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Colostrum plays an important role in the transmission of Johne’s disease to calves. Pasteurization of colostrum is an effective way to reduce this risk. Literature showed that a pasteurization at 60°C for 60 minutes gave the best results of reducing the concentration of Mycobacterium avium subspecies paratuberculosis and keeping IgG concentrations high. Most pasteurization studies use a culturing method for detecting differences in pre and post pasteurization samples. The disadvantage of this method is the duration of culture and the relatively high detection limit. Also PCR is used for detecting MAP in colostrum. The disadvantage of this method is the impossibility of making difference between viable and unviable MAP bacteria. In this research a fundament was established for creating a new diagnostic method, based on the differentiation between the MAP specific F57 gene and MbtA gene, by using a restriction enzyme to cut the MbtA gene. The difference between MbtA/F57 ratio of pre and post pasteurization samples will tell the difference of viable/unviable MAP bacteria. Electrophoresis and rtPCR were used in the experiments. Two restriction enzymes proofed to be useful for cutting the pure MbtA gene: Not-1 and Hinf-1. The best results for cutting the pure MbtA gene were given by the Hinf-1 restriction enzyme. Further experiments are required for the development of a new diagnostic method to make the difference between viable and unviable MAP bacteria after pasteurization.

Uptake of Mycobacterium avium subsp. paratuberculosis (MAP) by calves in the first days of life from colostrum, milk and faeces is regarded an important moment of transmission. The objective of this study was to quantify theassociation between the MAP status of dams as determined by the presence of MAP DNA and antibody in colostrum and that of DNA in faeces and the environment with subsequent MAP shedding of their daughters. A cohort of 117dam-daughter pairs giving birth/being born on eight commercial dairy farms with endemic paratuberculosis was followed where colostrum, faecal and environmental samples (dust) were analysed for the presence of MAP using anIS900 real-time PCR. Antibodies in colostrum were measured by ELISA. Analysis of dust samples showed that on all farms environmental MAP exposure occurred continuously. In significantly more colostrum samples (48%) MAP DNAwas detected compared to faecal samples (37%). MAP specific antibodies were present in 34% of the colostrum samples. In total MAP DNA was present in faecal samples of 41% of the daughters at least once during the samplingperiod. The association between faecal shedding in the offspring and the dam MAP status defined by MAP PCR on colostrum, MAP PCR on faeces or ELISA on colostrum was determined by an exact cox regression analysis for discretedata. The model indicated that the hazard for faecal shedding in daughters born to MAP positive dams was not significantly different compared to daughters born to MAP negative dams. When born to a dam with DNA positivefaeces the HR was 1.05 (CI 0.6; 1.8) and with DNA positive colostrum the HR was 1.17 (CI 0.6; 2.3). When dam status was defined by a combination of both PCR outcomes (faeces and colostrum) and the ELISA outcome the HR was1.26 (CI 0.9; 1.9). Therefore, this study indicates that neither the presence of MAP DNA in colostrum, MAP DNA in faeces nor the presence of MAP antibodies in colostrum of the dam significantly influences the hazard of MAPsheddin

Tissue culture was carried out on 30 experimentally MAP inoculated calves in 5 different age groups (2w, 3m, 6m, 9m and 12m) with two different doses and on 3 uninfected calves (control), after euthanasia at 17 months of age. In total 21 tissues per animal were taken from different locations: duodenum, jejunum (mid and distal part), ileum (proximal, mid and distal part), ileocaecal valve, caecum, colon (spiral colon and transverse colon), rectum, lymph nodes corresponding with the previously mentioned gastro-intestinal tract samples (except for the spiral colon, transverse colon and rectum), hepatic lymphnode, tonsil, retropharyngeal lymph node and the superficial inguinal lymph node. After decontamination and 48 days of incubation at 37°C a confirmatory identification of MAP with F57 qPCR was performed. In total 19 of the 31 infected calves (61,3%) were tissue culture positive. The tissue with the highest probability for detection of an infected animal was the ileocaecal valve with 0.19, considered very low. In 45,2% of the infected animals MAP was found in the GI tract tissue, in 25,8% of the animals had positive tissues for the associated lymph nodes of the GI tract and for the other lymph nodes (hepatic, retropharyngeal and superficial inguinal) MAP was found in 12.9% of the animals. Out of the 19 tissue culture positive animals, MAP was detected in 73,7% of the animals with GI tract tissues. Considering that all MAP inoculated animals are successfully infected, which is perhaps not true, the results suggest that tissue culture, considered to be the gold standard, detects a lower percentage of infected animals than expected, this leads to underestimation of test agreements. Tissue samples from multiple sites are necessary to detect 100% of the infected animals. The probability of detection of an infected animal per location is too low to use as single detection method of an infected animal. Dissemination of MAP takes already place in the subclinical stage of JD

The aim of this study was to study which changes were caused in the acid-base balance and electrolytes during fasting. Five healthy lactating cows were selected for the longitudinal study to be monitored over five and a half day. This period was divided into a non fasting period and a fasting period. During the first day the cows could get used to the situation and acclimatize to the environment, on the second day baseline measurements were performed, day three and day four were the fasting period and day five and six were the follow up. Due to the fast, significant decreases were seen in pH and bicarbonate. A significant increase was seen in the total protein concentration. Changes in the electrolyte concentrations were seen but not all of them were significant. The conclusion of this study is that due to the unexpected dehydration a metabolic acidosis occurred. Electrolyte concentrations in plasma remain constant, probably due to the simultaneous occurrence of fasting and dehydration.

