Discipline: dairy research; Keywords: mastitis, AMR, liver fluke, milk flocculation, genetic and performance analysis.

 Author: Heinz Meissner, Milk SA Program Manager R & D, November 2018.   

1. Mastitis Program:

1.1. PRJ-0210 Antimicrobial resistance on dairy farms - Screening mastitis causing coliforms for the production of extended spectrum Beta-lactamases and Colistin resistance. Dr Inge-Marie Petzer.

The objective is to investigate the presence and antimicrobial resistance  patterns of E. coli strains isolated from clinical mastitis cases. To date a total  of 74 E. coli isolates have been collected, and collection is continuing. This  coincided with collection of information on birth weight, sex, history of  infection including diagnosis and treatment, SCC, and farm factors for all  positive animals identified. Resistance patterns of E.coli strains and Beta  -Lactamases antimicrobial susceptibility is now to be done. The antibiotics  cloxacillin, ampicillin, erythromycin, ceftriaxone, ciprofloxacin, ofloxacin,  gentamicin and kanamycin have been procured in the mean time.  

1.2. PRJ-0211: An investigation into the take-off time in milking machines in South Africa dairies. Dr Inge-Marie Petzer.

The objective is to quantify the relationship of take-off time with the  incidence of mastitis. The project is in the protocol development phase. To that effect a three-day trial was conducted where milking was evaluated with three Vadia machines on three consecutive days with only the take-off times altered. The preliminary data is being evaluated and will be used to complete the protocol.  

1.3. PRJ-0212: Non Staphylococcus aureus (coagulase negative staphylococci) (CNS) as a potential bacterial threat to udder health in South African dairy cows. Dr Inge-Marie Petzer.

The objective is to understand and quantify the potential threat of CNS. All CNS isolates used in this trial have been identified using the MaldiTof (HPLC) and API (biochemical test). A data bank was completed with cow information (days in milk, parity and milk yield) as obtained from producers, and SCC’s for each isolate. Antibiograms (5 different antibiotics) were performed on all isolates. The post graduate student received training in performing the biofilm test. Quite a number of isolates has already been tested for their ability to produce biofilm, despite the funds being received only in the second quarter.  

1.4. PRJ-0215: Bulk Tank Milk Testing. Dr Martin van der Leek/Dr Tracy Schmidt.

The objective is to demonstrate the use of routine bulk tank milk testing for the monitoring of bacterial counts in bulk milk combined with differential cultures and counts to identify the source of high bacterial counts. Testing commenced the first week in September with bulk tank samples being received from all participants. Monitoring is supported with data collected from the testing of all clinical mastitis cases as well as full herd testing as per the original project proposal. Additional test analyses, which were not initially included in the proposal, have been incorporated into the test portfolio to provide additional supportive data. These tests include antibiotic residue screening of BTM samples as well as Bactocount analysis of samples. Biannual microbiological testing of dairy water is also being scheduled. Furthermore, aliquots of all BTM samples have been retained for Mycoplasma bovis testing. A real-time PCR assay has been identified and the necessary test reagents have been procured.

1.5. PRJ-0207: Investigating alternative methods such as bacteriophages and bacteriocins to eliminate/control mastitis organisms. Dr Iona Basdew.

The objective is to support integrated management of bovine mastitis using bacteriophages and bacillomycin therapy, and targeted toward the major mastitic pathogens prevalent in South Africa. To date, the following strains have been positively typed using 16S sequencing and are in store: 5 Streptococcus uberis; 3 S. galactiae; 3 S. dysgalactiae. In addition, there are other cultures of the same species in storage that have been classified according to phenotypic characteristics only. Phages that have been isolated for the genomically characterized microbes are: 9 phages with lytic activity against all five S. uberis, 6 phages with lytic ability against all three strains of S. galactiae, and 8 phages with lytic capability against all three S. dygalactiae. In vitro screening of phages in respect of titer, shelf-life durability, temperature sensitivity, and formulation sensitivity is underway. It is likely that not all of the phages found with these wide host ranges will be robust enough to survive the sensitivity parameters. However, new phages are isolated on a routine basis in order to expand the phage bank, so there is a contingency in the event of the aforementioned occurring. MSc student, Mduduzi Shinga, has successfully completed his MSc study on Alternative Methods of Mastitis Diagnosis and will be graduating in April 2018. The Masters dissertations of Caleb Pillay and Mxolisi Ndlela, will be examined in November 2018.

