For three years Ruben worked for Bayer Mexico in the animal health area. He has worked in postpartum management of the cow and breeding. His focus is mastitis and the study of the disorders that cause this disease.

Ruben states that malnutrition occurs due to mastitis, a condition that leads to the disposal of numerous liters of milk because of antibiotic presence or elevated somatic cell counts. This reduces the milk supply. Thankfully, the presence of experts, essentially "doctors for the cows," in the barns helps identify these issues. Without their intervention, even more milk would go to waste. Milk containing coliforms can lead to stomach-related illnesses in humans. However, all of this can be prevented by ensuring proper pasteurization.

The milking routine begins when the cows are brought from the pens to the milking parlor by the herdsman. It is essential that the herdsman handles them with care, as stress can lead to various illnesses due to immunosuppression, making any bacteria capable of causing a disease. Therefore, rough or stressful treatment by the herdsman while moving the cows can compromise the milking routine. Then, in the holding areas, the cows cool down and thermal stress is reduced before entering the rotary milking system, which is the most common milking system in the Laguna region. Then, he describes the five-step milking protocol which includes: pre-seal application, foremilking, teat drying, attachment of the cluster unit that performs the milking, and post-milking teat seal.

To ensure that milk from cows with mastitis is safe for consumption and doesn't pose a risk to public health, certain steps are taken. These steps include:

·   Maintaining Clean Teat Cup Liners and Cold Tanks: Regularly cleaning the equipment used to milk the cows, such as the teat cup liners and cold storage tanks.

·   Thorough Cleaning After Milking: Implementing a proper and thorough cleaning routine after each milking session.

·   Keeping an appropriate temperature in the cold tank: Within the range of 2 to 4 degrees Celsius, to prevent proliferation in milk

·   Tank Sampling: Collecting samples from the milk storage tank every time it's emptied into the pipe that leads to the pasteurizer.

Monitoring somatic cell counts is also important:

·   Higher Somatic Cell Count, Higher Immunological Threat: A higher somatic cell count in the milk indicates a greater risk of potential immune system challenges.

·   Ideal Somatic Cell Range: Somatic cell counts should fall within the range of 200,000 to 250,000. If the count goes above 250,000, it may indicate underlying issues.

·   Low Somatic Cell Count: Conversely, a somatic cell count below 60,000 suggests minimal immunological threats or the possibility of an immune system malfunction.

The easiest and most efficient way to detect mastitis, without having to spend on laboratory tests, is at the fore milking. With this step, abnormalities in the characteristics of milk can be identified.

The primary treatment for mastitis is Masodine RTU (a ready-to-use solution for teat dip and spray), which doesn't leave any traces in the milk. When mastitis reaches higher levels, an additional treatment called neomycin is needed, which can be detected in the milk. In such cases, the cow needs to be identified and moved to an area where antibiotics are given and milk is segregated. The separated milk is often given to young calves. This is where antibiotic resistance can develop, as the milk retains antibiotic remnants.

At times, individuals without veterinary training diagnose mastitis and, having antibiotics on hand, misuse them to quickly address the issue.

Administering treatment through the udder (intramammary) is less intrusive than injections. It's better to keep stress levels low, and administering intramammary treatment is simpler.

Barns that encounter significant mastitis issues often lack proper hygiene practices. A lower level of technological advancement in the barn increases the likelihood of mastitis development.

Contribution

Discusses the outcomes resulting from cows suffering from mastitis. States that stress is a critical factor for cows to produce a good amount of milk. Explains the steps taken to guarantee the safety of milk in production for consumption. Highlights the significance of keeping track of the somatic cell count. Details the process of detecting mastitis.  Also, the treatments used in cows and how safety is ensured in terms of drug residues in milk. Explores the potential factors contributing to the rise of antibiotic resistance. Mentions the benefits of administering treatment through the intramammary route.

Has a master degree and PhD. in reproduction and genetics, and a doctorate. He graduated from the faculty of zootechnics of the Autonomous University of Chihuahua. His experience has been with reproduction mainly in beef cattle and animal selection. He currently works at Genex in New York State, USA, in the industry of dairy cattle.

From the perspective of animal husbandry, mastitis probably does not represent a problem for human health, but rather a challenge in terms of production and economics. Although the treatment itself is inexpensive, the resulting milk losses and additional expenses make it uneconomical. Milk is discarded due to the presence of antibiotics, which affects its quality. If a cow with mastitis or a high concentration of somatic cells is identified, buyers may reject it. Ultimately, the main obstacle lies in the economic aspect.

On a high-quality dairy, daily testing is common, using the milk from the storage bin for testing. If the results show a high somatic cell count, the price of the milk may be lowered or rejected, to prevent the contaminated milk from reaching the consumer. Sick cows are milked separately from healthy ones, highlighting the importance of good milking practices. If there is contaminated milk, you can opt for pasteurization or ultra-pasteurization. Daily testing is essential, using the contents of the storage container for testing.

In the milking process on a farm, several steps are followed. It begins with pre-dipping, where an iodine solution is applied to the teat for aseptic purposes. Then, the milker proceeds with foremilking, checking for any potential clots or blood. Subsequently, a machine with controlled pulsation is used to extract the milk. The machine detects when to stop to prevent drawing blood or causing harm to the cow. Finally, a sealing solution is applied.

Technicians must adhere to strict cleaning protocols and consistently sanitize their hands, as they could potentially transmit mastitis during the milking routine. It is crucial for the staff to understand the importance of correctly following hygiene protocols.

Detecting early stages of mastitis is challenging as they are not visible to the naked eye. Random samplings and somatic cell tests are conducted on milk before loading it onto the buyer's tank. Subclinical mastitis is hard to detect, hence daily testing is performed. Control can be achieved by recording milked cows in each tank and counting somatic cells to quickly identify the sick ones.

