6

REVIEW OF LITERATURE

India ranks first in the world in milk production and dairying in India are a

classic example of production by masses rather than mass production. Due to changes

in human food consumption patterns, demands for fruits, vegetables, milk and milk

products, meat, poultry and fisheries have been increasing over the period in recent

years. Among the different food sectors, the growth in dairy sector has been

commendable (National Academy of Agricultural Sciences, 2013). The rate of growth

in milk production in India is also substantially higher (3.6 per cent) than the world

average of 1.5 per cent. However, the total projected demand of milk by the year 2030

would be about 200 million tonnes, depending on assumptions about income,

population, urban growth, and expenditure elasticity parameters, which would imply

an annual increase of around 4 million tonnes during the next two decades (N.D.R.I,

2011). At the existing rate of growth in milk production, supply is likely to fall short

of the demand in next ten years. Among the several barriers in achieving the

production targets, mastitis continues to remain as a challenging impediment, since

the affected quarters may have 30 per cent less productivity and cow may lose about

15 per cent production (Radistitis et al., 2000). Mastitis in dairy animals is considered

as one of the most important economic diseases resulting into huge economic loss to

the country. Globally, mastitis accounts for about 38 per cent of the total direct costs

of the common production diseases (Kossaibati and Esslemont, 1997). In India, the

economic losses due to mastitis have increased about 115 folds in last five decades

(Dua, 2001). Lack of awareness, delay in detection of sub-clinical mastitis, lack of

markers for detecting ensuing mastitis, unhygienic milking practices, diverse

production systems, inadequate treatment etc. are some of the important contributing

factors in higher incidence of mastitis.

7

Epidemiological status of bovine mastitis (Global vis-à-vis India)

The lactation process has been remarkably successful since the earliest

mammals, allowing them to occupy a vast range of ecological niches. However,

lactation is seriously impacted by the development of mastitis among most, if not all,

mammalian species (Michie et al., 2003). This condition alters milk composition and

reduces milk secretion, facts that impair infant/offspring growth and development. In

food animal species, it is one of the diseases with highest economic impact and a

major animal welfare concern. A broad definition of mastitis is inflammation of the

mammary gland, including not only intramammary tissues but also related anatomical

structures such as nipples, mammary areolas, milk ducts, etc. In veterinary medicine,

mastitis is referred to an intramammary inflammatory reaction caused by an infectious

agent (Fetherston, 2001).

With the increase in milk production, the incidence of mastitis has also

increased. Surveys on the prevalence of mastitis in most of the countries, irrespective

of the cause, show a comparable figure of 50 per cent among dairy cows (Radistitis et

al., 200). Subclinical mastitis which is believed to be more prevalent rather than

clinical in most countries ranged from 19 to 78 per cent (Tuteja et al., 1993).

Although controlled studies involving large sample sizes are very few in the country,

the available reports suggest same pattern. Analysis of 513, 1707 and 1115 lactation

records of Sahiwal and crossbred cows, and Murrah buffaloes, respectively in an

organized farm in northern India over a period of 9 years revealed that overall

incidence of mastitis was 13 per cent with significant difference between the breeds.

Sahiwal cows had higher incidence (20.66 per cent) compared to crossbred cows

(14.18 per cent) or Murrah buffaloes (7.44 per cent). An influence of season on

8

disease incidence was also observed in both cows and buffaloes in the same study. In

other studies, it has been shown that the incidence was the highest among pure-breed

Holsteins and Jerseys but the lowest in local cattle and buffaloes. In Haryana and

Rajasthan, the prevalence has been reported to be 36.69 per cent and 60.25 per cent,

respectively (Sudhan and Neelesh, 2010). In several studies, it has been reported that

subclinical mastitis was 15 to 40 times more prevalent than the clinical form and was

of longer duration, difficult to detect, adversely affected milk quality and quantity. It

constitutes a reservoir of microorganisms that lead to cross-infection of other animals

within the herd. Based on the published reports, it is evident that the average

prevalence of mastitis in 1960s to early 1990s, was not more than 30 per cent but

increased afterwards to even more than 60 per cent (Sharma et al., 2012). Two

decades ago, the mean incidence of clinical mastitis in India was 1-10 per cent with

subclinical mastitis ranging from 10 50 per cent in cows and 5-20 per cent in

buffaloes, while recent studies showed higher incidence of subclinical mastitis

ranging from 20 to 83 per cent in cows and 45 per cent in buffaloes (Sharma, 2007).

