Neospora caninum and Ehrlichia canis co‐infection in a dog with meningoencephalitis

An 8‐year‐old mixed‐breed dog was presented for acute, progressive weakness and ataxia, inappetence, and weight loss. The patient was mentally normal, but nonambulatory, with a right head tilt, right positional ventral strabismus, and slight head tremors. A neurologic lesion was localized to the cerebellum and right brainstem. Cerebrospinal fluid (CSF) analysis showed a markedly increased protein concentration and mixed pleocytosis, with eosinophil predominance (44%), intracytoplasmic inclusions within eosinophils, consistent with Ehrlichia canis (E canis) morulae, and Toxoplasma gondii (T gondii) or Neospora caninum (N caninum) tachyzoites within eosinophils and monocytes. A serum indirect immunofluorescent antibody test was positive for N caninum (titer 1:12 800) and negative for T gondii. Both blood and CSF PCR results were N caninum‐ and E canis‐positive and T gondii‐ and Anaplasma phagocytophilum‐negative, and blood PCR, but not CSF PCR, was Hepatozoon canis‐positive. The dog was treated for 30 days with clindamycin, sulfamethoxazole‐trimethoprim, doxycycline, prednisone, and cephalosporin, but did not improve neurologically, and was euthanized. Brain histopathology showed moderate multifocal, subacute meningoencephalitis with necrosis and gliosis. The neurologic disease was mostly attributed to central nervous system (CNS) neosporosis, with the possible contribution of ehrlichiosis, which was likely a manifestation of blood‐brain barrier disruption. Hepatozoonosis was probably a result or cause of underlying immunosuppression. To our knowledge, this is the first report of CNSN caninum and E canis co‐infection detected by both CSF PCR and cytology and E canis morulae identified within CSF eosinophils.     

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.     

Eosinophilic intestinal diseases of the horse

Eosinophilic intestinal diseases of the horse are not commonly reported. Similar diseases are reported in man, but for either species, these eosinophilic diseases, are poorly understood. Eosinophilic intestinal disease may be accompanied by eosinophilic disease of other organs, or eosinophilia may be restricted to the intestine. When infiltration of eosinophils is restricted to the intestine, horses have a good prognosis for resolution of clinical signs, even though the treatment may include celiotomy with intestinal decompression or resection and administration of a corticosteroid. When eosinophilic intestinal disease is accompanied by infiltration of other organs, such as skin, pancreas, lungs or liver, the horse's prognosis for survival is very poor.     

Anti‐allergic effects of Lactobacillus crispatus KT‐11 strain on ovalbumin‐sensitized BALB/c mice

In this study, we investigated the effects of oral ingestion of Lactobacillus crispatus KT‐11 strain (KT‐11) on the immune response in an allergic rhinitis mouse model, ovalbumin (OVA)‐sensitized BALB/c mice. Sneezing activity in mice that were administered a KT‐11‐supplemented diet was significantly lower than that in mice administered a KT‐11‐free diet (control diet) at age 11 weeks. We found that serum OVA‐specific immunoglobulin E (IgE) levels and total number of interleukin (IL)‐4⁺CD4⁺ spleen cells in mice that were administered a KT‐11‐supplemented diet were significantly lower than in mice administered a control diet. The ratio of spleen interferon‐γ⁺CD4⁺/IL‐4⁺CD4⁺ cells was higher in the mice administered the KT‐11‐supplemented diet compared to that in mice administered the control or L. rhamnosus GG‐supplemented diet. In contrast, the number of CD11b⁺CD80⁺ and FcεRIα⁺CD117⁺ cells was significantly lower in mice administered the KT‐11‐supplemented diet. These results suggested that KT‐11 reduced OVA‐induced allergic symptoms in BALB/c mice via the adjustment of the T helper type 1/T helper type 2 balance, and a decrease in the number of antigen‐presenting cells and high affinity IgE receptor‐positive mast cells.     

