Subclinical Mastitis Dynamics in Response to the Use of Autogenous Vaccine

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Lactating dairy cows were enrolled into two groups: Vaccinated (n = 300) and Control (n = 300). To identify the herd mastitis microbiological profile, CMT test were conducted, and positive milk samples were submitted to microbiological culture. Corynebacterium bovis, Escherichia coli, Staphylococcus chromogenes, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis showed a high isolation rate and were used in autogenous vaccine manufacturing. The first vaccination occurred in March 2022 and the booster 30 days later. Blood samples from 10% of each group were collected every 30 days for seroconversion analysis by ELISA. Data of monthly clinical mastitis and individual SCC were evaluated by T-test (GraphPad Prism; P < 0.05). Vaccination stimulated the cows’ immune system to produce specific antibodies against vaccine bacteria. The individual monthly SCC average were lower for Vaccinated than in Control group. In subclinical mastitis dynamics, Vaccinated group showed a higher number of healthy and cured cows, and a lower number of cows with new infections and chronic cows. No difference was observed in clinical mastitis rate among groups. In conclusion, autogenous vaccine appropriately stimulates immune system, reduces the individual monthly SCC average, increases the number of healthy and cured cows and reduces the number of cows with new infections and chronic cows in a crossbred dairy herd. autogenous vaccine adaptative immunity mastitis dairy cows Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Bovine mastitis is a disease that leads to many economic losses for dairy farms, which are related to treatments costs, milk discharge due to antimicrobials residues and animal culling. Mastitis also compromises milk quality and animal welfare (De Vliegher et al. 2012 ). Mastitis occurs in most of cases by mammary gland invasion by pathogenic microrganisms, which develops a systemic inflammatory response and milk production reduction is one of the major negative effects of this disease (Akers and Nickerson, 2011 ). Once mastitis is caused predominantly by bacterium, the primary treatment recommendation is based on use of intrammamary and/or systemic antimicrobials. The efficacy of mastitis treatment depends on many factors, such as bacterium specie, dose, interval and application route, treatment period, antimicrobials base association, etc. Therefore, the indiscriminate use of antimicrobials in dairy cows can lead to selection of multi resistant bacteria, which in general is detected by low clinical and microbiological cure rates (Sol et al. 1997 , Sears and McCarthy, 2003 ). Due to the importance of rational use of antimicrobial in relation to one health, which involves human, animal, and environment health (Lees and Aliabadi, 2002 ), the implementation of strategies for control and prevention of mastitis is essential to reduce the negative impacts of this disease. Mastitis prevention by vaccination can be more efficient and less expensive alternative than treatment. Vaccination objectives is to reduce the severity of cases and infection pressure in the herd, also reduces the demand of antimicrobials treatments (Kurtyak et al., 2021 ). Actually, there are commercial and autogenous vaccines available in veterinary market for mastitis prevention in dairy cattle. According to Grein et al. ( 2022 ), autogenous vaccines have gained increasing importance over the years, especially due to efforts to reduce the use of antimicrobials. The differential of autogenous vaccines refers to the reduction of antigenic variability from pathogenic bacteria causing mastitis. The bacterins used in autogenous vaccines are originate from bacterial strains that circulates and causes mastitis in determinate dairy herd. Once mastitis is a high prevalence disease in dairy herds and it causes significant losses for dairy industry. Therefore, our objective was to evaluate subclinical mastitis dynamics and clinical mastitis occurrence rate in response to use of an autogenous vaccine in a crossbred dairy herd. Material and methods Farm, animals, and nutritional and sanitary management This experiment was conducted during nine months (December 2021 to August 2022) in a commercial dairy farm located in Triângulo Mineiro region of Minas Gerais State, Brazil. The herd was composed by Girolando (crossbred Holstein/Gyr) animals with 1,085 lactating dairy cows, producing 30.3 kg of milk/cow/day. Cows were housed in compost barns with sawdust bedding. Cooling systems were disposed in feeder line and waiting room of milking parlour. Lactating dairy cows were divided according to milk production, receiving total mixed ration (TMR) composed of corn silage, concentrate, and minerals, three times per day. All cows had ad libitum access to water and the diet was formulated in accordance with the National Research Council’s recommendations (NRC 2001 ). The vaccination program of the farm included the mandatory vaccines and vaccines against reproductive diseases. Cows were treated with bovine somatotropin (bST Lactotropin®, Agener União, Brazil) every 14 days, starting from 60 days in milk (DIM) until the cows reached 200 days of gestation. Autogenous vaccine manufacturing With the aim to identify the microbiological profile of mastitis at the herd, a general sampling of the herd was conducted in December 2021. All cows were submitted to the California Mastitis Test (CMT) and the milk samples with any level of reactivity (+, ++ or +++) were collected for microbiological culture, totalizing 429 samples. At the laboratory, milk samples were seeded using disposable bacteriological loops in bipartite Petri dishes containing blood and chromogenic agar, both non-selective. After seeding, the Petri dishes were incubated at 37°C for 48 hours and reading was performed for identification of suspicious colonies according to those characteristics in both culture media. Later, interesting colonies were peaked in blood, chromogenic, mannitol (suspicion of Staphylococcus spp.) and MacConkey (suspicion of Klebsiella spp.) agar and Petri dishes were incubated again at 37°C for 24 hours. After this period, some tests were performed to confirm the suspicions, such as catalase, oxidase and Gram stain, among other biochemical tests. For some bacterium, the Polymerase Chain Reaction (PCR) was used as a confirmation test, once some biochemical tests showed variable results. As a result, the isolation rate obtained by milk samples microbiological culture was described in Table 1 . Table 1 Isolation rate of bacterium identified in milk samples from general collection. Bacterium specie Isolation rate (n) Staphylococcus non aureus (SNA) 11.7% (50) Corynebacterium bovis 6.5% (28) Streptococcus uberis 5.4% (23) Streptococcus agalactiae 4.2% (18) Escherichia coli 4.0% (17) Contaminated samples 1.9% (8) Bacillus spp. 1.6% (7) Klebsiella spp. 1.6% (7) Streptococcus dysgalactiae 1.2% (5) More than 1 bacterium 1.2% (5) Staphylococcus aureus 0.5% (2) Lactococcus spp. 0.2% (1) Negative culture 60.1% (258) Contaminated samples 1 1.9% (8) More than one specie 2 1.2% (5) 1 Milk samples with ≥ 4 species of bacteria; 2 More than one specie of bacteria from the same dairy cow. The bacterium species identified and isolated from milk samples collected at the herd originated the seeds that were lately used in the production of autogenous vaccine. The criteria adopted for selection of bacterium species, which were included in the autogenous vaccine, were the isolation rate (Table 1 ) and the historic of onfarm microbiological culture of clinical mastitis cases. Thus, the seeds of Staphylococcus chromogenes, Corynebacterium bovis, Streptococcus uberis, Streptococcus agalactiae, Escherichia coli and Streptococcus dysgalactiae were selected for the composition of autogenous vaccine. Before the onset of autogenous vaccine production was the solicitation of manufacturing to the Ministério da Agricultura, Pecuária e Abastecimento (MAPA), as determined by the Normative Instruction #31 from May 20, 2003. Once authorized by MAPA, which takes around two or three days, the vaccine manufacturing was started at Inata Biológicos vaccine factory. To produce the autogenous vaccine, we grow all bacteria of interest in modified tryptic soy broth at 37°C with shaking