Postpartum Complications in the Buffalo

Parturition is considered a clean contaminated event. Problems following parturition such as retained placenta [1,2] , endometritis [3,4] and genital prolapse [5] have negative effects on the ovarian function of postpartum buffalo cows whereas problems such as metabolic disorders [6] and mastitis result in lowered overall production [7]. The immediate few days or weeks postpartum can be particularly problematic in cattle which readily develop diseases related to placental expulsion, metabolism, mammary glands and reproductive tract [8] and similar problems may appear in the buffalo. The complications of the reproductive tract during the postpartum period include hemorrhage, vulval hematoma, lacerations, reproductive organ (vagina, uterus) rupture, perineal injuries, distal displacement of pelvic organs (vagina, uterus, urinary bladder, perivaginal fat) and retained placenta [9] whereas those of metabolism include milk fever, hypophosphatemia and ketosis [10]. The complications of mammary gland include conditions such as udder edema, hemolactia, hypogalactia and mastitis [11]. Some of these problems can arise even after a normal parturition while others follow an abnormal parturition. Parturient injuries of the reproductive tract are less prevalent in the buffalo [9] probably due to a lower frequency of improper cervical dilation even in buffaloes suffering from dystocia [12,13]; the same is true for metabolic disorders [6,10] and mammary disorders [11].

Season affects the incidence of reproductive disorders [14] and similar effects are observed in relation to body weight changes at parturition (negative energy balance) [15] and agro-ecologic location [16]. There is a significant effect of management practices on the incidence of postpartum reproductive disorders [17]. Body condition score at calving can affect the incidence of calving disorders in buffaloes [18]. Pluriparous buffaloes with a medium condition score show the least incidence of disorders compared to thin and fat primiparous and pluriparous buffaloes. The period of calving and parity have a significant effect on postpartum complications such as pyometra and endometritis [19-21]. Similar effects of environmental temperature were recorded in the buffalo [22].

Different research analyses/surveys have mentioned the relative frequency of reproductive tract disorders during the postpartum period (Table 1) in buffaloes [20], however, disorders related to metabolism and the mammary gland are usually not mentioned in these reports.

The postpartum complications of the reproductive tract such as retained placenta and uterine infections have been described in previous chapters [27,28] and likewise those involving metabolism have been previously described [6]. In this chapter, the authors briefly describe the postpartum complications related to the reproductive tract (hemorrhage, rupture, distal displacement, retained placenta, genital infections) and metabolism whereas the mammary gland pathologies are described in detail.

1. Reproductive Tract Complications

Postpartum reproductive tract complications include hemorrhage, trauma (lacerations, perineal injuries), distal displacement of the reproductive tract (prolapse), retained placenta and genital infections.

1.1. Postpartum Hemorrhage

Postpartum hemorrhage may be due to trauma, lacerations or tearing of tissues. Hemorrhage may be due to injury by forceful handling of a retained placenta (Fig. 1) or because of intrauterine manipulations [29]. Hemorrhages were observed in Bulgarian Murrah buffaloes suffering from dystocia and maintained on slippery floors [30]. Some degree of postpartum hemorrhage is inevitable, however, profuse expulsion of fresh blood 24-36 h following parturition, is potentially serious and usually a result of breakage of maternal cotyledon, blood vessel or trauma to the birth canal subsequent to dystocia handling [29]. Prompt attention to such a condition is necessary to avoid development of shock and eventual death of the animal. It is suggested to inspect the birth canal using a vaginoscope or vaginal speculum to locate the traumatic lesion and ligate the bleeding points if possible. Under serious conditions of blood loss through the birth canal, introduction of ice or infusion of cold normal saline along with epinephrine is suggested. Administration of 20-50 U of oxytocin IM or IV and IV infusion of 250-500 mL of calcium borogluconate is often helpful. Severe blood loss rapidly results in shock with a weak pulse, cyanotic mucus membranes, cold extremities, increased respiratory rate and decreased urinary output being clinically evident [29]. It is suggested to administer fluid replacements with hemostats, corticosteroids and antibiotics. The administration of iron-dextran [31] and iron-sorbitol citric acid complex [32] has been suggested for hemorrhagic complications.

Cervical bleeding is expected in cases of cervical dilation failure; large blood vessels of the birth canal may rupture due to forced traction. Cervical bleeding can be prevented by local pressure, cold fomentation and ligation. Retraction of the cervix is suggested for ligation of large blood vessels.

Figure 1. Severe postpartum hemorrhage in a parturient buffalo with genital prolapse immediately postpartum.

1.2. Hematoma/Edema

Hematomas of the birth canal or vulva are rare in buffalo. They are usually left untreated with overall supplementation of anti-inflammatory and analgesic drugs [29]. These local injuries are absorbed generally within 5 to 10 days. However, if too large and cumbersome, they can be incised evacuating clots and suturing the site. Vulvar edema is the result of dystocia handling or a consequence of truss application for prevention of a vaginal prolapse (Fig. 2). The edema usually resolves spontaneously, however, cold fomentation, diuretics and anti-inflammatory drugs are suggested for 3-5 days if the edema is extensive. Vulvar abscess (Fig. 3) in buffaloes can occur a few days after parturition and can be treated by the administration of antibiotics or by surgical excision.

Figure 2. Severe vulvar edema in parturient buffaloes.

Figure 3. Vulvar abscess in a parturient buffalo.

1.3. Lacerations of the Birth Canal

Vulvar lacerations can occur due to forceful extraction of a fetus from a buffalo without vulvar relaxation (Fig. 4, Fig. 5). However, they are uncommon in the buffalo. Extensive lacerations require careful suturing whereas minor lacerations heal spontaneously with little care.

Figure 4. Vulvar laceration in a parturient buffalo.

Figure 5. Vulvar laceration in a parturient buffalo.

