Mollicutes which include the genus Mycoplasma are wall-less bacteria that are found in avian, insect, mammalian and reptilian hosts¹¹. Until now, more than 15 species have been isolated or detected in dogs5. In spite of the fact that mycoplasmas can be found in healthy and diseased dogs, some studies have associated the presence of these agents with infertility, respiratory disease, arthritis and colitis5. Considering that information about the association of canine mycoplasmas with infections in dogs is scarce, this short communication describes a case of fertility disorders and other anomalies attributed to Mycoplasma spumans and Mycoplasma maculosum in a Bernese Mountain dog kennel.

The Bernese Mountain dog kennel was located in the city of Mar del Plata (Buenos Aires province). It has been oper-ating normally for 21 years, with high rates of pregnancy, producing two to three litters per year. The kennel was con-sidered to be free of brucellosis and leptospirosis because of the lack of antecedents of these diseases and the neg-ative serology of the dogs, which were tested regularly. Dogs were vaccinated with the sextuple vaccine (polyva-lent vaccine that confers immunity against canine distemper virus, canine parvovirus, Leptospira interrogans serovars canicola and icterohaemorrhagiae, canine adenovirus type 2 and canine parainfluenza virus) and dewormed regularly 2-4 times a year. In October 2017 a female (Catalina) and a male (Gucci) dogs were imported from Brazil and incorporated into the breeding stock. These dogs were tested only against Brucella (immunochromatographic rapid test, FASTest® BRUCELLA canis and blood culture), giving negative results. Thereafter, fertility disorders and other anomalies began to be observed: (i) after two unsuccessful matings between a female named Wish and Gucci, a singleton puppy with tracheal aplasia was born, (ii) Gucci’s sperm analysis showed low sperm counts (1.5mill/ml), without motility, 100% of dead forms and 70% of abnormalities in head, midpiece and tail, reason enough for being excluded from the kennel, (iii) after a male called Egmont mated Catalina, a puppy died of a fatal pneumonia after an unsuccessful treatment with amoxicillin-clavulanic acid. By June 2019, all the bitches still had pregnancy loses. After having ruled out several infectious agents by bacte-riology (Brucella sp., Escherichia coli, Citrobacter freundii and beta-hemolytic streptococci) and serology (microscopic agglutination test for Leptospira sp.) in all the dogs, the owners sent vaginal swabs from three non-pregnant bitches (Catalina, Clara and Troya) for Mycoplasma diagnosis. Once in the laboratory, DNA extraction from vaginal swabs was performed using the Puriprep-S commercial kit (Inbio High-way, Argentina) following the manufacturer’s instructions. For Mycoplasma spp. detection, a nested PCR targeting the 16S-23S rRNA intergenic spacer region (ITS) was per-formed under the conditions reported by the authors13. Only Catalina rendered a positive result, with two differ-ent size bands (330 bp and 210 bp, approximately) observed in the agarose gel. In order to identify Mycoplasma spp., both PCR products were purified (Puriprep-GP Kit, Inbio Highway), quantified and sequenced (ABI 3130xl; Applied Biosystems) using the inner primers described by Tang et al.13 The sequences were curated using the BioEdit software8 and aligned against the database using nucleotide BLAST (http://www.ncbi.nlm.nih.gov/blast). The 16S-23S rRNA ITS sequence obtained from the 310 bp amplicon (GenBank accession number MW646397) showed 97.44% sim-ilarity with the same region of Mycoplasma maculosum strains NCTC10168 (LR215037.1), Skotti B (FJ595090.1), PG15 (AF443610.1), ATCC 19327 (AY973564.1), 97.12%o with Mycoplasma leopharyngis (AY762644.1) and 97.14% with Mycoplasma sp. isolate BA019827 (KX863544.1). The 214bp (GenBank accession number MW646398) sequence showed 98.1% and 97.63 similarity with M. spumans (AF538684.1 and AY762642.1) respectively. Based on these results, in July 2019, the 13 male and female adult dogs from the kennel (even Gucci, the male dog) were treated with doxycycline (500mg/day) for a 15-day period. Two months after the treatment with doxycycline, Gucci’s sperm analysis revealed the following parameters: normal sperm counts (52 mill/ml), 61% rapid progressive, 19% slow progressive and 12% in situ motility) with 10% of dead forms and 75% of normal forms. Despite the doxycycline treatment, four female dogs (Wish, Catalina, Eureka and Clara) could not get pregnant. For this reason, in August 2019, the owner sent vaginal swabs from two females to the laboratory (Catalina and Eureka). Both samples rendered positive results with one and two bands present in agarose gel, respectively. This time sequenc-ing was not performed. These females were treated with azithromycin (750mg/day) for a nine-day period starting on the day of mating. After that, Gucci mated successfully with Eureka and Troya and, Egmont mated Catalina, the three bitches became pregnant. In December 2019, Troya and Eureka delivered healthy puppies. Catalina could not deliver because during her pregnancy she suffered from a gastric torsion and needed surgery. After this episode, no repro-ductive disorders were observed. During 2020, 27 healthy puppies were born.