at 200 rpm for 48 h. The culture media was centrifuged at 3500 xg for 20 min, and then the bacterial pellet was washed 3 times with sterile phosphate-buffered saline (PBS). The bacteria were suspended in a solution of 1% formalin in PBS (10 8 colony-forming unit (CFU)/mL) and slowly shaken for 48 h at 25°C. The completely inactivated bacterial cells were washed 3 times with sterile PBS to remove formalin. Commercial adjuvant Montanide™ ISA 61 (Seppic, Paris, France) was emulsified with the inactivated bacteria at a volume ratio of 26% antigen to 74% adjuvant (10 8 CFU/mL) as the final concentration. Experimental groups and vaccination protocol For the trial 600 lactating dairy cows were paired according to DIM, parity, somatic cell count (SCC) and milk production (Table 2 ), and then the cows were randomly assigned in two groups: Vaccinated group (n = 300) and Control group (n = 300). During the experimental period, 31 animals were culled following the criteria stablished by the farm, 15 from Vaccinated group and 16 from Control group. Table 2 Number of animals, DIM, milk production, SCC and parity according to experimental groups. Experimental group Number of animals DIM Milk production (L/day) SCC (*1000 cells/mL of milk) Parity Vaccinated 300 116 35.1 285 1.95 Control 300 115 35.3 301 1.93 The vaccination protocol starts in March 2022. It consisted in an intramuscular application of one dose (2 mL) of autogenous vaccine. The second dose (booster) were performed 30 days after the first dose. The Control group received 2 mL of sterile saline (placebo) also by intramuscular route following the same protocol of Vaccinated group. Milk routine, clinical and subclinical mastitis diagnosis. Lactating dairy cows were mechanically milked three times per day (4:00 am, 12:00 pm, 8:00 pm). The machine has 24 milking clusters with automatic extraction. The average of SCC from the milk tank was 318 thousand cells per mL of milk during the trial period. The average of clinical mastitis monthly rate of the herd was 9.4% during the trial period. Clinical mastitis diagnosis was performed by forestripping all teats every milking, to observe abnormal milk (presence of clots, flakes, and/or any changes in the color and consistency) and signs of inflammation from a mammary quarter. A milk sample were collected from every clinical mastitis case for on farm microbiological culture. The data of clinical mastitis were registered by the milker leadership in specific spreadsheets, containing: cow ID, mastitis grade (1, 2 or 3; Santos and Laranja, 2019 ), diagnostic date, infected teat (s), the dates of onset and end of treatment, used drugs, antimicrobial withdrawal period and then the result of milk microbiological culture. For the monthly clinical mastitis rate calculation was considered the number of occurred cases in a month divided by the number of lactating dairy cows in that month for each experimental group. Individual quarter milk samples representative of the whole milking were collected from milk meters once a month, to diagnose subclinical mastitis by electronic SCC. The SCC threshold of 200,000 cells/mL of milk was used to define infected cows. In addition, the cow’s condition for the mastitis subclinical dynamics were evaluated comparing the result of the current month in relation to the result of the past month for each cow among each group. Thus, the distribution of cows in each condition (healthy, cured, new infection and chronic) was made between April and August 2022, according to Radostits et al. ( 2007 ) (Table 3 ). Table 3 Characterization of the cow's condition according to the dynamics of subclinical infection. SCC (*1000 cells/mL of milk) Classification for subclinical mastitis dynamics evaluation Previous month Current month ≤ 200 ≤ 200 Healthy > 200 ≤ 200 Cured ≤ 200 > 200 New infection > 200 > 200 Chronic Blood sampling for seroconversion by ELISA Blood samples from 10% of cows of each group were collected at the day of first vaccination (March - D0), at the day of vaccination booster (April - D30), and then in the next three following months (May - D60; June - D90; July - D120) after booster for seroconversion analyses. The seroconversion of IgG-specific antibodies by cows during the immunization time was examined by Enzyme-Linked Immuno Sorbent Assay (ELISA). The samples were analyzed individually and then the result was calculated by the average of the group for each day. Hight-affinity polystyrene 96-well microplates (Costar 3590) were coated with Streptococcus uberis, Streptococcus agalactie, Streptococcus dysgalactiae, Staphylococcus chromogenes, Corynebacterium bovis, Escherichia coli , individually, in carbonate-bicarbonate buffer (0.06 M, pH 9.6) and incubated overnight at 4°C. After washing the wells with PBS containing 0.05% Tween 20 (PBS-T), bovine serum albumin in PBS-T (5% PBS-T-BSA) was added as a blocking solution. The plate was incubated for 1h at 37°C. Serum samples of IgG (1:500) from cows were diluted in PBS-T-BSA 3%, added and incubated for 1h at 37°C. After washed with PBS-T, the secondary antibody anti-bovine IgG, produced in goat peroxidase conjugated, was diluted 1:15.000 in 1% PBS-T-BSA, added and incubated for 1h at 37°C. After washing, the reaction was developed by adding TBM Solution for 15 min. The reaction was stopped by adding 100 µL of Stop solution. Optical density (OD) was determined at 600 nm in an ELISA reader. Data were expressed as ELISA index (EI) as follows: EI = OD/cutoff of negative controls plus three standard deviations. To establish the cutoff values for the negative IgG controls, serum from unvaccinated cows (Control group) were used. Values of EI > 1.2 were considered positive. Statistical analysis All data were first checked for normal distribution and expressed as mean ± standard deviation. Clinical mastitis rate and the average of SCC of each group were evaluated monthly. For the dynamics of subclinical mastitis, the result of the current month was compared to the previous month, and the results were showed considering the average of the role analyzed period (April to August). For seroconvertion analysis by ELISA, samples were submitted at least three times in triplicate and the result was given by EI. Significance differences were determined by T-test, with multiple comparisons of Bonferroni test using the GraphPad Prism Software (version 6.01). Statistical significance of the analysis was defined as P ≤ 0.05, and a tendency was defined as 0.05 < P ≤ 0.10. Results With a seroconversion analysis using EI, it was found that the autogenous mastitis vaccine was able of stimulate the immune system of cows in the vaccinated group to produce specific antibodies against the agents present in the vaccine, as expected. In blood samples collected on D0, which correspond to the day of application of the first dose of the vaccine, no difference was detected between the vaccinated and control groups. From D30, which corresponds to the day of application of the vaccine booster, and on days 60, 90 and 120 after vaccination, there is a difference between the Vaccinated and Control groups, which varied according to each of the agents tested ( P < 0.05; Fig. 1 ). The effect of vaccination on the average individual SCC was verified in the present study. In all analyzed months, the average of the Vaccinated group was lower than the average of the Control group (Fig. 2 ). The dynamics of subclinical infections, evaluated in accordance with the proposed by Radostits et al. ( 2007 ), the Vaccinated group showed better results than the Control group. In the average calculated between the months of April and August, there was a greater number of cows in healthy ( P = 0.0392) and cured ( P = 0.0027) conditions in the Vaccinated group than in the Control group. In the same way, the vaccinated group also presented a smaller number of cows in the conditions of new ( P = 0.0080) and chronic ( P = 0.0002) infections compared to the Control group (Fig. 3 ). The rate of clinical mastitis in the Vaccinated group was numerically below the rate observed in the Control group between the months of May and August, however there was no statistical difference between the groups ( P > 0.05; Fig. 4 ). Discussion It was observed through seroconversion for specific IgG antibodies to the bacteria Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, Staphylococcus