UNIVERSIDADE ESTADUAL PAULISTA - UNESP 

CÂMPUS DE JABOTICABAL 

DIVERSIDADE GENÉTICA DE Mycoplasma hyopneumoniae 

ASSOCIADO AS LESÕES DE PLEURISIAS DETECTADAS 

AO ABATE 

 

 

 

Ana Karolina Panneitz 

                                                                                                  Médica Veterinária 

 

 

 

 

 

 

 

 

 

2025 



UNIVERSIDADE ESTADUAL PAULISTA - UNESP 

CÂMPUS DE JABOTICABAL 

DIVERSIDADE GENÉTICA DE Mycoplasma hyopneumoniae 

ASSOCIADO AS LESÕES DE PLEURISIAS DETECTADAS 

AO ABATE 

 

 

 

 

Discente: Ana Karolina Panneitz 

 

Orientador: Prof. Dr. Luís Guilherme de Oliveira 

 

 

Dissertação apresentada à Faculdade de 
Ciências Agrárias e Veterinárias – Unesp, 
Câmpus de Jaboticabal, como parte das 
exigências para a obtenção do título de 
Mestre em Ciências Veterinárias 

 

 

 

2025 



 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



REGISTRO DE IMPACTO 

 

O curso de mestrado promove avanço científico, formando profissionais qualificados 

para enfrentar desafios complexos. Esta dissertação destaca a importância da 

integração entre ciência e setor produtivo. O estudo investiga a diversidade genética 

de Mycoplasma hyopneumoniae, revelando diversidade genética do agente, 

reforçando a necessidade de aprimorar estratégias de controle do CDRS. 

 

 

 

 

 

 

 

 

 



 



DADOS CURRICULARES DO AUTOR 

 

Ana Karolina Panneitz – nascida em 26 de maio de 2000, na cidade de Rio 

Negrinho – Santa Catarina, Brasil. Médica Veterinária graduada pela Universidade 

Federal de Santa Catarina (UFSC) em 2023. Durante a graduação foi integrante do 

GESA – Grupo de Estudos em Suínos e Aves e do GEHisPa – Grupo de Estudos em 

Histologia e Patologia, participando da organização de eventos e projetos de 

extensão. Realizou estágios extracurriculares nas áreas de patologia animal, 

microbiologia veterinária e suinocultura. O Trabalho de Conclusão de Curso foi 

realizado na área de diagnóstico veterinário, com o tema “Avaliação de formação de 

biofilme de diferentes sorovares de Salmonella sp. isolados de frangos de corte”, sob 

orientação do Prof. Dr. Álvaro Menin, o estágio curricular obrigatório também foi 

realizado na área diagnóstico veterinário, no Instituto de Pesquisa e Diagnóstico 

Veterinário (VERTÀ Laboratórios), sob supervisão da Dra. Carolina Reck. Ingressou, 

em março de 2023, no Programa de Pós-graduação em Ciências Veterinárias, na 

FCAV – UNESP Jaboticabal, onde foi bolsista FAPESP, sob a orientação do Prof. Dr. 

Luís Guilherme de Oliveira. Durante o mestrado participou do grupo de estudos em 

Suínos (Suinesp) e também realizou estágio de pesquisa no exterior, por 6 meses, na 

Ghent University (Bélgica) sob a supervisão do Prof. Dr. Dominiek Maes. Possui 

experiência nas áreas de diagnóstico veterinário, sanidade de suínos e 

experimentação animal. 

 

 

 

 

 

 

 

 

 

 



EPÍGRAFE 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A vida não é fácil para nenhum de nós. Mas e 

daí? Nós devemos ter persistência e, acima de 

tudo, confiança em nós mesmos. Devemos 

acreditar que somos talentosos em alguma 

coisa, e que essa coisa, a qualquer custo, deve 

ser alcançada. (Marie Curie) 

https://www.pensador.com/autor/marie_curie/


DEDICATÓRIA 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Dedico este trabalho aos meus pais. Tenho 

muito orgulho de vocês e sou muito grata por 

sempre me apoiarem. 

 



AGRADECIMENTOS 

 

Agradeço: 

 

Aos meus pais, Rodolfo e Elenice, que sempre me incentivaram a estudar, 

possibilitaram que isso fosse uma realidade para mim e acima de tudo sempre 

apoiaram as minhas decisões. E que nos momentos de minha ausência, dedicados 

ao mestrado, sempre compreenderam e me ensinaram que precisamos construir o 

futuro a partir da dedicação no presente. 

Ao meu orientador Prof. Dr. Luís Guilherme, por não medir esforços para que 

eu pudesse aproveitar ao máximo as oportunidades do mestrado. 

A FCAV/Unesp pelo excelente programa de pós-graduação e estrutura que 

permitiu o desenvolvimento do projeto. 

A FAPESP, pela concessão da bolsa de mestrado regular e também pela bolsa 

de estágio no exterior (Processos nº 2023/01747-4 e 2023/17921-3). 

Ao Prof. Dr. Dominiek, pela supervisão na BEPE, por todas as oportunidades 

de desenvolvimento profissional e por toda a bondade, compreensão e confiança. 

Aos colegas do Laboratório de Medicina de Suínos, por todo suporte nas 

coletas e execução de experimentos. Especialmente a Cláudia e seus maravilhosos 

conselhos. 

Ao Prof. Dr. David e ao Jean por prontamente aceitarem a parceria e 

contribuírem com o trabalho. 

A Eduarda, o que eu vô fala ducê? Um dos maiores presentes do mestrado. Só 

tenho a agradecer por sua amizade, por todo o apoio, todos os conselhos, todas as 

horas e horas no laboratório, toda a confiança que você me transmitiu todos os roles 

aleatórios, todas as cervejas no Skina Bar. Você tornou esse período muito mais leve. 

A Gabrielle, nossa estagiária 10/10, amiga obrigada por todo o suporte nas 

coletas (com uma trilha sonora de respeito), companhia nos treinos e por me 

convencer que eu estava pronta para ser a mãe da Lady. 

Ao Fernando, e suas habilidades com fotoshop (KKKKcrying), parcerias de 

eurotrip (visitas a pietá) e conselhos (foram muitos “mano.....” durante esses anos). 

A Leila, que mesmo estando em outro estado (e por um tempo em outro 

continente) sempre sabia a hora que eu mais precisava conversar. Além disso muito 



obrigada por tomar conta de minha filha Lady. A Paola por estar sempre lá, pra tudo 

que é papo. A Stephanie, por ficar horas e horas em ligação de vídeo jogando 

conversa fora pra que eu não ficasse sozinha. Letícia, que sempre surge com updates 

(pra não falar fofocas). Andressa, que sempre me apoia e me lembra de acreditar em 

mim mesma. 

Amigos que a Unesp proporcionou, em especial ao Daniel, Lorena, Jaira, 

Ismael, Lari, Bárbara (e Maria) e Isa, vocês tornaram o período no JBK muito mais 

alegre. 

Aos amigos que a UGhent proporcionou. Especialmente a Karina (cuida só), 

tantas risadas, histórias boas e mesmo com muito trabalho sempre deixando tudo tão 

leve. Li, Let’s seeeee hahah . Também a Olivia, Rafa, Bruno, Pati, Mari, Mateuz, 

Renjie, Belle, Lotte, Fabiola, Marleen e Charlotte vocês foram muito importantes para 

eu me sentir em casa no BE. 

A Caroline, que faz uns 5 anos que me deu aula mas tem o cargo vitalício de 

prof Carol. Que mesmo de longe e quem diria de perto (e na europa kkk), sempre 

esteve disposta a ensinar e apoiar, obrigadaaa por ouvir meus áudios que são quase 

podcasts e sempre me aconselhar (“você já sabe o que eu vou dizer neh Ana, mas...”). 

Ao Prof. Álvaro e Drª Carolina, obrigada por sempre estarem lá pra apoiar, 

acreditar, aconselhar e ensinar. Vocês são exemplos, e não largo mesmo hahahah  

Aos meus demais amigos que estiveram de longe, ou que não vejo tanto quanto 

eu gostaria. E que, felizmente, a lista é extensa. Saibam que penso com muito carinho 

em todos vocês. Obrigada por contribuírem com a construção da pessoa que me 

tornei. 

