Use of on-farm data to guide treatment and control mastitis caused by Streptococcus uberis

Treatment of mastitis is the most common reason for use of antimicrobial agents in dairy cattle. The responsible use of antimicrobials could be strengthened by knowledge of predictors for cure, which would help to tailor treatment decisions. Ideally, to allow for widespread uptake, this would be achieved using data that are routinely available. To assess whether this is feasible in practice, farmers were invited to submit milk samples from mastitis cases to their veterinary practice for bacteriological culture. Among 624 culture-positive samples, 251 were positive for Streptococcus uberis. Using cow-level data, cases were classified as severe, first nonsevere, repeat, or subclinical. Additional data were collected at the cow level [somatic cell count (SCC), parity, lactation stage, milk yield, fat and protein contents, treatment] and at the herd level (housing, bedding, premilking teat disinfection, postmilking teat disinfection). Severe cases were overrepresented among heifers and animals in early lactation, and repeat cases were overrepresented in cows with 3 or more lactations. The probability of cure was higher among first- and second-parity animals than among older cows, and was higher in animals with a single elevated cow-level SCC than in animals with multiple high SCC records. Results obtained in the current study are similar to those previously described for Staphylococcus aureus mastitis. Thus, routinely available cow-level information can help to predict the outcome of antimicrobial treatment of the most common causes of gram-positive mastitis

Molecular and mathematical epidemiology of Staphylococcus aureus and Streptococcus uberis mastitis in dairy herds

Mastitis is the most common and costly production disease affecting dairy cows. Staphylococcus aureus and Streptococcus uberis are two major mastitis-causing pathogens. Staphylococcus aureus is traditionally classified as contagious pathogen, while Streptococcus uberis is classified as environmental pathogen. Infected udders form the main reservoir of contagious pathogens. Exposure to contagious pathogens is largely limited to the milking process. Environmental pathogens are present in the environment of the cow. Cows may be exposed at any time in life. We used a combination of molecular and mathematical methods to study whether S. aureus and S. uberis are indeed contagious and environmental pathogens, respectively. Pulsed-Field Gel Electrophoresis and binary typing were used for strain typing of S. aureus and had higher typeability and discriminatory power than phage typing. Comparison of clinical and epidemiological observations with typing results revealed significant associations between distinct genotypes and severity of disease, suggesting strain-specific virulence. Analysis of human and bovine S. aureus isolates showed that strains clustered predominantly in host specific clones. Circumstantial evidence suggested an environmental origin of one S. aureus strain. Teat skin did not appear to be an important source of intramammary S. aureus. Random-amplified polymorphic DNA typing was used to characterize S. uberis strains. Within two herds, a variety of strains were found in addition to a predominant strain, suggesting co-existence of strains with environmental and contagious epidemiologies. Infections with dominant strains lasted longer than infections with unique strains. One dominant strain caused an outbreak of mastitis that was limited to lactating cows. The strain was also isolated from the milking machine. The combined evidence strongly supports contagious transmission during milking. SIR-models were used to describe the spread of mastitis and simulate the impact of control measures. Prevalence of infection was a significant predictor for the number of new infections with S. uberis in one herd, as would be expected under the assumption of contagious transmission. Presence of other pathogens did not affect the susceptibility of udder quarters to new infection, but quarters that had recovered from infection were more susceptible than quarters that had not experienced mastitis. The same was true for S. aureus, as shown in a study of cow and quarter-level risk factors for new infections and by means of an SIR-model. Treatment of subclinical mastitis and prevention of influx of infected animals contribute to elimination of S. aureus mastitis from a herd under the assumption that S. aureus is spread solely through contagious transmission. However, even when R, the reproduction ratio, was lower than the threshold value of 1, indicating that contagious transmission was curbed, new infections continued to occur. Among other things, this may be the result of infections that originate from environmental sources. In summary, stain typing studies and mathematical models suggest that both S. aureus and S. uberis are genetically varied species that include strains with contagious transmission as well as strains with environmental epidemiology. This implies that both options have to be considered when studying mastitis epidemiology or choosing control measures for dairy herd managemen

Development and application of a prophage integrase typing scheme for group B Streptococcus

