Bovine mastitis due to coagulase-negative staphylococci and the role of minor pathogens on mastitis

Tese de Doutoramento em Ciências Veterinárias. Especialidade de Sanidade AnimalBovine mastitis is the most common disease of dairy cows. To implement efficient control measures, it is generally necessary to diagnose the relevant aetiologic agents. Coagulasenegative staphylococci (CNS) and Corynebacterium spp. are considered minor mastitis pathogens because of the mild impact they have on udder health. Despite that, they are the most common agents isolated from milk samples in several large scale surveys worldwide. The objectives of this study were to evaluate if there were differences in pathogenicity between individual CNS species, to evaluate if alternative sampling or diagnostic techniques could more accurately determine in which cases the aforementioned minor pathogens were responsible for mastitis, and to determine if treatment of CNS infected quarters was a costeffective control measure. We followed 111 intramammary infections due to CNS for up to 48 weeks in four commercial dairy farms. Duration of infection had a mean of 188 days and was not significantly different between CNS species; geometric mean quarter somatic cell count (SCC) overall was 132,000 cells/ml and was also not significantly different between CNS species. There were differences in diversity between CNS species, with Staphylococcus epidermidis and Staphylococcus simulans showing less diversity than Staphylococcus haemolyticus, the epidemiological significance of which is debated. Freezing milk samples overnight at -20ºC did not increase detection of intramammary bacteria in milk samples. Use of a real-time PCR-based test allowed for detection of udder pathogens beyond the ones identified using conventional bacteriology in milk samples with a high SCC that were culture negative or that yielded CNS or Corynebacterium bovis shown by conventional bacteriology. Single quarter milk samples were collected in duplicate from 132 dairy cows in a commercial dairy farm, with the standard technique and by use of a cannula surpassing the teat canal. There was a significant difference between the two sampling techniques for recovery of Corynebacterium spp. and for culture-negative samples. The observed difference could not be attributed to a particular sampling order and no significant change was observed in quarter SCC between the sampling day and seven days later, indicating iatrogenic IMI following use of the alternative technique was not an issue. Use of a deterministic economic model allowed to determine that in most situations lactational treatment of subclinical mastitis due to CNS would result in a net financial loss, on average of €38.74 per treated quarter. Taken together, these results indicate that CNS have a low impact in terms of udder health, with little differences between individual species. Treating CNS subclinical mastitis during the lactation is not cost-efficient and should therefore not be advised. Using a RTPCR and sampling with a teat cannula might improve diagnosis of mastitis etiology.RESUMO: MASTITE BOVINA POR STAPHYLOCOCCI COAGULASE-NEGATIVOS E O PAPEL DE AGENTES PATOGÉNICOS MENORES NA MASTITE - A mastite bovina é a doença mais comum das vacas leiteiras. Para implementar medidas de controlo eficientes, é geralmente necessário identificar os agentes etiológicos relevantes. Os staphylococci coagulase-negativos (SCN) e Corynebacterium spp. são considerados agentes patogénicos menores porque têm um impacto ligeiro na saúde do úbere. Apesar disso, são os agentes mais frequentemente isolados a partir de amostras de leite em vários estudos de larga escala, em diversos pontos do mundo. Os objectivos deste estudo foram avaliar se havia diferenças em termos de patogenicidade entre espécies individuais de SCN, avaliar se um método de colheita e de diagnóstico alternativos podiam determinar mais precisamente em que casos os agentes mencionados acima eram responsáveis por mastite, e determinar se o tratamento antimicrobiano durante a lactação de quartos infectados com SCN era uma medida de controlo eficiente em termos de custos. Acompanhámos 111 infecções intramamárias por SCN durante até 48 semanas em quatro explorações leiteiras comerciais. A duração média de infecção foi de 188 dias e não foi significativamente diferente entre espécies de SCN; a média geométrica de contagens de células somáticas (CCS) global foi de 132.000 células/ml de leite e também não foi significativamente diferente entre espécies de SCN. Houve diferenças na diversidade entre espécies de SCN, tendo Staphylococcus epidermidis e Staphylococcus simulans mostrando menor diversidade do que Staphylococcus haemolyticus, sendo discutida o possível significado epidemiológico destas observações. A congelação de amostras de leite durante a noite a -20ºC não permitiu o aumento da detecção de bactérias em amostras de infecções intramamárias. A utilização de um PCR em tempo real permitiu a detecção de agentes patogénicos mamários para além dos identificados após utilização de técnicas bacteriológicas convencionais em amostras de leite com CCS elevadas que se mostraram negativas após cultura ou que levaram ao isolamento de SCN ou Corynebacterium bovis. Foram colhidas amostras de leite de quartos individuais em duplicado de 132 vacas leiteiras numa vacaria comercial, com a técnica convencional e com a utilização de uma cânula permitindo ultrapassar o canal do teto. Observou-se uma diferença significativa entre as duas técnicas de colheita para a recolha de Corynebacterium spp. e para amostras sem crescimento após cultura. A diferença observada não era atribuível a uma ordem de colheita particular e não foram observadas alterações significativas na CCS dos quartos entre o dia de colheita e sete dias mais tarde, indicando que a infecção intramamária iatrogénica após a utilização da técnica alternativa não constituiu um problema. A utilização de um modelo económico determinístico permitiu determinar que na maioria das situações o tratamento durante a lactação de mastites subclínicas por SCN resultaria numa perda finaceira líquida, em média de €38.74 por quarto tratado. Considerados em conjunto, estes resultados indicam que os SCN têm um impacto baixo em termos de saúde do úbere, com poucas diferenças entre espécies individuais. Tratar mastites subclínicas por SCN durante a lactação não é eficiente em termos de custos e não deve portanto ser aconselhado. A utilização de um RT-PCR e a colheita de amostras com uma cânula de tetos pode melhorar o diagnóstico das causas de mastite