 

 

 

 

 

 

 



i 
 

SUMÁRIO 

Página 

CAPÍTULO 1 – Considerações gerais ......................................................................... 1 

1. Introdução ...................................................................................................... 1 

2. Revisão de Literatura ..................................................................................... 2 

3. Referências .................................................................................................... 6 

1CAPÍTULO 2 – Exploring the genetic diversity of Mycoplasma hyopneumoniae in pigs 

with pneumonia and pleurisy at slaughter ................................................................. 12 

1. Introduction .................................................................................................. 14 

2. Materials and Methods ................................................................................. 16 

2.1 Macroscopic evaluation of lungs and sample collection ............................ 16 

2.2. DNA extraction and conventional PCR (cPCR) for endogenous 

mammalian glycer-aldehyde 3-phosphate dehydrogenase (gapdh) gene .......... 17 

2.3. Real-time PCR (qPCR) for p102 gene of M. hyopneumoniae ............... 18 

2.5. Detection of bacterial co-infections by multiplex qPCR .......................... 19 

2.6. Histopathological and immunohistochemical evaluation ........................ 19 

2.7. Statistical analysis ..................................................................................... 20 

3. Results ......................................................................................................... 20 

3.1. Gross evaluation and qPCR for the detection of M. hyopneumoniae .... 21 

3.2. Characterization of the allelic profiles of M. hyopneumoniae ................. 21 

3.3. qPCR for the detection of bacterial co-infection ..................................... 24 

3.4. Severity of gross and histopathological lesions ..................................... 25 

4. Discussion .................................................................................................... 27 

5. Conclusions .................................................................................................. 29 

6. References ................................................................................................... 31 

 

 

 

 

 

  



 

CERTIFICADO DA COMISSÃO DE ÉTICA NO USO DE ANIMAIS 

 

 

 



 

DIVERSIDADE GENÉTICA DE Mycoplasma hyopneumoniae ASSOCIADO AS 

LESÕES DE PLEURISIAS DETECTADAS AO ABATE 

 

RESUMO - Na suinocultura as doenças respiratórias possuem alta prevalência 
e geram grande impacto econômico. O Mycoplasma (M.) hyopneumoniae é o principal 
patógeno envolvido no complexo das doenças respiratórias dos suínos (CDRS) e 
contribui para o desenvolvimento de pleurisias em suínos. Devido ao seu limitado 
metabolismo possui cultivo laborioso, tornando as ferramentas moleculares úteis para 
o diagnóstico da infecção e estudo de variabilidade de cepas. Com isso, este estudo 
investigou a diversidade genética de M. hyopneumoniae em suínos no momento do 
abate de acordo com a severidade das lesões de pneumonia e pleurisia observadas. 
Também foram avaliadas as co-infecções por Pasteurella (P.) multocida tipo A, 
Actinobacillus (A.) pleuropneumoniae e vírus da influenza suína A (sIVA). Pulmões 
(N=70) com diferentes graus de pleurisia e com lesões compatíveis com infecção por 
M. hyopneumoniae foram coletados por conveniência em abatedouro frigorífico. 
Foram realizadas avaliações macroscópicas e microscópicas. M. hyopneumoniae foi 
detectado por qPCR, e a técnica de MLST foi empregada para caracterização genética 
deste agente. As co-infecções com P. multocida tipo A e A. pleuropneumoniae também 
foram avaliadas por qPCR, enquanto imunohistoquímica foi usada para avaliar a 
infecção por sIVA. Todos os pulmões amostrados foram positivos para M. 
hyopneumoniae. A histopatologia confirmou as lesões associadas ao M. 
hyopneumoniae. A caracterização por sequência de múltiplos locus (MLST) foi 
possível em 25 pulmões e revelou 10 perfis alélicos distintos, nenhum correspondendo 
a tipos de sequência conhecidos no banco de dados público. Co-infecções foram 
detectadas em 40% das amostras com A. pleuropneumoniae e 32% com P. multocida, 
em 12% foram detectados ambos os patógenos, 52% das amostras apresentaram 
lesões microscópicas compatíveis com infecção por sIVA. Os diversos perfis genéticos 
encontrados ressaltam a necessidade do isolamento e caracterização das cepas de 
campo para compreensão de possíveis variações patogênicas. 
 

 

Palavras-chave: doenças respiratórias, hiperplasia de BALT, MLST, pneumonia, 
pneumonia enzoótica dos suínos 

 

 

 

 

 

 

 



 

GENETIC DIVERSITY OF Mycoplasma hyopneumoniae ASSOCIATED WITH 

PLEURISY LESIONS DETECTED AT SLAUGHTER  

 

ABSTRACT - In pig production, respiratory diseases have a high prevalence and 

generate great economic impact. Mycoplasma (M.) hyopneumoniae is the main 

pathogen involved in the porcine respiratory disease complex (PRDC) and contributes 

to the development of pleurisy in pigs. Due to limited metabolism, it has laborious 

cultivation, making molecular tools useful for diagnosing infection and studying strain 

variability. With this in mind, this study investigated the genetic diversity of M. 

hyopneumoniae in pigs at slaughter according to the severity of the pneumonia and 

pleurisy lesions observed. Co-infections with Pasteurella (P.) multocida type A, 

Actinobacillus (A.) pleuropneumoniae and Swine Influenza Virus A (sIVA) were also 

evaluated. Lungs (N=70) with different scores of pleurisy and lesions compatible with 

M. hyopneumoniae infection were collected for convenience at a slaughterhouse. 

Macroscopic and microscopic evaluations were carried out. M. hyopneumoniae was 

detected by qPCR, and the MLST technique was used for genetic characterization of 

this agent. Co-infections with P. multocida type A and A. pleuropneumoniae were also 

assessed by qPCR, while immunohistochemistry was used to assess sIVA infection. 

All the lungs sampled were positive for M. hyopneumoniae. Histopathology confirmed 

the lesions associated with M. hyopneumoniae. Multilocus sequence characterization 

(MLST) was possible in 25 lungs and revealed 10 distinct allelic profiles, none of which 

corresponded to known sequence types in the public database. Co-infections were 

detected in 40% of the samples with A. pleuropneumoniae and 32% with P. multocida, 

in 12% both pathogens were detected, 52% of the samples showed microscopic 

lesions compatible with sIVA infection. The different genetic profiles found highlight the 

need to isolate and characterize field strains in order to understand possible 

pathogenic variations. 

 

 

Keywords: BALT hyperplasia, MLST, pneumonia, swine enzootic pneumonia, 

respiratory diseases 

 

 

 

 

 



1 
 

 

CAPÍTULO 1 – Considerações gerais 

 

1. Introdução 

 

O complexo das doenças respiratórias dos suínos (CDRS) é um grande 

causador de perdas econômicas para a suinocultura mundial (MAES et al., 2023). 

Esse quadro envolve a infecção do sistema respiratório dos suínos por uma gama de 

agentes infecciosos, e, também é exacerbado por fatores estressantes e ambientais 

(BOETERS et al., 2023). O CDRS está associado a diminuição de índices 

zootécnicos, alta morbidade e mortalidade, aumento dos custos produtivos, devido 

compra de medicamentos e programas de controle e prevenção além da condenação 

de carcaças no frigorifico (MAES, D. et al., 2008). 

Um dos pontos chaves para o desenvolvimento do CDRS é a infecção por 

Mycoplasma (M.) hyopneumoniae (MAES et al., 2018). Essa bactéria é o agente 

etiológico da pneumonia enzoótica suína (PES), doença que afeta suínos no mundo 

todo, e é tida como endêmica na maioria dos rebanhos da suinocultura industrial no 

Brasil (ARRUDA et al., 2024; GABARDO et al., 2013). Animais de todas as idades 

podem ser afetados pela doença, entretanto os sinais clínicos são mais evidentes nas 

fases de crescimento e terminação (MAES, D. et al., 2018).  

A PES se caracteriza como broncopneumonia crônica, tendo como sinal clínico 

característico a manifestação de tosse seca e não produtiva. O diagnóstico definitivo 

da doença é realizado por meio da necropsia, histopatologia e ensaios moleculares 

(SIBILA et al., 2007). O M. hyopneumoniae é também um dos principais agentes 

envolvidos nos casos de pleurisias detectadas no frigorífico. Apesar de não ser o 

agente etiológico dessa lesão, que é atribuída as co-infecções por Pasteurella 

multocida (PM) e Actinobabillus pleuropneumoniae (APP) (PETRI et al., 2023; TURNI 

et al., 2021). 

Atualmente, as vacinas comerciais são ótimas aliadas visando a redução das 

lesões e sinais clínicos ocasionadas pela infecção por M. hyopneumoniae, e 

consequentes prejuízos zootécnicos causados pelos quadros de pneumonia e 

pleurisias. Entretanto as vacinas não previnem a colonização do trato respiratório dos 

suínos pelo M. hyopneumoniae, fazendo com que o sucesso no combate ao agente 



2 
 

 

envolva também os diversos elos das medidas de controle e biosseguridade 

(GALDEANO et al., 2019; SIBILA et al., 2007). 

O conhecimento dos fatores de patogenicidade e virulência do M. 

hyopneumoniae, bem como a variabilidade da bactéria a nível de campo ainda é 

escasso devido ao laborioso cultivo do agente. Entretanto são relatadas diferenças no 

curso clínico da doença bem como severidade das lesões, sendo essas relacionadas 

a tais características (BETLACH et al., 2019).  

Visando a melhor compreensão do envolvimento do M. hyopneumoniae nas 

lesões de pneumonia e pleurisias detectadas ao abate, levando em conta as 

características genéticas do agente. O objetivo do presente trabalho foi verificar a 

diversidade genética de M. hyopneumoniae associado a severidade das lesões 

pulmonares no momento do abate e a detecção de co-infecções em rebanhos de 

suínos do Brasil. 

 

2. Revisão de Literatura 

 

O Complexo da Doença Respiratória dos Suínos (CDRS) é um quadro clínico 

multifatorial complexo que afeta os suínos. Esse quadro é um dos problemas mais 

preocupantes na produção de suínos, é estimada uma perca de R$ 216 milhões por 

ano para a suinocultura brasileira decorrente de pleurisias, pneumonias e aderências 

(NASCIMENTO et al., 2018). Além disso o CDRS está associado a diminuição de 

índices zootécnicos, alta morbidade e mortalidade, aumento dos custos produtivos, 

devido aos altos custos de programas de controle e prevenção (MAES et al., 2023). 

