An insight of clinico-haemato-cytokine and milk alterations in clinical mastitis in goats

Introduction

Mastitis is a complex, multi-etiological illness characterized by inflammation of the parenchyma of the mammary glands along with pathological abnormalities in glandular tissues as well as physical, chemical, and typically bacterial changes in milk (Constable et al., 2017) [5] . The most prevalent and infectious ailment in dairy animals is mastitis. It has been estimated that mastitis is currently costing India Rs. 7165 crores in damages (Bansal and Gupta, 2009) [3] . Dairy industries suffer enormous financial losses as a result of the disease's effects on milk yield and milk quality, animal fatalities and culling, and associated treatment costs (Singh, 2021) [38] . Mastitis causes global profitable losses of USD 35 billion for the world's dairy industry and Rs. 6000 crores for India's dairy industry. It is crucial to identify mastitis early in order to minimise the financial damage to dairy farmers and dairy assiduity (Sathiyabarathi M. et al., 2016) [29] . Mastitis is more common in meat goats that have been grown using intense and semiintensive management techniques. Mastitis may cause producers to earn less money, depending on the disease's severity. Mastitis is typically linked to poor hygiene habits and brought on by the bruising of breast tissue or teats as a result of injuries, nursing, fly bites, or other skin wounds that compromise a vital infection barrier. Mastitis is also linked to toxins produced by bacteria, fungi, or viruses. A doe's immune system is weakened and struggles to defend against the invasion of foreign things that cause diseases like mastitis under stressful circumstances like severe temperatures, muddy and damp living conditions, or a sudden change in nutrition. Damage to the udder tissue causes altered concentrations of milk constituents and increased concentrations of sodium and chloride ions which raises the pH and electric conductivity of the milk. The blood-milk barrier is damaged as a result of inflammation, allowing serum electrolytes to enter the milk (mostly salt and chlorine), increasing electrical conductivity and decreasing electrical impedance. The high concentration of alkaline blood components like sodium and bicarbonate ions, which are caused by inflammation of the mammary gland and enhance the permeability of the blood capillaries, may be responsible for the elevated milk pH in subclinical mastitis (Guha et. al. 2010 & Sinha et al. 2018) [31,39] . Moreover, lower acidity, which is linked to reduced lactose concentrations in mastitic milk, may be the cause of the higher milk pH during mastitis. The electrical conductivity of milk is altered as a result of mammary gland inflammation in goats. Mastititic milk in goats has a higher electrical conductivity, especially when both mammary glands are affected. Examining the haematological and biochemical parameters of animals is a key factor in determining how accurate a diagnosis is. Deviation from normal hemato-biochemical indices is indicative of ill health, and they aid in diagnosis and evaluation of the severity and prognosis of the disease condition, as evident in the white blood cell accumulation in mastitic udder. The investigation of basic hematological and biochemical indicators contributes to the knowledge of metabolic profile and their possible disorder, whether of a latent or clinical nature (Mohammed 2016) [40] .

Aim/Objective of study

The present study was designed to evaluate the relationship of mastitis with clinical signs, milk & udder parameters, pH, Electrical Conductivity, Hematological Parameters & Cytokine Estimation in goats affected with mastitis, as due to inadequate study on inter-relationship of mastitis with hematological, cytokine & milk parameters in goats.

Materials & Methods

Selection of Goats based on Clinical Signs indicative of Mastitis

Goats presented to the Veterinary Clinical Complex, Anand, Gujarat, India with clinical signs like reduced milk yield, painful, hot, hard and swollen udders, abnormal milk were suspected for mastitis. Out of these, 30 positive cases were included in the therapeutic study. Healthy goats from Instructional Livestock Farm Complex of College of Veterinary Science & A. H. Anand, were selected as a control group.

pH & Electrical Conductivity of Milk

Milk samples were collected aseptically from the mastitis affected goats. The mid-stream milk was collected using sterile container. pH & Electrical conductivity of milk samples were recorded by Digital pH & Conductivity Meter (LAQUA Benchtop pH & Electrical Conductivity Analyzer). pH electrode was calibrated to 7.0 whereas EC electrode was calibrated to 10.1. Readings were taken on 'day 0' & 'day 5' of treatment. The results of electrical conductivity were expressed in milli Siemens per cm (mS/cm)

Hematological Parameters

Blood was collected aseptically in heparinized tubes from jugular veins of goats showing signs of mastitis & healthy goats as healthy control. Blood samples collected from the goats were used for hematological examination and blood parameters like Hemoglobin (g/dl), Packed cell volume (%), Total Erythrocyte Count (TEC) (x106 l), Total Leucocyte Count (TLC) (x103µ/ l),µ/ Differential Leucocyte Count (DLC) (%) like Lymphocyte%, Monocyte% and Neutrophil% were recorded.

