Effect of experimentally increased protein supply to postpartum dairy cows on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis.

The effect of experimentally increasing the postpartum protein supply on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis was studied using 8 periparturient Holstein cows in a complete randomized design. At calving, cows were assigned to abomasal infusion of water (CTRL) or casein (CAS) in addition to a lactation diet. Casein infusion was gradually decreased from 696 ± 1 g/d at +2 d relative to calving (DRTC) to 212 ± 10 g/d at +29 DRTC to avoid excessive supply. Synthesis rate of plasma proteins was measured at -14, +4, +15, and +29 DRTC by measuring [C]Phe isotopic enrichment in arterial plasma free Phe, total plasma proteins, and albumin after 3, 5, and 7 h of jugular ring[C]Phe infusion. Plasma volume was determined at +4 and +29 DRTC by dilution of a [I]BSA dose. Synthesis rate of tissue protein in biopsied rumen papillae was determined by measuring [C]Phe isotopic enrichment, and mRNA expression of selected genes was measured by real-time qPCR. Total and differential leukocyte counts were performed and immune responsiveness of monocytes was evaluated by tumor necrosis factor ɑ (TNFɑ) concentration on ex vivo whole blood stimulation with Escherichia coli lipopolysaccharide (LPS) and responsiveness of T-lymphocytes by interferon γ (IFNγ) concentration on stimulation with Staphylococcus aureus enterotoxin ß (SEB). Further, ELISA plasma concentrations of IgM, IgA, and IgG were determined. The DRTC affected the majority of investigated parameters as expected. The CAS treatment increased milk protein yield (P = 0.04), and tended to lower TNFɑ (P = 0.06), and lowered IFNγ (P = 0.03) responsiveness per monocyte and lymphocyte, respectively, compared with CTRL. Further, fractional synthesis rate of albumin was greater at +4 DRTC for CAS compared with CTRL but did not differ by +29 DRTC (interaction: P = 0.01). In rumen papillae, synthesis rate of tissue protein was greater for CAS compared with CTRL (P < 0.01) and mRNA expression of genes for cell proliferation tended to be or were greater for CAS compared with CTRL (P ≤ 0.07). In conclusion, increased postpartum protein supply seem to enhance vital body functions as interpreted from increased liver synthesis of albumin, increased rumen papillae proliferation, and stabilized the ex vivo inflammatory responsiveness of leukocytes. Further studies are needed to enlighten the importance of increased postpartum protein supply in periparturient cows.

Effects of combined liver and udder biopsying on the acute phase response of dairy cows with experimentally induced E. coli mastitis.

A minimally invasive biopsy technique was evaluated for udder tissue collection in dairy cows with Escherichia coli mastitis. Meanwhile, the effect of taking repeated liver and udder biopsies on the systemic and local acute phase response (APR) of the dairy cows was investigated during the disease. The cows were divided into a biopsy group (B) (n = 16) and a no-biopsy group (NB) (n = 16) and were sampled in the acute disease stage and in the recovery stage. The cows' pre-disease period served as a control period for establishing baseline values for the investigated parameters. A total of 32 Holstein-Friesian cows were inoculated with 20 to 40 colony-forming units (cfu) of E. coli in one front quarter at 0 hour. Liver biopsies were collected at -144, 12, 24 and 192 h, and udder biopsies were collected at 24 and 192 h post E. coli inoculation (PI) using a minimally invasive biopsy technique. Effects of combined biopsying were investigated by recording production traits, clinical response, and measuring inflammatory milk and blood parameters: E. coli, somatic cell count, milk amyloid A (MAA) levels, white blood cell count, polymorphonuclear neutrophilic leukocyte numbers and serum amyloid A levels at several time points. E. coli inoculation changed all production parameters and the clinical and inflammatory response in all cows except one that was not infected. Combined biopsying had no constant or transient effect on the daily feed intake, the clinical responsiveness or the blood parameters, but affected the daily milk yield and some milk parameters transiently, that is, the presence of blood in milk, increased E. coli counts and MAA levels during the acute disease stage. Combined biopsying had no effect on the parameters in the recovery stage apart from the presence of blood in the milk. In conclusion, although, a minimally invasive biopsy technique was used, tissue damages could not be avoided when biopsying and they transiently affected the inflammatory parameters in the mammary gland. Nevertheless, we believe combined biopsying of liver and udder is as an acceptable approach to study the systemic and local APR in dairy cows during E. coli mastitis, if the timing of biopsying and other types of sampling is planned accordingly.