1.4. Perineal Injuries

Although parturient perineal injuries are uncommon in the buffalo, there are two recent reports [33,34] and a previous study that described a utero-rectal fistula [35] due to the delivery of a fetus per rectum [36] and similar rare cases have been observed elsewhere (Fig. 6). The low frequency of such injuries in buffalo appears to be on account of proper relaxation of the birth canal at parturition [37] and a less forceful birth. Perineal hernia has also been recorded in the buffaloes [38-42]. Experimental exteriorization of the perineum [43] and repair of perineal lacerations [33,34] and perineal hernia have been described [38-42]. The surgical procedures for repair of perineum are delayed for at least 2 months post-calving except when they are minor degree and can be repaired immediately. The anesthesia for such procedures adopted is epidural infiltration anesthesia [34] and some experience to carry out these procedures is necessary.

Figure 6. Recto-vaginal fistula in a parturient buffalo leading to delivery of the fetus per rectum.

1.5. Nerve Damage and Paralysis

Reports on the nerve paralysis subsequent to calving are not available for buffaloes. It appears that, because of sufficient dilation of the birth canal at parturition in the buffalo [13], a spacious pelvis [12] and other anatomic differences, nerve damage during parturition is less likely. The ischium bone in the buffalo forms an angle of 70 degrees with the horizontal in buffalo compared to 50 degrees in cows [44]. The tuber ischii is large and "y" shaped in buffalo [44]. The distance between the acetabulum and tuber coxae, and between the tuber ischii and acetabulum is nearly equal (with difference of 4-5 cm) in cattle, whereas in buffaloes the difference of 8-10 cm exists [45]. The 5th sacral vertebra is loosely attached with the 4th sacral vertebra in buffalo whereas in the cow the 4th and 5th sacral vertebra are fused [45,46]. The body of the first sacral segment is very wide and is more flattened dorsoventrally in the buffalo [45]. Collectively these variations result in significantly greater transverse and sacropubic diameters, pelvic outlet, and pelvic inlet, vertical and diagonal pelvic diameters in the buffalo compared to cows [44] and thus there is less likelihood of parturient nerve damage in the buffalo.

1.6. Rupture of Pelvic Organs

Rupture of genital organs can occur as a result of dystocia handling or rarely spontaneously. The vagina, cervix and the uterus can rupture during or following parturition.

1.6.1. Vaginal and Uterine Rupture

Vaginal rupture has been sporadically reported in buffaloes [47-53]. Vaginal ruptures are sometimes associated with herniation of urinary bladder [53] and intestines [37,51,53,54]. Minor parturient vaginal ruptures may develop into inflammatory vaginitis and could heal with care. Extensive ruptures require suturing and application of emollient creams. For the more severe cases, reconstructive surgery is indicated [55]. Prolapse of abdominal organs through vaginal ruptures are difficult to repair surgically. A vaginal prolapse with a concurrent rectal prolapse has also been recorded in a buffalo [56]. A rare case of vaginal rupture following uterine torsion with subsequent intestinal prolapse has been recorded in a buffalo [57].

Uterine ruptures are usually the result of dystocia, uterine torsion and monstrosities [58-61]. A rare case of cervical rupture post-calving has been recorded in a buffalo [62]. A spontaneous uterine rupture with displacement of the fetus into the abdominal cavity was also recorded in a buffalo [63,64].

1.7. Postpartum Genital Prolapse

The extroversion of vagina/uterus beyond the vulvar labia is known as prolapse [65]. The basic reason for the prolapse of genitalia appears to be weakening or relaxation of constrictor vestibule muscles and the vulvar lips and atony of vaginal musculature [37]. Many case reports on postpartum uterine prolapse (Fig. 7) have appeared in the literature [5,54,66-79]. Similarly cervico-vaginal prolapse (Fig. 8) has also been recorded during the postpartum period [56,75,80-90] in buffaloes. The prolapse of the urinary bladder with retention of urine has rarely been recorded in buffaloes [91].

Figure 7. Complete uterine prolapse in parturient buffaloes.

Figure 8. Cervico vaginal prolapse in parturient buffaloes.

The overall incidence of genital prolapse in one study in Pakistan was 7.73% [88], whereas in Bulgarian Murrah buffaloes the incidence of uterine prolapse was 14.58% [30]. The relative incidence of uterine and vaginal prolapse in buffaloes in India was 19.27% and 80.73% respectively [92]. Data on 776 calvings revealed that postpartum vaginal prolapse was less common compared to prepartum prolapse in buffaloes [93]. The incidence of prolapse was significantly affected by the size of the dam and calf, length of gestation and gender of the calf [93]. It was found in Bulgarian Murrah buffaloes that dams with lower body weight before and after calving were more likely to suffer from uterine prolapse even if they bore an average sized calf [94]. Prolapse is rare in South America and Egypt whereas its incidence increased in Italy due to intensive management and feeding practices [65]. Deficiencies of serum calcium, phosphorous and other minerals are considered to increase the incidence of prolapse in buffaloes [95,96].

Deficiency of selenium was considered one probable reason for prolapse in buffaloes [97,98]. Uterine prolapse usually develops up to 48 h postpartum and the highest occurrence was recorded within 24 h of calving [30] probably due to severe uterine contractions at this time. The body shape and the shape of the pelvis as predisposing factors for prolapse were addressed in a previous study on Mediterranean buffaloes [65]. Animals with prolapse sometimes develop urinary infections [84]. Cervicotomy and trachelorrhaphy has been suggested to treat irreducible uterine prolapse in buffaloes [99] and likewise cervicopexy has been suggested for fixation of the prolapse [100].

There are strong indications that the prolapse of genitalia is a heritable trait in buffalo and repeatability of the trait is reported to be 0.424 [101]. The prepartum vaginal prolapse in buffaloes has been dealt with in a previous Chapter 13 of this Book; Maternal Complications of Gestation in The Buffalo: Etiology, Antenatal Diagnosis and Management [37] and further details of genital prolapse would be discussed in a separate Chapter 20 of this book Genital Prolapse in the Female Buffalo [102].

