In vitro generation of mature neutrophils from canine Lin- bone marrow cells

The major goal of this work was to describe the in vitro generation of mature functional neutrophils derived from a canine enriched haematopoietic progenitor cell population. We have utilised lineage depletion by immunomagnetic selection to isolate a canine haematopoietic progenitor cell population. The physical, immunological, metabolical and morphological methodologies employed in this study have permitted us to isolate and define a cell population enriched in Rh-123low and CD34+ cells. Irradiated pre-established long-term bone marrow cultures (LTBMC) were utilised to determine the self-renewal ability of lineage negative (Lin-) cells, as well as their capacity to differentiate into mature functional neutrophils. The authors demonstrate for the first time that canine neutrophils derived from Lin- cells are able to produce oxyradicals, express a specific neutrophil surface antigen, and contain gelatinase granules. These characteristics enable them to migrate through basement membranes to act as a first line defence mechanism. The fact that these cells are able to differentiate into functional mature cells, and give rise to long-term culture-initiating cells (LTC-IC) after 35 days of culture, allows the authors to assure that the isolated canine enriched haematopoietic cell population exhibit functional characteristics, associated with primitive haematopoietic cells.     

The host-parasite relationship in bovine neosporosis

Infection with the protozoan parasite Neospora caninum is thought to be a major cause of reproductive failure in cattle worldwide. Cattle infected with the parasite are three to seven times more likely to abort compared to uninfected cattle. The parasite may be transmitted to cattle through the ingestion of oocysts that are shed in the faeces of acutely infected dogs (definitive host of N. caninum) or by congenital infection from mother to foetus via the placenta. Interestingly, transplacental transmission can occur over consecutive pregnancies and congenitally infected heifers can transmit the parasite to their own offspring. This repeated vertical transmission observed in naturally infected cattle suggests that cattle do not easily develop effective immunity to the parasite, presenting a significant challenge to the development of a control strategy based on vaccination. Neosporosis is a disease of pregnancy and studying the bovine maternal and foetal immune responses during pregnancy will help us to understand the change in the balance between the parasite and the host that may result in disease of the foetus. Studies in non-pregnant cattle and in murine models of infection have shown the importance of T-helper 1-type immune responses involving pro-inflammatory cytokines, such as IFNgamma and IL-12, in limiting intracellular multiplication of the parasite. During pregnancy, changes occur in the immune system allowing the mother to accept the foetal allograft. Research in other species has stressed the crucial role of T-helper 2-type cytokines at the materno-foetal interface in maintaining the pregnancy and regulating the potentially damaging effect of Th-1 responses. Studies in cattle have shown that cell proliferation and IFNgamma responses may be significantly down-regulated around mid-gestation. This may mean that cattle are less able to cope with N. caninum infection at this time and are more likely to transmit the parasite to the foetus. Another important factor is the gestational age and hence immuno-competence of the foetus at the time of infection. Early in gestation, N. caninum infection of the placenta and subsequently the foetus usually proves fatal, whereas infection occurring in mid to late pregnancy may result in the birth of a congenitally infected but otherwise healthy calf. Studies of foetal immune responses have shown that at 14 weeks of gestation, lymphocytes only respond to mitogen, while by 24 weeks (mid-gestation), they respond to antigen by proliferating and releasing IFNgamma. Clearly, there are several factors influencing the outcome of N. caninum infection in pregnancy: the timing, quantity and duration of parasitaemia, the effectiveness of the maternal immune response and the ability of the foetus to mount an immune response against the parasite. The challenge is to design a vaccine that will prevent foetal infection by N. caninum. This is likely to involve a fine balancing act with the immune system that will allow intervention in a manner that will tip the host-parasite balance in favour of the host without compromising the pregnancy.     

Plasma endotoxin concentration in horses: a methods study

Background: Accurate determination of plasma endotoxin concentration is critical for ex vivo and in vitro cellular and molecular studies of endotoxemia in horses. However, reports are conflicting with respect to anticoagulant, handling, and sample preparation.

Objective:

Objective: The purpose of this study was to determine the effect of blood sample fraction and handling time on measurement of endotoxin concentration in horses.

