Calcium potentiates the effect of estradiol on PGF2α production in the bovine endometrium

Background

Estradiol (E₂) is required for luteolysis in cows and its injection stimulates prostaglandin F2α (PGF2α) release. The main goal of our study was to investigate the ability of endometrial explants and cells treated with E₂ and the calcium ionophore (CI) A23187 to synthesize PGF2α.

Results

Treatment with E₂in vivo resulted in a 48.4% increase of PGF2α production by endometrial explants treated in vitro with A23187. Production of PGF2α was better stimulated with A23187 at concentrations of 10^-6 and 10^-5 mol/L compared with other concentrations used. The concentration of PGF2α for untreated bovine endometrial cell cultures was 33.1 pg/mL, while for cultures treated with E₂, A23187, or a combination of E₂ and A23187, the PGF2α concentration was 32.5, 92.4 and 145.6 pg/mL, respectively.

Conclusions

Treatment with A23187 tended to stimulate PGF2α production. In the presence of E2, A23187 significantly stimulated PGF2α synthesis. It appears that A23187 potentiates the effects of E₂ with respect to synthesis of endometrial PGF2α in cattle.     

Fixed-time artificial insemination in beef cattle

Background

The study was designed to test the effect of fixed-time artificial insemination (fixed-AI) after the slightly modified Ovsynch protocol on the pregnancy rate in beef cattle in Finnish field conditions. The modification was aimed to optimize the number of offsprings per AI dose.

Methods

Ninety Charolais cows and heifers were entered into the program an average of 1.8 times. Thus, 164 animal cases were included. Animals were administered 10-12 μg of buserelin. Seven days later animals without a corpus luteum (CL) were rejected (20.7%) while the remaining 130 cases with a CL were administered prostaglandin F_2α, followed 48 h later with a second injection of buserelin (8-10 μg). Fixed-AI was performed 16-20 hours after the last injection.

Results

The pregnancy rate was 51.5% (67/130). The pregnancy rate after a short interval (50-70 d) from calving to entering the program was significantly higher than that after a long interval (>70 d).

Conclusion

This protocol seems to give acceptable pregnancy results in beef herds and its effect on saving labour is notable.     

Characterization of coagulase negative staphylococci from cases of subclinical mastitis in dairy cattle in Kampala,Uganda

Background

Coagulase negative staphylococci (CNS) are the most common pathogens leading to subclinical mastitis (SCM) in dairy cattle in Uganda. Coagulase negative staphylococci can vary between bacterial species in how they cause disease. The aim of the study was to characterize CNS, from cows with SCM in Uganda, at the species level.

Findings

Quarter milk samples (n = 166) were collected from 78 animals with SCM. Bacteriological analyses were carried out at Makerere University, Kampala, Uganda and at the National Veterinary Institute (SVA), Uppsala, Sweden. The most common pathogens found in milk samples from cows with SCM were CNS (31.7%). Two species of CNS were found, S. epidermidis (85%) and S. haemolyticus (15%). Of the CNS isolates, 16/20 (80%) were positive for β-lactamase production (β+).

Conclusions

In milk samples from cows with SCM caused by CNS, S. epidermidis was most prevalent, followed by S. haemolyticus.     

Blood concentrations of the cytokines IL-1beta,IL-6, IL-10, TNF-alpha and IFN-gamma during experimentally induced swine dysentery

Background

Knowledge of the cytokine response at infection with Brachyspira hyodysenteriae can help understanding disease mechanisme involved during swine dysentery. Since this knowledge is still limited the aim of the present study was to induce dysentery experimentally in pigs and to monitor the development of important immunoregulatory cytokines in blood collected at various stages of the disease.

Methods

Ten conventional pigs (~23 kg) were orally inoculated with Brachyspira hyodysenteriae B204^T. Eight animals developed muco-haemorrhagic diarrhoea with impaired general body condition. Blood was sampled before inoculation and repeatedly during acute dysentery and recovery periods and cytokine levels of IL-1β, IL-6, Il-10, TNF-α and IFN-γ were measured by ELISA.

