Diurnal differences in milk composition and its influence on in vitro growth of Staphylococcus aureus and Escherichia coli in bovine quarter milk.

In experimental intramammary inoculation studies, it has been observed that mastitis susceptibility is influenced, among others, by cow factors. To identify milk characteristics leading to these differences, quarter milk samples of morning and evening milk were collected and analyzed for their composition (protein, fat, lactose, urea, lactoferrin, lactoperoxidase, and ß-lactoglobulin concentrations), somatic cell count, and antibodies against Staphylococcus aureus. Furthermore, in vitro growth of S. aureus and Escherichia coli in fresh quarter milk samples was determined. All measured parameters differed significantly between quarters and also between morning and evening milk with the exception of lactose levels. In addition, quantitative growth of S. aureus and E. coli was significantly different in morning milk compared with evening milk. Mixed model analysis revealed that replication of S. aureus was negatively associated with the presence of fat, S. aureus-specific IgG1 antibodies, contamination of the milk sample and morning milk. Replication of E. coli was negatively associated with fat concentrations, and positively associated with morning milk. The significant difference between morning and evening milk supports the theory that changes in milk composition influence bacterial growth. Although all determined milk components differed significantly between quarters and in time no significant association with bacterial growth could be identified with the exception of fat for both studied species and IgG1 titers for S. aureus. The negative association of fat with bacterial growth was assumed to occur due to activation of lipolysis by milk handling and can most likely be neglected for in vivo relevance. The fact that S. aureus-specific IgG1 titers were negatively associated with S. aureus growth in vitro encourages the ongoing effort to develop a vaccine against S. aureus-induced mastitis.

Transmission of Actinobacillus pleuropneumoniae among weaned piglets on endemically infected farms.

Clinical outbreaks due to Actinobacillus pleuropneumoniae occur recurrently, despite the wide-scale use of antimicrobials or vaccination. Therefore, new approaches for the prevention and control of these outbreaks are necessary. For the development of alternative measures, more insight into the transmission of the bacterium on farms is necessary. The aim of this cohort study was to quantify transmission of A. pleuropneumoniae amongst weaned piglets on farms. We investigated three possible transmission routes: (i) indirect transmission by infected piglets within the same compartment, (ii) transmission by infected pigs in adjacent pens and (iii) transmission by direct contact within pens. Additionally, we evaluated the effect of independent litter characteristics on the probability of infection. Two farms participated in our study. Serum and tonsil brush samples were collected from sows pre-farrowing. Serum was analysed for antibodies against Apx toxins and Omp. Subsequently, tonsil brush samples were collected from all piglets from these dams (N=542) in three cohorts, 3 days before weaning and 6 weeks later. Tonsil samples were analysed by qPCR for the presence of the apxIVA gene of A. pleuropneumoniae. Before weaning, 25% of the piglets tested positive; 6 weeks later 47% tested positive. Regression and stochastic transmission models were used to assess the contribution of each of the three transmission routes and to estimate transmission rates. Transmission between piglets in adjacent pens did not differ significantly from that between non-adjacent pens. The transmission rate across pens was estimated to be 0.0058 day(-1) (95% CI: 0.0030-0.010), whereas the transmission rate within pens was ten times higher 0.059 day(-1) (95% CI: 0.048-0.072). Subsequently, the effects of parity and serological response of the dam and litter age at weaning on the probability of infection of pigs were evaluated by including these into the regression model. A higher dam ApxII antibody level was associated with a lower probability of infection of the pig after weaning; age at weaning was associated with a higher probability of infection of the pig after weaning. Finally, transmission rate estimates were used in a scenario study in which the litters within a compartment were mixed across pens at weaning instead of raising litter mates together in a pen. The results showed that the proportion of infected piglets increased to 69% if litters were mixed at weaning, indicating that farm management measures may affect spread of A. pleuropneumoniae.

A cohort study on Actinobacillus pleuropneumoniae colonisation in suckling piglets.