Analysis of the data from more than 100 recent studies spread over 21 States of India

indicate that the overall prevalence of mastitis ranged from 25 to 97 per cent with a

mean prevalence of about 50 per cent (Sharma, 2007). This clearly indicates the

drastic increase in the prevalence of mastitis especially the subclinical form of the

disease, which is an alarming situation for the dairy sector in the country.

Incidence of mastitis in dairy cows varies depending on geographical location

and housing environment. In North America and Europe, the incidence of clinical

mastitis in dairy cows ranges from 7 to 30 cases per 100 cows-years at risk (Erskine

et al., 1988; Olde et al., 2008; Barkema et al., 1998). In grazing herds from New

9

Zealand, a lower incidence rate of 10% and 19% was reported, but in one of the

studies only cows with less than 100 days after parturition were evaluated

(McDougall et al., 1999; Petrovski et al., 2009). In the New South Wales region of

Australia clinical mastitis incidence was 16% (Stevenson et al., 2000). Reports on

clinical mastitis incidence and prevalence from other countries are scarce. In Africa

for example, a study from Tanzania reported clinical mastitis incidence of 43 cases

per 100 cow-years at risk (Dohoo et al., 2011). Subclinical mastitis prevalence is

reported to be 31% in USA, 20 to 40% in Western Europe, and 29% in Australia

(Piepers et al., 2007; Rodrigues et al., 2005; Barkema et al., 1997; Roesch et al.,

2007; Plozza et al., 2011). In other countries with developing dairy industries,

subclinical mastitis prevalence is much higher. A survey from a dairy province in

China reported 54% prevalence at the cow level and 28% at the quarter level (Li et

al., 2009). Similarly, cow level prevalence in Brazil was 47% (Costa et al., 1998). All

these reported values should be considered historical, since the incidence and

prevalence of mastitis in dairy cows is largely determined by conditions in each

geographical region and individual herd and are constantly evolving. For example,

some studies demonstrated a direct relation between herds‟ bulk tank SCC and

clinical mastitis incidence. This approach reveals a higher incidence of clinical cases

or herds with lower SCC than those with high counts, possibly due to a reduction in

the total amount of immune cells in the mammary gland (Barkema et al., 1998).

10

Epidemiological factors involved in mastitis (Fig. 1)

Fig. 1 Epidemiological factors influencing mastitis etiology and pathophysiology

The course of mastitis cases is directly related to its clinical manifestations and

the etiological agent. For example, in periparturient and early lactation dairy cows,

intramammary infections (IMI) caused by coliform pathogens are often the cause of

peracute or acute mastitis. These cases have systemic symptoms that can be fatal or

lead to uarter losses (Burvenich et al., 2007). Both coliforms and Staphylococcus

aureus can cause chronic mastitis cases in dairy animals that are usually diagnosed by

high SCC and therefore considered subclinical mastitis. In the case of coliforms,

strains isolated from chronic cases have a better capacity for intracellular growth in

bovine mammary epithelial cells than strains isolated from acute clinical cases

(Almeida et al., 2011).

11

Mastitis may lead to clinical symptoms and, as a consequence, it is often

diagnosed directly by visual assessment of breast/udder inflammation or by changes

in milk‟s organoleptic properties. In addition, there are several ancillary tests that are

used to detect both clinical and especially subclinical mastitis and these include: SCC,

which is considered the standard method, milk microbiological cultures, pH, lactose

content, electrical conductivity, SFMT, flow measurements, and quantification of

acute phase proteins (Pyorala, 2003). The choice of one or more of these procedures

usually depends on the availability, proximity to a diagnosis laboratory, personal

experience and/or technical skills. Microbiological analysis of milk is the only method

that allows for an etiological diagnosis of mastitis, however, there are relevant factors

that have to be considered when using this diagnostic tool. These factors include: use

of standard protocols for milk sample collection, correct identification and

quantification of bacterial isolates, and the relevance of clinical symptoms in the

animal (Dohoo et al., 2011). The collection of a representative sample for microbial

analysis is of outmost importance for good etiological diagnosis. In food animal

medicine, there are standard protocols for milk sample collection proposed by the

National Mastitis Council (NMC) and the International Dairy Federation (IDF)

(Hogan et al., 1999; Goodridge et al., 2004).

Epidemiological Aspects of Mastitis udder health depends on a balanced

interaction between host and its microbiota, which may contain microorganisms

ranging from probiotic to potentially infectious. Obviously, there are relevant

differences among mammals regarding the number, size, position and structure of the

mammary glands. In addition, mammals (even within a same species) differ widely in

their ecosystems, management and use (e.g., milk producing versus meat-producing

12

domestic species). Therefore, there are many microbial, host and/or environmental

factors that may play important roles in the development of mastitis (Fig. 2) (Barkema

et al., 2006).

