A Case of Laying Hens Mycosis Caused by Fusarium proliferatum

In this article, we present the first case report of a chicken mycosis caused by F. proliferatum occurred on a private farm in the Russian Federation. Lesions on the skin of the legs and scallops were reported. The object of this study was samples of feed and pathological material from sick hens-layers. Mycological analysis included determination of the total number of fungi (TNF) and identification and determination of the toxicity and pathogenicity of the isolates. The identification of the isolate was carried out taking into account direct microscopy, morphological features, and the method of molecular genetic analysis. Microscopic fungi of the genus Penicillium and Rhizopus were isolated by mycological analysis of the feed. The test feed was nontoxic. Mycological examination of pathological material (scrapings from the combs and affected legs) identified an isolate of Fusarium proliferatum, which showed toxicity on biological objects (protozoa, rabbits) and pathogenicity (white mice). Dermal application of F. proliferatum suspension was accompanied by reddening of the rabbit skin. Intraperitoneal injection of fungal spores caused mycosis in white mice. Polymerase chain reaction (PCR) made it possible to identify this type of microscopic fungus (F. proliferatum) with high accuracy in the samples under study. The research results allow us to consider F. proliferatum as a cause of poultry disease against the background of predisposing factors in the form of desquamation of the stratum corneum of the skin against the background of immunosuppression and metabolic disorders caused by an imbalance in the diet.


Introduction
Fusarium is a group of the most common plant pathogens found throughout the world that are detrimental to crop production. F. proliferatum is a very important member of the Fusarium genus and is capable of infecting many economically important crops including wheat, peas, corn, rice, asparagus, date palm, garlic, onion, pineapple, and banana [1][2][3]. F. proliferatum spreads mainly with seeds and crop residues and biosynthesizes a large number of mycotoxins (bovericin, moniliformin, fusaric acid, and highly toxic fumonisins) [4][5][6].
Tis globally widespread fungal pathogen can survive in many ecological niches, but the optimal environment is a warm and humid climate, as well as loamy soils [7]. Such characteristics indicate the high plasticity and excellent adaptability of F. proliferatum to changing environmental conditions [8,9]. Fusarium infections are very difcult to fght as efective fungicides are not available, so multistep actions are required, including plant and soil protection and reduction of inoculation [10,11]. In addition, the accumulation of secondary metabolites of Fusarium in plant tissues and their possible harmful efects on human and animal health is an additional problem in efective plant protection [12,13]. Te best strategy for controlling an infection is to produce plants that are resistant to the pathogen [14,15]. Recently, reports have begun to appear in the literature that unusual fungal species, such as F. proliferatum and others, may be underestimated as agents, causing mycotic infections, including mycoses in animals and humans. Molecular phylogeny in a study [16] showed that 23 Fusarium species, distributed over eight species complexes, were associated with veterinary mycoses. Te most common infections in animals were eye infections, followed by dermatomycosis and onychomycosis. In addition, the authors conclude that F. redolens caused cutaneous granuloma in a cat in California, which is only the second report of this soil fungus associated with mycotic infection.
Two cases of noninvasive fungal rhinosinusitis are described [17], which developed due to the F. proliferatum disease. Tey were the frst to associate this fungal pathogen with maxillary fungus balls of odontogenic origin [18]. With an emphasis on genetic identifcation of F. proliferatum, the histopathological features of granulomatous feline pododermatitis with fusarium infection were frst described. Tere has also been a reported case of infection of F. proliferatum in humans via a punctured fnger from a plant [19]. In the article [20], F. proliferatum, identifed by EF-1ɑ DNA sequencing, is described as an important new species causing fungal keratitis in China, where it was isolated out of 877 cases with a frequency of 17.7%. However, the authors note that the role of F. proliferatum in this disease is not well understood. A case of nonhaual hyalogyphomycosis caused by Fusarium proliferatum has been reported in an immunocompetent patient. Irregular hyalophomycosis due to Fusarium spp. is rare, with an incidence of about 3% in Switzerland [21]. In Japan, out of 11 cases of hyalophomycosis caused by Fusarium spp., there were three infections with F. proliferatum, accompanied by the development of subungal onychomycosis [22].
Our paper indicates that F. proliferatum is capable of causing mycosis in chickens. However, summarizing the literature data, we can conclude that mycoses of animals and humans caused by F. proliferatum have not actually been studied.

