Milk pH of 21 dairy buffaloes suffering from clinical mastitis ranged from 7.00-8.50. Citrate content of mastitic milk ranged from 24.00-47.50 mg /100ml. Cultured milk yielded Staphylococci, Streptococci, Escherichia coli, Bacilli and Klebsiella. The animals were treated with 12gm and 30gm of tri-Sodium citrate in 250 ml of water orally once daily till recovery. With 12gm recovery period was 7-13 days and 30gm took 3-5 days. The pH ~6.50, citrate, lactose, total protein and fat content of milk were restored to normal levels, bacterial colonies markedly reduced after treatment. Citrate acts as a most effective buffer which regulates Ca2+ and H+ in the udder and maintains normal pH. Citrate also sequesters Calcium and inhibits its clumping. Hence, in citrate deficiency the free Ca2+ clump together and form flakes which behave like lime inflicting injury to the secretory tissue in the udder provoking inflammatory reaction rendering impervious tight junctions between blood and milk leaky. The swapping of ions between blood and milk then result in equalizing the pH of blood and milk i.e., 7.40 or higher. The injury inflicted by Ca and alkaline milieus providing conducive conditions for the establishment of environmental pathogens at the injury site producing infectious mastitis. Treatment with tri-Sodium citrate restores normal pH (~6.50) and scavenges the infectious organisms from the udder thus curing mastitis. Intravenous administration of tri-Sodium citrate as 5% in normal saline has been standardized for treatment of mastitis. The 50ml I/V doses of 5% solution of tri-Sodium citrate in sterilized normal saline given morning and evening cures the clinical cases in 1-3 days. The treatment with tri-Sodium citrate is also very effective in controlling mastitis even in cases refrectory to prevalent antibiotics. This treatment is safe, economical, no culling-replacement, milk discarding or withdrawal period and residual hazards in milk/meat. It is recommended that the pH of milk in dairy animals be recorded as a routine to monitor the prevalence of subclinical/ clinical mastitis on the dairy farms for devising proper control measures.
Mastitis is a perpetual problem of all milk producing animals. The conservative estimates of economic losses from this malady have been made almost in each and every state world-wide. Several groups of scientists are working disparately to find out the exact cause and effective treatment of this most formidable disease. Though much of the work on intricate biochemical interactions at the molecular level directed towards unveiling the nub of this malady are being elucidated but still the problem appears elusive.
Coming down to the versatile dairy animal, buffalo (Bubalis bubalis), the "Asian Black Gold" a population of about 130 million globally suffer extensively from mastitis (Fagiolo and Lai 2007). Despite the use of best available facilities at hand to understand the pathobiology of mastitis, the problem still remain economically most important to the dairy industry throughout the world. The ideal modus operandi to prevent or reduce the economic losses, the definite cause of mastitis must be identified and then possible control measures implemented. While scanning the literature on mastitis and biosynthesis of milk in the udder it became apparent that citrate plays a very crucial role in the lactogenesis and maintenance of udder health through ionic equilibration (Peaker and Linzel 1975, Hyvonen and others 2010). Citrate level is always low and concomitant pH high in mastitic milk (Dhillon and others 1989). It was hypothesized that replenishment of citrate deficiency with extraneous tri-sodium citrate might play some protective role against mastitis, hence, these studies were undertaken and the results are communicated in this paper.
MATERIALS AND METHODS
Twenty one buffaloes affected with mastitis were included in this investigation. Milk samples from six buffaloes were cultured and identification made from ensuing colonies. The number of colonies were counted before and after the treatment of the affected buffaloes. Physical examination of milk and udder was conducted and degree of mastitis was graded on the basis of the following scale:
+ Presence of flakes Fig 1. (Please see attached Doc)
++ Serosanguineus milk with admixture of flakes Fig2. (Please see attached Doc)
+++ Curdeled milk with admixture of blood clots Fig3. (Please see attached Doc)
++++- Frank blood with whitish tinge of milk Fig4. (Please see attached Doc)
Grading of milk was compared with the pH of milk from the affected quarters to qualitatively identify the severity of mastitis. Milk citrate content was determined by the method of White and Davies (1963) quantitatively before and after the treatment. Based on the appearance, consistency, pH and citrate content of milk were the main criteria for the diagnosis of mastitis and treatment with tri-Sodium citrate.
The treatment consisted of 12gm or 30gm of tri-Sodium citrate dissolved in 250ml of water daily as drench till recovery. No other treatment as antibiotic etc., was given. The consent of the owner of buffaloes affected with mastitis was obtained before the initiation of the treatment with tri-Sodium citrate.
