48.2% or 78.4%, respectively.Similar or higher prevalence rates were reported in Egypt (55.8%) [11], Iran (92.6%) [6], Tuscany/Italy (87.7%) [9], Umbria/Italy (71.7% in cattle aged between 1 and 6 months) [10], and Poland (58.8%) [13]; the latter three studies also included asymptomatic carrier animals.In contrast, a much lower prevalence of 1.6% was observed in Pakistan, likely reflecting the breeding system that is typical for the rural area investigated, with smallholdings of one or two cows of native breeds [25].
Although the present study demonstrated high agreement between the clinical assessment of skin lesions categorized as 'ringworm very likely' (category v) and laboratory results, a rather moderate agreement was observed between the clinical assessment of skin lesions categorized as 'ringworm likely' (category l) and laboratory examination (Table 3); this indicates that clinical cases of category l might require confirmatory laboratory tests if a justification of the exclusion of calves from auction is requested.In this category, microscopy showed a somewhat higher agreement with clinical assessment than nested PCR, which conflicts with previous reports where molecular assays (nested PCR, real-time PCR) showed significantly higher efficiencies in detecting dermatophytes than microscopy [11,13,15].Another limitation of microscopy is its inability to identify the species of dermatophyte present in the sample, which necessitates additional testing, such as cultural or molecular identification.A high congruency was also noticed between the clinical assessment of skin lesions categorized as 'ringworm unlikely' (category u) and laboratory test results, with only 2 out of 64 cases that have been clinically misdiagnosed but carrying T. verrucosum being detected through the use of nested PCR.
Our findings revealed that nested PCR and amplicon sequencing correctly identifies T. verrucosum, T. mentagrophytes, and M. canis in samples that were culture-positive (n = 36); this finding agrees with those of other studies, where the nested PCR results were compared with the cultural identification results [11,19].The present study also proved that nested PCR possesses an analytical sensitivity that is superior to that of cultures, with a 26.5% higher efficiency in detecting dermatophytes present in the samples (culture 21.7% vs. nested PCR 48.2%).Possible explanations for the lower sensitivity of the culture method are the overgrowth of non-dermatophyte molds (observed in 28 out of 44 nested PCR positive samples) known to prevent or obscure the development of slowly growing dermatophytes on agar plates; these lead to not-yet-positive cultures after 4 weeks of incubation, the presence of non-viable dermatophytes in samples from pretreated calves, or the entrapment of dermatophytes in the keratin [11,26].
Trichophyton verrucosum was the most frequent dermatophyte detected in our study through the use of nested PCR (n = 59), followed by T. mentagrophytes (n = 20) and M. canis (n = 1).T. verrucosum has been widely described as the predominant etiologic agent of bovine ringworm, with endemic occurrence in many countries worldwide and cattle representing the main reservoir and natural host of this dermatophyte species [4,13,27].For T. mentagrophytes and M. canis, on the other hand, wild rodents and dogs and cats in farms have been suggested to be the most likely sources of transmission to cattle [11].
In the present study, an MLST scheme was applied for the intraspecific differentiation of dermatophytes separating 27 T. verrucosum isolates into two MLST sequence types, and 8 T. mentagrophytes isolates into four MLST sequence types.This sequence-based approach appeared to have a similar or even higher discriminatory power compared to those of PCR fingerprinting methods (PCR-melting profile (PCR-MP) fingerprinting, microsatellite primed PCR (MSP-PCR) fingerprinting) used in previous studies to differentiate T. verrucosum or T. mentragrophytes isolates [4,28].The main advantage of MLST in comparison to DNA fingerprinting is, however, the unambiguity and transferability of sequence data, providing comparability between laboratories and with database entries.Altogether, the phylogenetic analyses suggest that ringworm in calves from Styria, Austria, is pre-dominantly caused by clonally related isolates of a single T. verrucosum strain circulating in the Styrian cattle population; however, different T. mentagrophytes strains may cause dermatophytosis in calves on occasion.

Conclusions
In conclusion, the clinical assessments of calves at auction were largely confirmed through laboratory diagnosis with nested PCR followed by amplicon sequencing providing rapid, sensitive, and species-specific detection of dermatophytes in samples from calves displaying skin lesions that are characteristic for ringworm; these seem to be primarily caused by a single T. verrucosum strain in calves gathered at Styrian auction markets.

Figure 1 .
Figure 1.Examples for skin lesions of (A) category 'ringworm very likely' presenting annular, crusty lesions, and skin thickening on the left ear; (B) category 'ringworm likely' with non-circular lesions, few crusts and flakes, and very mild thickening of the skin on the calf's head; (C) category 'ringworm unlikely' (no skin thickening, flakes or crusts, abrasive hair loss, lateral on the neck).

Figure 1 .
Figure 1.Examples for skin lesions of (A) category 'ringworm very likely' presenting annular, crusty lesions, and skin thickening on the left ear; (B) category 'ringworm likely' with non-circular lesions, few crusts and flakes, and very mild thickening of the skin on the calf's head; (C) category 'ringworm unlikely' (no skin thickening, flakes or crusts, abrasive hair loss, lateral on the neck).

Figure 2 .
Figure 2. Cultural and molecular detection of dermatophytes in samples taken from calves excluded from auction.(A) T. verrucosum producing slow growing, cream-colored colonies with a suede-like surface, a raised center, and submerged growth in the periphery after 21 days of incubation on Sabouraud dextrose agar.(B) T. mentagrophytes presenting with white colonies with a downy or fluffy-powdery surface texture, and brown reverse pigmentation (not shown) after 10 days of incubation on Sabouraud dextrose agar.(C) Direct nested PCR on samples producing amplicons at 400 bp for T. verrucosum (lanes 1, 3, and 4) and T. mentagrophytes (lanes 5-7), and a 420 bp amplicon for M. canis (lane 2).Lane M-molecular weight marker; lane 8-negative control.

Figure 2 .
Figure 2. Cultural and molecular detection of dermatophytes in samples taken from calves excluded from auction.(A) T. verrucosum producing slow growing, cream-colored colonies with a suede-like surface, a raised center, and submerged growth in the periphery after 21 days of incubation on Sabouraud dextrose agar.(B) T. mentagrophytes presenting with white colonies with a downy or fluffypowdery surface texture, and brown reverse pigmentation (not shown) after 10 days of incubation on Sabouraud dextrose agar.(C) Direct nested PCR on samples producing amplicons at 400 bp for T. verrucosum (lanes 1, 3, and 4) and T. mentagrophytes (lanes 5-7), and a 420 bp amplicon for M. canis (lane 2).Lane M-molecular weight marker; lane 8-negative control.

Table 1 .
Laboratory diagnostic results of microscopy, culture, and nested PCR for the detection of dermatophytes in 166 skin samples.

Table 2 .
Laboratory diagnostic results of microscopy, culture, and nested PCR for the detection of dermatophytes in skin samples of clinical categories v (n = 47), l (n = 55), and u (n = 64).

Table 2 .
Laboratory diagnostic results of microscopy, culture, and nested PCR for the detection of dermatophytes in skin samples of clinical categories v (n = 47), l (n = 55), and u (n = 64).