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Revista mexicana de ciencias pecuarias
versão On-line ISSN 2448-6698versão impressa ISSN 2007-1124
Rev. mex. de cienc. pecuarias vol.11 no.4 Mérida Out./Dez. 2020 Epub 02-Mar-2021
https://doi.org/10.22319/rmcp.v11i4.5124
Articles
Frequency of M. hyopneumoniae, M. hyorhinis and M. hyosynoviae in nasal and lung samples from pigs with symptoms of porcine enzootic pneumonia
a Universidad Nacional Autónoma de México. Facultad de Medicina Veterinaria y Zootecnia, Departamento de Microbiología e Inmunología. UNAM. Ciudad Universitaria, 04519, CDMX, México.
b Hospital General “Dr. Manuel Gea González”, Departamento de Biología Molecular e Histocompatibilidad. CDMX, México.
c Universidad Nacional Autónoma de México, Facultad de Medicina Veterinaria y Zootecnia, Departamento de Cerdos. CDMX, México.
d Universidad Nacional Autónoma de México, Facultad de Medicina Veterinaria y Zootecnia, Departamento de Patología. CDMX, México.
M. hyopneumoniae, M. hyorhinis and M. hyosynoviae are genetically related species of the genus Mycoplasma that affect pig production. The objective of this work was the isolation and identification by PCR of M. hyopneumoniae, M. hyorhinis and M. hyosynoviae from nasal swabs and lung samples of pigs from different regions of Mexico in order to determine the frequency of these species and to evaluate PCR as a diagnostic tool for PEP. Pigs aged 4 to 8 weeks with clinical diagnosis of PEP were included. Lung samples and nasal swabs were obtained for the isolation of the Mycoplasma in liquid Friis medium and identified by species-specific PCR based on the 16S rRNA subunit. Isolation was achieved in 37.11 % (36/97) of the samples. The three Mycoplasma species were identified in lung and nasal swab samples. Mycoplasma co-infection was identified in 27.77 % (10/36). The bacterial genera associated with Mycoplasma infections were E. coli, Bordetella, Enterobacter, SCN, Corynebacterium, Pasteurella, Streptococcus, Shigella and Klebsiella. Mixed infection was present in 26 nasal swabs (45.61 %) and absent in the lungs. It was concluded that the frequency of Mycoplasma on production farms was higher than expected (40.27 %). It was also identified other Mycoplasma species involved in the development of PEP. Therefore, surveillance through isolation and molecular techniques can be of great help to breeding stock providers, as well as for removing Mycoplasma from pig farms.
Key words Mycoplasmosis; M. hyopneumoniae; M. hyorhinis; M. hyosynoviae; Porcine enzootic pneumonia
M. hyopneumoniae, M. hyorhinis y M. hyopsynoviae son especies genéticamente relacionadas del género Mycoplasma que afectan la producción porcina. El objetivo de este trabajo fue el aislamiento e identificación por PCR de M. hyopneumoniae, M. hyorhinis y M. hyosynoviae a partir de hisopos nasales y muestras de pulmón de cerdos de diferentes regiones mexicanas para determinar la frecuencia de estas especies y evaluar la PCR como herramienta diagnóstica para NEP. Se incluyeron cerdos de 4 a 8 semanas con diagnóstico clínico de NEP. Se obtuvieron muestras de pulmón e hisopos nasales para el aislamiento de Mycoplasma en medio Friis líquido y se identificaron mediante la PCR especie-específica basada en la subunidad 16S rRNA. El aislamiento se logró en 37.11 % (36/97) muestras. Las tres especies de Mycoplasma se identificaron en muestras de pulmón e hisopos nasales. La co-infección por Mycoplasma se identificó en el 27.77 % (10/36). Los géneros bacterianos asociados a las infecciones por Mycoplasma fueron E. coli, Bordetella, Enterobacter, SCN, Corynebacterium, Pasteurella, Streptococcus, Shigella y Kebsiella. La infección mixta estuvo presente en 26 hisopos nasales (45.61 %) y ausente en pulmones. Se concluyó que la frecuencia de Mycoplasma en las fincas de producción fue mayor a la esperada (40.27 %). También se identificaron otras especies de Mycoplasma involucradas en el desarrollo de la NEP. Por lo tanto, la vigilancia asistida por el aislamiento y las técnicas moleculares pueden ser de gran ayuda para la eliminación de Mycoplasma de las explotaciones porcinas y para los proveedores de pie de cría.