The success of the antibiotic treatment used, the absence of other pathogens causing infertility, the detection of Mycoplasmas, the changes in Gucci’s sperm analysis and the recovery from the bitches’ previous subfertility after the antibiotic treatments, suggest that Mycoplasma species were associated with that subfertility episode in this dog kennel. Although the results of Gucci’s sperm anal-ysis improved after treatment with doxycycline, it was only after the treatment with azithromycin that success-ful pregnancies were achieved. In this regard, azithromycin, doxycycline, enrofloxacin, marbofloxacin, minocycline, orb-ifloxacin and pradofloxacin have been pointed out as effective against Mycoplasma-associated respiratory infec-tions in dogs9. However, azithromycin would be the most suitable antibiotic for the treatment of infections caused by Mycoplasma9. Although M. canis is the predominant species identified from the vaginas of fertile and infertile bitches6,10,12 and Mycoplasma cynos had been associated with pneumonia4,16, we did not find any of those species. M. spumans and M. maculosum have been identified from the respiratory and genitourinary systems of dogs with and with-out respiratory infection1,2,7 and from fertile and infertile dogs2,6,10. These antecedents might explain the subfertility episode in this kennel, the fatal pneumonia of Catalina’s puppy and even, the tracheal aplasia of Wish’s puppy. Fur-ther experimental studies are necessary to confirm our results. With regard to M. leopharyngis, we did not find any antecedents about its presence in dogs, since together with other six Mycoplasma species, it affects felines3. Con-sidering the high similarity (98.6%) in the 16S-23S rRNA ITS region between M. maculosum and M. leopharyngis14, the detected species could be M. maculosum. However, the presence of M. leopharyngis in the urogenital tract of dogs cannot be strictly ruled out. The analysis of other target sequences would have allowed the unambiguous identificaron of Mycoplasma species. Taking into account the phylogenetic relatedness and pairwise sequence similarities of the species within the family Mycoplasmataceae, a reli-able and useful taxonomic tool, analyzing 16S-23S rRNA ITS, RNA polymerase beta subunit (rpoB) and 16S rRNA genes, has been proposed15. Regrettably, rpoB and 16S rRNA genes were not addressed in this study. In this way, the analysis of the three-target sequence has been pointed out as a valuable tool for routine analyses15.

Unfortunately, veterinarians do not often consider Mycoplasma in the differential diagnosis of respiratory and reproductive diseases in dogs. For this reason, Mycoplasma detection either by culture or molecular-based tests is rarely requested. From a practical point of view, the detec-tion of these agents is important for the implementation of an adequate antibiotic treatment, beyond the identi-fication of the involved species. Due to the lack of cell wall in Mycoplasma, they are inherently resistant to p-lactam antibiotics. In this report, prolonged therapy with doxycycline and azithromycin demonstrated to be effec-tive against Mycoplasma infections, agreeing with previous data9. In spite of the limitation of this study, since Mycoplasma culture was not tried, the importance of the diagnosis of these agents was demonstrated.

Conflict of interest

The author declares that he has no conflicts of interest.

Acknowledgements

This study was financially supported in part by FONCyT-ANPCyT-MinCyT, República Argentina [PICT 02148/2018], PPI 2019-2021 SeCyT-UNRC and IDi division of ACERCA Laboratorio. The author is deeply grateful to the owners of the Bernese Mountain dog kennel, Silvina Otero and Gustavo Pisani and to the veterinarians Ricardo Abaurrea, Carlos Enrique Sorribas and Isabel Zubiros.