Prevalence of mastitis in “Recombinant Bovine Somatotropin (rbST)” treated herds of dairy buffaloes

The milk and other products of dairy industry are integral part of food chain. It is estimated that population of planet earth is increasing day by day and will reach nine billion in 2040-2050 (Anonymous, 2005). So, demand of milk is increasing with ever increasing population (Buzby et al., 2006; Bilal, 2008; Serbester, 2012). Although there is an increasingtrend of milk production since last several years but this is due to increase in number of animals, not due to production increase per animal (Otto et al., 2003). Increase in dairy production potential not only increases animal protein but also improve economic and social status of farmer (Sarwar et al., 2002).

This potential is available in our country in the form of buffaloes known as black gold of Pakistan (Khan, 1986). Bubalus bubalus (buffalo) belongs to family bovidae. Buffaloes and cattle in Pakistan contribute bulk supply of milk (95%) in market (Javaid et al., 2009). Nilli Ravi breed of country produce about 75% of all milk supply (Sharif et al., 2009) and consist of 76.7% of total buffaloes (Otto et al., 2003). Although Nilli- Ravi buffalo is best milk producer among other breeds of buffaloes in the world but per animal production is still squat as compared to cattle production of established dairy systems. It is most docile breed and offer unlimited dairy potential because of its milk and milk products are most favored by Pakistani consumer and consider it as a symbol of pride in society. The phenotypic beauty of Nilli-Ravi buffalo is matchless while well managed animals have 15-20 liters milk production per day (Ali et al., 2011).

Recombinant bovine somatotropin (rbST) is genetically engineered synthetic analogue of bST (bovine somatotropin), normally release from anterior lobe of pituitary and manufactured from recombinant DNA technology (Soliman and Barody, 2014). Milk yield response has been observed in many dairy breeds and animals having different genetic potential (Hartnell, 1995; Boonsanit et al., 2012). In a long term study with Holstein cows, bST treatment increase the fat corrected milk (FCM) in dose dependent fashion from 23 to 41% over control production 27.9 kg/day (Bauman et al., 1989). The use of this technology no doubt increase production and efficiency of dairy animals. For the evaluation of this technology the aspect of animal’s health should be also include (Bauman et al., 1992, Crooker et al., 1991). It is very difficult to see all possible effect on animal’s health because it required a large number of studies in different environmental and managemental conditions (Eppardet al., 1987).

Mastitis is one of the main health issues that affect mostly the high producing and poorly managed dairy herds. It is defined as inflammations of mammary gland that destroy the udder of animals subsequently depress in milk yield, thus affects the economics of dairy business. It may be clinical and subclinical. In clinical mastitis there is sudden swelling, redness, pain and altered milk secretion of the effected quarter. Milk may contain clots, flakes or it may be watery in consistency accompanied by fever, depression and anorexia also in some cases (Hillerton, 1999). There will be increase in pH, electrical conductivity and total dissolved substances while decrease in milk fat, lactose, protein and SNF in mastitic milk (Hussain et al., 2012). There is 16.72% and 21.08% clinical mastitis among cattle and buffaloes respectively (Bilal et al., 2004). There may be 10% or more milk loss on the quarter level (Dijkhuizen and Renkema, 1978) and 10% loss in remaining lactation after acute mastitis (Blowey, 1986).

In subclinical mastitis signs and symptoms don’t appear (Blosser, 1979) but somatic cell count gets higher (Radostits et al., 2007). It may be diagnosed by somatic cell counts, California Mastitis Test (CMT), White Side Test (WST) and Surf field mastitis test (Muhammad et al., 1995; Muhammad et al., 2010). It is more threatening because may drop in milk as much as 20% of the infected quarter (Schepers and Dijkhuizen, 1991) and 3 to 4 times more prevalent than clinical mastitis (Jasper et al., 1982).

Mastitis is outcome of various factors associated with the host, pathogen and environment. Association of host and management are key factor in mastitis occurrence. Among various infectious agents a vast majority of bovine mastitis belongs to bacterial origin. Staphylococci, Streptococci, Pseudomonas, Salmonellae, Bacillus, klebsiella, Enterococci and Crynobecterium are main isolations in bovine mastitis (Ali et al., 2011). Infectious agent may be contagious or environmental. Studies suggest that there is positive correlation between milk yield and mastitis and somatic cell count (Eppardet al., 1987, Shook, 1985). Other genetic studies indicate that annual gain in milk yield (45 to 60 kg/year) through genetic improvements there is 0.4 to 1.3 % increase in incidence of clinical mastitis per herd (Pell et al., 1992, Wiltone et al., 1972). The study findings which are reported in literature indicate that due to increase of milk yield there is increase in rate of incidence of mastitis. 