Effect of porcine glucagon‐like peptides‐2 on tight junction in GLP‐2R + IPEC‐J2 cell through the PI3k/Akt/mTOR/p70S6K signalling pathway

Because of rare glucagon‐like peptide‐2 (GLP‐2) receptor (+) cells within the gut mucosa, the molecular mechanisms transducing the diverse actions of GLP‐2 remain largely obscure. This research identified the naturally occurring intestinal cell lines that endogenously express GLP‐2R and determined the molecular mechanisms of the protective effects of GLP‐2‐mediated tight junctions (TJ) in GLP‐2R (+) cell line. (i) Immunohistochemistry results showed that GLP‐2R is localised to the epithelia, laminae propriae and muscle layers of the small and large bowels of newborn piglets. (ii) GLP‐2R expression was apparent in the cytoplasm of endocrine cells in IPEC‐J2 cell lines. (iii) The protein expressions of ZO‐1, claudin‐1, occludin, p‐PI₃K, p‐Akt, p‐mTOR and p‐p70^S6K significantly (p < 0.05) increased in GLP‐2‐treated IPEC‐J2 cells, and all of them significantly (p < 0.05) decreased when LY‐294002 or rapamycin was added. GLP‐2 improves intestinal TJ expression of GLP‐2R (+) cells through the PI₃k/Akt/mTOR/p70^S6K signalling pathway.     

Early and Cost‐Effective Identification of High Risk/Priority Control Areas in Foot‐and‐Mouth Disease Epidemics

Geo‐referenced data from the 2001 Uruguayan foot‐and‐mouth disease (FMD) epidemic were explored to assess whether spatial analysis could lead to cost‐benefit based policies. Four variables were analysed: (i) location and size of 4022 individual rural land parcels, of which 574 were infected over 60 days, (ii) animal density, (iii) percentage of dairy farms per county, and (iv) road density. Each variable was categorized into two to five classes (e.g. small/medium/large) and the proportion of cases per class reported at days 1–3 of the epidemic was compared with that reported at days 4–6. A higher proportion of cases was found at days 4–6 than at days 1–3 in areas with: small and medium size land parcels, high animal density, >20% farms specialized in dairy production, and high road density (P < 0.03 for each). Each of these classes showed a greater proportion of cases at days 7–60 than the proportion of the total territory covered by each class's area (early case concentration ratios: 1.14–1.37). Land parcel clusters were indicated by Moran's I‐test (P < 0.01). A new region was constructed by intersecting the four spatial classes associated with higher proportions of cases at days 4–6. At days 7–60, this region included 50.4% of all cases and represented 30.6% of the territory under study (final case concentration ratio: 1.65). The final area per case in this region was at least 33% lower and covered at least 45% less territory than any of the four single‐variable approaches. Bio‐statistical, multivariate spatial analysis of early cases may greatly increase the efficiency of epidemiologic policy.     

FC‐54 Evaluation of IgE‐mediated late‐phase reactions in the skin of normal placebo‐ and prednisolone‐treated dogs: cellular,cytokine and chemokine responses

IgE‐mediated late‐phase reactions can be induced in the skin of normal and atopic dogs by intradermal injections of anti‐IgE antibody. The histology of these reactions is very similar to that of naturally occurring atopic dermatitis. To characterize the cellular, cytokine and chemokine responses in the skin of placebo‐ and prednisolone‐treated dogs, normal beagles received either placebo or 0.5 mg/kg prednisolone twice daily for three days prior to intradermal injection of polyclonal rabbit anti‐canine IgE. Eight‐millimetre punch biopsy skin samples were taken before injection and at the injection sites after 6, 24 and 48 h. Histological and immunohistochemical examination revealed a rapid cellular influx. Eosinophil and neutrophil numbers increased from <1 to 61.4 ± 14.1, and from 7 to 62.2 ± 10.8 cells/mm², respectively, within 6 h after injection, and remained moderately elevated 48 h later. The numbers of CD1c+, CD3+ and CD4+ mononuclear cells were also increased by 6 h. Taqman analysis demonstrated 2.5‐ to 72‐fold increases in mRNA expression for IL‐13, IL‐5, MCP (CCL2), RANTES (CCL5) and TARC (CCL17). Levels of mRNA for IL‐2, IL‐4, IL‐6, and IFNγ remained negligible. Prednisolone administration suppressed the influx of neutrophils and eosinophils, and the expression of IL‐13, CCL2, CCL5 and CCL17 (33, 97, 58, 86, 73 and 90%, respectively), as well as the influx of CD1c+ and CD3+ cells. These data document the cytokine and chemokine response to anti‐IgE injection and demonstrate the anti‐inflammatory effect of prednisolone. Funding: Schering‐Plough Animal Health.     