Palabras clave Micoplasmosis; M. hyopneumoniae; M. hyorhinis; M. hyosynoviae; Neumonía enzoótica porcina
Introduction
The Swine Respiratory Disease Complex (PRDC) is a major health problem for the pig industry worldwide1. It is caused by the association of infections such as Mycoplasma, porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), Pasteurella multocida, Actinobacillus pleuropneumoniae, Streptococcus suis, Haemophilus parasuis, Bordetella bronchiseptica, and Arcanobacterium pyogenes1,2. A predisposing factor is porcine enzootic pneumonia (PEP), primarily caused by Mycoplasma hyopneumoniae3, which adheres to the respiratory epithelium, damages the ciliated cells of the trachea, bronchi and bronchioles4, and suppresses the immune response of the upper respiratory tract that favors the development of PRDC5,6.
PEP is a high-prevalence chronic respiratory disease with high morbidity and low mortality. between 30 to 80 % of the pig programmed for slaughter exhibit typical consolidation lesions7,8. Throughout the pig's productive life, the prevalence of M. hyopneumoniae increases until it reaches the age of slaughter, even in vaccinated animals9. Reproductive females are a reservoir that perpetuates the continuous circulation of respiratory pathogens associated with PEP10,11.
The severity of the disease differs among herds, with a high prevalence in conventional pig farms12. The most significant clinical sign of PEP is a chronic, dry, non-productive cough that occurs in fattening pigs aged 16 to 22 wk. The main macroscopic lesion is cranio-ventral pulmonary consolidation5, which is histologically characterized by broncho-interstitial pneumonia with hyperplasia of the bronchus-associated lymphoid tissue (BALT)13. The main risk factor for PEP is vertical transmission from sow to piglet during lactation, given that vaccination does not guarantee protection14 since M. hyopneumoniae can circulate in vaccinated animals15 and in free-living animals such as wild boar, with which vulnerability to M. hyopneumoniae is shared, and which can be a reservoir of these bacteria16. The severity of the disease at the time of slaughter may be predicted of the initial prevalence at weaning, based on the variables indicative of infection (average of lung lesions, percentage of lung tissue affected, presence of M. hyopneumoniae in the bronchial epithelium and seroconversion), as there is a positive correlation between these two variables17.
Most Mycoplasma infections remain subclinical18 and may involve other species of the same bacterial genus such as M. hyorhinis, a commensal inhabitant of the upper respiratory tract mucosa and tonsils19. M hyosynoviae, a species mainly associated with acute arthritis and, to a lesser extent, with suppurative pneumonia with severe pulmonary consolidation, and pleurisy20,21,22. M. hyopneumoniae, M. hyorhinis and M. hyosynoviae are genetically related species of porcine interest23, which can be discriminated by PCR based on the hypervariable regions of the 16S subunit of the genus23,24.
The objective of this work was the isolation and identification by PCR of M. hyopneumoniae, M. hyorhinis and M. hyosynoviae from nasal swabs and samples from pigs of different regions of the Mexican Republic, in order to determine the frequency of these species and to evaluate PCR as a diagnostic tool for enzootic swine pneumonia.
