Characterization of a novel nicotinamide adenine dinucleotide-cytochrome b5 reductase mutation associated with canine hereditary methemoglobinemia

Hereditary methemoglobinemia associated with nicotinamide adenine dinucleotide-cytochrome b5 reductase (b5R) deficiency is a rare autosomal recessive disorder in animals. Recently, nonsynonymous b5R gene (CYB5R3) variants have been reported to be associated with canine and feline hereditary methemoglobinemia. However, the underlying molecular mechanisms of canine and feline methemoglobinemia caused by these nonsynonymous variants have not yet been reported. Previously, we reported a Pomeranian dog family with hereditary methemoglobinemia, carrying CYB5R3 mutation of an A>C transition at codon 194 in exon 7, replacing an isoleucine residue with leucine (p.Ile194Leu). In this study, we investigated the enzymatic and structural properties of the soluble form of wild-type and Ile194Leu canine b5Rs to characterize the effects of this missense mutation. Our results showed that the kinetic properties of the mutant enzyme were not affected by this amino acid substitution. The secondary structure of the wild-type and Ile194Leu b5Rs detected by circular dichroism showed a similar pattern. However, the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin hydrolysis. Moreover, the thermostability and unfolding measurements indicated that the mutant enzyme was more sensitive to temperature-dependent denaturation than the wild-type b5R. We concluded from these results that unstable mutant enzyme properties with normal enzymatic activity would be associated with hereditary methemoglobinemia in the Pomeranian dog family.     

Comparison of wet-mount,Wright-Giemsa and Gram-stained urine sediment for predicting bacteriuria in dogs and cats

This study assessed the standard urinalysis technique and sediment stain techniques as predictors of bacterial culture results for canine and feline urine. Canine (n = 111) and feline (n = 79) urine samples were evaluated using unstained wet-mount and air-dried Gram and Wright-Giemsa stained sediment; results were compared to aerobic bacterial culture. Eleven canine and 7 feline urine samples were culture positive. Unstained wet-mount and stained sediment had sensitivities of 89% and 83% and specificities of 91% and 99%, respectively. The specificity of using either stain was higher (P < 0.01) than wet-mount examination for detecting bacteriuria. There were significant differences among 3 technologists in detecting true positives (P < 0.01). Association of sediment and culture results used 112 canine and 81 feline samples. There was a negative association (P < 0.01) between lipid detection and wet-mount identification of bacteria.     

Retrospective Biomolecular Investigation of Coxiella burnetii and Leptospira spp. DNA in Cases of Abortion,Stillbirth and Neonatal Mortality in Dogs and Cats

Abortion and neonatal mortality are events that can occur in breeding bitches and queens. It has been reported that up to 55% and 33% of these cases remain without a known cause, respectively, in canine and feline pregnancies. Unusual abortigenic and potentially zoonotic agents, including Coxiella burnetii and Leptospira spp., may be involved in these cases. C. burnetii is able to cause reproductive disorders in cattle, sheep and goats, and cases of abortion have been observed in dogs and cats. Moreover, several outbreaks of C. burnetii infection in humans have been caused by delivering bitches and queens, and some of these animals experienced abortion. Leptospira interrogans sensu lato is able to cause abortion or stillbirth in several animal species and its abortigenic role has occasionally been described in bitches and queens. The aim of this study was to search for C. burnetii and Leptospira spp. DNA in a retrospective series of 103 cases of canine and feline abortion, stillbirth, and neonatal mortality submitted for the identification of possible infectious agents. One hundred and fifty-one specimens were tested using PCR assays and found negative for C. burnetii and Leptospira DNA. However, in 49 samples (47.6%) other infectious causes of abortion, stillbirth, and neonatal mortality were identified. These results showed that C. burnetii and Leptospira spp. are probably not common abortigenic agents or causes of neonatal deaths in dogs. However, given the potential abortigentic and zoonotic role of these agents, surveillance of canine and feline abortion, stillbirth, and neonatal mortality could be advisable for a systematic investigation of these events.     

