Validation of the Sysmex XN-V Automated Nucleated Red Blood Cell Enumeration for Canine and Feline EDTA-Anticoagulated Blood

The enumeration of nRBCs (nucleated red blood cells) by manual counting is time-consuming and imprecise. As the first veterinary hematology analyzer, Sysmex XN-V provides automated nRBC counts. This study aimed to evaluate the performance of Sysmex XN-V in the enumeration of nRBCs for cats and dogs by comparing automated nRBC counts to manual counts from a total of 3810 canine and 2844 feline specimens. Repeatability, reproducibility, stability, carry-over, and linearity were assessed. The repeatability and reproducibility of Sysmex XN-V were good, with mean coefficients of variation (CV) of 4.5% and 5.4%, respectively. Bland–Altman difference analysis revealed mean biases shown as nRBCs/100 WBCs of 0.01 in dogs and 0.11 in cats with low nRBCs (20 nRBCs/100 WBCs). The total observable error was below 9% in both species and at all ranges. Overall concordance between methods was high (91% in canine and 93% in feline samples). The automated nRBC count by Sysmex XN-V was found to be accurate and precise and can replace manual counts for cat and dog samples. Non-statistical quality assurance by scattergram evaluation, re-gating, and confirmation by blood smear evaluation is, however, recommended, especially in cases with severe normoblastosis. This advancement will save time, reduce errors, and add prognostic value to hematological results for animal patients

Protective Immunity against Infection with <i>Mycoplasma haemofelis</i>

Hemoplasmas are potentially zoonotic mycoplasmal pathogens, which are not consistently cleared by antibiotic therapy. Mycoplasma haemofelis is the most pathogenic feline hemoplasma species. The aim of this study was to determine how cats previously infected with M. haemofelis that had recovered reacted when rechallenged with M. haemofelis and to characterize the immune response following de novo M. haemofelis infection and rechallenge. Five specific-pathogen-free (SPF)-derived naive cats (group A) and five cats that had recovered from M. haemofelis infection (group B) were inoculated subcutaneously with M. haemofelis. Blood M. haemofelis loads were measured by quantitative PCR (qPCR), antibody response to heat shock protein 70 (DnaK) by enzyme-linked immunosorbent assay (ELISA), blood lymphocyte cell subtypes by flow cytometry, and cytokine mRNA levels by quantitative reverse transcriptase PCR. Group A cats all became infected with high bacterial loads and seroconverted, while group B cats were protected from reinfection, thus providing the unique opportunity to study the immunological parameters associated with this protective immune response against M. haemofelis. First, a strong humoral response to DnaK was only observed in group A, demonstrating that an antibody response to DnaK is not important for protective immunity. Second, proinflammatory cytokine interleukin-6 (IL-6) mRNA levels appeared to increase rapidly postinoculation in group B, indicating a possible role in protective immunity. Third, an increase in IL-12p35 and -p40 mRNA and decrease in the Th2/Th1 ratio observed in group A suggest that a Th1-type response is important in primary infection. This is the first study to demonstrate protective immunity against M. haemofelis reinfection, and it provides important information for potential future hemoplasma vaccine design

Mycotoxins in Wheat and Mitigation Measures

Latest estimates for world cereal production in 2015 and EU‐28 production in 2014 are approximately 2540 and 323 mil tons, respectively. The FAO estimated that the global wheat consumption is about 66 kg/per capita. Among the most important risks associated with wheat consumption are mycotoxins. It has been estimated that up to 25% of the world\u27s crops grown for food and feed may be contaminated with mycotoxins. Despite efforts in controlling fungal growth, mycotoxin co‐contamination represents an unavoidable risk, occurring pre‐ and postharvest and resulting in reduced nutritional value and possible risks for human and animal health. In addition to health risks, mycotoxins have a detrimental effect on the quality and the processing performance of wheat. Mitigation measures to manage the challenge of mycotoxins in wheat include strategies at pre‐ and postharvest. Preharvest events are predominantly dictated by environmental factors and good agronomic/cultural practices, whereas storage and processing are the major areas where contamination can be prevented at postharvest. Integrating as many management options as possible may minimize the risk of mycotoxin contamination in wheat and wheat products

Canine tick-borne pathogens in Cyprus and a unique canine case of multiple co-infections

Canine tick-borne pathogens such as Ehrlichia canis and Hepatozoon canis are widespread in the Mediterranean basin but have never been reported or investigated in Cyprus. We describe herein the presence of canine tick-borne pathogens in three dogs with clinical signs compatible with vector-borne diseases from Paphos area of Cyprus. Molecular and phylogenetic analysis revealed the presence of E. canis, Anaplasma platys, H. canis, Babesia vogeli and Mycoplasma haemocanis in Cyprus. One dog co-infected with E. canis, H. canis, B. vogeli and M. haemocanis is, to the best of our knowledge, the first report of this multiple co-infection in dogs. The tick-borne pathogens reported in the current study should be considered in the differential diagnoses in dogs exposed to ticks in Cyprus