Effects of Bos taurus autosome 9-located quantitative trait loci haplotypes on the disease phenotypes of dairy cows with experimentally induced Escherichia coli mastitis.

Several quantitative trait loci (QTL) affecting mastitis incidence and mastitis-related traits such as somatic cell score exist in dairy cows. Previously, QTL haplotypes associated with susceptibility to Escherichia coli mastitis in Nordic Holstein-Friesian (HF) cows were identified on Bos taurus autosome 9. In the present study, we induced experimental E. coli mastitis in Danish HF cows to investigate the effect of 2 E. coli mastitis-associated QTL haplotypes on the cows' disease phenotypes and recovery in early lactation. Thirty-two cows were divided in 2 groups bearing haplotypes with either low (HL) or high (HH) susceptibility to E. coli. In addition, biopsies (liver and udder) were collected from half of the cows (n=16), resulting in a 2 × 2 factorial design, with haplotype being one factor (HL vs. HH) and biopsy being the other factor (biopsies vs. no biopsies). Each cow was inoculated with a low E. coli dose (20 to 40 cfu) in one front quarter at time 0 h. Liver biopsies were collected at -144, 12, 24, and 192 h; udder biopsies were collected at 24h and 192 h post-E. coli inoculation. The clinical parameters: feed intake, milk yield, body temperature, heart rate, respiration rate, rumen motility; and the paraclinical parameters: bacterial counts, somatic cell count (SCC), and milk amyloid A levels in milk; and white blood cell count, polymorphonuclear neutrophilic leukocyte (PMNL) count, and serum amyloid A levels in blood were recorded at different time points post-E. coli inoculation. Escherichia coli inoculation changed the clinical and paraclinical parameters in all cows except one that was not infected. Clinically, the HH group tended to have higher body temperature and heart rate than the HL group did. Paraclinically, the HL group had faster PMNL recruitment and SCC recovery than the HH group did. However, we also found interactions between the effects of haplotype and biopsy for body temperature, heart rate, and PMNL. In conclusion, when challenged with E. coli mastitis, HF cows with the specific Bos taurus autosome 9-located QTL haplotypes were associated with differences in leukocyte kinetics, with low-susceptibility cows having faster blood PMNL recruitment and SCC recovery and a tendency for a milder clinical response than the high-susceptibility cows did.

Gram-typing of mastitis bacteria in milk samples using flow cytometry.

Fast identification of pathogenic bacteria in milk samples from cows with clinical mastitis is central to proper treatment. In Denmark, time to bacterial diagnosis is typically 24 to 48 h when using traditional culturing methods. The PCR technique provides a faster and highly sensitive identification of bacterial pathogens, although shipment of samples to diagnostic laboratories delays treatment decisions. Due to the lack of fast on-site tests that can identify the causative pathogens, antibiotic treatments are often initiated before bacterial identification. The present study describes a flow cytometry-based method, which can detect and distinguish gram-negative and gram-positive bacteria in mastitis milk samples. The differentiation was based on bacterial fluorescence intensities upon labeling with biotin-conjugated wheat germ agglutinin and acridine orange. Initially 19 in-house bacterial cultures (4 gram-negative and 15 gram-positive strains) were analyzed, and biotin-conjugated wheat germ agglutinin and acridine orange florescence intensities were determined for gram-negative and gram-positive bacteria, respectively. Fluorescence cut-off values were established based on receiver operating characteristic curves for the 19 bacterial cultures. The method was then tested on 53 selected mastitis cases obtained from the department biobank (milk samples from 6 gram-negative and 47 gram-positive mastitis cases). Gram-negative bacteria in milk samples were detected with a sensitivity of 1 and a specificity of 0.74, when classification was based on the previously established cut-off values. However, when receiver operating characteristic curves were constructed for the 53 mastitis cases, results indicate that a sensitivity and specificity of 1 could be reached if cut-off values were reduced. This flow cytometry-based technique could potentially provide dairy farmers and attending veterinarians with on-site information on bacterial gram-type and prevent ineffective antimicrobial treatment in mastitis cases caused by gram-negative bacteria.