From different herds around the peri-urban area of district Faisalabad 300 lactating buffaloes were sampled randomly (150 receiving rbST treatments were included in treatment group while other 150 buffaloes were not receiving rbST treatment served as control group). Clinical cases of mastitis were differentiated based on signs and symptoms while screening for subclinical mastitis was done by Surf Field Mastitis Test (SFMT). A 3% solution of household detergent (Surf Excel®, Uniliver Pakistan) was prepared. The test was performed by adding equal volumes of test solution and milk and swirled up to one minute. The formation of gel and floccules depicted the positive samples (Muhammad et al., 1995; Muhammad et al., 2010). Milk samples were collected aseptically from 1200 quarters of 300 buffaloes. Immediately after collection of samples, stored in ice box and transported to Laboratory of Clinical Medicine & Surgery Department, University of Agriculture Faisalabad. In laboratory these samples were processed for Somatic Cell Count (SCC). In mastitis positive samples procedures described by National Mastitis Council Inc., USA (1987) were followed for culturing the milk samples and identification of mastitis pathogens Isolation and Identification was done. The prevalence of mastitis was determined by following formula 

Somatic cell count was performed by direct microscope somatic cell count (DMSCC) technique modified by Athar (2007) from Schalm et al. (1971). Somatic cell count was also be done by strips available as “The Porta SCC® Qiuck Test” (Porta Check 2010). Based on SFMT the positive samples will be subjected to bacteriological examination. The isolation of causative organisms will be done as per criteria recommended by National Mastitic Council Inc. USA (Anonymous, 1987). Total milk production was recorded of sampled buffaloes to determine the average milk yield per day per animal. Data thus generated was subjected to Chi square test and ANOVA was determined. The confidence interval and odd ratio where appropriate will be use (Steel et al., 1997). 

The prevalence of clinical mastitis was 9.3% and 8.7% and subclinical mastitis was 33.7% and 31.7% in rbST and non-rbST treated buffaloes respectively. Irrespective of numerical difference of prevalence values, statistically these were not significant as (P > 0.05). But in rbST treated buffaloes risk of clinical mastitis increased up to 8% and 19% more subclinical mastitis risk than buffaloes that were not under rbST treatment. Our study showed similar results studies done previously as in one study done on cattle, out of 87 clinically positive cases 47.1% were positive cases in control group and 52.9% were in treatment group although there was also numerical difference but this was not also statistically significant (Judge et al., 1997). On the other hand, 11 to 16% increase in milk also increase 25% more risk of clinical mastitis (Dohoo et al., 2003) and annual gain in milk yield (45 to 60 kg/year) through genetic improvements there is 0.4 to 1.3 % increase in incidence of clinical mastitis per herd (Pell et al., 1992, Wiltone et al., 1972) but this increase is less than that of reported. In another study, there was also increase in clinical mastitis done on rbST (Posilac) treated multiparous and primiparous and expected rate was determined by Linear Mixed Model analysis for a 252-day standardized treatment period were 0.23 and 0.28 for control and rbST treated primiparous cows and 0.38 and 0.50 for control and rbST treated multiparous cows. Statistically non significance showed this is not due to rbST but other factors. Clinical mastitis in buffaloes was 4% (khan and Muhammad, 2005), 7% in local Pakistani cattle and 12% in cross breeds (Akhtar et al., 2012). While subclinical mastitis prevalence was 21.7% (Dhakal, 2006), 51% (Sharif and Ahmed, 2007), 44% (Ali et al., 2011), 65.2% (Shahid et al., 2011) in buffaloes, 29% in local Pakistani cattle breeds and 41% in cress breed (Akhtar et al., 2012). This difference may be attributed to difference of cleanliness, managemental systems, production potential, teat structure and lack of mastitis control measures etc.

There was 20.7%, 25.0%, 30.4% and 23.9% quarter wise prevalence of mastitis in Left Front (LF), Right Front (RF), Left Rear (LR) and Right Rear (RR) quarter respectively of rbST treated buffaloes while 21.7%, 22.9%, 31.3% and 24.1% respectively in buffaloes that were not under rbST treatment (table 4.7). Results were analyzed by Chi-Square Test. Statistically difference of quarter wise prevalence was not (p > 0.05) significant (table 4.8). The prevalence of mastitis was more in hind quarters in both rbST treated and not treated buffaloes than front quarters. This difference is also similar to previous studies as in hind quarters prevalence was 73.3% LR and 63.1% RR and front quarters were 26.6% LF and 36.8% RF (Bilal and Muhammad, 2004), hind quarters 30.45% RR, 24.50% LR) and front quarters 23.84% LF, and 21.19 % LR (Sharif and Ahmed, 2007). Ali, 2009 also reported similar results. Didonet et al., 1986, and Adkinson et al., 1993 also reported higher incidence in hind quarters than fore quarter. These differences may be due to more milk production in hind quarters than front quarters and due to teat size and diameters in buffaloes (Ali, 2009).