chromogenes, Corynebacterium bovis a nd Escherichia coli , individually, that the vaccinated cows showed an immunological response due to the autogenous vaccine against mastitis (Fig. 1 ). A similar result was found by Pieres et al. (2017), in which animals vaccinated with a polyvalent commercial vaccine showed a higher concentration of specific antibodies compared to unvaccinated animals. Furthermore, with revaccination (booster dose) antibodies are produced quickly and in greater quantities, in addition to remaining longer and the phagocytic activity of neutrophils appears to increase, triggering a more efficient elimination of the infection (Moyuddin et al., 2020; Piepers et al., 2017 ). It is known that subclinical mastitis can progress to clinical mastitis (Santos and Laranja, 2019 ). Since the use of the autogenous mastitis vaccine promoted a reduction in the average individual SCC of the Vaccinated group in relation to the Control group (Fig. 2 ), the autogenous vaccination may be beneficial in preventing severe cases of subclinical mastitis, especially for chronic cases, although no reduction in clinical mastitis cases was detected in the present study (Fig. 4 ). Autogenous vaccination against mastitis contributed positively to the subclinical mastitis dynamics throughout the experimental period. It was observed an increase in the number of healthy and cured cows and a reduction in the number of new infections and chronic cows (Fig. 3 ), and the last condition was the most significant effect among the four evaluated. Animals with chronic infections are those that have persistent subclinical infection. Consequently, these cows have a reduction in milk production and become sources of contamination for healthy cows, and are animals with more chance of culling. These factors influences directly the profitability of dairy farms, and so the use of autogenous vaccines may be an alternative to improve the health of the mammary glands of lactating cows. The monthly clinical mastitis rate was similar among groups throughout the evaluated period (Fig. 4 ). This result can be explained by the fact that only one third of the herd was vaccinated. The other thirds were the Control group and the remaining cows of the herd that was not included in the trial. These animals shared the same housing and milking environments and they probably contributed to increase the infection pressure for the Vaccinated group, which resulted in a similar incidence of clinical mastitis among groups, despite the reduction in cases of subclinical mastitis. Schukken et al. ( 2014 ) found positive results in vaccinated animals, using vaccines against Staphylococcus aureus and coagulase-negative Staphylococcus . They observed a higher cure rate, lower transmission rate, significant decrease in the prevalence of S. aureus and a tendency of a lower rate of new infections in the vaccinated group. In relation to coagulase-negative Staphylococcus , there was no difference in cases of new infections comparing the vaccinated and control groups. Furthermore, the authors observed that the efficacy of the vaccine depended of parity, which primiparous cows showed better results compared to third-lactation cows. In the present study, this analysis was not performed because the cows were selected for the trial according to similar characteristics (Table 2 ). Kurtyak et al. ( 2021 ) analyzed a dairy herd of 600 cows and found positive and significant results of an autogenous vaccine against mastitis. The average clinical mastitis rate on the dairy herd was 12% and 25% for subclinical mastitis in the last three years before vaccination. After the application of the autogenous vaccine, these numbers decreased to 2% and 5%, respectively, which corresponds to a six-fold decrease in clinical mastitis cases and a five-fold decrease in subclinical mastitis cases. Furthermore, the authors observed a significant improvement in milk quality. Before vaccination, the number of cows with high SCC was 48% and after vaccination this number reduced to 2.6%. The authors concluded that the autogenous vaccine had satisfactory performance not only in mammary gland health, but also in milk quality and herd profitability. Corroborating these results, Mohyuddin, et al. ( 2020 ), also show that autogenous bacterial vaccines can be an excellent alternative to the use of antibiotics in preventing mastitis in dairy farms. Several studies evaluated the different types of mastitis vaccines available in veterinary market, as well as their effectiveness in preventing and controlling this disease. In a review by Rainard et al. ( 2021 ) it was shown a variety of results. Mastitis has a multifactorial etiology, the agents are not static, which increases the difficult to cure and prevent this disease by using commercial vaccines. Based on this fact, autogenous vaccines can be an efficient alternative, since it contains an antigenic fraction from the microorganisms that circulates in a determined herd (Moyuddin et al., 2020). However, it is important to highlight that the use of the autogenous vaccine against mastitis should be used as an additional tool for controlling the disease, associated with management measures to minimize mastitis transmission and keep the immune system of the cows working correctly. These management measures include an adequate milking routine, treatment of sick animals, segregation and/or culling of chronic infected animals, balanced nutrition, maintenance of animal comfort and well-being conditions, among others. In conclusion, autogenous vaccination against mastitis stimulates the cows' immune system to produce specific antibodies against the agents present in the vaccine. It also reduces the average individual SCC, and based in the dynamics of subclinical mastitis, increases the number of cows in healthy and cured conditions and reduces the number of cows in conditions of new and chronic infections in relation to the Control group in a crossbred dairy herd. Declarations Acknowledgements The authors acknowledge the Higher Education Personnel Improvement Coordination (CAPES; Brasilia, Brazil). Animal Rights In the conduct of this study, all rules, regulations, and ethical considerations recommended by Brazil and other reputable authorities have been carefully observed. Conflicts of interest Part of this study was financed by Inata Produtos Biológicos LTDA. Funding This work was supported in part by the Higher Education Personnel Improvement Coordination (CAPES; Brasilia, Brazil - Finance Code 001). Author R.M. Santos received scholarship from CNPq (PQ—Research productivity, Process no. 306873/2022-2) and author A. C. F. Faria has received scholarship by CAPES grant number 88887.480170/202000. The other authors have received research support from Inata Produtos Biológicos LTDA. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Authors Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Carla Cristian Campos, Felipe Zanforlin Freitas, José William Maluf de Paula, Isabela Pacheco Borges, Roberta Tomaz Botta França, Paulo César Franco Dutra and Ricarda Maria dos Santos. The first draft of the manuscript was written by Ana Cláudia Fagundes Faria and Carla Cristian Campos and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability The datasets generated during and/or analyzed during the current study are not publicly available due to involve data from the manufacture of the autogenous vaccine used in the study, but are available from the corresponding author on reasonable request. Ethics Approval The Council of Animal Care from the Federal University of Uberlândia - UFU (protocol number 23117.031273/2022-16) approved all animal procedures conducted during this experiment. References Akers, R.M. and Nickerson, S.C., 2011. Mastitis and its impact on structure and function in the ruminant mammary gland. Journal of mammary gland biology and neoplasia, 16, 275-289. https://doi.org/10.1007/s10911-011-9231-3 De Vliegher, S., Fox, L.K., Piepers, S, McDougall, S and Barkema, H.W., 2012. 