Sickness behavior in dairy cows during Escherichia coli mastitis.

The consequences of mastitis in terms of dairy cow behavior are relatively unknown. Future assessment of dairy cow welfare during mastitis will be facilitated by knowledge about the potential of mastitis to induce sickness behavior. Our aim was to examine behavior of dairy cows in the period from 2 d before (d -2 and -1) to 3 d (d 0, 1, and 2) after experimental intramammary challenge with Escherichia coli. Effects of experimentally induced mastitis on behavior were examined in 20 primiparous Danish Holstein-Friesian cows, all 3 to 6 wk after calving and kept in tie stalls. After evening milking on d 0, each cow received an intramammary infusion with 20 to 40 cfu of E. coli in 1 healthy front quarter. Paraclinical and bacteriological examinations were conducted to confirm infection. Half of the cows were subjected to liver and udder biopsies twice during the trial. Behavior was video-recorded on 5 consecutive days, d -2 to +2 after challenge when the cows were not disturbed by humans. The behavior of the animals was compared among all days. Infection with E. coli altered the behavior of the dairy cows. Time spent feeding was lower in the initial 24 h after infection compared with that on the other days (16.6±1.1, 16.5±1.0, 13.2±1.2, 18.1±1.1, and 16.0±0.8% of time for d -2, -1, 0, 1, and 2, respectively). The duration of standing idle increased on d 0 compared with that on the control days and d 1 and 2 (29.4±2.6, 28.0±2.3, 39.1±2.6, 31.4±3.8, and 25.9±2.6% of time for d -2, -1, 0, 1 and 2, respectively). The frequency of self-grooming behavior per hour decreased in the initial 24h compared with that on d -2, -1, and 2 (4.1±0.8, 5.4±1.9, 3.2±0.6, 3.6±0.6, and 4.8±1.0 for d -2, -1, 0, 1, and 2, respectively). Likewise, duration of rumination and frequency of turning the head against the udder decreased in the first days after infection (rumination: 32.2±1.6, 34.8±1.8, 27.9±1.7, 30.0±2.6, and 34.8±1.7% of time; and frequency of turning head: 0.6±0.1, 0.6±0.1, 0.3±0.1, 0.3±0.1, and 0.6±0.1 per hour for d -2, -1, 0, 1 and 2, respectively). The cows subjected to biopsies showed an overall decreased lying time during the entire observation period (36.3±1.5 vs. 46.1±2.2% of time) but not directly related to the period after the biopsies. Dairy cows show classic signs of illness behavior in the hours after intramammary challenge with E. coli. This knowledge can be useful for the development of welfare assessment protocols, early disease detection, and for future work aimed at understanding the behavioral needs of dairy cows suffering from mastitis.

Enzyme activity and acute phase proteins in milk utilized as indicators of acute clinical E. coli LPS-induced mastitis.

