Potential mechanism of action of J5 vaccine in protection against severe bovine coliform mastitis.

Coliform mastitis is one of the most difficult diseases to treat in the modern dairy industry. Curative therapy with antibiotics remains only moderately effective and depends on the stage at which the disease is treated, The most successful strategies for combating coliform. mastitis appear to be prevention by hygienic management or prophylactic immunization. The severity of clinical symptoms of coliform mastitis has been shown to be reduced by immunization with the Escherichia coli J5 vaccine. However, although the J5 vaccine has been licensed in the United States for about 10 years, the immunological basis of its mechanism of action is still unknown. Until now, protection by J5 vaccination has often been explained by a straight forward mechanism of enhanced antibody production resulting in increased opsonization of coliform bacteria and lipopolysaccharides (LPS). The possibility that J5 vaccination could decrease risk factors for coliform mastitis such as impaired blood polymorphonuclcar neutrophil leukocyte (PMN) diapedesis has never been investigated. This review provides arguments to support the hypothesis that J5 vaccination may reduce the severity of coliform mastitis by inducing a condition of mammary gland hyper-responsiveness, characterized by a T helper 1 (Th1) response and mediated by memory cells inside the mammary gland, finally resulting in enhanced PMN diapedesis upon an intramammary infection

Decreased neutrophil bactericidal activity during phagocytosis of a slime-producing Staphylococcus aureus strain.

Phagocytosis and intracellular killing by bovine polymorphonuclear leukocytes (PMN) are important host defence mechanisms against mastitis caused by Staphylococcus aureus. We compared the phagocytosis and overall killing of a non slime-producing (NSP) S. aureus and its slime-producing (SP) variant by blood PMN, using an in vitro bacteriological assay. Seven clinically healthy Holstein-Friesian dairy cows in mid-lactation stage were used for this purpose. The percentages of overall killing for the NSP and SP variant were 34 +/- 3% and 21 +/- 4% (P < 0.05) and the corresponding percentages of phagocytosis were 40 +/- 4% and 31 +/- 4%, respectively. A significant positive correlation (r = 0.79; P < 0.001) was found between phagocytosis and overall killing. These results suggest that the presence of slime was responsible for a decreased phagocytic ingestion and overall killing

Apoptosis and necrosis of blood and milk polymorphonuclear leukocytes in early and midlactating healthy cows.

Increased milk somatic cell counts (SCC) are used as an indicator for bovine mastitis. During mastitis, polymorphonuclear leukocytes (PMN) become the predominant cell type. Shortly after parturition, the severity of mastitis is increased and several PMN functions are downregulated. Apoptotic and necrotic processes of PMN could influence SCC and PMN functions. In this study, the percentages of apoptotic and necrotic PMN in blood and milk from early and midlactating healthy cows were compared. Apoptosis and necrosis of PMN were quantified using a dual-color flow cytometric procedure with fluorescein labeled annexin-V (green) and propidium iodide (red). Using this technique three different subpopulations of bovine PMN could be detected: apoptotic cells (high intensive green fluorescence), necrotic cells (high intensive green and high intensive red fluorescence) and viable cells (low intensive green and low intensive red fluorescence). Following a 4 h incubation of blood from both groups of cows at 37 degreesC to induce apoptosis, the mean percentage of apoptotic blood PMN was significantly higher (P<0.01) in early lactating cows (15.1%, n=9) compared with midlactating cows (5.3%, n=10). The mean percentage of necrotic PMN remained lower than 5% in all cows. In contrast to blood, no significant difference was found between the percentage of apoptotic PMN in milk from early (41.2%, n=7) and midlactating cows (34.0%, n=8). The percentage of necrotic PMN in milk from early lactating cows (25.9%, n=7) was significantly higher than that in midlactating cows (14.2%, n=8) (P<0.05). Higher percentages of apoptotic as well as necrotic PMN were consistently found in milk compared to blood in all cows. From these results, it can be concluded that spontaneously induced apoptosis was higher in blood PMN from early lactating cows than in blood PMN from midlactating cows. The higher percentage of necrotic milk PMN in early lactating cows than in midlactating cows could be explained by the induction of secondary necrosis