Chronic "Candidatus Mycoplasma turicensis" infection

"Candidatus Mycoplasma turicensis" infects felids. The pathogenesis of "Candidatus M. turicensis" chronic infection is poorly understood. The goals of the present study were to (1) induce reactivation of the infection in chronic carrier cats by attempted immunosuppression, (2) identify potential tissue sequestration using real-time TaqMan® PCR and (3) monitor the humoral immune response by DnaK enzyme-linked immunosorbent assay (ELISA). Ten specified pathogen-free cats that had ostensibly recovered from experimental "Candidatus M. turicensis" infection were used: five cats (group 1) received high dose methylprednisolone (attempted immunosuppression), while five cats served as untreated controls (group 2). Besides weekly blood samples, tissue samples were collected from bone marrow, kidney, liver and salivary glands at selected time points. The cats in group 1 had significantly lower lymphocyte counts and higher blood glucose levels after methylprednisolone administration than the controls. After methylprednisolone administration one blood and three tissue samples from cats in group 1 tested PCR-positive; before the administration, only one sample was positive. All other samples tested PCR-negative. All cats stayed seropositive; the antibody levels of the cats in group 1 showed a significant transient decrease after methylprednisolone administration. This is the first study to report the presence of "Candidatus M. turicensis" in tissues of chronically infected cats and the persistence of anti-feline hemoplasma antibodies in the absence of detectable bacteremia. Methylprednisolone administration did not lead to a significant reactivation of the infection. Our results enhance the knowledge of "Candidatus M. turicensis" infection pathogenesis and are clinically relevant to the prognosis of hemoplasma-infected cats

Autosomal recessive hyposegmentation of granulocytes in Australian Shepherd Dogs indicates a role for LMBR1L in myeloid leukocytes

Pelger-Huët anomaly (PHA) in humans is an autosomal dominant hematological phenotype without major clinical consequences. PHA involves a characteristic hyposegmentation of granulocytes (HG). Human PHA is caused by heterozygous loss of function variants in the LBR gene encoding lamin receptor B. Bi-allelic variants and complete deficiency of LBR cause the much more severe Greenberg skeletal dysplasia which is lethal in utero and characterized by massive skeletal malformation and gross fetal hydrops. HG phenotypes have also been described in domestic animals and homology to human PHA has been claimed in the literature. We studied a litter of Australian Shepherd Dogs with four stillborn puppies in which both parents had an HG phenotype. Linkage analysis excluded LBR as responsible gene for the stillborn puppies. We then investigated the HG phenotype in Australian Shepherd Dogs independently of the prenatal lethality. Genome-wide association mapped the HG locus to chromosome 27 and established an autosomal recessive mode of inheritance. Whole genome sequencing identified a splice site variant in LMBR1L, c.191+1G>A, as most likely causal variant for the HG phenotype. The mutant allele abrogates the expression of the longer X2 isoform but does not affect transcripts encoding the shorter X1 isoform of the LMBR1L protein. The homozygous mutant LMBR1L genotype associated with HG is common in Australian Shepherd Dogs and was found in 39 of 300 genotyped dogs (13%). Our results point to a previously unsuspected function of LMBR1L in the myeloid lineage of leukocytes

Lack of cross-protection against Mycoplasma haemofelis infection and signs of enhancement in "Candidatus Mycoplasma turicensis"-recovered cats

"Mycoplasma haemofelis" and "Candidatus Mycoplasma turicensis" are feline hemoplasmas that induce hemolytic anemia. Protection from homologous re-challenge was recently demonstrated in cats recovered from primary infection. Here, we determined if cats recovered from "Cand. M. turicensis" infection were protected against infections with the more pathogenic M. haemofelis. Ten specified pathogen-free cats were exposed to M. haemofelis. Five of the ten cats had recovered from "Cand. M. turicensis" bacteremia (group A), and five cats were naïve controls (group B). No cross-protection was observed. By contrast, the "Cand. M. turicensis"-recovered cats displayed faster M. haemofelis infection onset (earlier PCR-positive and anemic) than the controls. No "Cand. M. turicensis" was detected in any cat. M. haemofelis shedding was observed in saliva, feces and urine. In both groups, evidence of a Th1 response was observed (high IFN-γ, low IL-4), but IL-10 levels were also high. In group A, total, CD4+ and CD8+ T cells increased within days after M. haemofelis exposure. At times of maximal bacteremia, macrocytic hypochromic anemia, neutropenia, monocytosis and a decrease in leukocyte, eosinophil, and lymphocyte counts and subsets thereof (B- and T-cells, CD4+, CD8+ and CD4+CD25+ cells) were particularly significant in group A. Moreover, an increase in protein concentrations, hypoalbuminemia and a polyclonal hypergammaglobulinemia were observed. Five of ten M. haemofelis-infected cats subsequently cleared bacteremia without antibiotic treatment. In conclusion, the study suggests that a previous hemoplasma infection, even when the cat has ostensibly recovered, may influence subsequent infections, lead to an enhancement phenomenon and other differences in infection kinetics