Actinobacillus pleuropneumoniae causes respiratory disease in pigs and despite the use of preventive measures such as vaccination and antimicrobials clinical outbreaks still occur. At weaning often many piglets are not colonised. If differences in prevalence between litters are large and if factors were known that could explain these differences, this may provide an opportunity to raise groups of A. pleuropneumoniae free piglets. To this end, a cohort study was performed on two endemically infected farrow-to-finish farms. Seventy-six of 133 sows were selected using stratified random selection by parity. Farmers complied with a strict hygiene and animal management protocol to prevent transmission between litters. Tonsil brush and serum samples taken three weeks before parturition were tested for antigen with an apxIVA qPCR and antibodies with Apx and Omp ELISAs, respectively. Three days before weaning tonsil brush samples from all piglets (n=871) were collected and tested for antigen. Whereas all sows tested positive both in serology tests as well as qPCR, 0.41 of the litters tested fully negative and 0.73 of all piglets tested negative. The proportion of positively tested piglets in positive litters ranged from 0.08-1.0 (median=0.36). A grouped logistic regression model with a beta binomial distribution of the probability for piglets to become infected was fitted to the data and associations with explanatory variables were explored. To test the possibility that alternatively the clustering was caused by onwards transmission among the piglets, a transmission model was fitted to the data incorporating sow-piglet and piglet-piglet transmission, but this model did not fit better. The results of this study showed that the number of colonised suckling piglets was highly clustered and mainly attributable to the variability of infectiousness of the dam, but no dam related risk factor for colonisation status of litter or piglets within litters could be identified.

Detection of Actinobacillus pleuropneumoniae in pigs by real-time quantitative PCR for the apxIVA gene.

A real-time quantitative PCR (qPCR) for detection of the apxIVA gene of Actinobacillus pleuropneumoniae was validated using pure cultures of A. pleuropneumoniae and tonsillar and nasal swabs from experimentally inoculated Caesarean-derived/colostrum-deprived piglets and naturally infected conventional pigs. The analytical sensitivity was 5colony forming units/reaction. In comparison with selective bacterial examination using tonsillar samples from inoculated animals, the diagnostic sensitivity of the qPCR was 0.98 and the diagnostic specificity was 1.0. The qPCR showed consistent results in repeatedly sampled conventional pigs. Tonsillar brush samples and apxIVA qPCR analysis may be useful for further epidemiological studies and monitoring for A. pleuropneumoniae.

Homologous whole bacterin vaccination is not able to reduce Streptococcus suis serotype 9 strain 7997 transmission among pigs or colonization.

Streptococcus suis (S. suis) is an important porcine pathogen worldwide, and antibiotics are often applied to treat or prevent clinical signs. Vaccination could be an alternative measure to reduce the abundant use of antimicrobials. The aim of this study was to determine the effect of vaccination with homologues whole bacterin vaccine containing S. suis serotype 9 strain 7997 on transmission of this serotype among pigs and on mucosal colonization. Caesarean derived, colostrum deprived pigs (N=50) were housed pair wise. Thirteen pairs were vaccinated intramuscularly with 2-3×10(9) colony forming units (CFU) inactivated S. suis serotype 9 per dose and α-tocopherolactetaat as adjuvant at 3 and 5 weeks of age; twelve pairs served as non-vaccinated controls. At 7 weeks of age, one pig of each pair was intranasally inoculated with 1-2×10(9)CFU of the homologues strain, whereas the other pig of each pair was contact-exposed. Tonsil brushings and saliva swabs were collected for 4 weeks, and tested for the presence of S. suis by bacteriological culture. No differences in number of S. suis in the tonsils or saliva samples or in clinical signs were observed between vaccinated and control pigs. In all pairs, transmission between inoculated and contact exposed pigs occurred, and no difference was observed in rate at which this occurred. The estimated transmission rate parameter ß between vaccinated pigs was ß(v)=5.27/day, and for non-vaccinated pigs ß(nv)=2.77/day (P=0.18). It was concluded that vaccination against S. suis serotype 9 did not reduce transmission, nor colonization and that there were no indications that protection against clinical signs was induced.

Effect of Eimeria acervulina infection history on the immune response and transmission in broilers.