The optimum method of application for treating mastitis would be an intramammary infusion applied during drying. The crucial thing is to determine what depth is sought to be reached within the anatomy of the mammary gland. In general, the treatments remain in the cistern of the mammary gland. Industry technicians often use intramammary treatments, which facilitates their administration.

Contribution:

Emphasizes the economic impact rather than a human health concern. Milk quality can be compromised due to antibiotic residues, leading to potential rejection by buyers. Proper milking practices, daily testing, and hygiene protocols are highlighted to prevent mastitis. The milking process involves pre-dipping, machine-assisted milking, and sealing. Detecting mastitis early is challenging, requiring regular testing. Intramammary treatments are preferred, and industry technicians use such treatments for ease of administration.

Leonardo has primarily focused on dairy cattle, currently working in the milking and feeding areas, but he has also been involved in, reproduction, health, nursing, and breeding.

He mentions zoonotic diseases which can be transmitted by consuming milk contaminated with mastitis. These include brucellosis, tuberculosis, and leptospirosis, although they do not cause mastitis.

He explains three methods for early detection of mastitis. The most common is visual detection, where abnormal secretions from the mammary gland are examined. Also, the California test, and somatic cell count, are used.

During the milking process, it is essential to follow hygiene and sanitation protocols. This involves washing the equipment with alkaline, chlorine or acids. At the start of milking, the udders are disinfected with iodine, and the milking machine self-cleans. Hand disinfection is performed and gloves are used. Pasteurization is done to remove microorganisms from milk.

Cows with mastitis have a unique treatment process. Their infected milk is discarded and they're not milked with the rest. To avoid spreading the infection, these cows are kept apart from the herd and milked separately. Their milk could be discarded or given to calves.

Treatments for mastitis include determining the severity of the disease and administering anti-inflammatories to reduce inflammation. In rainy seasons, there are usually E. coli infections, so negative-spectrum antibiotics are administered. Intramammary tubes with the appropriate antibiotic are used, combined with anti-inflammatories, antihistamines and even modulators to stimulate the immune system. For less severe cases, the application method is through intramammary treatments, while in severe cases, intravenous, parenteral or intramuscular methods are used.

Contribution:

Discusses diseases that can spread from animals to humans through contaminated milk. Explains three ways to detect mastitis early. Describes hygiene and sanitation guidelines. Covers measures to control, monitor, and treat mastitis in cows to stop the infection from spreading.

Dr. Arroyo, a professional with over 40 years of experience in the dairy industry, discussed various aspects of dairy farming, particularly focusing on mastitis control through the milking process. It is necessary to establish a good milking process followed by five steps: preseal application to clean the udders with an iodine solution, foremilking (this step is crucial for detecting milk changes), teat drying, attachment of the cluster unit that performs the milking, and post-milking teat seal to protect the udder from microorganisms that could get in.

At DELMAR's cowshed, we could observe the only "carousel" system in México, which consists of a rotary parlor that makes the milking process easier for operators. Furthermore, in the conversation, he explained to us how heat stress can impact cows and lead to reduced milk production. Due to this, they choose to use cooling methods, such as fans and sprinklers.

Different mastitis-causing pathogens were mentioned, and the use of cultures to identify specific bacteria causing mastitis was emphasized. (To identify the bacteria causing mastitis, culturing is performed. There are over 40 pathogens known to cause mastitis, and a week-long sampling process is used to identify them.)

Overall, early detection, prevention, and responsible use of antibiotics are essential in managing mastitis and ensuring the well-being of dairy cows and the production of high-quality milk.

Contribution: 

He states regarding the parameters of Alpura, the optimum cell count for this dairy plant is 150,000 somatic cells per ml of milk. A few years ago, the parameter used to be 500,000 cells, considered a high cell count. Bacteria such as S. aureus and S. agalactiae cause high cell counts. When asking about the best application method to treat mastitis he mentioned that intramammary infusions are really common, and that it is important to note that there is no alternative treatment for mastitis other than antibiotics. Genetic studies can help predict if a cow is more prone to mastitis or resistant to it, but no other effective alternative treatment exists.

Enrique Rascón veterinary doctor of group LIDERLAC Zaragoza in Delicias, Chihuahua mentioned that the process followed when a cow is suspected of being infected is: took a sample of milk from the quarter that can be infected and is sent it to the laboratory. If the cow is infected an oxytetracycline is applied by intramammary way or parenteral, and also a support therapy of antihistamines, antiinflammatories, analgesics, and immunostimulator is given, he also mentioned that the most common bacterias that cause the infection are coliforms, S. aureus, and E. coli, not to mention that clostridium another bacteria which quickly develops infection and in that case is better to get rid of the cow, because there's no way to help the cow; that's why is so important to have lab results identifying the microorganism that is causing the mastitis and know with which microorganism are we fighting against.

Contribution:

He mentioned something that left us without words, “when the cow is infected by Staphylococcus aureus we immediately get rid of it no matter the condition, age, or reproductive condition of the cow”. Because it is such a complicated bacteria to fight against because it is persistent and spreads very quickly, this affects a lot because it leads to abortions, mastitis can be cured but it becomes a recurrence.

“I consider that the most effective treatment is oxytetracycline, and Mastillet an intramammary infusion”.

He also mentioned when we asked about alternative treatments, that there's a treatment based on oregano essences but the milk acquires the flavor of the oregano, also there's garlic based vaccines, so that makes them basically unfunctional.

Adrian is a teacher at CBTA 90, high school in the city of Cuauhtemoc, Chihuahua. He mentions that food management is something primordial while having cattle. If the diet is not correct or is restricted, it immediately reflects on the cows production. Hygiene, food and reproduction are the three things we must watch out for in a dairy farm.

