Luiz Francisco Zafalon

Mastitis is a major challenge for dairy producers, and infrared thermography (IRT) is a potential method for identifying affected cows. However, its reliability is influenced by various factors, including animal and environmental conditions. Machine learning techniques can enhance thermal diagnosis. This study aimed to assess IRT for predicting the infectious etiology of subclinical bovine mastitis using machine learning in a voluntary milking system for pasture-raised cows. Eighty-nine lactating cows from a farm in São Carlos, Brazil, underwent thermographic analyses of their udders over six months. Mean (TMED), minimum (TCOLD), and maximum (THOT) udder temperatures were recorded. Microbiological examination, fat level evaluation, and somatic cell count (SCC) in milk samples were performed. Additional data, such as lactation stage, breed, parity, milking time, cleanliness index, ambient temperature, and humidity, were collected. Supervised machine learning techniques were applied to predict mastitis and its infectious etiology. Logistic regression models showed that udder thermal parameters correlated with body temperature. The ROC curve (AUC) for mastitis prediction using IRT was 0.96. Multinomial logistic regression demonstrated high specificity (> 0.95) for identifying S. aureus, S. uberis , and S. dysgalactiae . The AUC ROC for predicting infectious etiology was 0.90. SCC significantly predicted mastitis and these pathogens, while fat levels were not significant. The Holstein breed was relevant in the multinomial model. IRT combined with machine learning shows promise for detecting subclinical mastitis in pasture-raised cows, optimizing pathogen identification in dairy herds.

Bovine subclinical mastitis (SCM) is the costliest disease for the dairy industry. Technologies aimed at the early diagnosis of this condition, such as infrared thermography (IRT), can be used to generate large amounts of data that provide valuable information when analyzed using learning techniques. The objective of this study was to evaluate and optimize the use of machine learning by applying the Extreme Gradient Boosting (XGBoost) algorithm in the diagnosis of bovine SCM, based on udder thermogram analysis. Over 14 months, a total of 1035 milk samples were collected from 97 dairy cows subjected to an automatic milking system. Somatic cell counts were performed by flow cytometry, and the health status of the mammary gland was determined based on a cutoff of 200,000 cells/mL of milk. The attributes analyzed collectively included air temperature, relative humidity, temperature‐humidity index, breed, body temperature, teat dirtiness score, parity, days in milk, mammary gland position, milk yield, electrical conductivity, milk fat, coldest and hottest points in the mammary gland region of interest, average mammary gland temperature, thermal amplitude, and the difference between the average temperature of the region of interest and the animal’s body temperature, as well as the microbiological evaluation of the milk. Using the XGBoost algorithm, the most relevant variables for solving the classification problem were identified and selected to construct the final model with the best fit and performance. The best area under the receiver operating characteristic curve (AUC: 0.843) and specificity (Sp: 93.3%) were obtained when using all thermographic variables. The coldest point in the region of interest was considered the most important for decision making in mastitis diagnosis. The use of XGBoost can enhance the diagnostic capability for SCM when IRT is employed. The developed optimized model can be used as a confirmatory mechanism for SCM.

Background: We analyzed the effects of a homeopathic therapies to control subclinical mastitis in two dairy herds. Two experimental herds were used, one in the municipality of São Carlos, SP (Herd A) and the other in Bagé, RS (Herd B), with 46 and 37 lactating animals, respectively. Milk yield, somatic cell count (SCC), and levels of fat, protein, lactose and total solids were evaluated. Milk samples were collected for microbiological confirmation of mastitis. The lactating cows were divided into four groups formed on each of the two farms: cows with mastitis, treated and untreated, and healthy cows, also with and without homeopathic treatment. The occurrence of mastitis was compared between groups using the chi-square test. Means of continuous variables were compared by means of the Tukey-Kramer test. The herd effect on SCC, milk yield, and other compositional aspects was determined in treated and untreated animals, using multiple correspondence analysis. Results: No difference was found in the occurrence of mastitis between treated and untreated animals in Herd B, but in Herd A, mastitic cows were more commonly cured in the untreated group (PConclusions: No correspondence was found between the treated and untreated groups. Overall, our findings suggest that homeopathic treatment did not improve milk quality and production of experimental animals.

