Improved diagnostics and further investigations of bovine mastitis caused by mollicutes

The ultimate aims of this thesis were to improve the diagnostics of mollicute-associated mastitis and confirm their pathogenic role in bovine mastitis. Three types of experiments were carried out on improving diagnostics of mollicute associated mastitis (PCR, real time PCR-high resolution melting curve analysis (HRM), and Enzyme linked immunosorbent assay (ELISA)) on 368 milk samples from a single commercial dairy farm in South Australia (Farm 1), and were compared to conventional culture. Samples were collected purposively only from cows with high somatic cell count and/or mastitis treatment failure. For some tests (e.g. ELISA) samples from a second farm (n=40) in South Australia were also used (Farm 2). Novel primers of 16S ribosomal RNA were used in the PCR and HRM methodologies. A phylogenetic relationship among field isolates of mycoplasmas and acholeplasmas was created based on 16S rRNA sequences. An indirect ELISA, based on a recombinant fragment of the Mycoplasma immunogenic lipase A (MilA) protein was tested in milk for M. bovis antibodies, and compared to PCR and culture. For estimation of the pathogenic role of mollicutes associated with bovine mastitis, milk herd test data was analysed from 7,560 cow-tests. Four types of media (glycerol (GLY) + dimethyl sulphoxide (DMSO), gelatine + DMSO, foetal bovine serum (FBS) + DMSO, and original milk (CON)) were used to test the survivability of Mycoplasma bovis over time at 1, 2, 4, 8 and 16 weeks freezing in domestic and -80º C freezers. Very high prevalence of mollicutes was detected in the 288 purposively sampled cows (76.7%) using species-specific PCR. Culture was inferior in detecting infected milk samples (25.1%). The novel universal PCR demonstrated best concordance with species-specific PCR (Cohen’s Kappa= 0.747 ± 0.031). The novel HRM analysis was able to discriminate between four of the field isolates of Mycoplasma spp. and Acholelasma laidlawii. Mycoplasma bovis antibodies were detected only in 68/291 samples (23.4%). The co-infection with two or more mollicutes had a similar effect on milk composition to other major mastitis pathogens. Long-term stored milk samples should be enriched with some of the cryopreservatives used in this thesis. All cryopreservatives improved the survivability of M. bovis in milk samples stored under freezing conditions. The combination of GLY and DMSO resulted in significantly higher recovery rates at week 16, compared to CON with 57.1% (95% CI = 21.43–133.34) and 19.1% (95% CI = 11.73–60.27), respectively. The use of GLY and DMSO should therefore be encouraged for use as a cryoprotectant for M. bovis at − 20 and − 80 °C. Microbiological and molecular techniques used in this thesis should result in improved diagnostics of mollicute-associated mastitis providing rapid and accurate screening techniques. This should become a cornerstone in control strategies of mollicute-associated bovine mastitis.Thesis (Ph.D.) -- University of Adelaide, School of Animal and Veterinary Sciences, 201

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Evaluation of effects of Mycoplasma mastitis on milk composition in dairy cattle from South Australia

Abstract Background Mycoplasma mastitis is increasingly posing significant impact on dairy industry. Although the effects of major conventional mastitis pathogens on milk components has been widely addressed in the literature, limited data on the effects of different Mycoplasma and Acholeplasma spp. on milk quality and quantity is available. The aim of this study was to determine the casual relationship of Mycoplasma spp. and A. laidlawii to mastitis and compare them to subclinical mastitis caused by conventional mastitis pathogens from a single dairy herd in South Australia; Mycoplasma spp. and A. laidlawii were detected using PCR applied directly to milk samples. The herd had mastitis problem with high somatic cell count and low response rate to conventional antimicrobial therapy. A total of 288 cow-level milk samples were collected aseptically and used in this study. Results Conventional culture showed a predominance of coagulase-negative staphylococci, followed by coagulase-positive staphylococci, Streptococcus spp., Enterococcus spp., E. coli, and Klebsiella spp. PCR results showed a high prevalence of mycoplasmas (76.7%), including A. laidlawii (10.8%), M. bovis (6.2%), M. bovirhinis (5.6%), M. arginini (2%), and (52.1%) of cows were co-infected with two or more Mycoplasma and Acholeplasma species. Mycoplasma co-infection significantly increased somatic cell counts (SCC) similar to conventional mastitis pathogens and compared to non-infected cows with 389.3, 550.3 and 67.3 respectively; and decreased the milk yield with 29.0, 29.9 and 34.4 l, respectively. Mycoplasma co-infection caused significant increase in protein percentage, and significant decrease in fat percentage and total milk solids, similar to other conventional mastitis pathogens. In contrast, changes in milk composition and yield caused by various individual Mycoplasma species were non-significant. Conclusions Mycoplasma mastitis had on-farm economic consequences similar to common conventional mastitis pathogens. Results of our study indicate that co-infection Mycoplasma mastitis caused similar effect on milk composition to other mastitis pathogens and we hope these findings raise the awareness of the importance of their detection on routine diagnostic panels

Discrimination between some Mycoplasma spp. and Acholeplasma laidlawii in bovine milk using high resolution melting curve analysis

Abstract Objectives This study aimed to provide a rapid, accurate and cost-effective diagnostic real time polymerase chain reaction-high resolution melting curve assay (PCR-HRM) to identify and distinguish between four different mycoplasmas and Acholeplasma laidlawii isolated at cow-level from a single commercial dairy farm in South Australia. One set of genus-level universal primers was designed targeting the 16S ribosomal RNA gene. Results Real time PCR-HRM analysis was able to identify and distinguish between five different mollicutes, namely A. laidlawii, M. arginini, M. bovirhinis, M. bovis and uncultured Mycoplasma. Results were confirmed through sequencing. Our developed assay provides rapid and accurate screening for Mycoplasma mastitis detection

Evaluation of three cryoprotectants used with bovine milk affected with Mycoplasma bovis in different freezing conditions

Abstract Objectives Currently, there is no consensus protocols regarding the combination of glycerol (GLY), gelatin or foetal bovine serum (FBS) with dimethyl sulphoxide (DMSO) as cryoprotectants for Mycoplasma bovis in bovine milk samples. This study aimed to compare different cryopreservation compounds and storage temperatures for M. bovis. Results There were significant differences in the survival of M. bovis on different media. Differences were also observed between different storage conditions. All additives improved the survival of M. bovis in comparison to control (CON). The combination of GLY and DMSO was shown to be significantly different to CON with 57.1% (95% CI = 21.43–133.34) and 19.1% (95% CI = 11.73–60.27), respectively at week 16, and its use should be encouraged as a cryoprotectant for M. bovis at − 20 and − 80 °C. GEL/DMSO showed the highest survival rate for M. bovis with 57.14% (95% CI = 21.43–133.34) at 4 °C in comparison with CON 14.29% (95% CI = 9.60–50.39). FBS/DMSO showed the highest survival rate for the short-term preservation similarly to other additives. The evaluated cryopreservative compounds would improve survivability of M. bovis in milk for both transport and long-term storage. Hence, it is recommended to use the mentioned methods for routine transportation or storage purposes for suspicious M. bovis milk samples

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical science. © The Author(s) 2019. Published by Oxford University Press