Infection following challenge of the lactating and dry udder of dairy cows with Actinomyces pyogenes and Peptostreptococcus indolicus

Challenge of 12 mammary glands of cows in mid-lactation with 10(7) colony forming units (cfu) of Peptostreptococcus indolicus on two occasions led to clinical mastitis in only four quarters. The bacteria were rarely recovered and disappeared from the secretion within 14 days. In challenges 7 days prior to drying off eight of 12 quarters became infected and at drying off all quarters challenged became infected. The infections established at drying off persisted well into the dry period. P. indolicus infection was also established in all of 12 dry glands challenged, but usually eliminated at calving or early in the next lactation. Isolation of P. indolicus was accompanied in about one-third of cases by changes in the appearance of the secretion. Intramammary challenge with Actinomyces (formally Corynebacterium) pyogenes led to clinical and subclinical infections in nine of 12 lactating glands and in all of six dry glands. Dry period infections with A. pyogenes were more severe and rarely eliminated even by antibiotic therapy. Infections during lactation were often eliminated either naturally or by antibiotic therapy. Intermittent recovery of A. pyogenes from the lactating mammary gland, without clinical signs of infection, was possible for up to 90 days after challenge. Combined infections with A. pyogenes and P. indolicus were clinically more severe with a higher frequency of systemic involvement. It was shown that in the non-lactating gland an acute mastitis, similar to 'summer mastitis' could be established either by simultaneous inoculation with A. pyogenes and P. indolicus or by subsequent inoculation of quarters excreting P. indolicus with A. pyogenes.     

The use of polyethylene intramammary device in protection of the lactating bovine udder against experimental infection with Staphylococcus aureus or Streptococcus agalactiae.

The susceptibility of lactating bovine udder quarters fitted with a polyethylene intramammary device to infection was investigated. Following experimental challenge with Streptococcus agalactiae or Staphylococcus aureus, the incidence of infection was significantly (p less than 0.05) lower in intramammary device-fitted quarters compared to control quarters. In general, total foremilk and strippings milk somatic cell counts for intramammary device-fitted and control quarters were not significantly (p less than 0.05) different. Differential foremilk and strippings milk somatic cell counts were significantly (p less than 0.05) higher in samples from intramammary device-fitted quarters compared to control quarters.     

Immunological responses following experimental endobronchial infection of badgers (Meles meles) with different doses of Mycobacterium bovis

The Eurasian badger (Meles meles) is a wildlife reservoir for Mycobacterium bovis infection in Ireland and Great Britain and has been implicated in the transmission of tuberculosis to cattle. Vaccination of badgers is an option that could be used as part of a strategy to control the disease. In this study we used an endobronchial infection procedure to inoculate groups of badgers with three different doses (3x10(3), 2x10(2) and <10 Colony Forming Units (CFUs)) of M. bovis. After 17 weeks the disease status of each animal was determined by post-mortem pathology and culture for M. bovis. Each of the inoculum doses resulted in establishment of infection in the badgers. The cell-mediated immune (CMI) responses were measured by lymphocyte transformation assay (LTA) of peripheral blood mononuclear cells (PBMCs) cultured with bovine tuberculin (PPD-B). In each infected group the CMI responses increased with a kinetic profile corresponding to the delivered dose and the post-mortem pathology. The serological responses were measured by ELISA and a multi-antigen print immunoassay (MAPIA) in order to investigate any changes in the antigenic repertoire associated with different infective doses. In contrast to the CMI responses, the ELISA and MAPIA showed that the recognition of antigens by the badgers was intermittent and not strongly influenced by the dose of M. bovis.     

Immunological and pathological responses of red deer resistant or susceptible genotypes, to experimental challenge with Mycobacterium avium subsp. paratuberculosis

This study aimed to monitor the clinical, immunological and pathological changes in red deer for 49 weeks after experimental oral challenge with Mycobacterium avium subsp. paratuberculosis (MAP) and to assess the heritability of resistance in the offspring of two red stags. Eighteen young deer, which were bred from unselected hinds and sired by two stags resistant (R) or susceptible (S) to paratuberculosis, were challenged with MAP and monitored for 49 weeks. Biopsy samples of the jejunal lymph node were collected at Weeks 4 and 13 and at necropsy after euthanasia of clinically affected animals or when electively killed at Week 49. Three animals (two S and one R) developed clinical disease and were euthanised. The nine S offspring had significantly more severe lesions than the nine R offspring (Mantel-Haenszel Chi-square P=0.017). The average Lesion Severity Score (LSS) of R offspring was 5.9 (mild), and 7/9 had no or very mild lesions. In contrast, the LSS of S offspring averaged 11.7 (severe), and 7/9 had severe lesions. Most of the resistant, but not the susceptible, animals showed evidence of resolving lesions and a reduction in the number of MAP between 13 and 49 weeks after challenge. One R offspring appeared to completely cure itself, and progressed from mild culture-positive paratuberculosis lesions at Week 13 to having no signs of disease or infection 36 weeks later. This study showed significant heritable resistance/susceptibility to paratuberculosis and key differences in immunological responses in the first 3 months after challenge, indicating different paths to relative success or failure to control MAP. In general, R deer had higher IFN-γ levels, low antibody titres and fewer MAP, while S deer had lower IFN-γ levels, higher antibody and more MAP.     