Cytokine Analysis

Blood was collected in serum clot activator tubes for separation of serum. Serum samples were separated by using centrifugation machine @ 3000 RPM for 10 minutes. These separated serum samples were aspirated with sterile pipette & collected in serum collecting vials which were stored at -20° centigrade till further use for serum cytokine estimation. Cytokines analysis was done by Sandwich ELISA (Fine test) Enzyme Linked Immuno Sorbent Assay using serum samples of goats positive for mastitis. IL-6 (Interlukin 6) as Pro-inflammatory cytokine & IL-10 as an Anti-Inflammatory Cytokine found in serum samples of goats were studied by Goat IL-6 ELISA Kit & Goat IL-10 ELISA Kit respectively.

Result & Discussions 4.1 General Clinical signs of Goats Affected with Mastitis

In present study, 30 goats showing clinical signs of mastitis regarding milk abnormalities like color & consistency of milk, udder abnormalities like unilateral or bilateral affection of udder, consistency of udder, signs of inflammation of udder like swollen, hot, painful, hyperemic), general signs like fever, depression, weakness, anorexia, reduced milk yield were recorded as per table no 1 & figure 1. [6] reported depression (65%), lack of appetite (75%), increase in body temperature (fever) of 40 °C and above (100%), PR >90 beats/min (80%), respiratory rate above 25 breaths/min (85%) and 40% recumbency. Gabli et. al., (2019) [11] studied prevalence of mastitis in dairy goats in which clinical examination of mammary glands showed 3.55% goats with clinical mastitis, 20% primiparous, 80% multiparous. 98.34% goats with half udder affected, 6.66% goats with both teats affected. [12] , Ya'u (2014) [36] , Peter (2015) [23] , Krishnappa (2016) [18] as Warm, swollen, hard, hyperemic, painful and hot affected udders with a grossly enlarged teat, palpation udder showed hard to touch, response to pain and the absence of milk from the affected quarter. Merz (2016) [20] , Singh (2018) [32] reported sudden onset of swelling and redness of udder, pain, and reduced and altered milk secretion from the affected quarters, clots, flakes, watery consistency of milk. Danmallam et. al., (2019) [6] , Garba (2019) [13] , Shaheen et. al., (2020) [30] and Poonia (2022) [24] recorded pain, heat, swelling, redness of udder with abnormal secretion of milk such as clots, flakes, or watery milk, Bluish-reddish tinge and spotty hemorrhages on udder, abnormalities of the milk like thick with foulsmelling yellow-green pus secretion, no milk letdown.

Milk & Udder Parameters in

Milk pH & Electrical Conductivity of Milk

In present study, the mean ±S.E. value of milk pH of mastitis affected goats was found to be higher (7.02±0.03) towards alkaline as compared to healthy goats (6.18±0.07) which was found to be decreased to 6.56±0.03 highly significantly (p<0.01) post treatment as shown in table & figure no. 3. The present research findings are in accordance with Fandialan and Davide (2001) [10] , Ghosh (2004) [15] , Samanta (2006) [28] , Sharma (2007) [41] , Garg and Singh (2008) [14] , Bagri (2018) [1] . A significant increase were recorded in pH and acidity of subclinical mastitis milk samples as compared to normal milk samples was recorded by Modh (2018) [42] & Sinha (2018) [31] also recorded elevation in milk pH value in Subclinical Mastitis (SCM) cows (7.1 ± 0.01) as compared to the healthy cows (6.65 ± 0.01). Poonia (2022) [24] recorded significantly (p<0.01) higher pH in mastitis animals towards alkalinity. Elevated milk pH during mastitis may be due to lower acidity which is associated with reduced lactose contents in mastitis milk. Increased milk pH in the mastitis may be related with high concentration of alkaline blood constituents such as sodium and bicarbonate ions which, due to inflammation of the mammary gland, increases permeability of the blood capillaries. Similarly, the mean ±S.E. value of Electrical Conductivity (EC) of milk of mastitis affected goats was found to be higher (7.96±0.12) as compared to healthy goats (6.55±0.08) which was found to be decreased (7.06±0.11) highly significantly (p<0.01) post treatment as shown in table & figure no. 3. Similar findings were recorded by Diaz (2012) [7] that, EC increased with infection in Saanen goats (from 5.6 mS/cm in healthy animals to 5.8 m S /cm in infected ones), whereas EC decreased with infection in Alpine goats (from 6.1 mS/cm for healthy goats to 5.4 mS/cm in infected ones). Romero (2012) [7] observed the higher milk electric conductivity value in the SCM affected animals as compared to healthy animals. Sinha (2018) [31] , Poonia et.al., (2022) [24] demonstrated statistically significant (p<0.01) increase in electric conductivity & pH. Gupta (2010) [43] reported a significant reduction in EC and pH values post treatment in subclinical mastitis affected animals. In present study, the electrical conductivity of milk is altered as a result of mammary gland inflammation in goats causing damage to the blood-milk barrier, allowing serum electrolytes to enter the milk (mostly salt and chlorine), increasing electrical conductivity.

