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Product Code: 00006135

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Mastitis is a detrimental disease that economically affects dairy farming by reducing milk quality and quantity produced, the sudden death of dairy cows, premature culling of dairy cows before attaining their maximum production ages, and the cost of disease prevention and treatments. In Sub-Sahara Africa, mastitis prevalence among smallholders’ dairy farmers have been recorded to exceed 50 percent, and it has continued to cause significant threats. In East Africa, subclinical mastitis prevalence varies between 16 and 80 percent among smallholders’ dairy farmers. Full udder quarters of 38 lactating crossbred dairy cows were cleaned with 75% alcohol and screened with the California mastitis test (CMT) kit for subclinical mastitis and somatic cell count (SCC). A total of 152 milk samples were collected aseptically from each quarter of 38 lactating dairy cows into screw-capped bottles `for direct microscopic somatic cell count analyses in the laboratory. Ninety-six blood samples from both lactating and non-lactating crossbred cattle were obtained for genomic DNA extractions. The genomic DNA was later run in polymerase chain reactions with two oligo primers of 252 bp (LGB) and 301bp (LTF) for beta-lactoglobulin lactoferrin genes, respectively. Results CMT scores carried out in the farm revealed that 55.01% of udder quarters were negative for subclinical mastitic, 43.99 % trace, and 1.32 % were positive for subclinical mastitis. The Least Square Difference (LSD) for pairwise comparison between CMT scores and lactation stage were significantly different between First and second lactation at 0.25±0.11, the second and third at 0.27±0.0118 at P≥0.05. The means of SCC among the breeds were significantly other at P≥0.05, for Ayshire and Friesian (68.055±18.82 cells/ml); Ayshire and Guernsey (71.976±23.844 cells/ml); Friesian and Jerseys (64.863±21.429 cells/ml); and Guernsey and Jersey (68.78±25.952 cells/ml). Results of PCR-DNA sequenced showed that there were several genetic variations in the nucleotide sequences. These were identified as Single Nucleotide Polymorphisms (SNPs) associated with mastitis resistance or tolerance. The genetic variations found among nucleotide sequences of both breeds positive for subclinical mastitis and those free from subclinical were oriented in either transitions or transversions models due to tautomeric shifts in the nucleotide sequences. They were either substituted or inserted, which eventually created variability among nucleotide base sequences. They could also be a result of point shift mutation in the nucleotide sequences. In conclusion, the study also found out that milk quality and somatic cell count were closely associated with the presence of beta-lactoglobulin and lactoferrin genes. The nucleotide sequences of genes have shown significant associations with milk quality and somatic cell counts.

Keyword: Crossbred, Gene, Somatic Cell Count, Mastitis resistant


1.1 Background information 1
1.2 Problem statement 3
1.3 Justifications 3
1.4 Objectives 4
1.4.1 Overall objective 4
1.4.2 Specific objectives 4
1.5 Hypotheses of the study 4

2.1 Mastitis in dairy cattle 5
2.1.1 Classification of mastitis based on clinical signs 6
2.1.2 Predisposing factors causing Mastitis 7
2.1.3 Mastitis based on the Mode of infections 7
2.2 California Mastitis Test (CMT) 8
2.3. Somatic Cell Count 11
2.3.1 Factors that affect the number of SCC 12
2.3.2 Association between Somatic cell counts and Mastitis infections 12
2.4 Somatic cell score 13
2.5. Genes involved in Immune response 14
2.5.1 Beta-lactoglobulin gene 14
2.5.2 Lactoferrin gene 15
2.6. Optimization of Polymerase Chain Reactions for amplification 16
2.6.1 Primers’ Design 16
2.6.2 Formation of DNA Template 17

3.1 Study Area 19
3.2 Sample collections 20
3.2.1 Milk samples’ collections 20
3.2.2 Blood samples’ collections 20
3.3 Microscopic Analysis of SCC 20
3.4 Statistical analysis 21
3.5 Genomic DNA Extraction from blood samples 22
3.6 Visualization of genomic DNA 22
3.7 PCR Amplification for DNA templates 22
3.8 Bioinformatics analysis of the DNA sequence data 24
3.8.1 Quality control of the raw DNA sequence data 24
3.8.2 Sequence alignments 24
3.8.3 Sequences Submission to Genbank 25

4.1 The California mastitis test scores for the udder quarters of lactating crossbred dairy cows 26
4.1.1 Association between the California mastitis test scores and somatic cell scores 27
4.2 Association between somatic cell scores and lactation stage 28
4.3 Somatic cell count 28
4.3.1 The average of somatic cell count in the udder quarter of four genotypes of the crossbred dairy cows 30
4.3 Gel Electrophoresis 32
4.4 Evolutionary relationship between Sequences of lactoferrin gene 34
4.5 Evolutionary relationship between Sequences of beta-lactoglobulin gene 36
4.6 The association between Somatic cell count and the lactoferrin gene 37
4.7 The association between Somatic cell count and beta-lactoglobulin gene 40

