Diagnosing intramammary infection: Controlling misclassification bias in longitudinal udder health studies.

Using imperfect tests may lead to biased estimates of disease frequency and of associations between risk factors and disease. For instance in longitudinal udder health studies, both quarters at risk and incident intramammary infections (IMI) can be wrongly identified, resulting in selection and misclassification bias, respectively. Diagnostic accuracy can possibly be improved by using duplicate or triplicate samples for identifying quarters at risk and, subsequently, incident IMI. The objectives of this study were to evaluate the relative impact of selection and misclassification biases resulting from IMI misclassification on measures of disease frequency (incidence) and of association with hypothetical exposures. The effect of improving the sampling strategy by collecting duplicate or triplicate samples at first or second sampling was also assessed. Data sets from a hypothetical cohort study were simulated and analyzed based on a separate scenario for two common mastitis pathogens representing two distinct prevailing patterns. Staphylococcus aureus, a relatively uncommon pathogen with a low incidence, is identified with excellent sensitivity and almost perfect specificity. Coagulase negative staphylococci (CNS) are more prevalent, with a high incidence, and with milk bacteriological culture having fair Se but excellent Sp. The generated data sets for each scenario were emulating a longitudinal cohort study with two milk samples collected one month apart from each quarter of a random sample of 30 cows/herd, from 100 herds, with a herd-level exposure having a known strength of association. Incidence of IMI and measure of association with exposure (odds ratio; OR) were estimated using Markov Chain Monte Carlo (MCMC) for each data set and using different sampling strategies (single, duplicate, triplicate samples with series or parallel interpretation) for identifying quarters at risk and incident IMI. For S. aureus biases were small with an observed incidence of 0.29 versus a true incidence of 0.25IMI/100 quarter-month. In the CNS scenario, diagnostic errors in the two samples led to important selection (40IMI/100 quarter-month) and misclassification (23IMI/100 quarter-month) biases for estimation of IMI incidence, respectively. These biases were in opposite direction and therefore the incidence measure obtained using single sampling on both the first and second test (29IMI/100 quarter-month) was exactly the true value. In the S. aureus scenario the OR for association with exposure showed little bias (observed OR of 3.1 versus true OR of 3.2). The CNS scenario revealed the presence of a large misclassification bias moving the association towards the null value (OR of 1.7 versus true OR of 2.6). Little improvement could be brought using different sampling strategies aiming at improving Se and/or Sp on first and/or second sampling or using a two out of three interpretation for IMI definition. Increasing number of samples or tests can prevent bias in some situations but efforts can be spared by holding to a single sampling approach in others. When designing longitudinal studies, evaluating potential biases and best sampling strategy is as critical as the choice of test.

Identification of subclinical mastitis caused by Mycoplasma spp. from screenings of bulk tanks / Identificação de mastite subclínica causada por Mycoplasma spp. de triagem de tanques de expansão

Mastitis caused by Mycoplasma spp., regardless of species, are considered highly contagious pathogens and, usually was not responsive to antimicrobial therapy. Five dairy herds, comprising 489 animals and 1,956 mammary glands, were used in this study. Milk samples were obtained from bulk tanks and subjected to polymerase chain reaction (PCR) for the identification of Mollicutes, Mycoplasma spp., and Mycoplasma bovis. Moreover, individual samples from cases of clinical and subclinical mastitis in quarters of the dairy herds' animals that yielded a positive PCR upon bulk tank analysis were subjected to molecular analysis. Only one bulk tank was positive for class Mollicutes by PCR. All positive samples classified as mastitis teats had their DNA extracted and tested by PCR for both class Mollicutes and M. bovis. Of these, two (2.08%) were positive for Mycoplasma genus, although none was positive for M. bovis. This result suggests that the PCR of bulk tanks is a viable tool in monitoring and preventing mastitis infections caused by Mycoplasma spp.(AU)

