Teat Health and Mastitis in Buffalo

Mastitis is the most commonly encountered and discussed problem in dairy animals that ensues when microorganisms overcome a three-tiered defense of udder immune system comprising of physical barriers and non-specific and specific immune responses to initiate inflammatory process (Sordillo et al., 2002). Physical barriers of the udder comprises of anatomic features of the teat and associated structures namely the teat skin, teat sphincter muscle and keratin plug that block the invading bacteria at the teat sphincter, the point of entry. The causative pathogen may be present near the opening of the teat canal, either through dirty and wet conditions at the teat end, through teat end lesions or colonization, on contaminated surfaces of milking units (liners or claws), or cow prep procedures. Teat health thus plays an important role in prevention of mastitis. When these pathogens enter mammary glands through teat canal, they colonize, proliferate and release toxins, damaging the mammary gland cells (Quirk et al., 2012) and initiating inflammatory reaction. Teat health or mastitis play important role in dairy farming it causes huge economic loss to the farmers. Therefore teat health is an important predisposing factor for mastitis and can be addressed for the prevention and control of mastitis. The present work was therefore designed to study the effect of teat health attributes as a predisposing factor in development of this disease with special emphasis on teat end microbiota and their molecular characterization.

Screening of buffaloes for sub clinical/clinical mastitis
 
Total 100 lactating dairy buffaloes of different parity were screened for mastitis. The animals were selected randomly and grouped on basis of Teat condition score. Thorough examination of udder was performed for the detection of any abnormality in the udder viz. presence of any lesion, pain, heat and swelling.  Milk from each quarter was striped out for detecting the abnormality in the milk like colour and consistency. Screening of buffaloes for mastitis was performed with the help of battery of tests namely: California Mastitis Test (CMT), White side teat (WST), pH and Somatic Cell Count (SCC) to detect  mastitis in and  samples showing strong positive reaction were selected. These examinations were continued until final selection of buffaloes (Radostits et al., 2010).
 
Teat health assessment
 
Teat health was assessed by scoring the teats for teat end condition, teat skin color, teat dryness, teat skin chapping and teat end bacterial analysis
 
Teat end condition was scored on a 1 to 4 scale, corresponding to the visibility of a hyperkeratosis ring. A score of 1 indicated no visible ring, 2 indicated moderate visibility, 3 indicated complete visibility and 4 indicated extreme ring thickness (NMC, 2007). 
       
Teat skin color was evaluated by examining any change in color tint or pigment. A score of 1 indicated a teat with normal coloring, 2 indicated a hint of blue or red coloring and a score of 3 indicated complete reddening of teat skin (NMC, 2007).
       
Teat dryness/roughness was evaluated using a friction test examining resistance upon touch determined by the scorer (Reeves et al., 2017). A score of 1 indicated a smooth surface with no frictional resistance involved, 2 indicated slight frictional resistance and slight roughness and a 3 indicated extreme roughness and a high frictional resistance level (NMC, 2007). 
       
Teat skin chapping was evaluated by observing cracks on the skin surface. A score of 1 indicated a smooth surface with no visible cracking, 2 indicated minor appearances of skin cracks and 3 indicated extensive and irritated skin cracking (NMC, 2007). 
 
Teat-end swabs collection and analysis of bacterial count
 
Two teat-end swabs from each buffalo were collected individually from both hind quarters by rotating a moistened cotton swab, covering an area of 2 cm² outside the teat orifice. The first swab was taken immediately after udder sanitization whereas the second swab was performed on another quarter after applying the udder wash followed with teat spray solution. Teats were sprayed with the oil and allowed contacting buffalo’s teat skin for 30 s and subsequently dried using a dry and single use sterile towel to remove the surplus oil. The teat-end swabs were placed in a separate test tube containing 0.1% peptone water and stored in ice container until analyzed following all aseptic measures. All teat-end swabs were analyzed for bacterial counts, i.e. total bacterial count (TBC), Staphylococcal count (STA), Streptococcal count (STR) and Coliform count (COL). The swab tubes were vigorously shaken using vortex mixer for 30s to extract the bacteria from the cotton swab and the cotton tips were then removed.
 
Molecular characterization of teat end swabs
 
Molecular characterization and PCR were carried out by Cytogene lab, Lucknow using Sanger’s Method.
 