However, increased incidence of mastitis appears to be more related to random effects than specifically to rbST (Pell et al., 1992). Thus, White et al., 1994 found no association between rbST treatment and the incidence or duration of clinical mastitis. They concluded that under normal conditions, there is a positive relationship between the incidence of mastitis and peak and total milk yield and that treatment with rbST did not alter this relationship. So, cows that produce more milk normally have a greater tendency to develop mastitis and this relationship exists regardless of the use of rbST (White et al., 1994). Accordingly, the incidence of mastitis in rbST-treated cows is due more to the increased milk yield than to any direct effects of rbST (Phillips, 1996).

Bovine mastitis is caused by different bacterial genera including Staphylococcus, Streptococcus and E coli etc. (Ahmad, 2001). Surrounding of animals including soil, bedding, utensils and manure are main pathogen sources and they may be infect the animals through infected utensils, cloths, milker’s hand and milking machine. The data of bacterial examination is presented in the Table 4.17. Among rbST and without rbST treated buffaloes out of 92 and 83 SFMT positive cases 9.8% and 8.4% gave growth that was not due to infection. There was 41.3%, 26.1%, 8.7% and 14.1% isolates of Staphylococcus, Streptococcus, E coli and others respectively of rbST treated buffaloes and 34.9%, 24.1%, 13.2% and 19.3% respectively isolates in buffaloes that were not under rbST treatment. This is statistically not significant (P > 0.05). There was 13.6% and 12.7% microbial growth in rbST and non rbST treated buffalo’s milk samples. The difference was negligible and study was coherent with previous study where microbial growth was 15.6% (Iqbal et al., 2004) but little difference with Pitkala et al., 2004 reported growth 21-33%. These differences may be due to season, managemental conditions, difference in sample handling and use of antibiotics.

In the present study Staphylococcus genera was the main pathogen followed by Streptococcus genera. These results were similar to previous studies like Staphylococcus was 50% and 53.85% in cattle and buffaloes respectively and Streptococcus was 27.78% and 23.07%. Hameed et al., 2008 Benites et al., 2003, Raza et al., 2013, Ali et al., 2008, Arshad et al., 2006 and in India Joshi and Gokhale, 2006 have similar results.

Somatic cell count (SCC) internationally is an indicator to check the quality of milk and its suitability for human consumption (Antunac et al., 1997). In buffaloes the normal somatic cell count ranges from 0.5 × 10⁵ to 3.75 × 10⁵ /ml (Silva and Silva, 1994). In the present study there was 3.588 × 10⁵ /ml and 3.467 × 10⁵ /ml somatic cell count in both rbST and without rbST treated buffaloes. The difference of somatic cell count is statistically not significant. Same results were reported in various other studies done on cattle (Zinn et al., 1993; white et al., 1994; Bauman, 1992; Lissemore, 1992). According to some other studies negative relation described between somatic cell count and rbST treatment (Phillips, 1996). There was no significant relationship between SCC and rbST treatment from 100 to 780 mg/day dose. Beyond this dose rbST may cause health problems (Pell et al., 1992). The sensitivity of Porta SCC® milk test was 66% compared to SFMT in buffaloes while in other study 74% was reported (Porta Check, 2010).

In proposed study there were 10.02 and 7.08 kg average milk production in rbST and without rbST treated buffaloes. There is 41% more milk production in rbST treated buffaloes and this was statistically significant (P < 0.05). In other studies, there was 44.15% in Egyptian buffaloes with rbST+ monensin (Helal and Nasheen, 2008), 25% in Murrah buffaloes @ 250mg/14 day rbST dose (Mishra and Shukla, 2004); 12.2 and 20.0% with 250 and 500 mg/14 days in cattle (Abdel-Rahman et al., 2010). In Murrah and Italian buffaloes there was 15.72% and 17% respectively (Jorge et al., 2002; Huber et al., 1997) while in Nilli Ravi Buffalo is 30% when given in combination with 20% enzose (Nisa et al., 2006) and in goat there was 14-29% increase in milk is reported (Baldi et al., 1999). In short, non-significant results of prevalence of mastitis and no effect on quality of milk in term of somatic cell count may indicate that rbST has not direct role in increase incidence of mastitis in buffaloes. 

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