Material and methods
Animals and sample collection
Pigs aged 4 to 8 wk diagnosed with PEP according to clinical signs and with gross lesions in the lung (purple to gray areas of tissue consolidation in the cranio-ventral lung lobe) were included in this study. 40 lung samples and 57 nasal swabs were aseptically obtained by pressing against the structural wall of the tissue25. Sample collection was conducted on farms in four regions of Mexico, from May 2015 to January 2016 (Table 1). Each sample was collected in duplicate for Mycoplasma isolation and for traditional bacteriology. All animal procedures were approved by the Institutional Committee for the Care and Use of Experimental Animals (CICUAE) of the National Autonomous University of Mexico, following international ethical standards.
Mycoplasma isolation
For Mycoplasma isolation, nasal swabs were resuspended in 2 ml of Friis medium. Lung samples were frozen at -20°C until they were followed up in the laboratory. Lung samples were routinely processed by maceration in 3 ml of Friis medium for isolation18,26,27. 200 μl of the suspension of each sample in Friis medium were inoculated in 1.8 ml of Friis medium supplemented with pig serum (10 %), horse serum (10 %), and penicillin (100 μg/mL) to optimize the recovery of M. hyopneumoniae28, and supplemented with L-arginine (0.05 %) for the recovery of M. hyosynoviae29. Subsequently, up to 10-6 serial dilutions were made, and, finally, 10 μL were plated onto Friis agar27. Tubes were incubated at 37 °C until a color change was observed in the medium, or up to 30 d, before being discarded. Positive samples were those that developed at least one unit of color change, while samples that had no color change after 30 d were considered negative. The agar plates were incubated at 37 °C with 5 % CO2 for 1 to 2 wk. Each isolated colony was further inoculated into 2 ml of Friis medium and incubated. After observing the color change, the cultures were evaluated to confirm their purity and subsequent use until PCR discrimination of the species.
Species-specific PCR for the identification of Mycoplasma
PCR based on the 16S rRNA subunit for the identification of the three Mycoplasma species was applied to each of the isolates. The reference strains M. hyopneumoniae ATCC 25617, M. hyorhinis ATCC 17981, M. hyosynoviae strain S-16, and M. bovis Donetta PG45-all kindly donated by Aarhus University, Aarhus, Denmark-were cultured in 50 ml medium, concentrated by centrifugation for DNA extraction according to the protocol with guanidinium thiocyanate30. Each isolate was also processed for DNA extraction and stored at -70 °C until further analysis.
Amplification of the 16S rRNA subunit was performed in a total reaction volume of 25 µL containing 0.25 µL of Taq PCR Reaction Mix (Sigma-Aldrich, Austria), 10 pmol of each sense and antisense initiator (Table 2)24, and 10 µl of DNA31. The reaction conditions were: initial denaturation at 96 °C, for 5 min, followed by 30 denaturation cycles at 94 °C for 45 s, alignment at 72 °C for 2 min, and extension at 72 °C for 4 min. DNA from pure cultures of M. hyopneumoniae ATCC 25617, M. hyorhinis ATCC 17981 and M. hyosynoviae strain S-16 were applied as positive controls, and M. bovis Donetta PG45, as negative control.
Table 2 PCR initiators based on 16S rRNA from M. hyopneumoniae, M. hyorhinis and M. hyosynoviae
Mycoplasma species |
Sequence (5 -3) | Product (bp) |
Reference |
---|---|---|---|
M. hyopneumoniae | F 5'-TTC AAA GGA GCC TTC AAG CTT C-3' R 5'-GAC GTC AAA TCA TCA TGC CTC T-3' |
1000 | 30 |
M. hyorhinis | F 5' CGGGATGTAGCAATACATTCAG 3' R 5' GACGTCAAATCATCATGCCTCT 3' |
1129 | 30 |
M. hyosynoviae | F 5' CAGGGCTCAACCCTGGCTCGC 3' R 5' GACGTCAAATCATCATGCCTCT 3' |
585 | This work Gen Bank Access No. NR029183. 1 |
Results
Mycoplasma isolation
97 samples were collected: 40 from lungs with typical Mycoplasma lesions suggestive of PEP (Figure 1) and 57 nasal swabs from pigs from different geographical regions of Mexico (Table 1). From the lung samples, 22.5 % (9/40) were positive to the isolation of Mycoplasma spp and 77.5 % (31/40) were negative. Of the nasal swabs, 47.36 % (27/57) were positive, and 52.63 % (30/57) were negative. In the positive samples, the color change of the culture medium was observed as early as the 5th d or until the 12th d. On average, the color change was observed on the 7th d. The remaining samples were considered as negative after 30 d without color change.