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Factors associated with bacteriological cure during lactation after therapy for subclinical mastitis caused by Staphylococcus aureus. Journal of Dairy Science, 80(11), 2803-2808. https://doi.org/10.3168/jds.S0022-0302(97)76243-X Supplementary Files CEUAExperimentomastite.pdf Cite Share Download PDF Status: Published Journal Publication published 30 Sep, 2025 Read the published version in Tropical Animal Health and Production → Version 1 posted Reviewers agreed at journal 01 Nov, 2024 Reviewers invited by journal 04 Aug, 2024 Editor assigned by journal 09 May, 2024 First submitted to journal 08 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4325121","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":335841667,"identity":"938a583b-9ff7-4098-9239-02094d5befa3","order_by":0,"name":"Ana Cláudia Fagundes Faria","email":"","orcid":"","institution":"Universidade Federal de Uberlandia - Campus Umuarama","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Cláudia Fagundes","lastName":"Faria","suffix":""},{"id":335841668,"identity":"44f0e760-57a6-4baf-9b98-f17c102d112b","order_by":1,"name":"Carla Campos","email":"data:image/png;base64,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","orcid":"","institution":"INATA Biologicos","correspondingAuthor":true,"prefix":"","firstName":"Carla","middleName":"","lastName":"Campos","suffix":""},{"id":335841669,"identity":"6de1b054-7d9a-4aae-b625-294c3378514c","order_by":2,"name":"Paulo César Franco Dutra","email":"","orcid":"","institution":"INATA Biológicos","correspondingAuthor":false,"prefix":"","firstName":"Paulo","middleName":"César Franco","lastName":"Dutra","suffix":""},{"id":335841670,"identity":"24e908e5-f379-4a79-a1f6-bb65cc25ec1b","order_by":3,"name":"Felipe Zanforlin","email":"","orcid":"","institution":"Evoluir Saúde do Leite","correspondingAuthor":false,"prefix":"","firstName":"Felipe","middleName":"","lastName":"Zanforlin","suffix":""},{"id":335841671,"identity":"f5250c02-21fa-4077-9a0a-e56d211a35ce","order_by":4,"name":"Roberta Tomaz Botta França","email":"","orcid":"","institution":"Universidade Federal de Uberlandia - Campus Umuarama","correspondingAuthor":false,"prefix":"","firstName":"Roberta","middleName":"Tomaz Botta","lastName":"França","suffix":""},{"id":335841672,"identity":"b2a1f30e-4bfd-4e9c-b2c6-6f5c65efc532","order_by":5,"name":"Isabela Pacheco Borges","email":"","orcid":"","institution":"Inata Biológicos","correspondingAuthor":false,"prefix":"","firstName":"Isabela","middleName":"Pacheco","lastName":"Borges","suffix":""},{"id":335841673,"identity":"bab24ce4-877f-4204-88b1-ee01a505378a","order_by":6,"name":"José William Maluf de Paula","email":"","orcid":"","institution":"INATA Biológicos","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"William Maluf","lastName":"de Paula","suffix":""},{"id":335841674,"identity":"7a002217-0c35-4a1b-819a-57e45f5d12ff","order_by":7,"name":"Ricarda Maria dos Santos","email":"","orcid":"","institution":"Universidade Federal de Uberlandia - Campus Umuarama","correspondingAuthor":false,"prefix":"","firstName":"Ricarda","middleName":"Maria dos","lastName":"Santos","suffix":""}],"badges":[],"createdAt":"2024-04-25 15:34:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4325121/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4325121/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11250-025-04613-2","type":"published","date":"2025-09-30T15:57:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63628308,"identity":"c37de5ce-ef5e-425f-bab5-b9980318208b","added_by":"auto","created_at":"2024-08-30 10:08:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":243757,"visible":true,"origin":"","legend":"\u003cp\u003eSeroconversion detected by ELISA for Control group (•) and for the bacteria \u003cem\u003eCorynebacterium bovis\u003c/em\u003e (■), \u003cem\u003eEscherichia coli\u003c/em\u003e (○), \u003cem\u003eStaphylococcus chromogenes\u003c/em\u003e (▲), \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e (♦), \u003cem\u003eStreptococcus dysgalactiae\u003c/em\u003e (*) e \u003cem\u003eStreptococcus uberis\u003c/em\u003e (x), on days 0, 30, 60, 90 and 120 related to the first dose of autogenous vaccine. The red dottle line represents the threshold of ELISA index.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4325121/v1/888ba0b20ad94978df207b1b.png"},{"id":63628307,"identity":"87aaeb73-4ae5-4b00-a6ed-ae4c1d084f3d","added_by":"auto","created_at":"2024-08-30 10:08:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":55571,"visible":true,"origin":"","legend":"\u003cp\u003eAverage of individual SCC of Vaccinated and Control groups between April and August 2022 (* \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; **\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001; *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4325121/v1/61cb76574983ac61dafb5b89.png"},{"id":63628305,"identity":"576f8d9f-8358-43d4-b22f-5304db041fe8","added_by":"auto","created_at":"2024-08-30 10:08:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":103958,"visible":true,"origin":"","legend":"\u003cp\u003eSubclinical infection dynamics: average of animals distributed in each condition in Vaccinated and Control groups between April and August 2022 (* \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; **\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001; *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4325121/v1/7e992aea766280e26ff6e803.png"},{"id":63628309,"identity":"7a31e1a9-e07b-4dfc-b58a-59cd19520e66","added_by":"auto","created_at":"2024-08-30 10:08:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":49333,"visible":true,"origin":"","legend":"\u003cp\u003eClinical mastitis monthly rate in Vaccinated and Control groups between April and August 2022 (* \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; **\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001; *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4325121/v1/0b4e048ce80f052080bbb61b.png"},{"id":92883730,"identity":"410a8dc8-403a-4ee7-aa3c-3ad6e6431307","added_by":"auto","created_at":"2025-10-06 16:08:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1119634,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4325121/v1/ef8c7f34-f39d-4b23-8917-86d1778adb08.pdf"},{"id":63628306,"identity":"231478f7-fb58-4264-890c-0e7248adff80","added_by":"auto","created_at":"2024-08-30 10:08:29","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":517832,"visible":true,"origin":"","legend":"","description":"","filename":"CEUAExperimentomastite.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4325121/v1/b6bb623d76e3094ec0d3eb6d.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eSubclinical Mastitis Dynamics in Response to the Use of Autogenous Vaccine\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBovine mastitis is a disease that leads to many economic losses for dairy farms, which are related to treatments costs, milk discharge due to antimicrobials residues and animal culling. Mastitis also compromises milk quality and animal welfare (De Vliegher et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMastitis occurs in most of cases by mammary gland invasion by pathogenic microrganisms, which develops a systemic inflammatory response and milk production reduction is one of the major negative effects of this disease (Akers and Nickerson, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Once mastitis is caused predominantly by bacterium, the primary treatment recommendation is based on use of intrammamary and/or systemic antimicrobials. The efficacy of mastitis treatment depends on many factors, such as bacterium specie, dose, interval and application route, treatment period, antimicrobials base association, etc. Therefore, the indiscriminate use of antimicrobials in dairy cows can lead to selection of multi resistant bacteria, which in general is detected by low clinical and microbiological cure rates (Sol et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Sears and McCarthy, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDue to the importance of rational use of antimicrobial in relation to one health, which involves human, animal, and environment health (Lees and Aliabadi, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), the implementation of strategies for control and prevention of mastitis is essential to reduce the negative impacts of this disease. Mastitis prevention by vaccination can be more efficient and less expensive alternative than treatment. Vaccination objectives is to reduce the severity of cases and infection pressure in the herd, also reduces the demand of antimicrobials treatments (Kurtyak et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eActually, there are commercial and autogenous vaccines available in veterinary market for mastitis prevention in dairy cattle. According to Grein et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), autogenous vaccines have gained increasing importance over the years, especially due to efforts to reduce the use of antimicrobials. The differential of autogenous vaccines refers to the reduction of antigenic variability from pathogenic bacteria causing mastitis. The bacterins used in autogenous vaccines are originate from bacterial strains that circulates and causes mastitis in determinate dairy herd.