Benzimidazole F167Y polymorphism in the canine hookworm, Ancylostoma caninum: Widespread geographic, seasonal, age, and breed distribution in United States and Canada dogs.

Surveillance data for Ancylostoma spp. and the A. caninum benzimidazole treatment resistance associated F167Y polymorphism using molecular diagnostics was obtained in a large population of dogs from the United States and Canada. Real-time PCR (qPCR) for Ancylostoma spp. and allele-specific qPCR detecting a single nucleotide polymorphism (SNP) F167Y was used in 262,872 canine stool samples collected between March and December of 2022. Ancylostoma spp. was found at an overall prevalence of 2.5% (6538/262,872), with the highest prevalence in the Southern US, 4.4% (4490/103,095), and the lowest prevalence in Canada 0.6% (101/15,829). The A. caninum F167Y polymorphism was found with the highest prevalence (13.4%, n = 46/343) in the Western US and the lowest in Canada at 4.1% (4/97). The F167Y polymorphism was detected every month over the 10-month collection period. Seasonal distribution showed a peak in June for both Ancylostoma spp. (3.08%, 547/17,775) and A. caninum F167Y (12.25%, 67/547). However, the A. caninum F167Y polymorphism prevalence was highest in September (13.9%, 119/856). Age analysis indicates a higher prevalence of both hookworm infections and occurrence of resistant isolates in puppies. The breeds with the highest F167Y polymorphism prevalence in Ancylostoma spp. detected samples were poodles (28.9%), followed by Bernese Mountain dogs (25%), Cocker spaniels (23.1%), and greyhounds (22.4%). Our data set describes widespread geographic distribution of the A. caninum benzimidazole resistance associated F167Y polymorphism in the United States and Canada, with no clear seasonality compared to the Ancylostoma spp. prevalence patterns. The F167 polymorphism was present in all geographic areas with detected hookworms, including Canada. Our study highlights that the F167Y polymorphism is represented in many dog breeds, including greyhounds.

Comparative study of a broad qPCR panel and centrifugal flotation for detection of gastrointestinal parasites in fecal samples from dogs and cats in the United States.

BACKGROUND: For decades, zinc sulfate centrifugal fecal flotation microscopy (ZCF) has been the mainstay technique for gastrointestinal (GI) parasite screening at veterinary clinics and laboratories. Elsewhere, PCR has replaced microscopy because of generally increased sensitivity and detection capabilities; however, until recently it has been unavailable commercially. Therefore, the primary aim of this study was to compare the performance of real-time PCR (qPCR) and ZCF for fecal parasite screening. Secondary aims included further characterization of markers for hookworm treatment resistance and Giardia spp. assemblages with zoonotic potential and qPCR optimization. METHODS: A convenience sampling of 931 canine/feline fecal samples submitted to a veterinary reference laboratory for routine ZCF from the Northeast US (11/2022) was subsequently evaluated by a broad qPCR panel following retention release. Detection frequency and agreement (kappa statistics) were evaluated between ZCF and qPCR for seven GI parasites [hookworm/(Ancylostoma spp.), roundworm/(Toxocara spp.), whipworm/(Trichuris spp.), Giardia duodenalis, Cystoisospora spp., Toxoplasma gondii, and Tritrichomonas blagburni] and detections per sample. Total detection frequencies were compared using a paired t-test; positive sample and co-infection frequencies were compared using Pearson's chi-squared test (p ≤ 0.05 significant) and qPCR frequency for hookworm benzimidazole (BZ) resistance (F167Y) and zoonotic Giardia spp. assemblage markers calculated. Confirmatory testing, characterization, and qPCR optimization were carried out with Sanger sequencing. RESULTS: qPCR detected a significantly higher overall parasite frequency (n = 679) compared to ZCF (n = 437) [p = < 0.0001, t = 14.38, degrees-of-freedom (df) = 930] and 2.6 × the co-infections [qPCR (n = 172) vs. ZCF (n = 66)], which was also significant (p = < 0.0001, X2 = 279.49; df = 1). While overall agreement of parasite detection was substantial [kappa = 0.74; (0.69-0.78], ZCF-undetected parasites reduced agreement for individual and co-infected samples. qPCR detected markers for Ancylostoma caninum BZ resistance (n = 5, 16.1%) and Giardia with zoonotic potential (n = 22, 9.1%) as well as two parasites undetected by ZCF (T. gondii/T. blagburni). Sanger sequencing detected novel roundworm species, and qPCR optimization provided detection beyond ZCF. CONCLUSIONS: These results demonstrate the statistically significant detection frequency advantage offered by qPCR compared to routine ZCF for both single and co-infections. While overall agreement was excellent, this rapid, commercially available qPCR panel offers benefits beyond ZCF with detection of markers for Giardia assemblages with zoonotic potential and hookworm (A. caninum) BZ resistance.

