Quantification of mucosa-adhered microbiota of lambs and calves by the use of culture methods and fluorescent in situ hybridization coupled with flow cytometry techniques

The intestinal mucosa-associated microbiota could play important biological roles due to its close proximity with the animal host, but knowledge on its composition is still limited. The aim of this study was to characterize the microbial communities tightly associated with different parts (rumen, duodenum and colon) of the gastrointestinal tract (GIT) of healthy lambs and calves by using both cultural, and fluorescent in situ hybridization-flow cytometry (FCM-FISH) techniques. Lactic acid bacteria genera were one of the predominant bacteria detected in lambs and calves by both methodologies, possibly constituting an index of their healthy status. The levels of Lactobacillus were significantly higher (p<0.05) in the rumen and duodenum of lambs, and in the rumen of calves. The levels of Bifidobacterium were significantly higher (p<0.05) in the colon of both animal species and the rumen of lambs. Significant differences (p<0.05) were found in counts of other microbial groups (yeast, Enterococcus, Propionibacterium, Bacteroides, Clostridium and Enterobacteriaceae) at diverse GI sections depending on the animal species. In general, microbial counts follow the same trends regardless the applied technique. The most remarkable differences were found in detection levels of Bacteroides and Clostridium, which tended to be significantly higher (p<0.05) when analysed by FCM-FISH. This technique also allowed the detection of quantitatively important bacteria (sulphate-reducing bacteria, Atopobium and Coriobacterium), which are difficult to cultivate in selective medium. Therefore, FCM-FISH has been proven to be a sensitive high throughput approach that provides additional information to that obtained by traditional culture techniques about the complexity of the GI ecosystem of these animal species.     

Evaluation of endotoxaemia in the prognosis and treatment of scouring merino lambs

This study looked at measurement of endotoxaemia as a tool in determining prognosis and probable response to treatment in scouring lambs. One hundred eighty-three lambs in the first 15-20 days of life, from eight Merino sheep farms located in the region of La Serena, south-west Spain, were used in this experiment. Scouring and normal/control lambs were selected following a clinical examination, the scouring group was further divided into subgroups, specifically those that did or did not survive 72 h following treatment. At the time of the clinical examination, faecal and blood samples were taken. Faecal culture and commercial faecal antigen tests for detection of enteropathogens in faeces and serum endotoxin measurement using chromogenic lymulus amoebocyte lysate (LAL) were carried out. Scouring lambs received 0.07 mg/kg liveweight halofuginone once a day for 3 days, a single oral dose of 0.20 mg/kg liveweight of spectinomycin and oral rehydration fluid. The pathogens isolated were Cryptosporidium spp. and Escherichia coli. The case fatality rate was 51% in the scouring lambs. Postmortem findings were consistent with enterotoxigenic E. coli infection. The concentration of endotoxin was 0.18 +/- 0.12 ng/ml in the control group, 0.35 +/- 0.17 ng/ml in the surviving lambs and 0.46 +/- 0.14 ng/ml in the non-surviving lambs. Significant differences between groups were found. Case fatality rate of the scouring lambs with endotoxaemia below 0.30 ng/ml was 0%, while it was 100% above 0.50 ng/ml. These results may be utilized as a prognostic indicator in lambs affected by E. coli and Cryptosporidium that will help aid in decision-making as to whether to treat a lamb or not based on its chances of survival.     