Results

IL-1β was increased at the beginning of the dysentery period and coincided with the appearance of Serum amyloid A and clinical signs of disease. TNF-α increased in all animals after inoculation, with a peak during dysentery, and IL-6 was found in 3 animals during dysentery and in the 2 animals that did not develop clinical signs of disease. IL-10 was found in all sick animals during the recovery period. IFN-γ was not detected on any occasion.

Conclusion

B. hyodysenteriae inoculation induced production of systemic levels of IL-1β during the dysentery period and increased levels of IL-10 coincided with recovery from dysentery.     

Circulating β-endorphin,adrenocorticotrophic hormone and cortisol levels of stallions before and after short road transport: stress effect of different distances

Background

Since transport evokes physiological adjustments that include endocrine responses, the objective of this study was to examine the responses of circulating β-endorphin, adrenocorticotrophic hormone (ACTH) and cortisol levels to transport stress in stallions.

Methods

Forty-two healthy Thoroughbred and crossbred stallions were studied before and after road transport over distances of 100, 200 and 300 km. Blood samples were collected from the jugular vein: first in a single box immediately before loading (pre-samples), then immediately after transport and unloading on arrival at the breeding stations (post-samples).

Results

An increase in circulating β-endorphin levels after transport of 100 km (P < 0.01), compared to basal values was observed. Circulating ACTH levels showed significant increases after transport of 100 km (P < 0.001) and 200 km (P < 0.001). Circulating cortisol levels showed significant increases after road transport over distances of 100, 200 and 300 km (P < 0.001). An effect of transport on β-endorphin, ACTH and cortisol variations was therefore evident for the different distances studied. No significant differences (P > 0.05) between horses of different ages and different breeds were observed for β-endorphin, ACTH and cortisol levels.

Conclusion

The results obtained for short term transportation of stallions showed a very strong reaction of the adrenocortical system. The lack of response of β-endorphin after transport of 200–300 km and of ACTH after transport of 300 km seems to suggest a soothing effect of negative feedback of ACTH and cortisol levels.     

Filarioid nematodes in cattle,sheep and horses in Finland

Background

In autumn 2006, Finnish meat inspection data revealed lesions in tendons, muscles and ligaments of bovine hind legs leading to partial condemnation of carcasses. In gross pathological examination at Finnish Food Safety Authority Evira, Oulu (now Fish and Wildlife Health) Research Unit, Onchocerca sp. (Filarioidea; Onchocercidae) nematodes were detected in lesions. Due to this, a pilot study was made in order to find out what filarioid nematodes do occur in cattle, horses and sheep in Finland.

Methods

Ventral skin biopsies from 209 dairy cattle and 42 horses, as well as blood samples from 209 cattle, 146 horses and 193 sheep, were collected from different parts of Finland and examined for microfilariae. Visceral organs and other tissues from 33 cattle with parasitic lesions were studied histopathologically.

Results

Onchocerca sp. microfilariae (mf), 240 μm long, range 225–260 μm, 5.4 μm thick, were found in 37% of the skin biopsies of cattle. All blood samples from cattle, horses and sheep and skin biopsies from horses were negative for mf. Ventral skin microfilaria prevalence in cattle was higher in southern Finland than in the North (p = 0.001). Animal age and sampling time was not associated with mf prevalence. The infection was evenly distributed among young and older animals. Macroscopic lesions on tissues included greenish-grey discolouration and often oedema. In most of the lesions, small pale nodules were seen on the fasciae. Histopathologic examination of the samples revealed mild to intense infiltration with eosinophilic granulocytes and multifocal nodular lymphoplasmacytic aggregations were seen. In some samples, there were granulomatotic lesions with central necrotic tissue and cell detritus, surrounded by eosinophilic granulocytes, lympho-, plasma- and histiocytes and some multinucleated giant cells. Around living nematodes no or only weak inflammatory changes were observed.

Conclusion

Onchocerca sp. infection in cattle was found to be common in Finland, but the amount of pathological changes leading to condemnation of infected parts is low compared to the mf prevalence. Pronounced pathological changes are distinct but rare and mild changes are difficult to distinguish. No other filarioid nematodes were observed from the animals and it appears that horses and sheep may be free from filarioid nematodes in Finland.     