Heterogeneity in exposure to Eimeria spp. of chickens in a flock will result in differences between individual birds in oocyst output and acquired immunity, which subsequently affects transmission of the parasite in the population. The aim of this study was to quantify effects of previous infection of broilers with Eimeria acervulina on immune responses, oocyst output and transmission. A transmission experiment was carried out with pair-wise housed broilers, that differed in infection history. This "infection history" was achieved by establishment of a primary infection by inoculation of birds with 50,000 sporulated E. acervulina oocysts at day 6 of age ("primed"); the other birds did not receive a primary infection ("naïve"). The actual transmission experiment started at day 24 of age: one bird (I) was inoculated with 50,000 sporulated oocysts and was housed together with a non-inoculated contact bird (C). Oocyst excretion and parameters describing transmission, i.e. the number of infected C birds and time passed before start of excretion of C birds, were determined from day 28 to day 50 for six pairs of four different combinations of I and C birds (I-C): naïve-naïve, naïve-primed, primed-naïve and primed-primed. Immune parameters, CD4(+), CD8(+), αßTCR(+) and γδTCR(+) T cells and macrophages in duodenum, were determined in an additional 25 non-primed, non-inoculated control birds, and in the naïve-naïve and naïve-primed groups, each group consisting of 25 pairs. Although the numbers of CD4(+) T cells and γδTCR(+) T cells increased after primary infection, none of the immunological cell types provided an indication of differences in infectivity, susceptibility or transmission between birds. Oocyst output was significantly reduced in primed I and C birds. Transmission was reduced most in the primed-primed group, but nonetheless transmission occurred in all groups. This study also showed that acquired immunity significantly reduced oocyst output after inoculation and contact-infection, but not sufficiently to prevent transmission to contact-exposed birds.

Blastocyst morphology, actin cytoskeleton quality and chromosome content are correlated with embryo quality in the pig.

Embryo survival rates obtained after transfer of in vitro produced porcine blastocysts are very poor. This is probably related to poor quality of the embryos. The aim of the present study was to determine markers for good quality blastocysts. Therefore, we tried to link blastocyst morphology to several morphological and cell biological properties, and evaluated the survival of in vitro produced, morphologically classified, blastocysts following non-surgical transfer. In vitro and in vivo produced blastocysts were allocated to two groups (classes A and B) on the basis of morphological characteristics. The quality of their actin cytoskeleton, their total cell number, their ability to re-expand after cytochalasin-B treatment and the occurrence of numerical chromosome aberrations were studied and compared. In vivo produced blastocysts were used as a control. Our results indicate that the ability of blastocysts to re-expand after cytochalasin-B-induced actin depolymerization was positively correlated with the morphology of the blastocyst, and associated with the quality of the actin cytoskeleton. Chromosome analysis revealed that mosaicism is inherent to the in vitro production of porcine embryos, but also that in vivo produced blastocysts contained some non-diploid cells. In non-surgical embryo transfer experiments more recipients receiving class A blastocysts were pregnant on Day 20 than those receiving class B blastocysts. One recipient gave birth to six piglets from class A in vitro produced blastocysts, providing a verification of the enhanced viability of blastocysts that were scored as 'good' on the basis of their morphology.

Expression of bone morphogenetic protein2 (BMP2), BMP4 and BMP receptors in the bovine ovary but absence of effects of BMP2 and BMP4 during IVM on bovine oocyte nuclear maturation and subsequent embryo development.

Bone morphogenetic proteins (BMPs) have been implicated in the regulation of ovarian follicular development and are promising candidates to apply in IVM and IVF protocols. We investigated the expression of BMP2, BMP4 and BMP receptors in bovine ovaries and the effects of BMP2 and BMP4 during oocyte maturation on bovine IVM. Reverse transcription polymerase chain reaction studies with antral follicles showed the expression of BMPR-IA, BMPR-IB, ActR-IA, ActR-IIB, BMPR-II and BMP4 mRNA in all follicular compartments, while BMP2 mRNA was generally restricted to theca and cumulus tissue. Immunohistochemistry demonstrated the presence of BMPR-II in oocytes and granulosa cells of preantral follicles but only in oocytes of antral follicles. The immunostaining of BMP2 and BMP4 was limited to theca interna and approximately 25% of oocytes of antral follicles. Exogenously added BMP2 or BMP4 to IVM medium did not affect oocyte nuclear maturation, cumulus cell expansion, nor blastocyst formation following IVF. It is concluded that a BMP-signaling system, consisting of BMP2, BMP4, type II and I receptors, is present in bovine antral follicles and that this system plays a role in development and functioning of these follicles rather than in final oocyte maturation and cumulus expansion.

