Data analysis workflow for the detection of canine vector-borne pathogens using 16 S rRNA Next-Generation Sequencing.

BACKGROUND: Vector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP . RESULTS: We tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample. CONCLUSIONS: Bloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine.

Ehrlichiosis and Anaplasmosis: An Update.

Canine ehrlichiosis and anaplasmosis are zoonotic tick-borne diseases with broad distribution. Advances in diagnostics have enhanced our understanding of the species of rickettsial organisms involved, their expanding geographic distribution, and their impact on the health of dogs, cats, and people. While clinical remission can be achieved with appropriate antimicrobial therapy, optimal treatment modalities for the elimination of infection remain somewhat uncertain. Protection through vaccines for ehrlichiosis or anaplasmosis remains elusive. This review provides practicing veterinarians with the most current information about the transmission, diagnosis, and management of ehrlichiosis and anaplasmosis in dogs and cats.

"Candidatus Neoehrlichia mikurensis" infection in a dog from Germany.

"Candidatus Neoehrlichia mikurensis" is a new intracellular pathogen associated with human infection and death. "Candidatus Neoehrlichia mikurensis" infection in a chronically neutropenic dog from Germany was confirmed by DNA sequencing. The same organism was previously described from ticks and two sick human beings from Germany.

Evaluation of peptide- and recombinant protein-based assays for detection of anti-Ehrlichia ewingii antibodies in experimentally and naturally infected dogs. [corrected]

OBJECTIVE: To evaluate microtiter-plate format ELISAs constructed by use of different diagnostic targets derived from the Ehrlichia ewingii p28 outer membrane protein for detection of E ewingii antibodies in experimentally and naturally infected dogs. SAMPLE POPULATION: Serum samples from 87 kenneled dogs, 9 dogs experimentally infected with anti-E ewingii, and 180 potentially naturally exposed dogs from Missouri. PROCEDURES: The capacities of the synthetic peptide and truncated recombinant protein to function as detection reagents in ELISAs were compared by use of PCR assay, western blot analysis, and a full-length recombinant protein ELISA. Diagnostic targets included an E ewingii synthetic peptide (EESP) and 2 recombinant proteins: a full-length E ewingii outer membrane protein (EEp28) and a truncated E ewingii outer membrane protein (EETp28) RESULTS: A subset of Ehrlichia canis-positive samples cross-reacted in the EEp28 ELISA; none were reactive in the EESP and EETp28 ELISAs. The EESP- and EETp28-based ELISAs detected E ewingii seroconversion at approximately the same time after infection as the EEp28 ELISAs. In afield population, each of the ELISAs identified the same 35 samples as reactive and 27 samples as nonreactive. Anaplasma and E can is peptides used in a commercially available ELISA platform did not detect anti-E ewingii antibodies in experimentally infected dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The EESP and EETp28 ELISAs were suitable for specifically detecting anti-E ewingii antibodies in experimentally and naturally infected dogs.

Use of broad range16S rDNA PCR in clinical microbiology.

Broad range 16S rDNA PCR can be used to facilitate the diagnosis of infectious diseases of bacterial origin by detecting 16S rDNA sequences in patient samples. Post amplification sequencing facilitates identification of the infecting organism, but may not allow for differentiation at the species or strain level. This review focuses on the historical use and current applications of broad range 16S rDNA PCR in the diagnosis of bacterial infection. Use of an enrichment liquid culture prior to PCR and the use of real time PCR are also considered. A review of the literature indicates that the diagnostic utility of broad range 16S rDNA PCR is enhanced substantially, if the detected organism is a well-documented pathogen. Frequent detection of environmental organisms of undetermined pathogenicity is currently a limitation. This review also examines weighted criteria developed by different researchers and proposes a decision making tree that establishes the relative importance of various criteria for attributing diagnostic relevance when evaluating individual patient samples. Based upon our review of the literature, a more uniform consensus on the accurate interpretation of broad range 16S rDNA PCR results are needed to improve the microbiological utility of this modality for the diagnosis of bacterial infections in animals and in human patients.

Infection and replication of Bartonella species within a tick cell line.