Cross-reactivity of commercially available anti-human monoclonal antibodies with canine cytokines: establishment of a reliable panel to detect the functional profile of peripheral blood lymphocytes by intracytoplasmic staining

Background

The process for obtaining monoclonal antibodies against a specific antigen is very laborious, involves sophisticated technologies and it is not available in most research laboratories. Considering that most cytokines remain partially conserved among species during evolution, the search for antibody cross-reactivity is an important strategy for immunological studies in veterinary medicine. In this context, the amino acid sequence from human and canine cytokines have demonstrated 49–96 % homology, suggesting high probability of cross-reactivity amongst monoclonal antibodies. For this, 17 commercially available anti-human monoclonal antibodies [IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-10, IL-12, IL-13, IL-17A, IFN-γ (#1, #2), TNF-α (#1, #2) and TGF-β], were evaluated in vitro for intracellular cytokine detection in a stimulated canine blood culture by flow cytometry and confocal microscopy. Lymphocytes from peripheral blood of healthy and two unhealthy dogs were analyzed.

Results

Eleven anti-human mAbs [IL-1α, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-12, IL-17A, TNF-α (#1, #2) and TGF-β] cross-reacted against canine intracellular cytokines. The specificity of the assays was not affected after Fc-blocking. Three anti-human cytokine mAbs [IL-4, IL-8 (#2) and TGF-β] when evaluated by confocal microscopy also cross-reacted with intracellular canine cytokines. The identification of human mAbs that cross-reacted with canine cytokines may support their use as immunological biomarkers in veterinary medicine studies.

Conclusion

The identification of these 11 anti-human cytokine mAbs that cross-reacted with canine cytokines will be useful immunological biomarkers for pathological conditions by flow cytometry and fluorescence microscopy in dogs.     

Expression of hypoxia-inducible factor-1α and vascular density in mammary adenomas and adenocarcinomas in bitches

Background

The study aimed at examining hypoxia-inducible factor (HIF)1α expression in adenocarcinomas and adenomas in bitches in regard to tumour malignancy grade, proliferation, apoptosis and vascularisation. Therefore, paraffin sections of 15 adenomas and 64 adenocarcinomas sampled from 79 dogs aged 6 to 16 years were analysed.

Results

A significantly higher HIF-1α expression was noted in adenocarcinomas in comparison to adenomas (P < 0.0004). Moreover, HIF-1α expression in adenocarcinomas correlated positively with tumour malignancy grade (r = 0.59, P < 0.05), Ki-67 antigen expression (r = 0.43; P < 0.0005), TUNEL-positive cells (r = 0.62, P < 0001) and tumour vascularity measured by quantification of vessels characterized by the expression of von Willebrand Factor (r = 0.57, P < 0.05).

Conclusion

Results of this study indicate a similar biological role of HIF-1α in dogs and in humans, which may confirm suitability of the animal model in investigations on progression of tumours in humans.     

Nematode control in suckler beef cattle over their first two grazing seasons using a targeted selective treatment approach

Background

With concerns over the development of anthelmintic resistance in cattle nematode populations, we must re-examine our approach to nematode control in cattle. Targeted selective treatments (TST), whereby individual animals are treated instead of entire groups, are being investigated as an alternative. The study objective was to determine if anthelmintic usage could be reduced using a TST-based approach to nematode control in spring-born suckler beef cattle over their first and second grazing seasons (SGS) without affecting performance. In the first grazing season (FGS), 99 calves with an initial mean (s.d.) calf age and live weight on day 0 (June 28^th 2012) of 107 (23.1) days and 160 (32.5) kg, respectively, were used. The study commenced on day 0 when calves were randomised and allocated to one of two treatments; 1), standard treatment (control) and 2), TST. Control calves were treated subcutaneously with ivermectin on days 0, 41 and 82 in the FGS. All calves were treated with ivermectin on day 124 and housed on day 133. In the SGS, only heifer calves from the FGS were used and control heifers were treated with ivermectin on day 393. Animals were weighed, blood and faecal sampled every three weeks. The TST animals were treated with ivermectin if thresholds based on a combination of plasma pepsinogen concentrations, faecal egg count and/or the presence of Dictyocaulus viviparus larvae in faeces (FGS only) were reached.

