Protection against Mycoplasma bovis infection in calves following intranasal vaccination with modified-live Mannheimia haemolytica expressing Mycoplasma antigens.

Novel live vaccine strains of Mannheimia haemolytica serotypes (St)1 and St6, expressing and secreting inactive yet immunogenic leukotoxin (leukotoxoid) fused to antigenic domains of Mycoplasma bovis Elongation Factor Tu (EFTu) and Heat shock protein (Hsp) 70 were constructed and tested for efficacy in cattle. Control calves were administered an intranasal mixture of M. haemolytica St1 and St6 mutants (ΔlktCAV4) expressing and secreting leukotoxoid while vaccinated calves were administered an intranasal mixture of like M. haemolytica St1 and St6 leukotoxoid mutants coupled to M. bovis antigens (EFTu-Hsp70-ΔlktCAV4). Both M. haemolytica strains were recovered from palatine tonsils up to 34 days post intranasal exposure. On day 35 all calves were exposed to bovine herpes virus-1, four days later lung challenged with virulent M. bovis, then euthanized up to 20 days post-challenge. Results showed all cattle produced systemic antibody responses against M. haemolytica. The vaccinates also produced systemic antibody responses to M. bovis antigen, and concurrent reductions in temperatures, middle ear infections, joint infection and lung lesions versus the control group. Notably, dramatically decreased lung loads of M. bovis were detected in the vaccinated cattle. These observations indicate that the attenuated M. haemolytica vaccine strains expressing Mycoplasma antigens can control M. bovis infection and disease symptoms in a controlled setting.

Serological evidence for historical and present-day exposure of North American bison to Mycoplasma bovis.

BACKGROUND: Mycoplasma bovis causes mastitis, otitis, pneumonia and arthritis in cattle and is a major contributor to bovine respiratory disease complex. Around the year 2000, it emerged as a significant threat to the health of North American bison. Whether healthy bison are carriers of M. bovis and when they were first exposed is not known. To investigate these questions we used a commercially available ELISA that detects antibodies to M. bovis to test 3295 sera collected from 1984 through 2019 from bison in the United States and Canada. RESULTS: We identified moderately to strongly seropositive bison from as long ago as the late 1980s. Average seroprevalence over the past 36 years is similar in the United States and Canada, but country-specific differences are evident when data are sorted by the era of collection. Seroprevalence in the United States during the pre-disease era (1999 and prior) was significantly higher than in Canada, but was significantly lower than in Canada during the years 2000-2019. Considering individual countries, seroprevalence in the United States since the year 2000 dropped significantly as compared to the years 1985-1999. In Canada the trend is reversed, with seroprevalence increasing significantly since the year 2000. ELISA scores for sera collected from free-ranging bison do not differ significantly from scores for sera from more intensively managed animals, regardless of the era in which they were collected. However, seroprevalence among intensively raised Canadian bison has nearly doubled since the year 2000 and average ELISA scores rose significantly. CONCLUSIONS: Our data provide the first evidence that North American bison were exposed to M. bovis many years prior to the emergence of M. bovis-related disease. Patterns of exposure inferred from these results differ in the United States and Canada, depending on the era under consideration. Our data further suggest that M. bovis may colonize healthy bison at a level sufficient to trigger antibody responses but without causing overt disease. These findings provide novel insights as to the history of M. bovis in bison and will be of value in formulating strategies to minimize the impact of mycoplasmosis on bison health and production.

Comparison of Two Multilocus Sequence Typing Schemes for Mycoplasma bovis and Revision of the PubMLST Reference Method.

Mycoplasma bovis causes pneumonia, pharyngitis, otitis, arthritis, mastitis, and reproductive disorders in cattle and bison. Two multilocus sequence typing (MLST) schemes have been developed for M. bovis, with one serving as the PubMLST reference method, but no comparison of the schemes has been undertaken. Although the PubMLST scheme has proven to be highly discriminatory and informative, the recent discovery of isolates missing one of the typing loci, adh-1, raises concern about its suitability for continued use. The goal of our study was to compare the performance of the two MLST schemes and identify a new reference scheme capable of fully typing all isolates. We evaluated 448 isolates from diverse geographic and anatomic sites that collectively represent cattle, bison, deer, and a goat. The discrimination indexes (DIs) for the PubMLST and the alternative scheme are 0.909 (91 sequence types [STs]) and 0.842 (77 STs), respectively. Although the PubMLST scheme outperformed the alternative scheme, the adh-1 locus must be retired from the PubMLST scheme if it is to be retained as a reference method. The DI obtained using the six remaining PubMLST loci (0.897, 79 STs) fails to reach the benchmark recommended for a reference method (0.900), mandating the addition of a seventh locus. Comparative analysis of genome sequences from the isolates used here identified the dnaA locus from the alternative scheme as the optimal replacement for adh-1 This revised scheme, which will be implemented as the new PubMLST reference method, has a DI of 0.914 and distinguishes 88 STs from the 448 isolates evaluated.

Prevalence of Mycoplasma spp. in the Respiratory Tract of Healthy North American Bison (Bison bison) and Comparison with Serum Antibody Status.