The importance of non-visual and on-line monitoring of udder health increases as the contact between humans and animals decreases, for example, in robotic milking systems. Several indicator systems have been introduced commercially, and a number of techniques are currently in use. This study describes the kinetics of seven indigenous milk parameters for monitoring udder inflammation in an Escherichia coli lipopolysaccharide (LPS, endotoxin)-induced mastitis model. Proportional milk from LPS-infused quarters was compared with milk from parallel quarters, which were placebo-treated with sterile 0.9% NaCl solution. Somatic cell counts (SCCs), the acute phase proteins (APP), that is, milk amyloid A (MAA) and haptoglobin (Hp), and the enzymes N-acetyl-ß-D-glucosaminidase (NAGase), lactate dehydrogenase (LDH), alkaline phosphatase (AP) and acid phosphatase (AcP) were measured at fixed intervals during the period from -2 to +5 days after LPS and NaCl infusions. All parameters responded significantly faster and were more pronounced to the LPS infusions compared with the NaCl infusions. All parameters were elevated in the proportional milk collected at the first milking 7 h after infusion and developed a monophasic response, except Hp and MAA that developed biphasic response. SCC, LDH, NAGase and Hp peaked at 21 h followed by AP, AcP and MAA peaking at 31 h with the highest fold changes seen for MAA (23 780×), LDH (126×), NAGase (50×) and Hp (16×). In the recovery phase, AP, AcP and Hp reached base levels first, at 117 h, whereas LDH, NAGase and MAA remained elevated following the pattern of SCC. Minor increases of the milk parameters were also seen in the neighboring (healthy) quarters. Distinction between inflamed and healthy quarters was possible for all the parameters, but only for a limited time frame for AP and AcP. Hence, when tested in an LPS mastitis model, the enzymes LDH, NAGase and AP in several aspects performed equally with SCC and APP as inflammatory milk indicators of mastitis. Furthermore, these enzymes appear potent in the assessment of a valuable time sequence of inflammation, a necessary ingredient in modeling of programs in in-line surveillance systems.

Cytokine and acute phase protein gene expression in repeated liver biopsies of dairy cows with a lipopolysaccharide-induced mastitis.

A minimally invasive liver biopsy technique was tested for its applicability to study the hepatic acute phase response (APR) in dairy cows with Escherichia coli lipopolysaccharide (LPS)-induced mastitis. The hepatic mRNA expression profiles of the inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), IL-1beta, IL-6, and IL-10, and the acute phase proteins serum amyloid A isoform 3 (SAA3), haptoglobin (Hp), and alpha(1)-acid glycoprotein (AGP) were determined by real-time reverse transcription-PCR. Fourteen primiparous cows in mid lactation were challenged with 200 microg of LPS (n = 8) or NaCl solution (n = 6) in 1 front quarter. Six repeated liver biopsies were collected at -22, 3, 6, 9, 12, and 48 h relative to LPS challenge in 4 LPS-infused cows and 3 NaCl-infused cows. The remaining cows had 3 liver biopsies taken at -22, 9, and 48 h. Production data and clinical signs were recorded and white blood cell counts and somatic cell counts (SCC) were analyzed to investigate the effect of repeated liver biopsies and verify the LPS model. Plasma concentrations of TNF-alpha, SAA3, Hp, and AGP were determined for comparison with the liver expression data. Repeated liver biopsies had no effects on the production data, clinical signs, or APR of dairy cows. Compared with the NaCl-infused cows the LPS-infused cows responded to the LPS treatment by increased body temperature (38.6 +/- 0.1 vs. 39.4 +/- 0.1 degrees C), short-term leukopenia followed by leukocytosis (6.44 +/- 0.4 vs. 5.69 +/- 0.3 x 10(6) cells/mL), an increased SCC (log(10) 2.1 +/- 0.1 vs. log(10) 2.8 +/- 0.1 x 10(3) cells/mL), heart rate (76 +/- 1 vs. 93 +/- 1 beats/min), and respiratory rate (32 +/- 2 vs. 36 +/- 1 breaths/min) in the acute phase of the disease. The LPS treatment upregulated the hepatic expression of TNF-alpha (103 +/- 24 vs. 255 +/- 18 units), IL-1beta (37 +/- 23 vs. 296 +/- 18 units), IL-6 (8 +/- 17 vs. 122 +/- 12 units), and IL-10 (130 +/- 66 vs. 541 +/- 50 units), and SAA3 (64 +/- 36 vs. 128 +/- 28 units) and Hp (9 +/- 82 vs. 762 +/- 65 units) reaching maximum levels at 3 to 6 h and 9 to 12 h postinfusion, respectively. Plasma concentrations of TNF-alpha (nondetectable vs. 1.9 +/- 0.3 ng/mL), SAA (19.8 +/- 19.4 vs. 149.7 +/- 15.5 microg/mL) and Hp (71.4 +/- 143.7 vs. 1,013.8 +/- 111.5 microg/mL) were elevated in the LPS-infused cows at 4 to 12 h, 8 to 120 h, and 24 to 120 h postinfusion, respectively. The hepatic expression of AGP and the AGP plasma concentration remained unaltered in LPS-induced cows. In conclusion, a minimally invasive liver biopsy technique can be used for studying the hepatic APR in diseased cattle. Lipopolysaccharide-induced mastitis resulted in a time-dependent production of inflammatory cytokines and SAA and Hp in the liver of dairy cows.

