Antimicrobial Susceptibility of Bovine Subclinical Mastitis and During Milk Separation Isolates and the Accompanying Hygienic Practices

Corresponding Author: Sultan Farag Nagati Department of Bacteriology, Animal Health Research Institute (AHRI), Agriculture Research Center (ARC), Egypt Email: sultan_farag99@yahoo.com Abstract: Milk is considered one of the most perfect foods for humans and milk contains many nutrients. Subclinical mastitis has been counted as a great problem for dairy manufacture. The disease causes severe economic losses due to lower milk production, treatment costs and increased labor and milk retained for human consumption after treatment and premature culling. A cross-sectional study was conducted in 3 villages of Tamia district, Fayoum governorate, Bacteriological examination, Antibiotic susceptibility test, to study the presence of sub-clinical mastitis and milk contamination during milk separation and milking process and to detect the most resistance isolate to antibacterial agents and find the suitable antibacterial and disinfectants. 25.7% of the milk samples were positive for the California test. Skim milk and cream showed the isolation of Staphylococcus aureus (S. aureus), Streptococcus aglacteae (S. aglacteae), Coliform bacteria, Arcanobacterium pyogenes (A. pyogenes) and Escherichia coli (E. coli). The microbiological quality of the two disinfectants tested was considered satisfactory, as no significant number of microbial contaminants were recovered from them following the sterility test. Only 26.7% of milkers use water and soap for washing their hands. The participants had a lack of information about subclinical mastitis and improper hygienic practices during the milking and milk-handling process.


Introduction
Milk is considered one of the most perfect foods for humans, milk contains many nutrients needed for growth and development and prevention of many nutritional disorders (Dhanashekar et al., 2012). Raw milk is still used by a large number of farm families, workers and growing sectors of the population because they believe this raw milk is not only safe, but pasteurization destroys the health benefits of milk (LeJeune and Rajala-Schultz., 2009) milk is rich in proteins, lipids and sugars, milk is an example of ideal culture medium for various pathogens, including bacteria, viruses, fungi and parasites, which can be important sources of foodborne pathogens. These pathogens could be found in milk by direct contact with the contaminated animal environment and secretion from the udder of infected animals including mastitis and subclinical mastitis (Dhanashekar et al., 2012;Zeinhom and Abdel-Latef, 2014).
Mastitis is the most common inflammatory disease of the mammary gland in dairy animals, which causes several changes in milk such as, physical, chemical and microbiological changes (Eshratkhah et al., 2012). Mastitis is categorized into two forms, clinical which is diagnosed clinically and subclinical which is mainly diagnosed by estimation of somatic cell count and bacterial examination (Blowey and Edmondson, 2010).
Over 100 diverse micro-organisms have been listed as a cause of intramammary infection in dairy animals, the main causative micro-organisms were classified into infectious pathogens including (Staphylococcus aureus and Streptococcus agalactiae) and environmental organisms most encountered are species of streptococci other than Streptococcus agalactiae and Gram-negative bacteria such as E. coli and Klebsiella (Dhanashekar et al., 2012;Zeinhom and Abdel-Latef, 2014;Fox and Gay, 1993).
Subclinical mastitis has been counted as a great problem for dairy manufacture; the infected quarter appears seemingly natural and could act as a hidden source of infection among dairy cattle (Sorensen et al., 2015). The disease causes severe economic losses due to lower milk production, treatment costs and increased labor and milk retained for human consumption after treatment and premature culling, therefore early detection of mastitis at the subclinical stage is important for most dairy farmers to take proper measures toward treatment and prevention of transmission of infection to another healthy one in the animal and thus reducing production losses. The aim of the study is pointed to study the presence of subclinical mastitis and milk contamination during milk separation and the milking process and to detect the most resistant isolate to antibacterial agents and find suitable antibacterial and disinfectants.

