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Katinka Bekő Veterinary Medical Research Institute, Hungária körút 21, H-1143 Budapest, Hungary

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Eszter Zsófia Nagy Veterinary Medical Research Institute, Hungária körút 21, H-1143 Budapest, Hungary

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Dénes Grózner Veterinary Medical Research Institute, Hungária körút 21, H-1143 Budapest, Hungary

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Zsuzsa Kreizinger Veterinary Medical Research Institute, Hungária körút 21, H-1143 Budapest, Hungary

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Miklós Gyuranecz Veterinary Medical Research Institute, Hungária körút 21, H-1143 Budapest, Hungary

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Abstract

Several Mycoplasma species can form biofilm, facilitating their survival in the environment, and shielding them from therapeutic agents. The aim of this study was to examine the biofilm-forming ability and its potential effects on environmental survival and antibiotic resistance in Mycoplasma anserisalpingitidis, the clinically and economically most important waterfowl Mycoplasma species. The biofilm-forming ability of 32 M. anserisalpingitidis strains was examined by crystal violet assay. Biofilms and planktonic cultures of the selected strains were exposed to a temperature of 50 °C (20 and 30 min), to desiccation at room temperature (16 and 24 h), or to various concentrations of eight different antibiotics. Crystal violet staining revealed great diversity in the biofilm-forming ability of the 32 tested M. anserisalpingitidis strains, with positive staining in more than half of them. Biofilms were found to be more resistant to heat and desiccation than planktonic cultures, while no correlation was shown between biofilm formation and antibiotic susceptibility. Our results indicate that M. anserisalpingitidis biofilms may contribute to the persistence of the organisms in the environment, which should be taken into account for proper management. Antibiotic susceptibility was not affected by biofilm formation; however, it is important to note that correlations were examined only in vitro.

Abstract

Several Mycoplasma species can form biofilm, facilitating their survival in the environment, and shielding them from therapeutic agents. The aim of this study was to examine the biofilm-forming ability and its potential effects on environmental survival and antibiotic resistance in Mycoplasma anserisalpingitidis, the clinically and economically most important waterfowl Mycoplasma species. The biofilm-forming ability of 32 M. anserisalpingitidis strains was examined by crystal violet assay. Biofilms and planktonic cultures of the selected strains were exposed to a temperature of 50 °C (20 and 30 min), to desiccation at room temperature (16 and 24 h), or to various concentrations of eight different antibiotics. Crystal violet staining revealed great diversity in the biofilm-forming ability of the 32 tested M. anserisalpingitidis strains, with positive staining in more than half of them. Biofilms were found to be more resistant to heat and desiccation than planktonic cultures, while no correlation was shown between biofilm formation and antibiotic susceptibility. Our results indicate that M. anserisalpingitidis biofilms may contribute to the persistence of the organisms in the environment, which should be taken into account for proper management. Antibiotic susceptibility was not affected by biofilm formation; however, it is important to note that correlations were examined only in vitro.

Introduction

Mycoplasma anserisalpingitidis is the most important waterfowl-pathogenic Mycoplasma species, both clinically and economically (Volokhov et al., 2020). It was first isolated in 1983 from a gander with phallus inflammation in Hungary (Stipkovits et al., 1984; Varga et al., 1986), and since then, it has also been confirmed to be present in other European and Asian countries as well (Stipkovits et al., 1986; Sprygin et al., 2012; Gyuranecz et al., 2020; Grózner et al., 2021). Mycoplasma anserisalpingitidis can cause chronic infections in geese and ducks (Razin and Jacobs, 1992; Grózner et al., 2019). Clinically manifested mycoplasmosis occurs during excessive stress with cloaca and phallus inflammation, salpingitis and testicular atrophy being the most frequent signs (Stipkovits et al., 1986; Hinz et al., 1994; Stipkovits and Kempf, 1996; Dobos-Kovács et al., 2009).

At present, control of the disease comprises the improvement of housing conditions and antibiotic therapy. Unfortunately, rapid development of multi-drug resistance in M. anserisalpingitidis has been described (Grózner et al., 2016; Gyuranecz et al., 2020). Bacterial biofilm formation is likely to contribute to an increase in the incidence of clinically untreatable infections (Reid, 1999; Daubenspeck et al., 2020).

Biofilm formation occurs when bacteria switch from a planktonic state to a surface-attached state (Coffey and Anderson, 2014). Bacterial biofilms consist of cells attached to a substratum or to each other, encased within an extracellular matrix composed of an aggregation of polysaccharides, polypeptides, nucleic acids and lipids (McAuliffe et al., 2006; Colvin et al., 2011; Daubenspeck et al., 2020). The physical and chemical properties of the extracellular matrix constituents coupled with their particular interactions allow the matrix to shield the biofilm cells from adverse environmental conditions (e.g. heat, desiccation, chemicals, invasion by other bacteria). The matrix also provides the mechanical properties to protect the cells from external forces and to ensure that the biofilm community remains attached to a surface (Yan and Bassler, 2019).

Compared with planktonic cells, biofilms are commonly found to be 10 to 1,000 times more resistant to antibiotics (Costerton et al., 1999; Mah and O'Toole, 2001). Besides the impeded antibiotic penetration into biofilms, increased antibiotic tolerance likely arises from the altered physiology of the biofilm cells. Cells dwelling inside thick biofilms could be in stationary phase, as penetration of nutrients and oxygen are known to be limited due to consumption by peripherally-located cells. Time-dependent antibiotic killing of a bacterial population shows that actively growing cells are killed first whereas cells in stationary phase are killed at a much lower rate (Yan and Bassler, 2019).

Several Mycoplasma species are able to form biofilm, including Mycoplasma agalactiae, Mycoplasma bovis (McAuliffe et al., 2006), Mycoplasma gallisepticum (Chen et al., 2012) and Mycoplasma genitalium (Daubenspeck et al., 2020). An explanation for the chronicity associated with mycoplasmal diseases is that the bacteria are thought to persist in the host by the formation of an adherent biofilm, shielding the organism from host immune components such as antibodies or phagocytes, and therapeutic agents as well (Hall-Stoodley et al., 2004; McAuliffe et al., 2006; Feng et al., 2020). Furthermore, McAuliffe et al. (2006) provided evidence that biofilm formation facilitates the survival of Mycoplasma species in the environment as well. In this study, the biofilm-forming ability of M. anserisalpingitidis was examined by the crystal violet assay (McAuliffe et al., 2006). Additionally, the effect of biofilm formation on the resistance of M. anserisalpingitidis strains against heat, desiccation and eight different antibiotic agents was also investigated.