Bartonella species are fastidious, gram negative bacteria, some of which are transmitted by arthropod vectors, including fleas, sandflies, and lice. There is very little information regarding the interaction and/or transmission capabilities of Bartonella species by ticks. In the present study, we demonstrate successful infection of the Amblyomma americanum cell line, AAE12, by seven Bartonella isolates and three Candidatus Bartonella species by electron or light microscopy. With the exception of Bartonella bovis, infection with all other examined Bartonella species induced cytopathic effects characterized by heavy cellular vacuolization and eventually cell lysis. Furthermore, using quantitative real time PCR (qPCR), we demonstrated significant amplification of two B. henselae genotype I isolates in the A. americanum cell line over a 5 days period. Ultimately, tick-cell derived Bartonella antigens may prove useful for the development of more sensitive diagnostic reagents and may assist in the development of an effective vaccine to prevent the further spread of disease caused by these organisms.

Duloxetine ingestion in 364 dogs.

OBJECTIVE: To describe abnormal clinical signs following duloxetine ingestion in dogs. ANIMALS: 364 client-owned dogs that ingested duloxetine. PROCEDURES: The American Society for the Prevention of Cruelty to Animals, Animal Poison Control Center electronic database was searched for records of dogs with duloxetine ingestion between January 2012 and December 2016. Data collected included age, body weight, breed, duloxetine exposure and dose, clinical signs, and overall outcome. Clinical signs were categorized as either neurologic, digestive, cardiovascular, respiratory, or metabolic and endocrine. Outcomes were categorized as no clinical signs, fully recovered, died, or unknown. RESULTS: Clinical signs developed in 55 of the 364 (15.1%) dogs with known ingestion of duloxetine. The most common clinical signs were lethargy (22/55 [40%]), mydriasis (18/55 [33%]), vomiting (11/55 [20%]), and trembling (6/55 [11%]). Dogs that ingested an estimated dose of duloxetine ≥ 20 mg/kg (9.1 mg/lb) were more likely to have had abnormal clinical signs than were dogs that ingested < 20 mg/kg. CONCLUSIONS AND CLINICAL RELEVANCE: Findings indicated that most dogs in the present study did not have clinical signs associated with ingestion of duloxetine and that development of clinical signs varied by individual dog. Further information is needed to determine toxic dose ranges for duloxetine ingestion in dogs. (J Am Vet Med Assoc 2019;255:1161-1166).

Molecular Characterization of Tandem Repeat Protein 36 Gene of Ehrlichia canis Detected in Naturally Infected Dogs from Peru.

Ehrlichia spp. are emerging infectious pathogens, especially in the Americas. Although Ehrlichia canis is primarily a parasite of dogs, polymerase chain reaction-confirmed human infections have been reported from Mexico, Venezuela, and Costa Rica. This study reports the presence of E. canis DNA in 13.7% of 205 dogs from urban areas in Peru and of those, five were analyzed for phylogenetic variation using the Tandem Repeat Protein 36 (TRP36) gene. The use of the TRP36 gene for such analysis was validated against 16S rRNA and heat shock protein genes using Shannon's entropy bioinformatic approach. When compared with other E. canis strains previously reported, three unique and novel E. canis strains were detected. In addition, the TRP36 amino acid tandem repeat sequences of the Peruvian strains share close similarity to an E. canis strain detected from four human blood bank samples in Costa Rica. This study reports for the first time domestic dogs infected with E. canis strains closely related to a zoonotic strain, which may be of public health concern as dogs can be chronically infected with this pathogen.

Isoniazid toxicosis in dogs: 137 cases (2004-2014).