2. Liver fluke Program:

2.1. PRJ-0204 : Fasciola hepatica: Impact on Dairy Production and Sustainable Management on Selected Farms in South Africa. Dr Jan van Wyk.

The objective is to study the life cycle and favoured environmental conditions  of the snail host, how it is infected by the fluke and as a result how to manage cattle infections more reliably on pasture farms. Over the period of the report a total of 353 further liver samples were analysed, thus totalling 1089 samples completed. A second set of soil samples have also been completed. In terms of statistical analysis of all data, re- formatting of the data, required by the statistician in preparation for the analysis has taken longer than anticipated, but will proceed soon. However, data is still accumulating, with the result that more than one “run” of the analyses will be required, a first aimed at finalising the statistical methods to be used, and re-runs as and when additional data is received. The exceptional duration of the on-farm survey in comparison with previous surveys globally presents an important opportunity for evaluating factors such as: (i) the large differences in rainfall between the different years of the project, from more or less normal in 2015, to a severe drought in 2017; and (ii) the fact that a number of the trial snail habitats included in the longitudinal survey were drained, provided the opportunity to study re-infestation rates. A fifth set of soil samples was collected as from the 9th October, at the time of snail and plant survey sampling, thus totalling three for the year. Laboratory snail culture is progressing very well, including that of the main intermediate host   of Fasciola hepatica, namely L. truncatula, which is considerably more difficult to breed in the laboratory than L. (P.) columella the other species reported to be susceptible to F. hepatica in Europe. Recently some F. hepatica ova and worms have also been obtained from the Tsitsikamma and these are ready for analysis. According to plan, the fluke ova are to be used for infecting principally L. truncatula snails and also some L. (P.) columella and for producing metacercariae (the infective stage of the parasite) for analysis and for ascertaining whether the latter snail species is susceptible to the local strain of F. hepatica populations. Sufficient material has been collected for initiating PCR testing of parasite antigen in the environment once the testing system has been initialised in the laboratory.

2.2. PRJ-0209: Integrated control of Faciolosis (liver flukes) of cattle. Prof Mark Laing/Matthew van Wyngaard.

The objective is to identify a biological control agent antagonistic to the aquatic snails which serve as intermediate host of liver fluke. Bacillus spp. is believed to be potential bacterial antagonists. The aim is therefore to screen a large volume of Bacillus isolates for molluscicidal activity. To this end a population of aquarium snails is maintained in five temperature -controlled breeding tanks. These aquarium snails are being applied as a proxy species to the Lymnae spp. serving as fluke intermediate hosts. Currently the screening of bacterial isolates is progressing with this aquarium snail population only, as Lymnae columnella, a probable host of liver fluke in South Africa, has proved difficult to breed in the laboratory. The bulk of the work currently being undertaken is focused on the large-scale screening of Bacillus spp. isolates from local watercourses in Pietermaritzburg (KZN) and surrounds, in order to test the efficacy of the Bacillus species on the aquatic snails in vitro.

3. Milk flocculation Program:

3.1. AP/NP/D/RM74 (WCDA): The effect of anion/cation difference and potassium content of the diet on heat stability of milk. Prof Robin Meeske.

The objective was to alter the potassium content of pasture to reflect either high or low K+ content by fertilization, as a previous controlled trial where the K content was varied in the concentrate portion of the diet of the cows, showed a high correlation with the alizarol test outcome. Unfortunately the difference between high and low K+ in the pasture was not sufficient to detect measurable differences. In addition it was confounded with interacting P and N levels. After consultation with the Program Manager R & D on the 17th of October, it was decided to abandon the trial.