![](/img/revistas/rmcp/v11n4//2448-6698-rmcp-11-04-946-gf3.png)
In (A) lung with typical PEP injury, distributed over all lobes of the lung, (B) approach to lung consolidation, (C) lung with higher degree of lung consolidation, (D) lung sequestration resulting from the evolution of the injury, (E) evidence of scarring in the lung tissue.
Figure 1 Typical Mycoplasma lesions in the lungs collected for this study
PCR results
Amplified fragments of 1,000 bp of M. hyopneumoniae, 1,129 bp of M. hyorhinis, and 585 bp of M. hyosynoviae using the reference strains (ATCC 25617, ATCC 17981, and M. hyosynoviae strain S-16), were visualized by 1.5% agarose gel electrophoresis at 80 V for 60 min, stained with ethidium bromide and displayed on a UV transilluminator, as shown in Figure 2. 22 % (2/9) of the lung sample isolates (LSIs) of Mycoplasma tested positive for M. hyopneumoniae; 55.5 % (5/9), for M. hyorhinis, and 44 % (4/9), for M. hyosynoviae. 44 % (4/9) of the LSIs tested negative with species-specific PCR. 7.40 % (2/27) of the nasal swab isolates (NSIs) tested positive for M. hyopneumoniae; 51.85% (14/27), for M. hyorhinis, and 33.3 % (9/27), for M. hyosynoviae. 22.22 % (6/27) of the NSIs tested negative with species-specific PCR (6/27) (Table 3). Despite having been successfully isolated, four LSIs and six NSIs remained unidentified with the species-specific PCR.
![](/img/revistas/rmcp/v11n4//2448-6698-rmcp-11-04-946-gf4.png)
Lane 1, Molecular Weight Marker (1 Kb plus Invitrogen), Lane 2, M. hyopneumoniae ATCC 25617, 1000 bp; Lane 3. M. bovis Donetta PG45, donated by the University of Aarhus, Denmark, Lane 4, M. hyorhinis, ATCC17981, 585 bp, Lane 6, Unrelated product with 685 bp of p97 protein from M. hyopneumoniae, ATCC25617, Lane 7, M. hyosynoviae, strain S-16, 1129 bp, also donated by the University of Aarhus, Denmark, Lane 8, Molecular Weight Marker (1 Kb plus Invitrogen).
Figure 2 Electrophoretic profiles of the amplified fragments of M. hyopneumoniae, M. hyorhinis and 16S rRNA
Table 3 List of isolates identified by species-specific PCR for M. hyopneumoniae, M. hyorhinis, and M. hyosynoviae
Sample | Positive isolation (%) | |||
---|---|---|---|---|
M. hyopneumoniae |
M. hyorhinis |
M. hyosynoviae |
Mycoplasma spp isolates |
|
Lung | 2/9 (22.0) | 5/9 (55.5) | 4/9 (44.0) | 4/9 (44.0) |
Nasal swab | 2/27 (7.4) | 14/27 (51.8) | 9/27 (33.3) | 6/27 (22.2) |
Total | 4/36 (11.1) | 19/36 (52.7) | 13/36 (36.1) | 10/36 (27.7) |
The coexistence of M. hyopneumoniae, M. hyorhinis and M. hyosynoviae was detected in ten samples representing 27.77 % (10/36): in two lungs all three species, in two other lungs and five nasal swabs M. hyorhinis and M. hyosynoviae were identified, and only one swab contained M. hyopneumoniae and M. hyorhinis (Complementary Table 1). Additionally, the associated bacterial genera identified by general bacteriology in nasal swabs were E. coli, Enterobacter, coagulase-negative Staphylococcus, Klebsiella, Bordetella, Corynebacterium, Pasteurella, Shigella and Streptococcus. No bacterial growth was identified in lung samples.