Experimentally induced Escherichia coli mastitis in lactating primiparous cows

Escherichia coli (E. coli) is één van de voornaamste oorzaken van klinische mastitis bij hoogproductieve melkkoeien. De bacterie bezit geen specifieke virulentiefactoren met een rol in de pathogenese van de aandoening. De afweerreactie na experimenteel geïnduceerde E. coli mastitis werd reeds uitgebreid onderzocht bij multipare dieren tijdens de vroege post-partum periode, doch de gevoeligheid en reactiviteit tegenover E. coli van primipare koeien, een niet onbelangrijk deel van de populatie op het melkveebedrijf, werd tot op heden nauwelijks bestudeerd. De doelstelling van dit proefschrift was de studie van de ontstekingsreactie na intramammaire E. coli inoculatie bij primipare koeien. Daarnaast werd het modulerend effect op de ontstekingsreactie door een variatie in inoculum dosis, inhibitie van de prostaglandine synthese en vaccinatie tegenover het endotoxine in hetzelfde experimenteel model onderzocht. Validatie van drie melkstaalname technieken toonde aan dat beperkte bacteriële contaminatie tijdens de bemonstering geen significante invloed had op de verschillende klassieke in vitro functietesten op boviene neutrofielen. Daaruit bleek tevens dat de manuele melkstaalname voldoende garanties zou bieden voor een representatieve staalname tijdens E. coli experimenten. Na de beschrijving van materiaal en methoden aangewend in de verschillende experimentele infecties werd de invloed van pariteit op de ernst van de ontstekingsreactie na experimentele intramammaire E. coli challenge bij primipare koeien onderzocht. Primipare koeien reageerden als ‘moderate responders’, op basis van zowel de kwartiermelkproductie in de niet-geïnfecteerde kwartieren op d+2 na infectie als de klinische ‘severity’ score. De melkproductiedaling was zeer kortstondig en snel herstel van de locale ontsteking werd waargenomen. Een variatie in de inoculum dosis (1 x 104 en 1 x 106 kve E. coli) had een modulerend effect op de ontstekingsreactie, zowel wat betreft de klinische symptomen als de kinetiek van cytokines en merkers van de vroege immuunrespons (IL-8, C5a, sCD14 en LBP). Inoculatie met de hoogste dosis leidde tot een vroegere activatie van de immuunrespons, alhoewel het herstel van de melkproductie niet verschilde tussen beide inoculum doses. Inhibitie van de prostaglandine synthese via de toediening van een COX-2 inhibitor leidde na experimenteel geïnduceerde E. coli mastitis tot een verbetering van de algemene klinische conditie, voornamelijk door een snelle reductie van de koorts en herstel van de voormagenmotiliteit. Daarnaast werd de eicosanoid productie (PGE2 en TXB2) in de geïnfecteerde kwartieren verlaagd. Profylactische vaccinatie tegenover het endotoxine tijdens de late dracht (7 – 9 mnd) gaf geen merkbare verbetering van de klinische conditie, niettegenstaande de gunstige evolutie van de circulerende bloedleukocyten. Vaccinatie was amper in staat om de ontstekingsreactie na E. coli mastitis te moduleren. Primipare koeien tijdens de peripartum periode kunnen als laag risico kandidaten op ernstige klinische mastitis, ten gevolge van E. coli, aanzien worden. De ontstekingsreactie na experimenteel geïnduceerde E. coli mastitis is kortdurend, afgelijnd en geneest zonder behandeling. Een beperkte modulatie van de ontstekingsreactie is mogelijk door variatie van de inoculum dosis of inhibitie van de prostaglandine synthese, terwijl profylactische vaccinatie tegenover het endotoxine weinig effect had in dit infectiemodel

Supplementation of a beta-mannanase enzyme reduces post-weaning diarrhea and antibiotic use in piglets on an alternative diet with additional soybean meal

