Discussion
Although M. bovis is a global menace yet, there are no trade restrictions for any of the markets in Egypt presenting a trade risk.Mycoplasma bovis does not cause disease in humans and is not a notifiable disease (AHDB, 2020) although it was previously cultured from the sputum of a patient with lobar pneumonia (Madoff et al., 1979) and it is one of the major causative agents of bovine mycoplasmosis. Infection with the organism is associated with a broad range of clinical manifestations including bronchopneumonia, treatment-resistant mastitis, otitis, meningitis, and genital disorders, tenosynovitis, keratoconjunctivitis, chronic pneumonia, arthritis, polyarthritis with high morbidity and late-termabortions (Bürki et al., 2015; Hananehet al., 2018).
The disease may be dormant in an animal – causing no symptoms (Maunsell et al., 2011). In times of stress the animal may shed bacteria in milk and nasal secretions. As a result, other animals may be infected and become ill or carriers themselves (Calcutt et al., 2018). In our case, the camels are subjected to severe stress during transportation on foot covering thousands of kilometers or when they are transported by rail vehicles. Effects of transportation and movement include (FAO, 2001): stress, bruising, trampling, suffocation, heart failure, heat stroke, sun burn, bloat, poisoning, predation, dehydration, exhaustion, injuries and fighting. Although the circumstances of how or/and where camels in this study became infected are unknown, it is possible that potential infection includes the importation of live camel by vehicles which have been used for the transportation of the animals. Contact between infected and non-infected animals when it occurs in confined spaces increases, as during the transport of the camels from Aswan to Cairo by rail truck, the risk of “nose-to-nose” transmission becomes unavoidable. When the camels reach Cairo they are transported on foot to Birqash Market, the largest camel market in Egypt, where they are sold for slaughter, farm work, tourism and transport. The farm equipment are additional factors that play important roles in the spread of the disease, especially those that come into direct contact with infected animals.
In this study we have extended the arsenal of factors implicated in the pathogenicity of M. bovis in addition to those reported previously (Großhennig et al., 2016). The overlapping but distinct effects of H2S indicate that the bacteria possess a set of virulence determinants that together allow the bacteria getting an efficient access to the host’s resources. Although the production of H2S as a virulence factor has not been observed for pathogens causing lung infections before with the exception of two studies that have reported (Großhennig et al., 2016) that H2S is an additional factor implicated in the cytotoxicity and virulence of M. pneumonia and a very recent study on M. arginini (Osman et al., 2020). This is consistent with the results we obtained for M. bovis in this study.
One aspect of pathogenesis that warrants further comment is the ability of many Mycoplasma species to form biofilms, with M. bovisrepresenting one of the prolific biofilm producers among a survey of species tested (McAuliffe et al., 2006). However, the formation of biofilms by many Mycoplasma species has mainly been demonstrated in vitro (McAuliffe et al., 2006; Simmons et al., 2013; Wang et al., 2017). The role of bacterially derived biofilms in causing human disease has been known for some time (Wilson, 2001), and an increasing appreciation of biofilms in bovine mastitis is emerging (Melchior et al., 2006; Gomes et al., 2016). It is therefore plausible that biofilms elaborated by M. bovis may influence some aspect of the disease course or pathogenicity in camels. Unfortunately, due to the absence of the vsp gene in 11 out of the 13 isolates in this study, we were unable to compare different M. bovis isolates for a correlation analysis as shown by Calcutt et al. (2018) who demonstrated the biofilm production and correspondingvsp expression profile. The aforementioned ability of M. bovis to survive in bedding (Justice-Allen et al., 2010) may be explained by the presence of biofilms, which is important in other sand-containing environments (Whitman et al., 2014). In vitro , biofilm production conferred greater resistance to heat and desiccation than was exhibited by planktonic M. bovis cells (McAuliffe et al., 2006), raising the possibility that this capability may contribute to the observed environmental persistence and perhaps to chronic infection in the bovine host (Calcutt et al., 2018) which could consequently also convey this characteristic to the desert dwelling animal, the camel.
Antibiotic resistance is an ongoing one of the most pressing threats in the world. The World Health Organization recently recognised antibiotic resistance as a serious global problem, not only in terms of human health but also for the animals (both domestic and wildlife) and the environment (Gibbs, 2014). However, the role of wild animals as a reservoir of antibiotic resistant bacteria has been acquiring attention in recent years (Finley et al., 2013; Smith et al., 2014; Dias et al., 2018).
