A core genome multilocus sequence typing (cgMLST) analysis of Mycoplasma bovis isolates.

Mycoplasma bovis (M. bovis) is an emerging major bovine pathogen, causing economic losses worldwide in the dairy and beef industry. Whole-genome sequencing (WGS) now allows high resolution for tracing clonal populations. Based on WGS, we developed the core genome multilocus sequence typing (cgMLST) scheme and applied it onto 151 genomes of clonal and non-clonal strains of M. bovis isolated from China, Australia, Israel, Denmark, Canada, and the USA. We used the complete genome of M. bovis PG45 as the reference genome. The pairwise genome comparison of these 151 genome sequences resulted in 478 cgMLST gene targets present in > 99.0 % clonal and non-clonal isolates with 100 % overlap and > 90 % sequence similarity. A total of 478 core genes were retained as cgMLST target genes of which an average of 90.4-99 % were present in 151 M. bovis genomes, while M. agalactiae (PG2) had 17.0 % and M. mycoides subsp. capri (PG3), M. ovipneumoniae (Y98), and M. arginine resulted in 0.0 % of good targets. When tested against the clonal and non-clonal strains, we found cgMLST clusters were congruent with the MLST-defined clonal groups, which had various degrees of diversity at the whole-genome level. Notably, cgMLST could distinguish between clonal and epidemiologically unrelated strains of the same clonal group, which could not be achieved using traditional MLST schemes. Our results showed that ninety-two M. bovis genomes from clonal group isolates had > 10 allele differences and unambiguously differentiated from unrelated outgroup strains. Additionally, cgMLST revealed that there might be several sub-clones of the emerging ST-52 clone. The cgMLST phylogenetic analysis results showed substantial agreement with geographical and temporal information. cgMLST enables the use of next-generation sequencing technology to bovine mycoplasma epidemiology at both the local and global levels. In conclusion, the novel cgMLST scheme not only showed discrimination resolution highly as compared with MLST and SNP cgMLST in sub-typing but also indicated the capability to reveal more population structure characteristics than MLST.

Oral supplementation of alkaline phosphatase in poultry and swine.

The importance of intestinal alkaline phosphatase (IAP) in maintaining gut health and intestinal homeostasis is well established. The objective of this study was to investigate the tolerance of poultry and swine to dietary supplementation of a novel microbial-derived alkaline phosphatase (AP; E.C. 3.1.3.1 produced by Paenibacillus lentus strain CMG3709). Studies were conducted on day-old Ross 308 chicken (n = 1,000; Study 1) and weaned piglets (n = 180; Study 2) for a duration of 42 d; and consisted of four treatment groups (TG) based on the concentration of microbial-derived AP supplemented in their diet at 0; 12,000; 20,000; and 200,000 U/kg of feed. Parameters such as animal survival, hematology, coagulation, and biochemical indices were assessed at the end of the study. The effect of microbial AP on nutrient absorption through skin pigmentation and intestinal permeability were also investigated in broilers (n = 600; Study 3). In poultry (Study 1), there were no statistically significant differences between control and TG for any of the hematological and biochemical parameters, except for a marginal increase (P < 0.05) in serum phosphorus at the highest dose. This variation was not dose-dependent, was well within the reference range, and was not associated with any clinical correlates. In swine (Study 2), hematological parameters such as leukocyte, basophil, and lymphocyte counts were lower (P < 0.05) for the two highest doses but were traced back to individual variations within the group. The biochemical indices in piglets showed no significant differences between control and supplemental groups except for glucose (P = 0.0005), which showed a high effect (P = 0.008) of the random blood collection order. Nonetheless, glucose was within the normal reference range, and were not related to in-feed supplementation of AP as they had no biological significance. The survival rate in all three studies was over 98%. Dietary supplementation of microbial-derived AP up to 16.7 times the intended use (12,000 U/kg feed) level had no negative effects in both poultry and swine. In-feed supplementation of microbial-derived AP for 28 d improved intestinal pigment absorption (P < 0.0001) and reduced intestinal paracellular permeability (P = 0.0001) in broilers (Study 3). Based on these results, it can be concluded that oral supplementation of microbial-derived AP is safe for poultry and swine and effective at improving gut health in poultry.

Development of a qPCR for the detection and quantification of Salmonella spp. in sheep feces and tissues.

Salmonella spp. are common causes of disease in intensive livestock production systems, and contamination of foodstuffs is of significant concern for public health. Therefore, the identification and quantification of Salmonella spp. is important for monitoring the level of fecal shedding or tissue colonization in infected animals and animal products. We developed and evaluated a quantitative PCR (qPCR) method on spiked sheep tissue and fecal samples for the detection and quantification of Salmonella spp. Without the use of a pre-enrichment step, the qPCR limit of detection (LOD) results for sheep fecal (4 × 104-6 × 103 cfu/g) and tissue (4 × 105-4 × 103 cfu/g) samples were not adequate for detection purposes. With the inclusion of a 6-h pre-enrichment step in buffered peptone water (BPW), the LOD was 9 cfu/g (2.57 × 101 copies/g) in sheep feces, and 5.4 cfu/g (3.22 copies/g) sheep tissue. Comparison of the 6-h BPW qPCR method with a 24-h mannitol-selenite-cystine broth enrichment culture method using spiked samples revealed a sensitivity of 91% and 92%, respectively, and a specificity of 100% for both methods. The correlation was significant between the quantity (copies/mL) of Salmonella spp. in BPW at 6 h and at 0 h, allowing semiquantitative analysis. Our results demonstrate that, following inclusion of a 6-h pre-enrichment step in BPW, qPCR is semiquantitative with improved LODs of Salmonella spp. in sheep fecal and tissue samples.