Materials and methods

Growth conditions and analysis of biofilm-forming ability of M. anserisalpingitidis

In total, 32 M. anserisalpingitidis strains were used in this study, including the ATCC BAA-2147 type strain, two Polish and 29 Hungarian field isolates (Table 1). All strains were stored at −70 °C until use. The medium used for the propagation of the organism consisted of Mycoplasma broth medium (pH 7.8) (Thermo Fisher Scientific Inc./Oxoid Inc., Waltham, MA, USA) supplemented with 0.5% (w/v) sodium pyruvate, 0.5% (w/v) glucose, 0.005% (w/v) phenol red and 0.15% (w/v) L-arginine hydrochloride. The number of microorganisms in the cultures used for the tests was determined by broth microdilution method (Hannan, 2000) and standardised (106 colour changing units, CCU mL−1) in order to obtain comparable results. The bacterial cultures were inoculated at 1:10 ratio in the broth medium in duplicates (cultures B and P), and were incubated at 37 °C for 48 h. Cultures B were left intact to grow biofilm, while cultures P were disrupted by vortexing, suspension and scratching the wall and bottom of the tubes three times daily, in order to prevent biofilm growth and remain in the form of planktonic cells in the broth. Analysis of biofilm growth was performed by crystal violet staining as described previously with minor modifications (McAuliffe et al., 2006). Tubes were emptied by discarding the broth cultures carefully, washed twice in tap water to remove non-adherent cells and stained with 0.5% crystal violet solution for 30 min. Washing steps were repeated before being left to dry at room temperature.

Table 1.

Background information and biofilm-forming ability of the 32 tested Mycoplasma anserisalpingitidis strains

ID Host Sample Origin Year of isolation Biofilm formationa
ATCC BAA-2147 goose phallus lymph Hungary 1983
MYCAV47 duck lung and air sacs Tázlár, Hungary 2012 ++
MYCAV50 goose phallus Cered, Hungary 2013 ++
MYCAV55 goose ovarian follicle Kiskunmajsa, Hungary 2013
MYCAV61 goose phallus lymph Tatárszentgyörgy, Hungary 2013
MYCAV63 goose trachea Sükösd, Hungary 2013
MYCAV66 goose phallus lymph Tiszaföldvár, Hungary 2014
MYCAV67 goose phallus lymph Szentes, Hungary 2014 ++
MYCAV68 goose phallus lymph Érpatak, Hungary 2014
MYCAV70 goose phallus lymph Cered, Hungary 2014 ++
MYCAV75 goose phallus lymph Dömsöd, Hungary 2014
MYCAV76 goose phallus lymph Tiszabábolna, Hungary 2014 ++
MYCAV91 goose phallus Hajdúsámson, Hungary 2011 ++
MYCAV94 goose cloaca Tiszabábolna, Hungary 2012
MYCAV160 goose phallus lymph Érpatak, Hungary 2015 ++
MYCAV161 goose phallus lymph Szilaspogony, Hungary 2015 ++
MYCAV162 goose phallus lymph Encsencs, Hungary 2015 ++
MYCAV176 goose phallus Cered, Hungary 2015
MYCAV177 goose phallus Cered, Hungary 2015 +
MYCAV178 goose ovarian follicle Cered, Hungary 2015 +
MYCAV179 goose trachea Apátfalva, Hungary 2015
MYCAV180 goose phallus Kisbér, Hungary 2015
MYCAV270 goose cloaca Szentes, Hungary 2016 ++
MYCAV271 goose phallus lymph Szentes, Hungary 2016 +
MYCAV415 goose phallus lymph Hungary 2017 ++
MYCAV494 goose phallus lymph Hajdúsámson, Hungary 2018 +
MYCAV668 goose cloaca Starogard Gdański, Poland 1985 ++
MYCAV671 goose semen Gödöllő, Hungary 2019 +
MYCAV675 goose cloaca Zielona Góra, Poland 1986 +
MYCAV688 goose cloaca Rém, Hungary 2019
MYCAV929 goose cloaca Cered, Hungary 2020 +
MYCAV967 goose cloaca Érpatak, Hungary 2021

a no staining (−), slight staining (+), or strong staining (++) in the crystal violet assay.

Investigation of the effect of biofilm formation on heat resistance

To investigate the effect of biofilm formation on environmental survival, three Hungarian M. anserisalpingitidis strains with pronounced biofilm-forming ability (high-level staining in the crystal violet assay) were selected randomly (MYCAV70, MYCAV270, MYCAV415). As control, a clinical isolate exhibiting no biofilm formation was used (MYCAV55). After 48-h incubation at 37 °C, M. anserisalpingitidis biofilms and planktonic cell cultures were exposed to a temperature of 50 °C for 20 min or 30 min, then incubated at 37 °C for 1 h. After destroying the structure of biofilm by vortexing, suspension and scratching the wall and bottom of the tubes, the broth cultures were mixed well, divided into 500-µl aliquots, and stored at −70 °C until the counting of viable bacteria was performed. The number of colour changing units of the cultures was determined at 37 °C on 96-well microtitre plates with broth microdilution method by titrating a 10-fold dilution series of the Mycoplasma suspension. The plates were checked daily and the final results were read after 14 days. The highest dilution that showed a colour change was considered to contain 10° CCU mL−1 (Hannan, 2000).

Investigation of the effect of biofilm formation on resistance to desiccation

After 48-h incubation at 37 °C, biofilms and planktonic cell cultures of the selected four M. anserisalpingitidis strains (MYCAV70, MYCAV270, MYCAV415, and MYCAV55 as control) were harvested by centrifugation at 9,000×g for 9 min and the supernatant was removed. The pellet was exposed to desiccation at room temperature for 16 h or 24 h, then suspended in fresh broth and incubated at 37 °C for 1 h. After destroying the structure of biofilm by vortexing, suspension and scratching the wall and bottom of the tubes, the broth cultures were mixed well, divided into 500-µl aliquots, and stored at −70 °C until the counting of viable bacteria was performed as described above.