Enzyme supplementation with a beta-mannanase to degrade beta-mannan fibers present in the diet has been shown to restore and improve performance in swine. The current study was conducted on a farm which had historical episodes of post-weaning diarrhea. In total, 896 newly weaned piglets were enrolled in two consecutive trials. Each trial consisted of 32 pens of 14 piglets housed in one large post-weaning compartment. Piglets at the same feeder were randomly assigned to the two treatment groups. The study compared the performance of post-weaned piglets fed either a commercial 3-phase nursery diet (Control) or an adapted diet supplemented with a beta-mannanase (Hemicell HT; Elanco) (Enzyme), with some of the more expensive proteins replaced by soy bean meal in phase 1 and 2, and net energy (NE) content reduced by 65 kcal/kg in phase 3. All data analyses were performed using R version 3.6.3 (R Core Team, 2020). All tests were performed at the 5% level of significance. When multiple testing was involved, the nominal 5% Familywise Error Rate (FWER) was used. The study showed similar performance on the alternative diet with beta-mannanase and the common commercial diets (P > 0.05). However, the Enzyme treated group had a significantly better general clinical score. Moreover, the number of individual treatments was a factor exp(0.69441) or 2 (CI 95% [1.46; 2.74]) higher (P < 0.001) in the Control group as compared to the Enzyme treated group. The number of treated animals was a factor exp(0.62861) or 1.87 (CI 95% [1.43; 2.53]) higher (P < 0.001) and the number of pigs with a repeated treatment was a factor exp(0.9293) or 2.53 (CI 95% [1.26; 5.09]) higher (P = 0.009) in the Control group as compared to the Enzyme treated group. In total, 7 (1.56%) piglets died in the Control group, whereas only 2 (0.45%) piglets died in the Enzyme treated group. The hazard ratio for mortality in the Control group relative to the Enzyme treated group was and estimated as 1.74 (CI 95% [0.51; 5.96]). Thus, the Control group had a non-significantly (P = 0.375) increased mortality. In conclusion, the results suggest that the use of an exogenous heat-tolerant beta-mannanase allowed reduced levels of expensive protein sources to be used in the first two diets fed post-weaning, and 65 kcal/kg lower net energy content to be used in the third diet without adverse effects on intestinal health or overall performance. In fact, the occurrence of PWD and number of individual treatments during the post-weaning period were significantly reduced on the beta-mannanase supplemented diets

Differential leukocyte count method for bovine low somatic cell count milk

Whereas many differential leukocyte count methods for high somatic cell count (SCC) milk from mastitic cows are available, only a few have been developed for low SCC milk. We have developed a flow cytometric differential leukocyte count method for low SCC milk. The procedure consists of 1) 1.5 ml of diluted milk sample (30%, vol/vol dilution with PBS), 2) centrifugation, 3) leukocyte labeling with SYTO 13 and 4) flow cytometric analysis. Four major leukocyte populations can be clearly identified in the green fluorescence-side scatter dot plot: lymphocytes and monocytes (LM), polymorphonuclear neutrophils (PMN), mature macrophages (MO), and cells with apoptotic features based on chromatin condensation and nuclear fragmentation. The optimal processing temperature was 20degreesC. Significant differences among samples with similar differential leukocyte counts were found. Storage of milk samples during 2 d at 7degreesC had no effect on differential leukocyte count. Using the new method, differential leukocyte count was performed in low SCC milk samples from cows in early, mid, and late lactation. In accordance with previous studies, PMN and M P percentages were lower and LM percentages were higher in early lactation than in the other stages of lactation. The percentage of cells with apoptotic features was higher in early lactation than in mid and late lactation. In conclusion, a rapid, simple, accurate, and reproducible standard procedure was developed to determine the differential leukocyte count (MO, PMN, LM, and cells with apoptotic features) of bovine low SCC milk

Moderate inflammatory reaction during experimental Escherichia coli mastitis in primiparous cows

