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.