5.1 The California mastitis test scores 42
5.2 The role of somatic cell scores in the udder quarters 43
5.3 Somatic cell count as an indicator of milk quality 43
5.4 The significances of the lactoferrin and the beta-lactoglobulin genes 45
5.5 Genetic parameters underlying similarities between nucleotide sequences 46

6.1 Conclusions 48
6.2 Recommendations 49


Figure 1:  A Map showing Kanyariri Veterinary Farm – University of Nairobi 19

Figure 2: Infection status of the udder quarters for lactating dairy cattle genotypes. 26

Figure 3: The image of amplified PCR Products of Beta-lactoglobulin gene 33

Figure 4: the image of amplified PCR Products for Lactoferrin gene 34

Figure 5: The maximum likelihood tree for 31 lactoferrin gene sequences 35

Figure 6: The maximum likelihood tree for 27 beta-lactoglobulin gene sequences 37

Table 1. The interpretation of California Mastitis Test in regards to somatic cell count 9
Table 2: The two pairs of primers which were used in the amplification of Beta-lactoglobulin and Lactoferrin genes respectively 24
Table 3: Multiple Comparison of the effect CMT and SCS across udder’s quarters of the crossbred dairy cattle genotypes 27
Table 4: Mean difference between Somatic Cell Score and Lactation stages of the crossbred dairy cattle. 28
Table 5: Means separation of somatic cell counts between four genotypes of crossbred dairy cows 30
Table 6: SCC means across the udder quarters of the four genotypes of crossbred dairy cows 31
Table 7: Correlation between somatic cell score and the udder quarters of crossbred dairy cattle genotypes. 32
Table 8: Accession Numbers for Different Sequences of Crossbred Dairy Cattle and Nucleotide base sequence variations from 50 to 100 base pairs for Lactoferrin gene 39
Table 9: Accession Numbers for Different Sequences of Crossbred Dairy Cattle and Nucleotide base variations from 09 to 195 base pairs for the beta-lactoglobulin gene 41

Appendix 1: Consumables used during DNA extractions 70
Appendix 2: DNA Mini Extraction kit 70
Appendix 3: vacutainers of blood samples 71
Appendix 4: Milk Samples in sterilized bottles 71
Appendix 5: Gel electrophoresis tank 72
Appendix 6: Some of Crossbred dairy cattle recruited in study 72
Appendix 7: A Polymerases Chain Reactions. 73
Appendix 8: A California Mastitis Test Kit (Paddle) 73
Appendix 8: Heat block which used for incubation of blood samples before extraction of Genomic DNA (ThermoStat plus) 73


𝜇l Microliter
g/L gram per litre
ml millilitre
A Adenine
ANOVA Analysis of Variances
BLAST Basic Local Alignment Search Tool
Blastn Basic Local Alignment Tool against known nucleotides
C Cytosine
CM Clinical Mastitis
CMT California Mastitis Test
DCT Dry Cow Therapy
DF Degree of freedom
DNA Deoxyribonucleic Acid
DMSCC Microscopic Somatic Cell Count
EDTA Ethylene diamine tetra acetic Acid
FAO Food and Agricultural Organization
G Guanine
G-C Guanine - Cytosine
GDP Gross Domestic Products
HSD Honest Significant Differences
MEGA Molecular Evolutionary Genetic Analysis 
NCBI National Centre for Biotechnology Information
LSCS Lactation Somatic Cell Scores
LF Left Fore
LGB Beta-lactoglobulin gene
LH Left Hind
LTF Lacto-transferrin/Lactoferrin
LSD Least Significant Difference
PBS Phosphate Buffered Saline
PCR Polymerase Chain Reactions
RF Right Front
RH Right Hind
SC Somatic Cells
SCC Somatic Cell Counts
SCS Somatic Cell Scores
SPSS Statistical Package for Social Science
T Thymine
Ta Annealing Temperature
Tm Melting Temperature
U Uracil
UoJ University of Juba
USAID United States Agency for International Development
UV Ultra- Violet