3.2. PRJ-0217: Further studies to determine the effect of proteolytic enzymes in raw milk on flocculation and gelation. Dr Koos Myburgh.

The objective is to obtain more information on the activation of plasminogen to plasmin, during the reporting period by specifically paying attention to the role of fat and certain free fatty acids (FFA). It was previously found that the by-products (fatty acids) of fat hydrolysis from Pseudomonas lipase enzyme treatment activate the inactive plasminogen to plasmin in homogenized and un-homogenized raw full-cream milk. However, it was not known which of the fatty acids in milk are responsible for this activation. Therefore, the fatty acids naturally present in milk were identified and from that information, various commercial pure free-fatty acids were evaluated to study their individual and combined roles   on the activation of plasminogen to plasmin which is a major cause of age gelation in milk. From the results, it was evident that the FFA’s: butyric acid (C4), hexanoic acid (C6), 2-keto-D-gluconic acid (C6), octanoic acid (C8), decanoic acid (C10), DNP-ε-amino-N-caproic acid (C12), linoleic acid (C18), mystiric acid (C14), palmitic acid (C16), oleic acid (C18:1), stearic acid (C18), and the cocktail of all the free-fatty acids significantly activated plasminogen to plasmin protease. This information is critical to the UHT industry because it means that after UHT treatment when milk is sterile the plasminogen can survive the treatment as well as the FFA`s. Thus within weeks, the plasminogen can be activated and gelation can occur at room temperature. The impact of refrigeration temperatures (4-5°C) on the activation of plasminogen to plasmin protease was also investigated. It was previously established by RP-HPLC and the alizarol test that the presence of plasmin protease and potassium iodate (KIO3), which is the plasminogen activator in raw full-cream milk results in milk flocculation and gelation as early as 24 hour to over 96 hour incubation at refrigeration temperatures (5°C) and at room temperatures (25°C) respectively. In this experiment, full cream milk treated KOI3 and commercial plasmin was incubated at refrigeration temperature (5°C) and also at room temperature (25°C) for several days. Surprisingly more proteolysis occurred in milk during refrigeration temperature compared to milk stored under room temperature according to the RP-HPLC data. The reason for this phenomenon is not clear but it could be that the casein structure shrinks under low temperature which exposes the β-Casein, allowing the plasmin to easily ‘cut it off’, even at very low temperatures. This implies that enormous damage could be inflicted to the casein molecule during milk storage at the farm due to proteinase and lipase activity caused by psychrotolerant bacteria at 5oC. These products of hydrolysis can easily survive UHT and therefore may be responsible for plasminogen activation that digests the UHT milk to cause gelation.

4. Genetic and Performance Monitoring:

4.1. PRJ-0216: Alternative approaches for analysis of production performance from automatic milking systems in SA. Prof Esté van Marle-Köster/Anton Gresse.

The objective of the study was to perform production analyses with the primary aim of constructing a template for extracting and analysing herd performance data from producers employing automatic systems. Two large herd dairy producers representing a total mixed ration (TMR) system, and a pasture-based production system participated in the study. By extracting retrospective animal records from multiple years, comprehensive data tables were constructed for different production analyses. Data was extracted from the AfiFarm herd management software from S.A.E Afikim, Kibbutz, Israel. Analyses included time-trend evaluation of herd numbers, mean production and reproduction performance at the heifer and cow level, distribution of exit reasons and assessing the relationship between the genetic merit of sires and the mean performance of progeny. Findings in this study confirmed that automated management systems permit extraction and analyses of multiple variables imperative to dairy management at the herd and cow level. The software used in these systems has the potential to serve as a platform to add a vast number of dairy cow performance records for future analyses. The results can serve as a template for other producers. A MSc Animal Science thesis was obtained from the investigation.