Transgenic cows that produce recombinant human lactoferrin in milk are not protected from experimental Escherichia coli intramammary infection.

This is the first study describing an experimental mastitis model using transgenic cows expressing recombinant human lactoferrin (rhLf) in their milk. The aim of the study was to investigate the concentrations in milk and protective effects of bovine and recombinant human lactoferrin in experimental Escherichia coli mastitis. Experimental intramammary infection was induced in one udder quarter of seven first-lactating rhLf-transgenic cows and six normal cows, using an E. coli strain isolated from cows with clinical mastitis and known to be susceptible to Lf in vitro. Clinical signs were recorded during the experimental period, concentrations of human and bovine Lf and indicators of inflammation and bacterial counts were determined for milk, and concentrations of acute-phase proteins and tumor necrosis factor alpha were determined for sera and milk. Serum cortisol and blood hematological and biochemical parameters were also determined. Expression levels of rhLf in the milk of transgenic cows remained constant throughout the experiment (mean, 2.9 mg/ml). The high Lf concentrations in the milk of transgenic cows did not protect them from intramammary infection. All cows became infected and developed clinical mastitis. The rhLf-transgenic cows showed milder systemic signs and lower serum cortisol and haptoglobin concentrations than did controls. This may be explained by lipopolysaccharide-neutralizing and immunomodulatory effects of the high Lf concentrations in their milk. However, Lf does not seem to be a very efficient protein for genetic engineering to enhance the mastitis resistance of dairy cows.

Immune response to a killed infectious bursal disease virus vaccine in inbred chicken lines with different major histocompatibility complex haplotypes.

The influence of MHC on antibody responses to killed infectious bursal disease virus (IBDV) vaccine was investigated in several MHC inbred chicken lines. We found a notable MHC haplotype effect on the specific antibody response against IBDV as measured by ELISA. Some MHC haplotypes were high responders (B201, B4, and BR5), whereas other MHC haplotypes were low responders (B19, B12 and BW3). The humoral response of 1 pair of recombinants isolated from a Red Jungle Fowl (BW3 and BW4) being identical on BF and BG, but different on BL, indicated that part of the primary vaccine response was an MHC II restricted T-cell dependent response. The humoral response in another pair of recombinant haplotypes originating in 2 different White Leghorn chickens being BF21, BL21, BG15 (BR4) and BF15, BL15, BG21 (BR5) on the MHC locus indicated that the BG locus may perform an adjuvant effect on the antibody response as well. Vaccination of chickens at different ages and in lines with different origin indicated that age and background genes also influence the specific antibody response against inactivated IBDV vaccine.