Hematological Parameters

In the present hematological study, the mean ± SE value of hemoglobin (g/dl), PCV (%), TEC (x10 In present study, there was a significant decrease in the mean ± SE value of hemoglobin in mastitis affected goats than healthy goats. This decrease in hemoglobin level may be due to the defensive mechanism of mononuclear phagocyte system which increases iron trapping & storage, and reduces iron availability to microorganism causing mastitis which results in decrease in hemoglobin synthesis.

Decrease in packed cell volume of goats affected with mastitis in this study could be due to the decreased production of RBCs indicating micocrocytic hypochromic anaemia which is common in inflammatory disorders like mastitis. Total Erythrocyte Count (TECx10 6 /µl) decreased in mastitis affected goats as compared to the healthy goats. The substantial decrease in TEC of goats affected with mastitis is due to inflammatory condition and influence of inflammatory cytokines responsible for mastitis, which causes iron trapping & storage leading less availability of iron to bacteria ultimately results into decrease in production of RBCs, i.e., decrease in TEC. Findings of present study are in accordance with El-Latif et al. (2010) [8] , Yusuf et al.

(2013) [37] , Sadek et al.( 2017) [44] , Garba et al. (2019) [13] and Saleh et al. (2022) [27] who reported significant decrease (p<0.05) in hemoglobin%, packed cell volume%, significant decrease (p<0.05) in TEC of goats affected with mastitis of goats affected with mastitis. However, Garba et al. (2019) [13] recorded non-significant (p>0.05) effect on PCV (27.96±1.31%) in mastitis affected goats as compared to healthy goats (29.59±0.68%).

The TLC value of mastitis affected goats in all 30 goats was found to be higher as compared to healthy goats. Similarly, Garba et al., (2019) [13] reported significant increase (p<0.05) in TLC values of goats affected with mastitis. Increased level of total leucocyte count in present study is due to migration of inflammatory cells which is usually associated with acute inflammatory reaction like mastitis.

The mean (± SE) value of lymphocytes (%) was decreased in mastitis affected goats as compared to the healthy goats. Significant decrease in lymphocyte% found in mastitis affected goats in present study i.e. lymphopenia is most likely the result of degree of inflammatory response at the systemic level, and stress induced increase in blood cortisol level, which have a secondary role in redistribution of recirculating lymphocytes. The mean ± SE value of monocytes (%) was found to be decreased in mastitis affected goats as compared to the healthy goats. However, El-Latif et al. (2010) [8] , recorded significant increase in monocyte count in goats affected with clinical mastitis. The lack of significant differences in monocyte count could be attributed to phase & progression of disease as mastitis. The mean value of neutrophils (%) was found to be increased in mastitis affected goats as compared to the healthy goats. The results of present hematological investigations were in accordance with Radostits et al. (2007) [25] , who stated marked leucopenia, neutropenia and degenerative shift to left in mastitis. El-Latif et al. (2010) [8] , Yusuf et al.

(2013) [37] & Koop et al.( 2016) [17] reported neutrophilia and lymphopenia in mastitis goat. Similar findings were recorded by Krishnappa et al.( 2016) [18] , Tripathy (2018) [34] , Gabli et al.( 2019) [11] , Garba et al.( 2019) [13] , and Saleh et al.( 2022) [27] , who observed significant increase (p<0.05) in neutrophil count. In present study, increased level of neutrophils found may be caused by various immunomodulatory effects due to increased demand of leucocyte in infected udder. Neutrophillia & degenerative shift to left occurs due to migration of large number of neutrophils into the affected udder. Stress associated condition could have also played role in development of neutrophillia. In this study, neutrophillia could be due to degree of inflammatory responses at the systemic levels shown by goats infected with mastitis.