Mastites bovinas causadas por Mycoplasma spp., independentemente da espécie causadora, são consideradas de alta contagiosidade e geralmente não responsivas à terapia antimicrobiana. Cinco propriedades leiteiras foram utilizadas neste estudo, totalizando 489 animais e 1956 quartos mamários. As amostras de leite foram obtidas de tanques de expansão e submetidas à reação em cadeia da polimerase (PCR) para pesquisa de Mollicutes, Mycoplasma spp. e Mycoplasma bovis. Apenas um tanque de uma propriedade foi positivo na PCR para a classe Mollicutes. Amostras individuais de casos de mastite subclínica provenientes de propriedade com tanque positivo também foram submetidas à análise molecular; dessas, duas delas (2,08%) foram positivas para a classe Mollicutes e para o gênero Mycoplasma, entretanto nenhuma foi positiva para a espécie Mycoplasma bovis. Isso sugere que a PCR de tanques de expansão de propriedades leiteiras demonstra ser uma ferramenta viável no monitoramento e na prevenção de infecções por Mycoplasma spp.(AU)

Influência da mastite nas frações eletroforéticas do colostro de vacas da raça Holandesa / Influence of mastitis on electrophoretic fractions of colostrum from Holstein cows

O objetivo desse estudo foi o de avaliar as frações proteicas em secreções colostrais de vacas acometidas por mastite clínica imediatamente após o parto. Para tanto, foram utilizadas 30 vacas da raça Holandesa distribuídas em três grupos, a saber: Grupo I (GI)- 10 vacas pluríparas sadias, Grupo II (GII) 10 vacas pluríparas que pariram com mastite assintomática e Grupo III (GIII) 10 vacas pluríparas que pariram com mastite clínica. Foram avaliadas as concentrações de imunoglobulina a (IgA), lactoferrina (LF), albumina, imunoglobulina G (IgG), ß-lactoglobulina (ß-Lg) e α-lactoalbumina (α-La) por meio da eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio (SDS-PAGE).Observou-se que a IgG, LF e a albumina variaram entre as glândulas com mastite assintomática e clínica quando comparadas às glândulas sadias, e que a presença de um único microrganismo é capaz de promover alterações no proteinograma, com ou sem manifestações clínicas na glândula mamária.(AU)

The aim of this study was to evaluate the protein fractions in colostral secretions of cows affected by mastitis immediately after calving. Therefore, 30 Holstein cows were divided into three groups: Group I (GI) composed of ten multiparous cows calving without mastitis; Group II (GII) composed of ten multiparous cows calving with subclinical mastitis, and Group III (GIII) composed of ten multiparous cows calving with mastitis. The concentration of immunoglobulin A (IgA), lactoferrin (LF), albumin, immunoglobulin G (IgG), ß-lactoglobulin (ß-Lg) and α-lactoalbumin (α-La) was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the IgG, LF and albumin vary among glands of subclinical and clinical mastitis and healthy and that the presence of a bacteria in the mammary gland was the key role for changing of the pattern of serum protein source.(AU)

Perfil de imunoglobulinas, cura clínica e bacteriológica após diferentes tratamentos para a mastite clínica bovina / Profile of immunoglobulins, clinical and bacteriological cure after different treatment routes of clinical bovine mastitis