Phylogenetic analysis of feline panleukopenia virus (FPLV) strains in Korean cats

Sixteen Korean feline panleukopenia virus (FPLV) strains were compared with 48 non-Korean strains and two vaccine strains to conduct phylogenetic analysis of the FPLVs currently circulating among cats in Korea. Most of the residues that discriminate between FPLVs and canine parvoviruses (CPV-2, -2a, -2b, and -2c), including 80-Lys, 93-Lys, 103-Val, 323-Asp, 564-Asn, and 568-Ala, were conserved in the Korean FLPVs; however, exceptions were observed in two strains, namely K50/08 (80-Gln) and V142 (323-Asn). Phylogenetic analysis using the Bayesian inference and Neighbor-joining method showed that FPLVs were not segregated on a clear temporal or geographical basis. Three clusters (G1, G2, and G3) were formed by the VP2 nucleotide sequences analysed and Korean strains belonged to the G1 (n = 13) and G2 (n = 3) clusters. The ratio of non-synonymous to synonymous substitutions (dN/dS) revealed that purifying selection acts on the VP2 gene of Korean FPLVs.     

Conventional and molecular diagnostic testing for the acute neurologic patient

Objective – The aim of this review is to describe and evaluate both conventional and molecular diagnostic testing utilized in dogs and cats with acute neurologic diseases. Various types of polymerase chain reaction (PCR) are explored along with novel molecular diagnostic testing that ultimately may prove useful in the critical care setting. Data Sources – PUBMED was searched to obtain relevant references material using keywords: ‘canine OR feline meningitis AND meningoencephalitis,’‘feline infectious peritonitis,’‘canine distemper,’‘canine OR feline AND toxoplasma,’‘canine neospora,’‘canine OR feline AND rickettsia,’‘granulomatous meningoencephalitis,’‘steroid responsive meningitis arteritis,’‘necrotizing encephalitis,’‘novel neurodiagnostics,’‘canine OR feline AND CNS borrelia,’‘canine OR feline AND CNS bartonella,’‘canine OR feline AND CNS fungal,’‘nested OR multiplex OR degenerate OR consensus OR CODEHOP AND PCR.’ Research findings from the authors' laboratory and current veterinary textbooks also were utilized. Human Data Synthesis – Molecular diagnostic testing including conventional, real‐time, and consensus and degenerate PCR and microarray analysis are utilized routinely for the antemortem diagnosis of infectious meningoencephalitis (ME) in humans. Recently, PCR using consensus degenerate hybrid primers (CODEHOP) has been used to identify and characterize a number of novel human viruses. Veterinary Data Synthesis – Molecular diagnostic testing such as conventional and real‐time PCR aid in the diagnosis of several important central nervous system infectious agents including canine distemper virus, Toxoplasma gondii, Neospora caninum, rickettsial species, and others. Recently, broadly reactive consensus and degenerate PCR reactions have been applied to canine ME including assays for rickettsial organisms, Borrelia spp. and Bartonella spp., and various viral families. Conclusions – In the acute neurologic patient, there are several key infectious diseases that can be pursued by a combination of conventional and molecular diagnostic testing. It is important that the clinician understands the utility, as well as the limitations, of the various neurodiagnostic tests that are available.     

Duration of immunity for canine and feline vaccines: a review

In our studies aimed at assessing the minimum duration of vaccinal immunity (DOI), approximately 1000 dogs have been vaccinated with products from all the major US veterinary biological companies. The DOI for the various products is determined by antibody titers for all dogs and, by challenge studies in selected groups of dogs. Recently, all major companies that make canine vaccines for the U.S. market have completed their own studies; published data show a 3 years or longer minimum DOI for the canine core products, canine distemper virus (CDV), canine parvovirus type 2 (CPV-2), and canine adenovirus-2 (CAV-2). Studies with feline core vaccines - feline parvovirus (FPV), calicivirus (FCV) and herpes virus type I (FHV-1) have shown a minimum DOI of greater than 3 years. Based on these results, the current canine and feline guidelines (which recommend that the last dose of core vaccines be given to puppies and kittens > or =12 weeks of age or older, then revaccination again at 1 year, then not more often than every 3 years) should provide a level of protection equal to that achieved by annual revaccination. In contrast, the non-core canine and feline vaccines, perhaps with the exception of feline leukaemia vaccines, provide immunity for < or =1 year. In general the effectiveness of the non-core products is less than the core products. Thus, when required, non-core vaccines should be administered yearly, or even more frequently.     

