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First molecular investigation to detect avian Mycoplasma species in clinical samples from laying-hen farms in Tunisia

BMC Veterinary Research volume 20, Article number: 459 (2024) Cite this article

Abstract

Background

Avian mycoplasmas are known pathogens, which cause severe economic losses in poultry flocks. PCR is a rapid, sensitive, and less expensive diagnostic tool than culture for the identification of mycoplasmas in poultry farms. The objective of this study was to determine by PCR the presence of Mycoplasma spp., Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), and Mycoplasma pullorum (MP) in laying hens located in the Sfax region, in the South of Tunisia.

Results

A total of 781 tracheal swabs were collected from 13 laying-hen farms without clinical signs at the date of sampling. MP was detected by a newly described specific PCR assay. The prevalence calculated from PCR results at the flock level was 100% for Mycoplasma spp., 0% for MG, 84.6% for MS and 61.5% for MP. The overall prevalence at the animal level was 38.7% for Mycoplasma spp., 0% for MG, 25% for MS and 6.4% for MP. The overall prevalence of 100% of avian mycoplasmas in laying-hen farms (38.7% prevalence at the animal level) shows an alarming situation.

Conclusions

These results underline the importance of monitoring the emergence and spread of Mycoplasma strains in farms in order to decrease economic losses due to mycoplasmoses.

Peer Review reports

Background

Respiratory diseases are the leading cause of poultry morbidity worldwide. Avian mycoplasmas are highly contagious poultry pathogens that can cause acute and chronic diseases and are a major problem for all bird populations [1].

Avian mycoplasmosis was first described as a poultry respiratory disease in turkeys in 1926 and chickens in 1936 (reviewed by Charlton and collaborators [2]). About 120 different species of Mycoplasma have been identified to infect various organisms, of which only about 20 are adapted to birds [3]. Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), Mycoplasma meleagridis (MM), and Mycoplasma iowae (MI) are of major concern and have the greatest impact on the poultry industry [4, 5].

MG is the most pathogenic species, responsible for Chronic Respiratory Disease (CRD) in chickens and infectious sinusitis in turkeys. CRD is characterized by respiratory rales, coughing, nasal discharge, air sacculitis, swelling of infraorbital sinuses and conjunctivitis in commercial poultry [6]. MS can cause respiratory problems, infectious synovitis, typical eggshell apex abnormalities, and reduced egg production in chickens and turkeys [7]. MI and MM are specific pathogens of turkeys, although isolation from chickens has already been described [4, 8]. These Mycoplasma infections have a significant economic impact on poultry production, resulting in a 10-20% decrease in productivity and in a 5-10% increase in embryo mortality [9].

Mycoplasma pullorum (MP) can be isolated from laying hens [10, 11]. The pathogenic role of MP in chicken and turkey remains unclear despite several studies. Kleckner [12] showed that inoculation of two strains of MP isolated from chicken tracheal exudates into the sinuses or trachea of turkey poults did not induce lesions. Inoculation into the thoracic air sacs and tendon regions of the hock or leg in chicken or turkey did not result in aerosacculitis lesions. However, MP was isolated from turkey embryos from flocks with low hatchability in France [13] and was found to be pathogenic to chicken and turkey embryos [13, 14]. Inoculation of turkey poults with MP in the air sacs and infraorbital sinuses resulted in moderate aerosacculitis lesions [13].

Other Mycoplasma species, including Mycoplasma (M.) iners, M. gallinarum, M. gallinaceum, M. lipofaciens and M. glycophilum are isolated from poultry. These species are not considered to be pathogens of high concern to the poultry industry due to their very low or no pathogenicity risk [15].

Culture is the gold standard method for the detection of Mycoplasma species, but it is difficult due to their fastidious nature and can take 3-4 weeks. Serological testing is much faster and less expensive but has many disadvantages due to nonspecific and cross-reactions [16]. As an alternative to culture and serology, molecular methods are used to specifically detect Mycoplasma species by PCR, which is a widely used procedure and a faster and less expensive diagnostic tool than culture methods [17].

Avian mycoplasmoses are regulated diseases in Europe and the USA [18, 19] causing heavy economic losses in poultry farms. MG and MS are also listed as notifiable diseases for the World Organization for Animal Health (WOAH) [20]. However, despite the continued development of the poultry industry in Tunisia, very little information regarding epidemiological data on the prevalence of avian mycoplasmas in laying-hen flocks is available in Tunisia. Only one old study reported a seroprevalence of 52.4% for MG and 28.6% for MS in layers [21]. Production of laying hens in 2021 in the Sfax region was 3,926,415, representing 59.3% of national production whereas broiler production represented only 7.8%. The breakdown of table egg production by region showed that 62% of this production was located in southern Tunisia, particularly in the Sfax region, with production of 1,863 million units in 2020 (Groupement Interprofessionnel des Produits Avicoles et Cunicoles (GIPAC), [22]), of which 97% were produced intensively, while 7% corresponded to local production (rural farms). Thus, PCR tests to detect Mycoplasma genus [23], MS [24] and MG [25] were performed on samples from laying-hen flocks in this region to obtain data on Mycoplasma prevalence. A PCR assay to detect specifically MP was developed in the present work.