Many in vitro  studies have compared the susceptibility of M. bovis  against a range of antibiotics. Mycoplasmas are generally susceptible to antibiotics that affect protein (tetracyclines, macrolides, lincosamides, phenicols) or nucleic acid synthesis (fluoroquinolones) (Maunsell et al., 2011; Muller et al., 2019).M. bovis has developed antimicrobial resistance to many of the antimicrobial agents traditionally used in the therapy ofMycoplasma infections; in particular oxytetracyclines, tilmicosin and spectinomycin (Ayling et al., 2000; Nicholas et al., 2000; Sulyok et al., 2014; Calcutt et al., 2018). Acquired resistance to macrolides in M. bovis  is a widely known phenomenon. Gerchman et al. (2009) reported marked differences in susceptibility profiles to tylosin inM. bovis from different geographical regions, including Western Canada, Israel, Britain, Hungary, Japan, USA and France (Lysnyansky and Ayling, 2016). High level of resistance to macrolides has been reported by others (Ayling et al., 2000; Rosenbusch et al., 2003; Gerchman et al., 2009; Uemura et al., 2010; Gautier-Bouchardon et al., 2014; Sulyok et al., 2014) with the indication that macrolides have lost their efficacy on mycoplasmas. Our results provide further evidence for this phenomenon with 10 isolates being resistant to erythromycin only while the other two antibiotics belonging to the Macrolides tylosin and spiramycin were on the contrary, effective in this study (Table 1).
However, in contrast to other studies that reported increased resistance to antibiotics commonly used for the therapy of Mycoplasmainfections, including tetracyclines, phenicol and lincosamide, the majority of M. bovis isolates in this study were susceptible to florfenicolor lincomycin. Heterogenic susceptibility of M. bovisto tetracyclines is widely reported (Gourlay et al., 1989; Ayling et al., 2000; Rosenbusch et al., 2003; Gerchman et al., 2009; Uemura et al., 2010; Gautier-Bouchardon et al., 2014). Consistent with our results, increased resistance to oxytetracycline was reported previously in the UK, The Netherlands, North America, Israel, Belgium, Hungary, Japan and France (Lysnyansky and Ayling, 2016).
Although the most effective antibiotics tested in vitro for the treatment of M. bovis infections were fluoroquinolones (Lysnyansky and Ayling, 2016; Ayling et al., 2000; Rosenbusch et al., 2005;Francoz et al., 2005; Gerchman et al., 2009; Uemura et al., 2010; Soehnlen et al., 2011; Kroemer et al., 2012; Gautier-Bouchardon et al., 2014) yet, some in vitro  resistance to fluoroquinolones has also been reported (Gerchman et al., 2009). In this study all thirteenM. bovis isolates recovered from camels were resistant to ciprofloxacin, which is consistent with previously published data in cattle (Gerchman et al., 2009). Globally, the reports on susceptibility profiles of M. bovis to fluoroquinolones display extensive discrepancies that vary considerably from one country to another (Khalil et al., 2016).
M. bovis isolated from the camels in Egypt, may have been transmitted from cattle from other regions based on 16S ribosomal RNA sequence data. M. bovis has been reported to be transmitted across species from cattle, camel and other livestock in Eritrea, East Africa due to inter-species herd mixing at water points, resting areas as well as due to migration and uncontrolled livestock movement (Ghebremariam et al, 2018). Egypt has also recently increased the number of cattle it has imported from other countries, mainly from Brazil, Spain, Sudan, Colombia, Hungary, The Netherlands, Italy and Uruguay (Roushdy, 2018).
Alarmingly, our research identifies widespread resistance in camel to several first-line antimicrobials used in human medicine. Our results highlight camel in the wildlife as important host reservoirs and vectors for the spread of a virulent, multidrug-resistant M. bovis and genetic determinants of resistance. The inability of M. bovis to form biofilms should decrease their persistence. Taking into account that camel isolates do not contact directly with antibiotics, the resistance observed among the studied M. bovis is alarming and that measures are necessary to monitor this alarming phenomenon (Lysnyansky and Ayling, 2016). This could be related to the overuse/misuse of the antibiotics in human and veterinary medicine, with a consequent spread of resistance genes to the environment.