Fig. 2 Mastitis classification across species

Losses due to mastitis

The losses are either due to temporary or permanent loss of milk production,

poor milk quality, discarding of milk from affected animals prior to or after antibiotic

treatment and pre-mature culling of the cow or reduced productive life of animals.

The loss due to subclinical mastitis overweighs the loss associated with clinical

mastitis. Since mastitis affects the milk quality, its consequences are not restricted

only to but beyond the dairy farm. Increasing concerns among the consumers about

13

the antimicrobial residues, antimicrobial resistance, milk quality and animal welfare

further demand proper policies for an effective prevention and control of mastitis.

The loss of milk production is not just restricted during the course of the

disease but may continue throughout the life of the animal because of the permanent

damage that mastitis can cause to the mammary secretary tissues. The milk from the

suffering animal generally carries microbial load that renders it unsuitable for human

consumption. There is a considerable increase in the somatic cell count in milk of

cows and buffaloes suffering from mastitis. It is, therefore, important that India

undertakes a nationwide plan to prevent and control mastitis

In the affected animals, the milk yield is reduced considerably. Estimates of

milk yield loss by different workers range from 100 to 500 kg/cow per lactation.

When clinical mastitis occurs, additional costs result from discard of abnormal milk,

cost of drugs and veterinary services. According to a study, the estimated loss

following clinical mastitis in cows was almost 700 kg in first lactation and 1,200 kg in

the second or higher lactation (Wilson et al., 2004). Several studies conducted at the

United States show that costs related to mastitis on dairy farms are approximately U$

200 per cow/year. This gives an annual loss of 2 billion dollars for dairy industry

(Bogni et al., 2011). It is generally agreed National Academy of Agricultural Sciences

5 that at least 70 per cent of economic loss is due to reduction in milk production and

discard of milk from sick animals. Other causes are the elimination of milk containing

residues of antibiotics used in treating sick animals, loss of genetic stock by culling

cows early and therefore more expensive replacement, veterinary fees, cost of

medicines and payment of extra labour hours (Sharma et al., 2012). In India, annual

14

economic loss incurred by dairy industry on account of udder infections is estimated

to be Rs. 6053.21 crores and out of which loss of Rs. 4365.32 crore (70 - 80 per cent)

has been attributed to sub-clinical mastitis (Dua, K., 2001). In another report from

India, the annual economic loss due to mastitis has been calculated to be Rs. 7165.51

crores; losses being almost same for cows (3649.56 crores) and buffaloes (3515.95

crores). Subclinical mastitis has been estimated to account for 57.93 per cent (4151.16

crores) of the total economic loss due to mastitis (PDADMAS, 2011).

Etiology of Mastitis

Mastitis is the outcome of interaction of various factors associated with the

host, pathogen(s) and the environment. Association of some host and managerial and

housing determinants with mastitis is well established. At least 137 species of

microorganisms from a broad phylogenetic spectrum, including bacteria, yeast, fungi

and algae, are able to cause bovine mastitis. However, amongst these, only 5 species

of bacteria account for the bulk of bovine mastitis cases (Rinaldia et al., 2010) but

dominant causal agents may have some geographical signatures, as the distribution of

pathogenic bacteria displays a substantial geographic variation. Causal pathogens can

be divided into two groups based on their source: environmental pathogens and

contagious pathogens. Coliform organisms (Escherichia coli, Klebsiella sp etc.) and

Streptococcal organisms (Streptococcus uberis, S. bovis and S. dysgalactiae) are the

important environmental pathogens.

Environmental mastitis is caused by potential pathogens found generally in the

digestive tract (referred to as “coliforms”) of cattle or their surroundings such as

faeces, soil, bedding material and manure (Jones, 2006). These microorganisms

15

generally proliferate substantially in bedding (approximately 1,000,000 or more cells

per gram of bedding). This increases the probability of infection of mammary glands

leading to clinical mastitis (Bradley and Green, 1997). There is a positive correlation

between the number of coliforms present in the bedding material and the bacterial

load on the teat ends as well as the occurrence rates of clinical mastitis (Hogan et al.,

1989). Coliforms- particularly Escherichia coli, Enterobacter aerogenes, Klebsiella

pneumonia, Serratia marcescens and a Streptococcus spp., Streptococcus uberis are

the chief organisms found to cause environmental mastitis. Environmental mastitis

has previously constituted less than 10% of total mastitis cases, but more recently

there has been an increase in the incidence of environmental mastitis [Bradley and

Green, 1997; Boyer, 1997) particularly associated with S. uberis infection. This

pathogen is most often associated with chronic mastitis, which does not respond to

antibiotic treatment (Jones GM, 2006).

