Molecular identification of Ehrlichia ewingii in a polyarthritic Texas dog

An 8‐year‐old, neutered male, Golden Retriever presented for bilateral carpal joint effusion. A complete blood count revealed mild leukopenia and marked thrombocytopenia. Samples were sent to the Texas A&M Veterinary Medical Diagnostic Laboratory for blood smear review and serologic testing for tick‐borne diseases. Numerous morulae were observed within neutrophils, and antibodies against Ehrlichia canis were detected at a 1:512 dilution via the indirect fluorescent antibody (IFA) test. As neutrophilic morulae are morphologically indistinguishable between Ehrlichia ewingii and Anaplasma phagocytophilum, and genus‐wide cross‐reactivity is possible with serologic testing, additional molecular testing was performed. Quantitative real‐time polymerase chain reaction (qPCR) followed by conventional PCR and Sanger sequencing were performed on serum identified with E ewingii as the sole disease‐causing agent. Three months after diagnosis and treatment, no morulae were found, molecular testing for E ewingii detected no DNA, and convalescent IFA testing demonstrated a continued detection of antibodies for E canis at a 1:512 dilution. To the authors’ knowledge, this is the first reported case of E ewingii confirmed with molecular diagnostics in a Texas dog. The zoonotic transmission potential of E ewingii should be noted as Texas supports competent tick vectors, and dogs represent effective sentinels for human ehrlichiosis. This report also highlights the utility of molecular diagnostics when serologic and microscopic evaluations are not sufficient in providing the species‐level identity of a causative agent.     

Evidence for vertical transmission of Mycoplasma haemocanis,but not Ehrlichia ewingii,in a dog

A 2‐year‐old female intact pregnant Beagle was evaluated after the owner surrendered her to a shelter. Prepartum and 2 months postpartum at the time of routine spay, the dam was whole‐blood polymerase chain reaction (PCR) positive for Ehrlichia ewingii. She was also whole‐blood PCR positive for Mycoplasma haemocanis prepartum and continuously for 5 months thereafter. The dam delivered 5 healthy puppies, 1 of which was whole‐blood PCR positive for M. haemocanis. All 5 puppies had antibodies against Ehrlichia spp. at 1 month of age but not thereafter, and all puppies were Ehrlichia spp. PCR negative for 5 months of follow‐up. Therefore, this study supports a potential role for vertical transmission in the maintenance of M. haemocanis in dogs as reservoir hosts. In contrast, in this case there was no evidence that E. ewingii was transmitted transplacentally or during the perinatal period.     

Gastrointestinal pythiosis with concurrent presumptive gastrointestinal basidiobolomycosis in a Boxer dog

A 2‐year‐old female spayed Boxer dog was presented for a 1‐month history of progressive hemorrhagic diarrhea with tenesmus and weight loss despite trial courses of antibiotics and diet change. Abdominal ultrasound revealed severe, focal thickening, and loss of normal architecture of the colonic wall with abdominal lymphadenomegaly. Dry‐mount fecal cytology, performed on several consecutive days, consistently revealed numerous, round, 16‐20 μm structures with basophilic, granular content, and a thin cell wall. Transmission electron microscopy identified these structures as fungi. Culture, polymerase chain reaction (PCR), and sequencing of the internal transcribed spacer, D1/D2 regions, and DNA‐directed RNA polymerase II core subunit (RPB2) confirmed the presence of Basidiobolus microsporus in the feces. Biopsies collected via ileocolonoscopy revealed marked, multifocal, chronic, neutrophilic, and eosinophilic ileitis and colitis with ulceration, granulation tissue, and intralesional hyphae (identified with Gomori methenamine silver stain). A Pythium enzyme‐linked immunosorbent assay and Pythium‐specific PCR performed on the formalin‐fixed paraffin‐embedded biopsy specimens were positive while Basidiobolus‐specific PCR was negative, thus confirming a diagnosis of pythiosis. This report describes a fatal case of colonic and intestinal pythiosis with the presence of fecal Basidiobolus sp. spores, suggestive of concurrent gastrointestinal basidiobolomycosis.     