O presente estudo objetivou avaliar o perfil das Ig durante os diferentes tratamentos de vacas com mastite clínica. Para isso, 30 vacas com mastite clínica em um quarto mamário foram utilizadas e divididas em três grupos. O primeiro grupo foi composto por 10 animais submetidos ao tratamento combinado com infusão intramamária de 8,5mg de sulfato de cefquinoma após cada ordenha, totalizando três aplicações e administração intramuscular de 2,5mg/kg de enrofloxacina por três dias. O segundo grupo foi composto por 10 animais submetidos ao tratamento intramamário, com infusão intramamária de 8,5mg de sulfato de cefquinoma, após cada ordenha, totalizando três aplicações. O terceiro grupo foi composto por 10 animais submetidos ao tratamento sistêmico, com 2,5mg/kg de enrofloxacina, durante três dias. As amostras de leite foram coletadas de todos os animais antes dos tratamentos (momento 0), no segundo (momento 1), no quinto (momento 2) e no 12º dia (momento 3) após o término dos tratamentos. Estas foram submetidas à contagem de células somáticas, ao California Mastitis Test (CMT), ao exame bacteriológico e à quantificação das IgG1, IgG2, IgA e IgM. O tratamento combinado foi mais eficaz e precoce na taxa de cura clínica, na redução dos escores de CMT e da contagem de células somáticas. Além disso, os resultados do presente estudo demonstraram que as concentrações lácteas das diferentes classes de Ig, apesar de sua importância biológica, não estão relacionadas ao prognóstico da mastite clínica bovina, ou seja, não podem ser consideradas marcadores robustos associados à cura clínica e/ou bacteriológica da infecção intramamária.(AU)

The present study aimed to evaluate the profile of immunoglobins profile, clinical and bacteriological cure after different treatment routes of clinical bovine mastitis. Here, 30 Holstein cows with clinical mastitis in one quarter were uniformly divided into: 10 dairy cows that received 8.5mg of cefquinome sulphate administrated intramammarily during three consecutive milkings and 2.5mg/kg of enrofloxacin administrated parenterally during three consecutive days (Group 1); 10 dairy cows that received 8.5mg of cefquinome sulphate administrated intramammarily during three consecutive milkings (Group 2); and 10 dairy cows that received 2.5mg/kg of enrofloxacin administrated parenterally during three consecutive days (Group 3). Milk samples for somatic cell count, California Mastitis Test (CMT), bacteriological culture and quantification of IgG1, IgG2, IgM and IgA were collected before antimicrobial treatment, and after two, five and 12 days after the antimicrobial treatment. The combined treatment was more effective in the clinical cure rate, reduction of somatic cell count and CMT scores. Furthermore, the results demonstrated that milk concentration of different Igs classes were not related to prognosis of bovine clinical mastitis, and then cannot be considered as robust markers associated with clinical and bacteriological cures.(AU)

Mastite bovina por Prototheca zopfii na região oeste do Paraná: Relato de casos / Bovine mastitis by Prototheca zopfii in western Paraná: Case reports / Mastitis bovina por Prototheca zopfii en la región oeste de Paraná: Relato de casos

Relatam-se três casos de mastite clínica em vacas da raça Jersey de uma propriedade localizada na região Oeste do estado do Paraná, refratários ao tratamento com antimicrobianos. Após realização de cultura das amostras de leite, foi identificada Prototheca zopfii, uma alga aclorofilada, unicelular, presente em matéria orgânica e resistente a antimicrobianos. Foi indicado o descarte dos animais positivos e, como medida de profilaxia, a higienização de caixas de abastecimento de água e bebedouros, uma vez que o manejo da ordenha e higienização da ordenhadeira era realizada de maneira adequada na propriedade. De acordo com a literatura consultada, este é o primeiro relato de mastite por Prototheca zopfii na região Oeste do Paraná, salientando a importância da realização da cultura de amostras de leite de vacas portadoras de mastite, especialmente em casos refratários ao tratamento.(AU)

Three cases of clinical mastitis were reported in Jersey cows at a farm located in western Parana, with a history of refractory treatment with antimicrobials. Milk sample cultures presented Prototheca zopfii, an aclorophylated, unicellular algae, present in organic matter and resistant to antimicrobial treatment. The disposal of positive animals was indicated, and the prophylactic measure of cleaning the water storage and drinking containers were encouraged, since the milking and cleaning of the milking machine were properly performed at the farm. According to the literature, this is the first report of mastitis by Prototheca zopfii in western Parana, thus emphasizing the importance of performing cultures to the milk samples of cows presenting mastitis, especially in cases that are refractory to treatment.(AU)