Group B Streptococcus (GBS) is a gram-positive pathogen mainly affecting humans, cattle, and fishes. Mobile genetic elements play an important role in the evolution of GBS, its adaptation to host species and niches, and its pathogenicity. In particular, lysogenic prophages have been associated with a high virulence of certain strains and with their ability to cause invasive infections in humans. It is therefore important to be able to accurately detect and classify prophages in GBS genomes. Several bioinformatic tools for the identification of prophages in bacterial genomes are available on-line. However, genome searches for most of these programs are affected by the composition of their reference database. Lack of databases specific to GBS results in failure to recognize all prophages in the species. Additionally, performance of these programs is affected by genome fragmentation in the case of draft genomes, leading to underestimation of the number of phages. They also prove impractical when dealing with large genome datasets and they do not offer a quick way of classifying bacteriophages. We developed a GBS-specific method to screen genome assemblies for the presence of prophages and to classify them based on a reproducible typing scheme. This was achieved through an extensive search of a vast number of high-quality GBS sequences (n = 572) originating from different host species and countries in order to build a database of phage integrase types, on which the scheme is based. The proposed typing scheme comprises 12 integration sites and sixteen prophage integrase types, including multiple subtypes per integration site and integrase genes that were not site-specific. Two putative phage-inducible chromosomal islands (PICI) and their insertion sites were also identified during the course of these analyses. Phages were common and diverse in all major clonal complexes associated with human disease and detected in isolates from every animal species and continent included in the study. This database will facilitate further work on the prevalence and role of prophages in GBS evolution, and identifies the roles of PICIs in GBS and of prophage in hypervirulent ST283 as areas for further research

Association between genotypic diversity and biofilm production in group B Streptococcus

Background: Group B Streptococcus (GBS) is a leading cause of sepsis and meningitis and an important factor in premature and stillbirths. Biofilm production has been suggested to be important for GBS pathogenesis alongside many other elements, including phylogenetic lineage and virulence factors, such as pili and capsule type. A complete understanding of the confluence of these components, however, is lacking. To identify associations between biofilm phenotype, pilus profile and lineage, 293 strains from asymptomatic carriers, invasive disease cases, and bovine mastitis cases, were assessed for biofilm production using an in vitro assay. Results: Multilocus sequence type (ST) profile, pilus island profile, and isolate source were associated with biofilm production. Strains from invasive disease cases and/or belonging to the ST-17 and ST-19 lineages were significantly more likely to form weak biofilms, whereas strains producing strong biofilms were recovered more frequently from individuals with asymptomatic colonization. Conclusions: These data suggest that biofilm production is a lineage-specific trait in GBS and may promote colonization of strains representing lineages other than STs 17 and 19. The findings herein also demonstrate that biofilms must be considered in the treatment of pregnant women, particularly for women with heavy GBS colonization

Validation of tDNA-intergenic spacer PCR for species level identification of bovine coagulase-negative staphylococci

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Bovine viral diarrhoea virus loses quasispecies diversity rapidly in culture

Bovine viral diarrhoea (BVD) is an important disease of cattle, with significant impacts on animal health and welfare. The wide host range of the causative pestiviruses may lead to formation of virus reservoirs in other ruminant or wildlife species, presenting a concern for the long-term success of BVD eradication campaigns. It is likely that the quasispecies nature of these RNA viruses contributes to their interspecies transmission by providing genetic plasticity. Understanding the spectrum of sequence variants present in persistently infected (PI) animals is, therefore, essential for studies of virus transmission. To analyse quasispecies diversity without amplification bias, we extracted viral RNA from the serum of a PI cow, and from cell culture fluid after three passages of the same virus in culture, to produce cDNA without amplification. Sequencing of this material using Illumina 250 bp paired-read technology produced full-length virus consensus sequences from both sources and demonstrated the quasispecies diversity of this pestivirus A genotype 1a field strain within serum and after culture. We report the distribution and diversity of over 800 SNPs and provide evidence for a loss of diversity after only three passages in cell culture, implying that cultured viruses cannot be used to understand quasispecies diversity and may not provide reliable molecular markers for source tracing or transmission studies. Additionally, both serum and cultured viruses could be sequenced as a set of 25 overlapping PCR amplicons that demonstrated the same consensus sequences and the presence of many of the same quasispecies variants. The observation that aspects of the quasispecies structure revealed by massively parallel sequencing are also detected after PCR and Sanger sequencing suggests that this approach may be useful for small or difficult to analyse samples

Mastitomics, the integrated omics of bovine milk in an experimental model of Streptococcus uberis mastitis: 1. High abundance proteins, acute phase proteins and peptidomics

A peptidomic investigation of milk from an experimental model of Streptococcus uberis mastitis in dairy cows has incorporated a study of milk high abundance and acute phase (APP) proteins as well as analysis of low molecular weight peptide biomarkers. Intramammary infection (IMI) with S. uberis caused a shift in abundance from caseins, β-lactoglobulin and α-lactalbumin to albumin, lactoferrin and IgG with the increase in lactoferrin occurring last. The APP response of haptoglobin, mammary associated serum amyloid A3 and C-reactive protein occurred between 30–48 hours post challenge with peak concentrations of APPs at 72–96 hours post challenge and declined thereafter at a rate resembling the fall in bacterial count rather than the somatic cell count. A peptide biomarker panel for IMI based on capillary electrophoresis and mass spectrometry was developed. It comprised 77 identified peptides (IMI77) composed mainly of casein derived peptides but also including peptides of glycosylation dependent cell adhesion molecule and serum amyloid A. The panel had a biomarker classification score that increased from 36 hour to 81 hour post challenge, significantly differentiating infected from non-infected milk, thus suggesting potential as a peptide biomarker panel of bovine mastitis and specifically that of S. uberis origin. The use of omic technology has shown a multifactorial cross system reaction in high and low abundance proteins and their peptide derivatives with changes of over a thousand fold in analyte levels in response to S. uberis infection