OBJECTIVE To establish the minimum toxic dose of isoniazid in dogs, characterize the clinical signs and outcomes for dogs following isoniazid ingestion, and determine whether IV administration of pyridoxine to dogs with isoniazid toxicosis is protective against death. DESIGN Retrospective case series. ANIMALS 137 dogs with isoniazid toxicosis. PROCEDURES The electronic database of the American Society for the Prevention of Cruelty to Animals Animal Poison Control Center was reviewed from January 2004 through December 2014 to identify dogs with isoniazid toxicosis. For each dog identified, information extracted from the medical record included signalment, estimated dose of isoniazid ingested, clinical signs, treatment, and outcome. Follow-up communication with pet owners or primary care veterinarians was performed when necessary to obtain missing information. RESULTS Clinical signs of isoniazid toxicosis were observed in 134 of 137 (98%) dogs and included seizures (n = 104), CNS signs without seizures (94), and gastrointestinal (41), cardiovascular (19), urogenital (4), and respiratory (1) abnormalities. Of the 87 dogs for which the outcome was available, 61 survived, 18 died, and 8 were euthanized. Probability of survival was positively associated with body weight and IV administration of pyridoxine and negatively associated with dose of isoniazid ingested and presence of seizures. Dogs that received pyridoxine IV were 29 times as likely to survive as dogs that did not receive pyridoxine IV. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated rapid diagnosis of isoniazid toxicosis and prompt treatment of affected dogs with pyridoxine and other supportive care were imperative for achieving a successful outcome.

Impact of queen infection on kitten susceptibility to different strains of Bartonella henselae.

Domestic cats are the natural reservoir of Bartonella henselae, the agent of cat scratch disease in humans. In kittens, maternal IgG antibodies are detectable within two weeks postpartum, weaning in six to ten weeks postpartum and kittens as young as six to eight weeks old can become bacteremic in a natural environment. The study's objective was to evaluate if maternal antibodies against a specific B. henselae strain protect kittens from infection with the same strain or a different strain from the same genotype. Three seronegative and Bartonella-free pregnant queens were infected with the same strain of B. henselae genotype II during pregnancy. Kittens from queens #1 and #2 were challenged with the same strain used to infect the queens while kittens from queen #3 were challenged with a different genotype II strain. All queens gave birth to non-bacteremic kittens. After challenge, all kittens from queens infected with the same strain seroconverted, with six out of the seven kittens presenting no to very low levels of transitory bacteremia. Conversely, all four kittens challenged with a different strain developed high bacteremia (average 47,900 CFU/mL by blood culture and 146,893 bacteria/mL by quantitative PCR). Overall, qPCR and bacterial culture were in good agreement for all kittens (Kappa Cohen's agreement of 0.78). This study demonstrated that young kittens can easily be infected with a different strain of B. henselae at a very young age, even in the presence of maternal antibodies, underlining the importance of flea control in pregnant queens and young kittens.

An unmatched case controlled study of clinicopathologic abnormalities in dogs with Bartonella infection.

We compared clinicopathologic findings in dogs with Bartonella infection to Bartonella spp. negative dogs suspected of a vector-borne disease. Cases (n=47) and controls (n=93) were selected on the basis of positive or negative enrichment culture PCR results, respectively. Signalment, clinicopathologic findings and treatments were extracted from medical records. DNA sequencing identified Bartonella henselae (n=28, 59.6%), Bartonella vinsonii subsp. berkhoffii (n=20, 42.6%), Bartonella koehlerae (n=3, 6.4%), Bartonella volans-like (n=3, 6.4%) and Bartonella bovis (n=1, 2.1%). There were no significant differences in age, breed, size, sex or neuter status between cases and controls. Dogs infected with Bartonella sp. often had a history of weight loss [OR=2.82; 95% CI: 1.08-7.56] and were hypoglobulinemic [OR=4.26; 95% CI: 1.31-14.41]. With the exception of weight loss and hypoglobulinemia, clinicopathologic abnormalities in Bartonella-infected dogs in this study were similar to dogs suspected of other vector-borne infections.

Infection of domestic dogs in peru by zoonotic bartonella species: a cross-sectional prevalence study of 219 asymptomatic dogs.