Effects of diet energy density and milking frequency in early lactation on tumor necrosis factor-alpha responsiveness in dairy cows.

A whole blood stimulation assay (WBA) with Escherichia coli lipopolysaccharide (LPS) and an enzyme-linked immunosorbent assay (ELISA) were established to measure the production of tumor necrosis factor-alpha (TNF-alpha) in bovine plasma. The assays were used to study the effect of time around parturition, and diet energy density, and milking frequency on TNF-alpha responsiveness of dairy cows in early lactation. Forty cows were included in a 2 x 2 factorial block design. One factor was high (H) versus low (L) diet energy density and the other factor was two versus three daily milkings. Blood samples were collected in weeks -3, -1, 2, 3, 5, 9, and 13 around parturition, and investigated for the TNF-alpha production ex vivo and CD14+ monocytes. The TNF-alpha response, CD14+ monocyte number, and CD14 expression level on monocytes were significantly increased in the weeks close to parturition. However, dips of varying sizes were observed for the measured parameters in week 3 after calving. Diet and milking frequency had no effect on the TNF-alpha response ex vivo or CD14 expression level on monocytes, but cows fed diet H had significantly higher numbers of CD14+ monocytes than cows fed diet L. The WBA with LPS was a fast reliable method for repeated measurements of TNF-alpha responsiveness in cattle. Previous findings of increased TNF-alpha responses in periparturient cows were confirmed, whereas diet energy concentration and milking frequency had no effect on the TNF-alpha responsiveness in early lactation.

Leukocytes from heifers at different ages express insulin and insulin-like growth factor-1 (IGF-1) receptors.

The objectives of this study were to investigate if insulin receptors (IR) and insulin-like growth factor-1 receptors (IGF-1R) could be detected on bovine leukocytes using fluorescence antibodies and flowcytometry, and use this method to investigate whether the amount of receptors differed among heifers at different ages. Twenty Danish Holstein heifers in the following three age groups were included in the investigation: (1) heifers aged 25-45 days (n = 8), (2) heifers aged 185-205 days (n = 7), and (3) heifers aged 780-900 days (approximately one month prepartum; n = 5). Antibodies against human IR and IGF-1R were used for indirect immunofluorescence staining of cells, and were found to cross-react with the bovine receptors. IR were found on lymphocytes, monocytes, and neutrophils in all animals, while IGF-1R were found only on monocytes and neutrophils. The percentage of IR+ lymphocytes was almost doubled from stage 1 to 3 (34% versus 57%) and IR expression on lymphocytes and monocytes increased significantly (P<0.01) from 6.80 and 13.60 to 8.24 and 17.50 in heifers aged 185-205 days and heifers aged 780-900 days, respectively. No differences were observed for neutrophil IR or IGF-1R expression. In conclusion, surface IR and IGF-1R can be detected on bovine leukocytes by flowcytometry. Interestingly, the highest numbers of IR were found in heifers one month prepartum. The regulation of IR and IGF-1R expression on bovine leukocytes, and their role in host immunity, needs further investigation.

Early pathogenesis and inflammatory response in experimental bovine mastitis due to Streptococcus uberis.

A generally similar clinical response was observed in six lactating Holstein-Friesian cows after intramammary inoculation with approximately 10(7) colony-forming units of Streptococcus uberis. Increased concentrations of serum amyloid A (SAA) were measured in both milk and serum taken 6 and 11h after inoculation, respectively. In contrast, increased concentrations of haptoglobin were detected after 10h of infection, in milk only. In the blood, tumour necrosis factor-alpha (TFN-alpha) was detected (0.503 ng/ml) in only one animal, at the time of euthanasia (10h after infection). Interferon-gamma (IFN-gamma), like haptoglobin, was not detected in blood. Parallel to the development of inflammation and influx of inflammatory cells into the udder tissue, a marked decrease in the number of monocytes and neutrophils in blood was observed. Bacteria were found both intracellularly (macrophages and neutrophils) and within the lumen of ducts and alveoli. Lesions developed progressively in an ascending manner and became widespread throughout the mammary gland in less than 8h. The parallel development of inflammation and increased concentrations of SAA and haptoglobin in milk points to these acute phase proteins as potential diagnostic markers for the early detection of S. uberis -associated mastitis.

