Molecular methods for species-level identification have been developed for several other genera of mastitis pathogens, including
Prototheca, a group of yeast-like micro-algae that have been described as a cause of mastitis in Japan [
139], Europe [
5,
89,
160] and North and South America [
6,
28]. Several methods have been used for identification of species and subspecies genotypes of
Prototheca. Genotype-specific PCR and RFLP and 18S rDNA sequence analysis are used to identify
P. zopfii genotype 1 and genotype 2 and
P. blaschkae, formerly known as
P. zopfii genotype 3, [
5,
89,
139]. Real-time PCR can identify
P. zopfii genotype 2,
P. blaschkae and
P. wickerhamii [
161]. When combined with DNA resolution melting analysis (qPCR/RMA), real-time PCR also allows for identification
P. zopfii genotype 1,
P. stagnora and
P. ulmea [
161].
Prototheca zopfii genotype 2 is the most common genotype in milk (Table
2). Some authors suggest that this indicates increased ability to cause mastitis because
P. zopfii genotype 2 is rare in the environment compared to
P. zopfii genotype 1 [
139]. Other authors find
P. zopfii genotype 2 as the most common genotype in milk as well as the environment [
160]. Development of additional molecular methods, especially at the subspecies level, may aid in studies of the epidemiology of
Prototheca mastitis.
Table 2
Distribution of Prototheca isolates from milk and environmental samples over species and genotypes
Japan | 0 | 67 | 0 | 29 | 3 | 0 | |
Germany | 2 | 177 | 21 | n.t. | n.t. | n.t. | |
Poland | 0 | 43 | 1 | n.t. | n.t | n.t. | |
Italy | 0 | 105 | 3 | 0 | 45 | 8 | |
Mycoplasma spp. are mollicutes, cell wall-less, slow growing organisms that require special culture media and growth conditions. For
Mycoplasma spp., as for
Prototheca spp., many molecular studies focus on detection and species identification, especially because molecular diagnostics are faster than culture [
8,
81,
164,
165]. As for other bacterial mastitis pathogens, outbreak investigations and routes of transmission are the main subjects of molecular epidemiological studies. In contrast to most other mastitis pathogens,
Mycoplasma spp. may affect multiple organ systems. Asymptomatic carriage in the ears and respiratory tract has been described, as well as otitis, pneumonia and arthritis, primarily in calves, and mastitis in prepubertal calves and adult cattle [
61‐
63,
116]. The occurrence of various non-clinical and clinical manifestations adds new angles to
Mycoplasma transmission studies because multiple age groups, carrier states and disease syndromes may act as source for mastitis outbreaks [
152]. In addition, dissemination of the pathogen within the individual host, probably via hematogenous or lymphatic spread, may occur [
61,
62]. The molecular epidemiology of respiratory
M. bovis has been investigated with AFLP, PFGE and RAPD typing [
116]. AFLP [
102], PFGE [
14] and restriction enzyme analysis (REA) [
40] have been used to study the molecular epidemiology of mastitis-associated
Mycoplasma spp., including
M. bovis, M. californicum and
Mycoplasma sp. bovine group 7. Within individual animals, multiple strains of the same
Mycoplasma species can be found at different body sites or even within the mammary gland [
14,
63,
102]. In most instances, however,
M. bovis or
M. californicum isolates found in milk, udder parenchyma and supramammary lymph nodes belong to the same PFGE type [
14]. Furthermore, 90% of isolates from eyes, ears, feces, joints, the urogenital system and internal organs belong to the same PFGE type as the mammary strains [
14], which could be explained by hematogenous spread. In the respiratory tract, only 40% of isolates belonged to PFGE types found in the mammary system and the rest of the body [
14].
Mycoplasma is more heterogeneous in the respiratory tract and it is also found more commonly in the nose than other body sites [
152]. Thus, it seems likely that colonization of the respiratory tract by a heterogeneous
Mycoplasma population is occasionally followed by systemic dissemination of one or a few strains.
Mycoplasma spp. may be transmitted vertically from dam to calf or horizontally via nasal discharge or from cow to cow at milking [
40,
63]. Transmission and dissemination of
Mycoplasma via extra mammary routes explains why control of
Mycoplasma mastitis can fail when it is solely based on detection of intramammary infections and prevention of cow-to-cow transmission at milking [
61,
62]. In Australia,
Mycoplasma sp. bovine group 7, a member of the
M. mycoides cluster, has been associated with mastitis. An REA-based study of 24 epidemiologically related strains and 36 epidemiologically unrelated strains from multiple herds, countries and continents showed that all epidemiologically related isolates belonged to the same strain, whereas 28 different strains were detected among 32 unrelated isolates [
40]. REA did not have perfect typeability, but it did show good discriminatory power and excellent epidemiological concordance [
180]. Without demonstration of discriminatory power, homogeneity of strains within an outbreak would not have been epidemiologically meaningful. As in the studies of
M. bovis, multiple organs within an individual animal and multiple animals within a herd were infected by the same strain. This included calves, cows and aborted fetuses [
40]. Both
M. bovis and
Mycoplasma sp. bovine group 7 may persist in a herd for a long time, with documented persistence of a year and 18 months, respectively [
40,
152]. Regional persistence of
Mycoplasma strains for even longer periods may also occur. In Denmark, a single AFLP type was responsible for 2 cases of calf pneumonia and 2 multi-herd outbreaks of
M. bovis mastitis. The cases of pneumoniae were detected at intervals of 10 years (1981 and 1991) in one region. The multi-herd outbreaks of mastitis occurred in 1984 and 1986/7 in two different regions [
102]. The suggestion was made that the first pneumonia isolate gave rise to the first mastitis outbreak, which then led to the second mastitis outbreak. Contact between farms could have occurred via purchase of colonized or infected animals, attendance at animal shows or cross-contamination by service personnel visiting multiple farms [
102]. The same report describes that
M. bovis isolates obtained in the 1990s showed greater heterogeneity than those from the 1980s. Strains from the 1990s were primarily obtained from lung samples whereas strains from 1980 were largely obtained from mammary glands. It is unclear whether heterogeneity is associated with the different decades or the different organ systems. Considering that lung isolates were more heterogeneous than mastitis isolates, one could argue that the mastitis strain was particularly well suited to survival in the bovine mammary gland and that homogeneity indicated host adaptation of the strain rather than epidemiological connections between farms. As in many situations, this example illustrates that one has to be careful with interpretation of molecular data and that epidemiological data must be taken into consideration to identify and differentiate possible and likely scenarios [
212]. The recent publication of the complete genome of
M. bovis type strain PG45 [
209] and the emergence of
Mycoplasma mastitis in multiple countries [
135] can be expected to lead to development of additional typing methods and to further studies of the epidemiology, pathogenesis and control of
Mycoplasma mastitis. Given that MLST has already been developed for
M. agalactiae, a mastitis pathogen of sheep and goats, and for
M. hyopneumoniae, it seems only a matter of time until an MLST scheme for
M. bovis is published [
114,
117].