Ehrlichia canis infection in the cerebrospinal fluid of a dog characterized by morulae within monocytes and neutrophils

An 8-year-old neutered female English Pointer was referred to a veterinary referral center (southwest of England) with a 4-5-month history of fecal incontinence and no evidence of urinary incontinence. Blood and free-catch urine samples were collected and sent to an off-site laboratory. Further investigations were postponed until laboratory results were available. Blood results showed a mild leukopenia, mild nonregenerative anemia, moderate to marked thrombocytopenia, and a mild increase in ALT and ALP activities. The primary veterinarian and client did not proceed with any further investigations for thrombocytopenia. Three weeks after the initial presentation, there was considerable clinical deterioration and progression of neurologic signs. Thoracic radiographs and an abdominal ultrasonographic examination were unremarkable. Magnetic resonance imaging (MRI) of the brain and spinal cord revealed an intramedullary lesion at the level of the C7 vertebra, a cystic lesion in the forebrain, and a bilateral lesion in the thalamus. A lumbar cerebrospinal fluid (CSF) was collected. CSF analysis showed a robustly increased protein concentration and marked pleocytosis. The cytologic evaluation revealed a mixed cellular population. Occasional neutrophils and monocytoid cells showed purple spherical intracellular inclusions, resembling Ehrlichia morulae. An aliquot of CSF was used off-label with a dot ELISA test, which showed a strong positive result for antibodies against Ehrlichia canis/Ehrlichia ewingii. PCR identified these morulae to be E canis. To best of the authors’ knowledge, this is the first case of ehrlichial infection in canine CSF where Ehrlichia sub-species morulae present within neutrophils were confirmed to be Ehrlichia canis using PCR.     

Suppurative spinal meningomyelitis in a dog with intra-neutrophilic cerebrospinal fluid cells Ehrlichia canis morulae

A 2-year-old castrated male Creole Shepherd mixed dog was presented for non-ambulatory paraparesis of the pelvic limbs. The magnetic resonance imaging and cerebrospinal fluid (CSF) analysis were consistent with meningomyelitis. Positive serology for Ehrlichia canis/Ehrlichia ewingii suggested exposure to a pathogen; qPCR on the serum and the CSF confirmed active infection. Ehrlichial morulae were observed within CSF and peripheral blood polymorphonuclear neutrophils; a species-specific PCR confirmed E. canis infection. This is an interesting report of E. canis infection in a dog with morulae observed in neutrophils both in the peripheral blood and CSF.     

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

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

Effects of recent Leptospira vaccination on whole blood real-time PCR testing in healthy client-owned dogs

Background

Bacterin‐based canine Leptospira vaccines could present a challenge for the use of whole blood real‐time polymerase chain reaction (PCR) as a diagnostic tool. Recent vaccination could induce positive results if the targeted DNA fragment is present within the vaccine and in the blood of the recently vaccinated dog.

Objectives

The objective of this study was to assess whether 2 available 4‐serovar vaccines induce a positive real‐time PCR reaction in the blood of healthy recently vaccinated dogs.

Animals

Twenty healthy dogs.

Methods

This was a prospective study. Dogs were assigned to 1 of 2 vaccine groups. Both vaccines were culture‐based and include Leptospira interrogans serovars Pomona, Canicola, and Icterohaemorrhagiae and Leptospira kirschneri serovar Grippotyphosa. Whole blood for real‐time PCR and serum for the microscopic agglutination test (MAT) were collected prior to and 3 and 7 days after vaccination and weekly thereafter for 8 weeks. Two real‐time PCR tests targeting 2 different genes were performed independently in a blinded fashion.

Results

Both Leptospira vaccines produced positive real‐time PCR reactions when assayed undiluted or diluted 1 : 100 in canine blood. However, blood samples drawn from all dogs at all time points after vaccination were negative on PCR. All dogs developed MAT titers.

Conclusions and Clinical Importance

Recent vaccination with 2 commercially available vaccines does not interfere with the use of real‐time PCR for the identification of acute Leptospira infection in dogs.     