Research Objects.
Te studies were carried out in the laboratory of mycotoxins of the Federal State Budgetary Scientifc Institution "Federal Center for Toxicological, Radiation, and Biological Safety" (Russian Federation). Te objects of the study were samples of feed (compound feed PK 1-2-63) and pathological material from sick and dead birds from a private poultry farm. Laying hens of the LOHMANN cross at the age of 38-64 weeks were kept in cages of 12 individuals each. Te composition of the diet is presented in Table 1.

Determination of Microscopic Fungi.
Isolation of microscopic fungi from the feed was carried out by the method of successive dilutions with further inoculation on an agar Czapek medium (glucose 30 g, sodium nitrate 2.0 g, monosubstituted potassium phosphate 1 g, magnesium sulfate 0.5 g, potassium chloride 0.5 g, ferrous sulfate 0.01 g, distilled water 1 l, and agar-agar 20 g). Isolation of microscopic fungi from pathological material was carried out by inoculation on Sabouraud agar medium (fermentative dry peptone-7.0 g, soy four hydrolyzate-3.0 g, hydrated crystalline glucose-40.0 g, clarifed autolyzed yeast extract-4.0 g, and microbiological agar for dense medium-12.0 g).
For mycological analysis, food was taken after uniform mixing 10.0 g of the prepared food was placed in a fask with 100 ml of sterile 0.1% solution of the surfactant Tween-80 in distilled water. Te resulting suspension with the ratio of feed and solvent 1 : 10 was shaken for 15-20 min on a shaker. Ten, they were diluted with sterile distilled water (the ratio of feed and water is 1 : 10, 1 : 100, 1 : 1000, and 1 : 10000), and 1 ml of each of these dilutions was added to the surface of the nutrient medium in Petri dishes. Te inoculations were incubated in a thermostat at a temperature of 22-25°C for 5-7 days. Te number of spores in 1 g of feed and the species composition of fungi were determined. Te total number of colonies grown in the dishes of the main dilution was taken as 100% and the percentage of individual species of fungi was calculated accordingly.
Te calculation of the total amount of fungi in 1 g of the product was carried out according to the following formula: where X is the total number of fungi, expressed by the number of colony-forming units (CFU) in 1 g of the product; c-the sum of colonies on all plates, calculated in the inoculations of all three consecutively diluted suspensions; V 1 -volume of suspension 1 (dilution 10 −1 ); V 2 -volume of suspension 2 (dilution 10 −2 ); V 3 -suspension volume 3 (dilution 10 −3 ). Scrapings were made from the afected foci of legs and scallops in birds using a sterile scalpel (at the sites of infection on the border of healthy and afected parts of the keratinized skin). Te previously afected area was treated with 96% ethyl alcohol. Keratinized elements from legs and scallops were applied to agar media of Sabouraud and Czapek. Mycological studies of feed included identifcation, determination of the toxicity of isolated isolates on the test culture of Paramecium caudatum and on the skin of a rabbit, and determination of pathogenicity was carried out on white mice.