O CDRS ocorre pela combinação de fatores infecciosos e ambientais. Dentre 

os agentes infecciosos associados a esse quadro destacam-se as bactérias 

Mycoplasma (M.) hyopneumoniae, Pasteurella (P.) multocida Actinobacillus (A) 

pleuropneumoniae, Glaesserella (G.) parasuis e Streptococcus (S.) suis, e agentes 

virais como circo vírus suíno tipo 2 (PCV-2), vírus da influenza suína (SIV) e o vírus 

da síndrome reprodutiva e respiratória dos suínos (PRRSV) (o qual o Brasil é livre) 

(DE CONTI et al., 2021; OPRIESSNIG; GIMÉNEZ-LIROLA; HALBUR, 2011) 

A interação entre esses patógenos pode ocasionar o quadro de pleurisia, ou 

pleurite, inflamação na pleura. Muitas vezes é assintomático, sendo detectado apenas 

no momento do abate. Por causar aderências na carcaça a lesão muitas vezes cursa 



3 
 

 

com condenação, agravando os prejuízos para a suinocultura devido lesões 

pulmonares (MAES et al., 2023). No Brasil, aproximadamente 50% dos animais 

desviados para o Departamento de Inspeção Final (DIF) no frigorífico, apresentam 

alguma lesão pulmonar (ALBERTON; MORES, 2008). Mesmo os quadros de 

pneumonia e pleurisia em suínos sendo ocasionados por interações complexas e na 

maioria das vezes envolvendo origem multifatorial, diversos estudos apontam o M. 

hyopneumoniae como agente primário, desencadeando co-infecções e agravamento 

dos quadros clínicos (HANSEN et al., 2010). 

M. hyopneumoniae, é uma bactéria espécie especifica para suínos domésticos 

(Sus scrofa domesticus) e javalis (Sus scrofa scrofa) (LEAL ZIMMER et al., 2020). É 

agente primário da pneumonia enzoótica dos suínos (PES) e envolvido no CDRS 

(MAES et al., 2023). O M. hyopneumoniae pertence ao filo Firmicutes, classe 

Mollicutes e a Família Mycoplasmataceae (TOLEDO et al., 2023). Em 2018, foi 

proposta uma reclassificação taxonômica, alterando o nome de Mycoplasma 

hyopneumoniae para Mesomycoplasma hyopneumoniae (GUPTA et al., 2018). Na 

literatura científica o termo Mycoplasma hyopneumoniae ainda é usado com 

frequência. Além disso, o artigo dessa dissertação foi publicado com o nome antigo, 

para fins de consistência, usaremos a terminologia Mycoplasma hyopneumoniae no 

decorrer de toda dissertação. 

As bactérias pertencentes a esse gênero distinguem-se fenotipicamente de 

outras bactérias pela ausência da parede celular e são reconhecidos por serem os 

menores microrganismos autorreplicantes já descritos (RAZIN, 1973). Devida à 

ausência da parede celular, possuem morfologia pleomórfica apresentando entre 0,2 

a 0,8 µm de diâmetro, genoma pequeno, 893 a 926 kbp que codificam 500 a 1000 

genes, com baixo conteúdo de guanina + citosina (23-40%) (GAUTIER-

BOUCHARDON, 2018; SIMIONATTO et al., 2013). Com isso, possuem um 

metabolismo limitado e poucas vias biossintéticas.  A limitada capacidade de 

biossíntese faz com que a bactéria precise obter aminoácidos, purinas, pirimidinas e 

componentes da membrana celular do ambiente de crescimento, o que torna o seu 

requerimento nutricional complexo e dependente do hospedeiro (RAZIN, 1973; 

SIMIONATTO et al., 2013). Essa característica explica a grande dificuldade do cultivo 



4 
 

 

in vitro, com a necessidade de produção de um meio de cultivo rico e complexo, 

tornando o isolamento do agente laborioso (GOODWIN; HURRELL, 1970).  

Baseado no sequênciamento do rRNA gene 16S M. hyopneumoniae é 

classificado no grupo Hominis dos micoplasmas, sendo filogeneticamente próximo de 

M. flocculare e M. hyorhinis, micoplasmas comensais do trato respiratório de suínos. 

Entretanto não é próximo de M. pneumoniae e M. pulmonis, que causam doença 

similar em humanos e ratos, respectivamente (PETERS et al., 2008). 

O M. hyopneumoniae é um patógeno globalmente distribuído (LEAL ZIMMER 

et al., 2020). A infecção é altamente prevalente, sendo cerca de 70% das fazendas 

endemicamente infectadas em todo mundo (MAES, D. et al., 2018). No Brasil recentes 

trabalhos reportaram prevalência de 97,7% e 92,45% em amostras de pulmão 

coletadas no momento do abate e submetidas a qPCR (ARRUDA et al., 2024; PETRI 

et al., 2023). 

O patógeno pode ser introduzido no rebanho através da compra de marrãs com 

infecção subclínica, transmissão pelo ar ou por fômites (FANO; PIJOAN; DEE, 2005). 

Sendo que foi demostrado que a bactéria pode ser detectada no ar em distâncias de 

4,7 e 9,2 km (DEE et al., 2009; OTAKE et al., 2010). Uma vez introduzida no rebanho 

M. hyopneumoniae pode se disseminar através da transmissão horizontal, por meio 

de contato nariz com nariz entre matriz e leitão, e também por aerossóis. O agente 

não se dissemina pela via intrauterina, nem pelo colostro ou leite (VILLARREAL et al., 

2010). A transmissão indireta via fômites é possível, mas acredita-se que de menor 

importância no campo (BATISTA et al., 2004; PITKIN et al., 2011). A doença se espalha 

lentamente. Em um ambiente experimental, MEYNS et al., (2004) mostraram que um 

suíno infectado antes do desmame infectará outro animal na mesma baia durante o 

período de creche.  

A patogênese da infecção por M. hyopneumoniae é complexa, se caracteriza 

primariamente pela colonização do epitélio do trato respiratório dos suínos, resultando 

em ciliostase, perda de cílios e até mesmo a morte de células do epitélio respiratório 

tornando o aparato mucociliar menos eficiente (MAES, D. et al., 2018). A partir disso, 

a bactéria induz uma reação inflamatória exacerbada e suprime o sistema imune do 

hospedeiro, o que facilita a invasão de patógenos secundários, culminando no CDRS 

(SIMIONATTO et al., 2013). 



5 
 

 

Diferentes estudos demonstraram variabilidade entre cepas de M. 

hyopneumoniae nos níveos antigênico, proteômico, de transcriptoma, patogenicidade 

e também a nível genômico (BETLACH et al., 2019). Diversas técnicas moleculares 

foram padronizadas para caracterizar cepas de M. hyopneumoniae. Como o 

Polimorfismo de Comprimento de Fragmento Amplificado (AFLP); Análise de DNA 

Polimórfico Amplificado por Restrição (RAPD); Eletroforese em gel de campo pulsado, 

(PFGE); Análise de DNA polimórfico amplificado ao acaso (RFLP); Análise de 

microssatélite de DNA; Tipagem por sequenciamento de múltiplos locus (MLST), 

Análise do polimorfismo numérico de sequências repetitivas em múltiplos loci (MLVA), 

sequenciamento parcial de genes e sequenciamento completo do genoma (BETLACH 

et al., 2019).  

É importante salientar que cada método molecular apresenta suas vantagens 

e também desvantagens, principalmente levando em conta reprodutibilidade e 

comparação de resultados interlaboratoriais. Entretanto, dada a dificuldade de se 

realizar o isolamento de cepas de campo, ferramentas moleculares são extremamente 

uteis para a caracterização e estudo do patógeno. 

Nos recentes anos foram realizados diferentes estudos buscando a 

caracterização de cepas e compressão da dinâmica de infecção das diferentes cepas. 

No Brasil, já foram analisados genótipos de M. hyopneumoniae através de MLVA, que 

demonstraram alta diversidade dentro e entre as granjas de suínos (DOS SANTOS et 

al., 2015). Andrade et al., (2023), observou em trabalho realizado com amostras de 

pulmão oriundas de Minas Gerais que diferentes genótipos de M. hyopneumoniae 

estão distribuídos aleatoriamente em várias regiões do estado, sem um padrão de 

estrutura populacional e geográfica específico, sendo identificados 43 genótipos no 

estado, e a maioria das granjas (60%) apresentou mais de um genótipo. Takeuti et al., 

(2017) também observou diversidade genética dentro do mesmo rebanho, o que pode 

indicar a relação do genótipo com a severidade do quadro clínico. Entretanto ainda 

não há banco de dados para compartilhar as classificações encontradas nos estudos 

que utilizam essa técnica o que o que dificulta comparação de resultados e 

acompanhamento epidemiológico do M. hyopneumoniae (BETLACH et al., 2019; 

CALUS et al., 2007; MAYOR et al., 2008; SOSA et al., 2019).  