Galleria mellonella as an infection model for the multi-host pathogen Streptococcus agalactiae reflects hypervirulence of strains associated with human invasive disease

Streptococcus agalactiae, or group B Streptococcus (GBS), infects diverse hosts including humans and economically important species such as cattle and fishes. In the context of human health, GBS is a major cause of neonatal infections and an emerging cause of invasive disease in adults and of foodborne disease in Southeast Asia. Here we show that GBS is able to establish a systemic infection in Galleria mellonella larvae that is associated with extensive bacterial replication and dose-dependent larval survival. This infection model is suitable for use with GBS isolates from both homeothermic and poikilothermic hosts. Hypervirulent sequence types (ST) associated with invasive human disease in neonates (ST17) or adults (ST283) show increased virulence in this model, indicating it may be useful in studying GBS virulence determinants, albeit with limitations for some host-specific virulence factors. In addition, we demonstrate that larval survival can be afforded by antibiotic treatment and so the model may also be useful in the development of novel anti-GBS strategies. The use of G. mellonella in GBS research has the potential to provide a low-cost infection model that could reduce the number of vertebrates used in the study of GBS infection

Food Safety, Health Management, and Biosecurity Characteristics of Poultry Farms in Arusha City, Northern Tanzania, Along a Gradient of Intensification

Background: With the growth, urbanisation, and changing consumption patterns of Tanzania’s human population, new livestock production systems are emerging. Intensification of poultry production may result in opportunities and threats for food safety, such as improved awareness of biosecurity or increasing prevalence of foodborne pathogens including nontyphoidal Salmonella or Campylobacter spp. We conducted a semiquantitative analysis of poultry production systems in northern Tanzania, with emphasis on biosecurity, health management practices, and prevalence of foodborne pathogens, to gain insight into potential associations between intensification and food safety.   Methods: Interviews were conducted with managers of 40 poultry farms, with equal representation of 4 production systems (extensive, semi-intensive, or intensive production with indigenous chickens, and broiler farming). Per farm, up to 10 birds (total, 386) were tested for cloacal shedding of nontyphoidal Salmonella, with a subset of farms tested for Campylobacter. Data were analysed using univariate statistics, and results were discussed during feedback workshops with participating farmers and extension officers.   Results: Clear differences existed between farm types with regard to implementation of biosecurity and health management practices and use of extension services. By contrast, prevalence of foodborne pathogens (6 of 40 farms or 15% for nontyphoidal Salmonella and 13 of 26 farms or 50% for Campylobacter spp.) was not farm-type specific, indicating that it is driven by other factors. Across farming systems, knowledge and awareness of the presence of antimicrobials in poultry feed and the need to abide by post-treatment withdrawal times were limited, as was access to impartial professional advice regarding treatment.   Conclusion: Different control measures may be needed to protect poultry health compared to public health, and improvements in information provision may be needed for both

Bovine milk microbiome : a more complex issue than expected

Abstract The aim of this study was to analyze bacterial profiles of bovine mastitic milk samples and samples from healthy quarters using Next Generation Sequencing of amplicons from 16S rRNA genes and to compare results with microbiological results by PCR assays of the same samples. A total of 49 samples were collected from one single dairy herd during the same day. The samples were divided in two sample sets, which were used in this study. The DNA extraction as well as the library preparation and sequencing of these two sets were performed separately, and results of the two datasets were then compared. The vast majority of genera detected appeared with low read numbers and/or in only a few samples. Results of PCR and microbiome analyses of samples infected with major pathogens Staphylococcus aureus or Streptococcus uberis were consistent as these genera also covered the majority of reads detected in the microbiome analysis. Analysis of alpha diversity revealed a much higher species richness in set 1 than in set 2. The dominating bacterial genera with the highest read numbers clearly differed between datasets, especially in PCR negative samples and samples positive for minor pathogens. In addition to this, linear discriminant analysis (LDA) was conducted between the two sets to identify significantly different genera/family level microbes. The genus Methylobacterium was much more common in set 2 compared to set 1, and genus Streptococcus more common in set 1. Our results indicate amplification of contaminating bacteria in excess in samples with no or minor amounts of pathogen DNA in dataset 2. There is a need for critical assessment of results of milk microbiome analyses