Investigation of the effect of biofilm formation on antibiotic susceptibility

At first, possible correlations between biofilm-forming ability and the initial or final minimal inhibitory concentration (MICi or MICf) values were analysed. MIC values against the 32 examined M. anserisalpingitidis strains were previously determined (Grózner et al., 2016, 2022). The MICi of each strain was defined as the lowest concentration of the antibiotic that completely inhibited the growth at the time of colour change in the growth control (broth culture without antibiotics), while MICf was evaluated at the end of the incubation period (14 days). Additionally, the effect of preventing biofilm formation on antibiotic susceptibility in five selected M. anserisalpingitidis strains with biofilm-forming ability and high MIC values of certain antibiotics was investigated (MYCAV47, MYCAV67, MYCAV70, MYCAV178, MYCAV271). After 48-h incubation at 37 °C, M. anserisalpingitidis biofilms and planktonic cell cultures were supplemented with fresh broth in order to normalise pH and nutrient content. The cultures were then exposed to various concentrations of the following antibiotics: enrofloxacin (Batch No.: BCBZ6597), oxytetracycline (Batch No.: BCBR8034V), doxycycline (Batch No.: BCBS3626V), tiamulin (Batch No.: BCBW6530), lincomycin (Batch No.: BCBW4661), tylosin (Batch No.: BCBX0715), tilmicosin (Batch No.: BCBT8086), and tylvalosin (Batch No.: TVN1906054). All products originated from VETRANAL (Sigma-Aldrich Inc., St. Louis, USA) except for tylvalosin (Aivlosin), which was purchased from ECO Animal Health Ltd. (London, UK). The tested concentrations were selected based on the results of our previous studies: 0.078–10 μg mL−1 for enrofloxacin, doxycycline and tiamulin, and 0.125–64 μg mL−1 for the other antibiotics (Grózner et al., 2016, 2022). Initial MIC values of each strain were determined by broth microdilution method (Hannan, 2000) with a small modification (using Eppendorf tubes instead of microtitre plates). Cultures were incubated at 37 °C and checked three times daily until acidic colour change or 14 days.

Results

Biofilm-forming ability of M. anserisalpingitidis

Crystal violet staining revealed great diversity in the ability of the 32 tested M. anserisalpingitidis strains to form biofilm (Table 1). Three categories were distinguished based on the observed strength of staining. Some strains (n = 12) showed strong staining below the air/liquid interface. Other strains (n = 7) showed only slight staining regarded as weaker biofilm formation, while numerous strains (n = 13) including the ATCC BAA-2147 type strain exhibited no staining (i.e. no biofilm formation) at all (Fig. 1).

Fig. 1.
Fig. 1.

Biofilm-forming ability of the 32 tested M. anserisalpingitidis strains (in triplicate) by crystal violet staining. Abbreviations: numbers: ‘MYCAV’ strain ID numbers; ref: type strain BAA-2147; neg: negative control (no bacteria)

Citation: Acta Veterinaria Hungarica 70, 3; 10.1556/004.2022.00029

Effect of biofilm formation on heat resistance

Biofilm-forming M. anserisalpingitidis cultures (cultures B) of the strains MYCAV70, MYCAV270 and MYCAV415 were found to be more resistant to heat (50 °C) than their cultures consisting of planktonic cells only (cultures P) (Table 2). At least ten times more viable cells of each strain were detected in cultures B compared to cultures P, except for MYCAV270 in which no difference was observed between cultures B and P after 20 min of heat exposure. In case of the strain MYCAV55, which was found to be unable to form biofilm, no difference could be observed between cultures B and P in the 20-min heat assay, while culture P contained more viable cells in the 30th minute of heating.

Table 2.

Viable count (CCU mL−1) of four tested Mycoplasma anserisalpingitidis strains after exposure to a temperature of 50 °C for 20 or 30 min, or to desiccation for 16 or 24 h

Strain ID Culture type Heat 20 min Heat 30 min Dryness 16 h Dryness 24 h
MYCAV70 culture B 103 102 103 102
culture P 102 101 101 101
MYCAV270 culture B 102 102 102 102
culture P 102 100 101 100
MYCAV415 culture B 103 102 102 101
culture P 101 100 0 0
MYCAV55 culture B 102 100 101 100
culture P 102 101 101 100

Culture B: biofilm-forming culture; culture P: planktonic cell form culture.

Effect of biofilm formation on resistance to desiccation

In agreement with the results of the heat assay, cultures B of the biofilm-forming MYCAV70, MYCAV270 and MYCAV415 strains were more resistant to drying than their cultures P (Table 2). At least a tenfold difference was detected in the number of viable cells between cultures B and P of each strain. In case of MYCAV415, no surviving planktonic cells could be detected after 16-h drying, whereas biofilm cells were still viable even after 24 h. No difference could be observed between the survival rates of cultures B and P of the MYCAV55 strain that was unable to form biofilm.

Effect of biofilm formation on antibiotic susceptibility

No correlation was observed between biofilm-forming ability and the MIC values determined previously (Grózner et al., 2016, 2022) in the 32 examined M. anserisalpingitidis strains, as high MIC values of each antibiotic were determined against strains lacking this ability as well (Table 3). Accordingly, the antibiotic susceptibility profiles of B and P cultures of the five tested M. anserisalpingitidis strains were highly similar (Table 4), and these strains were mostly inhibited by a concentration close to the previously determined MIC value (no more than one dilution step difference). A fourfold initial MIC difference was observed in three cases: enrofloxacin with MYCAV47 P and oxytetracycline with MYCAV70 B and MYCAV178 P. However, no greater than one dilution step difference was observed between cultures B and P of each strain, and this difference could not be consistently attributed to either biofilms or planktonic cultures.

Table 3.

Previously determined (Grózner et al., 2016, 2022) initial (MICi) and final (MICf) minimal inhibitory concentration values (µg mL−1) of the 32 tested Mycoplasma anserisalpingitidis strains