Cytokine Analysis

The mean ± S.E. value of IL- [26] , El-Deeb (2013) [45] , Bayoumi et. al., (2016) [4] , Nayan (2012) [22] , Saleh (2022) [27] , Vitenberga et. al., (2022) [35] who also observed significant (p≤0.05) increase in the levels of IL-6 in mastitis affected animals. Significant rise in the level of IL-6 in the serum samples of goats affected with mastitis in the present study established the presence of the inflammatory cytokine IL-6 to be immediately associated with inflamed mammary glands at the onset of mastitis. The mean ±S.E. value of IL-10 was found to be lower in mastitis affected (556.17±6.99) goats on 'day 0' as compared to the mean value of IL-10 in healthy goats (773.06±19.60) which was found to be increased (643.54±7.63) highly significantly (p<0.01) post treatment on 'day 5'. The present findings are coincident with the results of Bannerman (2009) and Shaheen (2020) [30] recorded marked reduction (p<0.001) in IL-10 levels in clinical mastitis when compared to normal lactating cows however, Faaz and Abdullah (2022) [9] recorded significantly higher (p<0.001) expression of the IL-10 gene in mastitis-affected cows than non-infected animals in the investigation of IL-10 cytokine genes in mastitis originated from bacteria. Sulabh (2022) [33] identified the interleukin 10 (IL-10) as an important cytokine for the control of immune response. In this study, IL-10 as an anti-inflammatory cytokine countered the effects of pro-inflammatory cytokines and caused their excessive release which resulted in giving protection to the tissues against harmful effects of pro-inflammatory cytokine release and thus helped in a more balanced regulation by shifting the immune response towards a more controlled outcome.

Conclusion

Acknowledgement

AcknowledgementThe authors are thankful to Department of Veterinary Medicine & Principal, College of Veterinary Science & Animal Husbandry, Kamdhenu University, Anand, Gujarat, for providing necessary support for conducting this study.