Supplementary table 1 Identification of Mycoplasma isolates by species-specific PCR
Number | Description | Type of sample* |
M. hyop |
M. hyor |
M. hyos |
Bacterial Genera |
---|---|---|---|---|---|---|
1 | 111 | NS | - | + | - | CNS |
2 | 112 | NS | - | + | + | E. coli , Shigella |
3 | 113 | NS | - | - | - | Pasteurella |
4 | 114 | NS | - | - | - | Klebsiella, CNS |
5 | 115 | NS | - | + | + | Klebsiella, E. coli, CNS |
6 | 116 | NS | - | + | - | E. coli, CNS, Bordetella, Corynebacterium |
7 | 117 | NS | - | + | - | CNS, Corynebacterium |
8 | 118 | NS | - | + | - | CNS, Corynebacterium |
9 | 119 | NS | - | - | + | Enterobacter |
10 | 120 | NS | - | + | - | Corynebacterium |
11 | 121 | NS | - | - | - | Klebsiella, CNS |
12 | 122 | NS | - | + | + | Corynebacterium |
13 | 123 | NS | - | - | + | Klebsiella, CNS |
14 | 124 | NS | - | - | + | CNS |
15 | 125 | NS | - | + | - | Corynebacterium |
16 | 126 | NS | - | - | - | Corynebacterium |
17 | 127 | NS | - | - | - | Klebsiella, Corynebacterium |
18 | 130 | NS | + | - | - | CNS |
19 | 133 | NS | - | - | - | E. coli |
20 | 148 | NS | - | + | - | CNS |
21 | 159 | NS | + | + | - | No bacterial growth |
22 | 160 | NS | - | + | - | CNS |
23 | 161 | NS | - | - | + | Pasteurella |
24 | 162 | NS | - | + | + | E. coli, CNS |
25 | 165 | NS | - | + | - | Streptococcus, CNS |
26 | 168 | NS | - | + | - | E. coli |
27 | 170 | NS | - | + | + | CNS |
28 | 182 | L | - | + | + | No bacterial growth |
29 | 183 | L | - | + | + | No bacterial growth |
30 | 186 | L | - | - | - | No bacterial growth |
31 | 188 | L | - | - | - | No bacterial growth |
32 | 194 | L | + | + | + | No bacterial growth |
33 | 206 | L | + | + | + | No bacterial growth |
34 | 207 | L | - | + | - | No bacterial growth |
35 | 208 | L | - | - | - | No bacterial growth |
36 | 210 | L | - | - | - | No bacterial growth |
M. hyop = M hyopneumoniae: M. hyor= M. hyorhinis; M. hyos= M. hyosynoviae; *NS= nasal swab, L= lung, CNS= Coagulase-negative Staphylococcus
Discussion
In Mexico there are few studies on the association of these three Mycoplasma species with PEP, mainly due to the difficulties for their isolation and to those inherent in the biological sample. The concentration of microorganisms is often below the detection limit as a result of the widespread use of antibiotics for the control of porcine mycoplasmosis. Therefore, isolation procedures are necessary to encourage their growth and identification for research and surveillance purposes. The procedure used allowed the association identification of the three species related to pig production.
M. hyopneumoniae is the most frequently isolated species of Mycoplasma from pigs with clinical signs of pneumonia and has a low transmission rate. However, in association can increase the severity of infections caused by viruses and bacteria32.