Mastitis causes approximately 30% of the loss in production of a cow, being this one of the most expensive diseases to treat. Gram negative bacterias are not that common in mastitis. The most common treatment is penicillin.  Cephalosporins are also used commonly because they don’t present residues in milk, however nowadays are the ones that most resistance is causing. Rainy seasons are when t most cases of mastitis are presented. The contaminated milk isn’t given to the calves, it is thrown away.

Contribution:

During the interview and farm visit, the key takeaway was gaining firsthand knowledge about the cow milking process and how to identify cows with mastitis using the CMT. We also learned about the antibiotics in use and their administration methods. Notably, a combination of systemic and intramammary infusions is employed for cases of clinical mastitis.

Imelda started working in the dairy cattle industry in 1985. She focused on the mastitis laboratory and treatments. Later, she helped a veterinarian at a nationally recognized dairy company. She has worked in the area of calf rearing, production and mastitis. Currently she is dedicated to teaching, her projects include production, reproduction and animal health. She supports her students to do professional internships.

She stated that the main diseases that dairy cattle present include mastitis, problems related to the hooves and metabolic problems after giving birth or postpartum.

Highlights the importance of making staff aware of the protocols for handling the udder of cows during milking. Emphasis is placed on creating a cohesive team and performing a proper milking routine. She mentions the need for a calm process to stimulate the production of oxytocin, avoiding stress in the cows. The milking process is detailed, including cleaning, disinfection and stimulation of the udder. The importance of properly positioning the milking machine and ensuring that the milk flows correctly is highlighted. The milking process ends by applying the post-milking teat seal composed of 7% iodine and high amounts of glycerin so that the nipple does not dry out. It is important to offer fresh feed after milking to keep the cow standing and allow the sphincter to close effectively.

She discussed the relationship between clinical and subclinical mastitis in cows. For each cow with visible mastitis signs, like redness and warmth, there are 18 cows with subclinical mastitis. Regular evaluations using qualitative tests like the California test or quantitative tests like the modified Wisconsin test are recommended monthly.

During the rainy season, there is a higher proliferation of gram-negative or coliform bacteria. This is where the most severe cases occur. Coliforms possess a toxin that not only remains in the mammary gland but also travels, causing a systemic issue. It can even lead to the death of a cow.

S. aureus is destructive and resistant, infiltrating and damaging alveoli. Treating this bacterium might not be cost-effective. S. agalactiae rapidly increases somatic cell count, up to 1 million somatic cells overnight it is usually treated with penicillin.

The use of antibiotics worries her due to two reasons. First, the required milk withdrawal  and second, for how long it will be used. An antibiotic should be used for a maximum of 5 days and a minimum of 5 days to avoid generating resistance to antibiotics. Milk from cows with mastitis must be discarded. Mastitis-related milk loss is 5-10% varying with case severity.

Contribution:

Discusses the primary diseases affecting cows. Mentions the hygiene and sanitation protocols that are followed during milking. Provides statistical data regarding the occurrence of both clinical and subclinical mastitis. The microorganisms responsible for mastitis and their impact on cows are mentioned. Addresses the issue of infections caused by gram-negative bacteria in the udder of cows. States her concern regarding the utilization of antibiotics.

Carlos has spent 20 years dedicating himself to dairy cows in the private sector and now to the UACH in Delicias. He worked for the government for 3 years in the strategy to support technicians for small producers in the central/southern region of the state of Chihuahua. His experience has always been in dairy cattle.

He states that the main diseases occurring in high-production dairy cattle are mastitis, followed by reproductive issues and diseases such as metritis, all due to the stress factors that a cow can experience. Many cows are lost due to mastitis, which is a consequence of all the stress factors a cow can experience, including heat, lack of shade, cattle handling, bedding management, cows' resting time, and quantity and quality of water.

The control of mastitis is carried out by managing the beds, especially in the rainy season. Another important factor is the benefit of the sealant, which produces a little keratin plug.

The approximate amount of milk loss in cows can be calculated as follows. A cow produces an average of 20 liters of milk. In the case of antibiotic treatments lasting 5 days, plus the 4 days of milk withdrawal due to antibiotics, it totals 9 days of milk withdrawal. This results in a total of 180 liters of milk loss in a single cow.

Refloxaxin, an antibiotic that was used due to its affordability and availability, has encountered significant antimicrobial resistance. In the U.S., its usage is no longer permitted. Resistance is of concern as it is on the rise due to a decreasing number of effective drugs and an increasing prevalence of microorganisms displaying various defense mechanisms. It is recommended to combine multiple types of antibiotics to prevent antibiotic resistance.

Another strategy for mitigating microbial resistance is rotating antibiotics and trying to administer them locally. In their experience, antibiotic resistance tends to develop more when medications are administered via parenteral means, as it triggers the animal's defense system. When dealing with animals that experience recurrent mastitis, medication rotation should involve conducting a culture. Based on the results, a new medication can be prescribed.

Every day, the milking clusters are sanitized. They use both acidic and alkaline solutions along with temperature control. First, they apply the alkaline solution at 50°C for 6 to 8 minutes. Then, they rinse with room temperature water. After that, the acidic solution is applied at room temperature. They follow it with a water rinse, then chlorine, and finally another water rinse.

Contribution:

Discusses mastitis as the primary disease in dairy cattle and identifies its contributing factors. He estimates the milk loss resulting from mastitis. He also touches on treatments facing antimicrobial resistance and outlines efforts to minimize this issue. In his experience, parenteral administration causes greater antimicrobial resistance. Mentions the milking cluster cleaning process with acid and alkaline.

Her experience ranges from serving as a veterinarian for domestic animals to working in laboratory distribution in Queretaro for the past six years, particularly within the dairy cattle industry. We addressed various symptoms of clinical and subclinical mastitis, and she highlighted that antibiotic resistance is a growing concern, posing challenges in the treatment of mastitis. To ensure consumer safety, milk with antibiotic residues is withdrawn, and tests are conducted. Non-antibiotic treatments are also less effective. Furthermore, she mentioned that E. coli is the predominant bacteria causing mastitis, whereas S. aureus presents greater difficulty in treatment.