An experimental infection model for reproduction of calf pneumonia with bovine respiratory syncytial virus (BRSV) based on one combined exposure of calves.

Bovine respiratory syncytial virus (BRSV) has been recognised as an important pathogen in calf pneumonia for 30 years, but surprisingly few effective infection models for studies of the immune response and the pathogenesis in the natural host have been established. We present a reproducible experimental infection model for BRSV in 2-5-month-old, conventionally reared Jersey calves. Thirty-four colostrum-fed calves were inoculated once by aerosol and intratracheal injection with BRSV. Respiratory disease was recorded in 91% of the BRSV-inoculated calves, 72% had an accompanying rise in rectal temperature and 83% exhibited >5% consolidation of the lung tissue. The disease closely resembled natural outbreaks of BRSV-related pneumonia, and detection of BRSV in nasal secretions and lung tissues confirmed the primary role of BRSV. Nine mock-inoculated control calves failed to develop respiratory disease. This model is a valuable tool for the study of the pathogenesis of BRSV and for vaccine efficacy studies.

Major histocompatibility complex-linked immune response of young chickens vaccinated with an attenuated live infectious bursal disease virus vaccine followed by an infection.

The influence of the MHC on infectious bursal disease virus (IBDV) vaccine response in chickens was investigated in three different chicken lines containing four different MHC haplotypes. Two MHC haplotypes were present in all three lines with one haplotype (B19) shared between the lines. Line 1 further contains the BW1 haplotype isolated from a Red Jungle Fowl. Line 131 further contains the B131 haplotype isolated from a meat-type chicken. Finally, Line 21 further contains the international B21 haplotype. The chickens were vaccinated with live attenuated commercial IBDV vaccine at 3 wk of age, followed by a challenge with virulent IBDV at 6 wk of age. In this study, we found a notable MHC haplotype effect on the specific antibody response against IBDV, as measured by ELISA. The BW1 haplotype was found to have a significantly higher serum antibody titer against IBDV (7,872) than haplotypes B19 (mean 5,243), B21 (5,570), and B131 (5,333) at 8 d postinfection. However, a virus-neutralizing antibody test did not reflect this result. Nevertheless, the MHC haplotype-associated protective immunity was further supported by the bursa of Fabricius (bursa) recovery from the disease, as measured by histological scorings of the bursa. Chickens carrying the BW1 haplotype had a significantly lower bursa lesion score (1.7) than the haplotypes B19 (mean 3.8), B21 (3.6), and B131 (4.3) 8 d postinfection. Furthermore, multiple line effects were found in other variables when comparing Day 6 with Day 8. Body weight, relative weights of the bursa and the spleen, percentage and relative number of MHC II molecules on MHC II-positive lymphocytes, percentage and relative number of CD4 molecules on CD4-positive lymphocytes, and the specific antibody response all differed significantly among lines. Line 1, with Red Jungle Fowl genes, was clearly differentiated from the other two investigated lines. These results suggest an MHC II restricted T-cell dependent secondary antibody response against IBDV.

Increased pulmonary secretion of tumor necrosis factor-alpha in calves experimentally infected with bovine respiratory syncytial virus.