Sequence alignment and data analyses
 
The obtained forward and reverse sequences were aligned using online pairwise alignment tool BioEdit. The query sequences were identified considering E value a <1 × 10^-5 and maximum hits (99 or 100%) with a species in the reference database NCBI. In addition to BLAST, MEGA X was used for phylogenetic tree analysis employing Maximum Likelihood (ML) method.
 
Stastical analysis
 
Statistical analysis of data was done by using SPSS 20 software.

Influence of teat health on prevalence of mastitis
 
Teat end condition
 
Prevalence was studied for bufflaoes with different teat end scores. The prevalence in buffaloes with teat end score 1 was 45.94% (Sub clinical: 43.24% and clinical: 2.70%) which was 23.28% lower than buffaloes with teat end score 2 i.e. 69.23% (Sub clinical: 46.15% and clinical: 23.08%). The prevalence of clinical mastitis was much higher in animals with teat end score 2. A significant correlation (P<0.05) was established between teat end score and prevalence as well as with somatic cell count. Significant correlation (P<0.01) was established between teat end score and all other teat parameters namely teat skin color score, teat roughness, teat chapping and teat condition score.
 
Teat skin chapping
 
Teat skin chapping was evaluated by observing cracks on the skin surface with increased score referring to more visible cracks (Fig 1). Highest prevalence was recorded in buffaloes with teat chapping score 3 (80%) with prevalence of clinical mastitis also highest in this group of buffaloes (20%). The prevalence of subclinical mastitis was also highest in group 3 (60%). Cracks on the skin harbor the dirt/milk after milking which in turn increases the microbial load, a predominant cause of mastitis. No significant correlation could however be established between teat chapping and status of mastitis. Teat chapping however had significant correlation (P<0.01) with teat roughness, teat skin color score, teat end score and teat condition score.
 

 
Teat roughness score
 
Teat roughness had a significant role in prevalence with buffaloes with teat roughness score 3 having highest overall prevalence (87.5%) followed by teat roughness score 2 buffaloes (58.82%) and 36% in buffaloes with score 1. Maximum number of clinically affected buffaloes were from score 3 (37.5%). The prevalence of subclinical mastitis was maximum in score 2 (52.94%), 2.94% higher that score 3 (50%). This increase can be attributed to more number of buffaloes screened with score 2 (17) in comparison to score 3 (8). Group 1 had least prevalence as the skin surface was smooth and healthy.  A significant correlation (P<0.05) was established between teat roughness score and prevalence as well as with somatic cell count. Significant correlation (P<0.01) could also be established between teat roughness and all other teat parameters namely teat skin color score, teat end score, teat chapping and teat condition score.
 
Teat skin color score
 
Color was evaluated by examining any change in color tint or pigment (Fig 1). In the present study maximum buffaloes had a score of 1 having normal coloring followed by score 2 buffaloes and score 3 buffaloes. All the buffaloes with score 3 had only clinical mastitis (100%) followed by score 2 buffaloes (overall prevalence 73.33%, Sub clinical mastitis 46.67% and Clinical mastitis 26.67%) and score 1 buffaloes that had overall prevalence 37.5% with no cases of clinical mastitis. Red discoloration is an indication of genesis of inflammation and thus score 3 buffaloes were clinically affected buffaloes. A significant correlation (P<0.01) was established between teat skin color score, teat end score, teat roughness, teat chapping, teat condition score, status of mastitis as well as with somatic cell count.
 
Teat condition score
 
Buffaloes with teat condition score 3 had highest overall prevalence (71.43%) with highest prevalence of clinical mastitis (42.86%). Sub clinical mastitis was highest in score 2 buffaloes with prevalence 55.56% and overall prevalence of 61.11%. Teat condition score had a significant correlation (P<0.01) with all teat parameters discussed, status of mastitis as well as with somatic cell count (Table 1). 
 

       
All teat-end swabs were analyzed for bacterial counts, i.e. total bacterial count (TBC), staphylococcal count (STA), streptococcal count (STR) and coliform count (COL). The total bacterial count declined significantly (P<0.01) after spraying with oil from log₁₀TBC of 8.55 to 4.29 after 15 minutes of application of oil.in control untreated quarter no decline in the TBC was recorded. The E. coli count also declined significantly (P<0.01) from log₁₀COL 8.26 to 4.88 in O-4 treated group. In control group the log₁₀COL increased from 8.27 to 8.46. Significant decline of Log₁₀ S. aureus count and Streptococcal count was also recorded after application of oil from 7.01 to 4.83 and 7.13 to 4.91.
 