In this issue – January/February 2010

Septic osteomyelitis in foals  ·  Partial pressure of arterial carbon dioxide during general anaesthesia in horses  ·  Mastocytoma in digital flexor tendons of a horse  ·  Immune-mediated haemolytic anaemia in dogs  ·  Spotted fever group rickettsial antibodies in cats and dogs in Tasmania  ·  Consequences of false-positive results for Chlamydophila abortus in Australian sheep for the rejection of breeder sheep for export  ·  Femoral diaphysis fractures in cattle  ·  Haemolytic anaemia in cattle in New South Wales associated with Theileria infections  ·  Stimulation test for determining the steroidogenic capacity of the parrot testis  ·  Seminoma in a black rhinoceros     

Eosinophils of the horse: Part I: Development,distribution, structure and biochemical mediators

Eosinophils are becoming the target of increasing research interest as recent studies suggest that their role in immune homoeostasis and the immune response to disease is far more complex than previously understood. Historically, the horse eosinophil has been used to study basic eosinophil biology because of the considerable volume of blood required to obtain enough viable cells for reliable, repeatable experiments. This resulted in a large but disseminated body of literature pertaining to the structure and function of the horse eosinophil. More recently, equine clinicians have produced case reports and clinical studies in an effort to define the role of the eosinophil in diseases of the horse. A thorough review of the equine eosinophil incorporating both bench research and clinical reports does not exist. The objective of this two-part review is to fill this need by integrating the basic science and clinical research into a comprehensive body of work on what is known specifically about the horse eosinophil, and its role in equine health and disease. Part I summarises the development and tissue distribution of eosinophils in the normal horse, and presents what is known about the cell structure, migration and biochemical mediators of the horse eosinophil. Part II reviews the role of the eosinophil in diseases of the horse, and concludes with a summary of knowledge gaps and open research questions to benefit both those who wish to use the equine eosinophil as a model for basic science research, and those whose primary interest lies with diseases of horse.     

Generation of α‐1,3‐Galactosyltransferase‐Deficient Porcine Embryonic Fibroblasts by CRISPR/Cas9‐Mediated Knock‐in of a Small Mutated Sequence and a Targeted Toxin‐Based Selection System

The CRISPR/Cas9 system has enabled the editing of mammalian genomes; however, its applicability and efficiency in the pig genome has not been studied in depth. The α‐gal epitope synthesized by α‐1,3‐galactosyltransferase gene (GGTA1) is known as a xenoantigen obtained upon pig‐to‐human xenotransplantation. We here employed the CRISPR/Cas9 system‐mediated knock‐in of endogenous GGTA1 via targeted homologous recombination (HR). Linearized donors with ~800‐bp homology flanking the CRISPR/Cas9 target site [exon 4 (containing ATG) of GGTA1] served as a template for gene targeting by HR. Using a targeted toxin strategy to select clones lacking α‐gal epitope expression, we successfully obtained several knock‐in clones within 3 weeks of initial transfection. These results suggest that the use of CRISPR/Cas9‐mediated HR to knock‐in a mutated fragment at defined loci represents an efficient strategy to achieve the rapid modulation of genes of interest in swine cells and is a promising tool for the creation of KO piglets.     

State‐of‐the‐Art‐Review: Immunomodulatory effects of opioids

Objective – To review the immunomodulatory effects of opioids. Data Sources – Original research publications and review articles using the PubMed search engine with the following keywords – opioids, morphine, immuomodulation, and immunosuppression. Veterinary and Human Data Synthesis – Opioids have been shown to modulate the immune system in animal models by affecting both the acquired and innate arms of the immune system. Natural killer cell activity, T‐cell proliferation, antibody production, phagocytic cell function, and cytokine production have all been shown to be affected by opioids. Many of these effects are reversed by opioid antagonists. Opioids have also been shown to induce sepsis in laboratory animals. Opioid administration alters immune parameters in healthy humans at analgesic doses and may increase the risk of infection in some patient populations. Conclusions – While opioids remain the most powerful and widely used analgesics available, their negative effects on the immune system are well established in the laboratory setting. Thoughtful consideration should be given to the use of certain opioids in critically ill patients, especially those with pre‐existing immunocompromise.     

