How To Treat Mastitis In Cows Naturally

• Journal List
• J Vet Res
• 5.64(four); 2020 Dec
• PMC7734678

J Vet Res. 2020 December; 64(iv): 523–529.

Efficacy of Natural Formulations in Bovine Mastitis Pathology: Culling Solution to Antibiotic Handling

Claudia Pașca

¹Section of Apiculture and Sericulture, Kinesthesia of Brute Science and Biotechnologies, Cluj-Napoca, Romania

Liviu Alexandru Mărghitaș

^aneSection of Apiculture and Sericulture, Kinesthesia of Brute Science and Biotechnologies, Cluj-Napoca, Romania

Daniel Severus Dezmirean

¹Department of Beekeeping and Sericulture, Faculty of Fauna Science and Biotechnologies, Cluj-Napoca, Romania

Ioana Adriana Matei

²Section of Microbiology, Cluj-Napoca, Romania

Victorița Bonta

^threeLife Scientific discipline Constitute "King Michael I of Romania", University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, 400372, Romania

Ioan Pașca

⁴Department of Fauna Product and Food Safety, Faculty of Veterinarian Medicine, Cluj-Napoca, Romania

Flore Chirilă

ⁱⁱDepartment of Microbiology, Cluj-Napoca, Romania

Adrian Cîmpean

^ivDepartment of Animal Production and Food Safety, Faculty of Veterinary Medicine, Cluj-Napoca, Romania

Nicodim Iosif Fiț

²Department of Microbiology, Cluj-Napoca, Romania

Received 2020 Mar 19; Accepted 2020 Oct five.

Abstruse

Introduction

Bovine mastitis is an inflammatory affliction of the udder that causes important economic losses in the creature breeding and dairy product industries. Present, the conventional livestock antibiotic treatments are slowly beingness replaced by alternative treatments. In this context, the primary aim of this study was to evaluate the efficacy of natural products in alternative treatment of bovine mastitis.

Textile and Methods

2 natural formulations with previously suggested in vitro antimicrobial event were tested in vivo on mastitic cows. Animals with a positive diagnosis for mastitis (n = xx) were divided into 3 treatment groups: two groups (n = eight) were administered formulations of propolis, alcoholic extracts of Brewers Gold and Perle hops, plum lichen, common mallow, marigold, absinthe wormwood, blackness poplar buds, lemon balm, and essential oils of oregano, lavender, and rosemary designated R4 and R7 (differing only in the latter beingness more concentrated) and one grouping (n = 4) a conventional antibiotic mixture. In vivo efficacy of treatments was evaluated past somatic cell and standard plate counts, the treatment being considered efficacious when both parameters were under the maximum limit.

Results

R7 was constructive in the nearly cases, being therapeutically bactericidal in vi out of eight cows, while R4 gave skillful results in three out of eight cows, and conventional antibiotics cured i out of iv.

Conclusion

These results advise the possible therapeutic potential of these natural products in bovine mastitis.

Keywords: cows, mastitis, alternative treatment, in vivo testing, natural products

Introduction

Bovine mastitis is a major infectious illness that causes the largest economic losses in the fauna breeding and dairy industries. Negative financial impacts from mastitis on dairy farms were reported in many parts of the world, including the U.s., Europe, Commonwealth of australia, and Due south Africa (23). It is difficult to estimate the losses associated with clinical mastitis, and fifty-fifty more difficult to quantify those associated with the sub-clinical form. In European countries, losses due to mastitis per cow per year are estimated at betwixt 100 and 300 USD equivalent (nine). The sizeable economical brunt includes the direct costs, namely the price of treatment (drugs and veterinary fees), discharged milk, labour cost, fatalities, and repeated cases of mastitis, and the indirect repercussions, which are the decrease in milk yield, milk quality changes (compositional changes, poorer hygienic quality of milk, and public health considerations), culling and replacement toll, pre-term drying off, the creature welfare aspect of mastitis, and associated health problems (23).

Although mastitis is a complex disease involving many factors, and technically, the term could be used to describe any udder injury that may consequence in inflammation, information technology is generally accustomed equally a reference to the inflammatory reaction with microorganic causative agents that accept grown too abundantly or entered into the udder quarter canal and mammary tissue causing an intramammary infection (IMI) (16). Despite the definition of IMI beingness unclear, mammary gland health is often defined based on its bacteriology (the presence or absence of an IMI) or based on quarter-level or composite somatic jail cell count (SCC) (27). IMIs are generally detected through milk culturing. This enables the monitoring of udder health and identification of the aetiological agents of the disease and ensures targeted specific antimicrobial therapy (six).