Mastitis is one of the major diseases in dairy cattle, as it causes great economic losses to producers due to the reduction of milk production and changes in the quality of the product. The disease is mainly caused by bacteria of the genus Staphylococcus spp., these microorganisms can express various virulence factors, such as biofilms for example. In herds with organic management, producers and technicians use unconventional ways to treat and control the disease, such as homeopathy. However, it is not known if this type of treatment is able to control pathogenic bacteria such as those of the genus Staphylococcus, of relevance to animal and human health. Thus, the objective of this study was to investigate the production of biofilm in vitro and its genes by Staphylococcus spp. isolated in the milk of cows treated with homeopathy, as well as the persistence of microorganisms in animals. Ninety-nine isolates of Staphylococcus spp. from cows treated and not treated with homeopathy were identified by internal transcribed space-polymerase chain reaction and investigated for the presence of the icaABCD, bap, aap, atlE, and bhp genes and in vitro biofilm production using the adhesion method on polystyrene plates. The enzyme restriction profile was determined by Pulsed-Field Gel Electrophoresis. Clusters of S. aureus and S. epidermidis with three or more isolates had an isolate selected for Multilocus Sequence Typing. The frequency of S. aureus isolations was similar in treated and untreated cows, while 71.4% of the coagulase-negative identified were isolated in cows treated with homeopathy. The distribution of the operon ica genes was similar in animals with and without treatment, except for the icaD gene, more frequent in treated cows. Production of biofilm was associated with presence of one or more genes from the icaADBC operon. S. aureus revealed a greater diversity and greater dissemination in cows treated and not treated with homeopathy. Sequence Types ST1, ST5, and ST126 were identified in S. aureus. The presence of biofilm-associated genes and the in vitro production of biofilms, combined with the persistence of clonal profiles of Staphylococcus spp. demonstrate other forms of control for bovine mastitis should be researched for organic production herds.

Fifty-two Staphylococcus aureus recovered from papillary ostium and milk samples collected from cows with subclinical mastitis and milking environments in three small dairy herds located in southeastern Brazil were subjected to PCR identification based on the thermonuclease (nuc) gene. All the strains were submitted to in vitro antimicrobial susceptibility testing, and we investigated the sequence types (STs), agr groups (I-IV), virulence genes encoding for Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs), biofilm-associated proteins, bi-component toxins, pyrogenic toxin superantigens, and enterotoxins. Screening for oxacillin resistance (2-6 μg/ml oxacillin), beta-lactamase activity assays, and PCR for the mecA/mecC genes detected 26 methicillin-susceptible S. aureus (MSSA) and 26 mec-independent oxacillin-nonsusceptible S. aureus (MIONSA). While MSSA isolates were found to be susceptible to all antimicrobial agents tested, or only resistant to penicillin and ampicillin, MIONSA isolates were multidrug-resistant. ST126-agr group II MSSA isolates were prevalent in milk (n=14) and carried a broad set of virulence genes (clfA, clfB, eno, fnbA, fiB, icaA, icaD, lukED, hla, and hlb), as well as the ST126-agr group II MIONSA isolated from milking liners (n=1), which also carried the eta gene. ST1-agr group III MIONSA isolates (n=4) were found in papillary ostium and milk, but most MIONSA isolates (n=21), which were identified in both papillary ostium and milking liners, were agr-negative and assigned to ST126. The agr-negative and agr group III lineages showed a low potential for virulence. Studies on the characterization of bovine-associated MSSA/MIONSA are essential to reduce S. aureus mastitis to prevent economic losses in dairy production and also to monitor the zoonotic potential of these pathogens associated with invasive infections and treatment failures in healthcare.

The aim of this study was to determinate whether coagulase-negative staphylococci (CNS) from buffalo milk or the milking environment possess virulence factors that are associated with intramanimary infections or antimicrobial resistance. Milk samples (n = 320) from 80 lactating buffalo were evaluated for clinical and subclinical mastitis by physical examination, the strip cup test, California Mastitis Test (CMT), and somatic cell count (SCC) over a 4-mo period. In addition, swabs were obtained from the hands of consenting milkers (16), liners (64), and from the mouths (15) and nostrils (15) of buffalo calves. No clinical cases of mastitis were observed; however, CMT together with SCC results indicated that 8 animals had subclinical mastitis. Eighty-four CNS isolates were identified by MALDI-TOF MS and cydB real-time PCR (qPCR) and then evaluated by qPCR for presence of the eta, etb, sea, sec, cna, seb, sei, seq, sem, seg, see, and tst toxin genes, adhesion- and biofilm-associated genes (eno, ebps, fib, fnbA, coa), and the methicillin resistance gene (mecA). Resistance to antibiotics commonly used for mastitis treatment in Brazil was determined using the Kirby-Bauer test. Two strains were positive for the see and eta toxin genes; and mecA (1), eno (27), ebps (10), fnbA (10), and coa (5) genes were also detected. A notable number of isolates were resistant to erythromycin (30), penicillin (26), and cotrimoxazole (18); importantly, 10 vancornycin-resistant isolates were also detected. A smaller number of isolates were resistant to rifampicin (8), oxacillin (7), clindamycin (5), cefepime (4), tetracycline (3), ciprofloxacin (2), and chloramphenicol (1), and none were resistant to gentamicin or ciprofloxacin. Isolates with resistance to 2 (13 isolates), 3 (3), 4 (3), 5 (1), and 6 (1) antibiotics were detected. Taken together, our findings suggest that CNS isolates may not be a significant cause of clinical or even subclinical mastitis in buffaloes, but they may be a reservoir of virulence and antibiotic resistance genes.