Nineteen primiparous cows were experimentally infected in 2 quarters with 1 x 10(4) (group A) or 1 x 10(6) (group B) cfu of Escherichia coli P4:O32 per quarter within 2 to 4 wk after parturition. Blood and milk samples were collected from all primiparous cows at regular time intervals from d -4 to d +3 relative to inoculation. Milk production rapidly decreased in both groups during E. coli mastitis, but recovery appeared to be faster in group B at d + 1 postinfusion (p.i.). The milk production losses in the noninfected quarters were substantial on the day of inoculation, which is probably due to pronounced systemic effects. However, on d + 2 p.i. milk production in the noninfected quarters nearly reached preinfection levels, indicating a moderate clinical severity following intramammary inoculation. None of the other severity criteria evolved towards a severe response pattern. Reticulorumen motility was inhibited in both groups during E. coli mastitis. The clinical episode was short lasting in both groups. Rectal temperature, heart rate, blood leukocyte count, number of colony-forming units, milk somatic cell count and several indicators for the disintegration of the blood-milk barrier returned to normal values within 24 to 72 h p.i. Primiparous cows reacted with a moderate inflammatory response following intramammary infusion with a relatively high dose of E. cola. Despite the use of a high inoculum dose, primiparous cows in both groups showed pronounced resistance against severe intramammary E. coli infection. A possible effect of the inoculum dose could be present, however, further research into the effect of the inoculum dose on the inflammatory response should be performed

Serosurvey for viruses associated with reproductive failure in newly introduced gilts and in multiparous sows in Belgian sow herds

A serosurvey for viruses associated with reproductive disorders was conducted in 25 conventional Belgian farms. Serum antibody titers for porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine enteroviruses (PEV) and swine influenza viruses (SIV) were determined in gilts and sows. All the animals were seropositive for PCV2 and >95% were seropositive for all 4 embryopathogenic PEV serotypes. Consequently, special preventive measures appear to be unnecessary for these viruses. In I farm, non-vaccinated gilts were found to run a risk of developing PPV-induced reproductive disorders. Vaccination against PPV could exclude this risk. In 10 farms, gilts seronegative for one or more specific SIV subtypes were introduced into a herd that had previously been infected with the same subtypes. Vaccination of gilts against SIV may prevent reproductive disorders in gilts and respiratory problems in their offspring. In I farm, newly purchased gilts that were possibly shedding PRRSV were introduced into a PRRSV seronegative sow herd. Serological screening prior to purchase or vaccination of the sows could have resolved this dangerous situation

Use of trachea-bronchial swab qPCR testing to confirm Mycoplasma hyopneumoniae seropositivity in an SPF breeding herd

Background: A dedicated program to monitor for freedom of several economically important diseases is present within most of the breeding companies that currently deliver high health breeding animals to their customers. Serology is therefore the preferential approach in order to screen for most of these diseases, including Mycoplasma hyopneumoniae (M. hyopneumoniae). However, in case of positive serology, further decisions on farm health status and the related consequences should be based on additional confirmation tests. Case presentation: The current case report demonstrates that tracheo-bronchial swab (TBS) sampling is a suitable alternative to confirm a suspect M. hyopneumoniae-seropositive situation. A Central-European SPF herd was shown positive (90% positive, 10% suspect; n = 10) for M. hyopneumoniae using the conventional ELISA serology (Idexx HerdChek Mhyo ELISA) and a second ELISA test (IDEIA (TM) Mycoplasma hyopneumoniae EIA kit) did not exclude potential M. hyopneumoniae infection (10% positive, 70% suspect; n = 10). Further follow-up remained inconclusive on both tests. Throughout the entire monitoring period of 6 months, no coughing, necropsy lesions or lesions at slaughter could be detected which could confirm the M. hyopneumoniae health status. TBS sampling was used to confirm the health status for M. hyopneumoniae. In total, 162 samples were collected at different ages (n = 18 per age category): piglets at 3-6-9-12 and 15 wks of age, rearing gilts at 18-21-24 and 27 weeks of age. Collected TBS samples were negative for M. hyopneumoniae until 15 wks of age, but rearing gilts were highly M. hyopneumoniae-positive from 18 wks onwards with 87-100% M. hyopneumoniae-positive animals and PCR Ct-values between 25 and 33. Conclusions: This case report shows that collection of TBS samples to confirm the M. hyopneumoniae infection status of a breeding herd was able to provide additional information to serology in order to make crucial decisions concerning health management and eradication strategies within the breeding herd