Study Design and Setting
A cross-sectional study was conducted in 3 villages of Tamia district, Fayoum governorate. Fayoum governorate is located in the southwest of Cairo in Egypt. The information on animals being studied was collected from the owners. The owners of the animals are individual farmers every farmer had 1 to 3 cows or buffaloes. The sampled milking animals were; Egyptian buffaloes and local or crossbred cows. The farmers are mainly keeping their animals in the back yard of their house. The backyard units are connected directly to the owner's home. In Fayoum, the diet of the animals (home-prepared concentrates) is not devised according to the physiological needs, neither in terms of quality nor quantity. The animals are taken to the field early morning after milking every day for feeding by green ration and return home just before sunset. At home, the concentrates (1-3 kg/animal) are provided once daily, in addition to dry wheat hay. The average daily milk yields from buffalo ranged from 15 to 18 L/d and for local cow 10 to 13L/d crossbreed cow from 19 to 22 L/d. Most of the sampled animals were in the mid-lactation stage (from 3 to <6 months) based on the age of the calves. The target group was the milk separator machine owners and people using the milk separator machine for their animals (6 machine owners and all the women who milk animals and come to separate the milk with the separation machine which is operated manually). The data were collected and analyzed over 6 months from June to September 2020 Table 1.
The study was designed as follow: a. Cows and buffalo milk under manufacturing was tested for subclinical mastitis using the California test b. Milk samples were collected from cows and buffaloes milk before and after separation (skim milk) c. Hand swabs from workers and milking persons were collected before using the separator machine directly. Dorsal and palmar surfaces and fingertips were swabbed using a moistened cotton sterile swab (using sterile ringer's solution) which was rubbed gently against surfaces d. Swabs were collected from the separator machine before and after the operation e. Samples were collected from cream f. All samples were submitted for isolation of bacteria, followed by a susceptibility test for different antimicrobial agents g. Study the effect of the used two disinfectants on bacterial isolates h. An interview structured questionnaire was designed to assess the knowledge of farmers on clinical and subclinical mastitis and to identify their hygienic practices during the milking process. The data of the study were collected by the authors California Mastitis Test (CMT): (Leach et al., 2008) A small sample of milk (approximately 2 mL) per liter was collected in a plastic paddle of four shallow cups marked A, B, C and D with an equal amount of CMT reagent added to the milk. To mix the content, the paddle was rotated and after a few seconds (about 20 seconds) the result was read. The test was performed daily to support the data obtained with an accurate somatic cell count Table 2.

Bacteriological Examination
Samples were submitted for isolation and identification of different bacteria causing mastitis (the milk sample was collected from the four-quarter and considered as one sample) (Quinn et al., 2002;Abera et al., 2010), by plating on the following culture media plates (Oxoid): Sheep blood agar, MacConkey agar mannitol salt agar, Staph-Strept, media, Aloa agar, XLD, CN Pseudomonas-specific media Morphological characterization of the colonies, the effectiveness of hemolysis on sheep blood agar, microscopic morphology evaluation on Gram Stained samples and biochemical characterization by oxidase test, catalase test and Staphtect (Oxoid) or by commercial API (Biomèurieux).
Milk, swabs and cream samples were cultured on blood agar plates, incubated at 37°C for 16-24 h. The growth on the plate was confirmed by additional laboratory tests according to routine in vitro procedures. Staphylococcus aureus was recognized by the typical colony morphology, alpha and beta hemolysis, or by coagulant reaction (positive coagulase) when typical hemolysis regions were not present. Coagulase-negative staphylococcus was recognized by typical colony morphology and coagulation reaction. Streptococcus agalactia was determined by colony morphology and CAMP-reaction and biochemical reactions were used for typing to the species level. Enterococci were confirmed by Gram staining and the growth of model colonies on SlaBa plates (Oxoid Ltd. Basingstoke, England). Gramnegative bacteria of typical colony shape, p-nitrophenylb-D-Glucupyranosiduronic Acid (PGUA) and indole were considered E. coli. For other Gram-negative bacteria, the oxidase reaction and API 20 E biochemical test profile (BioMérieux) was used.
Assessment of the microbiological quality of two disinfectants (A and B) used on surface and farm equipment (Samson et al., 2017).