Results

No TST calves reached the treatment threshold criteria in the FGS. The FGS average daily live weight gain (ADG ± s.e.m.) for control and TST group calves was 0.89 ± 0.02 kg and 0.94 ± 0.02 kg day⁻¹, respectively (P = 0.17). In the SGS, all heifers were treated with ivermectin on day 431 due to clinical signs of respiratory disease. The ADG for control and TST heifers from turnout on day 321 to day 431 was 0.90 ± 0.04 and 0.80 ± 0.04 kg day⁻¹, respectively (P = 0.03).

Conclusions

Spring-born FGS suckler beef calves require minimal anthelmintic treatment to maintain performance. In contrast, clinical parasitic disease may develop in the SGS unless appropriate anthelmintic treatment is provided.     

Ultrasonography of the reticulum in 30 healthy Saanen goats

Background

The reticulum plays a crucial role in the ruminant digestive tract because the primary cycle of rumen motility always starts with a reticular contraction. In contrast to cattle, there are only few results on the ultrasonographic examination of the reticulum in goats. Therefore, it was the goal of the present study, to describe the results of ultrasonography of the reticulum of 30 healthy Saanen goats.

Methods

Ultrasonography was carried out on standing, non-sedated animals using a 5.0 MHz linear transducer. The shape, contour and motility of the reticulum were investigated. A nine-minute video recording of the reticulum was made for each goat and the frequency, duration and amplitude of reticular contractions were calculated as described for cattle.

Results

The reticulum appeared as a crescent-shaped structure with a smooth contour located immediately adjacent to the diaphragm. 0.8 to 2.1 (1.41 ± 0.31) reticular contractions were seen per minute. In all goats, biphasic reticular contractions were observed. 90% of the goats also had monophasic reticular contractions, and two had triphasic contractions. During the nine-minute observation periods, there were 0 to 6 monophasic reticular contractions and 6 to 15 biphasic contractions per goat. The duration of the biphasic contractions was 6.56 ± 0.74 s, which was significantly longer than the monophasic contractions at 4.31 ± 0.81 s. The average interval between two reticular contractions was 45.06 ± 12.57 s.

Conclusion

Ultrasonography of the reticulum in goats is a valuable tool to characterise the appearance and motility of this organ. In addition to the biphasic motility pattern seen in cattle the reticular motility of goats is characterized by monophasic reticular contractions. The results of the present study are an important contribution for better understanding of the reticular motility in goats.     

Seminal plasma did not influence the presence of transforming growth factor-β1, interleukine-10 and interleukin-6 in porcine follicles shortly after insemination

Background

The effects of seminal plasma on the presence of the cytokines transforming growth factor (TGF)-β1, interleukin (IL)-10 and IL-6 in ovarian follicles and follicular fluid were studied shortly after insemination in gilts.Ovaries from gilts were sampled 5–6 h after insemination with either seminal plasma (SP), fresh semen in extender (Beltsville thawing solution, BTS), spermatozoa in extender (Spz), or only BTS (control).

Results

Immunohistochemical (IHC) labeling of TGF-β1, IL-10 and IL-6 was evident in the ovarian oocytes and granulosa cells independent of stage of follicular development (antral follicles). Theca interna cells were labeled to a high degree in mature follicles. No consistent differences between treatment groups could be observed for any of the cytokines.In follicular fluid, high concentrations of TGF-β1 were found while the levels of IL-10 and IL-6 were low. There were no differences between treatment groups.

Conclusions

Our results show a presence of the cytokines TGF-β1, IL-6 and IL-10 in oocytes, granulosa and theca cells, as well as in the fluid of mature follicles suggesting a role of these cytokines in intra-ovarian cell communication. However, treatment (SP, fresh semen in BTS, spermatozoa in BTS or BTS) did not influence the IHC-labeling pattern or the levels of these cytokines in follicular fluid shortly after insemination.     

Antimicrobial resistant bacteria in wild mammals and birds: a coincidence or cause for concern?

Background

The emergence and dissemination of antimicrobial resistance (AMR) is a growing concern to public and animal health. The contribution attributable to wildlife remains unclear. In this study two unrelated wildlife species herring gulls (Larus argentatus) and a hybrid deer (Cervus elaphus x Cervus nippon) were investigated for the presence of Escherichia coli expressing an AMR phenotype.