Materials and methods

Field sample collection

This study was conducted from March to April 2022 in the Sfax region, located in the southeast of Tunisia and concentrating the largest part of layer farms.

In total, 781 tracheal swabs from 13 farms (12 commercial and one rural farms) of non-vaccinated laying hens of 18 to 106 weeks of age from different geographical areas of the Sfax region were tested for the presence of Mycoplasma species (Fig. 1; Table 1). Hens had been treated once or twice (except the rural farm) with antibiotics (tylosin, tylvalosin or tiamulin) as a preventative measure, particularly when chicks or pullets have been moved into their new buildings, but these treatments were carried out at least two weeks before the tracheal samples were taken.

Fig. 1
figure 1

Map of the region of Sfax (Tunisia) showing all farm locations and the percentage of Mycoplasma spp.-positive tracheal samples per farm

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Table 1 Information on tracheal swabs collected in commercial and rural layer flocks in the Sfax region (Tunisia)
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Tracheal samples were collected with a cotton swab by veterinarians during official controls of farms and in collaboration with Veterinary Services (Regional Agricultural Development Commission, Sfax, Tunisia). Swabs were placed into 2.0 mL of Frey’s broth medium [26] supplemented with antimicrobials (2.5 μg/mL of Amphotericin B (Sigma-Aldrich, St. Quentin Fallavier, France), 1 mg/mL of Ampicillin (Sigma-Aldrich) and 7.5 µg/mL of Colistin (Sigma-Aldrich)) to obtain initial suspensions. These suspensions were vortexed after a 60-90 min incubation at 37 °C ± 2 °C. From each sample in Frey’s medium, a 500 µL aliquot was taken for DNA extraction and the rest was stored at <-70 °C.

DNA extraction

DNA was extracted from clinical samples by a simple method following the protocol described by Ben Abdelmoumen and collaborators [27] with minor modifications. Briefly, 500 µL of broth sample was centrifuged at 14,000 x g for 30 min. The pellet was suspended in 100 µL of phosphate-buffered saline (PBS) and 100 µL of non-ionic detergent mixture solution (0.45% Nonidet P-40, 0.45% Tween 20, and 100 g/mL proteinase K) and incubated at 60 °C for 1 h, boiled for 15 min, and then centrifuged at 14,000 x g for 15 min. The supernatant was stored at -20 °C until use.

Detection of Mycoplasma spp., MG and MS by PCR

The presence of Mycoplasma spp. DNA in each sample was detected by a 16S rRNA genus-specific PCR as previously described [23] in a final volume of 50 μL. Briefly, the PCR mixture contained 2 mM of MgCl₂ (Biobasic, Markham, Canada), 5 μL of 10X PCR buffer (Biobasic), 400 nM of primers (Bio Basic) (Table 2), 200 μM of each desoxyribonucleotide triphosphate (dNTP) (Biobasic), 1.25 units of Taq polymerase (Biobasic), 10 μL of DNA samples and nuclease-free water (Bio Basic) to reach the final volume of 50 µL (Table S1). PCR amplification was performed using an automated thermal cycler (VWR we Enable science UNO 96, Berkshire, England) as described in Table 3. Standard samples of MS WVU1853 strain (ATCC 25204) and MG S6 (ATCC 15302) were used as positive controls and a DNA-free mixture reaction (with nuclease-free water) was used as negative control under the same conditions.

Table 2 Primer sequences used for the detection and identification of Mycoplasma species
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Table 3 Conditions for PCR assays
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Samples that tested positive for the Mycoplasma genus were then tested using species-specific primers.

The presence of MS and MG in samples was detected by MS-specific [24] and MG-specific [25, 28] PCR assays in a final volume of 50 μL. Briefly, the reaction mixture contained 5 µL of 10X PCR buffer (Roche, Sigma-Aldrich, Saint-Quentin-Fallavier, France), 2 mM of MgCl₂ (Bio-Rad, Marnes-la-Coquette, France), 400 nM of MS-specific primers or 800 nM of MG-specific primers (Sigma-Aldrich) (Table 2), 200 µM of each dNTP (Eurobio, Les Ulis, France), 1.25 U of Roche Taq DNAPolymerase for MS or 1 U of Roche Taq DNA Polymerase (Sigma-Aldrich) and 5 µM of tetramethyl ammoniun chloride (TMAC) (Sigma-Aldrich) for MG, 5 µL of DNA samples and nuclease-free water (Eurobio) to reach the final volume of 50 µL (Table S1). Standard samples of MS WVU1853 strain (ATCC 25204) and MG S6 (ATCC 15302) were used as positive controls and a DNA-free mixture reaction (with nuclease free water) was used as negative control under the same conditions.

PCR amplifications were performed using a T100™ thermal cycler (Bio-Rad) as described in Table 3.

Development of a MP-Specific PCR Assay.

Representative sequences of MP 16S rRNA were selected in GenBank (https://www.ncbi.nlm.nih.gov/genbank) and aligned with 20 different species of avian Mycoplasma using the ClustalW software available on the NPS@ website (https://npsa-pbil.ibcp.fr) to ensure the absence of significant homology with other Mycoplasma-species sequences. Species-specific primers for the detection of MP were designed and submitted to the online BLASTn tool available on the NCBI website (https://blast.ncbi.nlm.nih.gov) for final selection (Table 2). PCR primers were designed to exhibit optimal biophysical properties with no dimer formation with Primer3Plus (https://primer3plus.com). The selected MP-specific primers were subjected to PCR standardization to optimize hybridization, specificity and sensitivity.