Molecular characterization of Teat end surface microflora
 
Teat end swabs were collected asceptically to isolate the common micro organisms present and to assess the total bacterial count. The common bacteria isolated were E. coli, S. aureus and Streptococcus sp. The log10TBC was 8.55. The log₁₀ E. coli count (log₁₀COL) was 8.26, Log₁₀ S. aureus count and Streptococcal count was 7.01 and 7.13 respectively.
 
Bacterial counts
 
The isolates (06) were subjected for molecular characterization to identify the strains present in this area. The amplification of 16S r DNA by PCR with universal primer 27F-1492 R which span nearly full length of 16 SrRNA gene with expected amplicon of 144-1500 bp, the band was excised from gel and purified for sequencing (Fig 2). Phylogenetic tree was constructed and five different organisms were identified with highest percentage identity (Fig 3, 4, 5, 6, 7, 8).
 

 

 

 

 

 

 

       
The possible association between teat morphometric traits and subclinical mastitis (SCM) in dairy buffaloes was studied by Kaur et al., (2018). They concluded that teat morphometric traits seem to be associated with indicators of udder health in buffaloes, thus, their inclusion in breeding programmes for selection against undesirable dairy type traits may be of value in reducing susceptibility to intramammary infections in Indian buffaloes. Fox and Norell, (1994) reported that teat and udder skin should be healthy before milking and free of sores, wounds, or chapping where S. aureus could colonize the teat end and surrounding skin, one of the most predominant cause of mastitis.
       
Fox (1992) and Fox et al., (1991) demonstrated a correlation between teat skin condition and colonization of skin by Staphylococcus aureus and stated that rough or chapped skin will provide more places for bacteria to attach and survive. An impact on udder health and mastitis can be anticipated. McKinzie and Hemling (1996) showed an impact of teat skin condition on milk yield and milk out time. In the present study the use of pre milking wash and post milking essential oil combination spray significantly reduced the bacterial counts and it is therefore hypothesized to be of potent use in prevention of mastitis and its efficacy was further assessed changes in milk profile and teat health. Zecconi et al., (2006), found a significant increase in new intramammary infections with an increase of 5% in the thickness of the edges of teats. Santos and Fonseca (2007) opined that mainting the teat ends in good conditions is of primary importance because its sphincter muscle plays a crucial role in teat canal contraction keeping it closed between milkings, thereby preventing the entry of pathogens into the mam­mary gland. This action is aided by mature keratin cells present in the teat canal and together they represent the primary resis­tance barrier to mastitis. Kaur et al., (2018) thus were of the view that teat morphometric traits seem to be associated with indicators of udder health in buffaloes, thus, their inclusion in breeding programmes for selection against undesirable dairy type traits may be of value in reducing susceptibility to intramammary infections in Indian buffaloes. Prior to its use awareness campaigns were conducted among dairy owners screening their buffaloes milk and briefing about the ill effects of mastitis. Demonstration of the best package of practices and of the developed kit was done to sensitize the villagers and the farmers got convinced the kit was distributed which becomes popularize day by day among them.

The greatest way to prevent mastitis is to make changes to the environment, host and microorganisms on all three levels. As a result of the interaction between these elements, illness results. Farmers needed to be made more aware of the disease’s development and prevention because it was more prevalent in animals kept under poor management techniques. Awareness amongst farmers needs to be created if effective prevention is desired. It was assumed that if the microbial load is reduced, the chances of gaining infection too decreases especially if the host and environment factors especially udder hygiene are also taken care. The udder health kit prepared with indigenously developed pre and post milking dips and cleaning products  reduced the bacterial load, improved teat health score and milk profile and can thus be propagated for use in field condition.  

SV: Researcher executed the experiment, JP and Vibha Yadav: Designed the research, Dinesh Yadav, RK: Analyzed the samples, DP:  Critically revised the manuscript, Dinesh Yadav, Rakesh Gupta: Analysed the data.

None.