Bacterial growth during the early phase of infection determines the severity of experimental Escherichia coli mastitis in dairy cows.

The aim of this study was to investigate the importance of bacterial growth for the severity of experimental Escherichia coli mastitis, indirectly expressed as the area under the curve of bacterial counts in milk over time. The association of pre-infusion somatic cell count and post-infusion influx of inflammatory cells in milk with severity of infection was also examined. Bacterial growth was studied through culture in milk samples (in vitro) and through monitoring of bacterial counts in milk during the early phase of infection (in vivo) in 36 cows. Individual variation in bacterial counts was more than 2 x 10(2)-fold after 6 h of in vitro incubation, and more than 8 x 10(2)-fold 6 h after intramammary infusion. In vitro growth in milk was not associated with in vivo growth during the early phase of infection, nor with severity of E. coli mastitis. Somatic cell count before experimental E. coli mastitis was negatively associated with in vivo bacterial growth during the early phase of infection (R2 = 0.28), but was not associated with severity of E. coli mastitis (R2 = 0.06). In vivo bacterial growth during the early phase of infection (positive association; R2 = 0.41), together with influx of inflammatory cells in milk, expressed as mean hourly increase of somatic cell count between 6 and 12 h post-infusion (negative association; R2 = 0.11), are major determinants for the severity of experimental E. coli mastitis (R2 = 0.56).

In vitro growth of mastitis-inducing Escherichia coli in milk and milk fractions of dairy cows.

The outcome of E. coli mastitis in cows ranges from mild to severe in individual animals. This study explored the hypothesis that milk from individual cows differs in its growth medium properties for E. coli, and whether possible variation could be related to specific milk constituents. To mimic the early phase of intramammary E. coli infection, a low inoculum size and a short incubation period were used. Cell-reduced, cell- and fat-free (skim) and cell- and fat-free and protein-reduced (whey) fractions were prepared from whole milk samples (n=18). Ten ml of whole milk, milk fractions and brain heart infusion broth (BHI) were inoculated with approximately 100cfu E. coli. After 6h of incubation, bacterial counts were assessed by dilution plating in triplicate. Bacterial counts in whole milk differed up to a 100-fold between cows, which was not associated with SCC. Bacterial counts were significantly higher in whey fractions than in whole milk, cell-reduced and skim fractions and variation in whey was smaller, indicating that the acid-precipitable protein fraction contains the milk constituents of major relevance for inhibition of and variation in bacterial growth. The presence of fat and cells added to bacterial growth inhibition to a lesser extent. In conclusion, in vitro growth of E. coli in milk differs substantially between individual cows within an incubation period comparable with the early phase of intramammary infection. This suggests that the growth medium properties of milk could be of importance in the pathogenesis of E. coli mastitis and subsequent outcome of disease.

Beta-hydroxybutyrate levels in peripheral blood and ketone bodies supplemented in culture media affect the in vitro chemotaxis of bovine leukocytes.

The role of ketone bodies on chemotactic capacities of leukocytes was characterized in two experiments. Experiment I was performed to investigate the association between serum beta-hydroxybutyrate concentrations (BHB) and in vitro chemotaxis of leukocytes. Cows were divided into low-BHB, medium-BHB, and high-BHB ones and classified according to their BHB. Leukocytes from high-BHB cows had a significantly lower chemotactic differential than leukocytes from low-BHB cows (p < 0.01). The effect of adding ketone bodies into in vitro chemotaxis cultures on leukocytes chemotaxis was studied in Experiment II. Either individual or a combination of commercial ketone bodies - sodium salts of BHB (BHBA), lithium salt of acetoacetate (ACAC), and acetone (Acetone) - were diluted in culture media and divided into eight concentrations corresponding to concentrations of bovine subclinical and clinical ketosis. For leukocytes from medium- and high-BHB cow, the chemotactic indexes of leukocytes were reduced by ACAC and Acetone. Chemotactic differentials of cultures with ACAC and acetone supplementation from both sources of leukocytes were significantly lower than that of the control culture (p < 0.05). For leukocytes from high-BHB cows, chemotactic indexes were suppressed in a ketone-body environment. In conclusion, leukocytes from naturally-occurring ketotic cows have lower chemotactic differentials than those from non-ketotic cows, and a chemotactic capacity indicated by a chemotactic differential is impaired when leukocytes migrate in an environment with ketone bodies in vitro.