A review of mycoplasma diagnostics in cattle.

Mycoplasma species have a global distribution causing serious diseases in cattle worldwide including mastitis, arthritis, pneumonia, otitis media and reproductive disorders. Mycoplasma species are typically highly contagious, are capable of causing severe disease, and are difficult infections to resolve requiring rapid and accurate diagnosis to prevent and control disease outbreaks. This review discusses the development and use of different diagnostic methods to identify Mycoplasma species relevant to cattle, with a particular focus on Mycoplasma bovis. Traditionally, the identification and diagnosis of mycoplasma has been performed via microbial culture. More recently, the use of polymerase chain reaction to detect Mycoplasma species from various bovine samples has increased. Polymerase chain reaction has a higher efficiency, specificity, and sensitivity for laboratory diagnosis when compared with conventional culture-based methods. Several tools are now available for typing Mycoplasma spp. isolates, allowing for genetic characterization in disease outbreak investigations. Serological diagnosis through the use of indirect ELISA allows the detection of antimycoplasma antibodies in sera and milk, with their use demonstrated on individual animal samples as well as BTM samples. While each testing method has strengths and limitations, their combined use provides complementary information, which when interpreted in conjunction with clinical signs and herd history, facilitates pathogen detection, and characterization of the disease status of cattle populations.

Comparison of culture and a multiplex probe PCR for identifying Mycoplasma species in bovine milk, semen and swab samples.

Mycoplasma spp. are a major cause of mastitis, arthritis and pneumonia in cattle, and have been associated with reproductive disorders in cows. While culture is the traditional method of identification the use of PCR has become more common. Several investigators have developed PCR protocols to detect M. bovis in milk, yet few studies have evaluated other sample types or other important Mycoplasma species. Therefore the objective of this study was to develop a multiplex PCR assay to detect M. bovis, M. californicum and M. bovigenitalium, and evaluate its analytical performance against traditional culture of bovine milk, semen and swab samples. The PCR specificity was determined and the limit of detection evaluated in spiked milk, semen and swabs. The PCR was then compared to culture on 474 field samples from individual milk, bulk tank milk (BTM), semen and swab (vaginal, preputial, nose and eye) samples. Specificity analysis produced appropriate amplification for all M. bovis, M. californicum and M. bovigenitalium isolates. Amplification was not seen for any of the other Mollicutes or eubacterial isolates. The limit of detection of the PCR was best in milk, followed by semen and swabs. When all three Mycoplasma species were present in a sample, the limit of detection increased. When comparing culture and PCR, overall there was no significant difference in the proportion of culture and PCR positive samples. Culture could detect significantly more positive swab samples. No significant differences were identified for semen, individual milk or BTM samples. PCR identified five samples with two species present. Culture followed by 16S-23S rRNA sequencing did not enable identification of more than one species. Therefore, the superior method for identification of M. bovis, M. californicum and M. bovigenitalium may be dependent on the sample type being analysed, and whether the identification of multiple target species is required.

Genetic characterization of Australian Mycoplasma bovis isolates through whole genome sequencing analysis.

Mycoplasma bovis is a major pathogen in cattle causing mastitis, arthritis and pneumonia. First isolated in Australian cattle in 1970, M. bovis has persisted causing serious disease in infected herds. To date, genetic analysis of Australian M. bovis isolates has not been performed. With whole genome sequencing (WGS) becoming a common tool for genetic characterization, this method was utilized to determine the degree of genetic diversity among Australian M. bovis isolates collected over a nine year period (2006-2015) from various geographical locations, anatomical sites, and from clinically affected and non-clinical carrier animals. Eighty-two M. bovis isolates underwent WGS from which single nucleotide polymorphism (SNP) analysis, comparative genomics and analysis of virulence genes was completed. SNP analysis identified a single M. bovis strain circulating throughout Australia with marked genomic similarity. Comparative genomics suggested minimal variation in gene content between isolates from clinical and carrier animals, and between isolates recovered from different anatomical sites. A total of 50 virulence genes from the virulence factors database (VFDB) were identified as highly similar in the Australian isolates, while the presence of variable surface lipoprotein (vsp) genes was greatly reduced compared to reference strain M. bovis PG45. These results highlight that, while the introduction of multiple M. bovis strains has been prevented, elimination of the current strain has not been successful. The persistence of this strain may be due to the significant role that carrier animals play in harboring the pathogen. The similarity of clinical and non-clinical isolates suggests host and environmental factors play a significant role in determining host pathogen outcomes.