The port, a point of entry : the design of a new cruise liner terminal at the point, for Durban.

Thesis (M.A.)-University of KwaZulu-Natal, 2008.This research examines the complex factors that influence the design of a new cruise liner terminal for Durban. Secondary and primary data collection methods will be used in the research. Secondary data collection forms the majority of the information gathered. This method will focus on precedent and case studies from which conclusions will be drawn and a design brief for the cruise terminal derived. Cruise liners typically spend periods of less than a week in port, during which time passengers will visit local tourist attractions. Durban's cruise season lasts from mid-November till mid-April. The local cruise industry has seen a steady increase in passenger numbers over the years. This has seen the MSC Melody cruise ship being procured to meet the demands of the industry. As a consequence of this increase the current cruise liner terminal, the N-Shed, has been placed under increasing pressure to provide a world class facility and service experience, expectations which it is ill equipped to deliver. Through this study key aspects, specific to the cruise liner terminal design, are investigated. These include passenger movement optimisation, response to local conditions, form development, waterfront regeneration and the concept of the terminal as a gateway to the city of Durban. In developing the architecture of the cruise terminal, the understanding of these factors is vital if the designer is to make a meaningful contribution to society though his building . With the cruise liner terminal being a unique building typology, in the context of Durban, an opportunity arises to reconnect the city with its harbour edge

Bimodal milk flow and overmilking in dairy cattle : risk factors and consequences

Research Areas: Agriculture ; Veterinary SciencesTo maximise the return on capital invested in the milking parlour, the largest number of cows should be milked gently and completely in the shortest possible time. Bimodal milk flow and overmilking negatively influence the efficiency of the milk removal process and teat health. This observational study had the objective of investigating the prevalence of bimodal milk flow and overmilking, determining which individual and farm-related variables are associated with these occurrences, and determining the association of overmilking and bimodal milk flow with milk yield and with short- and long-term teat changes. Twenty-one farms were visited once during the study period, wherein the milking routine was timed, the teat condition was assessed, and dynamic evaluation of the milking vacuum was performed. A total of 606 vacuum graphic records were obtained, with an average of 29 ± 3 records per farm, in order to indirectly evaluate the milk flow and thus determine the occurrence of bimodal milking and overmilking time. The average percentage of bimodality per farm was 41.7%. The median overmilking time was 59 seconds, and on average, 78.3% of the cows in a herd were overmilked longer than 30 seconds. An association was found at cow level between the occurrence of bimodal milk flow and days in milk, the total stimulation time, parity, and the preparation lag time. The increase in the mean total stimulation time and the number of passes during preparation were associated with a decrease in the proportion of bimodality in the herd. Parity, reattachment of the milking unit and milking in manual mode were associated with an increase in overmilking time of an individual cow. The presence of a clogged air bleed hole in the claw and the reduction of the cluster removal milk flow threshold were associated with an increase in the herd’s median overmilking time. The average milk flow decreased with the increase in overmilking time and with the occurrence of bimodal milk flow. An association was also found between the occurrence of bimodal milk flow and decreased milk yield. A mean of 78.4% of cows per farm had shortterm teat changes in at least one teat, and 33.6% of evaluated cows per farm displayed at least one teat with hyperkeratosis. These results emphasise the association of bimodality and overmilking on milking efficiency and reinforce the importance of the milkers’ actions and the functioning of the milking parlour for its preventioninfo:eu-repo/semantics/publishedVersio