Preinfection in vitro chemotaxis, phagocytosis, oxidative burst, and expression of CD11/CD18 receptors and their predictive capacity on the outcome of mastitis induced in dairy cows with Escherichia coli.

Four to 6 wk after parturition, 12 cows in second, fourth, or fifth lactation were experimentally infected in one gland with Escherichia coli. The capacity of chemotaxis, phagocytosis, oxidative burst, and expression of CD11/CD18 receptors to predict the severity of IMI was measured. Bacterial counts in the infected quarter, expressed as area under the curve, and residual milk production in the uninfected quarters were compared to determine severity of the infection. Although these two outcome parameters were highly negatively correlated, regression models with preinfection tests for leukocyte function fitted best with bacterial counts as an outcome parameter. Of the preinfection tests for leukocyte function, chemotaxis best predicted the outcome of the IMI that had been experimentally induced by E. coli. The number of circulating peripheral leukocytes just prior to inoculation was used to predict 52 and 45% of the severity of IMI for bacterial counts and residual milk production, respectively. As a categorical variable, parity predicted 75 and 56% of the severity of IMI expressed as bacterial counts and residual milk production, respectively. Because of the strong effect of parity on the outcome of the experimentally induced mastitis, analysis was performed to discriminate between second parity cows and older cows. Significant differences were found for the number of circulating peripheral leukocytes and for the expression of CD11b/CD18 and CD11c/CD18 receptors between younger and older cows.

Within day and between day variation of the in vitro under agarose chemotaxis assay in bovine.

The present study was conducted to determine within day variation (experiment I) and between day variation (experiment II) of the in vitro under agarose chemotaxis assay. Further, results from experiment II were used to estimate a more stable immunological parameter for the chemotactic activity. In experiment I, blood samples of eight cows were taken every 4 h starting at 0800 during a 24 h period. This procedure was replicated on three different days with peripheral white blood cells of lactating bovine. Chemotactic differential showed variation within a day. The differences between samplings were not constant over the days, but varied randomly from day to day. In experiment II, 12 cows were followed for 8 consecutive days and blood samples for chemotaxis assay were taken once a day at 0730. Differences between the days were significant. With a conditional auto regression model of the first order adjusted least squares means of each cow were estimated over the 8 consecutive days. The chemotactic value of a day was used to estimate the value of the next day. Expanding the model with more previous days did not improve the model. The results of this study indicate that blood samples for chemotaxis should be taken at the same time of the day to control for within day variation. If a sequence of chemotactic values is available we strongly suggest working with adjusted least square means of chemotactic differentials. These adjusted means show less random variation and are a more stable parameter for chemotactic activity.

Onset of transcription in Patella vulgata coincides with cell cycle elongation and expression of tubulin genes.

In this study we show that the onset of embryonic transcription in the marine snail Patella vulgata coincides with the start of the sixth cleavage, when the cell-cycle elongates and divisions become asynchronous. Changes in mRNA content before and after onset of transcription were initially demonstrated by in vitro translation of isolated mRNA from different stages. Before the sixth cleavage, three major mRNAs encoding proteins of 36, 50 and 52 kDa were present. These proteins probably correspond to cyclin A and B and ribonucleotide reductase. After this stage, three major proteins with molecular weights of 36.5, 52.5 and 53 kDa were found after in vitro translation. Via hybrid selected translation and differential screening cDNAs corresponding to the 52.5 and 53 kDa proteins were cloned. The encoded proteins resemble tubulins from other animals to a high extent (between 96.5 and 93.1% identity for α-tubulin and 97.9 and 75.9% for ß-tubulin). The 36.5 kDa protein is the previously described actin. Both tubulins were expressed at or shortly after the first asynchronous division after the fifth cleavage.

Blood polymorphonuclear leukocyte chemotaxis during experimental Escherichia coli bovine mastitis.