Tres casos de mastitis clínica se ha relatado en vacas de la raza Jersey, de una propiedad ubicada en la región Oeste del Estado de Paraná, resistentes al tratamiento con antimicrobianos. Después de la realización de cultura de las muestras de leche, se identificó Prototheca zopfii, un alga aclorofilada, unicelular, presente en materia orgánica y resistente a antimicrobianos. Se aconsejó el descarte de los animales positivos y, como medida de profilaxis, la higienización de las cajas de abastecimiento de agua y bebederos, una vez que el manejo de ordeña e higienización de la ordeñadera era realizada de forma adecuada en la propiedad. De acuerdo con la literatura consultada, este es el primer relato de mastitis por Prototheca zopfii en la región Oeste de Paraná, resaltando la importancia de realización de cultura de las muestras de leche de vacas portadoras de mastitis, especialmente en casos resistentes al tratamiento.(AU)

Comparison of disease severity scoring systems for dairy cattle with acute coliform mastitis.

OBJECTIVE: To compare use of 4 disease severity scoring systems to predict bacteremia (yes vs no) and outcome (survived vs died or culled) in dairy cows with acute coliform mastitis (ACM). DESIGN: Retrospective cohort study. ANIMALS: 99 dairy cows with ACM. PROCEDURES: Cows were classified as having mild, moderate, or severe disease with a scoring system based on systemic disease signs alone (systemic severity score [SSS] system), a system based on local disease signs alone (local severity score [LSS] system), and 2 previously described systems based on a combination of local and systemic signs (local-systemic score 1 [LS1] and local-systemic score 2 [LS2] systems). Test performance was calculated to determine whether a severe disease classification could be used to predict bacteremia or outcome. RESULTS: 21%, 53%, 63%, and 38% of cows were classified as having severe disease with the SSS, LSS, LS1, and LS2 systems, respectively. For both bacteremia and outcome, sensitivity was highest for the LS1 system, but specificity and accuracy were highest for the SSS system. Examination of a scatterplot of true-positive rate versus false-positive rate for each of the scoring systems indicated that the SSS and LS2 systems were similar in their ability to correctly identify cows with bacteremia or an adverse outcome. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the SSS scoring system was better for identifying cows with bacteremia or an adverse outcome than was the LSS system and that the LS1 and LS2 systems were intermediate in their discriminatory abilities.

Severity of E. coli mastitis is mainly determined by cow factors.

Intramammary infections of dairy cows with Gram-positive bacteria such as Staphylococcus aureus (major cause of mastitis) have received a lot of attention because of their major economic impact on the dairy farm through production losses induced by an increase in somatic cell count. Management strategies, including greater awareness for efficient milking and hygienic measures, have limited the spread of Gram-positive bacteria and resulted in a significant decrease of proportion of S. aureus isolates and subclinical mastitis worldwide. Other organisms such as coliform subspecies and Streptococcus uberis, both environmental bacteria that cause clinical mastitis, have received less attention. Escherichia coli causes inflammation of the mammary gland in dairy cows around parturition and during early lactation with striking local and sometimes severe systemic clinical symptoms. This disease affects many high producing cows in dairy herds and may cause several cases of death per year in the most severe cases. It is well known that bacterial, cow and environmental factors are interdependent and influence mastitis susceptibility. Many studies, executed during the last decade, indicate that the severity of E. coli mastitis is mainly determined by cow factors rather than by E. coli pathogenicity. During E. coli mastitis, the host defense status is a cardinal factor determining the outcome of the disease. Today, we know that the neutrophil is a key factor in the cows' defense against intramammary infection with E. coli. Effective elimination of the pathogen by neutrophils is important for the resolution of infection and the outcome of E. coli mastitis. This review is a compilation of some major findings over the last 15 years concerning mainly host factors that modulate and influence neutrophil function and the mammary inflammatory reaction. The individual chapters address: virulence factors of E. coli strains, how neutrophils kill E. coli, connection between endotoxins, tumor necrosis factor-alpha and nitric oxide, severity classification of E. coli mastitis, lifespan of neutrophils, host factors that influence severity, tissue damage and production loss.