Types of Disinfectants Used and Their Neutralizers:
1. Type B disinfectant (Combination of four Quaternary Ammonium (QAC), glutaraldehyde and two ter) pene derivatives it neutralizer is lecithin 2. Type A disinfectant contains peracetic acid, its neutralizer is glycine

Sterility Check of Test Disinfectants
0.1 mL sample of each disinfectant was added to 0.9 mL sterile diluents, 0.02 mL volume of the diluted disinfectant was placed on each Nutrient Agar (NA) and Sabour Dextrose Agar (SDA) plates prepared. The NA plate was incubated at 37°C for 3 days; the SDA plate was incubated at 25°C (room temperature) for 7 days. Five or more colonies on either plate indicate contamination of the test disinfectant. Fungal isolates were identified on the basis of microscopic (using Lactophenol cotton blue staining) and macroscopic characteristics.

Standardization of Isolates
A single isolated colony of each isolate was obtained from Tryptic Soy Agar (TSA) plates and inoculated separately in 10 mL of Tryptic Soy Broth (TSB) and incubated for 24 h at 37°C. After incubation, the 24 h broth culture was filtered with a saline pre-wet filter paper to remove slime and centrifuged at 2000 rpm for 20 min. The cell pellets were washed with 10 mL TSB. Then the population density of the bacterial suspensions in the TSB (about 10 7 CFU/mL) was adjusted by matching 0.5 McFarland Standard (10 5 CFU/mL) then diluting 1:100 in sterile TSB.

Quantitative Suspension Test (QST)
0.1 mL of the standard bacterial suspension will be added to 0.9 mL of the disinfectant solutions and softly mingle at room temperature for contact times of 0, 1, 3, 5, 10 and 15 min. The timer was started when the bacterial test suspension was combined with the disinfectant. Then, at time 15 min, the specified contact time, 0.1 mL of the disinfectant-organism mixture was removed and transferred to a tube containing 0.9 mL neutralizer (tube A) and mix thoroughly. Within 5 min of transfer to the neutralizer tube, three additional ten-fold dilutions in saline blanks shall be made to achieve 10 1 , 10 2 and 10 3 dilutions (Tube B, Tube C and Tube D, respectively). 0.1 mL of each dilution was plated onto TSA plates in duplicate by the spread-plate technique and incubated at 37°C for 24 h. After incubation, TSA plates were noted for no visible growth. The surviving microbial colonies were enumerated, multiplied with factor hundred (100) and expressed as Colony-Forming Units per milliliter (CFU/mL). Controls were set for all organisms tested to indicate the neutralizer activity. For control, 0.1 mL each of 0.5 McFarland broth from the test object was mixed with 0.9 mL of neutral in separate tubes and so transferred to TSB, as a procedure described with disinfectants. Later, all the controls were striped onto TSA plates. The presence of growth indicates that the neutralizer is not inhibiting the microbes tested. Similarly, 0.1 mL of every disinfectant was mixed with 0.9 mL neutralizer, then 0.1 mL suspension of the test organism (0.5 McFarland standard) was added to every tube, later, they are directly transferred and incubated in TSB and streaked onto TSA plates. Growth on TSA plates shows effective neutralization of the disinfectant activity (USEPA, 2014).

Data Analysis
Data were collected, coded and analyzed using Statistical Package for Social Science (SPSS version 16. Chicago IL, USA). Simple descriptive analysis in the form of frequencies and percentages were performed.