Identifcation of Fungi.
Te genus of the isolate was determined taking into account the morphological characters using a light microscope (Olimpus CX23), which were compared with those in the fungus identifer, as well as with our long-term photo collection of fungi. Te preparations were prepared from a 5-day old culture of the isolate grown on agar medium [25]. A drop of fxing liquid (Amman lactophenol: distilled water 1 part, lactic acid 1 part, glycerin 2 parts, and phenol 1 part) was applied to a glass-alcohol-free glass slide using a microsyringe, then a piece of mycelium was taken with a mycological hook and layered on it, covered with a cover glass, then looked under a microscope at ×10 and ×40 magnifcations.
At the next stage, the species was confrmed by PCR. When creating a tool for the indication of microscopic fungi of the Fusarium genus by PCR, the following resources were used: National Center for Biotechnology Information resources (https://www.ncbi.nlm.nih.gov); "BLAST," basic local alignment search tool utility (https://blast.ncbi.nlm. nih.gov/Blast.cgi); Vector NTI 9.1.0 program, bioinformatics analysis methodology is similar to our earlier papers [26,27]. Te material for the research was the washings from the nutrient media of colonies of microscopic fungi. For the study, 500 μL of wash was taken, which was centrifuged at 5,000 rpm in a mini spin Eppendorf centrifuge; for further isolation of nucleic acids, 200 μL of sediment was taken. Isolation of nucleic acids (NC) was carried out by the method of magnetic sorption with a set of reagents "MAGNO-sorb" option 100-200 ("AmpliSens") according to the manufacturer's instructions. For each PCR amplifcation, the following composition of the reaction mixture was used, per sample: 1.5 μl of 25 mM MgCl 2 solution; 1.5 μl of 2.5 mM dNTP solution; 1.5 μl 10x PCR bufer; 0.5 μl of 10 pM PCR probe solution; 10 pM solution of forward and reverse primers, 0.5 μl; 0.5 μl Taq polymerase; 5 μl DNA and 3.5 deionized water. For PCR, we used reagents manufactured. Te fnal volume of the reaction mixture was 15 μL.
Oligonucleotide primers for amplifcation of this locus have the additional designation "α." RT-PCR was performed using the following primers: forward primer F pro ITS-AGCGGAGGGATCATTACCGAGTTTAC, reverse primer F pro ITS-AAAGTTTTGATTTATTTATGGTTTTACTC AGAAGTTACAT, and probe F pro ITS-Cy5-ATTGT TGCCTCGGCGGATCAGCC-BHQ3; forward primer F pro α-TGCGGTGGTATCGACAAGCGA, reverse primer F pro α-GAGCGGGGTAGCAGGCACGT, and probe F pro α-Rox-CTCTGCCCACCGATTTCACTTGCGATT-BHQ2. All oligonucleotide primers for determining marker loci of Fusarium proliferatum have the designation (except for indicating the type of oligonucleotide primer) "F pro." RT-PCR (real-time PCR) was carried out on a C1000 amplifer with a CFX96 optical unit. Te amplifcation program was as follows: (i) DNA denaturation at 95°C for 2 minutes; (ii) 5 cycles consisting of 10 seconds at 95°C and 30 seconds at 60.5°C; (iii) 40 cycles consisting of 10 seconds at 95°C and 30 seconds at 60.5°C; detection of the PCR result (fuorescence) occurs in each of 40 cycles of the third stage of PCR, at 60.5°C through the Rox channels and Cy5.

Biotesting on Protozoa Paramecium caudatum.
Te express method for determining the toxicity of fungal isolates on protozoa is based on the extraction of toxic substances with the subsequent efect of these extracts on the Paramecium. Te mycelium of micromycetes (5 days old) was poured with distilled water in a ratio of 1 : 1 by volume, and then mixed and incubated at a temperature of 10°C for 24 hours. Two drops of the extract from fungal cultures were applied to a glass slide using a Pasteur pipette, and one drop of the medium with protozoa was added. Te behavior of the Paramecia was observed under a Carl Zeiss Stemi 2,000 C stereoscopic microscope. If the protozoa did not die on the slide within 3-5 minutes, they were placed in a Petri dish on a circle with flter paper moistened with water to prevent the droplets from drying out.
Te criterion for determining the sensitivity of paramecium to toxic substances is the time from the onset of exposure to the test extract to the death of protozoa, which is ascertained on the basis of cessation of movement, deformation, or decay. If there is no death of the Paramecia, then they are monitored for no more than 2 hours. In the case when at least a part of the Paramecia during this time stops moving, the observation continues until the moment of death of all specimens. Many toxic metabolites cause the death of protozoa in the period from 1 to 20-30 minutes, low-toxic ones-up to 1-2 hours. Te survival rate of Paramecia was determined by the following formula: where N is the arithmetic mean (of fve tests) value of the amount of paramecium at the end of the experiment after 1 hour of exposure, pcs; N1 is the arithmetic mean (of fve tests) value of the number of parameters at the beginning of the experiment, pcs; 100 is the translation of the result into percent.
An isolate is considered toxic if 0 to 39% of protozoa die; slightly toxic from 4069%, nontoxic from 70100%.