PCR assays were performed using dreamTaq Green PCR Master mix (Fisher Scientific, Illkirch, France) according to the manufacturer's instructions. Briefly, a 25 µL reaction mixture contained 12.5 µL of dreamTaq PCR Master Mix, 400 nM of each primer (Sigma-Aldrich) (Table 2), 2 µL of DNA sample to be tested and nuclease-free water (Eurobio) to reach the final volume of 25 µL (Table S1). Nucleic acid extracts for optimization tests were quantified by spectrophotometry (Qubit fluorometer, Invitrogen by Life Technologies, Saint-Aubin, France): 50 ng of DNA and 10 µL of nuclease-free water were added to the mixture. Amplification, using a T100™ thermal cycler (Bio-Rad) was performed as described in Table 3 with hybridization for 40 s using a temperature gradient of 54 to 55 °C.

The specificity of the MP primers was confirmed with strains of MP, other avian Mycoplasma species and other bacteria (Table 4), as well as with positive and negative field samples stored in the ANSES laboratory. To determine the sensitivity of the PCR assay to detect MP, concentrations of 100 ng to 10 fg of DNA were used as template DNA.

Table 4 Mycoplasma and other bacterial used to establish the specificity and sensitivity of the Mycoplasma pullorum-specific PCR
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After optimization, the protocol described in Table 3 was selected, with a hybridization at 54.6 °C.

Detection of Amplified DNA

The PCR products were detected by electrophoresis at 120 V (Mycoplasma spp.), 115 V (MG) or 125 V (MS, MP) on a 2% agarose gel in Tris-Borate-EDTA buffer (TBE) (90 mM Tris, 90 mM borate, 2.5 mM EDTA pH 8.0). Amplified products were detected by ultraviolet transillumination (Quantum ST4 device and VisionCapt software, Vilbert Lourmat, Fisher Scientific, Illkirch-Graffenstaden, France) with ethidium bromide staining (2 mg/L, Fisher Scientific). A GeneRuler 1 kb DNA Ladder (Fisher Scientific) was used as a molecular size standard: the expected sizes of Mycoplasma spp., MS, MG, and MP amplicons were 280 pb, 207 bp, 330 bp and 304 bp, respectively.

Results

Development of the Mycoplasma pullorum-specific PCR test

The primer sequences for the new MP-specific PCR, designed on the 16S rRNA gene and the amplicon size are summarised in Table 2. The best amplification results were obtained with a temperature of 54.6 °C, with a sensitivity up to 10 pg (Figure S1).

After optimizing PCR conditions, primer specificity was tested: no cross amplification was observed when testing the microorganisms listed in Table 3. A clear and well-defined specific band of approximately 304 bp was visualized only from MP strains or MP-positive samples, and not from the other Mycoplasma or bacterial species tested (Figures S2 and S3).

Prevalence of Avian Mycoplasma Species in Layer Farms

For detection of Mycoplasma spp. in the samples from laying hens, the genus-specific PCR method was used. All thirteen sites (12 commercial and one rural farms) visited in the Sfax region were tested positive for Mycoplasma spp. (Table 1, Fig. 1), giving a prevalence of Mycoplasma-infected flocks of 100%. Out of the 781 samples collected, 302 (38.7%) samples were PCR-positive for Mycoplasma spp. Prevalence of positive tracheal swabs varied between farms, from 9.3% to 86.0% (Table 1, Figs. 1 and 2).

Fig. 2
figure 2

Detection of Mycoplasma spp., MS, MP and MG by specific PCR in tracheal samples collected from 13 laying-hen farms (781 hens) in the Sfax region of Tunisia. MS: Mycoplasma (M.) synoviae; MP: M. pullorum ; MG: M. gallisepticum

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Using species-specific PCR assays, 11/13, 8/13 and 0/13 flocks were tested positive for MS, MP and MG, respectively, giving a prevalence of 84.6%, 61.5% and 0%. At the animal level, out of 781 tracheal swabs collected, 195 (25.0%) and 50 (6.4%) samples were positive for MS and MP, respectively and all swabs were negative for MG (Table 1, Fig. 2).

Finally, co-infection with MS and MP was observed in 25 hens (3.2%) belonging to five (38.5%) flocks (Table 1).

Discussion

This study provides for the first time molecular data on the presence of different avian Mycoplasma species in layer farms in the Sfax region (South of Tunisia), which is the most productive poultry district in Tunisia [22].

To our knowledge, it is the first time that such a prevalence study is performed in Tunisia on layer flocks with PCR. Results showed a prevalence at flock level of 100%, 84.6%, 61.5% and 0% for Mycoplasma spp., MS, MP and MG, respectively; and a prevalence at animal level of 38.7%, 25.2%, 6.4% and 0%.

Our results are not in accordance with results of the only Tunisian study reporting a much higher flock prevalence of 52.4% for MG and a lower prevalence of 28.6% for MS in layers [21]. However, this previous study was based on serology and not PCR. Moreover, the Tunisian poultry production has evolved since 1997 and controls and biosecurity measures have been put in place on breeder farms to limit infections by MG, the most pathogenic species of avian mycoplasmas.