Emergence of canine hookworm treatment resistance: Novel detection of Ancylostoma caninum anthelmintic resistance markers by fecal PCR in 11 dogs from Canada.

OBJECTIVE: To describe dogs with detected Ancylostoma caninum anthelmintic treatment resistance markers in Canada. ANIMALS: 11 client-owned dogs with fecal quantitative PCR (qPCR) assay detected A caninum with benzimidazole (BZ) resistance genotypic markers. METHODS: Signalment, presenting concern, duration of clinical signs, fecal testing, treatment, and outcomes were obtained. Where available, follow-up data were collected via telephone or email with the primary veterinarian. RESULTS: Ancylostoma spp was detected from 184/32,205 dog fecal samples by reference laboratory qPCR surveillance, between May 15, 2022, and April 26, 2023. 11 of these 184 samples had A caninum with genetic BZ F167Y resistance marker detection. 4 dogs had not traveled outside Canada, 6 had been imported from the US, and the travel history was unclear in 1 dog. 7 of the dogs had gastro-intestinal signs (diarrhea or soft stool) on initial presentation. Clinical improvement was reported in 6 of these dogs (resolution of diarrhea and soft stool), with 1 dog lost to follow-up. All 11 dogs received anthelmintic treatment (varied drugs and duration). CLINICAL RELEVANCE: Identification of genetic markers of BZ resistance raises concerns about the potential animal and human impacts of resistant hookworms. 4 dogs lacked an origin from or travel history to the US, indicating true emergence and/or novel spread within Canada, not just importation from an area where resistance has been reported. Fecal surveillance was performed with a qPCR test incorporating treatment (BZ) resistance markers. There is a need to raise clinician awareness around treatment-resistant hookworm in dogs and the capability of fecal surveillance for genotypic and phenotypic resistance.

Novel molecular diagnostic (PCR) diagnosis and outcome of intestinal Echinococcus multilocularis in a dog from western Canada.

OBJECTIVE: To describe the novel PCR diagnosis and outcome of intestinal Echinococcus multilocularis in a dog. ANIMAL: A 13-month-old female intact dog with naturally occurring intestinal E multilocularis. CLINICAL PRESENTATION, PROGRESSION, AND PROCEDURES: The 13-month-old dog initially presented with a reduced appetite and weight loss and then developed hematochezia. The clinical history included a lack of endoparasite preventive care (fecal testing, deworming), exposure to coyotes, fox, sheep, and rodents and the dog had intermittently been fed a raw food diet. Physical examination revealed a thin dog, with a 2/9 body condition score, that was otherwise clinically unremarkable. A fecal sample was submitted for screening for gastrointestinal parasites as part of an infectious disease assessment. The fecal PCR test reported detection of E multilocularis. This result was sequenced as the European haplotype E3/E4. Centrifugal flotation (same sample) did not detect taeniid eggs. TREATMENT AND OUTCOME: The dog was treated with metronidazole, maropitant, and milbemycin oxime/praziquantel. Clinical improvement was noted within 48 hours. No DNA of E multilocularis was detected in a fecal sample collected approximately 10 days after treatment. The dog's owner was advised to provide monthly deworming (praziquantel) for all dogs on the property and to contact their human health-care provider due to potential zoonotic exposure risk. CLINICAL RELEVANCE: Increasing detection of E multilocularis is occurring in dogs in Canada and the US. Alveolar echinococcosis can cause severe disease in dogs and humans. Fecal PCR detection and surveillance may alert practitioners to canine intestinal cases and allow dogs to serve as sentinels for human exposure risk.