Pathogens causing mastitis may have different origins, existence classified according to the source as contagious, ecology, or opportunistic (sixteen). Contagious microorganisms are usually found on the udder or teat surface of infected cows and are the principal source of infection. Staphylococcus aureus is the species most oft isolated, followed by Streptococcus agalactiae and the less common pathogens such as Corynebacterium bovis and Mycoplasma bovis (xvi). Environmental pathogens are found in the immediate surroundings of the cow, in the sawdust and bedding, manure, and soil. Bacteria in this classification include streptococcal strains such as Streptococcus dysgalactiae, South. uberis, S. bovis, Enterococcus faecium, and E. faecalis, and coliforms such every bit Escherichia coli, Klebsiella pneumonia, and Enterobacter aerogenes (xvi). They crusade mastitis if the allowed arrangement of the host is compromised or if sanitation and hygiene is not fairly practiced (24). Opportunistic pathogens issue in balmy forms of mastitis and include coagulase-negative staphylococci (CoNS). These staphylococci may be isolated from milk, are commensal and usually imply a minor immune response in cattle and only cause mild infections. They include Southward. epidermidis, Southward. saprophyticus, S. simulans, Due south. chromogenes, S. hyicus, South. warneri, S. sciuri, and Southward. xylosus (xvi, 18).

Handling of mastitis usually involves intramammary assistants of antibiotics to clear the quarter of the causative organisms. A clinical response must be perceptible inside 5–vii days; otherwise, the case is usually considered a therapeutic failure (ii). The commercial intramammary antimicrobial products available in Europe contain either unmarried antibiotics such as cephalexin benzathine, cephalonium, cephapirin benzathine, cephazolin, cloxacillin benzathine, or rifaximin, or a mixture of two or 3 antibiotics. Products combining multiple antibiotics contain oxacillin benzathine and oxacillin sodium, framycetin sulphate and erythromycin stearate, neomycin sulphate and spiramycin, penicillin G procaine and novobiocin sodium, cloxacillin benzathine and neomycin sulphate, penicillin G procaine and neomycin sulphate, cloxacillin benzathine and colistin sulphate, or penicillin G procaine, nafcillin and dihydrostreptomycin sulphate (two, 11). Withal, the extensive use of antibiotics in the treatment and control of mastitis is not ever constructive and develops resistance and consequent non-responsiveness to antibiotic therapy (7). Other important issues are the possible implications for man health through an increased chance of antibiotic-resistant strains of bacteria emerging that may then enter the food chain and the increased risk of antibiotic residues in milk (29). Related to these risks, there is continuous pressure to reduce the apply of antibiotics in the treatment of food-producing animals and a dramatic increment in organic milk production, which has led to research into finding alternative antimicrobial agents.

Although there are several studies focused on the antimicrobial effects of plant products (ten, 22, thirty) or bee products (1, 8, 26) on pathogens isolated from milk from mastitic cows, there are only few data regarding in vivo testing. In the context of increased multidrug resistance and demand for organic products, the high costs of treating mastitis and of the development of efficient new synthetic drugs, the low manufacturing cost of plant products, and the credible lack of antimicrobial resistance to these phytochemicals, the aim of this study was to examination in vivo two plant-based and propolis-containing products with previously proven in vitro efficacy (21).

Fabric and Methods

Animals and samples. The study was conducted on a dairy farm located in Cluj County (46°44′54″N 23°50′0″E), Transylvanian Plainly, Romania, on a herd of 220 local-breed cows (Bălțata Românească) which were v years old and at the second or third calving. All the cows were tested for mastitis using the California mastitis test (CMT) correlated with clinical and subclinical signs. Clinical signs included lack of appetite, reduction in milk quantity, and milk with whey and fibrin clots. Additionally, somatic cell count (SCC) and bacterial cell count (BCC)/standard plate count (SPC) confirmed the diagnosis. Positive results for mastitis were obtained for twenty cows.

Milk sample collection and analysis. Samples were collected using the recommendations of the U.s. National Mastitis Council (19). The udders of the subjects were done with abundant clean water and wiped with paper towels, and the teats were surface-disinfected with swabs containing fourscore% ethanol. After discarding the first milk stream, approximately xl mL of quarter milk was collected into l mL sterilised tubes and stored at iv°C until the hygiene assay was performed in the reference laboratory of the Milk Quality Control Foundation, Cluj-Napoca, in the adjacent hour after collection.