Effect of retinoic acid on gene expression profiles of bovine intramuscular preadipocytes during adipogenesis

To investigate genes involved in intramuscular adipogenesis in ruminants, 16 genes with dramatic variable expression were selected. These were selected from the differentiation‐ and proliferation‐phase libraries of our previous serial analysis of gene expression (SAGE) studies of a clonal bovine intramuscular preadipocyte (BIP) cell line. We harvested the BIP cells over 12 days after adipogenic stimulation with all‐trans retinoic acid (ATRA). Quantitative real‐time PCR confirmed the earlier SAGE study results of the expression patterns of 15 of the genes. On day 6, TG accumulation increased significantly in the BIP cells but was completely inhibited in the 3T3‐L1 cells (the monogastric reference). ATRA enhanced expression levels of six genes whereas it suppressed expression of eight genes on day 3 of adipogenesis in the BIP cells. Forty‐eight hours after transfection, the messenger RNA expression level of the adipose differentiation‐related protein (ADFP), encoded by one of the upregulated genes, in the ADFP small interference RNA (siRNA)‐transfected cells was 3.5% of that in negative control‐transfected cells. Also, 6 days after induction the TG level in the ADFP siRNA‐transfected cells was 21.8% lower than that in negative control‐transfected cells. This analysis of gene expression profiles after ATRA treatment will contribute to our understanding of the molecular mechanisms involved in bovine intramuscular adipogenesis.     

Clinical Characteristics of Dogs with Progressive Myelomalacia Following Acute Intervertebral Disc Extrusion

Background

Progressive myelomalacia (PMM) is a catastrophic disease associated with acute intervertebral disc extrusion (IVDE). Published data on the clinical characteristics of this disease are limited.

Objective

To describe the onset and progression of clinical signs of PMM in a large case cohort.

Animals

Fifty‐one dogs, 18 with histopathologically confirmed PMM, 33 presumptively diagnosed based on clinical signs and diagnostic imaging.

Methods

Retrospective study. Dogs with confirmed IVDE and either a histopathologic diagnosis of PMM or a high clinical suspicion were identified by medical record search. Data on nature and progression of signs were extracted.

Results

Twenty‐four of 51 dogs were Dachshunds. T12–T13 was the most common site of disc extrusion (12 of 56), and 18 of 55 of mid‐to‐caudal lumbar discs (between L3 and L6) were affected. Onset of PMM signs ranged from present at first evaluation (17/51) to 5 days after presentation, with 25 of 51 cases developing signs within 48 hours. Progression of signs from onset of PMM to euthanasia or death, excluding 7 cases euthanized at presentation, ranged from 1 to 13 days with 23 being euthanized within 3 days. Nonspecific systemic signs were documented in 30 of 51 dogs.

Conclusion and Clinical Importance

The majority of dogs developed PMM within 2 days of presentation and was euthanized within another 3 days. However, onset can be delayed up to 5 days after presentation with progression to euthanasia taking as long as 2 weeks. Mid‐to‐caudal lumbar discs might be associated with an increased risk of PMM.     

Parasitemia due to Sarcocystis neurona‐like infection in a clinically ill domestic cat

An 8‐year‐old, 6‐kg, male neutered Domestic Shorthair cat was presented to The Ohio State University Veterinary Medical Center (OSU‐VMC) for difficulty breathing. Physical examination and thoracic radiographs indicated pneumonia, a soft‐tissue mass in the left caudal lung lobe, and diffuse pleural effusion. The effusion was classified as modified transudate. Rare extracellular elongated (~5–7 μm × 1–2 μm) zoites with a central round to oval‐shaped purple to deep purple vesicular nucleus with coarsely stippled chromatin and light blue cytoplasm were seen on a peripheral blood smear. Serum IgG and IgM were positive for Sarcocystis sp. antibodies and negative for Toxoplasma gondii antibodies, suggesting that the infection was acute rather than a recrudescence of prior infection. This organism was most consistent with either Sarcocystis neurona or Sarcocystis dasypi based on DNA sequence analysis of PCR products using COC ssRNA, ITS‐1, snSAG2, and JNB25/JD396 primer sets. This is the first report to visualize by light microscopy circulating Sarcocystis sp. merozoites in the peripheral blood of a domestic cat. Therefore, Sarcocystis should be considered as a differential diagnosis in cats with suspected systemic protozoal infection.     