Emergence of Ancylostoma caninum parasites with the benzimidazole resistance F167Y polymorphism in the US dog population.

BACKGROUND: Anthelmintic resistance to benzimidazole has been detected in the canine hookworm, Ancylostoma caninum. Benzimidazole resistance is believed to have developed originally in greyhounds, but has also been detected in non-greyhound pet dogs. The aim of this study was to validate a probe-based allele-specific real-time PCR tests for the F167Y polymorphism on the ß-tubulin isotype-1 gene and to determine the geographic distribution. METHODS: Allele-specific real-time PCR tests were established and validated to detect the codon 167 polymorphism in the Ancylostoma caninum ß-tubulin isotype-1gene. Additionally, real-time PCR tests were validated for Ancylostoma spp. and Uncinaria stenocephala. Two nucleic acid extraction protocols were validated including mechanical disruption of parasite structures in stool. The frequency of the F167Y single nucleotide polymorphism (SNP) was determined in hookworm confirmed stool samples. Samples with the resistant 167Y genotype were confirmed by ß-tubulin gene sequencing and allele frequencies were determined. RESULTS: The Ancylostoma spp. and A. caninum F167Y allele-specific real-time PCR tests were highly sensitive and specific when tested against synthetic DNA, spiked samples, and characterized parasites. Using an optimized total nucleic acid extraction protocol, 54 of 511 (10.6%) were found to contain the benzimidazole resistance allele. All 55 samples containing hookworms with the resistance mutation were confirmed by ß-tubulin gene sequencing. The majority of resistant hookworms (44 resistant, 183 tested; 24.4%) originated from Florida, five from California (103 tested, 4.9%), three from Idaho (40 tested, 7.5%), two from Nevada (22 tested, 9.1%), and one sample from Hawaii (13 tested, 7.7%). Resistant genotypes were found in 14 different dog breeds including eight in Greyhounds. Allele-frequency determination revealed resistance allele frequencies between 1 and 100% with 58% above 50%. CONCLUSIONS: This data strongly supports recent findings of benzimidazole resistant canine hookworms present throughout the general US pet dog population.

Role of Feline Coronavirus as Contributor to Diarrhea in Cats from Breeding Catteries.

(1) Background: Feline coronavirus infection (FCoV) is common in multi-cat environments. A role of FCoV in causing diarrhea is often assumed, but has not been proven. The aim of this study was to evaluate an association of FCoV infection with diarrhea in multi-cat environments. (2) Methods: The study included 234 cats from 37 catteries. Fecal samples were analyzed for FCoV RNA by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Potential co-infections were determined by applying a qPCR panel on different potential enteropathogens and fecal flotation. A fecal scoring system was used to categorize feces as diarrheic or non-diarrheic. (3) Results: Of the 234 cats included, 23 had diarrhea. The prevalence of FCoV infection was 87.0% in cats with and 58.8% in cats without diarrhea. FCoV infection was significantly associated with diarrhea (Odds Ratio (OR) 5.01; p = 0.008). In addition, presence of Clostridium perfringens α toxin (OR 6.93; p = 0.032) and feline panleukopenia virus (OR 13.74; p = 0.004) were associated with an increased risk of diarrhea. There was no correlation between FCoV load and fecal score. FCoV-positive cats with co-infections were not more likely to have diarrhea than FCoV-positive cats without co-infections (p = 0.455). (4) Conclusions: FCoV infection is common in cats from catteries and can be associated with diarrhea.

