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Veterinary Microbiology 231 (2019) 33–39 Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic High frequency and molecular characterization of porcine hemotrophic mycoplasmas in Brazil T Igor Renan Honorato Gattoa, Karina Sonálioa, Renan Bressianini do Amarala, Nelson Morésb, ⁎ Osmar Antonio Dalla Costab, Marcos Rogério Andréa, Luís Guilherme de Oliveiraa, a São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal, São Paulo, Via de Acesso Prof. Paulo Donato Castellane s/n, Jaboticabal, SP 14884-900, Brazil b Embrapa Swine and Poultry, Animal Health Laboratory, BR 153, Km 110, P.O. Box 21, Distrito de Tamanduá, Concórdia, CEP 89.700-000, Santa Catarina, Brazil ARTICLE INFO ABSTRACT Keywords: Mycoplasma suis and Mycoplasma parvum are the two hemotrophic mycoplasmas species described in pigs. M. suis Hemotrophic mycoplasmas is involved in infectious anemia, while M parvum infection is commonly subclinical. The objectives of this study Sows were twofold: (i) to investigate the prevalence of porcine hemotrophic mycoplasmas in sows from the southern Mycoplasma suis region of Brazil by quantitative real-time PCR (qPCR) and (ii) to genetically characterize a subset of the samples Mycoplasma parvum based on the 16S rRNA gene. A total of 429 blood samples were evaluated from 53 different farm sites. Porcine Infectious diseases hemoplasmas was detected at all the 53 tested sites and in 79.72% of the samples (342/429). Two sequences were obtained for Mycoplasma spp. The phylogenetic analysis based on the 16S rRNA gene (900 bp) showed that the Mycoplasma sequences were closely related to the M. suis cluster and that one sequence was positioned in the M. parvum cluster. In conclusion, porcine hemoplasmas have a high rate of prevalence in sows from commercial farms in the southern region of Brazil. This study demonstrated the first molecular detection and characterization of partial 16S rRNA gene of M. parvum in Brazil. 1. Introduction the clinical signs associated with infection in humans remain unknown. M. suis, the primary agent of infectious anemia in pigs (IAP) (Stadler Hemotrophic mycoplasmas (HMs) (class Mollicutes) are pleomorphic et al., 2014), can lead to economic losses in swine production uncultivable bacteria with small genomes that lack cell walls and have (Ritzmann et al., 2009). The process of adhesion to and invasion of an unique tropism for red blood cells (RBCs). So far, only two HMs that RBCs damages the cell membrane, inducing eryptosis and anemia infect swine have been described, namely, Mycoplasma suis and (Felder et al., 2011). Furthermore, disease severity is correlated with Mycoplasma parvum. These hemoplasmas have been shown to cause strain virulence (Felder et al., 2011; Stadler et al., 2014). While the single or coinfection in pigs (Fu et al., 2017). In phylogenetic analyses acute phase of swine hemoplasmosis is characterized by hemolytic based on the 16S rRNA gene, these species clustered into different ge- anemia, high fever, icterus and hemorrhagic diathesis, the chronic netic groups (Watanabe et al., 2011). While M. suis is considered the phase usually is characterized by the absence of anemia, retarded main etiological agent of infectious anemia in pigs around the world, M. growth in piglets and decreased reproductive efficiency in sows (Henry, parvum infection is usually characterized by the absence of clinical 1979; Messick, 2004; Hoelzle, 2008). Recently, a study showed that signs. These agents adhere to RBCs, triggering the cell's death and chronic infection interferes with the transcription of the host pathways dodging the immune system (Hoelzle, 2008; Groebel et al., 2009). frequently used by biomedical researchers and affects the scientific These organisms are also known for their ability to maintain a persis- results of studies using porcine models (do Nascimento et al., 2018). tent infection, despite antimicrobial treatment (Smith, 1992). The detection of M. suis in domestic pigs of different ages has been M. suis, the most characterized hemoplasmas species affecting pigs, reported around the world. The molecular prevalence in growing pigs has been detected in four mammalian species: pigs (Guimaraes et al., and sows ranges from 5% to 80% and has been reported in several 2009), humans (Yuan et al., 2009), dogs (Mascarelli et al., 2016) and countries (Table 1). Nevertheless, some authors have assessed the wild boars (Sus scrofa scrofa)(Hornok et al., 2018). Although the zoo- phylogenetic positioning of the different strains of M. suis to establish notic potential of M. suis has been reported in China (Yuan et al., 2009), the relationship among the other hemoplasmas species and M. suis, ⁎ Corresponding author. E-mail address: [email protected] (L.G. de Oliveira). https://doi.org/10.1016/j.vetmic.2019.02.024 Received 13 December 2018; Received in revised form 6 February 2019; Accepted 19 February 2019 0378-1135/ © 2019 Elsevier B.V. All rights reserved. I.R.H. Gatto, et al. Veterinary Microbiology 231 (2019) 