Contagious mastitis is caused by bacterial pathogens that thrive on the udder

skin and lesions of teat. They cannot survive for long periods in the environment and

generally are transmitted from one cow to another by the milking machine, the hands

of milkers, milk contaminated fomites or the sponge used while milking (Harmon,

1994; APHIS, 2008). The pathogens mainly associated with contagious mastitis are

Staphylococcus aureus and Streptococcus agalactiae (Smith and Hogan, 1995;

Barkema et al., 2006; Monecke et al., 2007). Although Streptococcus dysgalactiae is

considered as an environmental pathogen, there is evidence of its transmission from

cow to cow as a contagious pathogen causing mastitis (Smith and Hogan, 1995).

Mycoplasma species also cause contagious mastitis. Mycoplasma bovis is the

predominant species sometimes leading to severe problems like sudden onset, rapid

16

transmission and reduction in milk yield and lack of response to treatment (Harmon,

1994). However, the most recognised pathogen in the majority of clinical and

subclinical mastitis cases in most countries is Staphylococcus aureus [Workineh et

al., 2002; Barrett et al., 2005; Middleton, 2008). These bacteria are of immense

importance, causing over 25% of intra-mammary infections and adversely affecting

the quality of milk in a large number of clinical cases (Owens et al., 1988; Haveri,

2008). They are also considered the emerging pathogens causing bovine mastitis since

they are the most commonly isolated bacterial pathogens.

In addition to Staphylococcus spp., Corynebacterium spp. constitutes some of

the emerging pathogens causing bovine mastitis. Corynebacterium bovis is frequently

isolated from milk in many dairy farms and causes moderate inflammation of the

mammary gland (Hommez et al., 1999; Haltia et al., 2006). These infections result in

a slight increase in bulk tank somatic cell counts, changes in the composition of milk,

sudden reduction in milk production and clinical mastitis (Harmon, 1994). Four

species of non-lipophilic Corynebacteria found to cause clinical and sub-clinical

mastitis are C. amycolatum, C. mulcerans, C. pseudotuberculosis, and C.

minutissimum (Hommez et al., 1999). Other species of Corynbacterium isolated from

cases of clinical mastitis in sheep are C. mastiditis and C. camporealensis (Fernandez

et al., 1997).

Recent studies have revealed that coagulase negative staphylococci (CNS)

isolated from teat skin and teat canal, as well as from the coat and the nostril

comprises a major interest area of mastitis causal organisms (Pitkälä et al., 2004;

Tenhagen et al., 2006). Mastitis in heifers at calving is mainly caused by CNS. More

17

than 50 species and subspecies are included in this group (Pyörälä and Taponen,

2009). Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus

saprophyticus, Staphylococcus hyicus, Staphylococcus warneri, Staphylococcus

chromogenes, Staphylococcus sciuri and Staphylococcus xylosus are the commonly

encountered species of CNS in bovine mastitis (Rupp et al., 2000). The various

species of CNS isolated from bovine mastitis cases show varied pathogenicity,

antimicrobial susceptibility and virulence factors (Zadoks and Schukken, 2006;

Taponen and Pyöröla, 2009).

The major pathogens involved in mastitis are Streptococcus agalactiae,

Staphylococcus aureus, Corynebacterium bovis and Mycoplasma spp. The

distribution of pathogens varies among countries and even within country, production

systems, farms and individual animals. For example, Staphylococcus aureus is most

frequently encountered in clinical mastitis, followed by Streptococcus dysgalactiae in

Norway (Waage et al., 1999) while in Midwestern United States, coliforms are the

most frequently isolated bacteria. In Europe, clinical Klebsiella mastitis occurs less

frequently than clinical E. coli mastitis. In contrast, coliforms are less important and

Streptococcus uberis is the main concern in both clinical and subclinical mastitis in

New Zealand (Zadoks et al., 2011).