Challenges in navigating molecular diagnostics for common equine respiratory viruses.

Equine respiratory viruses remain a leading cause of equine morbidity and mortality, with the resurgence of certain infections, an increasing population of elderly, more susceptible horses, the growth of international equine commerce, and an expansion in geographic distribution of pathogens. The focus of rapid diagnosis of infectious diseases has also shifted recently, with the appearance and increasing importance of nucleic acid amplification-based techniques, primarily polymerase chain reaction (PCR), at the expense of traditional methods such as clinical microbiology. While PCR is fast, reliable, cost-effective, and more sensitive than conventional detection methods, careful interpretation of diagnostic test results is required, taking into account the clinical status of the patient, sample type, assay used and biological relevance of the detected viruses. The interpretation of common equine respiratory viruses such as influenza virus (EIV), alpha herpesviruses (EHV-1, EHV-4), arteritis virus (EAV) and rhinoviruses (ERAV, ERBV) is straight forward as causality can generally be established. However, the testing of less-characterized viruses, such as the gamma herpesviruses (EHV-2, EHV-5), may be confusing, considering their well-established host relationship and frequent detection in both diseased and healthy horses. For selected viruses, absolute quantitation (EHV-1 and EHV-4) and genotyping (EIV and EHV-1) has allowed additional information to be gained regarding viral state and virulence, respectively. This information is relevant when managing outbreaks so that adequate biosecurity measures can be instituted and medical interventions can be considered. The goal of this review is to help the equine practitioner navigate through the rapidly expanding field of molecular diagnostics for respiratory viruses and facilitate the interpretation of results.

Diagnostic Value of Detecting Feline Coronavirus RNA and Spike Gene Mutations in Cerebrospinal Fluid to Confirm Feline Infectious Peritonitis.

BACKGROUND: Cats with neurologic feline infectious peritonitis (FIP) are difficult to diagnose. Aim of this study was to evaluate the diagnostic value of detecting feline coronavirus (FCoV) RNA and spike (S) gene mutations in cerebrospinal fluid (CSF). METHODS: The study included 30 cats with confirmed FIP (six with neurological signs) and 29 control cats (eleven with neurological signs) with other diseases resulting in similar clinical signs. CSF was tested for FCoV RNA by 7b-RT-qPCR in all cats. In RT-qPCR-positive cases, S-RT-qPCR was additionally performed to identify spike gene mutations. RESULTS: Nine cats with FIP (9/30, 30%), but none of the control cats were positive for FCoV RNA in CSF. Sensitivity of 7b-RT-qPCR in CSF was higher for cats with neurological FIP (83.3%; 95% confidence interval (95% CI) 41.8-98.9) than for cats with non-neurological FIP (16.7%; 95% CI 6.1-36.5). Spike gene mutations were rarely detected. CONCLUSIONS: FCoV RNA was frequently present in CSF of cats with neurological FIP, but only rarely in cats with non-neurological FIP. Screening for spike gene mutations did not enhance specificity in this patient group. Larger populations of cats with neurological FIP should be explored in future studies.

Correlation of Feline Coronavirus Shedding in Feces with Coronavirus Antibody Titer.