6 
 

 

Com isso, a técnica molecular MLST vem sendo utilizada para estudos 

epidemiológicos de tipagem molecular de patógenos, incluindo M. hyopneumoniae 

(MAYOR et al., 2008). Este método se baseia no sequenciamento de genes 

housekeeping, que são responsáveis por codificar funções vitais para célula (LUIZ, 

2012). Por ser baseado na sequência dos nucleotídeos, fornece resultados 

padronizados e específicos, podendo discriminar linhagens com uma única alteração 

nucleotídica em um dos genes analisados. Além disso, os resultados são 

padronizados e podem ser comparados facilmente entre laboratórios através do banco 

de dados público: pubmlst.org (JOLLEY; BRAY; MAIDEN, 2024). O MLST para M. 

hyopneumoniae foi estabelecido para avaliação convencional de 3 genes (adk, rpoB, 

e tpiA), nos últimos anos há trabalhos em diversas regiões do mundo utilizando a 

técnica para compreender as características das cepas circulantes. Entretanto os 

dados para comparação ainda são escassos (FELDE et al., 2018; MAYOR et al., 

2008). Atualmente apenas Balestrin et al., (2019) desenvolveram estudos com 

amostras clínicas brasileiras utilizando essa técnica. Em estudo realizado por Zhang 

et al., (2021) na China reportou 60 novos genótipos de M. hyopneumoniae naquele 

país.  

Por fim, este projeto de pesquisa objetivou verificar a diversidade genética de 

M. hyopneumoniae associado a severidade das lesões pulmonares no momento do 

abate em rebanhos suínos do Brasil, buscando melhor compreensão da dinâmica de 

infecção e associação desse agente com demais patógenos envolvidos no CDRS, 

permitindo que possamos melhorar as medidas de controle de infecção do M. 

hyopneumoniae, consequentemente amenizando as percas causadas por 

pneumonias e pleurisias.  

 

3. Referências 

 

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ferramenta de diagnóstico das doenças respiratórias dos suínos. Acta Scientiae 
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ANDRADE, M. R. et al. Genetic diversity of Mycoplasma hyopneumoniae in finishing 
pigs in Minas Gerais. Pesq. Vet. Bras., v. 5150, p. 1–11, 2023. 
 



7 
 

 

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BALESTRIN, E. et al. Clonality of Mycoplasma hyopneumoniae in swine farms from 
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BETLACH, A. M. et al. Mycoplasma hyopneumoniae variability: Current trends and 
proposed terminology for genomic classification. [S. l.]: Blackwell Publishing Ltd, 
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maio 2024. 
 
 
CALUS, D. et al. Protein variability among Mycoplasma hyopneumoniae isolates. 
Veterinary Microbiology, v. 120, p. 284–291, 2007.  
 
 
DE CONTI, E. R. et al. Agents of pneumonia in slaughtered pigs in southern Brazil. 
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8 
 

 

FELDE, O. et al. Genotyping Mycoplasma hyopneumoniae isolates based on multi-
locus sequence typing , multiple-locus variable-number tandem repeat analysis and 
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GABARDO, M.; ZANDONAI, A.; CAMARGO, M.; GAVA, A.; CRISTANI, J.; 
TRAVERSO, S. Caracterização Sanitária de Suínos Criados Em Sistema “Wean-to-
Finish.” Medicina Veterinária 2013, 7, 23–31. 
 
 
GALDEANO, J. V. B. et al. Cross-sectional study of seropositivity, lung lesions and 
associated risk factors of the main pathogens of Porcine Respiratory Diseases 
Complex (PRDC) in Goiás, Brazil. Porcine Health Management, [s. l.], v. 5, n. 1, p. 
1–10, 2019.  
 
 
GAUTIER-BOUCHARDON, A. V. Antimicrobial Resistance in Mycoplasma spp . 
Microbiology Spectrum, v. 6, n. 4, 27 jul. 2018. 
 
 
GOODWIN, R. F. W.; HURRELL, J. M. W. Further observations on the problem of 
isolating Mycoplasma suipneutnoniae from field cases of enzootic pneumonia in pigs. 
Journal of Hygiene, v. 68, n. 2, p. 313–325, 1970. 
 
 
GUPTA, R. S. et al. Phylogenetic framework for the phylum Tenericutes based on 
genome sequence data: proposal for the creation of a new order Mycoplasmoidales 
ord. nov., containing two new families Mycoplasmoidaceae fam. nov. and 
Metamycoplasmataceae fam. nov. harbouring Eperythrozoon, Ureaplasma and five 
novel genera. Antonie van Leeuwenhoek, International Journal of General and 
Molecular Microbiology, v. 111, n. 9, p. 1583–1630, 1 set. 2018. 
 
 
HANSEN, M. S. et al. An investigation of the pathology and pathogens associated with 
porcine respiratory disease complex in Denmark. Journal of Comparative 
Pathology, [s. l.], v. 143, n. 2–3, p. 120–131, 2010.  
 
 
JOLLEY, K. A.; BRAY, J. E.; MAIDEN, M. C. J. Open-access bacterial population 
genomics : BIGSdb software , the PubMLST . org website and their applications. 
Wellcome Open Res, p. 1–20, 2024.  
 
 
LEAL ZIMMER, F. M. A. et al. Pathogenicity & virulence of Mycoplasma 
hyopneumoniae. VirulenceBellwether Publishing, Ltd., , 2020. 
 
 
 
 



9 
 

 

LUIZ, L. M. P. Identificação e agrupamento genético por MLST e PFGE de isolados 
bacterianos obtidos de fazendas da região do Campo das Vertentes, Minas Gerais. 
107 f.  2012. - UFMG, [s. l.], 2012.  
 
 
MAES, D. et al. Control of Mycoplasma hyopneumoniae infections in pigs. Veterinary 
Microbiology, 2008.   
 
 
MAES, D. et al. Update on Mycoplasma hyopneumoniae infections in pigs: Knowledge 
gaps for improved disease control. Transboundary and Emerging Diseases, [s. l.], 
v. 65, p. 110–124, 2018. 
 
 
MAES, Dominiek et al. Review on the methodology to assess respiratory tract lesions 
in pigs and their production impact. Veterinary Research, 2023.  
 
 
MAYOR, D. et al. Multilocus sequence typing (MLST) of Mycoplasma hyopneumoniae: 
A diverse pathogen with limited clonality. Veterinary Microbiology, [s. l.], v. 127, n. 1–
2, p. 63–72, 2008.  
 
 
MEYNS, T. et al. Quantification of the spread of Mycoplasma hyopneumoniae in 
nursery pigs using transmission experiments. Preventive Veterinary Medicine, v. 66, 
n. 1–4, p. 265–275, 15 dez. 2004. 
 
 
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OPRIESSNIG, T.; GIMÉNEZ-LIROLA, L. G.; HALBUR, P. G. Polymicrobial respiratory 
disease in pigs. Anim Health Res Rev, 2011.  
 
 
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Mycoplasma hyopneumoniae from a swine population infected with multiple viral 
variants. Veterinary Microbiology, v. 145, n. 3–4, p. 198–208, 26 out. 2010. 
 
 
PETERS, I. R. et al. RNase P RNA gene (rnpB) phylogeny of hemoplasmas and other 
Mycoplasma species. Journal of Clinical Microbiology, v. 46, n. 5, p. 1873– 1877, 
maio 2008. 
 
 
 
 



10 
 

 

PETRI, F. A. M. et al. Associations between Pleurisy and the Main Bacterial Pathogens 
of the Porcine Respiratory Diseases Complex (PRDC). Animals, v. 13, n. 9, 1 maio 
2023. 
 
 
PITKIN, A. et al. A one-night downtime period prevents the spread of porcine 
reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae by 
personnel and fomites (boots and coveralls). Journal of Swine Health and 
Production, 2011. 
 
 
RAZIN, S. Physiology of Mycoplasmas. Advances in Microbial Physiology, v. 10, p. 
1–80, 1973. 
 
 
SIBILA, M. et al. Chronological study of Mycoplasma hyopneumoniae infection, 
seroconversion and associated lung lesions in vaccinated and non-vaccinated pigs. 
Veterinary Microbiology, [s. l.], v. 122, n. 1–2, p. 97–107, 2007. 
 
 
SIMIONATTO, S. et al. Mycoplasma hyopneumoniae: From disease to vaccine 
development. Veterinary Microbiology, 2013.  
 
 
SOSA, C. et al. Genetic diversity of Mycoplasma hyopneumoniae in Mendoza 
province. Revista Argentina de Microbiología. v. 51, n. 3, p. 229–233, 2019.  
 
 
TAKEUTI, K. L. et al. Infection dynamics and genetic variability of Mycoplasma 
hyopneumoniae in self-replacement gilts. Veterinary Microbiology, [s. l.], v. 208, p. 
18–24, 2017.  
 
 
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Mycoplasma hyopneumoniae. Frontiers in Microbiology, v. 14, 2023. 
 
 
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Queensland Australia. Australian Veterinary Journal, v. 99, n. 5, p. 163–171, 1 maio 
2021. 
 
 
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Veterinární medicína, 2010. 
 
 
 



11 
 

 

ZHANG, H. et al. Genotype diversity of Mycoplasma Hyopneumoniae in Chinese swine 
herds based on multilocus sequence typing. BMC Veterinary Research, [s. l.], v. 17, 
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12 
 

1Este capítulo corresponde ao artigo científico publicado na revista Microorganisms 
2024, 12, 1988. (https://doi.org/10.3390/microorganisms12101988) 
 

1CAPÍTULO 2 – Exploring the genetic diversity of Mycoplasma hyopneumoniae 

in pigs with pneumonia and pleurisy at slaughter 

 

Ana Karolina Panneitz1, Eduarda Ribeiro Braga1, Fernando Antônio Moreira Petri1, 

Jean Carlo Olivo Menegatt2, David Driemeier2, Dominiek Maes3, Luís Guilherme de 

Oliveira1* 

1 Swine Medicine Laboratory, School of Agricultural and Veterinary Sciences, São Paulo State 

University (Unesp), Jaboticabal 14884-900, São Paulo, Brazil; 

2 Veterinary Pathology Department, Federal University of Rio Grande do Sul (UFRGS), Porto 

Alegre 91540-000, Brazil; 

3 Unit of Porcine Health Management, Faculty of Veterinary Medicine, Ghent University, 

Merelbeke 9820, Belgium.; 

 

Abstract: Mycoplasma (M.) hyopneumoniae is the key pathogen of the porcine 

respiratory disease complex (PRDC) and contribute to pleurisy in pigs. Due to its 

limited metabolism and laborious cultivation, molecular tools are useful for diagnostic. 