There is very high variability in results reported in the literature regarding avian mycoplasma infections in poultry farms worldwide [29]. Most prevalence studies performed in other North African neighbouring countries were carried out on samples taken from flocks showing clinical signs (which leads to an overestimation of the prevalence of pathogenic bacteria) and can hardly be compared to our study performed on flocks without apparent clinical signs. Two recent Egyptian studies calculated an individual prevalence of 40% for MG [30] and 50% for MS [31] in layer farms. A third one determined that 57.6% of the isolates were MG and 30.7% were MS in diseased and apparently healthy layers, but PCR assays were only carried out on isolates after culture and not directly on samples like our study, which may underestimate the percentage of infected hens [32]. An Algerian study, based on serology and isolation of mycoplasmas by culture, showed that 100% of laying hen flocks were MG-positive (serology) and MG was isolated by culture in 2.1% of hens [33]. Sid and colleagues [34] also showed that MG was endemic in Algeria. Finally, a study in Sicily showed an individual prevalence of 12.5% for MG and 23.2% for MS, and a flock-level prevalence of 28 to 40% for MG and 42.8 to 44% for MS [35]. Even if the techniques and methods used differ, the comparison of these studies seems to show that the prevalence of MG in layer flocks in Tunisia (0% at flock and individual levels) is significantly lower than in neighbouring countries. These results are in accordance with those reported in France or in Belgium for MG in layer flocks (0 and 0.2%, respectively; [36, 37]. The absence of MG in the farms visited in the Sfax region, even in hens from the rural farm, might be explained by the constant control measures adopted in breeding farms in Tunisia against this well-known pathogenic Mycoplasma, thus leading to a reduction of vertical transmission and of the presence of MG in the environment (horizontal transmission), and consequently to a reduction in the frequency of MG infections in commercial and rural flocks. It would be interesting to carry out another study including both commercial flocks without apparent clinical signs and others with respiratory clinical signs to determine if MG can be isolated from diseased flocks. This low prevalence might also be due to the preventive and curative antimicrobial treatments applied during the hens’ lifespan. In fact, an antibiotic treatment can reduce the number of live mycoplasmas present in the trachea and reduce the chances of detecting them by PCR. However, other mycoplasmal species were also detected in tracheal swabs despite these treatments.

The MS prevalence of 25.2% at the animal level determined in our study agrees with that reported in Sicily (23.2%) and in Egypt (30.7%) [32, 35] by PCR also. The very high prevalence at flock level (84.6%) is also in accordance with several studies in France (68%), Germany (75.0%) and Spain (95.0%) [36, 38, 39]. However, lower flock prevalences were found in Poland (29%) and Sicily (42.8-44%) [35, 40]. The persistence of this high level of MS contamination on farms may be explained by the recognized high capacity for vertical and horizontal transmission of MS [7]. MS infection in breeding flocks of laying hens can promote vertical transmission of the infectious agent to subsequent chicks [15]. In addition, MS can remain viable for long periods of up to 41 days on fomites (feathers, food or droppings) [41] and is able to infect other birds introduced in this MS-contaminated environment [42]. This can lead to the infection of new flocks introduced in the farm, especially if the cleaning-disinfection and emptiness steps are not well respected and in the case of multiple-age farms. Furthermore, laying hens remain on farms for long periods, at various stages of production. They are therefore more likely to be infected by different pathogens that can negatively affect their immune defences and make them more vulnerable to outbreaks of infection.

MS has been considered less important than MG in poultry for many decades. One of the main characteristics of MS infection is that it is mostly asymptomatic, resulting in a chronic subclinical upper respiratory infection [5], with generally little effect on the performance of layers [36]. However, it could promote infections by other Mycoplasma species, bacteria or viruses, and eventually have a negative effect on the host's immune defences [43, 44]. There is an increased consciousness to generate MS-free poultry [44, 45]. In countries with a well-developed poultry industry, the primary breeding stocks are free of MG and MS and the commercial breeding stocks are free of MG (compulsory slaughter of infected breeding stocks). In addition, studies documenting the high worldwide prevalence of MS (as documented in this study) stress the need for an update on its prevention and control. MS control and eradication programmes have been implemented for a long time in the USA, and the Dutch poultry industry implemented a mandatory control and an eradication programme for MS in 2013 [44]. However, control and eradication programmes are not compulsory in other European and many other countries. The MS-infection status of Tunisian breeding flocks is not known, which suggests that another study should be carried out, this time on breeding flocks instead of commercial ones to have data on the MS prevalence. Given its vertical and horizontal transmission, its faster dissemination than MG [5] and its persistence in the environment [10, 15, 42], the prevalence of this Mycoplasma species is likely to increase, resulting in greater economic losses for the local poultry sector.