Contribution:

"If your product works, it's going to be something big"
-Mireya Campos 

“When the product is released, I am willing to try it, as long as it is free of charge” -Johan Thiessen

"When is the product released? Will you bring it to me tomorrow?" -Franz

The experts posed various critical questions: Characterization of proteins: pH and temperature of action, particle size tests, cytotoxicity, and elimination times. Testing methods: In vitro testing and stability of proteins post-pasteurization. Product formulation information and impact on withdrawal period for milk. Safety measures and effect of endolysins on other microorganisms. Influence on organoleptic characteristics of milk and potential economic losses in the dairy industry. Clarification on endolysins' mode of action compared to antibiotics. Considerations on stability and potential resistance issues. They also highlighted the importance of accurate terminology regarding mastitis, the need to simulate in vivo conditions for testing, and the role of microorganisms in inflammation and resistance development. Additionally, they provided insights into pH variations based on cow health and microorganisms present in the udder.

The main station is located in Juarez City, and it was first founded in 1956 as the Hermanos Zaragoza cowshed. He mentioned that in Zaragoza, the milking process is carried out four times throughout the day. He also stated that mastitis is the primary disease in the cowshed. He emphasized that the cows require special care to maintain good milk production and mentioned that they use a cooler in the pens to prevent heat stress. He further noted that mastitis results in economic losses and reduced production.

Contribution:

He mentioned that the cows require special care to maintain good milk production, and that mastitis causes significant economic losses and reduced milk production.

Los pinos, a recognized cheese factory in México, has over 2000 cows in Delicias, Chihuahua. with 1100 involved in the milking process. These cows are milked three times daily, yielding 30,000 to 40,000 liters of milk daily, primarily used for cheese production. The fabric has its laboratory to monitor milk quality parameters such as somatic cells, fat, proteins, solids, and antibiotics. They follow a certified cheese-making process.Environmental commitment is evident as they use solar panels and have a biodigester that produces 99% of the gas required from sewage water. They also utilize whey, a byproduct from cheese production, to create dairy formulas for babies.

Cows with low-grade mastitis contribute milk to the tank, and the milk undergoes filtration to remove lumps. Severely ill cows are separated, and their milk isn't used for human consumption.

Contribution:

He mentioned that training programs ensure milkers treat cows kindly and correctly, which is an important factor in preventing mastitis because cows can suffer from immunosuppression. He also mentioned rigorous cleaning protocols are crucial to prevent the infection.

The Milk Collection center april 2th is a milk collection center where producers from the Julimes region in Chihuahua sell their milk. The tests conducted on the milk for it to be accepted include antibiotic testing and somatic cell count (less than 450,000 somatic cells per milliliter is considered good quality milk). A report is generated for each product, and when it is determined that the milk is not of optimal quality, they trace its source and contact the producer to identify the sick cow and its degree of illness for treatment. If the milk has a high somatic cell count, the collection center reduces the price paid per liter, whereas if antibiotics are detected, the milk is not accepted. In cases of concern, California tests are conducted. Three years ago, a program was initiated to assist producers in tackling mastitis as they were experiencing many cases, and the situation was critical. Upon arrival, the milk must be stored in a stainless steel container and refrigerated from the moment it is milked. When the milk has a high somatic cell count, it is separated into a different container.

Contribution:

This center plays an important role in assessing the quality of the milk produced in the region. It conducts antibiotic tests and somatic cell counts in the milk to determine its quality. If the milk does not meet quality standards, its origin is traced, and the producer is contacted to address health issues in the cows. Additionally, when milk has a high somatic cell count, its price is reduced, and it is not accepted if antibiotics are detected.

Contribution:

Mentions zoonotic diseases that can arise from consuming milk contaminated with mastitis. He also highlighted the importance of maintaining proper hygiene in the pen and during the milking process as essential measures to prevent mastitis. In addition, he provided us with information on a screening method to identify the presence of mastitis. He detailed the treatments he uses and how he applies them in practice. He also provided us with information on the acceptable limits of somatic cells.

The chemical environmental engineer, Heber Ramírez specializes in food analysis and safety. Currently, he works at the Zaragoza Hermanos cowshed laboratory (dairy cattle cowshed) located in Delicias, Chihuahua, where he mainly conducts microbiology studies.

He mentioned that to attack both gram-positive and negative microorganisms, broad-spectrum antibiotics are extensively used.

S. aureus, a Gram-positive microorganism, is one of the most significant. It must be eradicated because it causes chronic mastitis due to its ability to encapsulate. It is identified by its morphology, colonies of 2 to 5mm, golden or beige in color. A distinctive feature is its production of beta and alpha toxins, causing hemolysis in the agar. To confirm that it is S. aureus, a coagulase test is performed with rabbit plasma. If coagulation occurs within 2-3 hours, it confirms it is S. aureus. Cows with mastitis caused by S. aureus are sent to the slaughterhouse, and microbial tests are taken while the cows are fresh and young to initiate eradication. After administering medication, resampling is done two or three days later.

Microbiological knowledge can be applied to develop specific management strategies that reduce incidence with hazard analysis and critical control point assessments, evaluating the entire milking process to prevent cross-contamination through improper hand disinfection or the use of dirty towels.

To identify the causal agent of the infection, a sample of milk is taken directly from the udder quarter with suspected mastitis and sent to the laboratory. It is cultured on three agars: blood agar (for morphological observation), mycoplasma agar (to rule out mycoplasma), and MacConkey agar (to ensure it's a coliform).

S. agalactiae is highly contagious, and cows are sent to slaughterhouse within this cowshed, as it represents a significant contamination source.

E. coli has a short lifespan within the udder, with a high incidence but a brief infection. The main characteristic in the sample is that the milk turns yellow. Sometimes, the bacteria are no longer present after milking the cow because they generate antibodies to attack the bacteria.