BACKGROUND: Feline coronavirus (FCoV) infection is ubiquitous in multi-cat households. Responsible for the continuous presence are cats that are chronically shedding a high load of FCoV. The aim of the study was to determine a possible correlation between FCoV antibody titer and frequency and load of fecal FCoV shedding in cats from catteries. METHODS: Four fecal samples from each of 82 cats originating from 19 German catteries were examined for FCoV viral loads by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). Additionally, antibody titers were determined by an immunofluorescence assay. RESULTS: Cats with antibodies were more likely to be FCoV shedders than non-shedders, and there was a weak positive correlation between antibody titer and mean fecal virus load (Spearman r = 0.2984; p = 0.0072). Antibody titers were significantly higher if cats shed FCoV more frequently throughout the study period (p = 0.0063). When analyzing only FCoV shedders, cats that were RT-qPCR-positive in all four samples had significantly higher antibody titers (p = 0.0014) and significantly higher mean fecal virus loads (p = 0.0475) than cats that were RT-qPCR-positive in only one, two, or three samples. CONCLUSIONS: The cats' antibody titers correlate with the likelihood and frequency of FCoV shedding and fecal virus load. Chronic shedders have higher antibody titers and shed more virus. This knowledge is important for the management of FCoV infections in multi-cat environments, but the results indicate that antibody measurement cannot replace fecal RT-qPCR.

Effects of famciclovir in cats with spontaneous acute upper respiratory tract disease.

OBJECTIVES: The aim of this study was to assess the effects of famciclovir administration in cats with spontaneously acquired acute upper respiratory tract disease. METHODS: Twenty-four kittens with clinical signs of acute upper respiratory tract disease were randomly allocated to receive doxycycline (5 mg/kg PO q12h) alone (group D; n = 12) or with famciclovir (90 mg/kg PO q12h; group DF; n = 12) for up to 3 weeks. Clinical disease severity was scored at study entry and daily thereafter. Oculo-oropharyngeal swabs collected at study entry and exit were assessed using quantitative PCR for nucleic acids of feline herpesvirus type 1 (FHV-1), feline calicivirus (FCV), Chlamydia felis, Bordetella bronchiseptica and Mycoplasma felis. RESULTS: The median (range) age of cats was 1.5 (1-6) months in group D vs 1.6 (1-5) months in group DF (P = 0.54). Pathogens detected in oculo-oropharyngeal swabs at study entry included FCV (n = 13/24; 54%), M felis (n = 8/24; 33%), FHV-1 (n = 7/24; 29%), C felis (n = 7/24; 29%) and B bronchiseptica (n = 3/24; 12%). Median (range) duration of clinical signs was 11.5 (3-21) days in group DF and 11 (3-21) days in group D (P = 0.75). Median (range) total disease score at the end of the study did not differ between groups (group D 1 [1-1] vs group DF 1 [1-3]; P = 0.08). CONCLUSIONS AND RELEVANCE: This study revealed no significant difference in response to therapy between cats treated with doxycycline alone or with famciclovir; cats improved rapidly in both groups. However, identification of FHV-1 DNA was relatively uncommon in this study and clinical trials focused on FHV-1-infected cats are warranted to better evaluate famciclovir efficacy.

Evaluation of a quantitative enzyme-linked immunosorbent assay for feline leukemia virus p27 antigen and comparison to proviral DNA loads by real-time polymerase chain reaction.

Feline leukemia virus (FeLV) is an oncogenic retrovirus of cats. While higher viral RNA and proviral DNA loads have been correlated with progressive infections and disease, a similar correlation has been suggested for p27 antigen concentrations. This analytical study compared the results of a quantitative ELISA for p27 antigen with quantitative real-time PCR results for FeLV proviral DNA in patient samples. A significant positive correlation between copies of proviral DNA and the concentration of p27 antigen was identified (r = 0.761, P < 0.0001). Samples with high proviral DNA loads, at least 1 × 106 copies/mL of whole blood, typically had p27 antigen concentrations greater than 30 ng/mL in plasma. Samples with proviral DNA loads below this level all had concentrations of p27 antigen in plasma that were less than 10 ng/mL. Given this correlation, it is hypothesized that the concentration of p27 antigen at a given point in time may help to indicate the likelihood of a progressive or regressive infection similar to what has been demonstrated for proviral DNA loads.

Associations between clinical canine leishmaniosis and multiple vector-borne co-infections: a case-control serological study.