Bartonella species are emerging infectious organisms transmitted by arthropods capable of causing long-lasting infection in mammalian hosts. Among over 30 species described from four continents to date, 15 are known to infect humans, with eight of these capable of infecting dogs as well. B. bacilliformis is the only species described infecting humans in Peru; however, several other Bartonella species were detected in small mammals, bats, ticks, and fleas in that country. The objective of this study was to determine the serological and/or molecular prevalence of Bartonella species in asymptomatic dogs in Peru in order to indirectly evaluate the potential for human exposure to zoonotic Bartonella species. A convenient sample of 219 healthy dogs was obtained from five cities and three villages in Peru. EDTA-blood samples were collected from 205 dogs, whereas serum samples were available from 108 dogs. The EDTA-blood samples were screened by PCR followed by nucleotide sequencing for species identification. Antibodies against B. vinsonii berkhoffii and B. rochalimae were detected by IFA (cut-off of 1∶64). Bartonella DNA was detected in 21 of the 205 dogs (10%). Fifteen dogs were infected with B. rochalimae, while six dogs were infected with B. v. berkhoffii genotype III. Seropositivity for B. rochalimae was detected in 67 dogs (62%), and for B. v. berkhoffii in 43 (40%) of the 108 dogs. Reciprocal titers ≥1∶256 for B. rochalimae were detected in 19% of dogs, and for B. v. berkhoffii in 6.5% of dogs. This study identifies for the first time a population of dogs exposed to or infected with zoonotic Bartonella species, suggesting that domestic dogs may be the natural reservoir of these zoonotic organisms. Since dogs are epidemiological sentinels, Peruvian humans may be exposed to infections with B. rochalimae or B. v. berkhoffii.

Vector-borne diseases in client-owned and stray cats from Madrid, Spain.

The role of various vector-borne pathogens as a cause of disease in cats has not been clearly determined. The current study evaluated risk factors, clinical and laboratory abnormalities associated with Ehrlichia spp., Anaplasma spp., Neorickettsia spp., Leishmania spp., and Bartonella spp. infection or exposure in 680 client-owned and stray cats from Madrid, Spain. Our results indicate that a large portion (35.1%) of the cat population of Madrid, Spain, is exposed to at least one of the five vector-borne pathogens tested. We found seroreactivity to Bartonella henselae in 23.8%, to Ehrlichia canis in 9.9%, to Anaplasma phagocytophilum in 8.4%, to Leishmania infantum in 3.7%, and to Neorickettsia risticii in 1% of the feline study population. About 9.9% of cats had antibody reactivity to more than one agent. L. infantum DNA was amplified from four cats (0.6%), B. henselae DNA from one cat (0.15%), and B. clarridgeiae DNA from another cat (0.15%).

Re-emergence of Babesia conradae and effective treatment of infected dogs with atovaquone and azithromycin.

Babesia conradae (B. conradae) causes hemolytic anemia in dogs. This organism has not been reported clinically since it was originally described in southern California in 1991. To date, no anti-protozoal therapies have been associated with clearance of B. conradae. This report describes the use of atovaquone and azithromycin for the treatment of dogs naturally infected with B. conradae and report the re-emergence of B. conradae in southern California. Twelve dogs naturally infected with B. conradae were identified by practicing veterinarians and public health officials in southern California. Treatments consisted of a 10 day course of atovaquone (13.3mg/kg PO q 8h) and azithromycin (10-12.5mg/kg PO q 24h). Four dogs were treated in a randomized blinded placebo-controlled fashion, four additional cases were treated in a non-random, non-blinded fashion and one dog received no treatment. All dogs were tested for B. conradae DNA by polymerase chain reaction (PCR) initially and then once or 3 times post treatment (60-210 days). B. conradae infected dogs that received treatment did not have any detectable Babesia DNA by PCR after treatment. In contrast, dogs receiving placebo had detectable Babesia DNA by PCR throughout the study period. Combination therapy with atovaquone and azithromycin appears to be effective for acute and chronic babesiosis caused by B. conradae.

Feline coronavirus in multicat environments.

Feline infectious peritonitis (FIP), a fatal disease in cats worldwide, is caused by FCoV infection, which commonly occurs in multicat environments. The enteric FCoV, referred to as feline enteric virus (FECV), is considered a mostly benign biotype infecting the gut, whereas the FIP virus biotype is considered the highly pathogenic etiologic agent for FIP. Current laboratory tests are unable to distinguish between virus biotypes of FCoV. FECV is highly contagious and easily spreads in multicat environments; therefore, the challenges to animal shelters are tremendous. This review summarizes interdisciplinary current knowledge in regard to virology, immunology, pathology, diagnostics, and treatment options in the context of multicat environments.