Somatic jail cell count. The SCC was determined by the fluoro-opto-electronic method on a Fossomatic 90 Mastitis & Milk Quality Tester/Somatic Cell Counter (Foss, Denmark), co-ordinate to the HRN ISO 133662:2006 standard (13). A result of 100,000 cells/mL was taken to be a low SCC, since this is generally considered to reflect a healthy mammary gland (25). A loftier SCC was >200,000 cells/mL, equally this is a rational threshold for the presence of infection (v). The number of mesophilic aerobic bacteria was assessed indirectly based on the number of colonies generated by the cells of the microorganism present in the milk sample.

Standard plate count. The total number of bacteria (BCC) was determined by the flow cytometric method on a BactoScan FC milk bacteria analyser (Foss), according to the ISO 21187:2004 standard (fourteen). The BactoScan analyser was calibrated on the ground of colony number, which was determined by the reference method (counting the colonies of bacteria on a plate as a standard plate count – SPC), according to the HRN ISO 4833:2003 standard (15). For this count, two sterile Petri plates were inoculated by flooding with 1 mL of milk. 1 Petri plate was likewise inoculated with each of at least three decimal dilutions (10^−ane–x⁻ⁱⁱⁱ). The prepared plates were incubated at 37°C for 48 h. After incubation, plates having a number of colonies ranging from 25 to 250 were chosen for counting and the colony forming unit of measurement (CFU) number was calculated considering the dilution for the private plate. Results were expressed in CFU/mL of milk. A total number of leaner >100,000 jail cell/mL was considered an indication of mastitis.

Microbiological analysis of milk. For the positive animals, the pathogen analysis (genus or species identification) was performed in the Microbiology Laboratory of the University of Agricultural Sciences and Veterinarian Medicine in Cluj-Napoca, by cultivation on specific medium (Muller–Hinton, blood and MacConkey agar). Plate incubation was performed at 37°C for 24 h under aerobic conditions (12). After incubation, morphological analysis of colonies and bacterial cells was performed by Gram staining. For each colony, preliminary tests were performed in society to select the type of belittling profile alphabetize (API) microgallery.

The microMérieux API germ identification galleries (bioMérieux Inc., USA) are a bacterial classification system based on a series of experiments. They consist of 20, 32 or 50 tests, each contained in minor reaction tubes that include a nutrient substrate. This organisation was developed for the rapid identification of clinically relevant species. The interpretation of the results considers which of the tubes gave a positive reaction, and these data are then compiled into a number that is verified with a database containing all known microorganisms.

In order to confirm the identities of the pathogenic bacteria to the species level, Staphylococcus API 20 STAPH, Streptococcus API 20 STREP, and Bacillus API 50 CH were used for the respective genera (Table 1).

Tabular array 1

Pathogens identified in normal and mastitic milk

Identified microorganism	Gram +/−	Kit type	Code contour
Staphylococcus epidermidis	+	API Staph	6 seven seven iii i five one
Staphylococcus aureus	+	API Staph	ATCC 6538P
Staphylococcus schleiferi	+	API Staph	6 7 iii ii five 5 3
Staphylococcus hominis	+	API Staph	6 7 three 2 1 1 2
Aerococcus viridans	+	VITEK Identification	0625703821500211
Aerococcus viridans	+	VITEK Identification	0625707930700413
Staphylococcus aureus	+	VITEK Identification	0209610546712053
Bacillus cereus	+	API 50 CHB	7212
Corynebacterium spp.	+	VITEK Identification	4407611554521210
Micrococcus spp.	+	VITEK Identification	0625703050500211
Streptococcus agalactiae	+	VITEK Identification	0205610554226611

Bacterial strains were also identified by the VITEK two automated identification system (bioMérieux) (Tabular array 1). The system builds a strain's biochemical profile past interpreting seventy–80 tests impregnated on cards. The identification normally takes 3–4 h, beingness much more accurate and working with a large database according to the standards used (ISO 17025:2017).

The samples were processed following the identification of the bacterial flora, and then were tested for sensitivity to bee and found natural products as described past Markey et al. (17).