References

1. Bagri DK, Pandey RK, Bagri GK, Kumari R, Bagdi DL. Effect of subclinical mastitis on milk composition in lactating cows. J Entomol Zool Stud. 2018;6(5):231236.
2. Bannerman DD. Pathogen-dependent induction of cytokines and other soluble inflammatory mediators during intramammary infection of dairy cows. J Anim Sci. 2009;87:10-25.
3. Bansal BK, Gupta DK. Economic analysis of bovine mastitis in India and Punjab-A review. Indian J Dairy Sci. 2009;67:337-345.
4. Bayoumi MA, Amer IH, Mansour MA, Handousa SA. Comparative evaluation of Interleukin-6, somatic cell count, and electrical conductivity as predictive tools for subclinical mastitis in dairy buffaloes. Jpn J Vet Res. 2016;64(Suppl 2):S155-160.
5. Constable PD, Hinchcliff KW, Done SH, Grunberg W. Veterinary Medicine-A Textbook of Diseases of Cattle, Horses, Sheep, Pigs and Goats. 11th ed. Vol. 1. Elsevier; 2017.
6. Danmallam FA, Pimenov NV. Study on prevalence, clinical presentation, and associated bacterial pathogens of goat mastitis in Bauchi, Plateau, and Edo states, Nigeria. Vet World. 2019;12(5):638-645.
7. Diaz JR, Romero G, Muelas R, Alejandro M, Peris C. Effect of intramammary infection on milk electrical conductivity in Murciano-Granadina goats. J Dairy Sci. 2012;95:718-726.
8. El-Latif MMAE, Moustafa AH, El-Ramady RA. Some bacteriological, hematological, and biochemical changes on clinical mastitis in goat. Assiut Vet Med J. 2010;56(125):1-12.
9. Faaz RA, Abdullah FA. Expressions of IL4, IL10, and IFNγ cytokines genes during bacterial mastitis. J Adv Vet Anim Res. 2022;9(1):42-52.
10. Fandialan MM, Davide CL. Goat milk: The relationship of its pH with total titratable acidity. Dairy Technol. 2001; ISBN 971-8538-67-4:154.
11. Gabli Z, Djerrou Z, Gabli AE, Bensalem M. Prevalence of mastitis in dairy goat farms in Eastern Algeria. Vet World. 2019;12(10):1563-1572.
12. Ganguly S. A comprehensive and illustrious review on clinical and diagnostic aspects of mastitis infection in high yielding lactating cows. World J Pharma Res. 2014;3(9):352-360.
13. Garba B, Habibullah SA, Saidu B, Suleiman N. Effect of mastitis on some hematological and biochemical parameters of Red Sokoto goats. Vet World. 2019;12(4):572-577.
14. Garg A, Singh M. Risk assessment factors for detection of sub-clinical mastitis in crossbred cattle. Indian J Dairy Sci. 2008;61(6):465-467.
15. Ghosh CP, Nagpaul PK, Prasad S. Factors affecting subclinical mastitis in Sahiwal cows. Indian J Dairy Sci. 2004;57(2):127-131.
16. Gupta A, Mahajan S, Sharma R. Evaluation of antimicrobial activity of Curcuma longa rhizome extract against Staphylococcus aureus. Biotechnol Rep. 2015;6:51-55.
17. Koop G, Islam MN, Rahman MM, Khatun M, Ferdous J, Sayeed MA. Risk factors and therapy for goat mastitis in a hospital based case-control study in Bangladesh. Prev Vet Med. 2016;124:52-57.
18. Krishnappa S, Satyanarayana ML, Narayanaswamy HD, Rao S, Yathiraj S, Isloor S, Mukartal SY, Gupta S, Singh SV, Elattuvalappil AM, Dhama K, Mallaiah S. Haemato-biochemical profile and milk leukocyte count in subclinical and clinical mastitis affected buffaloes. Adv Anim Vet Sci. 2016;4(12):642-647.
19. Kumhar DL, Shukla PC, Singh B. Comparative efficacy of Levofloxacin and Ashwagandha in treatment of subclinical mastitis in lactating goats. Int J Sci Nature. 2017;8(3):635-638.
20. Merz A, Stephan R, Johler S. Staphylococcus aureus isolates from goat and sheep milk seem to be closely related and differ from isolates detected from bovine milk. Front Microbiol. 2016;7(319):1-7.
21. Modi RH, Islam MM, Nauriyal DS, Modi RJ, Wadhwani KN. Study on pH and somatic cell count in milk of sub-clinical mastitic cows in association with udder and teat shape. Indian J Anim Prod Mgmt. 2018;34(1-2):75-79.
22. Nayan V, Phulia SK, Bhardwaj A, Jerome A, Singh D. Transcripts of interleukins in mastitis affected and oxytocin administered buffaloes: Clinical significance and prognostic value. J Biotechnol Biomat. 2012;S1:2155-952X-S1.021-27/Special Issue. doi:10.4172/2155-952X.S1.021.
23. Peter ID, Dauda Y, Thlama PB, Ndahi JJ, Madziga HA, Stephen J, Mustapha A. A retrospective study of small ruminant diseases identified at the state veterinary hospital Maiduguri, Nigeria. J Anim Health Prod. 2015;3(4):88-93.
24. Poonia MP, Sharma A, Rajoria S, Nanda B, Sharma S, Singh N. Evaluation of milk constituents, electric conductivity, and milk pH as a pre-indicator of mastitis in Sirohi goats. Pharma Innovation J. 2022;SP11(7):3390-3395.