Concurrent infection with Trichinella spiralis and other helminths in pigs

The aim of this study was to investigate possible influence of different helmintosis in the development of Trichinella spiralis in experimental infected pigs. Forty-two Iberian pigs were allocated to six groups. Three groups were single inoculated with Ascaris suum, Metastrongylus apri or T. spiralis, respectively. Two groups were co-infected with T. spiralis and A. suum or T. spiralis and M. apri, respectively, while the last group included uninfected control pigs. Clinical signs were only observed in pigs with single or concurrent M. apri infections, with more severe respiratory symptoms in pigs with mixed M. apri infection. The number of A. suum and M. apri lung larvae, intestinal larvae of A. suum and adult M. apri were reduced in pigs with mixed Trichinella infections compared to pigs with single infections. In contrast, the number of liver white spots was higher in pigs with mixed infections. While T. spiralis muscular larval burdens were increased in pigs concomitantly infected with M. apri, they were reduced in pigs concomitantly infected with A. suum, compared to pigs receiving single infections with either of these helminths. Pigs with single or mixed A. suum infections showed higher eosinophil levels compared to the remaining groups. IgGt, IgG1, IgG2 and IgM against T. spiralis antigen could not be detected in pigs with single Ascaris or Metastrongylus infections, indicating that no cross-antibodies were produced. IgGt, IgG1 and IgM antibodies were detected earlier and generally at higher levels in mixed T. spiralis infections compared to single T. spiralis infections. The results suggest that T. spiralis had a low synergistic interaction with M. apri in concomitantly infected pigs, and an antagonistic interaction in concurrent infection with A. suum.     

Antiapoptotic activity of bovine herpesvirus type-1 (BHV-1) UL14 protein

Viruses have evolved different strategies to interfere with apoptotic pathways in order to halt cellular responses to infection. One previous study showed that transient transfection of bovine herpesvirus type-1 (BHV-1) UL14 protein is efficient in protecting Madin Darby kidney (MDBK) and human chronic myelogenous leukemia (K562) cells from sorbitol-induced apoptosis. This protein corresponds to a putative protein of BHV-1, which shares aminoacid sequence with a part of the peptide-binding domain conserved in human heat shock protein (HSP70) family. The pBK-CMV-UL14 plasmid transfected MDBK cells treated with sorbitol did not show caspase-3 and caspase-9 activation with respect to non-transfected MDBK cells (UL14 negative). Furthermore, we report that the expression of the full length sequence of BHV-1 UL14 is evident after 7 h of infection of BHV-1 on MDBK cells which were then treated with sorbitol. These results indicate that UL14 gene product has important implications to enhance cell survival in response to apoptotic stimuli.     

Total and Differential Leucocyte Counts and Lymphocyte Subpopulations in Lymph, Afferent and Efferent to the Supramammary Lymph Node, During Endotoxin-Induced Bovine Mastitis

Leucocyte trafficking in afferent and efferent mammary lymph and the supramammary lymph node in cows was examined during 4 h after intramammary infusion of endotoxin from Escherichia coli. Total and differential leucocyte counts were measured in milk, blood and lymph. The proportions of CD4(+), CD8(+), major histocompatibility complex (MHC) class II(+) and IgM(+) lymphocytes were examined in the lymph and lymph node. At post-infusion hour (PIH) 4, the flow rates of both lymph fluids had increased approximately eightfold. Total leucocyte concentration increased in afferent lymph, but decreased in efferent lymph. Neutrophils increased in afferent lymph at PIH 2 and in efferent lymph and milk at PIH 4. The predominant cell type in afferent lymph shifted from lymphocyte to neutrophil while lymphocyte was still at PIH 4 the predominant type in efferent lymph. Among the lymphocytes, B cells were predominant in afferent lymph and lymph node at PIH 4 while T cells, mainly CD4(+) cells, were predominant in efferent lymph both at PIH 0 and PIH 4. The CD4 : CD8 ratio was higher in efferent lymph and the challenged lymph node than in afferent lymph and the control node, respectively. There was a significant difference in proportions of each lymphocyte subpopulation except for IgM(+) cells, between afferent and efferent lymph after infusion. According to the results, there was already during the first hours of the immune response, a non-random trafficking of neutrophils and lymphocyte subpopulations resulting in a changed distribution of cells in afferent and efferent lymph and a difference in lymphocyte reactivity between the two lymph fluids.     

Surgical repair of femoral fractures in New World camelids: five cases (1996-2003)

Five New World camelids were admitted to the Western College of Veterinary Medicine between 1996 and 2003 for evaluation of femoral fractures. There were three alpacas and two llamas. Four of the animals were female and three were less than 3 months of age. Fracture configurations consisted of distal physeal fractures (three), a comminuted diaphyseal/metaphyseal fracture, and a transverse diaphyseal fracture. Fractures were diagnosed with a combination of physical examination and radiographs in all cases. All five fractures were repaired with internal fixation and three animals were discharged from the hospital with fractures that healed. One cria underwent successful internal fixation but died from pulmonary oedema during recovery from anaesthesia. Postoperative complications were rare and limited to inadequate fracture stability in one alpaca and prolonged recovery to weight bearing in another. One llama with a comminuted metaphyseal fracture, repaired with a 4.5 mm dynamic compression plate, subsequently had catastrophic failure of the bone 17 days after surgery. Overall the clients were pleased with the outcome of discharged animals. Although femoral fractures are considered rare, they pose a unique opportunity for the large animal veterinarian to successfully achieve fracture union with the aid of internal fixation.     