1.8. Placenta Retention

Retention of placenta (Fig. 9) appears to be the most frequent postpartum complication among buffaloes [7,23,25,26]. Delay in placental expulsion beyond six hours of fetal birth is considered retention of placenta [29], however, the range of placental expulsion in buffaloes varies from 30 min to 8 h [27,103]. Separation of placenta depends upon dilatation of maternal caruncular crypts and shrinking of fetal chorionic villi. The placenta plays a vital role during pregnancy but should be expelled subsequent to fetal delivery. The incidence of retention of placenta is 80% due to failure of separation of fetomaternal junctions and 20% due to lack of myometrial contractions [29]. The incidence of placental retention varies from 3% to 35.8%. If a farm has an incidence higher than 7% of placenta retention cases then they should look into their management practices. Lower glucose and calcium blood levels and other minerals have been mentioned as the predisposing factors for placenta retention [104-107] as well as deficiencies of vitamin E and selenium [108,109]. More detailed information on this subject can be found in Chapter 19 on Etiopathology and Therapy of Placental Retention and Postpartum Uterine Infections in the Buffalo in this book.

Figure 9. Parturient buffaloes with retained placenta.

1.9. Genital Infections

Genital infections are common during the postpartum period. The incidence of postpartum metritis varies from 6.98% to 25.22% [7,23,24,26,110]. Buffaloes with genital rugae of the vulva (Fig. 10) and loose vulva (Fig. 11) are more prone to developing postpartum genital infections. Other infections that occur within a few weeks of parturition include endometritis and pyometra (Fig. 12). More detailed information on this subject can be found in Chapter 19 on Etiopathology and Therapy of Placental Retention and Postpartum Uterine Infections in the Buffalo in this book and Chapter 24 on Genital Tract Affections in the Female Buffalo in this book.

Figure 10. A parturient buffalo with extensive vulvar rugae.

Figure 11. A buffalo with a loose vulva.

Figure 12. A buffalo with pyometra. The pus discharge can be seen at the vulvar lips.

2. Metabolic Complications

Metabolic disorders are uncommon in the buffalo. A recent study analyzed the data of 10 years of a referral center and an organized buffalo farm. The incidence of milk fever, ketosis and parturient hemoglobinuria was 2.60%, 16.59% and 20.2% respectively for 729 buffaloes presented to the referral center during 2004-2013. However at the organized farm (Surti buffaloes), the analysis of data for 529 calvings (2001-2011) revealed no incidence of any of these metabolic disorders [10]. The study also reflected that with proper management, buffaloes seldom develop metabolic disorders. Hypomagnesemia is rare in the parturient buffalo [6]. When it does occur, it seems to be due to a transitory decline in plasma concentrations of calcium and phosphorous at parturition in buffaloes [111,112], whereas the concentration of magnesium is only slightly decreased. More details on the various metabolic disorders in the buffalo can be found in Chapter 21 Metabolic Disorders in The Parturient Buffalo. Two recent studies suggested the incorporation of blood transfusion along with sodium acid phosphate for therapy of parturient hemoglobinuria in buffaloes [113], and the use of anabolic steroids for the management of ketosis in buffalo [114]. Buffaloes with both poor (Fig. 13) and fatty body condition scores are more prone to development of metabolic disorders.

Figure 13. Extremely poor body condition score in a parturient buffalo.

3. Complications of the Mammary Gland

Mammary gland pathologies such as clinical mastitis are not a frequent disorder of parturient buffaloes [7]. The low incidence of mammary gland pathologies may be due to the anatomic and physiologic differences of buffalo compared with cattle [115]. The disorders recorded for buffaloes include hemolactia [116,117], udder edema [118], hypogalactia [119] and mastitis [120]. Mastitis has been widely recorded in buffaloes at nearly all geographic locations with incidence ranging from 2% to 25.12% for clinical and 1.7% to 48.9% for subclinical mastitis and Staphylococcus spp. are the most frequent causative microbes [120]. Diagnostic approaches for clinical and subclinical mastitis in buffalo utilize the same tools as for cattle and somatic cell counts are the most frequent test used in buffaloes. In this chapter the parturition related mammary gland pathologies described include hemolactia, udder edema, hypogalactia and mastitis.

3.1 The Bubaline Mammary Gland and Milk Secretion

The bubaline mammary gland appears in prenatal fetuses at 90-109 days of pregnancy and comprises 4 mammary anlages (a mammary anlage is the first discernible group of cells destined to become the mammary gland) with a centrally located sprout embedded into mesenchymal tissue [121]. These develop into fully differentiated duct systems of the mammary gland at 251-254 days of gestation [121]. At birth the mammary gland consists of a small teat with a gland cistern and extensive duct system which develop progressively during the prepubertal period with significantly higher glandular tissue being formed at lactation [122]. Before parturition, under the influence of hormones and other factors, the bubaline mammary glands enlarge and are fully functional and developed before parturition [123]. During early lactation the cellular junctions in the mammary alveoli are 19.78≥0.99 nm wide and 174.16≥22.36 nm long and the gap junctions between external and internal nuclear membranes are 36.39≥1.75 nm in fully lactating cells [124].

The teat dimensions and milk outflow in buffaloes is similar to cattle yet there are subtle differences in some aspects of morphology and function. In Murrah buffaloes the fore and hind teat length ranged from 5 to 14 cm and 8 to 16 cm, respectively [115]. The teat girth (thickness) ranged from 7 to 14 cm and 8 to 16 cm, respectively [115]. Upon milk ejection there was more than a 10% increase in the teat length and teat girth [115]. Buffaloes had a mean total cisternal area (teat and gland) of around 22 cm2 for a single quarter which is less than half of what is seen in cows (40–45 cm2) and the cisterns were significantly larger in early compared to late lactation. The cavity area in the teat and gland regions was similar [115]. The teat canal length in buffaloes was around 3 cm and was much longer compared to the 0.5 to 1.5 length reported in cows [115]. Comparative abattoir studies in cattle and Egyptian buffaloes indicated that teat canals were 30 to 40% longer in buffaloes compared to Holstein cows [125]. It has also been recorded that the epithelial thickness of the buffalo streak canal was about 10% greater and the thickness of the sphincter was 13% greater compared to crossbred Holstein cows [122,126].