Strain ID Biofilma enrofloxacin oxytetracycline doxycycline tiamulin lincomycin tylosin tilmicosin tylvalosin
MICi MICf MICi MICf MICi MICf MICi MICf MICi MICf MICi MICf MICi MICf MICi MICf
MYCAV47 ++ 10 >10 n.a. >64 0.312 5 0.625 2.5 >64 >64 4 16 64 >64 n.a. 1
MYCAV50 ++ n.a. >10 n.a. >64 1.25 5 0.078 0.625 n.a. 4 0.5 2 0.5 2 0.25 0.25
MYCAV67 ++ n.a. 5 >64 >64 0.312 5 0.625 2.5 >64 >64 >64 >64 >64 >64 8 16
MYCAV70 ++ n.a. >10 32 >64 n.a. >10 n.a. 0.625 2 4 4 16 64 >64 0.25 1
MYCAV76 ++ 2.5 5 16 64 1.25 5 0.312 1.25 8 8 1 8 16 >64 0.25 0.5
MYCAV91 ++ n.a. 10 n.a. 64 0.625 2.5 n.a. 0.625 n.a. 8 0.25 0.25 0.25 0.25 0.25 0.25
MYCAV160 ++ 5 >10 64 >64 0.625 10 n.a. 0.625 4 4 n.a. >64 n.a. >64 1 2
MYCAV161 ++ 5 >10 64 >64 0.312 >10 0.312 0.625 4 4 4 16 >64 >64 n.a. 0.5
MYCAV162 ++ 1.25 2.5 32 >64 0.039 5 n.a. 0.625 2 4 8 16 64 >64 n.a. 0.5
MYCAV270 ++ n.a. n.a. n.a. n.a. 1.25 10 0.312 0.625 0.5 4 8 64 >64 >64 0.5 2
MYCAV415 ++ n.a. n.a. n.a. n.a. 1.25 10 0.625 2.5 n.a. n.a. n.a. n.a. >64 >64 n.a. n.a.
MYCAV668 ++ 0.312 0.625 0.25 2 0.078 0.312 0.156 0.312 0.25 1 0.25 0.25 0.25 0.25 0.25 0.25
MYCAV177 + 2.5 >10 >64 >64 n.a. 10 n.a. 0.625 2 4 n.a. >64 n.a. >64 n.a. 4
MYCAV178 + 5 5 >64 >64 1.25 5 n.a. 0.312 n.a. 2 0.25 4 0.25 >64 n.a. 0.5
MYCAV271 + n.a. n.a. n.a. n.a. 1.25 10 0.625 n.a. 1 4 n.a. 64 >64 >64 0.5 8
MYCAV494 + 5 10 n.a. n.a. 0.078 0.312 0.312 1.25 0.25 1 2 4 >64 >64 0.25 0.25
MYCAV671 + n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.
MYCAV675 + 0.156 1.25 0.25 1 0.039 0.312 0.312 1.25 0.5 4 8 64 >64 >64 0.5 2
MYCAV929 + n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.
MYCAV55 2.5 10 1 8 0.078 0.312 0.156 0.625 1 4 0.25 0.25 0.25 0.25 0.25 0.25
MYCAV61 5 5 0.5 2 0.039 0.078 0.312 0.132 2 2 0.25 0.25 0.25 0.25 0.25 0.25
MYCAV63 0.625 1.25 0.5 4 0.039 0.312 n.a. 0.156 0.5 2 n.a. 4 n.a. 64 0.25 0.25
MYCAV66 1.25 5 >64 >64 >10 >10 0.156 0.625 1 4 0.25 0.25 0.25 0.25 0.25 0.25
MYCAV68 2.5 5 64 >64 0.625 10 n.a. 5 >64 >64 n.a. >64 n.a. >64 8 16
MYCAV75 n.a. 5 32 >64 n.a. 10 0.156 0.625 2 4 0.25 0.25 0.25 0.25 0.25 0.25
MYCAV94 2.5 2.5 >64 >64 5 >10 n.a. 0.625 2 4 0.25 0.25 0.25 0.25 0.25 0.25
MYCAV176 n.a. 10 >64 >64 n.a. 5 n.a. 0.625 2 4 n.a. 64 n.a. >64 n.a. 4
MYCAV179 n.a. 10 1 4 0.156 0.312 0.312 1.25 4 4 n.a. 4 1 4 0.25 0.5
MYCAV180 5 5 n.a. 4 0.039 0.312 0.625 1.25 n.a. 4 n.a. 32 64 >64 0.25 1
MYCAV688 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.
MYCAV967 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.
BAA-2147 0.312 1.25 16 >64 5 >10 0.312 1.25 1 2 0.25 0.5 0.25 0.25 0.25 0.25

a no staining (−), slight staining (+), or strong staining (++) in the crystal violet assay; n.a.: data not available.

Table 4.

Initial minimal inhibitory concentration values (MICi, µg mL−1) of different antibiotics against biofilm and planktonic cell cultures of five Mycoplasma anserisalpingitidis strains

Strain ID Culture type enro oxy doxy tiam linco tylo tilm tylv
MYCAV47 culture B 5 64 0.312 0.625 >64 2 64 0.25
culture P 2.5 64 0.312 0.625 >64 4 64 0.25
MYCAV67 culture B 5 >64 0.312 0.312 >64 >64 >64 8
culture P 5 >64 0.312 0.625 >64 >64 >64 8
MYCAV70 culture B 10 8 0.312 0.312 2 4 64 0.25
culture P 10 16 0.312 0.312 2 8 64 0.25
MYCAV178 culture B 5 64 1.25 0.156 1 0.25 0.25 0.25
culture P 5 32 1.25 0.156 1 0.25 0.25 0.5
MYCAV271 culture B 5 64 1.25 0.625 1 32 >64 0.5
culture P 5 64 1.25 0.312 1 32 >64 0.5

enro: enrofloxacin; oxy: oxytetracycline; doxy: doxycycline; tiam: tiamulin; linco: lincomycin; tylo: tylosin; tilm: tilmicosin; tylv: tylvalosin; culture B: biofilm-forming culture; culture P: planktonic cell form culture.

Discussion

Crystal violet staining revealed great diversity in the biofilm-forming ability of the 32 tested M. anserisalpingitidis strains. Exploring the reason for the observed diversity within this species requires further investigations, with special attention to the genetic background. Mycoplasmas lack almost all genes commonly associated with biofilm formation in other bacterial species (McAuliffe et al., 2006). Nevertheless, in addition to participating in various attachment and binding processes, variable surface proteins have been found to be involved in biofilm formation in M. bovis (Thomas et al., 2003; Sachse et al., 1996, 2000). Likewise, polysaccharide capsule production may also influence biofilm formation and adhesion, and this phenotype may even differ among closely related mycoplasmas, such as Mycoplasma mycoides subsp. mycoides SC and LC (McAuliffe et al., 2006).

Biofilm-forming M. anserisalpingitidis cultures were found to be more resistant to heat at 50 °C than their cultures consisting of planktonic cells only. In line with the results of the heat assay, cultures B of the examined M. anserisalpingitidis strains were more resistant to desiccation than their cultures P. Mycoplasmas are usually considered to be of low resilience due to their reduced metabolic pathways and lack of cell wall. However, in agreement with the findings of McAuliffe et al. (2006) in ruminant-pathogenic Mycoplasma species, biofilm cultures of the tested M. anserisalpingitidis strains were found to be quite resistant to heat and desiccation. These results indicate that biofilm formation of M. anserisalpingitidis may contribute to the persistence of the organisms in the environment. This phenomenon is of great importance in the control of M. anserisalpingitidis infection, as increased environmental survival of their biofilms should be taken into account during the housing management and disinfection procedures.