Tritrichomonas foetus and Mycoplasma felis coinfection in the upper respiratory tract of a cat with chronic purulent nasal discharge

A 5‐year‐old indoor male neutered Siamese cat was presented with clinical signs of sneezing and chronic bilateral purulent nasal discharge. Multiple nasal cavity swabs were submitted for bacterial cultures, Mycoplasma felis‐DNA qPCR, and cytology. M felisqPCR was positive and cytomorphologic diagnosis was severe, acute, purulent, rhinitis with intralesional protozoal microorganisms consistent with a Trichomonas spp. Nested PCR (nPCR) confirmed the diagnosis of Tritrichomonas foetus. Systemic therapy with doxycycline for M felis and metronidazole for T foetus was started with remission of clinical signs within 2 weeks; however, symptoms relapsed shortly after therapy was discontinued. This study represents the first documented case of T foetus associated with chronic nasal discharge in a cat, which supports the hypothesis that T foetus can live in the nasal cavity. It is also the first reported case of M felis and T foetus coinfection, which indicates that with mycoplasmal feline upper respiratory tract infections, T foetus should be considered as a coinfecting agent.     

Persistent Ehrlichia ewingii Infection in Dogs after Natural Tick Infestation

Background

Ehrlichia ewingii, which causes disease in dogs and people, is the most common Ehrlichia spp. infecting dogs in the United States, but little is known about how long E. ewingii infection persists in dogs.

Hypothesis/Objectives

To evaluate the persistence of natural infection with E. ewingii in dogs.

Animals

Four Class A Beagles; no previous exposure to ticks or tick‐borne infectious agents.

Methods

Dogs were exposed to ticks by weekly walks through tick habitat in north central Oklahoma; dogs positive for infection with Ehrlichia spp. by sequence‐confirmed PCR and peptide‐specific serology were evaluated for 733 days (D). Whole blood was collected once weekly for PCR, and serum was collected once monthly for detection of antibodies to Ehrlichia canis (peptide p16), Ehrlichia chaffeensis (indirect fluorescence antibody [IFA] and variable‐length PCR target [VLPT]), and E. ewingii (peptide p28).

Results

All dogs (4/4) became infected with Ehrlichia spp. as evidenced by seroconversion on IFA to E. chaffeensis (4/4); PCR detection of E. ewingii (4/4) and E. chaffeensis (2/4) DNA using both nested and real‐time assays; and presence of specific antibodies to E. ewingii (4/4) and E. chaffeensis (2/4). Infection with E. chaffeensis was not detected after D55. Intermittent E. ewingii rickettsemia persisted in 3 of 4 dogs for as long as 733 days.

Conclusions and Clinical Importance

Our data demonstrate that dogs infected with E. ewingii from tick feeding are capable of maintaining infection with this pathogen long‐term, and may serve as a reservoir host for the maintenance of E. ewingii in nature.     

Necrotising fasciitis in a domestic shorthair cat – negative pressure wound therapy assisted debridement and reconstruction

A 10‐year‐old, domestic shorthair cat was presented for acute lameness of the left forelimb accompanied by severe pain, swelling, skin necrosis, malodorous discharge and pyrexia. Following a presumptive diagnosis of necrotising fasciitis aggressive surgical debridement of the affected soft tissues of the antebrachium and negative pressure wound treatment of the open defect were performed. Surgical findings supported the tentative diagnosis of necrotising fasciitis and Streptococcus canis was isolated from the wound. A free skin graft was performed 29 days after admission, and augmented by 3 days of negative pressure wound therapy to facilitate graft incorporation. Healing was achieved without complications and no functional or aesthetic abnormalities remained.     

Efficacy of Minocycline in Naturally Occurring Nonacute Ehrlichia canis Infection in Dogs

Background

Minocycline has been used in the treatment of Ehrlichia canis infection in dogs as an alternative to doxycycline, the recommended treatment. However, efficacy of this alternative therapy is unknown.

Objective

To assess the efficacy of minocycline in the treatment of natural occurring E. canis infection in dogs.

Animals

Ten privately owned dogs of mixed breed positive for E. canis by blood PCR.