Here, we present data on the complete genome sequences of 11 Staphylococcus sp. isolates (three S. chromogenes isolates and one isolate each of S. saprophyticus , S. xylosus , S. hominis , S. agnetis , S. caprae , S. aureus , and S. warneri ), obtained as part of a mastitis study of buffalo milk (from healthy animals and from those with subclinical mastitis) and milkers’ hands.

In face of the few reports found in national literature analyzing the potential influence of parturition number in serum proteinogram and biochemical profile in the peripartum period of high yielding dairy cows, the aim of the present study was to comparatively evaluate the dynamics of these serum constituents’ concentrations in blood samples obtained from primiparous and multiparous Holstein cows, 60 and 30 days prepartum and in the day of parturition. Data were analyzed by repeated measures variance analysis (ANOVA) and differences between groups and moments were analyzed by Tukey’s test. Results were considered significant when P<0.05. Parity influenced levels of total protein, albumin, globulins, magnesium, cholesterol, which were higher in multiparous cows, as well as concentrations of ceruloplasmin, total calcium, chloride and alkaline phosphatase activity, which were higher in primiparous cows. Parturition influenced serum concentrations of ceruloplasmin (+58%), transferrin (-25%), haptoglobin (+33%), total protein (-17%), globulins (-25%), immunoglobulin A (-43%), immunoglobulin G (-24%), total calcium (-12%), inorganic phosphorus (-10%), chloride (+5%), sodium (+4%), cholesterol (-23%), triglycerides (-38.6%), as well as activities of aspartate aminotransferase (+14%) and alkaline phosphatase (+28%). A decrease in serum levels of total calcium, inorganic phosphorus, cholesterol and triglycerides was more pronounced in multiparous than in primiparous cows. These results demonstrate that the interpretation of proteinogram and serum constituents should take into consideration lactation number and the moment of parturition as relevant factors in high yielding dairy cows in the transition period.

The aims of this study were to determine the occurrence of subclinical mastitis in sheep of different breeds and the values for somatic cell count (SCC) in milk for the diagnosis of the disease at lactation and weaning, a fundamental prerequisite for identifying animals in need of control measures. Milk samples were obtained from 1,457 mammary halves of Santa Inês, Texel, Ile de France, and Dorper sheep at two different periods, during the second week of lactation and at weaning. After teats antisepsis, the samples were collected, and identification of the infectious etiology of mastitis and determination of SCC were performed. Microorganisms were identified in 117/762 (15.3%) mammary halves in the second week of lactation and in 86/694 (12.4%) at weaning. Coagulase-negative staphylococci (CoNS) were the etiological agents with the highest incidence alone and in association with other microorganisms, with percentages of 58.1% and 60.6%, respectively. The Santa Inês presented a higher incidence of subclinical mastitis when compared to the other breeds. The cut-off values of SCC for subclinical mastitis were determined at both sampling periods and varied according to stage of lactation, as well breed. These results illustrate the lack of a universal value that can be used for the diagnosis of mastitis and suggests the need for permanent follow-up in herds in order to control the disease.

Coagulase-negative Staphylococcus spp. (CNS) are the main microorganisms involved in ovine mastitis. Treatment at the end of lactation can contribute towards cure and prevention of subclinical cases during the subsequent lactation. However, virulence factors and resistance mechanisms presented by CNS can decrease cure rates. The aims of the study were to identify the species of CNS in milk of mastitic ewes with and without antimicrobial treatment, and to investigate the presence of genes relating to resistance of β-lactam antimicrobials, formation of biofilms, production of enterotoxins and production of the toxic shock syndrome toxin. Cases of failure in the treatment were related with the presence/absence of the respective genes. Sixty sheep were divided into three groups: G1, without treatment; G2, animals treated via the intramammary route with 100mg of cloxacillin during drying off; and G3, sheep treated via the intramammary route with 50 mg of nanoparticulate cloxacillin. Milk samples were gathered during drying off and 15 and 30 days after the parturition of the subsequent lactation. The analyses to identify the species of CNS were carried out by means of the internal transcribe spacer technique and the investigation of the genes responsible for the virulence factors and resistance to oxacillin was performed using the polymerase chain reaction (PCR) technique. No sample was positive for the mecA gene. The only gene relating to production of enterotoxins was sec. Among the genes relating to production of biofilm, icaD was the only one identified in the three experimental groups. Staphylococcus warneri was the main species of CNS isolated during the pre and post-partum periods of the sheep. The species carrying genes relating to production of enterotoxins and biofilms were present in uncured sheep.