Increased resistance of biofilms to antibiotics has been described previously in many studies (Costerton et al., 1999; Reid, 1999; Mah and O'Toole, 2001; Stewart, 2002; Sharma et al., 2019; Yan and Bassler, 2019; Daubenspeck et al., 2020). However, no correlation was shown in this study between biofilm-forming ability and the minimal inhibitory concentration values determined previously (Grózner et al., 2016, 2022) in the 32 examined M. anserisalpingitidis strains. In case of other bacteria, the nutrient constitution of the growth media was described to affect inhibitory antibiotic concentrations of sessile bacteria (Chen et al., 2020). This phenomenon may have contributed to the observed lack of correlation between biofilm-forming ability and antibiotic susceptibility in M. anserisalpingitidis strains, although propagation and examinations were carried out in the nutrient-rich medium which is also used for isolation from clinical samples. Nevertheless, MIC values are primarily affected by resistance-associated genes and mutations, and other resistance mechanisms, such as extracellular vesicles or efflux pumps may also play a role (Medvedeva et al., 2014; Antunes et al., 2015; Grózner et al., 2022). Antibiotic susceptibility of the five tested biofilm-forming M. anserisalpingitidis strains was not clearly affected by the experimental prevention of biofilm formation, in the same way as in the study of McAuliffe et al. (2006) with ruminant-pathogenic Mycoplasma species. However, it is important to note that these studies examined the effect of biofilm formation on the susceptibility to antibiotics under in vitro conditions, and the effect of the constitution of growth media was not evaluated.

At present, the control of mycoplasmosis in waterfowl consists of appropriate animal hygiene measures and antibiotic treatment, while elevated MIC values have been detected against this pathogen in many antimicrobials. Therefore, discovering the factors responsible for increased resistance to these actions is essential. This is the first study demonstrating the biofilm-forming ability of M. anserisalpingitidis and confirming the protective effect of this characteristic on environmental survival of the pathogen.

Acknowledgements

The Polish strains were kindly provided by Anna Sawicka. This work was supported by the KKP19 (129751) grant of the National Research, Development and Innovation Office, Hungary, the SA-27/2021 grant of the Eötvös Loránd Research Network, the Project no. RRF-2.3.1-21-2022-00001 which has been implemented with the support provided by the Recovery and Resilience Facility (RRF), financed under the National Recovery Fund budget estimate, RRF-2.3.1-21 funding scheme and the support provided by the Ministry of Innovation and Technology of Hungary (legal successor: Ministry of Culture and Innovation of Hungary) from the National Research, Development and Innovation Fund, financed under the TKP2021-EGA-01 funding scheme of the National Research, Development and Innovation Office.

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    • Search Google Scholar
    • Export Citation
  • Chen, X. , Thomsen, T. R. , Winkler, H. and Xu, Y. (2020): Influence of biofilm growth age, media, antibiotic concentration and exposure time on Staphylococcus aureus and Pseudomonas aeruginosa biofilm removal in vitro. BMC Microbiol. 20 ,111.

    • Search Google Scholar
    • Export Citation
  • Coffey, B. M. and Anderson, G. G. (2014): Biofilm formation in the 96-well microtiter plate. Methods Mol. Biol. 1149 ,631641.

  • Colvin, K. M. , Gordon, V. D. , Murakami, K. , Borlee, B. R. , Wozniak, D. J. , Wong, G. C. and Parsek, M. R. (2011): The pel polysaccharide can serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa. PLoS Pathog. 7 ,1001264.

    • Search Google Scholar
    • Export Citation
  • Costerton, J. , Stewart, W. P. S. and Greenberg, E. P. (1999): Bacterial biofilms: a common cause of persistent infections. Science 284 ,13181322.

  • Daubenspeck, J. M. , Totten, A. H. , Needham, J. , Feng, M. , Balish, M. F. , Atkinson, T. P. and Dybvig, K. (2020): Mycoplasma genitalium biofilms contain poly-GlcNAc and contribute to antibiotic resistance. Front Microbiol. 11 ,585524.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Dobos-Kovács, M. , Varga, Z. , Czifra, G. and Stipkovits, L. (2009): Salpingitis in geese associated with Mycoplasma sp. strain 1220. Avian Pathol. 38 ,239243.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Feng, M. , Schaff, A. C. and Balish, M. F. (2020): Mycoplasma pneumoniae biofilms grown in vitro: traits associated with persistence and cytotoxicity. Microbiology 166 ,629640.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grózner, D. , Bekő, K. , Kovács, Á. B. , Mitter, A. , Hrivnák, V. , Sawicka, A. , Tomczyk, G. , Bányai, K. , Jánosi, S. , Kreizinger, Z. and Gyuranecz, M. (2022): Identification and detection of mutations potentially associated with decreased susceptibility to macrolides and lincomycin in Mycoplasma anserisalpingitidis isolates. Vet. Microbiol. 266 ,109362.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grózner, D. , Kovács, Á. B. , Wehmann, E. , Kreizinger, Z. , Bekő, K. , Mitter, A. , Sawicka, A. , Jánosi, S. , Tomczyk, G. , Morrow, C. J. , Bányai, K. and Gyuranecz, M. (2021): Multilocus sequence typing of the goose pathogen Mycoplasma anserisalpingitidis. Vet. Microbiol. 254 ,108972.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grózner, D. , Kreizinger, Z. , Sulyok, K. M. , Rónai, Z. , Hrivnák, V. , Turcsányi, I. , Jánosi, S. and Gyuranecz, M. (2016): Antibiotic susceptibility profiles of Mycoplasma sp. 1220 strains isolated from geese in Hungary. BMC Vet. Res. 12 ,170.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grózner, D. , Sulyok, K. M. , Kreizinger, Z. , Rónai, Z. , Jánosi, S. , Turcsányi, I. , Károlyi, H. F. , Kovács, Á. B. , Kiss, M. J. , Volokhov, D. and Gyuranecz, M. (2019): Detection of Mycoplasma anatis, M. anseris, M. cloacale and Mycoplasma sp. 1220 in waterfowl using species-specific PCR assays. PLoS One 14 ,e0219071.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gyuranecz, M. , Mitter, A. , Kovács, Á. B. , Grózner, D. , Kreizinger, Z. , Bali, K. , Bányai, K. and Morrow, C. J. (2020): Isolation of Mycoplasma anserisalpingitidis from swan goose (Anser cygnoides) in China. BMC Vet. Res. 16 ,178.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hall-Stoodley, L. , Costerton, J. W. and Stoodley, P. (2004): Bacterial biofilms: from the natural environment to infectious diseases. Nat. Rev. Microbiol. 2 ,95108.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hannan, P. C. (2000): Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary Mycoplasma species. International Research Programme on Comparative Mycoplasmology. Vet. Res. 31 ,373395.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hinz, K. H. , Pfützner, H. and Behr, K. P. (1994): Isolation of mycoplasmas from clinically healthy adult breeding geese in Germany. J. Vet. Med. B 41 ,145147.