Maternal and foetal immune responses of cattle following an experimental challenge with Neospora caninum at day 70 of gestation

The immune responses of pregnant cattle and their foetuses were examined following inoculation on day 70 of gestation either intravenously (iv) (group 1) or subcutaneously (sc) (group 2) with live NC1 strain tachyzoites or with Vero cells (control) (group 3). Peripheral blood mononuclear cell (PBMC) responses to Neospora antigen and foetal viability were assessed throughout the experiment. Two animals from each group were sacrificed at 14, 28, 42 and 56 days post inoculation (pi). At post mortem, maternal lymph nodes, spleen and PBMC and when possible foetal spleen, thymus and PBMC samples were collected for analysis. Inoculation with NC1 (iv and sc) lead to foetal deaths in all group 1 dams (6/6) and in 3/6 group 2 dams from day 28pi; statistically significant (p ≤ 0.05) increases in cell-mediated immune (CMI) responses including antigen-specific cell proliferation and IFN-γ production as well as increased levels of IL-4, IL-10 and IL-12 were observed in challenged dams compared to the group 3 animals. Lymph node samples from the group 2 animals carrying live foetuses showed greater levels of cellular proliferation as well as significantly (p ≤ 0.05) higher levels of IFN-γ compared to the dams in group 2 carrying dead foetuses. Foetal spleen, thymus and PBMC samples demonstrated cellular proliferation as well as IFN-γ, IL-4, IL-10 and IL-12 production following mitogenic stimulation with Con A from day 14pi (day 84 gestation) onwards. This study shows that the generation of robust peripheral and local maternal CMI responses (lymphoproliferation, IFN-γ) may inhibit the vertical transmission of the parasite.     

The susceptibility of bovine udder quarters colonized with Corynebacterium bovis to experimental infection with Staphylococcus aureus or Streptococcus agalactiae.

Twenty bovine udder quarters colonized with Corynebacterium bovis SR6 and 20 uncolonized quarters were challenged by inoculation of Staphylococcus aureus Newbould 305 (ATCC 29740) into the teat cistern. The percentage of infection in quarters colonized with C. bovis (50%) was significantly lower than that in controls (100%). By similar challenge no significant difference was observed between the percentage of infection with Streptococcus agalactiae ATCC 27956 in 33 quarters colonized with C. bovis (70%) compared to 33 controls (87.9%). A total of 37 quarters colonized with C. bovis and 37 control quarters were challenged with Staph. aureus Newbould 305 (ATCC 29740) and Maxi (ATCC 27543) and Strep. agalactiae (ATCC 27956) by exposure of the teat orifice. The percentage of teat ducts colonized with C. bovis which became infected with either pathogen was not different from that for controls.     

Immune cell kinetics in the ovine abomasal mucosa following hyperimmunization and challenge with Haemonchus contortus

Sheep were sensitized by repeated infection with Haemonchus contortus L3, followed by a 12 week rest period, and an abomasal cannula was surgically implanted in all sheep. Seven of the sensitized sheep were subsequently challenged with 50 000 H. contortus L3 while 4 control sheep were challenged with saline. Biopsy samples were taken using a fibreoptic endoscope on days 0, 1, 2, 3, 5, 7 and 28 after challenge and leukocyte subpopulations quantified by (immuno)histology. Differential blood cell counts were performed on the same days. At the end of the trial, sheep showed significantly reduced worm burdens compared to unsensitized control sheep, confirming their resistance status. Both blood and tissue eosinophils, as well as tissue γδ TCR⁺ cells were rapidly elevated by day 1 post L3 challenge (pc), peaking at day 3 pc. There was a slight increase in tissue CD4 T cells at day 2 pc, peaking at day 3 pc while no significant changes in CD8 T cells were observed. B cells (CD45R⁺) increased later into challenged tissues with a peak at 5 days pc. All tissue lymphocyte subpopulations as well as tissue and blood eosinophils were reduced by day 7 pc before increasing again at day 28 pc, suggesting separate responses to larval and adult antigens. In contrast, globule leukocytes and mucosal mast cells only showed one peak at day 5 pc and 28 pc, respectively. Unexpectedly, globule leukocytes correlated significantly with tissue eosinophils but not mucosal mast cells. The results are consistent with an early eosinophil-mediated killing of L3, possibly recruited by IL-5 produced by γδ T cells. In contrast to post-mortem studies, abomasal cannulation allowed sequential analysis of both early and late time points in the same animal, providing a more complete picture of cellular interactions at both peripheral and local sites, and their correlation with the different stages of parasite development.     