Eosinophils of the horse: Part II: Eosinophils in clinical diseases

Interpretation of eosinophilia in body fluids or tissues is often not straightforward. Eosinophil counts vary among clinically healthy individuals, and considerable overlap can occur between normal and affected animals in conditions such as allergic airway disease. Parasite exposure is a confounding factor when counts are increased, and in cases where very high counts and dramatic clinical signs make another disease process obvious, the underlying pathology may be uncertain and treatment difficult. Eosinophils are a component of the immune response in many diseases of the horse, but their specific role is often unknown and likely multifactorial. In helminth infections, eosinophils are assumed to be part of the normal host response to a pathogen, whereas in multisystemic eosinophilic epitheliotropic disease (MEED), the predominance of eosinophils likely represents a wildly dysregulated response, or an abnormal response altogether. This distinction is still not clear for other diseases. Understanding the pathways involved in recruitment, activation or suppression of eosinophils is required for more accurate diagnostics, effective therapeutics, and successful strategies for prevention of eosinophil associated diseases. Eosinophils of the horse: Part II reviews published observations on the eosinophil in clinical diseases of the horse. The behaviour of eosinophils in three common and relatively well-studied conditions is presented first, including gastrointestinal helminth infections, non-infectious respiratory disease, and insect bite hypersensitivity. The less common eosinophil-associated diseases such as eosinophilic disease confined to the intestine (EDCI) and MEED are considered, followed by a brief summary of the eosinophil in phycomycosis and neoplasia. In conclusion, a panoramic view of the equine eosinophil as presented in Parts I and II is placed in the larger context of current eosinophil research, and areas of study are identified that may improve our understanding of eosinophil biology in equine health and clinical disease.     

18F‐FDG‐PET/CT as adjunctive diagnostic modalities in canine fever of unknown origin

Fever of unknown origin (FUO) is a persistent or recurrent fever for which the underlying source has not been identified despite diagnostic investigation. In people, ¹⁸F‐fluoro‐2‐deoxyglucose positron emission tomography (¹⁸F‐FDG‐PET) alone or in combination with computed tomography (CT) is often beneficial in detecting the source of fever when other diagnostics have failed. Veterinary reports describing use of these modalities in animals with fever of unknown origin are currently lacking. Aims of this retrospective case series were to describe ¹⁸F‐FDG‐PET or ¹⁸F‐FDG‐PET/CT findings in a group of dogs with fever of unknown origin. Dogs presenting to a single center between April 2012 and August 2015 were included. A total of four dogs met inclusion criteria and underwent either positron emission tomography (n = 2) or positron emission tomography/CT (n = 2) as a part of their diagnostic investigation. All subjects underwent extensive diagnostic testing prior to ¹⁸F‐FDG‐PET/CT. Initial diagnostic evaluation failed to identify either a cause of fever or an anatomic location of disease in these four dogs. In each dog, positron emission tomography or positron emission tomography/CT was either able to localize or rule out the presence of focal lesion thereby allowing for directed sampling and/or informed disease treatment. Follow up ¹⁸F‐FDG‐PET/CT scans performed in two patients showed improvement of observed abnormalities (n = 1) or detected recurrence of disease allowing for repeated treatment before clinical signs recurred (n = 1). Fever resolved after specific treatment in each dog. Findings from the current study supported the use of positron emission tomography or positron emission tomography/CT as adjunctive imaging modalities for diagnosis and gauging response to therapy in dogs with fever of unknown origin.     

FC‐33 Expression of IL‐12 receptor β2 gene in atopic dogs

Atopic dermatitis (AD) is very common in dogs, but its pathogenesis is not yet fully understood. It has been suggested that a Th2‐dominant status may be associated with the occurrence of canine AD. IL‐12 is thought to be important for the differentiation of Th1 cells. The IL‐12 receptor β2 (IL‐12Rβ2) gene is considered to play a critical role in signal transduction and is attracting attention as one of the causative genes of AD in humans. The purpose of this study was to investigate the relationship between IL‐12Rβ2 gene expression and canine AD. The canine IL‐12Rβ2 gene was cloned by RT‐PCR and its nucleotide sequences were determined. Canine IL‐12Rβ2 showed 76.8% homology at the amino acid level with human IL‐12Rβ2, and its structural motifs were well conserved. cDNA with a 91 bp deletion including the transmembrane region was also cloned, which consequently produced a frame shift and an early stop codon. The deletion region corresponded to exon 14 of the human IL‐12Rβ2 gene on chromosome 1. The expression of deleted canine IL‐12Rβ2 mRNA in phytohemagglutinin‐stimulated peripheral blood mononuclear cells was examined in seven healthy dogs and 11 AD dogs. Both deleted and intact mRNAs were expressed at constant ratios in healthy and AD dogs. The results indicate that the deletion of the transmembrane region is not associated with the occurrence of AD, and that the expression of the deleted mRNA may be constitutive and produced by alternative splicing. Funding: Self‐funded.