1.1 Background information

Mastitis affects dairy production economically through its associated costs and production effects (Gupta et al., 2016; Martin et al., 2018). These costs may include: the price of drugs, reduced milk productions, and household income through the discarding of milk in times of treatment, sudden death, and premature dairy cows culling (Jingar et al., 2017). Mastitis is associated with declines in the quantity of milk produced due to high bacterial loads (Sharma et al., 2011; Jadhav et al., 2016) and the increase in milk somatic cells. These cells include; polymorphonuclear neutrophils, macrophages, lymphocytes, and mammary epithelial cells (Jadhav et al., 2016). The numbers of these cells in milk are used as an indicator to monitor mammary gland health, and their presence can be used to signal mastitic conditions in the udders of dairy cattle (Li et al., 2014; Jadhav et al., 2016).
In sub-Sahara Africa, the prevalence of mastitis among smallholder dairy farmers have been recorded to exceed 50 percent and continues to raise significant threats to dairy producers. In the great lakes region and western parts of Africa, milk production is mostly by smallholder dairy farmers (FAO, 2014). In East Africa, the prevalence of subclinical mastitis varies between 16 and 80 percent. To date, mastitis remains one of the significant factors limiting milk's maximum production by smallholder dairy farmers (FAO, 2014).
Mastitis prevalence has continued to increase despite practices adopted to manage it, such as continuous monitoring for subclinical mastitis, dry cow therapy, and culling of chronically infected cows. Udder health is a valuable trait in dairy cows that need to be maintained for high production of milk and better quality milk that meets consumers' demand in the market (Abebe et al., 2016). The udder health status of a dairy cow is a fundamental factor that boosts production outputs and increases the economic viability of a farm through income earned from the sales of dairy products and less money spent on disease control and prevention (Edwards et al., 2015). Immunity against mastitis is fundamental in addressing the economic challenges faced by smallholder dairy farmers (Fang et al., 2017). Traits associated with mastitis resistance may play a key role in improving milk quality and quantity. Information on systems that underlie these attributes is essential for smallholder dairy farmers' profitability and can be applied in designing selection criteria for dairy cows against mastitis (Peters et al., 2015; Ismail et al., 2018).
Generally, the presence of somatic cells in milk signifies deterioration in milk quality, and the SCCs are internationally used to evaluate milk quality (Sorensen et al., 2016). Breeds of dairy cattle with robust immune systems always produce significant amounts of somatic cells in the milk (Teresiah et al., 2016; Jadhav et al., 2016). However, in some instances, a high milk-producing breed of dairy cattle can produce many somatic cells due to inadequate milking and poor management (Gupta et al., 2016).
Beta-lactoglobulin and lactoferrin genes play significant roles concerning mastitis resistance and tolerance (Fang et al., 2017). The genes have a bactericidal and bacteriostatic action on pathogenic agents of mastitis. As compared to both SCC and SCS, mastitis has a low heritability making it a challenge to select mastitis resistance. Due to this, selective breeding can lower mastitis incidences in crossbred dairy cattle, where suitable candidate genes associated with mastitis resistance are identified (Cai et al., 2018).
Mastitis is a polygenic trait related to milk quality and production traits. These make it complicated for molecular markers to be used in selective breeding for mastitis resistance in dairy breeds of cattle (Tiezzil et al., 2015).
1.2 Problem statement

The low heritability of mastitis poses a significant challenge in selecting dairy cattle against the disease. Although traditional animal breeding and selection are broadly employed in the section for mastitis resistant dairy breeds, there has been no significant result (Curone et al., 2018). Smallholder dairy farmers make massive losses due to subclinical mastitis on their farms. Making dairy enterprises unprofitable (Fonseca et al., 2009; Cameron et al., 2015; Peters et al., 2015). Most of the losses are incurred due to the associated effects of the disease on the farm due to the challenge of diagnosing disease on farms. Inadequate awareness about the impact caused by the subclinical mastitis in dairy production is still a significant obstacle in smallholder dairy enterprise profitability

1.3 Justifications

Mastitis is a disease of economic importance in dairy through its associated effects on dairy production. The condition causes enormous production challenges like the death of dairy cows before attaining their maximum production ages, increased production cost, and declined in production, which in turn affects the returns earned. The frequent use of antibiotic drugs in mastitis treatment and prevention results in their accumulation as residues in animal bodies. These residues find their way into milk, making it unfit for human consumption raising public health concerns (Jadhav et al., 2016). This study was undertaken to evaluate genes associated with mastitis in crossbred dairy cattle, with a view of determining the relationship between California mastitis test scores and somatic cell scores in a sampling population, associations between somatic cell count and lactation stages, and to evaluate the associations between genes and milk quality in different crossbred genotypes of dairy cows.
1.4 Objectives
1.4.1 Overall objective

i. To determine genes associated with mastitis and Somatic Cell Counts in crossbred dairy cattle.

1.4.2 Specific objectives

i. To assess the relationship between California Mastitis Test scores (CMT) and Somatic Cell Counts in different crossbred dairy genotypes.
ii. To evaluate the relationship between genes associated with milk quality and Somatic Cell Counts in crossbred dairy cattle.

1.5 Hypotheses of the study

i. The presence of somatic cell count and California mastitis scores results are highly associated with the presence of subclinical mastitis in udder quarters of crossbred dairy genotypes.
ii. Mastitis is not only a factor that determines the level of somatic cell count present in milk and quality.

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