Development of maternal and foetal immune responses in cattle following experimental challenge with Neospora caninum at day 210 of gestation

This study examined the immunological responses of pregnant cattle and their foetuses following an experimental challenge with live Neospora caninum tachyzoites at day 210 of gestation. Animals were bled prior to and weekly throughout the experiment and sacrificed at 14, 28, 42 and 56 days post inoculation (dpi). At post mortem examination, samples of lymph nodes and spleen were collected from both dam and foetus for immunological analysis. Subcutaneous (sc) inoculation over the left prefemoral (LPF) lymph node of pregnant cattle at day 210 of gestation, led to the vertical transmission of parasites by 14 dpi, however no foetal deaths were observed in the infected animals. Foetuses from infected dams mounted Neospora-specific humoral and cell-mediated immune (CMI) responses by 14 dpi. These responses involved anti-Neospora IgG, antigen-specific lymphocyte proliferation, and the production of the cytokines IFN–γ, interleukin (IL)-4 and IL-10. There was also evidence of innate immunity during the response against Neospora from infected dams, with statistically significant (p < 0.05) increases in mean expression of toll like receptors (TLR)-2 on 56 dpi in maternal spleen, LPF, right prefemoral (RPF), left uterine (LUL) and right uterine (RUL) lymph nodes and TLR-9 in retropharyngeal (RLN), LPF and RPF lymph nodes from 28 dpi. Statistically significant (p < 0.05) increases in mean TLR-9 were detected in spleen samples from foetuses of infected dams, compared to the foetuses from control animals. Our results show that vertical transmission of the parasite occurred in all infected dams, with their foetuses showing effective Neospora-specific cell mediated, humoral and innate immune responses.     

Biofilm-forming ability profiling of Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates

Biofilm-forming ability has been increasingly recognized as an important virulence factor in Staphylococci, facilitating their persistence in the host, evading its defences and allowing bacterial survival at high antimicrobial concentrations. Staphylococcus aureus remains a major pathogen of chronic mastitis, but in the last years Staphylococcus epidermidis has emerged as a relevant mastitis pathogen. The present work aimed at the evaluation of the biofilm-forming ability of Staphylococci field isolates from bovine subclinical mastitis and at the development of a fluorescent in situ hybridisation (FISH) protocol that would allow the direct observation of biofilm formation in milk samples. The analysis of phenotypic expression in Congo Red Agar (CRA) and by FISH, showed that 37.5% of the S. aureus isolates produced biofilm, while by optical density measurement only 18.75% isolates revealed this phenotype. The results showed a fair agreement according to the kappa coefficient test (kappa = 0.259). Regarding S. epidermidis mastitis isolates, 37.5% revealed the ability to produce biofilm, but only four isolates were positive by all methods. This agreement was moderate (kappa = 0.467). The application of FISH to artificially contaminated milk samples allowed the direct observation of biofilm production by 37.5% isolates, showing total agreement with the CRA results. This method better mimics the in vivo conditions, especially in terms of the presence of calcium and iron, which in high concentrations, respectively, are known to inhibit or induce biofilm production.     

Transplacental infection of porcine fetuses following experimental challenge inoculation with encephalomyocarditis virus.

Ten multiparous sows were inoculated between 46 and 50 days of gestation with a fetal swine isolate of encephalomyocarditis virus (EMCV) to investigate the ability of the virus to cause transplacental infection and fetal death. Four sows (group 1) were inoculated IM with EMCV MN-25 that had been passaged 4 times on baby hamster kidney-21 line cell monolayers. Two sows were euthanatized at postinoculation (PI) day 23, and the other 2 sows at PI day 44. An additional 6 sows (group 2) were inoculated IM with the same virus that had been passaged 5 additional times in pigs. Two sows were euthanatized at 14 days, and the remaining 4 sows at PI day 28. Clinical signs were not observed in any of the sows, whereas all sows seroconverted to EMCV. In group 1, only 2 of 50 fetuses were mummified. Virus was not recovered, although EMCV antibodies were detected in the 2 mummified fetuses. In group 2, the 2 sows that were euthanatized at PI day 14 had 26 normal fetuses and there was no evidence of fetal infection. However, in the 4 sows euthanatized at PI day 28, 20 of 48 fetuses were mummified, hemorrhagic, or edematous. Encephalomyocarditis virus was recovered from 21 of 48 fetuses. Transplacental infection and fetal deaths in pregnant sows was achieved following infection with EMCV passaged in pigs.     