PCR testing for Mycoplasma spp. showed a prevalence of 38.7% (302/781) at the animal level, and a flock prevalence of 100%. Few studies reported results about Mycoplasma-genus prevalence: most studies only consider MG, MS or both species. Such a high prevalence was already described in Germany with 100% of Mycoplasma-infected flocks [38]. Differences in prevalence of Mycoplasma spp. observed between farms (Table 1, Fig. 1) might be due to less strict biosecurity and hygiene measures (poorly carried out cleaning/disinfection, duration of emptiness not respected) or contamination of hens via fomites, insects, rodents or staff working on site in the most infected farms. The presence of farms with much lower levels of contamination than others (5.5 versus 86.0% for the most distant values) suggests that it could be possible to improve significantly the situation with respect to mycoplasmosis in layer farms by informing and training all farm personnel about the importance of biosecurity and hygiene measures.

Among the 302 Mycoplasma-positive samples, only 195 samples were MS-positive. This suggested that other Mycoplasma species were probably present, and that there was a need to develop further specific PCRs to detect the presence of other Mycoplasma species. Since MP was found to be frequently associated with MS in laying hens by Cisneros-Tamayo and collaborators [11], we developed in the present study a rapid PCR test with high specificity and sensitivity to investigate the prevalence of MP in laying hens. Assays carried out on the samples with this new PCR revealed a MP prevalence of 6.4% (50/302) at the animal level and of 61.5% at flock level. Among the 50 MP-positive samples, 25 were co-infected with MS. This result is consistent with those of Cisneros-Tamayo and colleagues [11] who detected MP in 56.0% of the samples collected on commercial laying hen MS-positive flocks in France. To the best of our knowledge, no other study reported data on prevalence of MP in layers in Tunisia and other countries. However, other studies reported detection of this species in chickens [10], partridges, pheasants, turkeys [13, 46] and pigeons [10].

Finally, it should be noted that, in most farms (10/13, 76.9%), the percentage of tracheas infected with Mycoplasma spp. is higher than those infected by MS and/or MP. This suggests the presence of other Mycoplasma species that were not identified during this study with the three specific PCR assays. Other species can indeed be isolated from hens, such as M. gallinaceum, M. glycophilum, M. iners or M. gallinarum [4, 47]. Additional studies may be conducted on samples stored during this study to try to isolate and identify these species.

This study was carried out on a limited number of laying-hen flocks and in a single region of Tunisia (the Sfax region bringing together most layer flocks). Furthermore, this study was carried out on farms which did not present apparent clinical signs, which could lead to an underestimation of the prevalence of mycoplasmas, especially for MG which is the most pathogenic species. Other studies could be planned to confirm these figures, to sample farms with apparent clinical signs, and to extend it to other types of flocks (broilers, turkeys, breeding farms) and other regions of Tunisia.

Conclusion

Our results indicate that the Mycoplasma spp.-, MS- and MG-specific PCR assays are accurate tools for the molecular diagnosis of Mycoplasma infections in layer flocks directly from clinical samples. And the MP-specific PCR developed in the present study is reproducible and sensitive enough to detect concentration of 10 fg of MP DNA in clinical samples and may be used in the future for further epidemiological investigations of MP spread in poultry flocks.

The comparison of the prevalence values of the different species of mycoplasmas to that of other countries suggests that MG infections are well controlled in layer farms in the Sfax region of Tunisia, thanks to the control and eradication programmes for this species at the level of breeding flocks, or thanks to antibiotic treatments in layer farms which help keep Mycoplasma infections low. However, the rate of Mycoplasma-positive samples (38.7%) or flocks (100%) showed a worrying situation. Lack of biosecurity measures or hygiene in some farms may be one of the main reasons for the high prevalence of mycoplasmas in the Sfax region. The adoption of better hygiene and biosecurity practices, particularly in farms with high Mycoplasma prevalence, is recommended as a control strategy for mycoplasmosis in Tunisian farms.

Availability of data and materials

The datasets generated during the current study are available from the corresponding author upon reasonable request.

Abbreviations

CRD:

Chronic Respiratory Disease

dNTP:

Desoxyribonucleotide triphosphate

M.:

Mycoplasma

MG:

Mycoplasma gallisepticum

MI:

Mycoplasma iowae

MM:

Mycoplasma meleagridis

MP:

Mycoplasma pullorum

MS:

Mycoplasma synoviae

PBS:

Phosphate-buffured saline

TBE:

Tris-Borate-EDTA buffer

TMAC:

Tetramethyl ammoniun chloride

WOAH:

World Organization for Animal Health

References

  1. Ferguson-Noel N. Mycoplasmosis: Introduction. In: Swayne DE, editor. Diseases of Poultry. 14th ed. Wiley-Blackwell, Hoboken, NJ: USA; 2020. p. 907–11.

    Chapter  Google Scholar 

  2. Charlton BR, Bermudez AJ, Boulianne M, Eckroade RJ, Jeffrey JS, Newman LJ, et al. Avian mycoplasmosis. In: Charlton BR, editor., et al., Avian Disease Manual. American Association of Avian Pathologists, Kennett Square, PA: USA; 1996. p. 115–25.

    Google Scholar 

  3. Fraga AP, de Vargas T, Ikuta N, Fonseca ASK, Celmer ÁJ, Marques EK, et al. A multiplex real-time PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from Brazilian commercial poultry flocks. Brazilian J Microbiol. 2013;44(2):505–10. https://doi.org/10.1590/S1517-83822013000200028.