Analytical performance and method comparison of a quantitative point-of-care immunoassay for measurement of bile acids in cats and dogs

Point-of-care analyzers (POCAs) for quantitative assessment of bile acids (BAs) are scarce in veterinary medicine. We evaluated the Fuji Dri-Chem Immuno AU10V analyzer and v-BA test kit (Fujifilm) for detection of feline and canine total serum BA concentration. Results were compared with a 5th-generation assay as reference method and a 3rd-generation assay, both run on a bench-top analyzer. Analytical performance was assessed at 3 different concentration ranges, and with interferences. For method comparison, samples of 60 healthy and diseased cats and 64 dogs were included. Linearity was demonstrated for a BA concentration up to 130 µmol/L in cats (r = 0.99) and 110 µmol/L in dogs (r = 0.99). The analyzer showed high precision near the lower limit of quantification of 2 µmol/L reported by the manufacturer. Intra- and inter-assay coefficients of variation were < 5% for both species and all concentrations. Interferences were observed for bilirubin (800 mg/L) and lipid (4 g/L). There was excellent correlation with the reference method for feline (r_s = 0.98) and canine samples (r_s = 0.97), with proportional biases of 6.7% and −1.3%, respectively. However, a large bias (44.1%) was noted when the POCA was compared to the 3rd-generation assay. Total observed error was less than total allowable error at the 3 concentrations. The POCA reliably detected feline and canine BA in clinically relevant concentrations.     

Good agreement of conventional and gel-based direct agglutination test in immune-mediated haemolytic anaemia

Background

The aim of this study was to compare a gel-based test with the traditional direct agglutination test (DAT) for the diagnosis of immune-mediated haemolytic anaemia (IMHA).

Methods

Canine (n = 247) and feline (n = 74) blood samples were submitted for DAT testing to two laboratories. A subset of canine samples was categorized as having idiopathic IMHA, secondary IMHA, or no IMHA.

Results

The kappa values for agreement between the tests were in one laboratory 0.86 for canine and 0.58 for feline samples, and in the other 0.48 for canine samples. The lower agreement in the second laboratory was caused by a high number of positive canine DATs for which the gel test was negative. This group included significantly more dogs with secondary IMHA.

Conclusions

The gel test might be used as a screening test for idiopathic IMHA and is less often positive in secondary IMHA than the DAT.     

Detection of tumour necrosis factor-alpha in dogs with lymphoma(*)

Tumour necrosis factor‐alpha (TNF‐α) production by malignant lymphoblasts has been identified in vitro and in vivo in mice and humans, respectively. The goals of this study were (1) to evaluate a novel single‐sample TNF‐α assay and (2) to determine whether TNF‐α is increased in dogs with lymphoma prior to and following treatment. Canine TNF‐α was analysed concurrently using the novel Siemens Immulite^® single‐sample automated ELISA and the previously validated Quantikine^® standard ELISA. Serum from dogs with lymphoma and from breed‐, age‐ and gender‐matched control dogs was evaluated at two time points. Three of 25 (12%) dogs with lymphoma had detectable TNF‐α at diagnosis, whereas none had detectable TNF‐α following complete or partial remission. TNF‐α was not detectable in control dogs. Despite 91% homology between human and canine TNF‐α, the Immulite^® automated ELISA failed to detect canine TNF‐α. Serum TNF‐α appears to have limited value as a tumour marker in dogs with lymphoma.     