The relationship between the severity of experimental Escherichia coli mastitis and the chemotactic response of blood polymorphonuclear leukocytes was investigated before and during mastitis. Experimental E. coli mastitis was induced in 10 healthy cows by inoculation of the rear right quarters with 10(3) cfu of E. coli. Cows were classified into two groups based on the severity of the mastitis. Bacterial growth in the inoculated quarter was used as parameter that indicated severity. Before and during experimental mastitis, the chemotactic response and the number of circulating polymorphonuclear leukocytes were greater for the moderately diseased cows than for the severely diseased cows. During the first 24 h of the experimental mastitis, the chemotactic response of polymorphonuclear leukocytes decreased in both groups. Recovery of the chemotactic response of white blood cells was more rapid in moderately diseased cows than in severely diseased cows. Possibly, the larger proportion of band neutrophils (the less chemotactically active band neutrophils) partially accounts for the lower chemotactic response of the circulating polymorphonuclear leukocytes during experimental mastitis in the severely diseased cows.

Preinfection chemotactic response of blood polymorphonuclear leukocytes to predict severity of Escherichia coli mastitis.

Experimental mastitis was induced by inoculating rear right quarters of 10 healthy cows with 10(3) cfu of Escherichia coli. The chemotactic responses of peripheral blood polymorphonuclear leukocytes at d-6, -5, -2, -1, and immediately prior to inoculation were measured. Chemiluminescence of polymorphonuclear leukocytes was measured immediately prior to inoculation. Severity of the experimental mastitis was assessed by bacterial growth in the inoculated quarters. Results of this study indicated that severity of the experimental mastitis may be predicted by the chemotactic response in vitro of polymorphonuclear leukocytes isolated from the peripheral blood at d 2, d 1, and immediately prior to inoculation. The number of circulating polymorphonuclear leukocytes immediately prior to inoculation also showed a negative relationship with the severity of mastitis. No relationship existed between preinfection chemiluminescence of polymorphonuclear leukocytes and the severity of the experimental mastitis.

Actin genes expressed during early development ofPatella vulgata.

The actin gene family of the marine molluscPatella vulgata was chosen as a model system to study the regulation of genes expressed during early development in molluscs. Using a hamster actin cDNA clone as a probe, we isolated nine actin cDNA clones from trochophore larvae. The total nucleic acid sequence of three of these clones has been determined. Each clone contains the whole protein encoding region. The deduced amino acid sequences resemble actin proteins from other species to a high extent. The nucleotide sequence from the 3'UTR (UnTranslated Region) and 5'UTR from all nine clones has been resolved. In this way we could identify four different subtypes. Southern blots with genomic DNA were probed with different 3'UTR's corresponding to each subtype to determine the genomic organization. One 3'UTR detected one band probably corresponding with one gene. Another 3'UTR detected one or two genes and the third 3'UTR between two and four genes. Northern blots were used to detect the presence of actin mRNA during different stages of development. In the mature oocyte, actin mRNA is present in low amounts. The level of actin mRNA starts to rise steadily from 8 h after fertilization (88-cell stage) onwards. The level of the different subtype mRNAs, as specified by their 3'UTR rises at different developmental stages and to various extents. This indicates that the expression of each type is regulated independently and in relation to the developmental stage of the embryo.

Influence of a skin allograft on frequencies of CTL precursor in peripheral blood of the dog.

Alloantigen-specific cytotoxic T lymphocytes (CTL) and their precursors (CTL-P) have been determined in the peripheral blood of skin allografted dogs. CTL-P frequencies increased rapidly after transplantation and reached maximal values after complete rejection of the skin allograft. Differences in the time response kinetics of CTL-P frequencies between recipients were not correlated with the length of graft survival. The CTL-P frequencies declined after days 13-20 and appeared still to be elevated 30 days after rejection of the graft.

Cell-mediated cytotoxicity toward class I and class II antigens on canine T and B lymphoblasts.

Cellular cytotoxicity generated in vitro has been studied with canine T and B lymphoblasts as target cells. B lymphocytes were isolated by rosetting with protein A labeled sheep red blood cells and stimulated with sepharose bound protein A. Such highly purified stimulated B lymphoblasts appeared to be useful target cells for cellular cytotoxicity. Cold target inhibition studies and limiting dilution analysis revealed that B lymphocytes did not carry targets for cytotoxic T lymphocytes (CTL) which were not present on PHA stimulated T lymphoblasts.