\u003c/p\u003e \u003cp\u003eOnce mastitis is a high prevalence disease in dairy herds and it causes significant losses for dairy industry. Therefore, our objective was to evaluate subclinical mastitis dynamics and clinical mastitis occurrence rate in response to use of an autogenous vaccine in a crossbred dairy herd.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFarm, animals, and nutritional and sanitary management\u003c/h2\u003e \u003cp\u003eThis experiment was conducted during nine months (December 2021 to August 2022) in a commercial dairy farm located in Tri\u0026acirc;ngulo Mineiro region of Minas Gerais State, Brazil. The herd was composed by Girolando (crossbred Holstein/Gyr) animals with 1,085 lactating dairy cows, producing 30.3 kg of milk/cow/day. Cows were housed in compost barns with sawdust bedding. Cooling systems were disposed in feeder line and waiting room of milking parlour.\u003c/p\u003e \u003cp\u003eLactating dairy cows were divided according to milk production, receiving total mixed ration (TMR) composed of corn silage, concentrate, and minerals, three times per day. All cows had \u003cem\u003ead libitum\u003c/em\u003e access to water and the diet was formulated in accordance with the National Research Council\u0026rsquo;s recommendations (NRC \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe vaccination program of the farm included the mandatory vaccines and vaccines against reproductive diseases. Cows were treated with bovine somatotropin (bST Lactotropin\u0026reg;, Agener Uni\u0026atilde;o, Brazil) every 14 days, starting from 60 days in milk (DIM) until the cows reached 200 days of gestation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAutogenous vaccine manufacturing\u003c/h2\u003e \u003cp\u003eWith the aim to identify the microbiological profile of mastitis at the herd, a general sampling of the herd was conducted in December 2021. All cows were submitted to the California Mastitis Test (CMT) and the milk samples with any level of reactivity (+, ++ or +++) were collected for microbiological culture, totalizing 429 samples.\u003c/p\u003e \u003cp\u003eAt the laboratory, milk samples were seeded using disposable bacteriological loops in bipartite Petri dishes containing blood and chromogenic agar, both non-selective. After seeding, the Petri dishes were incubated at 37\u0026deg;C for 48 hours and reading was performed for identification of suspicious colonies according to those characteristics in both culture media. Later, interesting colonies were peaked in blood, chromogenic, mannitol (suspicion of \u003cem\u003eStaphylococcus\u003c/em\u003e spp.) and MacConkey (suspicion of \u003cem\u003eKlebsiella\u003c/em\u003e spp.) agar and Petri dishes were incubated again at 37\u0026deg;C for 24 hours. After this period, some tests were performed to confirm the suspicions, such as catalase, oxidase and Gram stain, among other biochemical tests. For some bacterium, the Polymerase Chain Reaction (PCR) was used as a confirmation test, once some biochemical tests showed variable results. As a result, the isolation rate obtained by milk samples microbiological culture was described in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIsolation rate of bacterium identified in milk samples from general collection.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBacterium specie\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsolation rate (n)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus\u003c/em\u003e non \u003cem\u003eaureus\u003c/em\u003e (SNA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.7% (50)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCorynebacterium bovis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.5% (28)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus uberis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.4% (23)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus agalactiae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.2% (18)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.0% (17)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eContaminated samples\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.9% (8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillus spp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.6% (7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eKlebsiella spp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.6% (7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus dysgalactiae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.2% (5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMore than 1 bacterium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.2% (5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5% (2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLactococcus spp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.2% (1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative culture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60.1% (258)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eContaminated samples\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.9% (8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMore than one specie\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.2% (5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eMilk samples with \u0026ge;\u0026thinsp;4 species of bacteria; \u003csup\u003e2\u003c/sup\u003eMore than one specie of bacteria from the same dairy cow.\u003c/p\u003e \u003cp\u003eThe bacterium species identified and isolated from milk samples collected at the herd originated the seeds that were lately used in the production of autogenous vaccine. The criteria adopted for selection of bacterium species, which were included in the autogenous vaccine, were the isolation rate (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and the historic of onfarm microbiological culture of clinical mastitis cases. Thus, the seeds of \u003cem\u003eStaphylococcus chromogenes, Corynebacterium bovis, Streptococcus uberis, Streptococcus agalactiae, Escherichia coli\u003c/em\u003e and \u003cem\u003eStreptococcus dysgalactiae\u003c/em\u003e were selected for the composition of autogenous vaccine.\u003c/p\u003e \u003cp\u003eBefore the onset of autogenous vaccine production was the solicitation of manufacturing to the Minist\u0026eacute;rio da Agricultura, Pecu\u0026aacute;ria e Abastecimento (MAPA), as determined by the Normative Instruction #31 from May 20, 2003. Once authorized by MAPA, which takes around two or three days, the vaccine manufacturing was started at Inata Biol\u0026oacute;gicos vaccine factory.