Evaluation of a hematology analyzer with canine and feline blood

A semiautomatic electronic blood cell counter (Sysmex F-800:Toa Medical Electronics Europa Gmbh, Hamburg, Germany) was evaluated using canine and feline blood, following the International Committee for Standardization in Hematology protocol (ICSH, 1984). Precision and overall reproducibility were acceptable for all the parameters studied except for the feline platelet count, in which overlapping of erythrocyte and platelet populations prohibited determination of an accurate platelet count. Since carry-over from canine hematocrit values and platelet counts and from feline hematocrit values was unsatisfactory, the use of a blank diluent sample between different analyses was necessary. Linearity of the analyzer was acceptable in the studied range. Thirty canine and feline blood samples were analyzed using the Sysmex F-800 and a manual method. Correlations between both methods were acceptable for all the parameters, except for feline platelet count and erythrocyte indices for both species. In the storage study, red blood cell count and hemoglobin concentration were the parameters with the longest stability (72 hours at 4 degrees C and 25 degrees C) in both species. A statistically significant increase in MCV was obtained at 12 hours post-extraction in canine samples stored at 25 degrees C and at 24 hours in refrigerated samples. Feline leucocyte counts showed a downward trend at 12 hours post-extraction at both temperatures. Canine platelet count decreased significantly at 6 hours post-extraction in samples stored at 4 degrees C. During the evaluation period, Sysmex F-800 was user friendly and appeared well suited for routine canine and feline blood cell analysis.     

In vitro evaluation of canine and feline calcium oxalate urolith fragility via shock wave lithotripsy

OBJECTIVE: To test the hypothesis that feline calcium oxalate uroliths are intrinsically more resistant to comminution via shock wave lithotripsy (SWL) than canine calcium oxalate uroliths through comparison of the fragility of canine and feline uroliths in a quantitative in vitro test system. SAMPLE POPULATION: Calcium oxalate uroliths (previously obtained from dogs and cats) were matched by size and mineral composition to create 7 pairs of uroliths (1 canine and 1 feline urolith/pair). PROCEDURE: Uroliths were treated in vitro with 100 shock waves (20 kV; 1 Hz) by use of an electrohydraulic lithotripter. Urolith fragmentation was quantitatively assessed via determination of the percentage increase in projected area (calculated from the digital image area of each urolith before and after SWL). RESULTS: After SWL, canine uroliths (n = 7) fragmented to produce a mean +/- SD increase in image area of 238 +/- 104%, whereas feline uroliths (7) underwent significantly less fragmentation (mean image area increase of 78 +/- 97%). The post-SWL increase in fragment image area in 4 of 7 feline uroliths was < 50%, whereas it was > 150% in 6 of 7 canine uroliths. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that feline calcium oxalate uroliths are less susceptible to fragmentation via SWL than canine calcium oxalate uroliths. In some cats, SWL may not be efficacious for fragmentation of calcium oxalate nephroliths or ureteroliths because the high numbers of shock waves required to adequately fragment the uroliths may cause renal injury.     

Simultaneous detection of the feline lungworms Troglostrongylus brevior and Aelurostrongylus abstrusus by a newly developed duplex-PCR

In addition to Aelurostrongylus abstrusus (Strongylida: Angiostrongylidae), referred to as the feline lungworm, Troglostrongylus brevior (Strongylida: Crenosomatidae) has recently been identified as an agent of bronco-pulmonary infestations in cats. These two parasites have a similar biology, share ecological niches, potentially co-infesting cats, but are difficult to be differentiated due to the morphological similarities of their first-stage larvae (L1). This paper describes a molecular tool, based on single-step duplex polymerase chain reaction (duplex-PCR) on the ribosomal internal transcribed spacer 2 region (ITS-2) for the simultaneous detection and differentiation of T. brevior and A. abstrusus. L1 of both species were collected from faecal samples, morphologically identified, and single larval specimens isolated. An aliquot of faeces was used as a test sample for a case of mixed natural infestation. The duplex-PCR was performed using species-specific forward primer sets for the ITS-2 region (i.e., A. abstrusus: 220 bp; T. brevior: 370 bp). The detection limit of the molecular assay was also assessed by serial dilutions of DNA from single larvae of both species (from ～4.0 to 4.0 × 10^?5 μg/μl). The duplex-PCR carried out on individual DNA samples was able to detect as low as 5.2 × 10^?3 μg/μl of DNA for A. abstrusus, 4.9 × 10^?3 μg/μl for T. brevior, and as low as 4.0 × 10^?3 μg/μl for samples containing both species. Species-specific bands of the expected sizes and two bands were simultaneously amplified from the faecal sample containing both species. The phylogenetic analyses of the ITS-2 sequences here examined and those available for other metastrongyloids were concordant in clustering them with those of other Troglostrongylus brevior and A. abstrusus sequences available in GenBank database. This molecular approach proved to be effective and highly sensitive for the simultaneous detection of the two lungworms species and it might be used for molecular epidemiological studies and for monitoring therapeutic protocols.     