S. uberis used to be an environmental agent only but is now one of the main causes of mastitis.

S. uberis and  S. agalactiae produce similar hemolysis. To identify which is which, a Camp medium culture is used to identify.  For precision, PCR, ELISA tests, or immunoassays can be conducted, but they are expensive. Using a culture medium takes more time but is cost-effective.

Contribution:

Knowledge about how mastitis is detected in a laboratory, the main changes in milk that microorganisms can cause, as well as specific data on why certain microorganisms are of greater concern in mastitis. The importance of having microbiological knowledge within the milking room, as mastitis can primarily be caused by cross-contamination during the milking process. Also mentions that the tests performed are mainly based on culturing microorganisms since molecular biology tests could be faster and more accurate but are extremely expensive. Additionally, it should be noted that when mastitis is caused by S. aureus and S. agalactiae, the cow is directly sent to the slaughterhouse, as S. aureus due to its encapsulation causes chronic mastitis,  and S. agalactiae is highly contagious.

Alejandra has been working for 9 years at an intensive production cowshed, specifically in the quality control laboratory, where milk is analyzed, and samples are taken to detect diseases within the herd. Additionally, she is pursuing a specialization in bovine production at UNAM.

At the cowshed where she works, cows primarily become ill due to bacteria, with one of the most common being E. coli. For diagnosing mastitis and identifying the microorganisms causing it, selective agar methods are mainly used as they are cost-effective.

The first detection of mastitis occurs during the milking routine. During foremilking, the first milk that is obtained is evaluated for any differences, changes in consistency, color, or even the presence of blood. If something abnormal is observed, the milker passes a cotton swab with alcohol over the teat and collects a milk sample in sterile tubes, collecting 1 mL.

Alejandra mentioned that some of the main agars used include blood agar, eosin and methylene blue agar (EMB, specific for coliform bacteria), and MacConkey agar.

To determine if it is a subclinical case, it can be detected using a Wisconsin test, which estimates the number of somatic cells, and the California test, which is mostly qualitative but allows identifying the affected quarter. Clinical mastitis can be easily detected as symptoms become apparent.

Identifying the causing microorganism is crucial as it allows for determining an effective treatment. In the case of microorganisms like S. aureus and S. agalactiae, the cow should be sent to the slaughterhouse, as there is a low recovery rate, and they are highly contagious. To recognize if it is Staphylococcus or Streptococcus, a catalase and amylase test is performed, where S. aureus gives a positive catalase reaction. Tests with Camp medium can also distinguish them since S. aureus naturally produces hemolysis, while S. agalactiae generates arrow-shaped hemolysis. Additionally, a coagulation test can be conducted where a colony is placed in a serum tube, and if it coagulates, it is positive for S. aureus.

The most effective and rapid tests are PCR, but these are more expensive. Some innovative tests for mastitis detection include measuring milk conductivity and changes in pH since milk tends to become more acidic.

To determine the appropriate antibiotic, an antibiogram is performed to determine which antibiotic the bacteria are sensitive to. This helps avoid antibiotic misuse and saves resources.

In general, the causing bacteria are categorized into environmental microorganisms, which do not pose many problems, and contagious microorganisms that can be transmitted through the milker's hand or the milking machine.

Contribution:

Alejandra discusses that the most common bacteria causing mastitis is E. coli. She explained that the tests that are performed mainly involve agar methods due to their lower cost compared to PCR, which is more effective and rapid but she mentioned that it is a good diagnostic tool. S. aureus and S. agalactiae are highly contagious, and these microorganisms lead to sending the cow to the slaughterhouse. The agars used for microorganism identification are primarily blood agar, eosin and methylene blue agar (EMB, specific for coliform bacteria), and MacConkey agar. To determine if it is S. aureus or S. agalactiae, a catalase and amylase test is performed. It is important to determine the causative microorganism to provide appropriate treatment. She mentioned that environmental microorganisms do not cause as many problems compared to contagious ones, which are mainly transmitted from the milker's hands or the milking machine.

Dr. Castro is a full-time teacher in zootechnics. Her thesis is focused on developing an intramammary sealant for bovine mastitis. The milk production process demands a high metabolic wear and tear on cows, especially during the transition period from a non-lactating pregnant cow to a lactating non-pregnant cow. There is no specific factor that determines if a cow is susceptible to mastitis, due to the multiple factors that can cause it. It's important to emphasize that the milk produced by animals is intended for human consumption. We must consider animal welfare and ensure that the plants are sustainable and environmentally friendly to guarantee the safety of what is being produced. In a dairy production system, several measures are taken to prevent mastitis, including strict hygiene protocols and livestock management. In dairies, insemination with frozen semen is used, where bulls resistant to mastitis are sought, but this is not reflected in the genotype tests. The primary microorganisms responsible for mastitis are S. agalactiae, S. uber, E. coli, and S. aureus. The last one is known for causing chronic mastitis due to biofilm production. Mastitis infections are caused from the outside to the inside, so the quarters are not infected among themselves. Nowadays there is an investigation with the objective of reducing the use of antibiotics due to growing resistance and its potential transfer to humans. The release of substances using nanoparticles or other technologies to achieve an antibiotic effect without the direct use of these drugs is investigated. She mentions the idea of developing a beta-lactam that prevents the synthesis of the microbial wall to treat chronic mastitis caused by S. Aureus and its biofilm production. In the field of biotechnology, dietary modifications and yeast feed additives have been developed to reduce mastitis. There are 137 microorganisms that can cause this disease, but so far, vaccines against them have not been successful. There are several studies in which bacteriophages have been attempted to be used to combat mastitis. However, their practical application is challenging because most of the tests are conducted in laboratory settings (in vitro). Additionally, finding farmers willing to offer their animals to test these products also poses difficulties. The use of sensors to detect mastitis is one of the most outstanding developments in this field. It is essential to take animal welfare into account and guarantee the sustainability of dairy plants, since the milk produced is for human consumption and its safety must be ensured.