    Article  CAS  Google Scholar 

  4. Ferguson-Noel N. Mycoplasmosis: Other mycoplasmal infections. In: Swayne DE, editor. Diseases of Poultry. 14th ed. Wiley-Blackwell, Hoboken, NJ: USA; 2020. p. 933–9.

    Google Scholar 

  5. Stipkovits L, Kempf I. Mycoplasmoses in poultry. Rev Sci Tech Off Int Epiz. 1996;15(4):1495–525.

    Article  CAS  Google Scholar 

  6. Armour NK. Mycoplasmosis: Mycoplasma gallisepticum infection. In: Swayne DE, editor. Diseases of Poultry. 14th ed. Wiley-Blackwell, Hoboken, NJ: USA; 2020. p. 911–23.

    Google Scholar 

  7. Ferguson-Noel N, Noormohammadi AH. Mycoplasmosis: Mycoplasma synoviae infection. In: Swayne DE, editor. Diseases of Poultry. 14th ed. Wiley-Blackwell, Hoboken, NJ: USA; 2020. p. 924–99.

    Google Scholar 

  8. El-Gazzar M, Bradbury JM. Mycoplasmosis: Mycoplasma iowae infection. In: Swayne DE, editor. Diseases of Poultry. 14th ed. Wiley-Blackwell, Hoboken, NJ: USA; 2020. p. 929–33.

    Google Scholar 

  9. Yadav JP, Tomar P, Singh Y, Khurana SK. Insights on Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry: a systematic review. Anim Biotechnol. 2021;33(7):1711–20. https://doi.org/10.1080/10495398.2021.1908316.

    Article  CAS  PubMed  Google Scholar 

  10. Poveda JB, Carranza J, Miranda A, Garrido A, Hermoso M, Fernandez A, et al. An epizootiological study of avian mycoplasmas in southern Spain. Avian Pathol. 1990;19(4):627–33. https://doi.org/10.1080/03079459008418718.

    Article  CAS  PubMed  Google Scholar 

  11. Cisneros-Tamayo M, Kempf I, Coton J, Michel V, Bougeard S, De Boisséson C, et al. Investigation on eggshell apex abnormality (EAA) syndrome in France: Isolation of Mycoplasma synoviae is frequently associated with Mycoplasma pullorum. BMC Vet Res. 2020;16(1):1–13. https://doi.org/10.1186/s12917-020-02487-0.

    Article  CAS  Google Scholar 

  12. Kleckner AL. Serotypes of avian pleuro-pneumoniae like organisms. Am J Vet Res. 1960;21:274–80.

    CAS  PubMed  Google Scholar 

  13. Kempf I, Gesbert F, Moalic P-Y, Guittet M, Bennejean G. Isolement d’une souche de Mycoplasma pullorum à partir d’œufs embryonnés de dindes. Bull Acad Vét France. 1996;149(2):197–203. https://doi.org/10.4267/2042/63840.

    Article  Google Scholar 

  14. Moalic PY, Kempf I, Gesbert F, Laigret F. Identification of two pathogenic avian mycoplasmas as strains of Mycoplasma pullorum. Int J Systematic Bacteriol. 1997;47(1):171–4.

    Article  CAS  Google Scholar 

  15. Nascimento E, Pereira V, Nascimento M, Barreto M. Avian mycoplasmosis update. Revista Brasileira de Ciência Avícola. 2005;7(1):1–9. https://doi.org/10.1590/s1516-635x2005000100001.

    Article  Google Scholar 

  16. Feberwee A, Mekkes DR, De Wit JJ, Hartman EG, Pijpers A. Comparison of culture, PCR, and different serologic tests for detection of Mycoplasma gallisepticum and Mycoplasma synoviae infections. Avian dis. 2005;49(2):260–8.

    Article  CAS  PubMed  Google Scholar 

  17. Yadav JP, Singh Y, Jindal N, Mahajan NK. Rapid and specific detection of Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry using single and duplex PCR assays. J Microbiol Methods. 2022;192: 106365. https://doi.org/10.1016/J.MIMET.2021.106365.

    Article  CAS  PubMed  Google Scholar 

  18. Council Directive 2009/158/EC. Animal health conditions governing intra-Community trade in, and imports from third countries of, poultry and hatching eggs. Official Journal of the European Union, L343/47-113. https://eur-lex.europa.eu/LexUriServ/ LexUriServ.do?uri=OJ:L:2009:343:0074:0113:EN:PDF (2009). Accessed 15 April 2024.

  19. National Poultry Improvement Plan: https://www.poultryimprovement.org/. Accessed 15 April 2024.

  20. World Organization for Animal Health. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, online version, Chapter 3.3.5.: Avian mycoplasmosis (Mycoplasma gallisepticum, M. synoviae). https://www.woah.org/fileadmin/Home/eng/Health_standards/tahm/3.03.05_AVIAN_MYCO.pdf (2021). Accessed 15 April 2024.

  21. Boussetta M, Chaouachi N, Mlik B. Etude sérologique et bactériologique des mycoplasmoses aviaires dans la région du Cap Bon en Tunisie. Rev Elev Méd Vét Pays Trop. 1997;50(2):93–6.

    Article  Google Scholar 

  22. Groupement Interprofessionnel des Produits Avicoles et Cunicoles (GIPAC). http://www.dataportal.gipac.tn/FreeProdReg.aspx (2021). Accessed 15 April 2024.