The arterial blood supply of lactating bubaline mammary glands arise from paired external pudic arteries that branch into cranial and caudal mammary arteries with additional caudal mammary arteries in high yielding buffaloes [127].

Figure 14. The bubaline mammary glands.

In the buffalo cisternal milk fraction was lower than that reported in cattle and goat (20 to 40%) and was only 5% of the total milk [115]. There was a close correlation between the cisternal area measured using ultrasound and measuring the cisternal milk yield [115]. During milk ejection, the cisternal area in the ultrasound cross sections increased significantly [115]. Buffalo teats were flaccid and empty due to the small cisternal fraction of milk prior to milk ejection, while during milk ejection; there was a remarkable increase in teat dimensions [115].

Studies have indicated that buffaloes store only a small fraction of milk in the cisternal cavities after a milking interval of 10 to 11 h [115]. In cows it has been demonstrated that an 8-h interval is optimum for measuring the cisternal size and cisternal fraction of milk [128]. In a previous study on the partitioning of milk in buffaloes, Aliev [129] studied different breeds, age, and stages of lactation and observed that there was almost no cisternal fraction found in buffaloes. This indicates that most of the milk synthesized between milking is stored in the alveolar lumen and the small ducts and there is very little drainage of milk into the cisternal cavities. The completeness of emptying the udder during milking of buffaloes is entirely dependent upon timing of oxytocin release in response to stimulation and its sustained release throughout the entire milking [130,131]. In buffaloes, milk ejection follows the same neurohormonal mechanism as in cattle, sheep, and goats where adequate stimulation cause an increase in blood oxytocin levels in one minute [115].

3.2. Blood in Milk (Hemolactia)

The presence of blood in milk is a state of physiological hyperemia of the mammary gland that is occasionally seen in buffaloes towards the end of gestation and for a short period just after parturition [116,117,132,133]. The presence of blood in milk normally persists no longer than 4 days. Around 30% of the buffaloes show blood in milk for 1 day only, 30% for 2-3 days and about 10% for 6 days while 30% of buffaloes develop mastitis [116]. Failure to milk out the udder may precipitate this condition at any stage during lactation [133]. During mid to late lactation failure to milk the udder invariably results in turgidity of the udder and diffuse reddening. Immediately after calving, hemolactia is usually caused by the rupture of many small congested blood vessels or seepage of blood into teat canals by diapedesis [133]. Severity varies from large clots and frank blood to a pale pink tinge of the milk [133].

The etiology of hemolactia often lies in the method of milking, feeding of certain feedstuffs, vitamin C deficiency, intrammamary infusion of irritants, chronic mastitis and some infections such as those with Leptospirosis [133].

One study examined the lactation records of 3184 lactations of buffaloes at the Military dairy farm and 2343 records of National Dairy Research Institute (NDRI), Karnal, India. The incidence of blood in milk at the military dairy farm was nil, whereas at the NDRI the incidence was 1.41% with 40% of buffaloes showing the condition during the first month of lactation [116].

Sometimes, erythrocytes in the milk are in sufficient numbers to impart a pinkish or reddish discoloration of the milk [117] resulting in a red cream and sediment. However, more frequently there is a mere trace of blood that can be demonstrated only upon centrifugation of the milk. The presence of small quantity of blood does not affect the flavor of the milk.

Extensive bleeding in the udder tissue may be due to penetrating lacerations, tread wounds, and whip injuries, horn pokes and straining of the tissues at milking [133]. As a result there are localized or widespread areas of blood diffusion into the connective tissue of the skin of the udder or in the wall of the cistern. In every instance of macroscopic abnormality observed when milking the udder, systemic and udder diseases as well as the effect of feed should be considered.

Therapy

A small amount of blood in the milk postpartum requires no specific treatment as it naturally disappears within two weeks. If bleeding is due to a slight trauma, it is not a matter of much concern. If the causative factor is feedstuff, it should be determined which is the causative agent and removed at once from the feed. Animals that pass blood into the milk should be milked out with as much care as possible avoiding intensive stripping [133]. Supportive treatment in the form of ice packs or cold-water sprays may be provided.

The extensive presence of blood in milk warrants immediate administration of hemostatics and parenteral coagulants along with intravenous administration of calcium preparations such as calcium borogluconate. Intravenous infusion of 5 mL (1%) epinephrine derivatives that produce vasoconstriction and increase the blood platelets are suggested [138]. Vitamin C preparations such as 1000 mg of ascorbic acid are suggested to be administered IM. In case of severe blood loss, fluid replacements are necessary. Homeopathic drugs Hamamelis Q and Silecea 6X administered mixed with 50 mL water at 2 h intervals orally was shown to solve the problem within 2-3 days of treatment [132]. Cold fomentations are often helpful in reducing the severity of hemolactia. Vitamin K injections are also helpful in the severe cases.

Blood in milk associated with chronic mastitis and bleeding granulation tissue first requires the control of the infection by suitable intracisternal medication followed by surgical removal of the granulation tissue. To preclude the development of ichoro-gangrenous mastitis (ichorous-watery blood tinged discharge), profuse administration of intra-cisternal antibiotics is required [133].

Milk mixed with blood is not fit for human consumption.

3.3. Udder Edema

Udder edema is a periparturient disorder characterized by excessive accumulation of fluids in the intercellular tissue spaces of the mammary gland [118]. The incidence appears to be higher in high producing dairy heifers at first calving; however, it is less frequent in the buffalo. The risk factors for this disorder appear to be obesity, high milk production, lack of exercise and longer gestation period. One recent study pointed out that udder edema in buffaloes was less severe in buffaloes fed low anionic or high anionic diets compared to those fed low cationic and medium cationic diet during the prepartum period [134]. Udder edema is a significant risk for development of mastitis, creates difficulty in milking, predisposes the teats and udder to injuries and is a major discomfort to the buffalo [118]. Edema is sometimes extensive, extending up to the navel producing difficulty in walking.