In India, Staphylococcus spp. have been reported to be the main etiological

agents of mastitis in cattle and buffaloes. However, there are no studies on nationwide

distribution of mastitis-causing bacteria in India. Apart from regional differences,

cows in tie-stalls have higher incidence of Staphylococcus aureus, Streptococcus

uberis, coagulase-negative staphylococci and other streptococcal infections compared

18

to those in free-stalls, where Klebsiella sp. and E. coli are main concerns (Olde et al.,

2008). Collectively, it suggests that distribution of organisms may vary between

regions and husbandry systems and it is important to pre-ascertain the

epidemiological pattern of mastitis pathogens in the implementation of management

strategies. In recent years, there have been changes in the relative and absolute

importance of different pathogens. In UK, during 1960s, it was observed that

Staphylococcus aureus was the most common organism in mastitis, but in 1980s, E.

coli was most commonly isolated from the milk of mastitis affected cows and the

same trend was also continued in 1990s. In several countries, S. aureus continues to

be the major cause of sub-clinical mastitis and the pathogens previously considered to

be purely environmental may also be capable of causing persistent infection. The

major objectives of the epidemiological investigations include the identification of

risk factors at farm level, major pathogens involved and the susceptibility of the host.

India being a large and diversified country with different farming systems and agro

climatic conditions, the prevalence of mastitis and the pathogens involved are likely

to vary with places and herds. Hence, obtaining the ground situation of the disease

and characterizations of epidemiological parameters to be intervened are of vital

importance.

In animals, clinical mastitis is characterized by signs of inflammation in the

mammary gland including hyperemia, pain, and increased gland size and density.

These symptoms may be accompanied or not by systemic signs, such as fever or

depression (Kemper and Gerjets, 2009).

19

Globally, staphylococci (S. aureus and CNS) are the most common mastitis-

causing agents in cows, buffaloes, ewes, does, dromedaries, rabbits, dolphins and

women (Rowan et al., 1996; Guliye et al., 2002; Moroni et al., 2006; Segura et al.,

20070. They are closely followed by Streptococci and E. coli which in some species

or settings may have a similar or higher prevalence than that of staphylococcal

mastitis. Less commonly, other Gram-positive (Actinomyces spp.,Corynebacterium

spp., Bacillus spp., Mycobacterium spp., Enterococcus spp., Clostridium spp.) and

Gram-negative (Klebsiella spp., Enterobacter spp., Citrobacter spp., Serratia spp.,

Proteus spp. and Pasteurella spp.) In dairy animals, mastitis causing pathogens are

classified depending on their epidemiological behaviour in contagious and

environmental (Eberhart et al., 1987). Among contagious pathogens S. aureus,

Streptococcus agalactiae, and Mycoplasma spp. are included. Contagious pathogens‟

main reservoir is the mammary gland; consequently, these bacteria are spread from

cow to cow or between quarters/ halfs of the same animal during the milking process.

In contrast, environmental pathogen reservoirs include bedding, pasture, and other

unanimated objects. These bacteria are isolated from the skin, teat ends, and teat

canals where they can gain access to the gland causing IMI. Streptococcus other than

agalactiae, Enterococcus sp, CNS, and coliform bacteria including Escherichia coli

and Klebsiella spp. are included in this group. It is important to note that certain

microbial, host and/or environmental factors may induce environmental agents to

behave as contagious pathogens or vice versa (Zadoks et al., 2001; Pyörälä et al.,

2009)

The mammalian ecosystem is hospitable, or at least receptive, to many

microorganisms including most of the bacterial groups that have potential to cause

20

mastitis. The evolutionary process has led to state of mutual acceptance or tolerance.

However, upon disturbance localized or disseminated invasive infections can occur.

Unfortunately, the exact causal events leading to the transition from colonization to

infection still are inaccurate in vivo (van et al., 2009). The ability to colonize and,

eventually, infect a host depends on several microbial characteristics, including the

expression of several virulence factors, e.g. the formation of biofilms, expression of

super antigens, and antimicrobial resistance. At the species level, pathogen-specific

transcriptomic disruptions are observed in the mammary gland as well as in peripheral

organs (Rinaldi et al., 2010). As an example, two major mastitis pathogens, such as

E.coli and S. aureus, elicit differential innate immune responses following IMI in

cows, which may determine the course and severity of the disease (Bannerman et al.,

2004). In relation to different Staphylococcal species, genomic analysis reveals that S.

aureus is more suited to develop acute infections than CNS. Conversely, CNS have a

tendency to cause sub-acute, subclinical or chronic infections. As an example,

members of the enterotoxin and exotoxin families, which function as super antigens

and inducers of a pro-inflammatory cytokine response, are unique to S. aureus and

have not been identified in characterized isolates of S. epidermidis or other CNS.