Methods:

Methods: Whole blood, anticoagulated with 3.8% (0.12 M) sodium citrate (9:1), was collected from 5 healthy horses. Whole blood (WB), platelet-rich plasma (PRP), and platelet-poor plasma (PPP) were spiked with endotoxin (2 EU/mL). Endotoxin-spiked WB samples were centrifuged immediately to generate PRP for measurement. Endotoxin concentration was subsequently measured by Limulus amebocyte assay at 0, 15, 30, 45, and 60 minutes. Assays were performed in triplicate and results were analyzed using Student's t -test, with significance set at P < .05.

Results:

Results: Mean endotoxin concentrations in 2 EU/mL-spiked WB were significantly different from those in PPP at all time points tested. Recovery of endotoxin in PRP generated from WB was significantly diminished after just 15 minutes.

Conclusion:

Conclusion: PRP generated from WB is significantly more reliable than PPP in determining endotoxin concentration ex vivo. Measurement of endotoxin in PRP generated from WB was significantly diminished after 15 min, identifying a time frame within which to process blood samples for endotoxin analysis.     

Proteome analysis of the sarcoplasmic fraction of pig semimembranosus muscle: implications on meat color development

Two-dimensional electrophoresis was used to investigate sarcoplasmic protein expression in pig Semimembranosus muscles sampled 20 min after slaughter. Two groups (light and dark) of 12 animals were selected from 1000 pigs, based on meat L values measured 36 h postmortem. Twenty-two proteins or fragments (p < 0.05) were differentially expressed. Muscles leading to darker meat had a more oxidative metabolism, indicated by more abundant mitochondrial enzymes of the respiratory chain, hemoglobin, and chaperone or regulator proteins (HSP27, alphaB-crystallin, and glucose-regulated protein 58 kDa). Conversely, enzymes of glycolysis were overexpressed in the lighter group. Such samples were also characterized by higher levels of glutathione S-transferase omega, which can activate the RyR calcium channels, and higher levels of cyclophilin D. This protein pattern is likely to have severe implications on postmortem metabolism, namely, acceleration of ATP depletion and pH fall and subsequent enhanced protein denaturation, well-known to induce discoloration.     

A comparison of extradural and intravenous methadone on intraoperative isoflurane and postoperative analgesia requirements in dogs

Objective To compare the effects of intravenous (IV) and extradural (ED) methadone on end‐tidal isoflurane concentration (Fe ′ISO) and postoperative analgesic requirements in dogs undergoing femoro‐tibial joint surgery. Study Design Randomized, blinded, clinical study. Animals Twenty‐four healthy client‐owned dogs undergoing surgical repair of ruptured cruciate ligaments. Methods Dogs were randomly assigned to two groups of 12 animals and received either ED or IV methadone (0.3 mg kg^?1 diluted with saline to 0.2 mL kg^?1). Pre‐anaesthetic medication was IV acepromazine (0.05 mg kg^?1). Anaesthesia was induced with propofol and maintained initially with an Fe ′ISO of 1.0% delivered in oxygen. Methadone was injected with the dogs in sternal recumbency; the observer was unaware of the administration route. At 10 minutes (stimulation 1) and 20 minutes (stimulation 2) after methadone administration pelvic limb reflexes were tested by digit‐clamping. The time at skin incision (stimulation 3), joint‐capsule incision (stimulation 4), tibial tuberosity drilling (stimulation 5), fabellar suturing (stimulation 6) and extracapsular tightening (stimulation 7) were noted. Changes in heart rate (HR) and respiratory rate and arterial blood pressure associated with surgery were recorded along with the corresponding Fe ′ISO. After 20 minutes of anaesthesia, Fe ′ISO was decreased to the minimum required to maintain stable anaesthesia. Immediately after tracheal extubation, 1, 2, 3 and 6 hours postoperatively and on the morning after surgery, the degree of pain present was assessed using a numerical rating scale. The HR, respiratory rates and blood pressure were also recorded at these times. Serum cortisol and blood glucose concentrations were measured before pre‐anaesthetic medication and at each postoperative pain scoring interval except at 1 and 2 hours. Ketoprofen (2 mg kg^?1), carprofen (4 mg kg^?1) or meloxicam (0.2 mg kg^?1) were given by subcutaneous injection whenever pain scoring indicated moderate discomfort was present. Results Controlled ventilation was required in six dogs which stopped breathing after IV methadone. The median Fe ′ISO at stimulus 5 was 1.0% in the IV and 0.83% in the ED group. At stimulus 6, Fe ′ISO was 1.0% in the IV and 0.8% in the ED group; the difference was statistically significant (p ≤ 0.05). There was no significant difference in the duration of postoperative analgesia associated with administration route. Conclusions Extradural methadone significantly reduces the isoflurane requirement compared with IV methadone during femoro‐tibial joint surgery in dogs. Clinical relevance Extradural methadone provides safe and effective pain relief in dogs undergoing cruciate ligament repair.     