This study investigated the genetic diversity of M. hyopneumoniae in slaughter pigs 

with pneumonia and pleurisy, and assessed co-infections by Pasteurella multocida 

type A (PM), Actinobacillus pleuropneumoniae (APP) and swine Influenza virus A 

(sIVA). Lungs (N=70) with different pleurisy scores and lesions compatible with M. 

hyopneumoniae infection were collected for convenience. Macroscopic and 

microscopic evaluations were performed. M. hyopneumoniae was detected using 

qPCR, and MLST was used for genetic charac-terization. Co-infections with PM and 

APP were also evaluated by qPCR, while immunohisto-chemistry assessed sIVA 

infection. All lungs were positive for M. hyopneumoniae. Histopathology confirmed M. 

hyopneumoniae associated lesions. MLST characterization was possible in 25 lungs, 

and revealed 10 distinct allelic profiles, none matching known sequence types in the 

public data-base. Co-infections were detected in 40% of the samples with APP and 2% 

with PM, with 12% showing both pathogens, 52% of the samples presented 

microscopic lesions compatible with sIVA infection. The diverse genetic profiles found  

underscores the necessity for research on isolation and potential pathogenic 

variations. 



13 
 

1Este capítulo corresponde ao artigo científico publicado na revista Microorganisms 
2024, 12, 1988. (https://doi.org/10.3390/microorganisms12101988) 
 

Keywords: alleles; BALT hyperplasia; MLST; pneumonia; porcine enzootic 

pneumonia; respiratory disease 

 



14 
 

 

1. Introduction 

Bacteria belonging to the genus Mycoplasma spp. are the smallest self-

replicating microorganisms and are phenotypically distinguished from other bacteria 

by the ab-sence of a cell wall [1]. These characteristics contribute to the difficulty of in 

vitro cultivating the bacteria belonging to this genus. Mycoplasma (M.) hyopneumoniae 

is the etiologic agent of porcine enzootic pneumonia (PEP) and one of the primary 

agents of the porcine respiratory disease complex (PRDC) [2]. The clinical 

manifestation of infection typically is characterized as chronic bronchopneumonia, with 

a non-productive cough being the most characteristic sign and is most evident in the 

growth and finishing phases [3]. The losses linked to M. hyopneumoniae infection are 

associated with a reduction in zootechnical indices, increased susceptibility to 

secondary infections that can lead to higher antibiotic use and carcasses being 

condemned in the slaughterhouse [2,4]. Ferraz et al. [5] estimated an economic impact 

of US$6.55 per affected pig slaughtered due to the M. hyopneumoniae infection. The 

definitive diagnosis of the disease is achieved by necropsy, histopathology, and 

molecular assays [6]. M. hyopneumoniae is also often detected in cases of pleurisy in 

slaughter pigs, although the pathogen itself does not induce this lesion, the 

microenvironment resulting from the lung lesions likely provides favorable conditions 

for co-infections like Pasteurella multocida type A (PM) and Actinobacillus 

pleuropneumoniae (APP) [7] Pleurisy is an important parameter for evaluation in 

slaughterhouses because it can lead to the condemnation of carcasses, generating a 

higher economic impact for the industry and challenging diagnoses [8,9]. Furthermore, 

previous studies indicate that the prevalence of this lesion in Brazilian herds is higher 

than 10% [4,8]. 

Knowledge about the pathogenicity and virulence factors of M. hyopneumoniae 

is scarce because of the in vitro growth characteristics [1]. However, there are 

differences in the clinical course of the disease and the severity of the lesions, which 

might be related to these characteristics [10]. Because it is a difficult bacterium to 

cultivate, different molecular biology tools are used for the epidemiological 

characterization of M. hyopneumoniae. Multi-locus Sequence Typing (MLST) and 

Multilocus Variable Number Tandem Repeats (MLVA) are the most widely used 

techniques due to their high power to discriminate variants. There are also other 



15 
 

 

methods for genotyping, such as pulsed-field gel electrophoresis (PFGE), random 

amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) 

and high-throughput sequencing [10,11]. Technological advances in next-generation 

sequencing platforms have the capacity to improve the accuracy of genetic diversity 

studies of microorganisms, but more studies are still needed to create robust 

databases [12]. 

The MLST technique is well-established and standardized for characterizing 

different species of bacteria. For M. hyopneumoniae several loci have been evaluated 

for this technique, both putative genes and house-keeping genes. Currently, for this 

pathogen, it analyses three target genes (adk, rpoB and tipA) [13]. One advantage of 

MLST is its public, online database for sharing and comparing results. So far, the MLST 

technique can discriminate 392 isolates in 469 Sequence Types (STs) 

(https://pubmlst.org/organisms/mycoplasma-hyopneumoniae accessed on 2 August 

2024). 

Some studies on the genetic diversity of M. hyopneumoniae in Brazil have 

shown high genetic diversity [14–16]. However, these studies used another tool for 

genetic characterization, which is a barrier to comparing results. Takeuti et al. (2017) 

[14] and dos Santos et al. (2015) [15] used the MLVA technique to characterize M. 

hyopneumoniae, while, Assao et al. (2019) [16] accessed the similarity according to 

the presence of some of the genes studied. Balestrin et al. [17] used the MLST 

approach to investigate the genetic diversity of M. hyopneumoniae strains in Brazil, but 

this study found a low genetic diversity between strains and also no relation with the 

geographical location. In that study, five STs were identified as circulating in Brazil, and 

most of the samples were identified as belonging to the ST-69 group (23-33-26). The 

contrast in results emphasizes the necessity of increasing the number of studies on 

this topic, to provide data for comparisons.  

Based on the high prevalence, the economic impact, and the differences in 

virulence and severity of the disease associated with the pathogen, our study 

investigated the genetic diversity of M. hyopneumoniae using the MLST technique in 

slaughter pigs with pneumonia and pleurisy and the occurrence of co-infections 

involving APP, PM and sIVA. 



16 
 

 

2. Materials and Methods 

2.1 Macroscopic evaluation of lungs and sample collection  

Between 2022 and 2023, lungs of pigs were collected for convenience in a 

slaughterhouse located in Guariba, São Paulo, Brazil. The lungs presenting different 

pleurisy scores and lesions suggestive of M. hyopneumoniae infection (cranioventral 

region of the lung showing areas of consolidation with purple to grey discoloration). 

Batches were sampled from four important pig-producing states in Brazil (Figure 1), 

including seven distinct cities: Uberlândia (Minas Gerais), São Gabriel do Oeste and 

Campo Grande (Mato Grosso do Sul), Cerqueira César (São Paulo), Guarujá do Sul, 

São José dos Cedros and Videira (Santa Catarina). 

 

Figure 1. Geographical distribution of batches sampled on the Brazilian map. Red dots 

represent the farms’ city. The allelic profile of M. hyopneunoniae from three states was 

evaluated. SC: Santa Catarina, MS: Mato Grosso do Sul, SP: São Paulo. The area 

marked in black in each state indicates the city from which the herds originated. * 

Figure created using QGIS software version 3.4.5. 

 

After evisceration, lungs were classified according to the severity of pleurisy 

using the Slaughterhouse Pleurisy Evaluation System (SPES), proposed by Dottori et 

al. (2007) [18]. Briefly, the classification is based on the severity of the pleurisy lesions: 



17 
 

 

score 0 in absence of pleurisy, score 1 pleurisy affecting the cranioventral portion of 

the lung, score 2 discrete unilateral pleurisy in caudal lobe, score 3 discrete bilateral 

pleurisy in the caudal lobes or extensive unilateral pleurisy in the caudal lobe and score 

4 resembles an extensive bilateral lesion bilateral pleurisy in the caudal lobes. From 

each batch, 10 pigs were sampled, namely 2 pigs per pleurisy score. (N=70). 

The severity of the pneumonia lesions was assessed following the methodology 

proposed by Madec and Kobisch (1982) [19]. Briefly, each lung lobe receives a score 

(0-4): (0) no lesion; (1) lesion affecting <25% of the lobe surface; (2) lesion affecting 

25-49% of the surface; (3) lesion affecting 50-74% of the surface; (4) lesion affecting 

≥ 75% of the surface. Points per lobe are summed to provide an overall area lung score 

(0-28). 

After the gross evaluation, fragments of lung tissue were collected from the 

apical-cardiac-diaphragmatic lobes at the edge between affected and non-affected 

tissue (including bronchi) for microscopic and molecular analysis. The samples for 

PCR analyses were stored in duplicate for molecular assays in RNAse and DNAse-

free cryogenic tubes and stored in a freezer at -80 ºC until laboratory analysis. The 

lung samples for histopathological and immunohistochemical analysis were stored in 

10% buffered formalin at a 1:10 tissue/formalin ratio. All the procedures described in 

this study were approved by the Animal Use Ethics Committee (CEUA) of FCAV/ 

Unesp-Campus Jaboticabal, under protocol number #001113/23. 