Aetiology of clinical mastitis in six Somerset dairy herds.

Clinical mastitis was monitored in six Somerset dairy herds for one year. The herds all had three-month geometric mean bulk milk somatic cell counts of less than 250,000 cells/ml. Escherichia coli was the predominant pathogen isolated on all the farms and in all months of the year. Environmental pathogens accounted for 61.4 per cent of all cases of clinical mastitis and for 79.3 per cent of the mastitis cases in which an aetiological agent was identified. The mean annual incidence was 41.6 cases per 100 cows (range 14 to 75). Affected cows suffered a mean of 1.5 cases and 16.4 per cent of quarters suffered at least one repeat case. Mastitis due to E. coli was more severe than mastitis due to other causes and it tended to be more severe in early lactation and during the housing period. Mastitis was significantly more severe (grades 2 and 3) in the herd with the lowest bulk milk somatic cell count and in the herd which was kept indoors throughout the year than in the other four herds. Mastitis was fatal in 2.2 per cent of cases and resulted in the death of 0.6 per cent of the lactating cows.

Application of fuzzy logic in automated cow status monitoring.

Sensors that measure yield, temperature, electrical conductivity of milk, and animal activity can be used for automated cow status monitoring. The occurrence of false-positive alerts, generated by a detection model, creates problems in practice. We used fuzzy logic to classify mastitis and estrus alerts; our objective was to reduce the number of false-positive alerts and not to change the level of detected cases of mastitis and estrus. Inputs for the fuzzy logic model were alerts from the detection model and additional information, such as the reproductive status. The output was a classification, true or false, of each alert. Only alerts that were classified true should be presented to the herd manager. Additional information was used to check whether deviating sensor measurements were caused by mastitis or estrus, or by other influences. A fuzzy logic model for the classification of mastitis alerts was tested on a data set from cows milked in an automatic milking system. All clinical cases without measurement errors were classified correctly. The number of false-positive alerts over time from a subset of 25 cows was reduced from 1266 to 64 by applying the fuzzy logic model. A fuzzy logic model for the classification of estrus alerts was tested on two data sets. The number of detected cases decreased slightly after classification, and the number of false-positive alerts decreased considerably. Classification by a fuzzy logic model proved to be very useful in increasing the applicability of automated cow status monitoring.

Use of systemic disease signs to assess disease severity in dairy cows with acute coliform mastitis.

OBJECTIVE: To evaluate the use of systemic disease signs for classifying severity of acute coliform mastitis in dairy cows. DESIGN: Prospective cohort study. ANIMALS: 144 dairy cows. PROCEDURE: Cows were examined at the time of initial identification of disease (time 0) and classified as having mild, moderate, or severe disease on the basis of rectal temperature, hydration status, rumen contraction rate, and attitude. A CBC and serum biochemical analyses were performed, and milk samples were submitted for bacterial culture at time 0 and 48 hours later. RESULTS: 69 cows were classified as having mild disease, 44 as having moderate disease, and 31 as having severe disease. Median WBC and neutrophil counts were significantly lower in cows with moderate or severe disease at time 0 than in cows with mild disease. Band neutrophil count was significantly higher at 48 hours and serum calcium concentration was significantly lower at time 0 and at 48 hours in cows with severe or moderate disease, compared with cows with mild disease. Twenty-eight, 51, and 77% of cows with mild, moderate, and severe disease, respectively, had > 100,000 colony-forming units/ml of milk at time 0. The odds that a cow with severe disease would die or be culled were 3.6 times the odds for a cow with moderate disease and 11.2 times the odds for a cow with mild disease. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that a classification scheme based on readily observable systemic disease signs can be used to classify disease severity in cows with acute coliform mastitis.