RESULTS AND DISCUSSION
Table 1 presents the data on the effect of 12 gm tri-Sodium citrate treatment, given orally, on various parameters of milk before and after treatment in mastitis affected buffaloes. It was observed that lowered citrate content was restored to normal levels after recovery. There was relative consistent lowering of udder milk pH of the affected quarters which came down to ~6.50 as the consistency of milk was also restored to normal at recovery which occurred within 4-13 days after the treatment. The treatment with 30gm of tri-Sodium citrate orally reduced the recovery period which was recorded to be 3-5days. Following treatment with both the dose levels there was rapid improvement in the levels of total proteins, lactose and fat (data not shown).
The organisms isolated from different milk samples were: Staphylococci, Streptococci, E coli, Bacilli and Klebsiella. The treatment also reduced the number of colonies in the culture at different dilutions of mastitic milk (table1). The treatment with tri-Sodium citrate proved very effective in controlling clinical mastitis in affected quarters. The treated animals did not show any untoward side effects.
MOST PROBABLE CAUSE(S) OF MASTITIS
Thus far, the most common causes of mastitis in dairy animals have been primarily imputed to the infectious agents (Zhao and Lacasse 2007). On the basis of infectious causes of mastitis a procession of drugs purported to be effective against these culprits emerged on the scene for controlling this ailment in dairy animals. In the beginning these drugs appeared specious. However, continual use of these chemicals proved palliative and confronted with biggest problem of drug resistance and milk and meat residue dangers for humans (Costa and others 1997). Moreover, the effectiveness of these antimicrobials was rarely more than 50% to control mastitis in dairy animals (Deluyker and others 2005). Different management practices e.g., dry-cow therapy, teat dipping, hygienic measures etc., were evolved to alleviate effects of this formidable problem, but the devil of mastitis is still starring and unrelenting. Nevertheless, delving disparately into the milk synthesis the mechanisms of injury to the parenchymatous tissue of the udder appears to becoming a bit clearer.
It has been widely demonstrated that citrate is the "harbinger of lactogenesis" (Peaker and Linzel 1975). They further reported that the level of citrate in udder of cow, goat and women shoots up 46 times around parturition. These findings enthuse one to speculate that citrate is apparently playing a pivotal role in milk synthesis and might be associated with mastitis in dairy animals.
It has been reported extensively that mastitic milk is significantly low in citrate (Oshima and Fuse 1981). Our investigations have also revealed that citrate levels are very low in milk of quarters affected with mastitis (33.71mg/ 100ml). A certain minimum concentration of citrate is essential for the normal synthesis of milk in the alveoli in the udder. Therefore, any in the citrate content would result in faulty synthesis of milk in a particular quarter(s) of the udder. We have observed that the affected quarters had very low concentration of citrate as compared with healthy quarters of the same animal (Dhillon and others 1989). The deficiency of citrate in a particular quarter may be due to nutritional, metabolic or some other intrinsic unknown factors which need further investigation.
The literature extant on mastitis have immensely revealed that mastitic milk is alkaline.The normal pH of milk in udder is ~6.50 which does not appear to be congenial for the growth of commonly isolated organisms from mastitic milk (Cruickshank and others 1970). Moreover, the philosophical postulation of invasion by environmental organisms through the teat canal and establish infections in udder seems untenable due to the presence of mechanical, chemical and immunological defense barriers throughout this route (Sordillo and Streicher 2002). Also it has been demonstrated histologically that 3.1% of samples, collected from the udders of slaughtered cows, from which microorganisms were isolated, did not show any histological changes (Benites and others 2002). Frost and others (1980) reported minimal damage to alveolar tissue after "moderate"cases of mastitis induced experimentally with E coli. Furthermore, the infectious cause of mastitis gets eclipsed by the studies of Newbould and Neave (1965) who could not establish 100% infections in udder through deliberate intra-mammary infusions with Staphylococcus aureus cultures. Several cases of clinical mastitis in bovines from which no infectious organism was isolated are on record (Wanasinghe and Frost, 1979; Bramley and others 1981; Gonzalez and others 1988).