Biotesting on Rabbits.
When setting a skin test on rabbits, the mycelial flm of the fungus grown on a nutrient medium was rubbed to a mushy state and applied with a glass rod to a clipped 6 × 6 cm area in the thigh area (the hair was cut of until completely exposed), rubbed lightly. For control, one bare skin area with a similar size 6 × 6 was used, to which the extract was not applied. In order to prevent licking of the extract applied to the skin, a collar was put on the rabbit's neck, which was removed 3 days after the frst application. On the next day after applying the extract, the reaction according to the state of the skin was taken into account: (+) First degree-redness of the skin, increased sensitivity-redness, soreness, and peeling; (++) Second degree-redness, soreness, peeling, thickening of the skin, and small rash in the form of bubbles of darkish coloration; (+++) Tird degree-redness, pronounced thickening of the skin, soreness and folding, superfcial necrosis, and scab formation; (++++) Fourth degree-severe swelling in the form of a roller on the lower border of the site, dry necrosis, and/or a continuous thick scab. Rabbits were not euthanized.

Biotesting on White Mice.
When setting up a bioassay on white mice, in order to determine pathogenicity, 2 groups of animals were formed (n � 10). Te control group was injected with physiological saline intraperitoneally in a volume of 0.5 ml. Te experimental animals were injected with 0.5 ml of a micellar suspension of a 5-day culture of the isolate with a concentration of 0.5 and 1 billion spores. Te animals were observed for 14 days. Te dead animals were dissected immediately; control and surviving mice were dissected at the end of the experiment using a humane method. Te surviving mice were euthanized by dislocation of the cervical vertebrae after light anesthesia with ether for anesthesia (inhalation). At autopsy, inoculations were made-prints from internal organs on Czapek agar to isolate micromycetes.

Statistical Analysis.
Statistical processing was performed using the STATISTICA 10 software (StatSoft, USA). Data are presented as the mean values ± standard deviation.