    • Search Google Scholar
    • Export Citation
  • Mah, T. F. and O’Toole, G. A. (2001): Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 9 ,3439.

  • McAuliffe, L. , Ellis, L. J. , Miles, K. , Ayling, R. D. and Nicholas, R. A. J. (2006): Biofilm formation by Mycoplasma species and its role in environmental persistence and survival. Microbiology 152 ,913922.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Medvedeva, E. S. , Baranova, N. B. , Mouzykantov, A. A. , Grigorieva, T. Y. , Davydova, M. N. , Trushin, M. V. , Chernova, O. A. and Chernov, V. M. (2014): Adaptation of mycoplasmas to antimicrobial agents: Acholeplasma laidlawii extracellular vesicles mediate the export of ciprofloxacin and a mutant gene related to the antibiotic target. Sci. World J. 2014 ,150615.

    • Search Google Scholar
    • Export Citation
  • Razin, S. and Jacobs, E. (1992): Mycoplasma adhesion. J. Gen. Microbiol. 138 ,407422.

  • Reid, G. (1999): Biofilms in infectious disease and on medical devices. Int. J. Antimicrob. Agents 11 ,223226.

  • Sachse, K. , Grajetzki, C. , Rosengarten, R. , Hanel, I. , Heller, M. and Pfutzner, H. (1996): Mechanisms and factors involved in Mycoplasma bovis adhesion to host cells. Zentralbl. Bakteriol. 284 ,8092.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sachse, K. , Helbig, J. H. , Lysnyansky, I. , Grajetzki, C. , Muller, W. , Jacobs, E. and Yogev, D. (2000): Epitope mapping of immunogenic and adhesive structures in repetitive domains of Mycoplasma bovis variable surface lipoproteins. Infect. Immun. 68 ,680687.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sharma, D. , Misba, L. and Khan, A. U. (2019): Antibiotics versus biofilm: an emerging battleground in microbial communities. Antimicrob. Resist. Infect. Control 8 ,76.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sprygin, A. , Volokhov, D. V. , Irza, V. N. and Drygin, V. (2012): Detection and genetic identification of Mycoplasma sp. 1220 in geese in the Russian Federation and Ukraine. Selskokhoziaistvennaia Biol. 2 ,8795.

    • Search Google Scholar
    • Export Citation
  • Stewart, P. S. (2002): Mechanisms of antibiotic resistance in bacterial biofilms. Int. J. Med. Microbiol. 292 ,107113.

  • Stipkovits, L. and Kempf, I. (1996): Mycoplasmoses in poultry. Rev. Sci. Tech. 15 ,14951525.

  • Stipkovits, L. , Varga, Z. , Czifra, G. and Dobos-Kovacs, M. (1986): Occurrence of mycoplasmas in geese affected with inflammation of the cloaca and phallus. Avian Pathol. 15 ,289299.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Stipkovits, L. , Varga, Z. , Dobos-Kovács, M. and Sántha, M. (1984): Biochemical and serological examination of some Mycoplasma strains of goose origin. Acta Vet. Hung. 32 ,117125.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Thomas, A. , Sachse, K. , Farnir, F. , Dizier, I. , Mainil, J. and Linden, A. (2003): Adherence of Mycoplasma bovis to bovine bronchial epithelial cells. Microb. Pathog. 34 ,141148.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Varga, Z. , Stipkovits, L. , Dobos-Kovács, M. and Sántha, M. (1986): Investigation of goose mycoplasmas. Arch. Exp. Veterinarmed. 40 ,105108.

  • Volokhov, D. V. , Grózner, D. , Gyuranecz, M. , Ferguson-Noel, N. , Gao, Y. , Bradbury, J. M. , Whittaker, P. , Chizhikov, V. E. , Szathmary, S. and Stipkovits, L. (2020): Mycoplasma anserisalpingitidis sp. nov., isolated from European domestic geese (Anser anser domesticus) with reproductive pathology. Int. J. Syst. Evol. Microbiol. 70 ,23692381.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yan, J. and Bassler, B. L. (2019): Surviving as a community: antibiotic tolerance and persistence in bacterial biofilms. Cell Host Microbe 26 ,1521.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Antunes, N. T. , Assuncao, P. , Poveda, J. B. and Tavío, M. M. (2015): Mechanisms involved in quinolone resistance in Mycoplasma mycoides subsp. capri. Vet. J. 204 ,327332.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chen, H. , Yu, S. , Hu, M. , Han, X. , Chen, D. , Qiu, X. and Ding, C. (2012): Identification of biofilm formation by Mycoplasma gallisepticum. Vet. Microbiol. 161 ,96103.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chen, X. , Thomsen, T. R. , Winkler, H. and Xu, Y. (2020): Influence of biofilm growth age, media, antibiotic concentration and exposure time on Staphylococcus aureus and Pseudomonas aeruginosa biofilm removal in vitro. BMC Microbiol. 20 ,111.

    • Search Google Scholar
    • Export Citation
  • Coffey, B. M. and Anderson, G. G. (2014): Biofilm formation in the 96-well microtiter plate. Methods Mol. Biol. 1149 ,631641.

  • Colvin, K. M. , Gordon, V. D. , Murakami, K. , Borlee, B. R. , Wozniak, D. J. , Wong, G. C. and Parsek, M. R. (2011): The pel polysaccharide can serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa. PLoS Pathog. 7 ,1001264.

    • Search Google Scholar
    • Export Citation
  • Costerton, J. , Stewart, W. P. S. and Greenberg, E. P. (1999): Bacterial biofilms: a common cause of persistent infections. Science 284 ,13181322.