Bovine respiratory syncytial virus (BRSV) is an important cause of respiratory disease among calves in the Danish cattle industry. An experimental BRSV infection model was used to study the pathogenesis of the disease in calves. Broncho alveolar lung lavage (BAL) was performed on 28 Jersey calves, of which 23 were experimentally infected with BRSV and five were given a mock inoculum. The presence of the cytokine tumor necrosis factor alpha (TNF-alpha) in the BAL fluids was detected and quantified by a capture ELISA. TNF-alpha was detected in 21 of the infected animals. The amount of TNF-alpha in the BAL fluid of calves killed post inoculation day (PID) 2 and 4 was at the same very low level as in the uninfected control animals. Large amounts of TNF-alpha were detected on PID 6, maximum levels of TNF-alpha were reached on PID 7, and smaller amounts of TNF-alpha were seen on PID 8. The high levels of TNF-alpha appeared on the days where severe lung lesions and clinical signs were obvious and the amounts of BRSV-antigen were at their greatest. Although Pasteurellaceae were isolated from some of the BRSV-infected calves, calves treated with antibiotics before and through the whole period of the infection, as well as BRSV-infected calves free of bacteria reached the same level of TNF-alpha as animals from which bacteria were isolated from the lungs. It is concluded that significant quantities of TNF-alpha are produced in the lungs of the calves on PID 6-7 of BRSV infection. The involvement of TNF-alpha in the pathogenesis of, as well as the anti-viral immune response against, BRSV infection is discussed.

Transtracheal administration of interleukin-12 induces neutrophil responses in the murine lung.

Although the roles of interleukin-12 (IL-12) in the immunomodulation of antigen-specific responses are well characterized, the effects of IL-12 on the respiratory tract following mucosal administration are not well defined. Therefore, we investigated changes in the murine lung shortly after intranasal (i.n.) administration of murine IL-12. We showed that IL-12 induced neutrophil influx to the murine lung in both C57BL/6 and BALB/c mice. Histologic examination revealed that intranasal administration of IL-12 with liposomes induced focal neutrophil infiltration into the alveoli and a significant increase in neutrophils in bronchoalveolar lavage fluids when compared with administration of liposomes alone. In vitro chemotaxis assays indicated that the observed pulmonary neutrophil response induced by IL-12 could have been due in part to the direct chemotactic activity of IL-12 for murine neutrophils.

Serological and genetic characterisation of bovine respiratory syncytial virus (BRSV) indicates that Danish isolates belong to the intermediate subgroup: no evidence of a selective effect on the variability of G protein nucleotide sequence by prior cell culture adaption and passages in cell culture or calves.

Danish isolates of bovine respiratory syncytial virus (BRSV) were characterised by nucleotide sequencing of the G glycoprotein and by their reactivity with a panel of monoclonal antibodies (MAbs). Among the six Danish isolates, the overall sequence divergence ranged between 0 and 3% at the nucleotide level and between 0 and 5% at the amino acid level. Sequence divergences of 7-8%, 8-9% and 2-3% (nucleotide) and 9-11%, 12-16% and 4-6% (amino acid) were obtained in the comparison made between the group of Danish isolates and the previously sequenced 391-2USA, 127UK and 220-69Bel isolates, respectively. Phylogenetic analysis showed that the Danish isolates formed three lineages within a separate branch of the phylogenetic tree. Nevertheless, the Danish isolates were closely related to the 220-69Bel isolate, the prototype of the intermediate antigenic subgroup. The sequencing of the extracellular part of the G gene of additional 11 field BRSV viruses, processed directly from lung samples without prior adaption to cell culture growth, revealed sequence variabilities in the range obtained with the propagated virus. In addition, several passages in cell culture and in calves had no major impact on the nucleotide sequence of the G protein. These findings indicated that the previously established variabilities of the G protein of RS virus isolates were not attributable to mutations induced during the propagation of the virus. The reactivity of the Danish isolates with G protein-specific MAbs were similar to that of the 220-69Bel isolate. Furthermore, the sequence of the immunodominant region was completely conserved among the Danish isolates on one side and the 220-69Bel isolate on the other. When combined, these data strongly suggested that the Danish isolates belong to the intermediate subgroup.