The invasiveness of Staphylococcus aureus and Staphylococcus epidermidis for the mammary gland of the mouse

The invasiveness of Staphylococcus aureus and Staphylococcus epidermidis for the mammary gland of the mouse was assessed by contaminating the damaged teats of suckling mice and sampling the mammary glands for the contaminating organism 48 hours later. Using this test system Staphylococcus aureus strain BB invaded 17 of 40 glands (42.5 %) and Staphylococcus epidermidis strain 279 invaded 2 of 40 glands (5.0 %). The histopathological changes in glands infected with Staphylococcus aureus were more severe than in those infected with Staphylococcus epidermidis.     

Bovine intestinal cellular responses following primary and challenge infections with Calicophoron microbothrium metacercariae

This study was carried out to establish whether cattle can develop resistance to re-infection by Calicophoron microbothrium by assessing the response of intestinal mucosal globule leukocytes, eosinophils, mast cells and basophils, and the establishment of the parasite in the host. A total of 24 1-year-old Tuli steers were randomly divided into four groups of six animals each and infected with C. microbothrium metacercariae. On the first day of the study, animals in Groups I and II were immunized with 5000 metacercariae and then challenged with 15,000 metacercariae on Day 150 post-immunization. Animals in Group III were immunized with 15,000 metacercariae at the same time that Groups I and II animals were challenged to act as a positive control group. Animals in Group IV were left uninfected and acted as a negative control group. Three animals from each group were slaughtered on Day 28 post-challenge and the remainder were slaughtered on Day 42 post-challenge. The established amphistomes were recovered and histopathological and cytological examinations were done on the jejunum, duodenum, abomasum and the rumen. The establishment rates of the challenge infection in the immunized and challenged groups were lower and ranged from 0 to 0.2% as compared to 6% from naive animals infected as positive controls. Animals immunized and then challenged with C. microbothrium had significantly higher eosinophil, mast cell and globule leukocytes counts in the intestinal mucosa (P < 0.05) as compared to those of the control group. The study indicates that cattle can develop resistance to C. microbothrium re-infection and that eosinophils and mast cells may be important cells in the rejection of the parasite.     

Inflammatory and metabolic responses to an intramammary lipopolysaccharide challenge in early lactating cows supplemented with conjugated linoleic acid

Supplementation of dairy cows with trans‐10, cis‐12 conjugated linoleic acid (CLA) allows nutrient repartitioning despite an energy deficiency in early lactation, which might be a benefit for the immune system, too. In this study, we investigated potential nutrient sparing effects of CLA in early lactating cows with low plasma glucose concentrations exposed to an intramammary lipopolysaccharide (LPS) challenge. Fifteen multiparous Holstein cows were exposed to an intramammary LPS challenge in week 4 p.p. Eight cows (CLA) were supplemented daily with 70 g of lipid‐encapsulated CLA (6.8 g trans‐10, cis‐12 and 6.6 g of the cis‐9, trans‐11 CLA isomer; CLA) and seven cows with 56 g of control fat (CON). Blood samples were obtained every 30 min along with rectal temperature, heart and respiratory rate, and milk samples were taken hourly until 10 hr after the LPS application. Plasma was analysed for concentrations of glucose, free fatty acids, beta‐hydroxybutyrate (BHB), cortisol, insulin and glucagon. In milk, somatic cell count and activity of lactate dehydrogenase were determined. Initial plasma glucose concentration was lower in CLA than in CON. During the immunostimulation, CLA had higher glucose concentrations than CON, and BHB decreased distinctly in CLA, whereas CON cows maintained BHB concentration at a lower level. Body temperature in CLA increased earlier, the difference between peak and basal temperature was higher, and the decline thereafter occurred earlier. In conclusion, CLA supplementation of early lactating cows exposed to an intramammary LPS challenge affected local and systemic immune responses. We assume that CLA supplementation triggered glycogen storage. Cows supplemented with CLA provided more glucose and preferentially used BHB as an energy source during the immune response. The more intense metabolic and more concentrated endocrine responses support an immunomodulatory effect of CLA supplementation.     