Results and Discussion
In the spring of 2019, on one of the private farms of the Russian Federation, the death of laying hens at the age of 6 months, ulcerative-necrotic lesions of the combs were reported on the toes with the formation of scab-like crusts, painful on palpation, and signs of pododermatitis. On clinical examination, lameness was noted. Te birds were losing weight. Te described clinical signs were progressive in nature;-they began with small pinpoint rashes and then intensifed. Te disease has been registered in poultry farms for more than 2 years, but less than 3 years. Figure 1 shows the afected combs and legs of birds.
We would call a problem considerable as the poultry farm contains more than 3 million layers at the same time. Clinical signs had been noted at the age of 120 days, though pathoanatomical changes in a liver and in kidneys could be visualized from 70-90 days age. Tus, it is possible to state pathology at 2.78 million layers, which is the prevalence approximately 92.7% of the livestock.
In addition to vivid signs of damage to the legs and scallops, changes were also noted in the internal organs. At autopsy, toxic liver degeneration was reported, the liver was fabby, yellow ("fatty liver"), the condition of the mucous membranes of the glandular and muscular stomachs was satisfactory, but the intestinal mucosa, especially the small intestine, was hyperemic, swollen, and easily detached; the pancreas was without visible changes. A decrease in the size of the spleen was a characteristic of the ureters-uric acid diathesis. Infectious (bacterial and viral) and invasive diseases (ectoparasites-ticks) were previously excluded in the interregional veterinary laboratory.
We conducted mycotoxicological and mycological studies of feed and pathological material. Taking into account the results of preliminary studies and clinical signs of the disease, we have chosen the mycological direction of searching for the cause of the disease.
During biotesting on paramecia and rabbit skin test, the studied food was nontoxic. Mycological analysis of the food revealed fungi of the genus Penicillium sp., Rhizopus sp. (Figure 3).
Te total number of fungi was 3.1 × 10 3 CFU/g of feed. Tis does not exceed the standard established in Russia (5.0 × 10 4 CFU/g of feed) and indicates the absence of mold growth. Te detected isolates of fungi of the genus Penicillium and Rhizopus did not have toxicity for P. caudatum; the survival rate of the test culture was 83.7 ± 2.5%. Te test on rabbit skin also did not reveal that their toxic propertiesskin redness, soreness, or peeling were not reported.
During mycological analysis of pathological material, F. proliferatum was isolated from the afected tissues of birds. It should be noted that other fungi were not found, when inoculated on nutrient media. Te mycelium of the culture was pink; the colonies of the fungus are clearly delineated, 4 Veterinary Medicine International with a uniform growth. Colonies are fast-growing; mycelium is branched on conidiophores. Microconidia are numerous, mostly pear-shaped, elongated, and truncated. Macroconidia are fusiform-sickle-shaped with a more pronounced pedicle at the base in the aerial mycelium, arranged in chains, the links of which are collected in false heads, oval in shape with 0-1 septum. Chlamydospores are intermediate, forming singly, in pairs in clusters and in chains. Te sclerotia had a plexus of hyphae of a horn-like consistency ( Figure 4). Genetic confrmation of the species F. proliferatum was performed by amplifcation of the "internal transcribed spacer" "translation elongation factor 1-alpha" genes ( Figure 5).
Each genetic marker was indicated by an individual fuorescent label, so the Cy5 detection channel was used for the "internal transcribed spacer" gene (shown in the graph as purple lines); for the translation elongation factor 1-alpha gene, the Rox detection channel was used (the graph shows in the form of orange lines). Te isolate of fungi of the Fusarium genus, identifed by PCR as Fusarium proliferatum, had toxicity, the death of protozoa was more than 91 ± 3.8%. Te reaction on the rabbit's skin was positiveredness, soreness, and peeling were observed. When administered intraperitoneally to white mice at a dose of 0.5 and 1 billion spores, it showed pathogenicity with the death of 75 and 90% of the animals within 7 days. Oppression of animals was noted, the coat was disheveled, the mice showed thirst, accompanied by abundant drinking, and refusal to feed. An autopsy was performed after the death of the animals. On visual inspection of the corpses, the coat is disheveled. Te mucous membranes of animals are pale pink with no visible changes. Te animal carcasses are exhausted; the heart and lungs are without visible changes. On examination of the stomach, small foci of hemorrhage are visible. When the stomach is cut, a small amount of fodder and mucus is observed. When examining the colon, foci of hemorrhage are also observed. Basically, the intestines are empty and contain no fodder. Te kidneys are bean-shaped; in the section, the border of the cortical and medullary layers is smoothed. Blood was inoculated on agarized Czapek media and by the method of imprinting a part of the parenchymal organs, spleen, and liver, followed by incubation at a temperature of 25°C for 5 days. An isolate of F. proliferatum was isolated in the organs. Probably, multiple passages of the mycosis pathogen from bird to bird that occurred naturally over a long period of time increased the contagiousness of the isolate.