Deterministic model to evaluate the impact of lactational treatment of subclinical mastitis due to coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) are the most frequently isolated bacteria from milk samples in several studies worldwide. Despite their relative frequency, specific measures aiming at their control are not well established. One possible measure to include in a control programme is lactational antimicrobial treatment. The decision to perform such treatment, as well as other actions on farm, should be based on the likelihood of financial return. A deterministic model was used to evaluate whether performing an antimicrobial treatment during the lactation for quarters infected with CNS was financially justifiable. Input variables for the impact of CNS on udder health were based on a previous study by the same authors and on available literature on the subject. Prices included in the model were based on 2009/2010 conditions in Portugal. The average result per antimicrobial treated quarter was a net loss of (sic)38.74. Performing a sensitivity analysis to evaluate how systematic variation of the input variables of the model would lead to outcome changes showed that variation in input variables nearly always led to a negative outcome, with the greatest variation in losses observed for variation in the length of treatment and milk withdrawal period (-(sic)46.26 to -(sic)28.49). The situations in which a net benefit was to be expected included the bulk tank somatic cell count decreasing to a level corresponding to a premium payment or to penalties being avoided, and the prevention of transmission of CNS in the milking parlour when the possibility of transmission was at its highest level. For most situations, lactational treatment of CNS subclinical mastitis was not financially justifiable

THE FUTURE OF VETERINARIANS IN DAIRY HERD HEALTH MANAGEMENT

The future of the Veterinary Practice in Dairy Health Management has changed and will change more drastically from our point of view in the next years. The consumer’s pressure and the Media are more and more concerned about animal welfare, traceability of animal products and safety of products of animal origin. On the other hand the Farmers in Europe have to produce under strong rules (competing with other countries outside Europe), which are normally very expensive to put in practice, and the veterinarians should adapt their knowledge to the new challenges, because without their work and cooperation, dairy farming will have no future. In that sense, the old veterinary practice has to go in other ways, otherwise the Veterinarians will loose clients and the animal population in Europe will be reduced. The Dairy farmers will ask for support in other areas besides clinical: efficacy, management, welfare, profitability, nutrition, prophylaxis, economics, reproduction, environmental protection, grassland management, etc.Cattle practitioners should be able to give answers in several subjects and this sets the challenge to our profession - Veterinary preparation has to be very strong in single animal species, particularly in Dairy or beef cows. The cattle practitioner has to look beyond, but he should never forget that “the single animal” has to be looked at as one unit of the herd, which means that without a very good knowledge of the single animal he will be insufficiently prepared to solve herd problems, and the Herd is the sum of several animals. We all know that very often one single animal allows us to implement herd strategies and develop prophylactic programs.We are convinced that the veterinary profession, and in our case the Cattle Medicine should have the ability to evolve, otherwise the Veterinarian as we know him will miss the train in the next years

THE FUTURE OF VETERINARIANS IN DAIRY HERD HEALTH MANAGEMENT

The future of the Veterinary Practice in Dairy Health Management has changed and will change more drastically from our point of view in the next years. The consumer’s pressure and the Media are more and more concerned about animal welfare, traceability of animal products and safety of products of animal origin. On the other hand the Farmers in Europe have to produce under strong rules (competing with other countries outside Europe), which are normally very expensive to put in practice, and the veterinarians should adapt their knowledge to the new challenges, because without their work and cooperation, dairy farming will have no future. In that sense, the old veterinary practice has to go in other ways, otherwise the Veterinarians will loose clients and the animal population in Europe will be reduced. The Dairy farmers will ask for support in other areas besides clinical: efficacy, management, welfare, profitability, nutrition, prophylaxis, economics, reproduction, environmental protection, grassland management, etc.Cattle practitioners should be able to give answers in several subjects and this sets the challenge to our profession - Veterinary preparation has to be very strong in single animal species, particularly in Dairy or beef cows. The cattle practitioner has to look beyond, but he should never forget that “the single animal” has to be looked at as one unit of the herd, which means that without a very good knowledge of the single animal he will be insufficiently prepared to solve herd problems, and the Herd is the sum of several animals. We all know that very often one single animal allows us to implement herd strategies and develop prophylactic programs.We are convinced that the veterinary profession, and in our case the Cattle Medicine should have the ability to evolve, otherwise the Veterinarian as we know him will miss the train in the next years

Dynamics of bovine intramammary infections due to coagulase-negative staphylococci on four farms

The objectives of this study were to compare the impact of different coagulase-negative species (CNS) on udder health measured in terms of individual quarter milk somatic cell count (SCC) and duration of intramammary infection, and to get some insight into most likely routes of infection for different CNS species. This longitudinal observational study was performed on four farms that were sampled at 4-week intervals for a total of 12 visits each. Quarters infected with CNS were followed through time with milk samples being submitted for bacteriological culture and SCC determination. PCR amplification of the internal transcribed spacer region and sequencing of the sodA and rpoB genes were used for species allocation. Pulsed-field gel electrophoresis (PFGE) was performed to assess strain identity. The percentage of quarters affected per farm varied between 6 and 35%, with the most frequently isolated CNS species being Staphylococcus epidermidis, followed by Staph. simulans, Staph. chromogenes and Staph. haemolyticus. It was possible to follow 111 intramammary infections due to CNS through time. Duration of infection had a mean of 188 d and was not significantly different between CNS species. Geometric mean quarter SCC overall was 132 000 cells/ml and was also not significantly different between CNS species. Despite the possibility of a different epidemiology of infection, the impact in terms of udder health seems to be similar for different CNS species