Class-specific antibody responses in pigs following immunization and challenge with sporocysts of Sarcocystis miescheriana

Sixteen pigs were each immunized by oral inoculation with 1000 sporocysts of Sarcocystis miescheriana at 8 weeks of age. Four equal groups were then challenged with 3 million sporocysts per animal at 40, 80, 120 or 160 days post-immunization (dpi). Host antibody responses were monitored using the enzyme-linked immunosorbent assay (ELISA) and the indirect fluorescent-antibody test (IFAT). When using class-specific ELISAs, dramatic increases in specific IgM-antibodies were observed at 21 dpi and increasing levels of IgG-antibodies were detected at 34 dpi. IgM-antibody titres dropped relatively quickly to insignificant levels, whereas IgG-antibody titres persisted at high levels until the end of the experiment. Following challenge, elevated levels of IgM-antibodies were detected, whereas IgG-antibody titres remained unchanged. The dynamics of the antibody titres detected by the IgG-IFAT closely corresponded to those detected by the IgG-ELISA. Despite the presence of specific antibodies at the time of challenge and the continued production of IgM-antibodies after challenge, the protective immunity decreased after 40 dpi and had disappeared by 120 dpi. Furthermore, none of the techniques used were suitable for the detection of specific antibodies during the early phase of acute sarcocystosis around 12 days after challenge.     

Clinical and immunological responses of ewes following vaccination with an experimental formalin-inactivated Chlamydia psittaci (ovis) vaccine and subsequent challenge with the live organism during pregnancy

The protection afforded by an experimental, killed, adjuvanted vaccine derived from the A22 strain of Chlamydia psittaci (ovis) against ovine enzootic abortion was studied. The vaccine was used undiluted (group A), at a dilution of 10(-3) (group B) and at a dilution of 10(-6) (group C). A fourth control group (group D) was inoculated with all components of the vaccine except the chlamydial antigen. A group of rams (group R) was also vaccinated with the chlamydial antigen diluted to 10(-3). Animals were challenged 70 days after mating with the A22 strain of C. psittaci (ovis) and were studied throughout pregnancy and the subsequent lambing period. Their cell-mediated immune responses were examined using a skin test and their humoral immune responses were studied using an ELISA. Tests for excretion of chlamydiae in their faeces and genital tract during pregnancy and after parturition and in the faeces of their lambs were made. The reproductive performance of the ewes was assessed by calculating the average weight of lambs produced per ewe in each group. The experimental vaccine protected the ewes in groups A and B against challenge with C. psittaci (ovis) as none showed clinical signs of OEA or excreted chlamydiae. The average weight of lambs produced per ewe in both groups was greater than 4 kg. Both groups seroconverted after vaccination but not all of them were positive to the skin test. The experimental vaccine at 10(-6) dilution of antigen did not protect the ewes as three of 10 ewes displayed clinical OEA and excreted chlamydiae in the products of parturition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time course of biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates

Biofilm formation is considered a selective advantage for staphylococci mastitis isolates, facilitating bacterial persistence in the udder. It requires attachment to mammary epithelium, proliferation and accumulation of cells in multilayers and enclosing in a polymeric matrix, being regulated by several loci. As biofilm formation can proceed through different pathways and time ranges, its detection may differ according to the time of observation. This study aimed at evaluating the time course evolution of biofilm production in Staphylococcus aureus (n = 26) and Staphylococcus epidermidis (n = 29) mastitis isolates by Fluorescent In Situ Hybridisation. Biofilm-forming ability increased with incubation time for both species: for S. aureus, 34.6%, 69.2% and 80.8% of the isolates were able to produce biofilm at 24, 48 and 72 h, respectively. For S. epidermidis, 44.8%, 62.1% and 75.9% of the isolates were biofilm-positive at 24, 48 and 72 h, respectively. No significant difference was found between species at each time point (Friedman's test, p > 0.05). For S. aureus, although a significant difference was found between 24 and 48 h (Wilcoxon matched paired test, p < 0.05), no significant difference was found between 24 and 48 h (p > 0.05). For S. epidermidis, significant differences were found between each time point (p < 0.05). Bacterial biofilms may impair eradication of chronic mastitis, rendering antibiotherapy less effective. Detection of biofilm-forming ability in mastitis isolates may provide useful information for the establishment of a more adequate therapeutic regimen, in view of the antimicrobial concentrations required for bacterial control. However, it is essential that biofilm formation time course is taken into consideration.