Findings

Bacterial isolates resistant to β-lactam compounds were identified in both animal species and the production of functional β-lactamase was confirmed using nitrocefin. The prevalence of resistant isolates was higher in herring gulls (87%) compared to deer (31%). Resistance to this class of antibiotic was found only in non-pathogenic E. coli in herring gulls and in both pathogenic and non-pathogenic E. coli strains in deer.

Conclusions

The presence of AMR in wildlife has implications for public health, food safety and potable water source protection among others.     

Immunity,safety and protection of an Adenovirus 5 prime - Modified Vaccinia virus Ankara boost subunit vaccine against Mycobacterium avium subspecies paratuberculosis infection in calves

Vaccination is the most cost effective control measure for Johne’s disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) but currently available whole cell killed formulations have limited efficacy and are incompatible with the diagnosis of bovine tuberculosis by tuberculin skin test. We have evaluated the utility of a viral delivery regimen of non-replicative human Adenovirus 5 and Modified Vaccinia virus Ankara recombinant for early entry MAP specific antigens (HAV) to show protection against challenge in a calf model and extensively screened for differential immunological markers associated with protection. We have shown that HAV vaccination was well tolerated, could be detected using a differentiation of infected and vaccinated animals (DIVA) test, showed no cross-reactivity with tuberculin and provided a degree of protection against challenge evidenced by a lack of faecal shedding in vaccinated animals that persisted throughout the 7 month infection period. Calves given HAV vaccination had significant priming and boosting of MAP derived antigen (PPD-J) specific CD4⁺, CD8⁺ IFN-γ producing T-cell populations and, upon challenge, developed early specific Th17 related immune responses, enhanced IFN-γ responses and retained a high MAP killing capacity in blood. During later phases post MAP challenge, PPD-J antigen specific IFN-γ and Th17 responses in HAV vaccinated animals corresponded with improvements in peripheral bacteraemia. By contrast a lack of IFN-γ, induction of FoxP3+ T cells and increased IL-1β and IL-10 secretion were indicative of progressive infection in Sham vaccinated animals. We conclude that HAV vaccination shows excellent promise as a new tool for improving control of MAP infection in cattle.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0112-9) contains supplementary material, which is available to authorized users.     

Impact of heparin and short term anesthesia on the quantification of cytokines in laboratory mouse plasma

Background

Studies have reported that heparin may be unsuitable as an anticoagulant in human plasma samples when quantifying cytokines using multiplex bead array assays. For mouse samples, multiplex assays have been validated for serum and EDTA-plasma, but it remains to be elucidated whether heparin influences the quantification of cytokines, and if so – to what extent. Furthermore, laboratory mice are often anesthetized for blood sampling, which causes acute stress that may influence circulating cytokine concentrations and thus bias experimental results. The objectives of the present study were to identify whether specific cytokine concentrations varied between heparin-plasma, serum, and EDTA-plasma, and whether short isoflurane anesthesia would influence the concentrations of these cytokines in the circulation. Twenty-three acute phase and pro-inflammatory cytokines were quantified in matched serum, EDTA-plasma, and heparin-plasma samples from anesthetized and unanesthetized male NMRI mice using a multiplex assay. In addition, samples from unanesthetized mice were spiked with three levels of heparin.

Results

The concentrations of five out of 23 cytokines were significantly different between sample types, but only one cytokine (IL-17A) differed between heparin-plasma and serum. When further spiking the heparin-plasma with increasing concentrations of heparin, there was a significant effect on 11 cytokines, where the cytokine recovery could be correlated to the heparin concentration for ten of these cytokines. Anesthesia resulted in lower concentrations of G-CSF, but had no significant impact on the concentrations of the other 22 cytokines.

Conclusion

In mice, heparin seems like a suitable anticoagulant for obtaining plasma for multiplex assays for the cytokines IL-1α, IL-1β, IL-2, IL-6, IL-9, IL-12p40, IL-12p70, IL-13, G-CSF, GM-CSF, IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, RANTES and TNFα, but an effect of heparin in high concentrations should be considered for the cytokines IL-9, IL-12p40, IL-12p70, KC, MCP-1, MIP-1β and RANTES. Short isoflurane anesthesia had significant impact on G-CSF, but none of the other cytokines.     