Detection of Subclinical Mastitic Milk (California Test)
Machines one and six used milk with subclinical mastitis of both types of animals (mixed), Machines two and four used milk of the same animal with no subclinical mastitis (cattle milk pure), Machine three used milk of the same animal with subclinical mastitis (cattle milk infected) and Machine 5 used milk both animals (mixed) with no subclinical mastitis (cattle milk pure). 74.3% of milk samples used in this study were negative to the California test, which clarified that most of the milk samples were non-mastitic milk, while 25.7% were positive for the California test Table 3.
The isolation of different pathogens in cow and buffalo`s milk (machines 1, 3 and 6) corroborate the results of the California test, which discovered the presence of a subclinical mastitis Tables 4 and 6.
Tables 5 and 6 illustrated the milk of both cows and buffalos were free from pathogens, in machines (2, 4 and 5) which also showed negative results with the California test. Table 5 showed the isolation of S. aureus, S. agalecteae and other coliforms bacteria from machine swabs collected before used and A. pyogenes, other coliforms bacteria and E. coli were isolated from one hand of the personal handle with the machine (1). In the case of a machine (4), S. aureus was isolated from the machine before used; the Hand swab showed the isolation of other coliforms, A. pyogenes and E. coli (50% for each). Skim milk and cream showed the isolation of S. aureus, S. aglacteae, other coliforms bacteria, A. pyogenes and E. coli Table 5.
In Table 6, coliform was isolated from both machine and hand swabs of a personal handle with the machine (5). Also, S. aureus and E. coli were isolated from the hand swab during the handle with a machine (5). The ratio of different isolates among different samples, where all isolates isolated from machine swabs, hand swabs and subclinical mastitis milk were isolated from skim milk and cream Table 7.
The microbiological quality of the two disinfectants tested was considered satisfactory, as no significant number of microbial contaminants were recovered from them following the sterility test. The bactericidal effect of B disinfectant (0.5%) and A disinfectant (2%) solution on isolates per contact time is presented in (Table 9, Figs. 1 and 2) respectively. On one hand, all isolates were grown when exposed for 3 min to both disinfectants except coliform no growth observed, on the other hand, A. pyogenes were grown when exposed to both disinfectants for 10 min. A period of ten minutes of exposure or contact was sufficient time for killing most isolates. Only A disinfectant has its biocides effect on A. pyogenes when exposed for 15 min.

Analysis of Data Collected from the Owners of the Milk Separator Machine and Milkers
All the owners of the milk processing machine use the machine for personal and business use. The machine is used for more than one type of animal by 100%. The milk used each time is a mixture of milk from both cows and buffaloes by 100%. No one of the owners knows subclinical mastitis. All the owners wash the machine. The machine is washed once a time at the end of the day by 100% of them. 50% of the owners use water only for cleaning and 50% use water and soap (Table 10).                The data were collected from 105 females aged from 18 to 55 years with a mean of 31.5±10.5 years. The majority (64.8%) were illiterate. Most of the participants (91.4%) milking more than one type of animal each time. Most of them (87.6%) had one or more animals with a history of the previous mastitis. 89.5% of milkers mix the milk from both quarters of the udder if there was no change of the milk color. Only, 11.4% of milkers using milk from infected udder with color milk change. No one of the milkers knows about subclinical mastitis. 100% of them have ever experienced a decrease in the production of animal milk. 68.1% of them asked veterinarians about low animal milk yield. All participants (100%) used milk in the presence of decreased animal milk production. The majority (83.8%) wash their hands before milking the animals. Only 26.7% of them use water and soap for hand-washing (Table 11).