  23. Ahani Azari A, Amanollahi R, Jafari Jozani R, Trott DJ, Hemmatzadeh F. High-resolution melting curve analysis: a novel method for identification of Mycoplasma species isolated from clinical cases of bovine and porcine respiratory disease. Tropical Animal Health and Production. 2020;52(3):1043–7. https://doi.org/10.1007/s11250-019-02098-4.

    Article  PubMed  Google Scholar 

  24. Lauerman LH, Hoerr FJ, Sharpton AR, Shah SM, van Santen VL. Development and application of a polymerase chain reaction assay for Mycoplasma synoviae. Avian Dis. 1993;37(3):829–34.

    Article  CAS  PubMed  Google Scholar 

  25. Kempf I, Blanchard A, Gesbert F, Guittet M, Bennejean G. The polymerase chain reaction for Mycoplasma gallisepticum detection. Avian Pathol. 1993;22(4):739–50. https://doi.org/10.1080/03079459308418961.

    Article  CAS  PubMed  Google Scholar 

  26. Frey ML. A medium for the isolation of avian mycoplasmas. Am J Vet Res. 1968;29:2163–71.

    CAS  PubMed  Google Scholar 

  27. Ben Abdelmoumen B, Ben Mohamed R, Gueriri I, Boughattas S, Mlik B. Duplex PCR to differentiate between Mycoplasma synoviae and Mycoplasma gallisepticum on the basis of conserved species-specific sequences of their hemagglutinin genes. J of Clin Microbiol. 2005;43(2):948–50. https://doi.org/10.1128/JCM.43.2.948-950.2005.

    Article  CAS  Google Scholar 

  28. Marois C, Dufour-Gesbert F, Kempf I. Polymerase chain reaction for detection of Mycoplasma gallisepticum in environmental samples. Avian Pathol. 2002;31(2):163–8.

    Article  PubMed  Google Scholar 

  29. Chaidez-Ibarra MA, Velazquez DZ, Enriquez-Verdugo I, Castro Del Campo N, Rodriguez-Gaxiola MA, Montero-Pardo A, et al. Pooled molecular occurrence of Mycoplasma gallisepticum and Mycoplasma synoviae in poultry: a systematic review and meta-analysis. Transbound Emerg Dis. 2022;69(5):2499–511. https://doi.org/10.1111/tbed.14302.

    Article  PubMed  Google Scholar 

  30. Marouf S, Khalf MA, Alorabi M, El-Shehawi AM, El-Tahan AM, El-Hack MEA, et al. Mycoplasma gallisepticum: a devastating organism for the poultry industry in Egypt. Poult Sci. 2022;101(3): 101658. https://doi.org/10.1016/j.psj.2021.101658.

    Article  CAS  PubMed  Google Scholar 

  31. Qoraa AM, Salem HM, Shakal M. The current status of Mycoplasma synoviae in broilers and laying chicken farms in some Egyptian governorates. Egypt J Vet Sci. 2023;54(5):805–13. https://doi.org/10.21608/EJVS.2023.206857.1491.

    Article  Google Scholar 

  32. Marouf S, Moussa IM, Salem H, Sedeik M, Elbestawy A, Hemeg HA, et al. A picture of Mycoplasma gallisepticum and Mycoplasma synoviae in poultry in Egypt: phenotypic and genotypic characterization. J. King Saud Univ. – Science. 2020;32:2263–8. https://doi.org/10.1016/j.jksus.2020.02.036.

  33. Heleili N, Mamache B, Chelihi A. Incidence of avian mycoplasmosis in the region of Batna, eastern Algeria. Vet World. 2011;4(3):101–5.

    Article  Google Scholar 

  34. Sid H, Benachour K, Rautenschlein S. Co-infection with multiple respiratory pathogens contributes to increased mortality rates in Algerian poultry flocks. Avian Dis. 2015;59(3):440–6. https://doi.org/10.1637/11063-031615-Case.1.

    Article  PubMed  Google Scholar 

  35. Galluzzo P, Migliore S, Galuppo L, Condorelli L, Hussein HA, Licitra F, et al. First molecular survey to detect Mycoplasma gallisepticum and Mycoplasma synoviae in poultry farms in a strategic production district of Sicily (South-Italy). Animals. 2022;12:962. https://doi.org/10.3390/ani12080962.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Dufour-Gesbert F, Dheilly A, Marois C, Kempf I. Epidemiological study on Mycoplasma synoviae infection in layers. Vet Microbiol. 2006;114(1–2):148–54. https://doi.org/10.1016/j.vetmic.2005.10.040.

    Article  PubMed  Google Scholar 

  37. Michiels T, Welby S, Vanrobaeys M, Quinet C, Rouffaer L, Lens L, et al. Prevalence of Mycoplasma gallisepticum and Mycoplasma synoviae in commercial poultry, racing pigeons and wild birds in Belgium. Avian Pathol. 2016;45(2):244–52. https://doi.org/10.1080/03079457.2016.1145354.

    Article  PubMed  Google Scholar 

  38. Köhn S, Spergser J, Ahlers C, Voss M, Bartels T, Rosengarten R, et al. Prevalence of mycoplasmas in commercial layer flocks during laying period. Berliner und Munchener Tierarztliche Wochenschrift. 2009;122(5–6):186–92.