High prevalence of tick-borne pathogens in dogs from an Indian reservation in northeastern Arizona.

We evaluated the serological and molecular prevalence of selected organisms in 145 dogs during late spring (May/June) of 2005 and in 88 dogs during winter (February) of 2007 from the Hopi Indian reservation. Additionally, in 2005, 442 ticks attached to dogs were collected and identified as Rhipicephalus sanguineus. Infection with or exposure to at least one organism was detected in 69% and 66% of the dogs in May/June 2005 and February 2007, respectively. Exposure to spotted fever group (SFG) rickettsiae was detected in 66.4% (2005) and 53.4% (2007) of dogs, but rickettsial DNA was not detected using polymerase chain reaction. Active Ehrlichia canis infection (by polymerase chain reaction) was identified in 36.6% (2005) and 36.3% (2007) of the dogs. E. canis infection was associated with SFG rickettsiae seroreactivity (p < 0.001). Anaplasma platys DNA was detected in 8.3% (2005) and 4.5% (2007) of the dogs. Babesia canis and Bartonella vinsonii berkhoffii seroprevalences were 6.7% and 1% in 2005, whereas in 2007 prevalences were 0% and 1.1%, respectively. No Bartonella spp., Ehrlichia chaffeensis, or Ehrlichia ewingii DNA was detected. Dogs on this Hopi Indian reservation were most frequently infected with E. canis or A. platys; however, more than half of the dogs were exposed to a SFG-Rickettsia species.

Serological and molecular prevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia species in dogs from Minnesota.

A population of 731 naturally exposed pet dogs examined at a private practice in Baxter, Minnesota, an area endemic for Lyme disease and anaplasmosis, was tested by serological and molecular methods for evidence of exposure to or infection with selected vector-borne pathogens. Serum samples were tested by enzyme-linked immunosorbent assay (ELISA) for Anaplasma phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies and for Dirofilaria immitis antigen. Blood samples from 273 dogs were also analyzed by polymerase chain reaction (PCR) for Anaplasma and Ehrlichia species DNA. Based on the owner history and the attending veterinarian's physical examination findings, dogs exhibiting illness compatible with anaplasmosis or borreliosis were considered clinical cases, and their results were compared to the healthy dog population. Antibodies to only A. phagocytophilum were detected in 217 (29%) dogs; to only B. burgdorferi, in 80 (11%) dogs; and seroreactivity to both organisms, in 188 (25%) dogs. Of 89 suspected cases of canine anaplasmosis or borreliosis, A. phagocytophilum or B. burgdorferi antibodies were detected in 22 dogs (25%) and 8 dogs (9%) respectively, whereas antibodies to both organisms were found in 38 dogs (43%). Ehrlichia canis antibodies and D. immitis antigen were each detected in 11 (1.5%) dogs. Anaplasma phagocytophilum DNA was amplified from 7 of 222 (3%) healthy dogs and 19 of 51 (37%) clinical cases. Seroreactivity to both A. phagocytophilum and B. burgdorferi was detected more frequently in suspected cases of anaplasmosis and/or borreliosis than seroreactivity to either organism alone. Based on PCR testing, A. phagocytophilum DNA was more prevalent in suspected cases of anaplasmosis or borreliosis than in healthy dogs from the same region.

Amphetamine poisoning in a dog: case report, literature review and veterinary medical perspectives.

Amphetamine abuse in human beings has increased, resulting in many reports of toxicity and death. In the US over 4 million people have abused amphetamines at least once, thus small animals are exposed to increased accidental poisoning risk. This report describes an acute amphetamine poisoning in a dog due to ingestion of 15 mg/kg fenproporex, leading to typical signs of catecholamines release and effects in different organ systems. Similar clinical and laboratory findings observed in human beings are reviewed and physiopathogenic mechanisms discussed, as well as the therapeutic approaches available in veterinary medicine.