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Mastitis in buffalo

Pathobiology, etiology and novel mastitis therapy in buffalo

Published: January 27, 2012
By: K. S. Dhillon, Ph.D and Jasmer Singh, Ph.D (College of Veterinary Science GADVASU)
Abstract            
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. 
INTRODUCTION
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 others2010). 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.
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 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  
Pathobiology, etiology and novel mastitis therapy in buffalo - Image 1
Pathobiology, etiology and novel mastitis therapy in buffalo - Image 2
Pathobiology, etiology and novel mastitis therapy in buffalo - Image 3
Pathobiology, etiology and novel mastitis therapy in buffalo - Image 4
Pathobiology, etiology and novel mastitis therapy in buffalo - Image 5
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Jasmer Singh
11 de septiembre de 2019

Hi Dr. Patil, Such cases does occur as peracute mastitis in almost all animals.
Pl record the pH of the milk, and physical appearance as to any clots, blood in milk, pain in udder and any other sign(s). Pl inform me such details of the case as to the stage of lactation any history of previous attacks and, if any, other which you might find.

Jasmer Singh.

Jasmer Singh
12 de mayo de 2015

Hello Dr. UD and Um,

As it is known for general information, Citrate is used as an anticoagulent in blood for transfusion @ three gms/Unit of blood and one unit of blood is equivalent to 450ml. When we use Trisodium citrate to treat mastitis it is used as 5% sterile sol. and as 50ml as one dose at a time and just imagine how much volume of blood is there in an adult cow or a buffalo and the dilution of citrate thereof. I think you got the answer.

Jasmer Singh Ph D

Umberto Francesa
12 de mayo de 2015
I am still wondering about the same question that Dr. Udhayashankar, posted almost a year ago. Umberto Francesa, DVM
Jasmer Singh
20 de septiembre de 2013

Hello Dr Habib, Did not hear from you for quite long. I am to request you for one favour, I need some data on the pH values of mare/jennies milk at different stages of lactation and any incidence of mastitis and its treatment. You can try this treatment with TSC at the same dose as in cow/buffalo. Furthermore, you can also record pH of pregnant mare teat secretions to identify the time of foaling. Also tell me about other cases of mastitis you have treated with TSC. Pl see the cases which are refrectory to TSC orally you should try I/V 5% TSC for better results.
Cieo Jasmer

Jasmer Singh
3 de junio de 2013
Hello Dr Habib, Thanks a lot for your reply. There are no side effects of TSC as the recommended dose. You can give 5% TSC in sterilized narmal saline I/V in some refrectory cases. You can give 50ml morning and evening doses as I/V and than orally, if required. Please intimate me the pH values and I would give you some more information regarding its prognosis. with regards Jasmer
Jasmer Singh
2 de junio de 2013
Hello Dr Habib, As you know citrate is an essential component of Tricarboxylic acid cycle and is obligatotry for the production of energy throughout the body for different systems to cope with sustenance of life. As pH is very important factor in maintaining various catalytic reactions in which citrate plays crucial roles, hence, its role in maintaining pH in different systems need not be overemphasized. I would request you to record pH of milk or any other secretion from udder on the spot, with graded pH paper strips. This procedure is very cheap, easy and reliable. Furthermore, if possible measure the citrate content of milk at different times of lactation and whenever there is increase in pH just give TSC @ 6gm for 3-5 days. Citrate can also be used as a preventative on dairy farms. Pl do communicate for further trials and any information. with regards Jasmer
Jasmer Singh
30 de abril de 2013

Hi Dr Shahnawaz, I would request you to use tri-Sodium citrate orally as well as I/V after recording the pH of milk on the spot with graded pH papers which may be procured from shops dealing with labortory chemicals in your area. I/V use of this salt hastens the recovery within usually 1-2 days. Morover, this treatment is most successfull against mastitis cases resistant to in vouge antibacterial drugs used to control mastitis. We have used this treatment not only in cow or buffalo but in other animals like goat/sheep successfully. The basic fact is that the process of milk secretion is remarkably similar in almost all the mammals (including humans) though the biochemical composition may vary slightly in different species.Therefore, any aberration in the synthesis of citrate in milk producing units-alveoli and lobulles would result in mastitis and other complications. Estimation of citrate in milk shall prove a boon to control mastitis prevalence on any established dairy farm of some standing with large number of quality animals. You or any of your colleagues can share results with me of this treatment with me for further discussions.
with regards Jasmer

Jasmer Singh
29 de abril de 2013

Hello Mr Mehta, You appear to be quite a progressive dairy farmer and I would advise a bit more. Mastitis is primarily due to deficiency of Citrate synthesis in udder which result in formation of flakse in the milk that behave like lime and cause injury to the secretory epithelium. Injury leads to inflammation and subsequent damaging effects in the udder. Administeration of tri-Sodium citrate @ six gms dissolved in about 250 ml of drinking water as a drench for 3-5 days cures the disease. tri-Sodium citrate can be procured from shops dealing in chemicals from your neighbouring areas. You should record pH of milk of affected animals and healthy ones to monitor if there is any chance of developing mastitis and preventive doses of tri-Sodium citrate can be given. Any further enquiry which you may need please don't hesitate to contact me and share the results of your implementation of this process at your farm. By the way how many dairy animals-cows and buffaloes are you maintaining at your farm/
With regards Jasmer

Jasmer Singh
2 de abril de 2013

Hello Dr Eman, Many people are working on these studies that appears to be of academic importance. However, in my opinon we should aim at investigations which could be of some use in the field for controling diseases of economic importance.

Jasmer

Jasmer Singh
9 de enero de 2013

Hello Mr Ceddon/Abbas

Citrate plays a very crucial role in the udder to maintain normal pH of milk i.e., ~ 6.50 (slightly acidic). At this pH the organisms usually isolated from mastitic milk can not grow and multiply. Citrate also sequester Calcium in udder and preserves its fluidity. Whenever there is disturbance in the synthesis of citrate in udder calcium ions become free which clumps together and form flakes that mocks lime and cause injury to the sorrounding tissues. The 'tight junctions' between secretory epithelium of udder become compromised due to swelling in the injured area resulting in swapping of ions like Na, K, Cl, Hco3 and citrate between blood and milk thus bringing the pH of udder equal to that of blood i.e., 7.40 or even higher (alkaline). Presence of injury caused by calcium clumps and alkaline pH at this site provides most conducive enviroments for the environmental organisms like Escherchia coli, Staphy, Strepto., etc., The inflammatory reaction attracts body defense cells particularly PML etc., through the leaky tight junctions and the situations becomes explosive depending upon the gravity of the injury. When tri-Sodium citrate is administered orally or I/V the normal environments of udder is restored and the infectious organisms are scavenged off and recovery is achieved.
Regarding estimation of citrate in milk I have read that some firm in Germany has marketed a kit for this purpose or otherwise some laboratry doing this job can be contacted.

with regards Jasmer

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