Therapeutic Management

Mild degree of edema resolves spontaneously or with cold fomentation. The more severe cases require administration of diuretics such as furosemide (1-2 mg/kg IM) and corticosteroids such as dexamethasone [118] and liver tonics [118].

Udder edema has been reported to respond favorably to homeopathic drug combinations of Phytolacca 200, Calcarea fluorica 200, Silecea 30, Belladona 30, Bryonia 30, Arruca 30, Conium 30 and Ipecacuanha 30, given orally within 2-5 days of parturition [135].

3.4. Hypogalactia

Poor milk production in parturient buffaloes has been documented [119] and is known as hypogalactia. Milk production is an interaction of various hormones, metabolites, ruminal function and environment. Buffaloes under thermal stress, low prolactin secretion, mild deficiency of calcium and phosphorous and negative energy balance during the immediate postpartum period are likely to suffer from hypogalactia. Dietary supplementation has a profound effect on milk production in buffaloes [136]. Periparturient diseases of reproductive tract such as retained fetal membranes or metritis [27] and metabolic conditions such as ketosis are likely to have negative effects on milk production [6].

Therapy

Improvement in milk production is likely with optimum nutrition [137] and absence of disease and stress. Buffaloes with low prolactin secretion are likely to benefit from the administration of prolactinemic drugs such as metoclopramide or domperidone. Many publications have addressed the usefulness of herbal galactagogues such as Leptadenia reticulata [138-140], Asparagus racemosus [118,138-144], fenugreek [140,145] and their combinations [146] on milk production improvement in buffaloes. However, milk production is a complex process that involves interplay of many hormones and other factors and thus no single therapeutic approach is likely to be successful without consideration of the management and nutrition.

3.5. Mastitis

Mastitis is defined as the inflammation of the parenchyma of the mammary gland. Inflammation can be infectious, traumatic or of toxic nature [147,148]. It is usually characterized by physical, chemical and bacteriological changes in the milk such as presence of blood, water, pus containing clots, flakes and shreds [149,150] and by pathological changes in the glandular udder tissue [152]. The disease has been recognized in most buffalo raising countries including India [151-157] , Pakistan [158-169], Iraq [170-173], Italy [147,174-176], Sri Lanka [177,178], Turkey [179,180], Nepal [181,182], Egypt [183-185], Portugal [186], Philippines [187], Spain [188], South America [189-192] and Bulgaria [193,194].

Similar to cattle, mastitis in buffaloes has been classified as clinical [148,195-197] and subclinical [198-200]. In the clinical form there are changes in the milk and inflammatory changes in the teats and udder [149,152,159,171,201-204] whereas subclinical mastitis is usually diagnosed employing a variety of direct and indirect tests such as California mastitis test, white side test, and somatic cell count performed on milk [205-211]. Clinical reports also classify clinical mastitis as peracute (with clinical signs of anorexia, pyrexia, decreased rumination, disinclination to move) [212,213], acute, subacute and chronic, depending on the intensity of clinical presentation [212,214-217]. A few reports described the occurrence of gangrenous mastitis in buffaloes [218-220] yet it appeared to be a clinical form of mastitis probably due to Staphylococci and E. coli [219]. Similar lesions were also observed in mastitis due to Clostridium perfringens in Egyptian buffaloes [203].

3.5.1. Etiology

Mastitis is considered to be a multifactorial disease caused by a group of pathogenic bacteria [221] and/or viruses [222-228]. A large number of bacteria including Staphylococci, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus pyogenes, E. coli, Pseudomonas, Proteus, Klebsiella, Diphtheroids, Mycoplasma, Clostridium perfringens and Listeria have been isolated from buffaloes suffering from mastitis [185,195,201,203,221,222,229-234]. Some unusual microorganisms isolated from buffaloes with mastitis in Brazil and Spain [188,189,235] include Lactococcus garviae. The most common bacterial isolates however, appear to be Staphylococci [134,200,221,236,237] and Streptococcus agalactiae [148] (Table 2). Isolation of yeasts such as Trichisporon and Cryptococcus has also been recorded in buffaloes with mastitis [222]. Staphylococcus aureus, Streptococcus agalactiae and Corynebacterium bovis are known as the contagious pathogen of mastitis in buffaloes [148,238] usually transmitted from an infected udder to a clean udder, contact with infected milk and through flies [148]. Environmental pathogens such as Coliforms (E. coli, Klebsiella), Streptococcus uberis, and Streptococcus dysgalactiae are transmitted between and during milking [148]. In a recent review it was mentioned that although Staphylococcus aureus had been the major mastitis pathogen in the past, coagulase negative Staphylococci have been emerging to be more common in recent years [120].

3.5.2. Incidence

Buffaloes have traditionally been considered to be less susceptible to mastitis compared to cattle and most studies conducted simultaneously in the two species, have confirmed that the incidence of mastitis is lower in the buffalo [153,169,177,183,193,198,199,239-242]. A nearly equal occurrence of mastitis in buffaloes and cattle was evident in a very few studies [183,241]. The udder in buffaloes is more pendulous and has longer teats compared to cattle. Buffaloes have a long, narrow teat canal and a tight sphincter with well-developed circular muscles which effectively prevent microorganisms from invading the udder and may be responsible for the lower incidence of mastitis in buffaloes [126,242]. It was considered that buffaloes were at a higher risk of teat injuries predisposing them to mastitis [243], yet this seems improbable. The use of the little known test Surf field test for detection of subclinical mastitis may be the reason why a higher incidence of mastitis was recorded in buffaloes compared to cattle in one study in Pakistan [165]. The pH of buffalo milk is more alkaline and buffalo species exhibit some degree of resistance towards subclinical mastitis [190].

Inflammation results from the introduction and multiplication of microorganisms into the mammary gland [243]. The incidence of subclinical mastitis is higher in buffaloes compared to clinical mastitis [164,165,170,171] and varies with breed, geographical location, management practices and other factors. The incidence of clinical mastitis in various studies varied from 2.7% to 40% [151,152,165,171,244] whereas that of subclinical mastitis ranged from 1.7% to 59.64% [169,200,245-248]. An exceptionally high incidence of clinical mastitis (72%) was recorded only in one study in Pakistan [249]. The incidence of subclinical mastitis varied with the type of test used for identification [250-252], stage of lactation [206] and sample processing [208]. Single quarter affection has been more prevalent for clinical mastitis in buffalo [197,253-255] and hindquarters were more affected [148,197,221,256,257].