Te principles of management of this farm are to exclude any components of animal origin ( Table 1). Defciency of animal proteins leads to metabolic disorders, disruption of normal liver function, including the function of synthesis of signifcant immunoglobulins, which creates the prerequisites for the development of immunosuppression, a decrease in the detoxifcation function of toxic and potentially toxic xenobiotics that enter the food. Violation of metabolic processes contributes to the violation of skin keratin, the stratum corneum becomes loose. Tis fungus was not found in feed; probably the causative agent of     disease also suggests a fungal origin of the disease. In confrmation of this, there is the fact that the owners have been practicing the exclusion of animal components from the diet for more than 7 years, but such a clinical picture has not been previously observed in birds. Some mycotoxins, such as trichothecenes, can cause both external damage to the skin and cause negative consequences in internal organs [28] which were observed during autopsy. Te state of immunosuppression of the body was confrmed by a decrease in the size of the spleen. In mycoses, characteristic destructive processes are often seen on the skin [29][30][31], which we observed on the limbs and scallops. Mycotoxins can alter the metabolism of the pathogen, which can change the outcome of an infectious disease [32,33]. Although there is information about the mutual enhancement of mycotoxins upon combined intake of animals [34,35], nevertheless, given the detected concentrations, in this case they cannot be considered as the cause of chicken disease.
By analogy with paper, we carried out a study using direct microscopy, further cultured and identifed the isolate morphologically and by PCR as for F. proliferatum. Direct microscopy revealed chlamydioconidia and hyphae in the same way as in the paper [36]. Te isolate had typical morphological properties. Te identifcation of Fusarium proliferatum is considered successful with positive amylation of both specifc markers. A similar approach for the indication of microscopic fungi (from two loci) is proposed [37,38]. Te clinical signs observed in this case, including emaciation and weight loss, suggest a decrease in immune function as a predisposing factor for infection, and the disturbance of the stratum corneum, in combination with crowded poultry and moisturizing of the wounds, enable the development of F. proliferatum [39].
According to Fusarium spp., an increasingly important cause of infections in people, especially those with weakened immune systems. A type of hyalogyphomycosis, fusarium can occur in both healthy and immunocompromised people. In healthy birds, microscopic fungi usually invade soft tissue by traumatic inoculation [40]. Tese infections take the form of onychomycosis, keratitis, endophthalmitis [41], and mycetoma [42]. In patients with neutropenia caused by cytotoxic chemotherapy, Fusarium spp. can cause systemic Fusarium. Skin infections associated with Fusarium spp. are rare in healthy people. As a rule, skin infections of Fusarium develop in immunocompromised patients, usually during a systemic infection with a virulent strain of Fusarium [43]. Moreover, the infection can progress both locally, causing extensive tissue destruction, and spread hematogenously. Our literary search showed only a few cases of onychomycosis in healthy people [44,45]. In these cases, F. proliferatum infection may have occured through contact with colonized plants or soil. We think that infections caused by Fusarium spp. are becoming an increasingly important cause of infections, especially not only in immunocompromised people but also in animals and poultry.
Violation of the norms for the stocking density of chickens in the workshops and the violation of air exchange, due to insufcient ventilation, led to injuries and the ingestion of spores of mold fungi of the Fusarium genus on the afected skin in the area of the legs. Te favorable temperature and humidity conditions for the feld isolates contributed to the rapid spread of the disease. Since almost all types of fungi are opportunistic pathogens, an important condition for the onset of the disease was a decrease in the body's resistance caused by feeding an unbalanced diet and violation of the conditions for keeping poultry.

Conclusion
Tus, given the danger of microscopic fungi of the Fusarium genus, capable of infecting organs and tissues, causing pathological processes with weakened immunity, it is necessary to observe zootechnical requirements, strictly take into account the norms of keeping birds, take preventive measures in a timely manner, control humidity and air exchange in workshops, and timely carry out mycological analysis of feed, as well as monitor the quality of storage of grain in bunkers in order to avoid mold growth. F. proliferatum infections present a difcult therapeutic challenge, even for an experienced mycologist. We also emphasize the importance of accurate species diagnosis for decision-making on further treatment strategies for such cases. Tere is a lot of information on fungal infections acquired as a result of trauma [44,45]; this report is, to our knowledge, the new case report associated with the occurrence of mycosis caused by F. proliferatum in chickens.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest associated with this publication.