BACKGROUND: Dogs that have clinical leishmaniosis (ClinL), caused by the parasite Leishmania infantum, are commonly co-infected with other pathogens, especially vector-borne pathogens (VBP). A recent PCR-based study found that ClinL dogs are more likely to be additionally infected with the rickettsial bacteria Ehrlichia canis. Further information on co-infections in ClinL cases with VBP, as assessed by serology, is required. The research described in this report determined if dogs with ClinL are at higher risk of exposure to VBP than healthy control dogs using a case-control serology study. RESULTS: Of the 47 dogs with ClinL, anti-E. canis/ Ehrlichia ewingii antibodies were detected in 17 (36.2%), anti-Anaplasma phagocytophilum/Anaplasma platys antibodies in 5 (10.6%) and antigen for Dirofilaria immitis in 2 (4.3%). Of the 87 control dogs, anti-E. canis/E. ewingii antibodies were detected in 14 (16.1%) and anti-A. phagocytophilum/A. platys antibodies in 2 (2.3%). No anti-Borrelia burgdorferi antibody tests were positive. No statistical differences between the ClinL dogs and control dogs regarding lifestyle or use of ectoparasitic prevention, were identified. The ClinL was significantly associated with anti-E. canis/E. ewingii antibodies (odds ratio = 2.9, 95% confidence interval: 1.3-6.7, P = 0.010) compared to controls by both multivariable logistic regression and structural equation modelling. CONCLUSIONS: It was demonstrated that an increased risk for E. canis/E. ewingii seropositivity is present in dogs with ClinL compared to clinically healthy control dogs, despite similar ectoparasitic prevention use and lifestyle. Based on these findings it is suggested that dogs with ClinL should not only be tested for E. canis co-infection using PCR but also serologically for E. canis/E. ewingii.

Lyon-IARC Polyomavirus DNA in Feces of Diarrheic Cats.

A viral metagenomic analysis of feces from an unexplained outbreak of feline diarrhea revealed the presence of Lyon-IARC polyomavirus (LIPyV) DNA. LIPyV, whose genome was originally sequenced from swabs of human skin, was fecally shed by three out of five diarrheic cats.

Nasal virome of dogs with respiratory infection signs include novel taupapillomaviruses.

Using viral metagenomics, we characterized the mammalian virome of nasal swabs from 57 dogs with unexplained signs of respiratory infection showing mostly negative results using the IDEXX Canine Respiratory Disease RealPCR™ Panel. We identified canine parainfluenza virus 5, canine respiratory coronavirus, carnivore bocaparvovirus 3, canine circovirus and canine papillomavirus 9. Novel canine taupapillomaviruses (CPV21-23) were also identified in 3 dogs and their complete genome sequenced showing L1 nucleotide identity ranging from 68.4 to 70.3% to their closest taupapillomavirus relative. Taupapillomavirus were the only mammalian viral nucleic acids detected in two affected dogs, while a third dog was coinfected with low levels of canine parainfluenza 5. A role for these taupapillomavirues in canine respiratory disease remains to be determined.

Detection of feline coronavirus mutations in paraffin-embedded tissues in cats with feline infectious peritonitis and controls.