33–39 Table 1 Summarized studies that assessed the molecular prevalence of porcine hemoplasmas around the world. Country Molecular test Category Prevalence (%) Reference † Brazil qPCR Wild boar 50 (7/14) Dias et al., 2019 Hungria Wild boar 47.1 (8/17) Hornok et al., 2018 China Growing pigs 42.4 (101/238) Fu et al., 2017 China Sows 70.9% (61/86) Fu et al., 2017 Brazil Non-technified pigs 76.2 (112/147) Toledo et al., 2016 Japan Growing pigs 5 (6/120) Watanabe et al., 2012 Japan Growing pigs 50 (6/12) Watanabe et al., 2011 Brazil Sows 80 (32/40) Guimaraes et al., 2011 United States Pigs 7.5 (3/40) Guimaraes et al., 2011 Germany Wild boar 10.03 (36/359) Hoelzle et al., 2010 Germany Growing pigs 13.9 (164/1176) Ritzmann et al., 2009 Switzerland Sows 19 (19/100) Hoelzle et al., 2007 Germany Growing pigs 10.6 (17/160) Hoelzle et al., 2007 ‡ Argentina cPCR Dog 2.9 (2/70) Mascarelli et al., 2016 Argentina Growing pigs 65.1 (155/284) Pereyra et al., 2010 China Growing pigs 86 (148/172) Yuan et al., 2009 China Humans 49 (32/65) Yuan et al., 2009 Brazil Sows 18.2 (22/121) Guimaraes et al., 2007 Brazil Piglets 0.61(1/61) Guimaraes et al., 2007 Brazil Boars 25 (1/4) Guimaraes et al., 2007 † quantative real time PCR (qPCR). ‡ Conventional PCR (cPCR). which also allows the description of novel hemoplasmas (Watanabe individual sow, and individuals were not resampled. This research was et al., 2011; Fu et al., 2017; Gonçalves et al., 2016). submitted to and approved by the CONCEA (National Council of Animal The information regarding the prevalence of M. suis infection within Experimentation Control) and the institution’s CEUA (Ethics sow herds in Brazil is scarce (Guimaraes et al., 2007; 2011). Since Brazil Commission for Animal Experimentation) under protocol number is the fourth largest pork producer in the world and has a great impact 001217/18. on pork meat exports, it is necessary to maintain the health status of the DNA extraction was performed from whole blood samples using the pig herds in the country. Accordingly, the objective of this study was protocol previously described by Kuramae-Izioka, 1997. The DNA twofold: (i) to investigate the prevalence of porcine hemotrophic my- concentration and quality (260/280 nm ratio) were measured using a coplasmas in sows from the southern region of Brazil with a quantita- Thermo Scientific NanoDrop 2000 Spectrophotometer (Thermo Fisher ® tive real-time PCR (qPCR) assay and (ii) to genetically characterize a Scientific , Wilmington, Delaware, USA). subset of the samples based on the 16S rRNA gene. 2.4. Conventional PCR for the endogenous gapdh gene 2. Materials and methods To avoid false-negative results in the qPCR assay for porcine he- 2.1. Study design moplasmas, all DNA samples were tested for the gapdh gene, using the primers gapdh-F (5′-CCTTCATTGACCTCAACTACAT-3′) and gapdh-R This work was designed as a cross-sectional study to assess the (5’-CCAAAGTTGTCATGGATGACC-3′), which flank a fragment of 437 molecular prevalence of porcine hemoplasmas in blood samples from base pairs (bp) (Birkenheuer et al., 2003). The conventional PCR sows. The sows originated from 53 commercial farm sites and 29 mu- (cPCR) was performed as previously described by Birkenheuer et al. nicipalities from three different states in Brazil (Paraná, Santa Catarina (2003), with some modifications (Toledo et al., 2016). and Rio Grande do Sul States), which comprise the major pig-production region (Fig. 1). Three slaughterhouses, located in the state of Santa Catarina, were conveniently selected to collect samples for this study. 2.5. qPCR for porcine hemoplasmas based on 16S rRNA gene All the pigs from the sampled farms were slaughtered between March and September, 2017. During the sampling period, blood samples from The DNA samples exhibiting positive results in the conventional all the slaughtered sows were collected. PCR for the gapdh gene underwent qPCR for porcine hemoplasmas based on the 16S rRNA gene using the primers F (5′-CCCTGATTGTAC TAATTGAATAAG-3′) and R (5’- GCGAACACTTGTTAAGCAAG-3′) and 2.2. Eligibility criteria the probe TaqMan (5′FAM- TGRATACACAYTTCAGMGBNFQ3′) (Thermo Fisher Scientific, Waltham, MA, USA) (Guimaraes et al., 2011; ’ The sampled animals consisted of sows. The animals origin data do Nascimento et al., 2014). All samples were tested in duplicate. When were collected from the mandatory Animal Transit Permits issued by the difference of Cq values obtained for the duplicates of the same ffi ffi the local o cial veterinary service o ces. sample was higher than 0.5, the sample was retested in triplicates. The amplification reaction was performed in a Thermal Cycler Model 96 ® 2.3. Samples and DNA extraction CFX (BioRad , Hercules, California, USA) using a final volume of 10 μL containing a mixture of 1 μL of DNA sample, 0.2 μM of each primer and ® A total of 429 blood samples were obtained from the three different probe, PCR buffer (GoTaq Probe qPCR Master Mix, Madison, WI, USA) ® states located in South Brazil.

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