Several virulence factors or toxins are included in certain pathogenicity genomic

islands. In S. aureus seven islands carry approximately one-half of the virulence

factors that have been identified. Variations in the pathogenic potential of strains

belonging to this species depends largely on allelic variation of the virulence genes,

on the presence or absence of individual and on the presence of single nucleotide

polymorphisms (SNPs) in key genes. Such differences are the most significant factor

contributing to observed variations in antibiotic resistance and virulence among S.

aureus strains (Gill et al., 2005).

21

Pathogenesis

A comprehensive understanding of the pathogenicity of mastitis is key for the

development of appropriate detection techniques. The primary cause of mastitis is a

wide spectrum of bacterial strains; however, incidences of viral, algal and fungal-

related mastitis were also reported (Pyorala, 2003). Normally, the teat canal is tightly

closed by sphincter muscles, preventing the entry of pathogens. It is lined with

keratin, a waxy material derived from stratified squamous epithelium that obstructs

the migration of bacteria and contains antimicrobial agents, such as long-chain fatty

acids, that assist in combating the infection. However, the efficiency of keratin is

restricted (Craven and Williams, 1985; Paulrud, 2005). Fluid accumulates within the

mammary gland as parturition approaches, resulting in increased intramammary

pressure (Paulrud, 2005) and mammary gland vulnerability caused by the dilation of

the teat canal and leakage of mammary secretions (Sordillo and Streicher, 2002).

Additionally, during milking, the keratin is flushed out and there is distention of the

teat canal (Rainard and Riollet, 2006). The sphincter requires nearly 2 hrs to return

back to the contracted position. Once inside the teat, bacteria must also elude the

cellular and humoral defence mechanisms of the udder (Sordillo and Streicher, 2002).

If they are not eliminated, they start multiplying in the mammary gland (Fig. 3). They

liberate toxins and induce leukocytes and epithelial cells to release chemo-attractants.

If the infection persists, internal swelling within the mammary epithelium, not

normally detectable by an external examination, can occur. The mammary gland

alveoli become damaged and start losing anatomical integrity (Fig. 3). The blood–

milk barrier is breached, causing extracellular fluid components, such as chloride,

sodium, hydrogen, potassium and hydroxide ions, to enter the gland and mix with the

22

milk (Zhao and Lacasse, 2008). When extensive damage to the blood–milk barrier has

occurred, blood might be detected in the milk. This leads to visible changes on the

udder, such as enhanced external swelling and reddening of the gland. Changes also

occur in the milk, including increased conductivity, increased pH, raised water

content and the presence of visible clots and flakes (Zhao and Lacasse, 2008; Kitchen,

1981). This marks the initial stage of clinical symptoms, and the most severe

infections might ultimately result in the death of the animal.

Fig. 3 Schematic representation of mastitis development in an infected udder.

Molecular identification of the mastitic pathogens

A reliable etiological analysis must identify the causing organism(s) at least at

the species level. Many phenotypic assays traditionally used for taxonomical

purposes, such as the carbohydrate fermentation pattern, are no longer valid due to

23

low specificity or non-conclusive results. Recently, the introduction of molecular

microbiology techniques such as PCR and matrix-assisted laser desorption/ionization

time-of-flight mass spectrometry (MALDI-TOF MS) have improved the sensitivity,

specificity, and processing time of bacterial identification procedures (Barreiro et al.,

2010). Furthermore, realtime PCR assays performed directly on milk samples

(without culturing) have benefits over conventional culture, including higher speed,

automated interpretation of results, and increased sensitivity (Taponen et al., 2009;

Koskinen et al., 2010). However, implementation of this technique must take into

account that, in addition to live bacteria, milk contains a high quantity of dead

bacteria and free bacterial DNA, thus reducing its specificity (Perez et al., 2007). As a

consequence, bacterial cultures are still required for routine etiological diagnosis of

mastitis. There is no doubt that ongoing and future improvements on the application

of Omics in mastitis diagnosis will provide powerful tools with high specificity and

sensitivity.

More recently, microbial identification techniques based on pyro-sequencing

of the 16S ribosomal RNA gene were used to obtain a description of the milk

microbes (Hunt et al., 2011). Results indicated that milk bacterial communities are

more complex than expected, with several genera representing greater than 5% of the

relative community abundance. Staphylococci (particularly Staphylococcus

epidermidis) and Streptococci were the dominant genera. Bacterial populations were

usually, although not always, stable over time within the same individual. These

findings open a new concept regarding the role of mammary microbiota in

maintaining mammary health in mammalian species.