Transplacental induction of fatty acid oxidation in term fetal pigs by the peroxisome proliferator-activated receptor alpha agonist clofibrate

Background

To induce peroxisomal proliferator-activated receptor α (PPARα) expression and increase milk fat utilization in pigs at birth, the effect of maternal feeding of the PPARα agonist, clofibrate (2-(4-chlorophenoxy)-2-methyl-propanoic acid, ethyl ester), on fatty acid oxidation was examined at full-term delivery (0 h) and 24 h after delivery in this study. Each group of pigs (n = 10) was delivered from pregnant sows fed a commercial diet with or without 0.8% clofibrate for the last 7 d of gestation. Blood samples were collected from the utero-ovarian artery of the sows and the umbilical cords of the pigs as they were removed from the sows by C-section on day 113 of gestation.

Results

HPLC analysis identified that clofibric acid was present in the plasma of the clofibrate-fed sow (~4.2 μg/mL) and its offspring (~1.5 μg/mL). Furthermore, the maternal-fed clofibrate had no impact on the liver weight of the pigs at 0 h and 24 h, but hepatic fatty acid oxidation examined in fresh homogenates showed that clofibrate increased (P < 0.01) ¹⁴C-accumulation in CO₂ and acid soluble products 2.9-fold from [1-¹⁴C]-oleic acid and 1.6-fold from [1-¹⁴C]-lignoceric acid respectively. Correspondingly, clofibrate increased fetal hepatic carnitine palmitoyltransferase (CPT) and acyl-CoA oxidase (ACO) activities by 36% and 42% over controls (P < 0.036). The mRNA abundance of CPT I was 20-fold higher in pigs exposed to clofibrate (P < 0.0001) but no differences were detected for ACO and PPARα mRNA between the two groups.

Conclusion

These data demonstrate that dietary clofibrate is absorbed by the sow, crosses the placental membrane, and enters fetal circulation to induce hepatic fatty acid oxidation by increasing the CPT and ACO activities of the newborn.     

Effect of sedation with detomidine and butorphanol on pulmonary gas exchange in the horse

Background

Sedation with α₂-agonists in the horse is reported to be accompanied by impairment of arterial oxygenation. The present study was undertaken to investigate pulmonary gas exchange using the Multiple Inert Gas Elimination Technique (MIGET), during sedation with the α₂-agonist detomidine alone and in combination with the opioid butorphanol.

Methods

Seven Standardbred trotter horses aged 3–7 years and weighing 380–520 kg, were studied. The protocol consisted of three consecutive measurements; in the unsedated horse, after intravenous administration of detomidine (0.02 mg/kg) and after subsequent butorphanol administration (0.025 mg/kg). Pulmonary function and haemodynamic effects were investigated. The distribution of ventilation-perfusion ratios (V_A/Q) was estimated with MIGET.

Results

During detomidine sedation, arterial oxygen tension (PaO₂) decreased (12.8 ± 0.7 to 10.8 ± 1.2 kPa) and arterial carbon dioxide tension (PaCO₂) increased (5.9 ± 0.3 to 6.1 ± 0.2 kPa) compared to measurements in the unsedated horse. Mismatch between ventilation and perfusion in the lungs was evident, but no increase in intrapulmonary shunt could be detected. Respiratory rate and minute ventilation did not change. Heart rate and cardiac output decreased, while pulmonary and systemic blood pressure and vascular resistance increased. Addition of butorphanol resulted in a significant decrease in ventilation and increase in PaCO₂. Alveolar-arterial oxygen content difference P(A-a)O₂ remained impaired after butorphanol administration, the V_A/Q distribution improved as the decreased ventilation and persistent low blood flow was well matched. Also after subsequent butorphanol no increase in intrapulmonary shunt was evident.

Conclusion

The results of the present study suggest that both pulmonary and cardiovascular factors contribute to the impaired pulmonary gas exchange during detomidine and butorphanol sedation in the horse.