 

2.2. DNA extraction and conventional PCR (cPCR) for endogenous 

mammalian glycer-aldehyde 3-phosphate dehydrogenase (gapdh) gene  

DNA was extracted from the lung samples using an in-house Tris-HCl protocol 

adapted from Kuramae-Izioka (1977) [20] The concentration and quality of the ex-

tracted DNA were measured using spectrophotometry (NanoDrop® One Spectropho-

tometer, Thermo Fisher Scientific, USA). 

To check for possible inhibitors in the extracted DNA samples and the 

occurrence of false negatives, all the samples were tested by conventional PCR 

(cPCR) as described by Birkenheuer et al.(2003) [21]. This cPCR detects the presence 

of a fragment of the endogenous mammalian glyceraldehyde 3-phosphate 



18 
 

 

dehydrogenase gene (gapdh). The samples were only submitted to qPCR if this gene 

was amplified, avoiding false negative results due to PCR inhibitors. 

 

2.3. Real-time PCR (qPCR) for p102 gene of M. hyopneumoniae 

The DNA extracted, positive for the presence of gapdh gene, was submitted to 

qPCR to detect the fragment of the p102 gene (adhesion protein) of M. 

hyopneumoniae, as described by Fourour et al. (2018) [22] and adapted by Almeida et 

al. (2020) [23]. Ta-ble S1 describes the target genes, the primers and probes and the 

amplicon size. 

 

2.4. Molecular characterization of M. hyopneumoniae 

The multilocus sequence typing (MLST) technique was performed as described 

by Mayor et al. (2008) [13] and preconized by the MLST public database 

(https://pubmlst.org/organisms/mycoplasma-hyopneumoniae). Amplifying via cPCR 

three housekeeping genes adk (encoding adenylate kinase), rpoB (coding for the b-

subunit of the RNA-Polymerase) and tpiA (encodes the triosephosphate Isomerase). 

Table S2 describes the target genes, the primers and the amplicon size. 

The cPCR amplified products were submitted to horizontal electrophoresis in a 

1.5% agarose gel stained with SYBR SAFE (10000X) (Thermo Fisher Scientific®, 

USA), using TBE pH 8.0 running buffer at 110V/500mA for 60 minutes. A molecular 

weight marker of 100 base pairs (Cellco Biotec, Brazil) was used in each 

electrophoresis, and the amplified fragments were visualized in an ultraviolet light 

transilluminator. The amplicons were purified enzymatically using ExoSAP-IT (Thermo 

Fisher Scientific®, USA) according to the manufacturer's instructions. The purified 

genetic material was quantified using spectrophotometry (NanoDrop® One 

Spectrophotometer, Thermo Fisher Scientific®, USA). 

Samples positive for the 3 target genes were submitted to Sanger sequencing 

(N=25). Approximately 10-20 ng of the purified PCR product was submitted to 

sequencing, using the same cPCR primers. The amplified products were purified with 

a commercial kit and sequenced using the BigDye™ Terminator v3.1 Cycle 

Sequencing Kit (Thermo Fisher Scientific™, USA) and ABI PRISM 310DNA Analyzer 

(Applied Bi-osystems™, USA). 



19 
 

 

Sequences were assembled, edited, and trimmed using BioNumerics version 

7.6.3 (Applied Maths, Belgium). Sequence types (ST) were assigned by BioNumerics 

using allelic profiles in the order adk-rpoB-tpiA genes. The same order was used to 

concatenate sequences for phylogenetic analysis, cluster analysis was performed 

using the Neighbor-Joining method. The evolutionary distances were computed using 

the p-distance method and are in the units of the number of base differences per site 

with Molecular Evolutionary Genetics Analysis (MEGA) software (version 11; https:// 

www. megasoftware.net/). 

 

2.5.  Detection of bacterial co-infections by multiplex qPCR 

A subset of positive samples for 3 target genes for MLST (N=25) was selected 

for the co-infection detection. The protocols for the detection of APP and PM were 

previously described by Sunaga et al. (2020) [7] and Goecke et al. (2021) [24] and 

adapted by Petri et al. (2023) [4]. The multiplex qPCR targets were the omlA gene 

(virulence protein) and the kmt1 gene (membrane lipoprotein) from APP and PM, 

respectively. Table S1 describes the target genes, the primers and probes and the 

amplicon size. 

 

2.6. Histopathological and immunohistochemical evaluation 

Lung samples positive for MLST target genes (N=25) were routinely processed 

and stained with hematoxylin and eosin (HE) for histopathological examination. M. 

hyopneumoniae lesions were systematically analyzed using a methodology adapted 

from Hansen et al. (2010) [25]. Bronchus-associated lymphoid tissue (BALT) was 

carefully examined and graded for the presence of hyperplasia as follows: (0) absent, 

in which no inflammatory infiltrate was observed in the peribronchial and 

peribronchiolar tis-sues and no evidence of lymphoid nodules; (+) mild diffuse 

inflammatory infiltrate of lymphocytes in the peribronchial and peribronchiolar tissues 

including the alveolar septa associated with the presence of a few lymphoid nodules; 

(++) moderate diffuse inflammatory infiltrate of lymphocytes in the peribronchial and 

peribronchiolar tissues including the alveolar septa associated with a marked number 

of lymphoid nodules; (+++) extensive number of lymphoid nodules with apparent 

occlusion of the air-way lumen.  



20 
 

 

Secondary bacterial lung lesions were classified according to airway exudates, 

as suppurative bronchopneumonia (a marked increase of neutrophils in the alveoli, 

bronchi, and bronchioles); necrosuppurative bronchopneumonia (a marked increase 

of neutrophils in the airway associated with areas of lung necrosis); and 

pleuropneumonia (acute/subacute pleuropneumonia: visceral pleura thickened by 

intense fibrin deposition, necrotic cellular debris, and many degenerate neutrophils, as 

well as areas of necrosuppurative bronchopneumonia; or chronic pleuropneumonia: 

characterized by extensive fibrous connective tissue proliferation on the pleura, fibrin 

deposition, and necrotic lung areas).  

Chronic pleural lesions (chronic pleuritis) were evaluated separately, 

characterized by thickening of the pleura due to the proliferation of fibrous connective 

tissue and blood vessels, and mild inflammatory infiltration of lymphocytes and macro-

phages. Additionally, non-specific lesions such as type II pneumocyte hyperplasia and 

alveolar edema were evaluated for their presence or absence.  

Immunohistochemistry (IHC) was applied to paraffin-embedded tissues in cases 

with histological lesions compatible with swine Influenza virus (sIVA) infection, as 

described by Watanabe et al. (2012) [26]. sIVA lesions were identified based on 

characteristic histological findings of lymphocytic bronchointerstitial pneumonia, 

accompanied by necrotic, proliferative, or obliterative bronchiolitis. 

 

2.7. Statistical analysis 

The variables were assessed for normality and homoscedasticity using the Shapiro-

Wilk test. Descriptive statistics are presented as mean and standard deviation for 

normally distributed data, and as a median with a range between first and third quartiles 

(IQR) when the data is not normally distributed. Parametric data were analysed using 

analysis of variance (ANOVA) followed Tukey’s test for multiple comparisons of means 

(p<0.05). Non-parametric data were analysed using the Mann-Whitney test for 

pairwise comparisons of medians (p<0.05). All data analyses were performed using 

the GraphPad Prism 10.2.2 software (La Jolla, USA). 

 

3. Results 

 



21 
 

 

3.1. Gross evaluation and qPCR for the detection of M. hyopneumoniae 

The lung consolidation score ranged from 0 to 28 points. Table S3 shows the 

consolidation points per lung lobe, as well as the total score per lung and the score of 

pleurisy. The median lung consolidation score across all lungs was 6 points, with an 

inter-quartile range (IQR) of 6 (3 – 9). There was a statistical difference (p-value 

0.0001) when comparing the median scores of lungs with pleurisy (7; IQR: 6 (4-10)) 

and without pleurisy lesions (3; IQR: 3.25 (1 - 4.25)). There was also a statistical 

difference in lung consolidation scores according to the severity of pleurisy (p-value 

0.0023), with a difference between the means of pleurisy score 0 (2.79 ±1.88) and 

scores 2, 3 and 4 (7.29 ±4.12; 8.17 ±4.76; 7.64±5.37) respectively.  

The results demonstrated the presence of DNA de M. hyopneumoniae in all 

collected lung samples (N=70). The mean Ct obtained was 26.69, without statistical 

difference when comparing the different severities of pleurisy (p-value 0.25). 

 

3.2. Characterization of the allelic profiles of M. hyopneumoniae 

The three housekeeping genes (adk, rpoB and tpiA) were detected in 25 of the 

70 samples positive for M. hyopneunoniae in the qPCR (35.71%). The mean Ct 

(23.16±3.43) for the detection of the p102 gene of M. hyopneumoniae was statistically 

lower (p-value <0.0001) in the samples where it was possible to amplify the three 

genes required to apply the MLST technique, compared to samples where only one or 

two of the maintenance genes were amplified (28.65±3.93) and the MLST technique 

could not be applied. Figure 2 shows the mean and standard deviation of the Cts of  

the lung samples targeting the p102 gene fragment eligible for the MLST 

technique. 

 



22 
 

 

 

Figure 2. Mean and standard deviation of M. hyopneumoniae detection Cts in qPCR 

targeting the p102 gene fragment. Comparing values of samples eligible or not for 

application of the MLST technique (p< 0.05). 