Experimental testing. Unlike found extracts, propolis extract and essential oils were incorporated into a natural soft gel mass (Xanthan gum). Eight formulations (R1–R8) were made according to a previous study (21), including aqueous excerpt of propolis, alcoholic extracts of Brewers Aureate and Perle hops, plum lichen, mutual mallow, marigold, absinthe wormwood, blackness poplar buds, lemon balm, and essential oil of oregano, lavander, and rosemary. These were tested in vitro separately and compared to seven antibiotics used in mastitis treatment (oxytetracycline, ceftriaxone, gentamicin, penicillin, florphenicol, enrofloxacin, and amoxicillin), besides equally described in the previous study (21). In that investigation, following the statistical analysis performed with Epi Info seven software (https://www.cdc.gov/epiinfo), the ii products R4 and R7 were observed to exert meaning effects on strains isolated from milk from mastitic cows, and these were chosen in the nowadays experiment for in vivo tests. These were administered intramammarily, in 10 mL volume. Each gel product contains aqueous extract of propolis, alcoholic extracts of Brewers Gilded and Perle hops, plum lichen, common mallow, marigold, absinthe wormwood, black poplar buds, lemon balm, and essential oil of oregano, lavender, and rosemary in dissimilar concentrations.

The cows diagnosed with clinical and subclinical mastitis (northward = twenty) were separated from healthy ones in another part of the shelter and divided into iii groups. Two of the groups (n = 8 in each) were treated with the two selected natural biological gels R4 and R7 chosen based on their in vitro antimicrobial activity published in a previous work (21). The two experimental groups were treated every twenty-four hours (morn and evening) for five days with gel administered intramammarily, gel R4 existence used on group 1 and gel R7 on group 2. The third group (n = 4) was treated intramammarily with a commercial conventional antibiotic (CA) product containing amoxicillin and clavulanic acid, post-obit the manufacturer'southward recommendations and indications.

The handling efficacy was evaluated by SCC and SPC. V milk samples were collected from each animal earlier treatment at 0 h; during treatment at 24 h, 48 h and 72 h and afterwards the concluding day of treatment at 120 h.

At the cease of the treatment, the two bee and plant natural products' (R4 and R7) effectiveness was confirmed in one case again in vitro on a pick of predominant bacterial strains identified from the same milk samples (Due south. intermedius, Southward. epidermidis, S. hominis, South. aureus, Bacillus spp., and Streptococcus agalactiae).

Statistical analysis. Statistical assay was performed with Epi Info vii software. The mean and standard deviation were calculated, and the differences between the averages were analysed using the ANOVA test (parametric exam for inequality of means), a value of P < 0.05 being considered statistically significant.

Results

Twenty out of 220 cows from the participating subcontract were diagnosed with mild clinical and subclinical mastitis based on CMT, SCC, SPC and clinical signs. In all cases, only Gram-positive bacteria were detected. In 15 out of xx cows, one bacterial species was identified, in four cows, co-infection with two species was discovered, and in one cow co-infection with 3 species was found. In 17 cows, different staphylococci strains were identified. In descending order of prevalence, the strains were S. epidermidis (in eleven cows), S. aureus (3), S. schleiferi (ii), and Southward. hominis (ane). Other identified Gram-positive cocci belonged to the Micrococcus (3), Streptococcus (i), and Aerococcus (1) genera. Gram-positive bacilli were identified in merely iii samples, being two Bacillus spp. and 1 Corynebacterium spp. Co-infection was recorded in five cases: two with Southward. schleiferi and Micrococcus spp.; one with S. epidermidis and S. hominis; another with South. aureus and Aerococcus spp.; and the final i with Staphylococcus spp., Micrococcus spp., and Corynebacterium spp.

Collection of mammary secretions was made earlier treatment (collection one) (Table 2), during handling: at 24 h (drove 2), 48 h (drove 3), and 72 h (collection iv), and afterwards the last twenty-four hours of treatment at 120 h (drove 5). In all treated cows, a decreasing number of leaner and somatic cells were observed between the first measurement (earlier handling) and the last measurement (subsequently the finish of treatment). Similarly, the mean number of bacteria (CFU/mL) in the fifth measurement was statistically significantly lower than the mean from the first measurement in both the group treated with R4 and the group treated with the commercial production (P = 0.00841 and P = 0.00301, respectively). In cows treated with R7, a significant deviation (P = 0.00872) was likewise observed for the fourth measurement (the terminal fabricated during the treatment) (Fig. 1).