Biosensors for On-Farm Diagnosis of Mastitis

Bovine mastitis is an inflammation of the mammary gland caused by a multitude of pathogens with devastating consequences for the dairy industry. Global annual losses are estimated to be around €30 bn and are caused by significant milk losses, poor milk quality, culling of chronically infected animals, and occasional deaths. Moreover, mastitis management routinely implies the administration of antibiotics to treat and prevent the disease which poses serious risks regarding the emergence of antibiotic resistance. Conventional diagnostic methods based on somatic cell counts (SCC) and plate-culture techniques are accurate in identifying the disease, the respective infectious agents and antibiotic resistant phenotypes. However, pressure exists to develop less lengthy approaches, capable of providing on-site information concerning the infection, and in this way, guide, and hasten the most adequate treatment. Biosensors are analytical tools that convert the presence of biological compounds into an electric signal. Benefitting from high signal-to-noise ratios and fast response times, when properly tuned, they can detect the presence of specific cells and cell markers with high sensitivity. In combination with microfluidics, they provide the means for development of automated and portable diagnostic devices. Still, while biosensors are growing at a fast pace in human diagnostics, applications for the veterinary market, and specifically, for the diagnosis of mastitis remain limited. This review highlights current approaches for mastitis diagnosis and describes the latest outcomes in biosensors and lab-on-chip devices with the potential to become real alternatives to standard practices. Focus is given to those technologies that, in a near future, will enable for an on-farm diagnosis of mastitisinfo:eu-repo/semantics/publishedVersio

New Insights on Streptococcus dysgalactiae subsp. dysgalactiae Isolates

Funding Information: This work was supported by the Unidade de Ciências Biomoleculares Aplicadas-UCIBIO, which is financed by Funding Information: Funding. This work was supported by the Unidade de Ci?ncias Biomoleculares Aplicadas-UCIBIO, which is financed by national funds from FCT/MEC (UIDP/04378/2020 and UIDB/04378/2020) and also by projects PTDC/CVT-EPI/4651/2012 and PTDC/CVT-EPI/6685/2014. FCT-MEC is also acknowledged for grant SFRH/BD/118350/2016 to CA-B. Publisher Copyright: © Copyright © 2021 Alves-Barroco, Caço, Roma-Rodrigues, Fernandes, Bexiga, Oliveira, Chambel, Tenreiro, Mato and Santos-Sanches.Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) has been considered a strict animal pathogen. Nevertheless, the recent reports of human infections suggest a niche expansion for this subspecies, which may be a consequence of the virulence gene acquisition that increases its pathogenicity. Previous studies reported the presence of virulence genes of Streptococcus pyogenes phages among bovine SDSD (collected in 2002–2003); however, the identity of these mobile genetic elements remains to be clarified. Thus, this study aimed to characterize the SDSD isolates collected in 2011–2013 and compare them with SDSD isolates collected in 2002–2003 and pyogenic streptococcus genomes available at the National Center for Biotechnology Information (NCBI) database, including human SDSD and S. dysgalactiae subsp. equisimilis (SDSE) strains to track temporal shifts on bovine SDSD genotypes. The very close genetic relationships between humans SDSD and SDSE were evident from the analysis of housekeeping genes, while bovine SDSD isolates seem more divergent. The results showed that all bovine SDSD harbor Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas IIA system. The widespread presence of this system among bovine SDSD isolates, high conservation of repeat sequences, and the polymorphism observed in spacer can be considered indicators of the system activity. Overall, comparative analysis shows that bovine SDSD isolates carry speK, speC, speL, speM, spd1, and sdn virulence genes of S. pyogenes prophages. Our data suggest that these genes are maintained over time and seem to be exclusively a property of bovine SDSD strains. Although the bovine SDSD genomes characterized in the present study were not sequenced, the data set, including the high homology of superantigens (SAgs) genes between bovine SDSD and S. pyogenes strains, may indicate that events of horizontal genetic transfer occurred before habitat separation. All bovine SDSD isolates were negative for genes of operon encoding streptolysin S, except for sagA gene, while the presence of this operon was detected in all SDSE and human SDSD strains. The data set of this study suggests that the separation between the subspecies “dysgalactiae” and “equisimilis” should be reconsidered. However, a study including the most comprehensive collection of strains from different environments would be required for definitive conclusions regarding the two taxa.publishersversionpublishe