  • Daubenspeck, J. M. , Totten, A. H. , Needham, J. , Feng, M. , Balish, M. F. , Atkinson, T. P. and Dybvig, K. (2020): Mycoplasma genitalium biofilms contain poly-GlcNAc and contribute to antibiotic resistance. Front Microbiol. 11 ,585524.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Dobos-Kovács, M. , Varga, Z. , Czifra, G. and Stipkovits, L. (2009): Salpingitis in geese associated with Mycoplasma sp. strain 1220. Avian Pathol. 38 ,239243.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Feng, M. , Schaff, A. C. and Balish, M. F. (2020): Mycoplasma pneumoniae biofilms grown in vitro: traits associated with persistence and cytotoxicity. Microbiology 166 ,629640.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grózner, D. , Bekő, K. , Kovács, Á. B. , Mitter, A. , Hrivnák, V. , Sawicka, A. , Tomczyk, G. , Bányai, K. , Jánosi, S. , Kreizinger, Z. and Gyuranecz, M. (2022): Identification and detection of mutations potentially associated with decreased susceptibility to macrolides and lincomycin in Mycoplasma anserisalpingitidis isolates. Vet. Microbiol. 266 ,109362.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grózner, D. , Kovács, Á. B. , Wehmann, E. , Kreizinger, Z. , Bekő, K. , Mitter, A. , Sawicka, A. , Jánosi, S. , Tomczyk, G. , Morrow, C. J. , Bányai, K. and Gyuranecz, M. (2021): Multilocus sequence typing of the goose pathogen Mycoplasma anserisalpingitidis. Vet. Microbiol. 254 ,108972.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grózner, D. , Kreizinger, Z. , Sulyok, K. M. , Rónai, Z. , Hrivnák, V. , Turcsányi, I. , Jánosi, S. and Gyuranecz, M. (2016): Antibiotic susceptibility profiles of Mycoplasma sp. 1220 strains isolated from geese in Hungary. BMC Vet. Res. 12 ,170.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Grózner, D. , Sulyok, K. M. , Kreizinger, Z. , Rónai, Z. , Jánosi, S. , Turcsányi, I. , Károlyi, H. F. , Kovács, Á. B. , Kiss, M. J. , Volokhov, D. and Gyuranecz, M. (2019): Detection of Mycoplasma anatis, M. anseris, M. cloacale and Mycoplasma sp. 1220 in waterfowl using species-specific PCR assays. PLoS One 14 ,e0219071.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gyuranecz, M. , Mitter, A. , Kovács, Á. B. , Grózner, D. , Kreizinger, Z. , Bali, K. , Bányai, K. and Morrow, C. J. (2020): Isolation of Mycoplasma anserisalpingitidis from swan goose (Anser cygnoides) in China. BMC Vet. Res. 16 ,178.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hall-Stoodley, L. , Costerton, J. W. and Stoodley, P. (2004): Bacterial biofilms: from the natural environment to infectious diseases. Nat. Rev. Microbiol. 2 ,95108.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hannan, P. C. (2000): Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary Mycoplasma species. International Research Programme on Comparative Mycoplasmology. Vet. Res. 31 ,373395.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hinz, K. H. , Pfützner, H. and Behr, K. P. (1994): Isolation of mycoplasmas from clinically healthy adult breeding geese in Germany. J. Vet. Med. B 41 ,145147.

    • Search Google Scholar
    • Export Citation
  • Mah, T. F. and O’Toole, G. A. (2001): Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 9 ,3439.

  • McAuliffe, L. , Ellis, L. J. , Miles, K. , Ayling, R. D. and Nicholas, R. A. J. (2006): Biofilm formation by Mycoplasma species and its role in environmental persistence and survival. Microbiology 152 ,913922.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Medvedeva, E. S. , Baranova, N. B. , Mouzykantov, A. A. , Grigorieva, T. Y. , Davydova, M. N. , Trushin, M. V. , Chernova, O. A. and Chernov, V. M. (2014): Adaptation of mycoplasmas to antimicrobial agents: Acholeplasma laidlawii extracellular vesicles mediate the export of ciprofloxacin and a mutant gene related to the antibiotic target. Sci. World J. 2014 ,150615.

    • Search Google Scholar
    • Export Citation
  • Razin, S. and Jacobs, E. (1992): Mycoplasma adhesion. J. Gen. Microbiol. 138 ,407422.

  • Reid, G. (1999): Biofilms in infectious disease and on medical devices. Int. J. Antimicrob. Agents 11 ,223226.

  • Sachse, K. , Grajetzki, C. , Rosengarten, R. , Hanel, I. , Heller, M. and Pfutzner, H. (1996): Mechanisms and factors involved in Mycoplasma bovis adhesion to host cells. Zentralbl. Bakteriol. 284 ,8092.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sachse, K. , Helbig, J. H. , Lysnyansky, I. , Grajetzki, C. , Muller, W. , Jacobs, E. and Yogev, D. (2000): Epitope mapping of immunogenic and adhesive structures in repetitive domains of Mycoplasma bovis variable surface lipoproteins. Infect. Immun. 68 ,680687.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sharma, D. , Misba, L. and Khan, A. U. (2019): Antibiotics versus biofilm: an emerging battleground in microbial communities. Antimicrob. Resist. Infect. Control 8 ,76.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sprygin, A. , Volokhov, D. V. , Irza, V. N. and Drygin, V. (2012): Detection and genetic identification of Mycoplasma sp. 1220 in geese in the Russian Federation and Ukraine. Selskokhoziaistvennaia Biol. 2 ,8795.

    • Search Google Scholar
    • Export Citation
  • Stewart, P. S. (2002): Mechanisms of antibiotic resistance in bacterial biofilms. Int. J. Med. Microbiol. 292 ,107113.

  • Stipkovits, L. and Kempf, I. (1996): Mycoplasmoses in poultry. Rev. Sci. Tech. 15 ,14951525.

  • Stipkovits, L. , Varga, Z. , Czifra, G. and Dobos-Kovacs, M. (1986): Occurrence of mycoplasmas in geese affected with inflammation of the cloaca and phallus. Avian Pathol. 15 ,289299.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Stipkovits, L. , Varga, Z. , Dobos-Kovács, M. and Sántha, M. (1984): Biochemical and serological examination of some Mycoplasma strains of goose origin. Acta Vet. Hung. 32 ,117125.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Thomas, A. , Sachse, K. , Farnir, F. , Dizier, I. , Mainil, J. and Linden, A. (2003): Adherence of Mycoplasma bovis to bovine bronchial epithelial cells. Microb. Pathog. 34 ,141148.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Varga, Z. , Stipkovits, L. , Dobos-Kovács, M. and Sántha, M. (1986): Investigation of goose mycoplasmas. Arch. Exp. Veterinarmed. 40 ,105108.

  • Volokhov, D. V. , Grózner, D. , Gyuranecz, M. , Ferguson-Noel, N. , Gao, Y. , Bradbury, J. M. , Whittaker, P. , Chizhikov, V. E. , Szathmary, S. and Stipkovits, L. (2020): Mycoplasma anserisalpingitidis sp. nov., isolated from European domestic geese (Anser anser domesticus) with reproductive pathology. Int. J. Syst. Evol. Microbiol. 70 ,23692381.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yan, J. and Bassler, B. L. (2019): Surviving as a community: antibiotic tolerance and persistence in bacterial biofilms. Cell Host Microbe 26 ,1521.