Table 2

Get-go collection of mammary secretion (time 0) from cows in experimental in vivo tests

Tested product	Patient	Predominant bacterial species isolated from milk sample	CFU/mL	SCC/mL
Group 1 treated with R4	1.	S. epidermidis	902,000	169,800
	2.	S. epidermidis	385,000	one,000,000
	three.	S. Micrococcus schleiferi, spp.	89,000	114,300
	4.	S. Micrococcus schleiferi, spp.	49,000	190,800
	5.	South. epidermidis	172,000	3,600
	six.	S. epidermidis	312,000	51,900
	7.	S. epidermidis	171,600	195,800
	viii.	Southward. epidermidis	364,000	240,000
Grouping two treated with R7	ix.	Due south. epidermidis	66,000	102,100
	10.	South. epidermidis	334,000	80,300
	eleven.	S. epidermidis, S. hominis	125,000	39,000
	12.	S. epidermidis	41,000	54,000
	13.	Bacillus spp.	48,000	105,900
	14.	South. epidermidis	37,000	10 000
	xv.	Due south. aureus	177,000	129,000
	16.	Streptococcus spp.	247,900	1,000,000
Control group treated with antibiotics	17.	Bacillus spp.	223,000	x,000
	18.	South. Aerococcus aureus, spp.	525,000	i,004,600
	19.	Southward. aureus	218,000	12,000
	twenty.	Corynebacterium spp. Staphylococcus spp. Micrococcus spp.	220,000	88,900

Number of colony-forming units (CFU) in milk from mastitic cows treated with two dissimilar natural plant-based formulations and conventional antibiotics

Among the cows treated with R4, in two cases a depression bacteria count (SPC <100000 CFU/mL) was recorded at 48 h during treatment, and in another three cases at the cease of treatment, while a high bacteria count was observed in three cases. In all these cases, S. epidermidis was isolated from the milk before the treatment. As well, a high SCC (SCC <200,000 cells/mL) was observed in three cows, among which two had a depression bacteria count (Fig. 2).

Somatic cells count (SCC) in quarter milk samples from cows treated with ii unlike natural plant-based formulations and conventional antibiotics

In the cows treated with R7, only 1 had a high bacteria count, and Streptococcus spp. was isolated from the milk of this cow. A low bacteria count was recorded in one cow at the third measurement, in another one at the fourth, and in v cows at the concluding measurement, after the end of treatment. A high SCC was recorded in 2 cows: the cow with a high leaner count and another.

Similarly, amidst the animals treated with CA, one presented a high bacterial count at the fifth measurement and the same fauna and other two cows had a loftier SCC. In the milk samples belonging to the cow with a persistent high bacterial count, Due south. aureus and Aerococcus spp. were detected.

If a treatment can be considered effective when both SCC and SPC are nether the maximum limit, R7 was effective in six out of 8 cows, R4 in three out of eight cows, and CA in 1 out of four cows. Still, the SCC count may take a longer time to fall to under the maximum limit since the treatments are aimed primarily at eliminating bacterial cells and the inflammation may also decrease under treatment only as a secondary effect. The selected predominant bacterial strains isolated from mastitis milk presented different values of inhibition expanse when tested in vitro (Fig. 2). Fifty-fifty with this ascertainment being made, the R7 product remains the most effective, conventional antibiotics beingness less effective and R4 the least.

Discussion

Handling of clinical and subclinical mastitis commonly involves antibiotic administration, to articulate the quarter of the causative organisms. Economic considerations require that the best possible cure should be obtained with the shortest withdrawal menses of the animal, so that the milk tin be marketed again (eleven). Antibody therapy cannot be discrete from costs for dairy farmers. For this reason, culling treatments are being adult, then that the concluding product (milk) is free of dangerous contaminants from the course of antibiotics which would otherwise be required.