Contribution:

Mastitis is a concern in milk production, especially during the transition from pregnancy to lactation. Multiple factors contribute to mastitis, making it hard to predict. Milk safety for human consumption is crucial. The main mastitis-causing microorganisms include S. aureus, responsible for chronic mastitis. Efforts are underway to reduce antibiotic use due to resistance. Nanoparticles are being explored as alternatives. Biotechnology offers dietary changes and yeast additives, but vaccines for the 137 mastitis-causing microorganisms remain elusive. Bacteriophages are studied but face practical challenges.

After obtaining her master's degree, she has been focused on working with nanoparticles in the field of nanotechnology, particularly focusing on Intelligent Delivery Vehicles. At the beginning of the interview, she mentions that the approval process for a new drug varies depending on the regulatory agencies of each country. In Mexico, it takes approximately 15 years and is regulated by COFEPRIS (acronym in Spanish for Federal Commission for the Protection against Sanitary Risks). The tests and clinical trials conducted during drug development are as follows. First, there are preclinical trials, where the proposed formulation is tested in the laboratory against a biological system such as cell culture. This first phase serves to assess the safety and efficacy of active ingredients while validating the expected response of the composition of the drug. Second, there is the clinical trial, where a small group of patients is involved to test safety and dosage. This is where the therapeutic dose is determined. Third, Phase 2 clinical trials, where the test population is expanded and side effects are analyzed. Finally, there are comparison and confirmation trials, where the drug's effect is studied in completely random patients and its impact is analyzed. If the drug shows good efficiency and yields significant data, the validation process with COFEPRIS begins, initiating the steps for commercialization approval. Even if a drug has been approved, research continues regarding possible allergic reactions and the presence of allergies. If it exhibits adverse effects, it must be withdrawn from the market. Medications, whether chemical or biological, must undergo various tests to ensure they maintain their stability and effectiveness during storage and use. An accelerated stability test involves exposing the medication to more adverse conditions than those on the shelf and observing the results. The real-time stability test involves subjecting the medication to conditions similar to those it will encounter in the target organism. Evaluating the physicochemical properties is related to packaging and labeling studies to determine if special packaging is required. Efficacy tests are conducted to verify if the medication truly fulfills its reported therapeutic action. Impurity tests analyze the amount of impurities generated during the medication synthesis process in both the upstream and downstream stages. The allowed purity limit is established by COFEPRIS. For biological medications, microbiological tests are performed to analyze the presence of endotoxins since products containing these compounds cannot be marketed. The speed and effectiveness of how a drug is absorbed depend on several factors, including the nature of the organ it is targeted at. For instance, mammary tissue contains a significant amount of fat, which favors the absorption of liposoluble drugs. The quantity of blood vessels in the tissue is also important, as greater vascularity allows for a quicker absorption of the drug. Other factors to consider include the permeability of cells in mammary tissue, the presence of the lymphatic system, hormonal influences, and the potential for interactions with other medications. Furthermore, the composition of the product also affects the speed and effectiveness of drug absorption. It is crucial to understand the polarity, size, pH, solubility, and other factors of the drug in question. To assess residues and ensure the safety of a new drug concerning food, samples are collected entirely at random. These samples undergo chemical analysis and evaluation methods to determine which components are present and in what quantity. Upon concluding this process, permissible levels of components are established, and an evaluation of potential risks involved is conducted. Selecting the type of packaging is a crucial step, as it could impact how the product is preserved over time. There are materials that have already been approved by various regulatory agencies such as the FDA and COFEPRIS. When assessing effectiveness in the laboratory, it's crucial to control the characteristics of our biological system to minimize potential influences from external variables. In addition to the aforementioned studies, it's essential to conduct research on how the drug behaves within the body (pharmacokinetics and dynamics), studies on appropriate dosing, safety tests, evaluations of skin irritability and sensitivity, analysis of the body's tolerance to reactions, and compatibility tests. When scaling up to larger production, it's necessary to have an easily reproducible protocol that complies with all regulations set by COFEPRIS. PhD. Francisca suggests that we keep the drug formulation as simple as possible, as this facilitates its approval by regulatory agencies. Opting for compounds that are already known and have been studied is a wise choice. It's also essential to ensure that any vehicle used is completely free of proteases.

Contribution:

Francisca discusses the drug approval process, clinical trials, stability testing, factors affecting drug absorption, safety evaluation, and packaging considerations. She emphasizes the importance of regulatory compliance, simplicity in formulation for approval, and thorough testing for stability, safety, and effectiveness. The absorption of drugs is influenced by factors such as organ nature and composition. Additionally, the need for compatibility testing, regulatory adherence, and avoidance of proteases in vehicles is highlighted.

Dr. Maria Luisa del Prado is an expert in chemical and materials engineering, with a master's degree and doctorate in materials science. She also has a postdoctoral degree in nanotechnologies for the controlled release of curcumin. During the interview, she highlighted three key aspects to designing an efficient and safe delivery system for veterinary medicines. First, size must be considered to evaluate the membranes it must pass through. Second, the surface area is crucial. Third, the capture percentage of the active compound must be analyzed. In Mexico, there is no specific regulation for unconventional vehicles, since there are few products that use nanomedicine. However, NOM-059 provides guidelines on medical devices, materials and procedures to follow. In her experience, Dr. Del Prado mentions that biopolymers are the most efficient delivery vehicles for materials such as proteins. This is because they are biocompatible, biodegradable and very resistant once the processes have been standardized. To launch a new drug product, it is essential to conduct preclinical and clinical trials that include both in-vitro and in-vivo testing. Additionally, stability testing must be performed on the final product to ensure long shelf life.