3.5.3. Risk Factors

Risk factors identified for the occurrence of mastitis in buffaloes include type of housing, method of milking, parity [148,165,211,221,233,238,257], month of lactation and rainfall [247]. It has been mentioned that farms with good management have a low incidence of mastitis in buffaloes [221]. A positive association of mastitis was observed with milk yield, pendulous udder, udder depth and dirty hind legs of buffaloes at Government and private farms in Pakistan [233]. Studies in Brazil observed that improper milking management such as lack of teat washing was considered a significant contributor to the occurrence of mastitis in buffaloes [258]. In the Philippines age and lactation length influenced the occurrence of subclinical mastitis in Bulgarian Murrah buffaloes [187]. Studies in Egypt pointed out that environmental microbes are a significant risk for mastitis and thus hygienic measures are extremely important for its prevention [259]. The season of calving [187,260] is a significant contributor towards development of mastitis with the incidence being higher during summer and rainy seasons [256]. The method of milking [249,261] significantly affects the incidence of mastitis with machine milking and hand milking with folded thumb being significant risks. Other risk factors (Fig. 15) include calf suckling [233], education of farmers and number of animals milked by the same milker [249]. Post milking teat dips using 0.5% chlorhexidine or 0.5% iodine significantly decreased the incidence of mastitis in buffaloes [262,263]. Calving related disorders such as retained placenta, vaginal prolapse and dystocia are a significant risk for the development of mastitis in buffaloes [249]; high milk yielding buffaloes are more prone to develop mastitis [238,257]; hot humid seasons favors the growth of microbes in the environment thus increasing the incidence of mastitis during summer and the rainy seasons [264,265]; close confinement [266] and the use of soil as bedding material increased the incidence of mastitis [165,257]. Teat injuries also increase the risk of mastitis in buffalo [165].

Figure 15. Risk factors for mastitis in buffaloes.

3.5.4. Bubaline Ulcerative Mammillitis

The occurrence of bovine herpes virus mammillitis caused by Bovine Herpes virus-2 (BoHV-2) was described in buffaloes for the first time in 1998 [223]. It was however, mentioned that the disease has been prevalent in buffaloes previously and has been named necrotic thelitis, ulcerative mammillitis and udder impetigo based on the clinical findings and not on the basis of virus isolation [280-286]. Later reports continued to name the disease with similar terminology [287-289]. Herpes virus mammillitis is manifested by severe ulceration of the skin of the teat and udder (Fig. 16). The affection usually affects buffaloes during their first lactation and the first two months of lactation [223,288]. There is acute enlargement of one or more teats, ulceration, necrosis, bluish black discoloration (Fig. 17) and sloughing of the affected teats [288,290]. The lesions may appear suddenly as multiple flattened nodules, varying from 1-4 cm in diameter on the udder and spreading to the teats. Some nodules become necrotic later and slough off [290]. There is no involvement of the mammary glandular tissue and milk remains normal at all stages of the disease [288]. Different mastitis tests revealed negative results on milk samples from buffaloes affected with ulcerative thelitis [289]. However, the lesions were painful and created difficulty in milking the udder. Thickening of the teat skin may lead to narrowing of the teat canal [289].

Confirmation of the virus can be done by electron microscopy of the vesicular fluid or scab tissue collected from ulcerative mammillitis cases [223,289]. Udder impetigo with identification of Staphylococcus aureus was recognized at one buffalo farm [287] and at another location; farms were not associated with each other [290]. The therapy for ulcerative mammillitis involves oral administration (1200 mg) and topical application (5%) of the antiviral drug acyclovir [289]. The simultaneous administration of antibiotics, antiallergics and anti-inflammatory drugs is suggested to speed up the recovery [287,289] and prevent secondary bacterial contamination.

Figure 16. Ulceration of the mammary gland in a buffalo with thelitis.

Figure 17. Gangrenous mastitis in a buffalo.

3.5.5. Diagnosis

The diagnosis of clinical mastitis is easily done based on clinical signs such as inflammatory changes in the udder, teats and milk. The diagnosis of subclinical mastitis is dependent upon a number of tests performed on the milk. In clinical mastitis, inflammatory swellings appear on one or multiple teats or the teats and the quarter. The swellings appear suddenly at or within few hours or a few days of parturition (Fig. 18) and changes appear in the milk quality [291]. In chronic mastitis the affected teat may regress (Fig. 19).

Figure 18. Mastitis in one teat of a buffalo.

Figure 19. Chronic mastitis in a buffalo, note the regressing teat.

Besides bacteriological culture of the milk for identification and isolation of microbes [166,200,203,230,259,292-294], a large number of indirect tests have been used for the detection of subclinical mastitis in buffaloes such as strips [205,247], California Mastitis Test [187,190,207,210,245,250,261], white side test [247,293], detection of electrical conductivity of milk [274,295,296], radial immunodiffusion test [297], lactose estimation [298], estimation of acute phase proteins [299] or PCR based assays for molecular characterization of Staphylococcus aureus [267,268].However, the most common test used in most studies appears to be the somatic cell count [176,198,205,207,210,252,300-304]. A new field test has been mentioned for the diagnosis of mastitis in buffaloes in Pakistan [305], however, its efficiency remains to be seen on a wider scale.

In the white side test (WST) and modified white side test (MWST), 4% NaOH and 0.5% alkyl arylsulfonate combined with 1.5% NaOH react with deoxyribonucleic acid in the somatic cells to form sodium salts and produce a precipitate [306]. Higher fat percent and larger lipid size in buffalo milk, however, result in a lower sensitivity of the test [287].