Citrate, indeed, is the main constituent of the buffer system responsible for the maintenance of pH (~6.50) in the udder and regulates the homeostasis between Ca2+ and H+ ions and is the mainstay for the fluidity of milk through its effect on casein micelles (Faulkner and Peaker, 1982; Shennan and Peaker 2000). Citrate in udder also ensures the sequestration of soluble Ca2+ in milk (Kon and Cowie, 1961) and there is significant synchronization between the two (Holt and Muir, 1979). Hence, deficiency of citrate in udder would lead to the "clumping" of Ca2+ which manifest as flakes in the mastitic milk. These flakes of Ca2+ behave like lime and probably injure the parenchymatous tissue in the udder alveoli due to reduced moderator effect of citrate. Following this injury the impermeable barriers to citrate in both directions between blood and milk is disrupted and the inflammatory reaction sets in leading to an array of subsequent events. Such injuries due to free Ca2+ has been reported in myocardium (Fleckenstein and others 1974 ; Singal and others 1979). It has also been recorded that a calcium-dependant endonuclease is associated with necrotic type changes in tissues (Arends and others 1990). Furthermore, important ions e.g., bicarbonate, chloride, sodium etc., transudates from blood into milk during mastitis due to permeability of tight blood-milk barriers. Thus, swapping of ions between blood and milk brings the pH of milk equal to that of blood or even higher and changes the pH of udder towards alkalinity i.e., 7.4 or higher. The lesions inflicted by free Ca+2 and most conducive alkaline milius in the udder prompts the environmental pathogens (commensals in the udder?) to invade and establish clinical/subclinical "infectious mastitis". Consequently pathogenesis is further exacerbated with the involvement of body defense mechanisms and severe inflammatory reaction ensues (Zhao and Lacasse 2007).
We have also reported that lactose, total proteins and fat are substantially lowered in mastitic milk. However, these constituents in milk increased markedly on recovery after tri-Sodium citrate therapy ( Dhillon and others 2000, Singh and others 2007). The increment in fat was spectacular (190%) because citrate plays an indirect role through NADPH de novo synthesis of fatty acids in the mammary gland (Garnsworthy and others 2006). There was significant reduction in the number of bacterial colonies after treatment with tri-Sodium citrate (Dhillon and others 1995). These observations substantiates that this treatment is radical and works at the root cause of mastitis resulting in remarkable cure of the malady without producing any side effects.
Altogether the findings of above investigations it can be concluded that the initial lesion in the pathogenesis of mastitis is caused by the disturbed homeostasis of citrate and Ca2+ in the udder. On the basis of this hypothesis we treated the clinical cases of mastitis in buffaloes by administering 12gm and 30gm of tri -Sodium citrate in 250ml of water daily as a drench. Similarly several workers have treated acute and/or sub-acute cases of mastitis in buffaloes with excellent results. They also compared it with other antimicrobials and reported that tri-Sodium citrate was superior as far as the restoration of normal pH and other constituents of milk in the udder was concerned (Yousaf and others 2010; Prakash and others 2010).
The treatment of mastitis with this salt has been standardized by enhanced doses to cut-short the recovery period (Dhillon and others 2007). The oral dose has been raised to 30gm in 250ml of water daily as a drench and recovery period cut-short to 3-5 days depending upon the severity of mastitis (Singh and others 2007). The disruption of the impermeable barrier between blood and milk in udder, as stated above, formed the basis of intravenous administration of tri-Sodium citrate. The dosage of this salt was standardized in cow-calves. Intravenous administration of medication directly reaches at the site of injury and normalizes the pH (~6.5) in the udder and the infectious agents are scavenged off and restoring ionic equilibrium. Moreover, the slightly acidic pH in normal udder is not suitable for generally isolated microbes from mastitic milk. With intravenous administration of tri-Sodium citrate in sterilized normal saline as 5% given morning and evening in 50ml doses the recovery period shortened to 1-3 days (Dhillon and Singh 2009, 2011). This treatment was safe, economical, very effective, avoided culling and discarding of milk with the minimal pain to the animal. Moreover, there is no withdrawal periods and hazards from residual problems in milk and meat. Presently, tri-Sodium citrate is employed extensively for the control of mastitis in dairy animals at farms with remarkable success. This treatment has also been found to be very effective in cases of mastitis refractory to antibiotics. On the basis of our investigations some pharmaceuticals have come-up with formulations intended for prevention and treatment of mastitis in dairy animals.
ARENDS, M. J., MORRIS, R.G. & WYLLIE, A. H. (1990). American Journal Pathology 136, 593
BENITES, N. R., GUERRA, J. L., MELVILLE, P. A. & da Costa, E.O. (2002). Journal Veterinary Medicine B 49, 366
BRAMLEY, A. J., GODINHO, K. S. & GRINDAL, R. J. (1981). Journal Dairy Research 48, 379
COSTA, E. O., GARINO, F., WATANABE Jr E. T., et al. (1997). Brazil. Proceedings 5th World Buffalo Congress, Caserta, Italy. 635
CRUICKSHANK, R., DUGUID, J. P. & SWAIN, R. H. A. (1970). Medical Microbiology: A Guide to the Laboratory Diagnosis and Control of Infection. The English Language Book Society and E & S Livingstone ,pp 39,101.