Mycoplasma bovis is a primary cause of respiratory and reproductive diseases in North American bison (Bison bison), with significant morbidity and mortality. The epidemiology of M. bovis in bison is poorly understood, hindering efforts to develop effective control measures. Our study considered whether healthy bison might be carriers of M. bovis, potentially serving as unrecognized sources of exposure. We used culture and PCR to identify mycoplasmas in the nasal cavity or tonsil of 499 healthy bison from 13 herds and two abattoirs in the US and Canada. Mycobacterium bovis was detected in 15 bison (3.0%) representing two herds in the US and one in Canada, while M. bovirhinis, M. bovoculi, M. arginini, or M. dispar was identified from an additional 155 bison (31.1%). Mycoplasma bovirhinis was identified most frequently, in 142 bison (28.5%) representing at least 10 herds. Of the 381 bison for which serum was available, only 6/13 positive for M. bovis (46.2%) tested positively with an M. bovis ELISA, as did 19/368 negative for M. bovis (5.2%). Our data reveal that M. bovis can be carried in the upper respiratory tract of healthy bison with no prior history or clinical signs of mycoplasmosis and that a large proportion of carriers may not produce detectable antibodies. Whether carriage of other mycoplasmas can trigger cross-reactive antibodies that may confound M. bovis serology requires further study.

Comparison of multilocus sequence types found among North American isolates of Mycoplasma bovis from cattle, bison, and deer, 2007-2017.

A prior multilocus sequence typing (MLST) study reported that Mycoplasma bovis isolates from North American bison possess sequence types (STs) different from those found among cattle. The 42 bison isolates evaluated were obtained in 2007 or later, whereas only 19 of 94 (~20%) of the available cattle isolates, with only 1 from North America, were from that same time. We compared STs of additional, contemporary, North American cattle isolates with those from bison, as well as isolates from 2 North American deer, all originating during the same timeframe, to more definitively assess potential strain-related host specificity and expand our understanding of the genetic diversity of M. bovis. From 307 isolates obtained between 2007 and 2017 (209 from cattle, 96 from bison, 2 from deer), we identified 49 STs, with 39 found exclusively in cattle and 5 exclusively in bison. Four STs were shared between bison and cattle isolates; one ST was found in cattle and in a deer. There was no clear association between ST and the health status of the animal of origin. An MLST-based phylogeny including 41 novel STs identified in our study reveals that STs found in bison fall within several divergent lineages that include STs found exclusively in cattle.

Mistaken identity of an open reading frame proposed for PCR-based identification of Mycoplasma bovis and the effect of polymorphisms and insertions on assay performance.

Mycoplasma bovis is an important cause of disease in cattle and bison. Because the bacterium requires specialized growth conditions, many diagnostic laboratories routinely use PCR to replace or complement conventional isolation and identification methods. A frequently used target of such assays is the uvrC gene, which has been shown to be highly conserved among isolates. We discovered that a previously described PCR putatively targeting the uvrC gene amplifies a fragment from an adjacent gene predicted to encode a lipoprotein. Comparison of the lipoprotein gene sequence from 211 isolates revealed several single nucleotide polymorphisms, 1 of which falls within a primer-binding sequence. Additionally, 3 isolates from this group were found to have a 1,658-bp transposase gene insertion within the amplified region that leads to a false-negative result. The insertion was not detected in a further 164 isolates. We found no evidence that the nucleotide substitution within the primer-binding region affects the assay sensitivity, performance, or limit of detection. Nonetheless, laboratories utilizing this method for identification of M. bovis should be aware that the region amplified may be prone to nucleotide substitutions and/or insertions relative to the sequence used for its design and that occasional false-negative results may be obtained.

Non-contiguous finished genome sequence of Ornithobacterium rhinotracheale strain H06-030791.

The Gram-negative, pleomorphic, rod-shaped bacterium Ornithobacterium rhinotracheale is a cause of pneumonia and airsacculitis in poultry. It is a member of the family Flavobacteriaceae of the phylum "Bacteroidetes". O. rhinotracheale strain H06-030791 was isolated from the lung of a turkey in North Carolina in 2006. Its genome consists of a circular chromosome of 2,319,034 bp in length with a total of 2243 protein-coding genes and nine RNA genes. Genome sequences are available for two additional strains of O. rhinotracheale, isolated in 1988 and 1995, the latter described in a companion genome report in this issue of SIGS. The genome sequence of O. rhinotracheale strain H06-030791, a more contemporary isolate, will be of value in establishing core and pan-genomes for O. rhinotracheale and elucidating its evolutionary history.

Complete genome sequence of Ornithobacterium rhinotracheale strain ORT-UMN 88.

Ornithobacterium rhinotracheale strain ORT-UMN 88 is a Gram-negative, pleomorphic, rod-shaped bacterium and an etiologic agent of pneumonia and airsacculitis in poultry. It is a member of the family Flavobacteriaceae of the phylum Bacteroidetes. O. rhinotracheale strain ORT-UMN 88 was isolated from the pneumonic lung of a turkey in 1995. It was the isolate first used to experimentally reproduce disease in turkeys and has since been the focus of investigations characterizing potential virulence factors of the bacterium. The genome of O. rhinotracheale strain ORT-UMN 88 consists of a circular chromosome of 2,397,867 bp with a total of 2300 protein-coding genes, nine RNA genes, and one noncoding RNA gene. A companion paper in this issue of SIGS reports the non-contiguous finished genome sequence of an additional strain of O. rhinotracheale, isolated in 2006.