M. hyorhinis has gone from being a secondary pathogen33,34, to being considered a causal agent of PEP and PRDC35. In this study, this species of Mycoplasma is the most prevalent in nasal swabs 51.85% (14/57); this observation can be explained by the success of the control measures that have been implemented in pig production farms. This study reports herein that M. hyosynoviae is in close interaction with the other two Mycoplasma species in lungs with typical PEP lesions. M. hyosynoviae was present in nasal swabs as a microorganism associated in a high percentage of the cases (33%, i.e. 9/27). Therefore, this commensal Mycoplasma species may have pathogenic potential, and further studies will be required to assess its role in the development of PEP.
Bacteriological culture is the "gold standard" for diagnosis. However, among its drawbacks, it is very laborious, it is seldom used as a routine method, and it does not distinguish between species associated with PEP. PCR based on the 16S rRNA subunit allowed to discern, quickly and precisely, between M. hyopneumoniae and M. hyorhinis. On the other hand, 10 cases were identified in which the species evaluated in this work were not involved. This result raises the possibility that other Mycoplasma species may be involved.
The collection method (nasal swab, tracheobronchial mucus, deep postmortem swab, bronchoalveolar lavage or lung tissue) has a significant effect on the frequency of M. hyopneumoniae, since the reported frequency varies between 3 and 40 %, according to the method used36. Hitherto, nasal swabs have been the method of antemortem sample collection in piglets at herd level28,37. Pieters et al38 suggest that laryngeal swabs are useful in the early stage of infection in piglets. Other authors state that the optimal sampling site for detection by molecular methods for M. hyopneumoniae is tracheobronchial mucus collection (TBMC), since its sensitivity is 3.5 times more sensitive in piglets aged under 25 d36.
The upper respiratory tract (nasal cavity and pharynx) plays an important role in monitoring and cleansing pathogenic microorganisms and also in inducing the appropriate immune response. M. hyopneumoniae mainly colonizes the cilia of the respiratory tract of the pigs39. In adult animals from production farms, TBMC becomes difficult and expensive to obtain. Animal handling is restricted in keeping with current swine influenza prevention measures, and, based on this experience, it is recommend the use of nasal swabs as the appropriate sampling technique.
The prevalence of M. hyopneumoniae in naturally infected sows is 36.4 %40; in piglets, it can vary from 3.6 to 16 %. The frequency of Mycoplasma determined here was higher than expected, namely: 37.11 % (36/97). Mycoplasma was present in the lungs of 22.50 % of the animals, (9/40), and in nasal swabs, in 47.36 % (27/57). Infection by a single Mycoplasma species was 44.44 % (16/36): in LSIs 11.11 % (1/9) and in NSIs 55.55 % (15/27). The association of more than one Mycoplasma species was present in 27.77 % (10/36): the association of the three species represented 2 % (2/10), and the association of M. hyorhinis and M. hyosynoviae 5.15% (5/10). Co-infection of M. hyopneumoniae and M. hyorhinis, and M. hyosynoviae and M. hyorhinis have previously been associated with joint problems. In this work, both associations were identified in the respiratory tract of animals in pig farms with PEP.
The rate of Mycoplasma associated with PEP is variable, regardless of whether the rate of mixed infections remains constant35. In this study, the bacterial genera associated in mixed infections were similar to those previously reported41. PCR can be complementary or alternative to histopathological diagnosis and represents an option for epidemiological surveillance and research. In addition, it can assist in the elimination of Mycoplasma spp from swine production farms, as it is the best long-term control strategy, so far, for many swine producers and breeding stock suppliers42.
Conclusions and implications
The frequency of Mycoplasma in pig farms in the states of Hidalgo, Guanajuato, Veracruz and Mexico was higher than expected (40.27 %). There are other Mycoplasma species that may be involved in the development of PEP, and this paper adds evidence of M. hyorhinis as a causal agent of PEP.
Acknowledgements
This work was supported by the Project DGAPA UNAM PAPITT IN 222515.
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Received: October 29, 2018; Accepted: September 09, 2019