DELUYKER, L. A., VAN OYE, S. N. & BOUCHER, J. F. (2005). Journal Dairy Science 88, 604
DHILLON , K. S. & SINGH, J. (2009). Veterinary Record, 8th August, in Letters.
.....................&................ (2011). Proceedings 30th World Veterinary Association Conference, Cape Town, Oct. 10-14, pp 42
..................,..............., & GILL, B. S.(1989). Journal Research, Punjab Agricultural Univ., 26, 477
..................., SINGH, S., VARINDRA & SINGH, T. J. (2000). Indian Journal Dairy Science 53, 32
...................., SINGH, T. J., SODHI, S. S., SANDHU, H. S., DWIVEDI, P. N., SINGH, J. & GILL, B. S. (1995). Indian Journal Animal Science 65, 9
....................,KUMAR, H., DHALIWAL, B. B. S., BAL, M. S., PANNU, M. S. & SINGH, J. (2007). PunjabVeterinary Journal 5, 55
FAGIOLO, A. & LAI, O. (2007). Italian Journal Animal Science 6(Suppl, 2), 200
FAULKNER, A. & PEAKER, M. (1982). Journal Dairy Research 49,159
FLECKENSTEIN, A., JANKE, J., DORING, H. J. & LEDER, D. (1974). Myocardial fiber necrosis due to intracellular Ca++ overload-a new prinicipal in cardiac pathophysiology. In: Recent Advances on Cardiac Structure Metabolism. 4:. Edited by N.S.Dhalla, University Park Press, Baltimore,pp. 563-580.
FROST, A. J., HILL, A. W. & BROOKER, B. E. (1980). Proceedings Royale. Society London B Biology Science 209, 419
GARNSWORTHY, P. C., MASSON, L.L., LOCK, A. L. & MOTTRAM, T. T. (2006). Journal Dairy Science 89, 1604
GONZALEZ, R. N., JASPER, D. E., FARVER, T. B., BUSHNEL, R. B. & FRANTIC, E. (1988). Journal American Veterinary Medical Association 193, 323
HOLT, C. & MUIR, D. D. (1979). Journal Dairy Research 46:433
HYVONEN, P., HAARAHILTUNEN, T., LEHTOLAINEN, T., HEIKKINEN, J., ISOMAKI, R. & PYORALA, S. (2010). Journal Dairy Research 77, 474
KON, S. K & COWIE, A. T. (1961). Milk: The mammary gland and its secretion. Vol. II Academic Press, New York and London, pp 216.
NEWBOULD, F. H. S. & NEAVE, F. K. (1965). Journal Dairy Research 32, 171
OSHIMA ,, M. & FUSE, H. (1981). Journal Dairy Research 48: 387
PEAKER, M . & LINZEL, J. L. (1975). Nature; 253: 464.
PRAKASH, V., YADAV, M. P. S. & SINGH, S.P. (2010). Asian Journal Animal Sciences 4,202
SHENNAN, D. B & PEAKER, M. (2000). Physiological Reviews 80, 925
SINGAL, P. K., MATSUKUBO, M. P. & DHALLA, N. S.(1979). British Journal Experimental Pathology,60: 96
SINGH, J., KAUR, K., MEENAKSHI., ALKA., VERMA, S., KUMAR, H & PANNU, M. S.(2007). Indian Journal Veterinary Medicine 72, 133
SORDILLO, L. M & STREICHER, K. L. (2002). Journal Mammary Gland Biology Neoplasia 7, 135
WHITE, J. C. D. & DAVIES, D. T. (1963). Journal Dairy Research30, 171
WANASINGHE, D. D & FROST, A. J. (1979). Australian Veterinary Journal 55,374
YOUSAF, A., SARFARAZ, I., ZAFAR, M. A., RAO ZAHID, A., HUSSAIN, A. & MANZOOR, A. (2010). Effect of treatment with tri-sodium citrate alone and in combination with Lavamisol Hcl on total bacterial count in Dairy Buffalo suffering from sub-clinical mastitis. Proc. 9th World Buffalo Congress, Buenos Aires, April 2010., pp 187-189. ( Invited Paper).
ZHAO, X. & LACASSE, P. (2007). Journal Animal Science 86,57
Table 1 Effect of mastitis on different parameters of milk from buffaloes before and after Treatment with tri-sodium citrate given daily @12gm in 250ml of water as drench