Diagnostic alternatives used by veterinary surgeons for the recording of udder diseases in the Norwegian Health Card System for Cattle.

A questionnaire, in which 7 cases of udder disease were described, was distributed to 890 veterinarians in Norway. They were requested to classify the cases according to the diagnostic alternatives listed in the Norwegian Health Card System for Cattle (NHCSC). The NHCSC recordings are used for progeny testing of bulls, for disease monitoring, and for research purposes. The aim of this study was to evaluate the quality of the recordings for udder diseases. The questionnaire was answered by 633 veterinary surgeons. Four cases of clinical mastitis (CM) with abnormal secretion as well as other clinical signs of inflammation were correctly classified as CM by almost 100% of the veterinary surgeons. A cow in the final stage of lactation, showing no clinical signs except for clots in the milk, was considered not to be a clinical case by more than 25% of the veterinary surgeons. A typical case of subclinical mastitis (SM) was reported as SM by 83% of the veterinary surgeons, and as CM by 16%. A subclinical case with a recent history of clots in the secretion was classified as SM by 66% of the veterinary surgeons, but almost 40% either reported CM as their sole diagnosis or considered the case to be CM in combination with SM. Of the clinical cases, those exhibiting marked local signs of inflammation and a systemic reaction were correctly classified as acute clinical mastitis (ACM) by 96%-98% of the veterinary surgeons. In the NHCSC, the diagnostic alternatives for cases of CM are ACM and chronic clinical mastitis (CCM). One case, for which the diagnosis subacute clinical mastitis was appropriate according to standard definitions, was classified as CCM by 66%, and as ACM by 6%. Based on the information given in the questionnaire, the diagnosis for 2 of the clinical cases could have been either ACM or CCM, and for both cases each of these 2 alternatives was reported by more than 43% of the veterinary surgeons. A teat lesion, which was present together with ACM in one cow, was reported by 91% of the veterinary surgeons.

Treatment of various forms of bovine mastitis with consideration of udder pathology and the pharmacokinetics of appropriate drugs: a review.

Various forms of clinical and subclinical mastitis occur in the bovine, and intramammary therapy alone or in combination with parenteral therapy must be implemented with due consideration of udder pathology and the pharmacokinetic properties of drugs used for the treatment of mastitis. Supportive therapy for cows with acute or peracute mastitis and optimal animal husbandry are of the utmost importance for a relatively good prognosis. The withholding period of milk for human consumption after the last intramammary antibiotic treatment as well as minimum intervals between antibiotic dry cow treatment and calving are given. The main reasons for mastitis therapy failure are udder pathology caused by the inflammatory process, the pharmacokinetic properties of mastitis drugs under those conditions, inadequate supportive therapy and poor animal husbandry.

[Investigations into the incidence of the various causative agents of mastitis in cattle. Studies done in the ambulatory clinic of the Faculty of Veterinary Medicine, Utrecht]. / Een onderzoek naar de frequentie van voorkomen van de verschillende mastitisverwekkers bij het rund. Een onderzoek in de buitenpraktijk van de Faculteit der Diergeneeskunde, Utrecht.

The clinical and bacteriological findings in every head of cattle treated for clinical mastitis in the Ambulatory Clinic of the Veterinary Faculty of Utrecht University were recorded over a period of twelve months. Cattle affected with mastitis were classified into a number of groups, a classification based on the clinical symptoms shown by the animals. In each of these groups the incidence of the various causative agents of mastitis was determined. Another subject of study was the distribution of the causative agents over the various groups. The high proportion (34 percent) of coliform micro-organisms, isolated from animals affected with acute mastitis and showing symptoms of generalized disease was a striking feature.