Objective This research was executed to examine what is happening with heart rhythm, heart rate, rectal temperature and respiration rate in dairy cattle who are fasting for 48 hours. This is one of two papers wrote about this research. The other paper gives some more information about the acid- bace balance and the electrolytes. Method Five healthy Holstein Frisian dairy cows (mean age 2, 9 years and mean days in lactation 274, 2) were followed for one week. Monday was adaptation day, on Tuesday the normal values were measured and on Wednesday and Thursday the cows were fasting. At Friday, the cows were eating again. Five times every day clinical examination was done: heart rate, respiration rate and rectal temperature were measured. One time a day urine was taken and also one time a day an ECG was made. The ECG was made to detect cardiac dysrhythmia’s. In urine was measured: pH, net acid excretion, S.G., creatinine and potassium. Three times a day blood was taken. In blood was measured: blood- gas values, electrolytes, hematocrit, total protein, BHBZ, creatinine and NEFA’s. Results Heart rate and respiration rate were significantly decreased during fasting. This returns very quickly to normal when the cows were eating again. Rectal temperature decreases also somewhat but this was not significant. At the ECG a prolonged ST segment, a prolonged TQ interval and a prolonged TP interval were seen. Conclusion The decreased heart rate was due to the increased influence of the parasymphatetic nervous system. And the increase of the parasymphatetic nervous system can have many reasons which are not become clear in this study. The abnormalities on the ECG’s were possibly due to a hypokalemia and/or a dysfunction of the potassium channels. The decrease in respiration rate was possibly due to a decrease in stomach filling.

Johne’s disease (JD), also known as paratuberculosis, causes a chronic intestinal infection in ruminants and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Due to Johne’s disease, there is a decrease in milk yield, which causes significant production losses. Since there is no cure available for animals infected with MAP, the main focus of management programs for Johne’s disease is prevention of infection and detecting infected animals with diagnostic tests. The issue with the current serological tests is that they have a low sensitivity. Lately, a paper has been published by Eda et al. (2005) in which a new test has been suggested: the Flow Cytometry Method (FCM). The objective of this study was to make a comparison between the described FCM and the ELISA (IDEXX) to determine the sensitivity and specificity of the FCM as an alternative diagnostic test for Johne’s disease. The hypothesis was that the FCM would have a sensitivity and specificity similar to the ELISA (IDEXX). FCM was optimized in our lab using samples from an infection trial as well as from herds with a known JD status. The results measured by flow cytometry are displayed in graphs. When a sample is negative, a peak in the histogram can be seen above the zero on the x-axis, and thus the histogram will be on the left side of the graph. When a sample is completely positive, the histogram will shift towards the right side of the graph. A wide variety of positivity can be seen in different samples. It was decided to use a cut-off of 70% for the FCM, since this cut-off produced the highest sensitivity (93%) and the highest specificity (84%) for the FCM. When the FCM was compared to the ELISA (IDEXX), the kappa value had an outcome of 0,7760. The positive predictive value is 85% and the negative predictive value is 93%. The FCM is a good candidate to be developed into a sensitive diagnostic test for detection of MAP infection, but further research still needs to be conducted.

Environmental samples were collected to investigate the spatial and temporal spread of Mycobacterium avium subsp. paratuberculosis (MAP) in a dairy cattle barn before and after the introduction of two groups of MAP-shedding animals. Samples collected off the floor of the barn reflected the moment of sampling whereas samples collected by microfiber wipes at a minimal of 3m height contained the accumulated settled dust over a 3-week period. Samples were analysed by IS900 qPCR for the presence of MAP DNA and by culture for viable MAP bacteria. MAP DNA was detected in a large number of sites both before and after introduction cattle. MAP DNA was detected inside the barn in floor and dust samples from cubicles and slatted floors and in settled dust samples located above the slatted floors and in the ventilation ridge opening. Outside the barn MAP DNA was detected by PCR in samples reflecting the walking path of the farmer despite hygiene measures. No viable MAP was detected before the introduction of shedder cattle. Three weeks later viable MAP was found inside the barn at 7/49 locations but not outside. Fifteen weeks later viable MAP was also detected in environmental samples outside the barn. In conclusion, introduction of MAP shedding cattle lead to widespread contamination of the internal and external environment of a dairy barn, including the presence of viable MAP in settled dust particles suggesting potential transmission of MAP infection through bio-aerosols.

Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease in cattle, was identified in settled-dust samples of Dutch commercial dairy farms, both in the dairy barn and in the young stock housing. Bioaerosols may play a role in within-farm M. avium subsp. paratuberculosis transmission.