Discussion
Subclinical mastitis is an annoying problem in dairy farms, it is responsible for virtually accounted for 70% of economic losses of mastitis cases and its frequency is about 15 to 40 times the frequency of clinical mastitis. (Losinger, 2005;El-Awady and Oudah, 2011). In the present study, a total of 71 samples were collected from six milk manual separating machine used for bacteriological isolation, 35 (24 cows and 11 buffaloes' milk before used), 12 milk byproduct (6 cream and 6 skimmed milk) and 24 swabs (6 swabs before using the machines, 6 swabs after using and 12 hand swabs from human) to determine the presence of the bacterial contamination of the milk byproduct. The hygienic measures taken by the handlers before, during and after milking have a greater effect on the safety and hygiene of milk and its products. Infected personnel involved in milk handling, containers used to put milk during milking, storage and delivery may be possible sources of contamination. Under bad sanitary conditions, milk can be easily contaminated by diverse microorganisms (Chatterjee et al., 2006). Studies exhibited that bovine clinical mastitis is currently one of the most serious problems found on dairy farms, where the disease is with the highest economic impact on milk production. The impact on public health should be taken into account as dairy animals produce milk and milk byproducts as a cream for consumption (Riekerink et al., 2008).
In this study, regarding the hygienic process of the owners of milk processing machine, all the owners used the machine for both types of animals (cows and buffalo) and all of them wash the machine once a day at the end of the milk operation process, no hygienic practices were followed between each time of work (100%) and none of them use disinfectants to clean the machine. These poor hygienic practices may be sources of contamination to milk and its products besides no one of the female milkers (100%) asked if the machine was cleaned or not. The majority (83.8%) wash hands before animal milking and 33.3% wash their hands after milking. 41% of them clean the udder before milking and only 26.7% of them use water and soap for washing the hands. These findings were similar to a study conducted in Ethiopia. All the milkers were also females, 76.5% of them practiced udder washing and drying before milking. However, 64.7% did not practice post milking udder wash. On the other hand, 58.8% practiced washing milking equipment with detergents before milking (Regasa et al., 2019), others reported that more than 60% of the milkers did not clean their hand nor cleaning the udder of the animal before milking, indicated that the microorganisms on hand could lead to contamination of the milk (Shija, 2013) Use of unsterilized containers and other practices such as milking with unsterilized bare hands and allowing calves to feed without cleaning udder nipples, exposing the milk to microbial contamination (Owusu-Kwarteng et al., 2020) and avoid crosscontamination proper cleaning and disinfectant of the containers is important (kivaria et al., 2006).
Mastitis constitutes one of the most important and expensive diseases of the dairy industry. It is difficult to discover Subclinical Mastitis (SCM) due to the absence of visible designation, in the mammary gland and milk. The alteration in milk yield and composition depends on the severity and duration of mammary gland infection (Oliver and Calvinho, 1995). In our findings, although no one of the milkers stated that they knew or heard of subclinical mastitis all of them (100%) reported that they had a reduction in animal milk production once and 58.1% asking vet advice on animal milk reduction and no one of them has done laboratory tests for their animals and this may reflect the high illiteracy rates and poverty in rural areas in Fayoum governorate (HDR, 2008). In India, (Sinha et al., 2014) revealed that economic losses due to mastitis were due to the reduced milk yield by 48.53% followed by veterinary expenses, which account for 36.57% of the total loss.
Our findings revealed that 25.7% of the milk samples were positive to the California test which revealed that their mastitic milk appeared before separating operation, this result may be explained by the fact that this study was conducted under high humid conditions during the summer months in Egypt, which increase the rate of mammary infections, this was similar to what reported by (Dang et al., 2013). We used the California Mastitic test in our study according to (Leach et al., 2008), the CMT was supported by (Galfi et al., 2017) who revealed that CMT represents a valuable diagnostic method in detecting subclinical mastitis in dairy cows and high sensitivity and specificity.
The isolation of different pathogens in cow and buffalos milk (machines 1, 3 and 6) corroborates the results of the California test which discovered the presence of subclinical mastitis. These results proved a possible explanation for this finding could be that most farmers in the study area do not run-through proper farming management and screen for mastitis at an earlier stage (Mpatswenumugabo et al., 2017;Bekuma and Ulfina, 2018).