The California Mastitis Test (CMT), which was developed as a cow side test for bovine milk to estimate the cell content, has also been used in buffalo milk [307-309]. It has been assumed that the interpretation of the test results should be the same as it is in bovine milk. In CMT, 3% triethanolamine sulfonate and bromocresol purple react with DNA in somatic cells rupturing the cell wall and forming a gel [297]. The formation of a gel indicates subclinical mastitis. The scoring is qualitative and subjective. CMT reflects the somatic cell count (SCC) level quite accurately and is a reliable indicator of severity of infection. CMT results alone should not be used for immediate treatment unless the specific pathogen is known or is performed in accordance with a treatment protocol recommended by a veterinarian.

The SCC in buffalo milk is performed utilizing the procedures mentioned for cattle [306] and generally the results are in the same proportion of samples with low and high SCC as reported for cows [310-313]. Briefly, 10 μL of thoroughly mixed milk sample is spread over 1 cm2 marked square area on a glass slide, making a thin film. The slide is dried at room temperature, fixed in methanol for 5 min, stained with Newman-Lampert stain for 2 min and dried at room temperature. The slide is then washed thrice in tap water and dried at room temperature. Somatic cells are counted at 1000 X magnification under a microscope using oil immersion [209]. The SCC per mL is calculated by multiplication of the working factor with the number of cells counted [209]. Alternatively automated optoflurometric cell counters can be used for estimation of SCC [314] although they are not popular among buffalo clinicians. SCC is recognized as a measure of udder inflammation and represents a sign of incorrect farm management [147]. The SCC includes the whole of leucocytes (WBC) and epithelial cells coming from the exfoliates of the udder existing in milk. More leucocytes mostly polymorphonuclear leucocytes (PMNs) infiltrate the mammary tissue as a response to the pathologic or stressing event [301].

Buffaloes regularly shed a small number of cells in the milk (macrophages, lymphocytes and neutrophils and cells lining the ducts) with a predominance of macrophages [315]. The cell count increases with advancing age and stage of lactation [174] with counts being higher around calving and towards the end of lactation [208,316-318]. The milking frequency and the method of milking (hand milking vs. machine milking) also affects the SCC to some extent [262,315,319] and minor changes also do occur during estrus [150]. SCC is also known to increase non-significantly from 1st to 4th parity [260] and is also significantly higher during summer compared to winter months [260]. In mastitis, the increase in SCC is due to the influx of neutrophils (PMNs) into the milk reaching 90% of the cell population [315]. The type of infection is known to affect the SCC. The highest SCC was observed in quarters infected with streptococci [252,320]. The severity of mastitis also affects the SCC [164].

There has been a lack of consensus on the threshold values of SCC for identification of subclinical mastitis in buffaloes. While many studies considered >200x103 cells/mL as being indicative of mastitis in buffaloes [178,206,209,252,302,307,308,321], others have considered at least >250x103 cells/mL [322] and >280x103 cells/mL [294]. A few recent studies have, on the other hand, considered >500x103 cells/mL as indicative of subclinical mastitis in buffalo [200,323,324]. Moreover, most of the studies in buffalo have utilized milk samples from individual quarters or buffaloes except a very few that utilized composite milk samples [176] or samples from bulk milk tanks [259].

Mastitic milk has a higher electrical conductivity compared to normal milk [325]. This is probably due to tissue damage and the subsequent increase in sodium and chloride ions in milk [274,296]. The normal electrical conductivity of buffalo milk ranged from 6.05 to 10.92 m-mhos with an average of 9.17 m-mhos and was not significantly different from cow milk [295]. The change in electrical conductivity is one of the earliest manifestations associated with new infections making the early detection and recording of mastitis [325]. Conductivity sensors can be incorporated in automated milking machines; however, such systems are not popular among buffalo production systems probably due to the high costs.

The comparative efficiency of different tests has been the subject of some studies in buffaloes and it has been pointed out that the efficiency of SCC was highest compared to other tests [210,250,251], however, it should be considered that no single test is 100% accurate. Several different tests should be performed simultaneously depending upon the requirements of the farm. SCC has been considered the gold standard for the diagnosis of subclinical mastitis [315] and its efficiency is improved significantly only when simultaneous organism identification tests are performed upon detection of mastitis. Efficiency of CMT was considered slightly higher in buffalo compared to cattle whereas SCC revealed comparable efficiency in both species [254,298].

3.5.6. Effects of Mastitis on Milk Quality

The milk yield is decreased in animals with mastitis and the quality of milk is also affected [326,327]. Mastitis impairs the coagulation properties of milk and results in a poor quality of cheese and curd [206,208]. The milk yield is negatively correlated with the total somatic cell count [328]. Significant increase in lactose and chloride content are also observed with the increasing total somatic cell counts [328]. The rennet coagulation properties and the clotting time are increased and the curd firming time and firmness are decreased [328]. Reduction in the quality of yoghurt and cheese [202] and reduction in the shelf life of processed milk [329] obtained from buffaloes with subclinical mastitis has also been recorded. Thus mastitis affects the milk production, composition and quality of the milk [202]. The microbial quality of the milk has public health significance [330] and hence a regular check-up for the presence of subclinical mastitis and effective therapies are desirable.

3.5.7. Therapy

The therapy for mastitis involves intramammary and intramuscular administration of antibiotics and anti-inflammatory drugs. Penicillins have traditionally been used for therapy of clinical mastitis in buffalo, however, some studies have shown ciprofloxacin to be an effective therapy [222] and similar results were mentioned for enrofloxacin [264,275,331,332]. A large number of antibiotics have been used intramammary and IM, alone or in combination, administered for 3-5 days (Table 3) with many studies suggesting a concomitant use to be beneficial.