Brucella canis induces canine CD4+ T cells multi-cytokine Th1/Th17 production via dendritic cell activation

Brucella canis is a small intracellular Gram-negative bacterium that frequently leads to chronic infections highly resistant to antibiotic therapy in dogs. Also, it causes mild human brucellosis compared to other zoonotic Brucella spp. Herein we characterize the cellular immune response elicited by B. canis by analysing human and canine CD4⁺ T cells after stimulation with autologous monocyte-derived dendritic cells (MoDCs). Human and canine B. canis-primed MoDCs stimulated autologous CD4⁺ T cells; however, a Th1 response was triggered by human MoDCs, whereas canine MoDCs induced Th1/Th17 responses, with increased CD4⁺ T cells producing IFN-γ and IL-17A simultaneously. Each pattern of cellular response may contribute to host susceptibility, helping to understand the differences in B. canis virulence between these two hosts. In addition, other aspects of canine immunology are unveiled by highlighting the participation of IL-17A-producing canine MoDCs and CD4⁺ T cells producing IFN-γ and IL-17A.     

Detection of bacterial contamination and DNA quantification in stored blood units in 2 veterinary hospital blood banks

Blood transfusions in veterinary medicine have become increasingly more common and are now an integral part of lifesaving and advanced treatment in small and large animals. Important risks associated with transfusion of blood products include the transmission of various infectious diseases. Several guidelines suggest what infectious agents to screen for in canine and feline transfusion medicine. However, while the risk of bacterial contamination of blood products during storage and administration has not been documented in veterinary medicine, it has emerged as a cause of morbidity and mortality in human transfusion medicine. Clinical experience shows that the majority of blood component bacterial contaminations are caused by only a few species. Unlike other types of bacteria, psychrotolerant species like Pseudomonas spp. and Serratia spp. can proliferate during the storage of blood units at 4°C from a very low titer at the time of blood collection to a clinically significant level (> 10⁵ CFU/mL) causing clinical sepsis resulting from red blood cell concentrate transfusions in human medicine. The purpose of this report was to describe the detection and quantification procedures applied in 4 cases of bacterial contamination of canine and feline blood units, which suggest the need for further investigations to optimize patients’ safety in veterinary transfusion medicine.     

Lamellar keratoplasty for the treatment of feline corneal sequestrum

A lamellar keratoplasty was used to treat corneal sequestrum in four Persian cats (six eyes). Following a superficial keratectomy, lamellar corneal allografts (feline corneal tissue) or heterografts (canine corneal tissue) which had been preserved at –20 °C were placed in the recipient cornea. All grafts became optically transparent within 2 months following surgery and no recurrences of the sequestrum have been noted during the follow-up period (4–30 months). We conclude that feline corneal sequestrum may be successfully treated with feline or canine donor corneal tissue using this technique.     

Development and use of real-time PCR to detect and quantify Mycoplasma haemocanis and “Candidatus Mycoplasma haematoparvum” in dogs