Evaluation of an antigen capture ELISA for the early diagnosis of Hypoderma lineatum in cattle under field conditions

An antigen capture or sandwich ELISA (sELISA) was evaluated for the diagnosis of Hypoderma lineatum in cattle under field conditions in northwestern Spain. The kinetics of circulating hypodermin C (HyC) and specific antibodies during the course of natural infestation were determined in a group of 10 Frisian calves. In addition, oesophagi and blood samples were taken from 105 cows at a slaughterhouse in order to compare three methods for the diagnosis of H. lineatum: sandwich ELISA for the detection of the antigen HyC (sELISA), indirect ELISA for the detection of antibodies anti-HyC (iELISA) and the detection of first instars (L1) in the oesophagus. In naturally infested cattle, HyC was present in circulation at low levels during the early and late phases of the infestation. However, in the middle phase, coinciding with the presence of L1 in the oesophagus, two peaks of increased HyC concentration were observed. Specific antibodies increased progressively until the first appearance of larvae in warbles on the back. There was no correlation between antigen or antibody levels and the number of grubs in the back. Prevalence of first instars in the oesophagi of slaughtered cows was 21.9% (23/105). The percentage of cattle that were positive for circulating antigen was slightly higher (24.8%), suggesting the recent destruction of migrating larvae in some animals. However, there was no correlation between the number of L1 and HyC levels. With the iELISA, 79% of the animals were positive to Hypoderma, which means that a high percentage of those animals have been exposed to the parasite but they had no apparent current infestation. The sELISA is a good tool to follow larval development within the host; however, the episodic elevation of HyC levels limits the usefulness of this test for the early diagnosis of Hypoderma under field conditions.     

Effects of 2 concentrations of sodium citrate on coagulation test results, von Willebrand factor concentration, and platelet function in dogs

BACKGROUND: Blood collection tubes containing 3.2% (0.109 M) sodium citrate, instead of 3.8% (0.129 M) sodium citrate, have recently become available in the United States. These tubes are visually indistinguishable from the traditional 3.8% sodium citrate tubes, except for wording on the label. Consequently, samples for hemostatic evaluation are frequently collected in tubes containing the lower concentration of sodium citrate. HYPOTHESIS: Results of hemostasis assays are different in samples collected in 3.2% versus 3.8% sodium citrate. ANIMALS: Twenty healthy dogs. METHODS: This study aimed at determining whether results of standard coagulation tests, von Willebrand factor concentration (vWF:Ag), and platelet function with the platelet function analyzer PFA-100a were affected by the different concentrations of sodium citrate. Blood samples were collected in tubes containing either 3.2% or 3.8% sodium citrate concentrations and processed routinely for coagulation assays (one-stage prothrombin time [OSPT], activated partial thromboplastin time [aPTT], fibrinogen concentration, and platelet count), vWF:Ag, and platelet function assays with a PFA-100. RESULTS: There was no significant difference between samples collected in 3.2% versus those collected in 3.8% sodium citrate for OSPT, aPTT, fibrinogen concentration, platelet count, or vWF:Ag. The closure times with collagen/adenosine diphosphate were significantly shorter (66 +/- 8.1 versus 74.8 +/- 9.7 seconds; P < .0001) with the 3.2% than with 3.8% sodium citrate concentration, and the hematocrit was significantly higher (47.9 +/- 5.6 versus 46.0 +/- 4.7 seconds; P = .03) in samples collected in 3.2% than in those collected in 3.8% sodium citrate. CONCLUSIONS AND CLINICAL IMPORTANCE: There is no clinically relevant effect of collection of blood into 3.2% or 3.8% sodium citrate.