OBJECTIVES: The amino acid substitutions M1058L and S1060A in the spike protein of feline coronavirus (FCoV) have been postulated to be responsible for the development of the pathogenic feline infectious peritonitis virus (FIPV), which causes feline infectious peritonitis (FIP). The aim of the following study was to investigate the presence of mutated virus in tissue samples of cats with and without FIP. METHODS: The study population consisted of 64 cats, 34 of which were diagnosed with FIP and 30 control cats. All cases underwent autopsy, histopathology and immunohistochemistry (IHC) for FCoV. Furthermore, a genotype-discriminating quantitative reverse transcriptase PCR (RT-qPCR) was performed on shavings of paraffin-embedded tissues to discriminate between cats with FIP and controls, and the sensitivity and specificity of this discriminating RT-qPCR were calculated using 95% confidence intervals (CIs). RESULTS: Specificity of genotype-discriminating RT-qPCR was 100.0% (95% CI 88.4-100.0), and sensitivity was 70.6% (95% CI 52.5-84.9). In cats with FIP, 24/34 tested positive for FIPV. In samples of three control cats and in seven cats with FIP, FCoV was found, but genotyping was not possible owing to low FCoV RNA concentrations. Out of the positive samples, 23 showed the amino acid substitution M1058L in the spike protein and none the substitution S1060A. One sample in a cat with FIP revealed a mixed population of non-mutated FCoV and FIPV (mixed genotype). For one sample genotyping was not possible despite high viral load, and two samples were negative for FCoV. CONCLUSIONS AND RELEVANCE: As none of the control animals showed FCoV amino acid substitutions previously demonstrated in cats with FIP, it can be presumed that the substitution M1058L correlates with the presence of FIP. FCoV was detected in low concentration in tissues of control animals, confirming the ability of FCoV to spread systemically. The fact that no negative controls were included in the IHC protocol could potentially lead to an underestimation of the sensitivity of the RT-qPCR.

Prophylactic and therapeutic effects of twice-daily famciclovir administration on infectious upper respiratory disease in shelter-housed cats.

OBJECTIVES: In humans with herpetic disease, early or pre-emptive famciclovir therapy reduces disease duration and severity. This prospective, masked, placebo-controlled study tested therapeutic and prophylactic effects of two famciclovir doses given to cats for 7 days following shelter entry. METHODS: Cats were assigned to prophylactic or therapeutic study arms based on clinical evidence of herpetic disease at study entry. Cats in the therapeutic arm received no treatment (n = 19), placebo (lactose; n = 18) or famciclovir at ~30 (n = 21) or ~90 mg/kg (n = 20) PO q12h for 7 days. Cats in the prophylactic arm received no treatment (n = 25) or famciclovir at ~30 (n = 28) or ~90 mg/kg (n = 27) PO q12h for 7 days. Disease scores, body weight, conjunctival feline herpesvirus 1 (FHV-1) shedding, and adoption rates were recorded on days 1 (admission), 8 (end of therapy) and 15 (1 week after cessation of therapy). RESULTS: No significant differences in clinical scores were observed among groups in the prophylactic or therapeutic arms at any of the three time points. However, within the therapeutic arm, viral shedding on day 8 was significantly higher in cats receiving no treatment than in those receiving ~30 or ~90 mg/kg famciclovir, and this effect persisted 1 week after famciclovir was stopped (day 15) only in cats receiving ~30 mg/kg, although this approached significance in cats receiving ~90 mg/kg. No significant differences in adoption rates were detected among groups in either arm throughout the study. CONCLUSIONS AND RELEVANCE: Although we did not demonstrate a statistically or clinically significant effect of famciclovir administration upon clinical signs of infectious upper respiratory disease or adoption, when it was administered at ~30 or ~90 mg/kg q12h for 1 week famciclovir reduced conjunctival FHV-1 shedding. This suggests a potential role in interrupting the infectious cycle within a shelter population; however, cost in time and resources, and stress and pathogen transmission induced by oral administration should be considered.

Prevalence of Clostridium perfringens netE and netF toxin genes in the feces of dogs with acute hemorrhagic diarrhea syndrome.