 

All the sequences corresponded to the allelic types previously deposited in the 

public database. Three allelic types were identified for the adk gene: 6; 13 and 23. For 

the rpoB gene, alleles 11; 18 and 33 were identified. For the tpiA gene, the alleles 

observed were 19; 26; 41; 60; 62; 79 and 88.  

When these alleles were combined (respecting the order of description of the 

target genes: adk, rpoB and tpiA), 10 different allelic profiles were observed as 

described in table 1. The allelic profiles found did not correspond to any sequence type 

already re-ported in the public database. Figure 3 shows the phylogenetic tree based 

on the sequence of the concatenated genes. The tree can be divided into 16 clades. 

Shorter branches are observed in the relationship between CG 9 and SG 7, as well as 

CG 1 and SG 4, these samples originated from the same producing state (Mato 

Grosso). However, the sequences SG 5 and SJ 10 form a very close pair, suggesting 

a high degree of similarity, even though their geographical origins are far apart.  

Regarding the geographical distribution, the most prevalent allelic profile (23-

18-26, found in 56% of the samples, 14/25) was exclusively observed in lung lesion 

samples from the central-west region. Additionally, allelic profiles 23-18-41; 13-18-26; 

23-18-60 and 23-11-26 were also found in this region. None of these allelic profiles 

were found in the southern region of Brazil, where the following profiles were observed: 

13-18-79; 13-18-88; 23-33-62 and 6-11-41. Moreover, a distinct profile from the other 



23 
 

 

regions was identified in the southeast region (23-11-19). Figure 1. Show the allelic 

profiles per state. 

 

Table 1. Allelic profiles found in this study (following the order adk, rpoB, tpiA) and their 

frequency (percentage and absolute number). 

Allelic profiles Samples 

23-18-26 56% (14/25) 

23-18-41 8% (2/25) 

13-18-79 8% (2/25) 

13-18-26 4% (1/25) 

23-18-60 4% (1/25) 

13-18-88 4% (1/25) 

23-11-19 4% (1/25) 

23-33-62 4% (1/25) 

6-11-41 4% (1/25) 

23-11-26 4% (1/25) 

 

 

 

Figure 3. Dendrogram created using the Neighbor-Joining method, based on the 

sequence of the concatenated genes following the order adk, rpoB and tpiA. The 



24 
 

 

distances were computed using the p-distance method and are in the units of the 

number of base differences per site. 

 

3.3. qPCR for the detection of bacterial co-infection 

In the detection of co-infection agents associated with pleurisy lesions, of the 25 

samples submitted to qPCR, 8 were positive for Pasteurella multocida type A (PM) 

(32%),10 for Actinobacillus pleuropneumoniae (APP) (40%), and three for both agents 

(12%). The qPCR Cts are summarized in Table 2. 

 

Table 2. Pig batch origin (City/state), allelic profile, pleurisy score, detection Cts of 

Mhyo: Mycoplasma hyopneumoniae, APP: Actinobacillus pleuropneumoniae, PM: 

Pasteurella multocida type A, and presence of microscopic lesions like sIVA infection* 

of each sampled lung. 

Sample Herd origin 
MLST allele profile Pleurisy 

Score  
Mhyo 

Ct  
APP Ct  

PM 
Ct  

Lesions 
like sIVA 
infection adk rpoB tpiA 

CG 1 
Campo Grande 

(MS) 
23 18 26 0 23.79 30.73 ND No 

CG 3 
Campo Grande 

(MS) 
23 18 26 1 18.59 38.00 ND Yes 

CG 4 
Campo Grande 

(MS) 
23 18 26 1 26.33 29.22 ND Yes 

SG 3 
São Gabriel do 

Oeste (MS) 
23 18 26 1 23.39 ND ND No 

SG 4 
São Gabriel do 

Oeste (MS) 
23 18 26 1 30.27 ND ND No 

CG 5 
Campo Grande 

(MS) 
23 18 26 2 28.54 31.71 ND No 

CG 6 
Campo Grande 

(MS) 
23 18 26 2 18.82 ND ND Yes 

SG 6 
São Gabriel do 

Oeste (MS) 
23 18 26 2 22.76 ND ND Yes 

CG 7 
Campo Grande 

(MS) 
23 18 26 3 21.78 27.96 ND No 

SG 7 
São Gabriel do 

Oeste (MS) 
23 18 26 3 20.43 ND ND No 

SG 8 
São Gabriel do 

Oeste (MS) 
23 18 26 3 23.45 35.20 ND Yes 

CG 9 
Campo Grande 

(MS) 
23 18 26 4 17.24 29.34 23.69 Yes 

SG 9 
São Gabriel do 

Oeste (MS) 
23 18 26 4 21.35 ND ND Yes 

SG 10 
São Gabriel do 

Oeste (MS) 
23 18 26 4 22.27 ND ND Yes 

SG 2 
São Gabriel do 

Oeste (MS) 
23 18 41 0 18.24 ND ND Yes 

SG 5 
São Gabriel do 

Oeste (MS) 
23 18 41 2 25.65 ND 28.60 Yes 

GS 9 
Guarujá do Sul 

(SC) 
13 18 79 4 21.52 ND 23.48 Yes 



25 
 

 

GS 10 
Guarujá do Sul 

(SC) 
13 18 79 4 24.68 ND 27.12 No 

CG 2 
Campo Grande 

(MS) 
13 18 26 0 24.94 29.00 ND No 

SG 1 
São Gabriel do 

Oeste (MS) 
23 18 60 0 27.04 ND ND Yes 

GS 6 
Guarujá do Sul 

(SC) 
13 18 88 2 25.00 ND 27.82 Yes 

CC 5 
Cerqueira César 

(SP) 
23 11 19 2 29.06 30.84 29.24 No 

VD 9 Videira (SC) 23 33 62 4 22.29 ND 36.92 No 

SJ 10 
São José do 
Cedro (SC) 

6 11 41 4 20.74 ND ND No 

CG 10 
Campo Grande 

(MS) 
23 11 26 4 20.83 20.72 17.46 No 

*Bronchiolitis (necrotic/proliferative or obliterative). 

 

3.4. Severity of gross and histopathological lesions 

Regarding the pleurisy scores assessed at slaughter, based on the genetically 

characterized samples (N=25), 4 lungs (16%) were classified with pleurisy scores 0 

and 1, 6 lungs (24%) with score 2, 3 lungs (12%) with score 3 and 8 lungs (32%) with 

score 4. Among these, score 0 showed 4 different allelic profiles, scores 1 and 3 had 

only 1 profile each, score 2 showed 4 different profiles, and score 4 presented 5 

different allelic profiles. Table S4 summarizes the macroscopic findings according to 

allelic profile found in this study, including the mean number of pulmonary consolidation 

points seen in the macroscopic evaluation and the pleurisy scores. 

In the microscopic analysis, all lungs showed histological lesions characteristic 

of M. hyopneumoniae infection (different scores of BALT hyperplasia). Secondary 

bacterial lesions were frequent, with histological findings of bronchopneumonia or 

pleuropneumonia observed in 14 out of 25 lungs evaluated (56%). Additionally, 

histological findings suggestive of sVIA co-infection were observed in 52% (13/25) of 

the samples, mainly proliferative or obliterative bronchiolitis, which were negative on 

IHC (N=13). Table 3 describes the severity of the microscopic lesions according to 

each allelic pro-file observed in this study. 

 



26 
 

 

Table 3. Histopathological findings in the 25 lung samples and for each allelic profile, the results are presented in percentages 

and absolute numbers. 

 

Histological lesion Total 23-18-26 23-18-41 13-18-79 13-18-26 23-18-60 13-18-88 23-11-19 23-33-62 6-11-41 23-11-26 

BALT*            

0  -   -   -   -   -   -   -   -   -   -   -  

 +  28 (7/25) 28.57 (4/14)  -  100 (2/2)  -   -   -  100 (1/1)  -   -   -  

 ++  56 (14/25) 57.14 (8/14) 50 (2/2)  -  100 (1/1) 100 (1/1) 100 (1/1)  -   -  100 (1/1)  -  

 +++  16 (4/25) 14.28 (2/14)  -   -   -   -   -   -  100 (1/1)  -  100 (1/1) 

Suppurative 
bronchopneumonia 

24 (6/25) 7.14 (1/14) 50 (1/2) 50 (1/2)  -   -  100 (1/1)  -  100 (1/1)  -  100 (1/1) 

Necrotizing 
bronchopneumonia 

24 (6/25) 28.57 (4/14) 50 (1/2)  -  100 (1/1)  -   -   -   -   -   -  

Pleuropneumonia 8 (2/25) 7.14 (1/14)  -   -   -   -   -   -   -   -  100 (1/1) 

Chronic pleuritis 80 (20/25) 78.57 (11/14) 50 (1/2) 100 (2/2)  -  100 (1/1) 100 (1/1) 100 (1/1) 100 (1/1) 100 (1/1) 100 (1/1) 

Pneumocyte 
hyperplasia 

84 (21/25) 85.71 (12/14) 100 (2/2) 50 (1/2) 100 (1/1) 100 (1/1) 100 (1/1)  -  100 (1/1) 100 (1/1) 100 (1/1) 

Alveolar edema 28 (7/25) 21.43 (3/14) 50 (2/2) 50 (1/2)  -   -  100 (1/1)  -   -   -   -  

Lesions like sIVA 
infection** 

52 (13/25) 57.14 (8/14) 100 (2/2) 50 (1/2) - 100 (1/1) 100 (1/1) - - - - 

*BALT: bronchial-associated lymphoid tissue hyperplasia; BALT hyperplasia was classified as absent (0), mild (+), moderate (++) or extensive (+ + +).  
**Bronchiolitis (necrotic/proliferative or obliterative). 