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Using natural polymers such as alginate and chitosan can be beneficial since they are compatible with living organisms. However, it is crucial to carefully evaluate whether sterilization of these particles will affect their physical and chemical characteristics. Additionally, working with alginate at a very small level (nanoscale) can be complicated. Finally, if chitosan has antimicrobial properties on its own, it could improve the effectiveness of the drug being proposed by the team.

He has 23 years of experience in the dairy cattle industry and currently works for the pharmaceutical company MSD, specializing in veterinary medicines. Given the significant issue of antibiotic resistance due to the extensive use of antibiotics, consuming milk from an unwell cow can contribute to this problem. Regarding necessary regulations, he mentioned a key rule in the drug market enforced by SAGARPA. This rule requires product registration, involving meeting specific parameters and conducting appropriate studies. Depending on the product, laboratories undergo this registration process. International pharmaceutical companies operate development departments dedicated to creating their unique molecules, expanding their product range according to their focus. These companies maintain research pipelines as well. In instances where an individual develops a novel product not yet in the market, pharmaceutical companies can obtain the patent for that particular product.

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He mentioned several interesting points. In the industry, pharmaceutical syringes are used, which deliver "multidrug" antibiotics targeting many microorganisms, often referred to as "antibiotic cocktails." This practice contributes to antibiotic resistance. When discussing alternative treatments, he emphasized options like homeopathic remedies such as oregano essences, immunostimulants that activate the immune system, and peroxide antiseptics with limited applications in certain types of mastitis. He also mentioned ongoing research involving bacteriophages, although currently, there are no patented products derived from bacteriophages. “In the mastistis treatments the most common method that is used is the intramammary infusion” - José Manuel Oropeza

Gabriela Mapes has over 30 years of experience in the dairy cattle sector, specializing in reproductive health and udder health. The interviewee emphasizes the importance of prevention and hygiene programs, immediate action upon suspecting mastitis signs, and collaboration between all stakeholders to reduce the impact on health and production.

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Mastitis has a significant impact on the dairy industry, causing major economic losses and reduced productivity. It stands out as the most costly disease in this sector. Treating mastitis costs between $95 and $211 per cow affected within the first 30 days of lactation. Introducing a new veterinary medicine to the market involves rigorous laboratory and clinical testing to establish efficacy, safety, and appropriate dosage. The process is regulated by both national and international guidelines, ensuring the quality of bovine mastitis treatment and milk. In addition to somatic cell count, conductivity tests can also serve as indicators of mastitis. Gabriela highlighted a genomic test that predicts a calf's susceptibility to diseases, leading to a remarkable 44% reduction in antibiotic use through targeted interventions.

The experts found our project to be highly interesting and relevant for our community. During the feedback session, they raised some important questions: How do you plan to control the endolysin release mechanism? Have you determined the thickness of the nanoparticles, considering its direct impact on the speed of endolysin release?
Contribution:
Additionally, they provided valuable suggestions: Consider the cost implications of the project. Acknowledge the openness of the Mennonite community towards our project. Elaborate more on the mechanism of action of endolysins. Allow the most capable presenter to handle the delivery of the presentation. Utilize pH variations in bactericidal experiments to assess the functionality of endolysins under different conditions. Include nanoparticles in bactericidal experiments. Clearly demonstrate the presence of endolysins, supplementing the SDS-PAGE gels. Determine the number of cows to which AureoBos would be applied, ensuring the trial's statistical reliability.

He has 24 years of experience at CIMAV (Center for Research in Advanced Materials S.C.). He pursued his master's and doctoral studies at another center called the Center for Applied Chemistry Research. His specialty is polymer creation, a vast field that cannot be fully encompassed. Polymers can be of natural, synthetic, or semi-synthetic origin. He specialized in a specific technique called emulsion polymerization. Over the last 5 years, he has been researching electrochemical sensors and drug delivery, as well as studying hydrogels made of biobased polymers such as cellulose. He believes that synthetic polymers can be more efficient as carriers for enzymes, as they can be designed to respond to stimuli such as changes in pH, temperature, or external applications like ultrasound, altering their structure. This ability has been demonstrated in numerous studies. In contrast, natural polymers like chitosan, guar gum, and cellulose are biodegradable and form gels. According to various bibliographic references, chitosan and alginate are biodegradable biopolymers compatible with the body, so he believes it is not necessary to repeat these tests if they have already been confirmed. He also mentions that his laboratory does not specialize in pharmaceutical tests, so he collaborates with expert laboratories in this field to assess the effectiveness of a drug.

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He considers synthetic polymers efficient carriers for enzymes, being able to respond to various stimuli, in contrast to natural polymers. He emphasizes the biodegradability and gel-forming properties of natural polymers. He relies on specialized laboratories to evaluate pharmaceutical effectiveness due to their laboratory's expertise.