24

Role of antibiotics in the treatment of bovine mastitis

The success of bovine mastitis therapy depends on the aetiology, clinical

presentation, and antimicrobial susceptibility of the aetiological agent among other

factors (Miltenburg et al., 1996). Therapy failure in the management of mastitis could

result from pathological changes that occur in the udder, aetiology related factors,

pharmacokinetic properties of the antimicrobial drugs, poor animal husbandry and

inadequate veterinary services. However, the control of mastitis has been successfully

achieved through the establishment of effective herd health control programs (Erskine

et al., 2002). Antimicrobial agents are the main therapeutic tools for the treatment and

control of mastitis. Among the main reasons of low efficacy of antibiotic treatment of

mastitis cases is the resistance of the bacteria to antimicrobials. Recently, several

studies have been conducted to determine the antibacterial susceptibility patterns of

mastitis pathogens isolated from clinical studies or submitted to diagnostic

laboratories (Brown and Scasserra, 1990; Gitau et al., 2003; Haile, 2004).

Antimicrobial agents are widely used for the treatment of bovine mastitis,

respiratory tract infections and diarrhoea in cattle. During acute infections and

outbreaks of infectious disease in groups or herds it is important to use an effective

antimicrobial treatment as early as possible. This empirical treatment is generally

based on knowledge of the resistance pattern of the different bacterial pathogens to

antimicrobial agents used in the particular animal species. Antimicrobial resistance is

an increasingly important problem among several bacterial species causing infection

in animals and humans in recent year. The problem for some bacterial species is so

critical that there is few treatment options left (Aarestrup et al., 1999; Levy et al.,

2001). The initial treatment of animals is commonly based on the experience

25

regarding the expected resistance of the infectious agent. The fact that occurrence of

antimicrobial resistance varies between countries and regions has the potential to

complicate that matter. Furthermore, knowledge of expected resistance is limited by

the small proportion of different bacterial pathogens from infected animals that

actually are investigated for their antimicrobial resistance pattern.

The evolution of antibiotic resistance in S. aureus strains is a serious cause of

concern in dairy animals (Wang et al., 2008). Strains of S. aureus resistant to β-

lactam antibiotics are known as methicillin-resistant S. aureus (MRSA). These strains

in intra-mammary dissemination often produce incurable severe intra-herd infections

(Moon et al., 2007; Kumar et al., 2010). MRSA strains have been observed to be

multi-drug resistant, such as aminoglycosides, macrolides, lincosamides,

streptogramins, tetracyclines, etc., which are often used in the treatment of mastitis

(Wang et al., 2008; Kumar et al., 2010). The transmission of bovine MRSA to

humans is possible and may contribute to outbreaks in animal and human populations

(Lee, 2003). Hence, it is necessary to know which endemic strains of S. aureus in

dairy cattle populations are highly pathogenic and methicillin-resistant

Milk

Milk may be defined as the whole, fresh, clean lacteal secretion obtained by

the complete milking of one or more healthy milchy animals, excluding that obtained

within 15 days before or 5 days after calving or such periods as may be necessary to

render the milk practically colostrum free, and containing the minimum prescribed

percentage of milk fat and milk-solids not fat. In India, the term „milk‟ when

unqualified, refers to cow or buffalo milk or a combination of the two.

26

Milk is one of the most important foods of human beings. It is universally

recognized as a complete diet due to its essential components and it is directly

available for consumption (Battaglia et al., 2007; Grimaud et al., 2009). Its role is to

nourish and provide immunological protection (Hemalatha et al., 2010). Milk has

distinct physical, chemical and biological characteristics, which justifies its high

quality for consumption. These characteristics present a favourable environment for

the multiplication of various bacteria. It is well known that freshly obtained milk

contains some bacteria and somatic cells, which represent the biological constituent of

the milk (Hemalatha et al., 2010). The biological constituents easily change

depending on production conditions, the health status of the cattle, hygiene practices

during milking, keeping and transportation of milk (Turner et al., 1990).

Many constituents of cows‟ milk can be broadly categorised according to their

physical properties or/and physiological functions. In this complex biological fluid,

minerals occur in chemical equilibrium between the free ions and complexes with

various components, such as protein, lipids, carbohydrates and low molecular weight

ligands like citrate and amino acids (Vegarud et al., 2000). Mineral and trace element

concentrations in raw cows‟ milk are not constant but mainly vary according to two

kinds of factors, those related with secretion from the mammary gland, such as the

lactation state, animal species and health status, and extrinsic factors, such as season,

dairy cattle ration (nutritional status of cow), environment (nature of soil and locality

of the farm). In this respect, several studies have been carried out to assess mineral

content of cows‟ milk from different areas (Licata et al., 2004; Muñiz-Naveiro et al.,

2005), as well as to evaluate preliminary correlations between animal feeding,

27

manufacturing process and elemental profile in cows‟ milk and dairy products (Coni,

et al., 1995).