disposition pattern of drugs administered IM has been studied in buffaloes. The disposition pattern of enrofloxacin in blood and milk of buffaloes suffering from clinical mastitis revealed that the peak concentration of the drug, when administered IM, was attained at 1 and 6 h and the minimum inhibitory concentration (MIC) in plasma and milk was maintained up to 24 h, suggesting that the IM administration is useful to treat mastitis [271]. Similarly, gentamicin (3 mg/Kg IM) reached a peak concentration within 2 h and was maintained up to 24 h, however; the drug was useful in buffaloes with mastitis caused by Gram negative bacteria such as Pseudomonas [365]. Based on disposition kinetics a single IM administration of 20 mg/Kg of long acting tetracycline was found to be useful to treat subclinical mastitis in buffaloes [366,367]. The peak concentration was attained within 4 h and the MIC of the drug was maintained in milk up to 84 h [366,367] and the cell counts were reduced significantly [367]. Single IV administration of ampicillin (6 mg/Kg) resulted in a peak concentration within 15 min and was maintained up to 6 h in milk of buffaloes, thus the repeated administration appears to be necessary for treatment [368]. Studies on disposition kinetics of IV administration of 100 mg/Kg of sulfonamides in buffaloes revealed that the excretion of sulfonamides was higher in saliva and urine than in milk thus limiting the usefulness of this drug in treating bubaline mastitis [369].

Many publications have addressed the antibiogram of microbes associated with mastitis in buffalo with diverse results [155,169,207,222,338,370-373]. It has also been suggested to administer the antibiotic on the basis of organism culture and in vitrosensitivity towards the antibiotics [261], however, the time needed for the results of culture and sensitivity warrant the administration of broad spectrum antibiotics immediately and then to review the clinical outcome upon receiving the culture and sensitivity reports. Moreover, the antibiotic sensitivity may vary depending upon the type and strain of bacteria, their virulence and time of initiation of therapy after infection.

The indiscriminate use of antibiotics for the therapy of mastitis is not recommended. There are only a few studies that have addressed the level of drug residues in milk [374]. Antibiotic residues in milk during therapy are a matter of public health; however, the milk withdrawal times during the treatment of mastitis in buffalo are neither addressed nor followed under most bubaline mastitis therapy programs. Indiscriminate use of antibiotics in buffaloes have probably led to development of resistance in mastitis causing microbes (Staphylococcus, Streptococcus and E. coli) towards commonly used antibiotics like gentamicin and enrofloxacin [250], streptomycin, penicillin and ampicillin [375], methicillin [376], cephradine [355] and many other antibiotics [253,377].

Buffaloes with subclinical mastitis often suffer from oxidative stress [378] hence the concurrent administration of antioxidants such as 1000 mg ascorbic acid (vitamin C) and vitamin A and vitamin E are suggested as they favor healing and recovery [273,379,380]. Supplementation of selenium and vitamin E is known to increase the polymorphonuclear cell phagocytosis and antioxidant levels in buffaloes affected with acute mastitis [333]. The usual supplementation rate of selenium in the buffalo is 0.3 ppm (as sodium selenite) [381,382] however, supplementation rates of 2.70 ppm for 2 months to buffalo calves did not result in significant effects on dry matter intake and nutrient utilization and did not result in any toxicity [383]. Adverse effects of selenium toxicity in buffalo calves grazing fodder with very high selenium appeared when the whole blood selenium concentrations increased above 2 μg/mL [384]. Similarly zinc supplementation improved recovery rates from subclinical mastitis in buffaloes [385]. Non-antibiotic alternatives suggested for the therapy of mastitis in buffaloes include the intramammary infusion of egg yolk immunoglobulins [386] or the administration of 12-30 g of trisodium citrate in 250 mL of distilled water IV for 3-5 days helps buffaloes recover from mastitis [216,270,363,364,387,388].

3.5.8. Prevention

Prevention strategies for mastitis include improvement in the hygiene and management of the animals. Daily cleaning of the teats with water and teat dips (0.5% Lugols solution) post milking helps in significantly reducing the incidence of mastitis in buffaloes [262,389]. Similarly, teat dips in 0.5% chlorhexidine was found effective [263]. Another suitable approach is the dry period therapy [390]. Administration of enrofloxacin 2.5 mg/Kg IM at 14 and 7 days prepartum or oxytetracycline 11 mg/Kg IM at 7 day prepartum [331], 200 mg ampicillin and 500 mg cloxacillin administered twice intramammary [263], 13.3 mg cloxacillin ointment thrice at 48 h interval few days before calving [391] or 200 mg cloxacillin thrice intramammary at drying off [309,320] have been suggested for the prevention of mastitis at calving [331]. A recent study mentioned the beneficial effects of IM administration of penethamate hydriodide (10 million IU) at 7 days precalving resulting in significant reduction of somatic cell counts at 10 and 30 days in milk in Mediterranean buffaloes [176].

Similar to reports on the use of Staphylococcal vaccines for prevention of mastitis in cows [392-394], one study documented the use of polyvalent vaccine in buffaloes [395]. Four different formalin-inactivated polyvalent mastitis vaccines containing Staphylococcus aureus, Streptococcus dysgalactiae and E. coli were evaluated for 6 months in 100 lactating buffaloes. Buffaloes vaccinated with adjuvanted vaccines evidenced significantly higher milk yield compared to unvaccinated controls [395]. Shakoor et al. [396] administered four different Staphylococcus aureus vaccines to healthy pregnant buffaloes 60 and 30 days before calving and recorded a significantly lower SCC in vaccinated buffaloes up to 4 months postpartum compared to unvaccinated buffaloes. However, the prospects of vaccinating lactating or pregnant buffaloes for mastitis prevention is not widely accepted because of failure of the vaccines to truly protect, given that there are too many different organisms involved and further large scale trials must be conducted to validate these results. A recent review in cattle showed that in a herd with 3% prevalence of Staphylococcus aureus and 30% prevalence of coagulase negative Staphylococci; the vaccine did not reduce the staphylococcal intramammary infection rate [397]. A recent study in buffaloes [356] revealed that antibiotic therapy in buffaloes affected with subclinical mastitis caused by Streptococcus agalactiae significantly reduced the SCC (3.29≥0.6x103 cells/mL) compared to SCC in buffaloes treated with a bivalent bacterin toxoid (5.73≥0.78 cells/mL). These results raise serious doubts on the routine use of vaccination for the prevention of mastitis in buffalo.