First report of Lagochilascaris (Nematoda: Ascarididae) eggs in a public park in Southern Brazil

Public parks, especially in developing countries, are places where stray animals run free and may contaminate the environment with different species of parasites. In an evaluation of environmental sanitation of these sites, soil samples were collected monthly from public parks in the Pelotas city, Rio Grande do Sul State, Southern Brazil. Eggs of the nematode Lagochilascaris sp. were found in a public playground. This is the first report of identification of this nematode in Southern Brazil.     

Quantification of transmission of livestock-associated methicillin resistant Staphylococcus aureus in pigs

Antimicrobial resistance in pigs becomes a public health issue when resistant organisms transfer from pigs to humans. Pigs are a large reservoir for livestock-associated (LA-)MRSA and people in contact with pigs are at risk for infection with LA-MRSA. Transmission and persistence of LA-MRSA within a pig population contributes to the maintenance of this zoonotic reservoir. Current knowledge on colonization and transmission of LA-MRSA in pigs is limited and mainly based on observational field surveys. Two experiments were performed to colonize pigs and quantify transmission of LA-MRSA between pigs. In the first experiment, colonization of six-week old piglets failed after intranasal inoculation, confirming the complexity of MRSA-colonization. In the second experiment, naive pigs got colonized after exposure to orally inoculated pigs. Subsequently, these contact-infected pigs transmitted MRSA to a new group of naive pigs. The reproduction ratio, R(0), was estimated with a SIS-model to quantify transmission between the first and second contact pigs as this resembles more the natural transmission. Two scenarios were evaluated, with different assumptions regarding infection status of individual pigs. R(0) varied between 3.7 and 4.3 and was significantly above 1, indicating a high probability of persistence of LA-MRSA, even without antimicrobial use.     

Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry

ABSTRACT: Understanding protein and gene function requires identifying interaction partners usingbiochemical, molecular or genetic tools. In plants, searching for novel protein-proteininteractions is limited to protein purification assays, heterologous in vivo systems such as the yeast-two-hybrid or mutant screens. Ideally one would be able to search for novel proteinpartners in living plant cells. We demonstrate that it is possible to screen for novel proteinproteininteractions from a random library in protoplasted Arabidopsis plant cells and recoversome of the interacting partners. Our screen is based on capturing the bi-molecularcomplementation of mYFP between an YN-bait fusion partner and a completely random preyYC-cDNA library with FACS. The candidate interactions were confirmed using in plantaBiFC assays and in planta FRET-FLIM assays. From this work, we show that the wellcharacterized protein Calcium Dependent Protein Kinase 3 (CPK3) interacts with APX3,HMGB5, ORP2A and a ricin B-related lectin domain containing protein At2g39050. This isone of the first random in planta screens to be successfully employed.     

Occurrence of the Microcystins MC-LW and MC-LF in Dutch Surface Waters and Their Contribution to Total Microcystin Toxicity

Microcystins (MCs) are the most frequently found cyanobacterial toxins in freshwater systems. Many MC variants have been identified and variants differ in their toxicity. Recent studies showed that the variants MC-LW and MC-LF might be more toxic than MC-LR, the variant that is most abundant and mostly used for risk assessments. As little is known about the presence of these two variants in The Netherlands, we determined their occurrence by analyzing 88 water samples and 10 scum samples for eight MC variants ((dm-7-)MC-RR, MC-YR, (dm-7-)MC-LR, MC-LY, MC-LW and MC-LF) by liquid chromatography with tandem mass spectrometry detection. All analyzed MC variants were detected, and MC-LW and/or MC-LF were present in 32% of the MC containing water samples. When MC-LW and MC-LF were present, they contributed to nearly 10% of the total MC concentrations, but due to their suspected high toxicity, their average contribution to the total MC toxicity was estimated to be at least 45%. Given the frequent occurrence and possible high toxicity of MC-LW and MC-LF, it seems better to base health risk assessments on the toxicity contributions of different MC variants than on MC-LR concentrations alone.