Association between canine leishmaniosis and Ehrlichia canis co-infection: a prospective case-control study

Abstract Background In the Mediterranean basin, Leishmania infantum is a major cause of disease in dogs, which are frequently co-infected with other vector-borne pathogens (VBP). However, the associations between dogs with clinical leishmaniosis (ClinL) and VBP co-infections have not been studied. We assessed the risk of VBP infections in dogs with ClinL and healthy controls. Methods We conducted a prospective case-control study of dogs with ClinL (positive qPCR and ELISA antibody for L. infantum on peripheral blood) and clinically healthy, ideally breed-, sex- and age-matched, control dogs (negative qPCR and ELISA antibody for L. infantum on peripheral blood) from Paphos, Cyprus. We obtained demographic data and all dogs underwent PCR on EDTA-blood extracted DNA for haemoplasma species, Ehrlichia/Anaplasma spp., Babesia spp., and Hepatozoon spp., with DNA sequencing to identify infecting species. We used logistic regression analysis and structural equation modelling (SEM) to evaluate the risk of VBP infections between ClinL cases and controls. Results From the 50 enrolled dogs with ClinL, DNA was detected in 24 (48%) for Hepatozoon spp., 14 (28%) for Mycoplasma haemocanis, 6 (12%) for Ehrlichia canis and 2 (4%) for Anaplasma platys. In the 92 enrolled control dogs, DNA was detected in 41 (45%) for Hepatozoon spp., 18 (20%) for M. haemocanis, 1 (1%) for E. canis and 3 (3%) for A. platys. No Babesia spp. or “Candidatus Mycoplasma haematoparvum” DNA was detected in any dog. No statistical differences were found between the ClinL and controls regarding age, sex, breed, lifestyle and use of ectoparasitic prevention. A significant association between ClinL and E. canis infection (OR = 12.4, 95% CI: 1.5–106.0, P = 0.022) was found compared to controls by multivariate logistic regression. This association was confirmed using SEM, which further identified that younger dogs were more likely to be infected with each of Hepatozoon spp. and M. haemocanis, and dogs with Hepatozoon spp. were more likely to be co-infected with M. haemocanis. Conclusions Dogs with ClinL are at a higher risk of co-infection with E. canis than clinically healthy dogs. We recommend that dogs diagnosed with ClinL should be tested for E. canis co-infection using PCR

Modified-live feline calicivirus vaccination elicits cellular immunity against a current feline calicivirus field strain in an experimental geline challenge study

Feline calicivirus (FCV) is a common cat virus associated with oral ulcerations and virulent-systemic disease. Efficacious FCV vaccines protect against severe disease but not against infection. The high genetic diversity of FCV poses a challenge in vaccine design. Protection against FCV has been related to humoral and cellular immunity; the latter has not been studied in detail. This study investigates the cellular and humoral immune response of specified pathogen-free (SPF) cats after modified-live FCV F9 vaccinations and two heterologous FCV challenges by the analysis of lymphocyte subsets, cytokine mRNA transcription levels, interferon (IFN)-γ release assays in peripheral blood mononuclear cells (PBMCs), anti-FCV antibodies, and neutralisation activity. Vaccinated cats developed a Th1 cytokine response after vaccination. Vaccination resulted in antibodies with neutralising activity against the vaccine but not the challenge viruses. Remarkably, IFN-γ-releasing PBMCs were detected in vaccinated cats upon stimulation with the vaccine strain and the first heterologous FCV challenge strain. After the first experimental infection, the mRNA transcription levels of perforin, granzyme B, INF-γ, and antiviral factor MX1 and the number of IFN-γ-releasing PBMCs when stimulated with the first challenge virus were higher in vaccinated cats compared to control cats. The first FCV challenge induced crossneutralising antibodies in all cats against the second challenge virus. Before the second challenge, vaccinated cats had a higher number of IFN-γ-releasing PBMCs when stimulated with the second challenge virus than control cats. After the second FCV challenge, there were less significant differences detected between the groups regarding lymphocyte subsets and cytokine mRNA transcription levels. In conclusion, modified-live FCV vaccination induced cellular but not humoral crossimmunity in SPF cats; innate immune mechanisms, secretory and membranolytic pathways, and IFN-γ-releasing PBMCs seem to be important in the host immune defence against FCV