    • PubMed
    • Search Google Scholar
    • Export Citation
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Senior editors

Editor-in-Chief: Ferenc BASKA

Editorial assistant: Szilvia PÁLINKÁS

 

Editorial Board

  • Mária BENKŐ (Acta Veterinaria Hungarica, Budapest, Hungary)
  • Gábor BODÓ (University of Veterinary Medicine, Budapest, Hungary)
  • Béla DÉNES (University of Veterinary Medicine, Budapest Hungary)
  • Edit ESZTERBAUER (Veterinary Medical Research Institute, Budapest, Hungary)
  • Hedvig FÉBEL (National Agricultural Innovation Centre, Herceghalom, Hungary)
  • László FODOR (University of Veterinary Medicine, Budapest, Hungary)
  • János GÁL (University of Veterinary Medicine, Budapest, Hungary)
  • Balázs HARRACH (Veterinary Medical Research Institute, Budapest, Hungary)
  • Peter MASSÁNYI (Slovak University of Agriculture in Nitra, Nitra, Slovak Republic)
  • Béla NAGY (Veterinary Medical Research Institute, Budapest, Hungary)
  • Tibor NÉMETH (University of Veterinary Medicine, Budapest, Hungary)
  • Zsuzsanna NEOGRÁDY (University of Veterinary Medicine, Budapest, Hungary)
  • Dušan PALIĆ (Ludwig Maximilian University, Munich, Germany)
  • Alessandra PELAGALLI (University of Naples Federico II, Naples, Italy)
  • Kurt PFISTER (Ludwig-Maximilians-University of Munich, Munich, Germany)
  • László SOLTI (University of Veterinary Medicine, Budapest, Hungary)
  • József SZABÓ (University of Veterinary Medicine, Budapest, Hungary)
  • Péter VAJDOVICH (University of Veterinary Medicine, Budapest, Hungary)
  • János VARGA (University of Veterinary Medicine, Budapest, Hungary)
  • Štefan VILČEK (University of Veterinary Medicine in Kosice, Kosice, Slovak Republic)
  • Károly VÖRÖS (University of Veterinary Medicine, Budapest, Hungary)
  • Herbert WEISSENBÖCK (University of Veterinary Medicine, Vienna, Austria)
  • Attila ZSARNOVSZKY (Szent István University, Gödöllő, Hungary)

ACTA VETERINARIA HUNGARICA
Institute for Veterinary Medical Research
Centre for Agricultural Research
Hungarian Academy of Sciences
P.O. Box 18, H-1581 Budapest, Hungary
Phone: (36 1) 287 7073 (ed.-in-chief) or (36 1) 467 4081 (editor)

E-mail: acta.veterinaria@univet.hu (ed.-in-chief)

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2022  
Web of Science  
Total Cites
WoS
972
Journal Impact Factor 0.900
Rank by Impact Factor

Veterinary Sciences 95/143

Impact Factor
without
Journal Self Cites
0.900
5 Year
Impact Factor
1.1
Journal Citation Indicator 0.47
Rank by Journal Citation Indicator

Veterinary Sciences 103/170

Scimago  
Scimago
H-index
38
Scimago
Journal Rank
0.277
Scimago Quartile Score

Veterinary (miscellaneous) Q2

Scopus  
Scopus
Cite Score
1.9
Scopus
CIte Score Rank
General Veterinary 76/186 (59th PCTL)
Scopus
SNIP
0.475

2021  
Web of Science  
Total Cites
WoS
1040
Journal Impact Factor 0,959
Rank by Impact Factor Veterinary Sciences 103/144
Impact Factor
without
Journal Self Cites
0,876
5 Year
Impact Factor
1,222
Journal Citation Indicator 0,48
Rank by Journal Citation Indicator Veterinary Sciences 106/168
Scimago  
Scimago
H-index
36
Scimago
Journal Rank
0,313
Scimago Quartile Score Veterinary (miscellaneous) (Q2)
Scopus  
Scopus
Cite Score
1,7
Scopus
CIte Score Rank
General Veterinary 79/183 (Q2)
Scopus
SNIP
0,610

2020  
Total Cites 987
WoS
Journal
Impact Factor
0,955
Rank by Veterinary Sciences 101/146 (Q3)
Impact Factor  
Impact Factor 0,920
without
Journal Self Cites
5 Year 1,164
Impact Factor
Journal  0,57
Citation Indicator  
Rank by Journal  Veterinary Sciences 93/166 (Q3)
Citation Indicator   
Citable 49
Items
Total 49
Articles
Total 0
Reviews
Scimago 33
H-index
Scimago 0,395
Journal Rank
Scimago Veterinary (miscellaneous) Q2
Quartile Score  
Scopus 355/217=1,6
Scite Score  
Scopus General Veterinary 73/183 (Q2)
Scite Score Rank  
Scopus 0,565
SNIP  
Days from  145
submission  
to acceptance  
Days from  150
acceptance  
to publication  
Acceptance 19%
Rate

 

2019  
Total Cites
WoS
798
Impact Factor 0,991
Impact Factor
without
Journal Self Cites
0,897
5 Year
Impact Factor
1,092
Immediacy
Index
0,119
Citable
Items
59
Total
Articles
59
Total
Reviews
0
Cited
Half-Life
9,1
Citing
Half-Life
9,2
Eigenfactor
Score
0,00080
Article Influence
Score
0,253
% Articles
in
Citable Items
100,00
Normalized
Eigenfactor
0,09791
Average
IF
Percentile
42,606
Scimago
H-index
32
Scimago
Journal Rank
0,372
Scopus
Scite Score
335/213=1,6
Scopus
Scite Score Rank
General Veterinary 62/178 (Q2)
Scopus
SNIP
0,634
Acceptance
Rate
18%

 

Acta Veterinaria Hungarica
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Acta Veterinaria Hungarica
Language English
Size A4
Year of
Foundation
1951
Volumes
per Year
1
Issues
per Year
4
Founder Magyar Tudományos Akadémia
Founder's
Address
H-1051 Budapest, Hungary, Széchenyi István tér 9.
Publisher Akadémiai Kiadó
Publisher's
Address
H-1117 Budapest, Hungary 1516 Budapest, PO Box 245.
Responsible
Publisher
Chief Executive Officer, Akadémiai Kiadó
ISSN 0236-6290 (Print)
ISSN 1588-2705 (Online)

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