In machines 2 and 4, the isolation of different microorganisms from hand swabs and machine swab (machine 2) revealed that bad cleaning of personal handling with machines and bad cleaning and no disinfecting of the machine (2) before use, which lead to the presence of all isolates present in cream and skimmed milk.
All isolates were isolated from machine swabs, hand swabs and subclinical mastitis milk from skim milk and cream. The dairy industry is known for its health policies, performance and high health standards. This industry is especially essential for good sanitation practices to ensure the safety and well-preservation of dairy products. (Marriott, 1997;Schlegelova et al., 2010).
Clean and sterile equipment and buildings are essential for the production, processing and distribution of healthy dairy products and it is important to use appropriate cleaning vehicles and equipment so that workers can clean the facility in a shorter period and with less work. (Marriott, 1997).
Enrofloxacin was the best antimicrobial agent for most isolates except for A. pyogenes and these results agree (Sripad et al., 2016). Most of the isolates displayed MDR to different classes of antimicrobial agents. The evidence for the effectiveness of antimicrobial treatments for mastitis is limited. Antibiotic resistance has turned into a considerable worry for human and animal health. The detection of MDR-from S. aureus and A. pyogenes in various samples is of concern and represents a major public health concern. These results agree with (Salauddin et al., 2020). Antiseptics used on equipment and surfaces should be tested periodically for effectiveness. Since some disinfectants lose their effectiveness upon standing in addition to organic matter, their effectiveness must be tested. While some methods help in choosing the right dilution of sanitizer for others to use, test the effectiveness of the sanitizer used. The present study evaluated the microbiological quality and efficacy of two common disinfectants (A and B) used to disinfect the environment, surfaces and equipment. The microbiological quality of the tested disinfectants. No bacterial or fungal growth of any of these isolates occurred after appropriate days of incubation on Nutrient Agar (NA) and Sabour Dextrose Agar (SDA). All isolates were grown upon exposure for 3 min to both antiseptics except coliform no growth was observed.
The period of ten minutes of exposure or contact was sufficient time for killing all isolates except A. pyogenes. Biocides (BC) resistance among bacterial pathogens is not frequent, but resistance may develop to user concentrations after exposure to sublethal concentrations of BC. (Sidhu et al., 2004). A. pyogenes were not grown when exposed to a disinfectant A for 15min. Hydrogen peroxide was the only disinfectant found to be effective versus all of the tested microorganisms. The biocidal effect of hydrogen peroxide is attributable to the-OH radical, formed by the decomposition of peroxide in the presence of catalysts, such as iron and copper ions, commonly found in microorganisms. The radical acts via an oxidative mechanism against the membrane, DNA and other cellular constituents of the microorganism (Montagna et al., 2019).
Selecting a disinfectant registered for a particular pathogen is crucial. It is also important to recognize that observed effects in laboratory studies of small increases in intolerance to some disinfectants and association with antibiotic resistance have to be balanced versus the general health benefits of using them (Gerba, 2015).

Conclusion
The results showed that the owners of the milk separation machine and small farmers, who account for the cattle milk production in Egypt, the participants had a lack of information about subclinical mastitis and improper hygienic practices during the milking and milk-handling process. The use of on-farm written protocols to treat mastitis encourages the rational use of antimicrobials and reduces antimicrobial use. The reason for the strains to remain after Cleaning and Disinfectant (C&D) is not resistant to the disinfectant, but could be due to insufficient C&D in critical locations and/or residual organic matter and/or other factors affecting the efficacy of disinfectants (such as diluting disinfectant with residual rinse water and environmental temperature. If this behavior continues and is consistent, this may lead to the presence of germs that are resistant to C&D in this area.

Ethical Approval and Consent to Participate
The study protocol was approved by the ethics committee of the animal health research institute at 27 Aught 2020 that complies with guidelines of the World Medical Association Declaration of Helsinki. Informed consent was obtained from all participants.