Vivi has worked at “Quesos Menonitas” since her professional internships. She is currently in charge of quality control and the laboratories. She has worked in various areas within the company, including sensory analysis, milk analysis, and microbiological analysis of the finished product. Her primary role at the cheese factory involves analyzing milk samples to determine if they meet quality standards. She frequently uses somatic cell count analysis to assess milk safety regarding mastitis. Furthermore, she's responsible for managing pest control, ensuring staff training, maintaining cleanliness, supervising veterinarians, and monitoring stable conditions. In the plant, she conducts environmental monitoring and handles documentation. The cheese factory has experienced significant growth, both in production quantity and quality. Currently, they produce 18 batches of cheese daily, which translates to more than 30 tons per day. It's important to highlight that the cheese factory complies with both national and international certifications. When the milk arrives at the cheese factory, it must go through several filters to be accepted. First, the temperature is checked, and it cannot exceed 7°C. Second, physical-chemical analyses are conducted to determine the fat, proteins, and caseins content. Third, the resazurin test is performed, where a dye is added to the milk and subjected to a specific temperature for a set time. At the beginning of this test, the sample is pink, and if it loses color, it indicates that the milk has a high microbial load. Fourth, the acidity of the milk is analyzed, which should fall within the range of 13-18 degrees Tornick. Fifth, the presence of neutralizers is checked to detect if any additives have been added to the milk. For example, some producers tend to add hydroxides or peroxides to reduce acidity and prevent the cheese factory from detecting improper milk handling in the barn. Sixth, the presence of inhibitors, such as antibiotics, beta-lactams, tetracyclines, and sulfonamides, is also assessed. Seventh, the presence of chlorine is also analyzed. Finally, somatic cell counting is conducted, which should be in the range of 200 to 400 thousand. Once the milk has passed through all the previously mentioned filters, the unloading process begins at the plant. Upon entry, it goes through a series of filters that remove any foreign materials, such as feces and flies. Then, it goes through a plate cooler until it reaches a temperature of 4.5°C. The milk is stored in silos and doesn't remain there for more than 24 hours. Finally, the milk enters the production process, where it passes through another plate exchanger that functions as a pasteurizer, ensuring that the milk is safe and of high quality for use in the final products. Each truck carries milk from multiple producers, so the drivers take a sample of milk from each producer. In the event that mastitis contamination is detected in a tanker, it is important to know which producer requires specific follow-up. The responsible veterinarian works directly with that particular barn. Currently, they continue to face mastitis issues with some producers, although the exact number affected is not known.

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For the milk to be accepted, it undergoes rigorous analysis that ensures its quality by evaluating its physicochemical characteristics and the presence of any contaminants. After meeting quality standards it enters the production phase. Vivi emphasizes safety and adherence to quality standards. Challenges with mastitis persist, necessitating continuous vigilance and collaboration with producers for effective follow-up.

Víctor oversees the milking process at Zaragoza. During our interview, he described the hygiene protocols they follow. Instead of using paper to clean the cows' udders, they opt for soft towels, aiming to minimize paper waste.

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He described the milk cooling process after milking. Initially, when the milk is extracted from the udder it is at 40°C and is cooled down to 1.5°C. Once cooled, it undergoes filtration before being transferred to the silos. A milk truck driver arrives to collect it. We inquired about the mastitis tests, and he mentioned that they conduct tests on all the cows every month to track their health. The samples collected are sent to the laboratory for analysis.

In livestock farming, there is a specific area for milk producers; However, there is no specialized sector that specifically focuses on addressing mastitis. He emphasized that the regulation and registration of new medications are in charge at the federal level, and it is suggested that we approach COESPRIS (for its acronym in English State Commission for the Protection against Health Risks) or SENASICA (for its acronym in Spanish for National Service of Health, Safety and Agri-Food Quality) in this regard. These federal institutions are in charge of determining whether or not a new drug should enter the market, since they evaluate the possible side effects that that drug could have. The importance of having a competitive advantage over any other product already existing on the market is highlighted. Upon learning about our AureoBos proposal, he found the project very interesting. He even offered to help us find farmers willing to test our product on their dairy herds and evaluate its effectiveness.

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There is absence of a specific sector focus on mastitis in livestock farming. He highlights the federal level's role in regulating and registering new medications. He recommended us to approach COESPRIS or SENASICA for guidance. The significance of a competitive edge in the market is underscored. Additionally, he mentions a positive reception of the AureoBos proposal, with an offer to facilitate product testing by farmers on their dairy herds to evaluate its efficacy.

COESPRIS, a state health authority, operates under state health legislation and the general health law. Various agreements, laws and regulations are derived from this last one. On the other hand, COFEPRIS, its counterpart at the federal level, delegates to COESPRIS the powers that they can exercise. COESPRIS' responsibilities are related to approximately 300 sectors, internally divided into various areas, the largest being food and beverages. Other areas include medications, healthcare services, and environmental health. In addition, it has programs to monitor brucellosis and another for bovine tuberculosis, specifically managed by the livestock committee. However, there is no specific program for mastitis. COESPRIS regulates establishments and everything related to the packaging, labeling and separation process, according to article 194 of the general health law. All primary activities related to the countryside are the responsibility of SENASICA and rural development. COESPRIS verifies that establishments such as cheese factories, supermarkets and collection centers use contaminant-free raw materials to produce different dairy products and comply with quality controls, achieving this through sampling. Medicines for animal use must have a health registration with SENASICA (for its acronym in Spanish for National Service of Health, Safety and Agri-Food Quality) to be able to go on sale and be marketed. During the interview, they told us that our project is very interesting to them, since it constitutes an effective alternative to antibiotic treatments. Opting for alternative treatments that do not involve the use of antibiotics significantly helps reduce indirect exposure to humans. They assure that by 2050, health problems related to antimicrobials will surpass those related to cancer. A product like AueroBos avoids economic losses and the risk of contaminating milk with antibiotics. In addition, they mention that they do not have updated data on the incidence of mastitis. The site with the largest amount of updated data in this regard is the INEGI.

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COESPRIS representatives in the interview inform us that there is no specific sector in charge of regulating and treating mastitis. They indicate that the most reliable database to measure the incidence of mastitis is that provided by INEGI. They also clarify that SENASICA is the government entity responsible for guaranteeing the implementation of good practices in the pens to obtain contaminant-free milk. In addition, they mention that medicines intended for use in animals must be registered with SENASICA.

Only 5% of dairy consumers know what bovine mastitis is. Everyone is concerned that dairy products are safe and of good quality. Specifically, they want to ensure that the products for sale are fresh and do not contain additives that could affect health. In our community, almost no dairy consumers trust sanitation and hygiene measures to ensure dairy products are free of chemicals such as antibiotics.

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Some dairy consumers suggest that products should have labels that report possible bacteria if they are not treated properly. Furthermore, they believe that there should be a suitable medication to treat mastitis in cows.