The identification of different staphylococci strainsin mastitic milk samples is consistent with that of other enquiry studies (28). In our previous study (21), 8 natural biological products were tested in vitro against pathogens isolated from mastitic milk. Among them, three presented inhibition areas comparable to those seen with both florphenicol and enrofloxacin and larger than those of penicillin, gentamicin, and amoxicillin (21). Because of the modest number of mastitis cases available on the tested subcontract, only ii of the natural products for mastitis treatment were included in this study. 3 products with in vitro efficacy from the previous study, including the two tested in the nowadays study, had the aforementioned natural ingredients but contained them at different concentrations. The agile ingredients included aqueous extract of propolis, alcoholic extracts of Brewers Golden and Perle hops, plum lichen, common mallow, marigold, absinthe wormwood, black poplar buds, lemon balm, and essential oil of oregano, lavander, and rosemary. The departure between the R4 and R7 formulations as tested in the present report was a college concentration of all the active ingredients in the latter. Although no statistically significant difference between the means of SSC and BCC amid the three groups (R4, R7, and CA) was reported, at five days subsequently the stop of treatment, a loftier proportion of cows treated with R7 had both parameters nether the limit (six out of viii), near half of the R4-treated cows (three out of viii) had such, and for the cows given CA, the proportion was a quarter (1 out of four). If only the full number of bacteria are considered, in the case of R7, seven out of eight cows had good BCC in their milk, three out of 4 did in the case of CA and five out of 8 for R4. These differences between BCC and SCC in the instance of CA, seen against the almost non-existent difference in the cases of R4 and R7, may propose that the gels besides take a part in the reduction of inflammation. This possible effect was previously suggested for several essential oils (Nepeta cataria, Rosmarinus officinalis, and Origanum vulgare) which contain various bioactive compounds with potent anti-inflammatory upshot including carvacrol, limonene, citronellal, and cinnamaldehyde (22).

Practiced BCC results were obtained in 7 out of eight cows treated with R7. In these seven cows, the bacteria South. epidermidis, S. hominis, S. aureus and Bacillus spp. were detected in the milk earlier treatment. As indicated by BCC, afterward the handling there was a decreasing of bacterial load suggesting its result on these species. The simply cow treated with R7 in which a high BCC was recorded also after the handling had Streptococcus spp. identified in its milk, suggesting a moderate consequence on this species. Similarly, in vitro testing of R7 showed good inhibitory activity on staphylococci (S. chromogenes, S. hyicus, South. intermedius, and South. xylosus), Lactococcus lactis, Kytococcus sedentarius, Bacillus cereus, and on the Gram-negative bacteria Vibrio fluvialis and Yersinia ruckeri (21). However, only moderate activity was recorded against Gram-negative bacteria such as Escherichia coli, Serratia liquefaciens, Enterobacter intermedius (Kluyvera intermedia), or Aeromonas hydrophila and A. caviae (21). The efficacy of R7 on Gram-negative bacteria was not evaluated in vivo, because these were not isolated from the milk samples from cows constitute positive for mastitis. However, other studies have shown skilful action confronting Gram-negative bacteria for preparations having some of the main ingredients of our gels. For instance, active compounds present in lichen extracts (in acetone, methanol, ethanol, water, diethyl ether, chloroform, and petroleum ether extraction) are bactericidal to Gram-negative microorganic pathogens like Aeromonas, Enterobacter, Helicobacter, Klebsiella, Pseudomonas and Proteus spp. (30). Similarly, ethanolic extract of propolis can be used against Gram-negative bacteria such as K. pneumoniae, Eastward. coli, or P. aeruginosa (1, eight, 26). An antimicrobial effect of Rosmarinus officinalis and Moringa oleifera was as well recorded for Gram-negative microorganisms isolated from milk (iv, 20). Based on these observations, by increasing the concentrations of the ingredients in the gels nether evaluation, a meliorate efficacy on Gram-negative bacteria could be obtained.

Compared to conventional therapy, the natural products used in this study (containing dissimilar establish extracts and propolis) suggest good efficacy in the treatment of mastitis acquired by Gram-positive bacteria. Medicinal plant extracts appear to be a condom, efficient, and depression-cost choice for treating this type of illness, minimising economic losses, and equally importantly, forestalling the development of drug resistance both in animals and in humans consuming animate being products. Further research is necessary to place all active compounds of the extracts, evaluate their effectiveness, and establish different safe indices (principally the potentially toxic concentration), and then are clinical trials.

Footnotes

Conflict of Interest

Disharmonize of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article.

Financial Disclosure Statement: The written report received the financial support of the (1) Project Partnerships in Priority Areas Plan - PN Two, adult with the support of MEN-UEFISCDI, project no. 148/2014; (2) Programme 2: Competitiveness of the Romanaian economy through enquiry, development and innovation: PN-III-P2-ii.1-CI-2018-1472 no. 252 CI from 03/09/2018. Project title: Biologically active product for controlling nosemosis in bees; and (iii) Subprogramme 1.2 - Institutional Performance - Projects for Financing the Excellence in CDI, Contract no. 37PFE/06.11.2018. Championship of the project: "Increasing the institutional performance through consolidation and development of enquiry directions within the USAMVCN".

Animal Rights Argument: None required.

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Articles from Journal of Veterinary Research are provided here courtesy of De Gruyter

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734678/

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