 

 

  



27 
 

 

4. Discussion 

The allelic profile analysis identified notable genetic diversity among the 

samples, with different allelic profiles geographically distributed, suggesting possible 

regional variations in the epidemiology of the infection. The housekeeping genes (adk, 

rpoB and tpiA) were detected in 35.71% of the samples (25/70). The success rate for 

using the technique is a challenge. Lung samples, especially from animals with chronic 

lesions often contain a low bacterial load. MLST is a technique that requires 

amplification of multiple loci and subsequent sequencing. This can be difficult to 

perform with clinical samples where the quantity and quality of the pathogen's DNA is 

limited. 

In a study carried out by Zhang et al. (2021) [27], in 47 out of 199 samples 

positive for M. hyopneumoniae in the nested-PCR, it was possible to use the MLST 

technique. The MLST technique usually requires the amplification of 7 target genes, 

but for M. hyopneumoniae the use of 3 target genes is sufficient to understand the 

relationship between the strains. Mayor et al. (2008) [13] demonstrated in her study 

that 3 genes have the same resolution when 7 genes are used in MLST analysis for 

M. hyopneumoniae. 

As the samples come from clinical specimens and not isolates, the presence of 

inhibitors and a greater amount of genetic material can hinder the performance of the 

technique [27]. However, considering the complexity and time involved in cultivating 

and isolating this agent, MLST is a useful tool with high discriminatory power and rapid 

execution for characterizing M. hyopneumoniae and understand infection dynamics 

[10,13]. 

Different strains of M. hyopneumoniae can lead to variable clinical signs and 

severity of lesions. In addition, a single batch of pigs can be infected by multiple strains 

with different genetic profile [28,29]. This highlights the importance of studies that aim 

to correlate the genetic characterization of strains with the severity of lesions, both for 

understanding the epidemiological scenario and potential response to vaccine 

antigens. In the present study, regardless of the observed allelic profile, the sampled 

animals exhibited histopathological lesions consistent with M. hyopneumoniae 

infection, with varying scores of pleurisy severity. However, we did not perform any 

correlations since the number of samples per allelic profile found was too limited. 



28 
 

 

Our study identified 10 new combinations of alleles not yet described in the Pub-

MLST database, but it was not possible to categorize the STs. This could have been 

because isolates with these genetic characteristics have not yet been described. 

Considering that only one other study has been carried out in Brazil using this 

technique, we can suggest that the allelic profiles observed in this study are 

characteristic of Brazil. Research in other countries, such as China, has also shown 

great genetic variation in M. hyopneumoniae, where sixteen new sequence types were 

found in 47 sequenced samples [27]. For a new ST to be incorporated into the 

database, data from an isolate or a complete genome is required 

(https://pubmlst.org/submit-data). The possible presence of multiple strains in the 

clinical sample can generate artefacts and difficult data analysis. Therefore, a careful 

evaluation of the electropherograms is essential to guarantee the quality of the results 

found. This underscores the need to increase efforts to characterize new strains 

currently circulating. Based on this, we can hypothesize that the samples belong to 

sequence types of characteristics of Brazil, especially given that only 2 Brazilian 

isolates are currently documented in the database.  

Additionally, the present study investigated the relationship between lung 

consolidation, the presence of M. hyopneumoniae DNA, and allelic profiles in lung 

samples from pigs with macroscopic lesions consistent with M. hyopneumoniae 

infection in Brazilian herds. Our findings revealed a significant correlation between the 

severity of pleurisy lesions and lung consolidation scores, indicating that M. 

hyopneumoniae infection may play a critical role in the pathogenesis of the observed 

lung lesions. M. hyopneumoniae presents high prevalence with 92.45% and 97.7% as 

reported in previous studies conducted in Brazil [4,8]. Marois et al. (2008) [30] has 

demonstrated that cross-contamination can occur in the scalding tank at the 

slaughterhouse, but in our study the macro and microscopic lesions support M. 

hyopneumoniae infection. This finding reinforces the relevance of M. hyopneumoniae 

as a primary pathogen in the complex of respiratory system diseases in pigs [31]. 

Co-infections between M. hyopneumoniae and PM (32%) and M. 

hyopneumoniae and APP (40%) emphasize the complexity of swine respiratory 

infections, where several pathogens may be involved. Arruda et al. (2024) found 10.1% 

of the cases exhibited co-identification involving M. hyopneumoniae, PM and APP [8]. 



29 
 

 

This percentage is similar to the 12% found in this study. The presence of coinfections 

can aggravate the se-verity of the disease, influence the response to treatment, and 

complicate control efforts [32]. In our study, no correlations were observed between 

the severity of the lesions and the presence of co-infections detected by qPCR. The 

clinical course in the pigs in the farms is unknown since the samples were obtained at 

the slaughterhouse. 

Furthermore, around half of the samples (13/25; 52%) exhibited chronic 

microscopic lesions suggestive of sIVA infection, even though they tested negative on 

IHC. However, chronic histological lesions caused by the sIVA are typical, and negative 

IHC results during this stage of infection are common, as reported by Menegatt et al. 

(2023) [33]. Additionally, it was noted that these samples had statistically lower Ct of 

detection for M. hyopneumoniae (p-value < 0.005). This can suggest a possible 

exacerbation of M. hyopneumoniae infection after lesions caused by sIVA, since 

previous sIVA infections can facilitate colonization of the respiratory tract by M. 

hyopneumoniae [34]. 

The sample size was low and can therefore be considered a limitation in this 

study. The MLST technique requires sufficient lung samples, and more animals per 

batch or parameter are needed to correlate the prevalence of a determined M. 

hyopneumoniae allelic profile. However, we can suggest that there is a great genetic 

diversity of M. hyopneumoniae strains circulating in Brazilian herds.  

 

5. Conclusions 

We found a high prevalence of M. hyopneumoniae using qPCR in lung samples 

with pneumonia lesions and different pleurisy scores. In addition, the most severe 

pleurisy lesions also had a higher lung consolidation score. BALT hyperplasia lesions 

associated with M. hyopneumoniae infection were the most prevalent microscopic 

findings. This study suggests a maximum Ct (28.65±3.93) for the detection of M. 

hyopneumoniae in qPCR using p102 gene how target to triage samples for the MLST 

technique. The allelic profile analysis identified notable genetic diversity among the 

samples, with different allelic profiles distributed in different regions of Brazil, 

suggesting possible regional variations in the epidemiology of the infection. These 

results contribute to a better understanding of the dynamics of M. hyopneumoniae 

infection in pigs and highlight the importance of considering both pathogen genetics 



30 
 

 

and the macroscopic and microscopic characteristics of lesions when assessing 

disease severity. For a better epidemiological understanding and future comparisons 

of the genetic profile of the strains more isolates of the agent should be characterized. 

 

Supplementary Materials: The following supporting information can be downloaded 

at https: //www.mdpi.com/article/10.3390/microorganisms12101988/s1. Table S1: 

Sequences of the primers and hydrolysis probes used for each target region of the 

multiplex qPCR: omIA gene (APP), p102 gene (M. hyopneumoniae) and kmt1 gene 

(PM), and the respective amplifier sizes (bp); Table S2: Sequences of the primers for 

M. hyopneumoniae MLST (amplification and sequencing); Table S3: Data for each lung 

evaluated; Table S4: Mean number of pulmonary consolidation points, total for each 

lung evaluated and per lung lobe, assessed in the macroscopic evaluation and the 

number of lungs per pleurisy score according to allelic profile. 

 

Author Contributions: Conceptualization, L.G.d.O; methodology, L.G.d.O.; formal 

analysis, A.K.P; investigation, A.K.P, E.R.B, J.C.O.M. and D.D; data curation, A.K.P 

and E.R.B; writ-ing—original draft preparation, A.K.P and L.G.d.O.; writing—review 

and editing, A.K.P, E.R.B, F.A.M.P, J.C.O.M, D.D, D.M, L.G.d.O.; visualization, 

L.G.d.O, D.M; supervision, L.G.d.O.; project administration, A.K.P and L.G.d.O.; 

funding acquisition, L.G.d.O. All authors have read and agreed to the published version 

of the manuscript. 

 

Funding: We are thankful to FAPESP (São Paulo Research Foundation) for the 

Master’s scholar-ship (FAPESP Process #2023/01747-4) and fellowship abroad 

(FAPESP Process #2023/17921-3) grant to A.K.P. We also thank the National Council 

for Scientific and Technological Development (CNPq) for the productivity grant to 

L.G.d.O. (CNPq Process #316447/2021-8). 

 

Data Availability Statement: The original contributions presented in the study are 

included in the article/Supplementary Materials, further inquiries can be directed to the 

corresponding author. 



31 
 

 

Acknowledgments: To the members of the Laboratory of Swine Medicine—

Unesp/FCAV for their support with the collections and assistance with laboratory 

analysis. And the Frigmel-Guariba/SP for facilitating the sample collection. 

 

Conflicts of Interest: The authors declare no conflicts of interest.  

 

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