\u003c/p\u003e \u003cp\u003eTo produce the autogenous vaccine, we grow all bacteria of interest in modified tryptic soy broth at 37\u0026deg;C with shaking at 200 rpm for 48 h. The culture media was centrifuged at 3500 xg for 20 min, and then the bacterial pellet was washed 3 times with sterile phosphate-buffered saline (PBS). The bacteria were suspended in a solution of 1% formalin in PBS (10\u003csup\u003e8\u003c/sup\u003e colony-forming unit (CFU)/mL) and slowly shaken for 48 h at 25\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe completely inactivated bacterial cells were washed 3 times with sterile PBS to remove formalin. Commercial adjuvant Montanide\u0026trade; ISA 61 (Seppic, Paris, France) was emulsified with the inactivated bacteria at a volume ratio of 26% antigen to 74% adjuvant (10\u003csup\u003e8\u003c/sup\u003e CFU/mL) as the final concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eExperimental groups and vaccination protocol\u003c/h2\u003e \u003cp\u003eFor the trial 600 lactating dairy cows were paired according to DIM, parity, somatic cell count (SCC) and milk production (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and then the cows were randomly assigned in two groups: Vaccinated group (n\u0026thinsp;=\u0026thinsp;300) and Control group (n\u0026thinsp;=\u0026thinsp;300). During the experimental period, 31 animals were culled following the criteria stablished by the farm, 15 from Vaccinated group and 16 from Control group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNumber of animals, DIM, milk production, SCC and parity according to experimental groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of animals\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDIM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMilk production (L/day)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSCC (*1000 cells/mL of milk)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eParity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVaccinated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe vaccination protocol starts in March 2022. It consisted in an intramuscular application of one dose (2 mL) of autogenous vaccine. The second dose (booster) were performed 30 days after the first dose. The Control group received 2 mL of sterile saline (placebo) also by intramuscular route following the same protocol of Vaccinated group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMilk routine, clinical and subclinical mastitis diagnosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eLactating dairy cows were mechanically milked three times per day (4:00 am, 12:00 pm, 8:00 pm). The machine has 24 milking clusters with automatic extraction. The average of SCC from the milk tank was 318 thousand cells per mL of milk during the trial period. The average of clinical mastitis monthly rate of the herd was 9.4% during the trial period.\u003c/p\u003e \u003cp\u003eClinical mastitis diagnosis was performed by forestripping all teats every milking, to observe abnormal milk (presence of clots, flakes, and/or any changes in the color and consistency) and signs of inflammation from a mammary quarter. A milk sample were collected from every clinical mastitis case for on farm microbiological culture. The data of clinical mastitis were registered by the milker leadership in specific spreadsheets, containing: cow ID, mastitis grade (1, 2 or 3; Santos and Laranja, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), diagnostic date, infected teat (s), the dates of onset and end of treatment, used drugs, antimicrobial withdrawal period and then the result of milk microbiological culture. For the monthly clinical mastitis rate calculation was considered the number of occurred cases in a month divided by the number of lactating dairy cows in that month for each experimental group.\u003c/p\u003e \u003cp\u003eIndividual quarter milk samples representative of the whole milking were collected from milk meters once a month, to diagnose subclinical mastitis by electronic SCC. The SCC threshold of 200,000 cells/mL of milk was used to define infected cows. In addition, the cow\u0026rsquo;s condition for the mastitis subclinical dynamics were evaluated comparing the result of the current month in relation to the result of the past month for each cow among each group. Thus, the distribution of cows in each condition (healthy, cured, new infection and chronic) was made between April and August 2022, according to Radostits et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacterization of the cow's condition according to the dynamics of subclinical infection.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eSCC (*1000 cells/mL of milk)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eClassification for subclinical mastitis dynamics evaluation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrevious month\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCurrent month\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHealthy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCured\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNew infection\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChronic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBlood sampling for seroconversion by ELISA\u003c/h2\u003e \u003cp\u003eBlood samples from 10% of cows of each group were collected at the day of first vaccination (March - D0), at the day of vaccination booster (April - D30), and then in the next three following months (May - D60; June - D90; July - D120) after booster for seroconversion analyses.\u003c/p\u003e \u003cp\u003eThe seroconversion of IgG-specific antibodies by cows during the immunization time was examined by Enzyme-Linked Immuno Sorbent Assay (ELISA). The samples were analyzed individually and then the result was calculated by the average of the group for each day. Hight-affinity polystyrene 96-well microplates (Costar 3590) were coated with \u003cem\u003eStreptococcus uberis, Streptococcus agalactie, Streptococcus dysgalactiae, Staphylococcus chromogenes, Corynebacterium bovis, Escherichia coli\u003c/em\u003e, individually, in carbonate-bicarbonate buffer (0.06 M, pH 9.6) and incubated overnight at 4\u0026deg;C. After washing the wells with PBS containing 0.05% Tween 20 (PBS-T), bovine serum albumin in PBS-T (5% PBS-T-BSA) was added as a blocking solution. The plate was incubated for 1h at 37\u0026deg;C. Serum samples of IgG (1:500) from cows were diluted in PBS-T-BSA 3%, added and incubated for 1h at 37\u0026deg;C. After washed with PBS-T, the secondary antibody anti-bovine IgG, produced in goat peroxidase conjugated, was diluted 1:15.000 in 1% PBS-T-BSA, added and incubated for 1h at 37\u0026deg;C. After washing, the reaction was developed by adding TBM Solution for 15 min. The reaction was stopped by adding 100 \u0026micro;L of Stop solution. Optical density (OD) was determined at 600 nm in an ELISA reader. Data were expressed as ELISA index (EI) as follows: EI\u0026thinsp;=\u0026thinsp;OD/cutoff of negative controls plus three standard deviations. To establish the cutoff values for the negative IgG controls, serum from unvaccinated cows (Control group) were used. Values of EI\u0026thinsp;\u0026gt;\u0026thinsp;1.2 were considered positive.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were first checked for normal distribution and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Clinical mastitis rate and the average of SCC of each group were evaluated monthly. For the dynamics of subclinical mastitis, the result of the current month was compared to the previous month, and the results were showed considering the average of the role analyzed period (April to August). For seroconvertion analysis by ELISA, samples were submitted at least three times in triplicate and the result was given by EI. Significance differences were determined by T-test, with multiple comparisons of Bonferroni test using the GraphPad Prism Software (version 6.01). Statistical significance of the analysis was defined as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05, and a tendency was defined as 0.05\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.10.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eWith a seroconversion analysis using EI, it was found that the autogenous mastitis vaccine was able of stimulate the immune system of cows in the vaccinated group to produce specific antibodies against the agents present in the vaccine, as expected. In blood samples collected on D0, which correspond to the day of application of the first dose of the vaccine, no difference was detected between the vaccinated and control groups. From D30, which corresponds to the day of application of the vaccine booster, and on days 60, 90 and 120 after vaccination, there is a difference between the Vaccinated and Control groups, which varied according to each of the agents tested (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe effect of vaccination on the average individual SCC was verified in the present study. In all analyzed months, the average of the Vaccinated group was lower than the average of the Control group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe dynamics of subclinical infections, evaluated in accordance with the proposed by Radostits et al. (\u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e), the Vaccinated group showed better results than the Control group. In the average calculated between the months of April and August, there was a greater number of cows in healthy (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0392) and cured (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0027) conditions in the Vaccinated group than in the Control group. In the same way, the vaccinated group also presented a smaller number of cows in the conditions of new (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0080) and chronic (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002) infections compared to the Control group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe rate of clinical mastitis in the Vaccinated group was numerically below the rate observed in the Control group between the months of May and August, however there was no statistical difference between the groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt was observed through seroconversion for specific IgG antibodies to the bacteria \u003cem\u003eStreptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, Staphylococcus chromogenes, Corynebacterium bovis a\u003c/em\u003end \u003cem\u003eEscherichia coli\u003c/em\u003e, individually, that the vaccinated cows showed an immunological response due to the autogenous vaccine against mastitis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A similar result was found by Pieres et al. (2017), in which animals vaccinated with a polyvalent commercial vaccine showed a higher concentration of specific antibodies compared to unvaccinated animals. Furthermore, with revaccination (booster dose) antibodies are produced quickly and in greater quantities, in addition to remaining longer and the phagocytic activity of neutrophils appears to increase, triggering a more efficient elimination of the infection (Moyuddin et al., 2020; Piepers et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is known that subclinical mastitis can progress to clinical mastitis (Santos and Laranja, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Since the use of the autogenous mastitis vaccine promoted a reduction in the average individual SCC of the Vaccinated group in relation to the Control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the autogenous vaccination may be beneficial in preventing severe cases of subclinical mastitis, especially for chronic cases, although no reduction in clinical mastitis cases was detected in the present study (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAutogenous vaccination against mastitis contributed positively to the subclinical mastitis dynamics throughout the experimental period. It was observed an increase in the number of healthy and cured cows and a reduction in the number of new infections and chronic cows (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), and the last condition was the most significant effect among the four evaluated. Animals with chronic infections are those that have persistent subclinical infection. Consequently, these cows have a reduction in milk production and become sources of contamination for healthy cows, and are animals with more chance of culling. These factors influences directly the profitability of dairy farms, and so the use of autogenous vaccines may be an alternative to improve the health of the mammary glands of lactating cows.\u003c/p\u003e \u003cp\u003eThe monthly clinical mastitis rate was similar among groups throughout the evaluated period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This result can be explained by the fact that only one third of the herd was vaccinated. The other thirds were the Control group and the remaining cows of the herd that was not included in the trial. These animals shared the same housing and milking environments and they probably contributed to increase the infection pressure for the Vaccinated group, which resulted in a similar incidence of clinical mastitis among groups, despite the reduction in cases of subclinical mastitis.\u003c/p\u003e \u003cp\u003eSchukken et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) found positive results in vaccinated animals, using vaccines against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and coagulase-negative \u003cem\u003eStaphylococcus\u003c/em\u003e. They observed a higher cure rate, lower transmission rate, significant decrease in the prevalence of \u003cem\u003eS. aureus\u003c/em\u003e and a tendency of a lower rate of new infections in the vaccinated group. In relation to coagulase-negative \u003cem\u003eStaphylococcus\u003c/em\u003e, there was no difference in cases of new infections comparing the vaccinated and control groups. Furthermore, the authors observed that the efficacy of the vaccine depended of parity, which primiparous cows showed better results compared to third-lactation cows. In the present study, this analysis was not performed because the cows were selected for the trial according to similar characteristics (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eKurtyak et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) analyzed a dairy herd of 600 cows and found positive and significant results of an autogenous vaccine against mastitis. The average clinical mastitis rate on the dairy herd was 12% and 25% for subclinical mastitis in the last three years before vaccination. After the application of the autogenous vaccine, these numbers decreased to 2% and 5%, respectively, which corresponds to a six-fold decrease in clinical mastitis cases and a five-fold decrease in subclinical mastitis cases. Furthermore, the authors observed a significant improvement in milk quality. Before vaccination, the number of cows with high SCC was 48% and after vaccination this number reduced to 2.6%. The authors concluded that the autogenous vaccine had satisfactory performance not only in mammary gland health, but also in milk quality and herd profitability. Corroborating these results, Mohyuddin, et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), also show that autogenous bacterial vaccines can be an excellent alternative to the use of antibiotics in preventing mastitis in dairy farms.\u003c/p\u003e \u003cp\u003eSeveral studies evaluated the different types of mastitis vaccines available in veterinary market, as well as their effectiveness in preventing and controlling this disease. In a review by Rainard et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) it was shown a variety of results. Mastitis has a multifactorial etiology, the agents are not static, which increases the difficult to cure and prevent this disease by using commercial vaccines. Based on this fact, autogenous vaccines can be an efficient alternative, since it contains an antigenic fraction from the microorganisms that circulates in a determined herd (Moyuddin et al., 2020). However, it is important to highlight that the use of the autogenous vaccine against mastitis should be used as an additional tool for controlling the disease, associated with management measures to minimize mastitis transmission and keep the immune system of the cows working correctly. These management measures include an adequate milking routine, treatment of sick animals, segregation and/or culling of chronic infected animals, balanced nutrition, maintenance of animal comfort and well-being conditions, among others.\u003c/p\u003e \u003cp\u003eIn conclusion, autogenous vaccination against mastitis stimulates the cows' immune system to produce specific antibodies against the agents present in the vaccine. It also reduces the average individual SCC, and based in the dynamics of subclinical mastitis, increases the number of cows in healthy and cured conditions and reduces the number of cows in conditions of new and chronic infections in relation to the Control group in a crossbred dairy herd.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the Higher Education Personnel Improvement Coordination (CAPES; Brasilia, Brazil).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Rights\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the conduct of this study, all rules, regulations, and ethical considerations recommended by Brazil and other reputable authorities have been carefully observed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePart of this study was financed by Inata Produtos Biol\u0026oacute;gicos LTDA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by\u0026nbsp;the Higher Education Personnel Improvement Coordination (CAPES; Brasilia, Brazil - Finance Code 001).