The world-wide contamination of milk with undesirable substances via animal

feeds, heavy metals, mycotoxins, dioxins and similar pollutants is considered to be of

great concern to public health due to their toxic effects on humans and wildlife. Milk

products are very important human nutrients and their consumption has increased in

recent years. It is known as an excellent source of calcium, magnesium and zinc and

hence supply very small amount of Fe and Cu (Ali et al., 2011)

Genetic diversity of bacteria

Several studies suggest that there are differences between strains isolated from

bovine and human hosts (Musser and Kapur, 1992; Kapur et al., 1995). Different

epidemiological studies of isolates from human and animals have been based on

phenotypic techniques, such as biotyping, bacteriophage typing and antibiotic

sensitivity testing (Tenover et al., 1994). Biotyping has been used as a simplified

method to differentiate isolates from humans and animals into host-specific (human,

bovine, poultry and ovine) or non-host specific biotypes (Devriese, 1984). Recently,

molecular DNA typing methods have been developed on the basis of genotype

characterization (Van et al., 1995). Random amplification of polymorphic DNA

(RAPD-PCR) has been applied extensively to distinguish different isolates (Van et

al., 1995). RAPD is an accessible and sensitive method based on the use of arbitrary

primers to amplify polymorphic segments of DNA. This technique has been widely

used in recent years for detection of diversity among isolates (Welsh et al., 1990;

Maslow et al., 1993). The modified polymerase chain reaction (PCR) with single

28

primers of arbitrary nucleotide sequence and requiring no prior sequence information

have proved useful in detecting intraspecific polymorphism among organisms (Welsh

and Mc Clelland, 1990; Williams et al., 1990). This amplification technique

(arbitrarily primed PCR on RAPD) can generate specific DNA fragments useful for

genome mapping, identification of isolates and application in molecular biology

(Hadrys et. al., 1992).

Molecular characterization of mastitic bacteria

Biofilm formation is another important virulence factor. The number of strains

carrying the biofilm related ica A and D gene and showing resistance to oxacillin,

erythromycin, clindamycin and mupirocin was significantly higher among mastitis

isolates (Delgado et al., 2009). Resistance to diverse antibiotics and a higher ability to

form biofilms found among mastitis specific strains may explain the chronic and

recurrent nature of this infectious condition. There are several other virulence factors

that modify bacterial ability to colonize and cause IMI but the combination of genes

associated to pathogenicity is strain-specific (Barkema et al., 2006; Monecke et al.,

2007, Wolf et al., 2011). In veterinary medicine, this could possibly lead to the use of

bacterial genetic characterization as a management tool. For instance, animal culling

or segregation could be determined on the bases of infectious status with strains

expressing specific virulence factors.

Although multiple bacterial and external factors influence attachment and

accumulation leading to biofilm formation (Gotz, 2002), the production of a

polysaccharide intercellular adhesin (PIA) or polymeric N-acetyl-glucosamine

(PNAG) by intercellular gene cluster adhesion (ica) operon-encoded enzymes (Mack

29

et al., 1996; Maira et al., 2002) is currently the best understood mechanism of biofilm

formation in staphylococci in vitro and in vivo (Rupp et al., 1999; Mack et al., 2004).

A great variety of Staphylococcus aureus strains carry the ica cluster, and some of

them constitute biofilm. Loss of the ica locus results in an incapacity to produce

polysaccharidic intercellular adhesin and to develop biofilms (Cramton et al., 2000).

Staphylococcal infections produced by ica carriers can be more problematic due to the

presence of methicillin and mupirocin resistance genes (Jones et al., 2001; Stewart

and Costerton, 2001). The rapid detection of the ica locus in Staphylococcal isolates,

will allow the development of prevention methods to reduce the bacterial capacity in

mastitis.

Therefore, based on the review of literature the present study was designed to

do survey of Bovine Mastitis in North Karnataka, isolation, identification and

antimicrobial susceptibility / resistance of bacterial isolates, effects on biochemical

parameters of the milk inoculated by mastitic pathogens and to do genetic diversity

and molecular characterization of the isolated mastitic bacteria.