Isolation and Identification of Mycoplasma Species in Dogs

It has been more than 80 years since Shoetensack first reported mycoplasma species in dogs. The initial studies were very slow and fruitless due to the challenge of growing mycoplasmas in samples contaminated by other bacteria. In addition, there were few techniques to isolate the mycoplasma mixtures available (Rosendal 1979). It is worth remembering that Watson and Crick (1953) published their studies on DNA structure in 1953, and that Mullis et al. (1986) only developed the Polymerase Chain Reaction (PCR) technique in the 1980s. Nevertheless, mycoplasmas in dogs were little studied until the 2000s, when several reports started to arise more often in many countries. However, many mycoplasma, acholeplasma and ureaplasma species had already been described, namely: Mycoplasma cynos, M. canis, M. edwardii, M. bovigenitalium, M. gateae, M. spumans, M. feliminutum, Acholeplasma laidlawii and Ureaplasma sp. The first three species were the most commonly reported in dogs. A new species, Mycoplasma mucosicanis SP. Nov., was isolated from both the mucosa and the urogenital tract of asymptomatic dogs (Spergser et al. 2011).

M. Canis and M. edwardii may often appear alone or combined with other mycoplasma species in the upper respiratory tract. However, these species do not seem to be associated with respiratory disease as a primary pathogen (Chalker et al. 2004; Johnson et al. 2013). On the other hand, M. cynos affects the lower respiratory tract, causing pneumonia alone or often combined with Canine Infectious Respiratory Disease Complex (CIRD) (Chalker et al. 2004; Johnson et al. 2013). The most common symptoms of this disease are dry cough, anorexia and apathy. Lesions caused by M. cynos similar to those caused by M. pulmonis infection and found in laboratory rodents (Barreto et al. 2003; Souza et al. 2016) — are pathognomonic for mycoplasma pneumonia (Hong and Kim 2012).

M. canis is the species most associated with infertility, mucopurulent discharges and cystitis in the urogenital system (L’Abee-Lund et al. 2003; Ulgen et al. 2006). However, other species, such as M. edwardii and M. spumans, may also be isolated in healthy animals (Maksimović et al. 2018).

Although the respiratory and urogenital tracts are affected by most isolated species in dogs, M. edwardii was isolated from a 12-year-old female dog that has presented acute polyarthritis followed by septicemia (Stenske et al. 2005). Moreover, a picture of purulent meningoencephalitis indicated a brain tissue condition in a six-week-old female dog (Ilha et al. 2010). Mycoplasma spp. was isolated from the ear canal of healthy dogs with external otitis for the first time in Paraíba State, Brazil (Santos et al. 2016). Mycoplasma canis and Mycoplasma spumans were isolated in a cat (Walker et al. 1995) after a dog bite; likewise, M. canis was isolated in a 62-year-old woman (Klein, Klotz, and Eigenbrod 2018). These findings suggest the existence of a new mode of mycoplasma transmission.

Isolation had originally prevailed as the diagnostic method, however, PCR has been the method of choice for detecting and typifying mycoplasma in dogs since 2000 (Chalker 2005; Janowski et al. 2008; L’Abee-Lund et al. 2003). This method has contributed to raise the number of reports. The difficulty in obtaining specific antisera for mycoplasma in dogs is an obstacle to serological typing (Chalker 2005). Alternatively, methods such as immunoperoxidase and immunofluorescence are extremely useful in typing mycoplasma isolates, mainly the canine ones (Nascimento et al. 2010).

Despite the onset of enzymatic and biomolecular techniques, isolation remains the gold standard for the diagnosis of animal mycoplasmosis such as the canine one. Therefore, isolation is essential for the validation of PCR methods (Chalker 2005) and to enable the application of immunoenzymatic techniques such as immunofluorescence and immunoperoxidase (Santos et al. 2010; Zeugswetter et al. 2007).

Laboratory challenges in the diagnosis of canine mycoplasmosis lead to underreported cases and to poorly informed veterinarians and breeders, who often use antibiotics that do not work for mycoplasma (Chalker 2005). This scenario results from factors inherent to microorganisms and hosts (Berčič et al. 2012; Maksimović et al. 2018; Mannering et al. 2009) involved in sample preparation and cultivation methods (Chalker 2005; L’Abee-Lund et al. 2003).

In Brazil, Oliveira, Costa, and Silva (1998) assessed the vaginal microbiota diversity of healthy female dogs, but did not isolate mycoplasma. However, Costa et al. (2004) and Nascimento et al. (2010) isolated Mycoplasma spp. from the respiratory and urogenital tracts of asymptomatic dogs. These mycoplasma species were then identified as M. canis and M. edwardii through Indirect Immunoperoxidase, thus confirming the presence of mixed infections in dogs (Nascimento et al. 2010).

Due to the difficulty of diagnosis and of contributing to the knowledge regarding the occurrence of canine mycoplasma. The aim of the present study was to assess the presence of Mycoplasma spp. in dogs with and without mycoplasmosis over a timeframe often years.