\u0026nbsp;Author R.M. Santos received scholarship from CNPq (PQ\u0026mdash;Research productivity, Process no. 306873/2022-2)\u0026nbsp;and author A. C. F. Faria has received scholarship by CAPES grant number\u0026nbsp;88887.480170/202000. The other authors\u0026nbsp;have received research support from Inata Produtos Biol\u0026oacute;gicos LTDA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Carla Cristian Campos,\u0026nbsp;Felipe Zanforlin Freitas, Jos\u0026eacute; William Maluf de Paula, Isabela Pacheco Borges, Roberta Tomaz Botta Fran\u0026ccedil;a, Paulo C\u0026eacute;sar Franco Dutra and Ricarda Maria dos Santos. The first draft of the manuscript was written by Ana\u0026nbsp;Cl\u0026aacute;udia Fagundes Faria\u0026nbsp;and Carla Cristian Campos and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are not publicly available due to involve data from the manufacture of the autogenous vaccine used in the study, but are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Council of Animal Care from the Federal University of Uberl\u0026acirc;ndia - UFU (protocol number 23117.031273/2022-16) approved all animal procedures conducted during this experiment.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkers, R.M. and Nickerson, S.C., 2011. Mastitis and its impact on structure and function in the ruminant mammary gland. Journal of mammary gland biology and neoplasia, 16, 275-289. https://doi.org/10.1007/s10911-011-9231-3\u003c/li\u003e\n\u003cli\u003eDe Vliegher, S., Fox, L.K., Piepers, S, McDougall, S and Barkema, H.W., 2012. Invited review: Mastitis in dairy heifers: Nature of the disease, potential impact, prevention, and control. Journal of dairy science, 95(3), 1025-1040. https://doi.org/10.3168/jds.2010-4074\u003c/li\u003e\n\u003cli\u003eGrein, K., Jungb\u0026auml;ck, C. and Kubiak, V., 2022. Autogenous vaccines: Quality of production and movement in a common market. Biologicals, 76, 36-41. https://doi.org/10.1016/j.biologicals.2022.01.003\u003c/li\u003e\n\u003cli\u003eKurtyak, B.M., Boyko, P.K., Boyko, O.P., Sobko, G.V., Romanovych, M.S., Pundyak, T.O., Mandygra, YuM. and Gutyj, B.V., 2021. Autogenous vaccines are an effective means of controlling the epizootic process of mastitis in cows. Ukrainian Journal of Ecology, 11(3), 145-152. https://doi.org/10.15421/2021_157 \u003c/li\u003e\n\u003cli\u003eLees, P. and Aliabadi, F.S., 2002. Rational dosing of antimicrobial drugs: animals versus humans. International journal of antimicrobial agents, 19(4), 269-284. https://doi.org/10.1016/S0924-8579(02)00025-0 \u003c/li\u003e\n\u003cli\u003eMohyuddin, M.T., Muhammad, G., Deeba, F. and Arshad, M.I., 2020. Therapeutic evaluation of autogenous mastitis vaccine alone and in combination with routine therapy in subclinical mastitis. Pak. J. Agri. Sci, 57(4), 957-961. https://doi.org/10.21162/PAKJAS/20.9040 \u003c/li\u003e\n\u003cli\u003eNRC. 2001. Nutrient Requirements for Dairy Cattle. Natl. Acad. Sci., Washington, DC.\u003c/li\u003e\n\u003cli\u003ePiepers, S., Prenafeta, A., Verbeke, J., De Visscher, A, March, R. and De Vliegher, S., 2017. Immune response after an experimental intramammary challenge with killed Staphylococcus aureus in cows and heifers vaccinated and not vaccinated with Startvac, a polyvalent mastitis vaccine. Journal of dairy science, 100(1), 769-782. https://doi.org/10.3168/jds.2016-11269\u003c/li\u003e\n\u003cli\u003eRadostits, O.M., Gay, C.C., Hinchcliff, K.W. and Constable, P.D., 2007. Veterinary medicine: a textbook of the diseases of cattle, horses, sheep, pigs, and goats. 10. ed. Spain: Saunders Elsevier, 2156p.\u003c/li\u003e\n\u003cli\u003eRainard, P., Gilbert, F. B., Germon, P., and Foucras, G., 2021. Invited review: a critical appraisal of mastitis vaccines for dairy cows. Journal of Dairy Science, 104(10), 10427-10448. https://doi.org/10.3168/jds.2021-20434 \u003c/li\u003e\n\u003cli\u003eSantos, M.V. and Laranja, L.F., 2019. Controle da Mastite e Qualidade do Leite: Desafios e Solu\u0026ccedil;\u0026otilde;es. Pirassununga-SP: Edi\u0026ccedil;\u0026atilde;o dos Autores, 1\u0026ordf; ed, 301p.\u003c/li\u003e\n\u003cli\u003eSchukken, Y.H., Bronzo, V., Locatelli, C., Pollera, C., Rota, N., Casula, A., Testa, F., Scaccabarozzi, L., March, R., Zalduendo, D., Guix, R. and Moroni, P., 2014. Efficacy of vaccination on \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and coagulase-negative staphylococci intramammary infection dynamics in two dairy herds. Journal of Dairy Science, 97(8), 5250-5264. https://doi.org/10.3168/jds.2014-8008\u003c/li\u003e\n\u003cli\u003eSears, P.M. and McCarthy, K.K., 2003. Management and treatment of staphylococcal mastitis. Veterinary Clinics: Food Animal Practice, 19(1), 171-185. https://doi.org/10.1016/S0749-0720(02)00079-8\u003c/li\u003e\n\u003cli\u003eSol, J., Sampimon, O.C., Snoep, J.J. and Schukken, Y.H., 1997. Factors associated with bacteriological cure during lactation after therapy for subclinical mastitis caused by Staphylococcus aureus. Journal of Dairy Science, 80(11), 2803-2808. https://doi.org/10.3168/jds.S0022-0302(97)76243-X\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"autogenous vaccine, adaptative immunity, mastitis, dairy cows","lastPublishedDoi":"10.21203/rs.3.rs-4325121/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4325121/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe objective was to evaluate subclinical mastitis dynamics and clinical mastitis monthly rate in response to use of an autogenous vaccine. Lactating dairy cows were enrolled into two groups: Vaccinated (n\u0026thinsp;=\u0026thinsp;300) and Control (n\u0026thinsp;=\u0026thinsp;300). To identify the herd mastitis microbiological profile, CMT test were conducted, and positive milk samples were submitted to microbiological culture. \u003cem\u003eCorynebacterium bovis, Escherichia coli, Staphylococcus chromogenes, Streptococcus agalactiae, Streptococcus dysgalactiae\u003c/em\u003e and \u003cem\u003eStreptococcus uberis\u003c/em\u003e showed a high isolation rate and were used in autogenous vaccine manufacturing. The first vaccination occurred in March 2022 and the booster 30 days later. Blood samples from 10% of each group were collected every 30 days for seroconversion analysis by ELISA. Data of monthly clinical mastitis and individual SCC were evaluated by T-test (GraphPad Prism; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Vaccination stimulated the cows\u0026rsquo; immune system to produce specific antibodies against vaccine bacteria. The individual monthly SCC average were lower for Vaccinated than in Control group. In subclinical mastitis dynamics, Vaccinated group showed a higher number of healthy and cured cows, and a lower number of cows with new infections and chronic cows. No difference was observed in clinical mastitis rate among groups. In conclusion, autogenous vaccine appropriately stimulates immune system, reduces the individual monthly SCC average, increases the number of healthy and cured cows and reduces the number of cows with new infections and chronic cows in a crossbred dairy herd.\u003c/p\u003e","manuscriptTitle":"Subclinical Mastitis Dynamics in Response to the Use of Autogenous Vaccine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-30 10:08:24","doi":"10.21203/rs.3.rs-4325121/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-11-01T08:02:44+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-04T22:18:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-09T06:22:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Animal Health and Production","date":"2024-05-08T09:21:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"757a4667-7903-494f-8b45-93448ec09a2c","owner":[],"postedDate":"August 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T16:00:55+00:00","versionOfRecord":{"articleIdentity":"rs-4325121","link":"https://doi.org/10.1007/s11250-025-04613-2","journal":{"identity":"tropical-animal-health-and-production","isVorOnly":false,"title":"Tropical Animal Health and Production"},"publishedOn":"2025-09-30 15:57:15","publishedOnDateReadable":"September 30th, 2025"},"versionCreatedAt":"2024-08-30 10:08:24","video":"","vorDoi":"10.1007/s11250-025-04613-2","vorDoiUrl":"https://doi.org/10.1007/s11250-025-04613-2","workflowStages":[]},"version":"v1","identity":"rs-4325121","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4325121","identity":"rs-4325121","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}