    PubMed  Google Scholar 

  39. Cortés V, Sevilla-Navarro S, García C, Tudón A, Marín C, Catalá-Gregori P. Seroprevalence and prevalence of Mycoplasma synoviae in laying hens and broiler breeders in Spain. Poultry Sci. 2021;100(3):100911. doi/https://doi.org/10.1016/j.psj.2020.11.076.

  40. Kursa O, Tomczyk G, Sawicka A. Prevalence and phylogenetic analysis of Mycoplasma synoviae strains isolated from Polish chicken layer flocks. J Vet Res (Poland). 2019;63(1):41–9. https://doi.org/10.2478/jvetres-2019-0010.

    Article  Google Scholar 

  41. Marois C, Dufour-Gesbert F, Kempf I. Detection of Mycoplasma synoviae in poultry environment samples by culture and polymerase chain reaction. Vet Microbiol. 2000;73:311–8.

    Article  CAS  PubMed  Google Scholar 

  42. Marois C, Picault J-P, Kobisch M, Kempf I. Experimental evidence of indirect transmission of Mycoplasma synoviae. Vet Res. 2005;36:759–69.

    Article  CAS  PubMed  Google Scholar 

  43. Buim MR, Mettifogo E, Timenetsky J, Kleven S, Ferreira AJP. Epidemiological survey on Mycoplasma gallisepticum and M. synoviae by multiplex PCR in commercial poultry. Pesquisa Veterinaria Brasileira. 2009;29(7):552–6. https://doi.org/10.1590/S0100-736X2009000700009.

  44. Landman WJ. Is Mycoplasma synoviae outrunning Mycoplasma gallisepticum? A viewpoint from the Netherlands. Avian Pathol. 2014;43(1):2–8. https://doi.org/10.1080/03079457.2014.881049.

    Article  PubMed  Google Scholar 

  45. Feberwee A, de Vries TS, Landman WJ. Seroprevalence of Mycoplasma synoviae in Dutch commercial poultry farms. Avian Pathol. 2008;37(6):629–33. https://doi.org/10.1080/03079450802484987.

    Article  PubMed  Google Scholar 

  46. Bradbury JM, Yavari CA, Dare CM. Mycoplasmas and respiratory disease in pheasants and partridges. Avian Pathol. 2001;30(4):391–6. https://doi.org/10.1080/03079450120066395.

    Article  CAS  PubMed  Google Scholar 

  47. Bencina D, Mrzel I, Tadina T, Dorrer D. Mycoplasma species in chicken flocks with different management systems. Avian Pathol. 1987;16:599–608. https://doi.org/10.1080/03079458708436409.

    Article  CAS  PubMed  Google Scholar 

  48. Décret n° 2009-2200 du 14 juillet 2009, fixant la nomenclature des maladies animales réglementées et édictant les mesures générales applicables à ces maladies. Journal Officiel de la République Tuisienne. 2009;59:2023-2026.

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Acknowledgments

The authors would like to thank Mathilde Le Tirant (ANSES) for her technical assistance, Riadh Bouaziz (Sfax University) for his help in creating maps, and Boutheina Mardassi for her welcome and help within the Pasteur Institute of Tunis. Authors would also like to thank veterinarians, technicians and farmers for their precious contribution to this work.

Funding

This research did not receive any external funding.

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Authors and Affiliations

  1. Laboratory Research of Toxicology Microbiology Environmental and Health, Faculty of Sciences of Sfax, Sfax University, BP 1171, 3000, Sfax, Tunisia

    Soumaya Sabri, Lamia Khannous & Radhouane Gdoura

  2. Mycoplasmology, Bacteriology and Antimicrobial Resistance Unit, Ploufragan-Plouzané-Niort Laboratory, French Agency for Food, Environmental and Occupational Health and Safety (ANSES), BP53, 22440, Ploufragan, France

    Soumaya Sabri, Séverine Ferré & Anne V. Gautier-Bouchardon

  3. Veterinary Services, Regional Agricultural Development Commission, Sfax, Tunisia

    Adel Souissi

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Contributions

RG, AB and LK conceived and designed the research. SS and SF conducted experiments and analyzed data. AS provided clinical samples. SS and AB wrote the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Anne V. Gautier-Bouchardon.

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Ethics approval and consent to participate

Samples were collected by veterinarians during official controls of poultry farms for mycoplasmoses (Mycoplasma gallisepticum and Mycoplasma synoviae are regulated diseases under regular surveillance according to Tunisian decree number 2200-2009 [48]) and in collaboration with Veterinary Services (Regional Agricultural Development Commission, Sfax, Tunisia). According to the Directive 2010/63/EU, ethical approval was not required for this study because samples were collected during “non-experimental agricultural practices” (article 1, 5a) by veterinarians with the consent of the farmers during official controls.

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Not applicable.

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The authors declare no competing interests.

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Sabri, S., Khannous, L., Ferré, S. et al. First molecular investigation to detect avian Mycoplasma species in clinical samples from laying-hen farms in Tunisia. BMC Vet Res 20, 459 (2024). https://doi.org/10.1186/s12917-024-04311-5

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BMC Veterinary Research

ISSN: 1746-6148

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