25. Radostits OM, Gray CC, Hinchcliff KW, Constable PD. Veterinary Medicine. A Text Book of the Disease of Cattle, Horse, Sheep, Pigs and Goats. 10th ed. Sounder; 2007. p. 673-749.
26. Sakemi Y, Tamura Y, Hagiwara K. Interleukin-6 in quarter milk as a further prediction marker for bovine subclinical mastitis. J Dairy Res. 2011;78:118-121.
27. Saleh N, Allam TS, Omran A, Abdelfattah M. Evaluation of changes in hemato-biochemical, inflammatory, and oxidative stress indices as reliable diagnostic biomarkers for subclinical mastitis in cows. Alexandria J Vet Sci. 2022;72(2):23-34.
28. Samanta A, Prasad S, Ghosh CP. Incidence of subclinical mastitis in Karan Swiss and Karan Eries crossbred cows. Indian J Dairy Sci. 2006;59(1):13-18.
29. Sathiyabarathi M, Jeyakumar S, Manimaran A, Jayaprakash G, Pushpadass HA, Sivaram M, Ramesha KP, Das DN, Kataktalware MA, Prakash MA, Kumar RD. Infrared thermography: A potential noninvasive tool to monitor udder health status in dairy cows. Vet World. 2016;9(10):1075-1081.
30. Shaheen T, Bilal Ahmad S, Rehman MU, Muzamil S, Bhat R, Hussain I, Bashir N, Mir MU, Paray BA, Dawood MAO. Investigations on cytokines and proteins in lactating cows with and without naturally occurring mastitis. J King Saud Univ Sci. 2020;32:2863-2867.
31. Sinha R, Bhakat MK, Mohanty TK, Ranjan A, Kumar R, Lone SA, Rahim A, Paray AR, Khosla K, Danish Z. Infrared thermography as non-invasive technique for early detection of mastitis in dairy animals-A review. Asian J Dairy Food Res. 2018;37(1):1-6.
32. Singh M, Kavitha K, Bharti D, Dixit SK, Mukherjee R, Soni S, Jagatap H, Gandhar JS. Clinical management of mastitis in goat: A case report. J Entomol Zool Stud. 2018;6(6):1163-1165.
33. Sulabh S, Panigrahi M, Varshney R, Gupta JP, Kumar S, Verma A, Kumar A, Muhasin Asaf MVN, Kumar P, Bhushan B. In-vitro analysis of Interleukin-10 expression in cell cultures of Crossbred cattle, Tharparkar cattle and Murrah buffalo in response to mastitis causing antigens derived from Staphylococcus aureus and Escherichia coli. Biol Rhythm Res. 2022;53(2):197-206.
34. Tripathy RK, Rath PK, Mishra BP, Panda SK, Jena B. Clinico-pathological and compositional changes in milk of mastitis cows. Int J Curr Microbiol Appl Sci. 2018;7(6):1680-1687.
35. Vitenberga VZ, Pilmane M, Serstn OK, Melderis I, Gontar Ł, Kocha M, Drutowska A, Maroti G, PrietoSimon B. Identification of inflammatory and regulatory cytokines IL-1α, IL-4, IL-6, IL-12, IL-13, IL-17A, TNF-α, and IFN-γ-producing cells in the milk of dairy cows with subclinical and clinical mastitis. Pathogens. 2022;11:372.
36. Ya'u I. Seroprevalence of Brucellosis in small ruminants in Bauchi State, Nigeria: Thesis. MSc Vet Med. Zaria: 2014. p. 78.
37. Yusuf A, Ikechuckwu OI, Lawan A, Ibrahim B. Alterations in hematological and serum biochemical parameters of Sahel goats with clinical mastitis. J Agric Vet Sci. 2013;4(4):74-77.
38. Singh H. Building effective blended learning programs. InChallenges and opportunities for the global implementation of e-learning frameworks 2021 (pp. 1523). IGI Global Scientific Publishing.
39. Guha S, Saha S. Fluoride ion sensing by an anion-π interaction. Journal of the American Chemical Society. 2010 Dec 22;132(50):17674-7.
40. Mohammed M, Khan MB, Bashier EB. Machine learning: algorithms and applications. Crc Press; 2016 Aug 19.
41. Sharma SV, Bell DW, Settleman J, Haber DA. Epidermal growth factor receptor mutations in lung cancer. Nature Reviews Cancer. 2007 Mar;7(3):169-81.
42. Modh A, Gayar OH, Elshaikh MA, Paulino AC, Siddiqui F. Pediatric head and neck squamous cell carcinoma: Patient demographics, treatment trends and outcomes. International journal of pediatric otorhinolaryngology. 2018 Mar 1;106:21-5.
43. Gupta A, editor. Transparency in global environmental governance: a coming of age?. Global Environmental Politics. 2010 Aug 1;10(3):1-9.
44. Sadek KM, Lebda MA, Nasr SM, Shoukry M. Spirulina platensis prevents hyperglycemia in rats by modulating gluconeogenesis and apoptosis via modification of oxidative stress and MAPK-pathways. Biomedicine & Pharmacotherapy. 2017 Aug 1;92:1085-94.
45. El-Deeb B, Fayez K, Gherbawy Y. Isolation and characterization of endophytic bacteria from Plectranthus tenuiflorus medicinal plant in Saudi Arabia desert and their antimicrobial activities. Journal of plant interactions. 2013 Mar 1;8(1):56-64.