BACKGROUND: Recently, novel pore-forming toxin genes designated netE and netF were identified in a Clostridium perfringens type A strain isolated from a dog with acute hemorrhagic diarrhea. OBJECTIVES: Pore-forming toxins could play an important role in the disease pattern of acute hemorrhagic diarrhea syndrome (AHDS) in dogs. Thus, we aimed to determine the prevalence of C. perfringens genes encoding for netE and netF in the feces of dogs with AHDS and to evaluate any association between selected clinical variables and the presence of these toxin genes. ANIMALS: In total, 174 dogs were included in the study. METHODS: Fecal samples of all dogs were tested by real-time polymerase chain reaction for netE and netF genes. Time to recovery, hospitalization time, and selected laboratory variables were compared between dogs with AHDS that were positive or negative for the toxin genes. RESULTS: A significant difference was found among the 3 groups in the prevalence of the pore-forming toxin genes netE and netF: dogs with AHDS: 26 of 54 (48.1%); dogs with canine parvovirus (CPV) infection: 0 of 54 (0%); and healthy dogs: 8 of 66 (12.1%; P < .001). In dogs with AHDS, no significant difference was detected in any variables evaluated between netE-positive and netF-positive and netE-negative and netF-negative dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: The prevalence of C. perfringens encoding for netE and netF is significantly higher in dogs with AHDS compared to control dogs. Further studies are warranted to evaluate whether these toxins are an inciting cause for AHDS in dogs.

First identification of a single amino acid change in the spike protein region of feline coronavirus detected from a coronavirus-associated cutaneous nodule in a cat.

CASE SUMMARY: A 32-month-old spayed female Singapura cat presented with a non-pruritic erythematous nodule on the upper lip. The cat also had multiple nodules in the liver but exhibited no other clinical signs consistent with classical feline infectious peritonitis (FIP), such as pleural effusion or ascites, uveitis or neurological symptoms. Histopathological and immunohistochemical analyses of the cutaneous nodule revealed pyogranulomatous dermatitis with intralesional macrophages laden with feline coronavirus (FCoV) antigen. Real-time reverse transcription (RT)-PCR of a cutaneous sample revealed a single nucleotide substitution in the spike protein gene of FCoV (mutation M1058L), which is consistent with an FCoV genotype commonly associated with FIP. The cat received a blood transfusion and supportive therapy, but the owner declined to continue the treatments owing to poor response. The cat was lost to follow-up 5 months after discharge. RELEVANCE AND NOVEL INFORMATION: This report describes a case of a coronavirus-associated cutaneous nodule in which the evidence of amino acid changes in the spike protein gene identified by RT-PCR were consistent with an FCoV genotype commonly seen in cases of FIP. To the best of our knowledge, this is the first report of a case of cutaneous disease associated with the mutated FCoV that was confirmed by molecular diagnostic testing.

Distribution and risk factors associated with Babesia spp. infection in hunting dogs from Southern Italy.

Canine babesiosis is caused by haemoprotozoan organisms of the genus Babesia which are transmitted by the bite of a hard tick. The aim of this survey was to determine the prevalence and risk factors associated with Babesia species infections in hunting dogs from Southern Italy. Blood samples were collected from 1311 healthy dogs in the Napoli, Avellino and Salerno provinces of the Campania region of Southern Italy. Serological testing was performed using two enzyme-linked immunosorbent assays (ELISA), with one designed to detect B. canis and B. vogeli antibodies, and the other designed to detect B. gibsoni antibodies. Blood samples were also tested by quantitative real-time polymerase chain reaction (qPCR) assays for amplification of B. canis, B. vogeli and B. gibsoni DNA. The overall seroprevalence for B. canis/B. vogeli was 14.0%, compared to 0.2% for B. gibsoni. B. canis and B. vogeli PCR positive rates were 0.15% and 1.1%, respectively. B. gibsoni DNA was not amplified by qPCR. Male gender (OR 1.85), increased age (OR 1.01), long hair coat (OR 1.61) and living in Salerno province (OR 1.71) represented risk factors for B. canis/B. vogeli seroreactivity. Hunting dogs in Southern Italy are often exposed to B. canis/B. vogeli, however Babesia spp. infection was infrequently detected using qPCR. Further studies are needed to determine the extent to which Babesia spp. cause clinical disease in hunting dogs, and to evaluate the potential epidemiological relationships between hunting dogs and wild animal populations sharing the same area.