Two canine haemoplasma species have been recognised to date; Mycoplasma haemocanis (Mhc), which has been associated with anaemia in splenectomised or immunocompromised dogs, and “Candidatus Mycoplasma haematoparvum” (CMhp), recently described in an anaemic splenectomised dog undergoing chemotherapy. The study aim was to develop quantitative real-time PCR assays (qPCRs) incorporating an endogenous internal control to detect Mhc and CMhp and to apply these assays to DNA samples extracted from canine blood collected in Northern Tanzania (n = 100) and from dogs presented to a Trinidadian veterinary hospital (n = 185).QPCRs specific for Mhc and CMhp were designed using 16S rRNA gene sequence data, and each was duplexed with an assay specific for canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The assays detected ≤10 copies of a sequence-specific haemoplasma plasmid per reaction and neither assay showed cross-reactivity with 10⁶ copies of the sequence-specific plasmid from the non-target canine haemoplasma species.Nineteen of the 100 Tanzanian samples (19%) were positive for Mhc alone and one (1%) was dually infected. One Trinidadian sample was negative for canine GAPDH DNA and was excluded from the study. Of the 184 remaining Trinidadian samples, nine (4.9%) were positive for Mhc alone, five (2.7%) for CMhp alone, and two (1.1%) dually infected.This is the first report of canine haemoplasma qPCR assays that use an internal control to confirm the presence of amplifiable sample DNA, and their application to prevalence studies. Mhc was the most commonly detected canine haemoplasma species.     

Prevalence of canine coronavirus (CCoV) in dog in Japan: detection of CCoV RNA and retrospective serological analysis

We collected rectal swabs from dogs in Japan during 2011 to 2014, and canine coronavirus (CCoV) nucleocapsid gene was detected by RT-PCR. The relationship between CCoV infection and the manifestation of diarrhea symptoms was investigated, and a correlation was noted (df=1, χ²=8.90, P<0.005). The types of CCoV detected in samples from CCoV-infected dogs were CCoV-I in 88.9% and CCoV-II in 7.4%, respectively. We retrospectively investigated the seroprevalence of CCoV-I in dogs in Japan during 1998 to 2006. The sera were tested with a neutralizing antibody test. In the absence of CCoV-I laboratory strain, we used feline coronavirus (FCoV)-I that shares high sequence homology in the S protein with CCoV-I. 77.7% of the sera were positive for neutralizing anti-FCoV-I antibodies.     

Activation of AKT in feline mammary carcinoma: a new prognostic factor for feline mammary tumours

The PI3K/AKT/PTEN pathway is involved in the pathogenesis of several human cancers. This study investigated the biological and prognostic value of PI3K/AKT/PTEN pathway dysregulation in feline mammary tumours. Expression of p-AKT, HER2, PTEN and steroid receptors was assessed by immunohistochemistry (IHC) in 27 malignant and 12 benign mammary tumours from 39 female cats followed up over a 24-month period. Feline mammary carcinoma (FMC) cell lines were analyzed by Western blot and the feline AKT gene sequence was characterized. p-AKT expression statistically correlated with tumour malignancy, histological dedifferentiation and clinical recurrence. The animals with tumours expressing p-AKT had a shorter disease-free period than those with p-AKT-negative tumours. AKT activation was associated with HER2 expression and PTEN down-regulation, as occurs in human breast cancer, and feline AKT sequencing showed high homology with the human AKT gene. No AKT activation was observed in relation to either oestrogen receptor α (ERα) or progesterone receptor expression. Taken together, these data offer an explanation for AKT signalling and its role in FMC pathogenesis and prognosis, shedding new light on similarities between feline mammary tumours and hormone-independent breast cancer.     

Prevalence and characterization of methicillin-resistant Staphylococcus pseudintermedius from symptomatic companion animals in Northern Italy: Clonal diversity and novel sequence types

The aim of this study was to assess the prevalence, the genotypic diversity, the antimicrobial resistance traits of canine and feline clinical methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolates in a diagnostic laboratory in Italy during 2015-2016.All isolates were characterized by multilocus sequence typing (MLST), staphylococcal cassette chromosome (SCC)-mec typing and staphylococcal protein A (spa)-typing. The resistance profiles were assessed by antimicrobial susceptibility testing and confirmed genotypically by the detection of mecA gene and by microarray analyses.The prevalence of MRSP isolates was high (31.6%). All the strains were multidrug resistant and the most frequent clone was ST71-SCCmec type II-III. These results confirm a high prevalence of MRSP amongst clinical samples from pets in Italy. These isolates show multidrug resistance features that are of concern both in veterinary and human medicine for clinical and epidemiological reasons.