Methods

Prospective, randomized clinical study. Dogs positive for E. canis by PCR were housed in a kennel environment and randomly allocated to receive doxycycline 10 mg/kg bodyweight PO once daily (“gold standard” control group) or minocycline (extralabel) 10 mg/kg bodyweight PO twice daily (treatment test group) for 28 days. Blood, analyzed by PCR to determine the presence or absence of E. canisDNA, was collected weekly during treatment starting on the first day of treatment and including through day 35, 7 days after the last treatment.

Results

In both groups, one dog tested negative after 7 days of treatment. For the doxycycline group, the latest time to a negative PCR test was after 3 weeks of treatment. For the minocycline group, the latest time was on day 28 of treatment. All dogs tested negative 7 days after the end of treatment.

Conclusion and Clinical Importance

Minocycline can be an effective alternative to doxycycline for clearing E. canis from the blood in nonacute infections.     

Diagnosis and management of Candida utilis infectious arthritis in a Standardbred filly

A 3‐year‐old Standardbred filly was admitted to the hospital for evaluation and management of previously diagnosed infectious arthritis of the right metacarpophalangeal joint (MCPJ). Candida utilis was isolated from multiple synovial samples submitted for bacterial culture and susceptibility. Following treatment with systemic and intra‐articular fluconazole and regional limb perfusion with amphotericin B and a second arthroscopic debridement the lameness improved and subsequent cultures were negative for bacterial or fungal growth. Infectious fungal arthritis should be a differential diagnosis for atypical or unresponsive joint infections especially in horses previously treated with a combination of intra‐articular corticosteroids and antibiotics.     

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

Thirty‐five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Paraná, Brazil. Twenty‐two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff’s method and processed for acid‐fast bacilli staining, culture in Stonebrink and Lowenstein‐Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR‐positive results (54.5%) was similar to that of culture‐positive results (51.5%, P > 0.05). The percentage of PCR‐positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR‐negative were reanalysed using 2.5 μl DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.     

A mycobacterial coinfection in a dog suspected on blood smear

A 4‐year‐old neutered female crossbred Shepherd was referred for a history of 10 days of anorexia, polyuria, polydipsia, polyadenomegaly, and diarrhea. On physical examination, the dog appeared quiet, responsive, and apyretic, with generalized and severe lymphadenomegaly. Hematologic abnormalities included neutrophilic leukocytosis with left shift, and lymphopenia. Blood smears revealed intracytoplasmic bacilli negatively stained with May–Grünwald–Giemsa in neutrophils and monocytes. Lymph node smears revealed pyogranulomatous adenitis with calcified deposits and many negative‐staining rod structures, both within the cytoplasm of neutrophils and macrophages, and free in the background. An acid‐fast stain (Ziehl–Neelsen) confirmed the diagnosis of mycobacterial infection. The dog was euthanized for public health and ethical reasons, and the postmortem examination revealed severe and generalized granulomatous and necrotizing lymphadenitis, panniculitis, and hepatitis, and infiltration of epithelioid macrophages in the lungs, colon, and spleen. Numerous acid‐fast bacilli, consistent with mycobacterial infection, were observed both in the cytoplasm of epithelioid macrophages and giant cells, and free in the background. Mycobacterium bovis was first confirmed by conventional PCR of organ extracts. Mycobacterium avium was detected in a culture of the same organs. Further PCR amplifications and sequencing revealed a coinfection with 2 different species of mycobacterium, one belonging to the Mycobacterium avium complex and the other to the Mycobacterium tuberculosis complex.     

Canine ocular thelaziosis caused by Thelazia callipaeda in Portugal

Ocular thelaziosis caused by Thelazia callipaeda is a vector‐borne disease affecting dogs and humans. We report a case of thelaziosis in a 10‐year‐old German Shepherd dog from Vila Real city (Portugal). Ophthalmological examination revealed bulbar and nictitating membrane conjunctival hyperemia with serous discharge noted at the left medial canthus and blepharitis. Schirmer tear test value and intraocular pressure were within the reference ranges in both eyes, and biomicroscopy showed a transparent cornea without lesions or edema and no inflammatory reaction in the anterior chamber. No funduscopic alterations were detected by direct and indirect ophthalmoscopic examination. When testing the nasolacrimal patency, two white worms were observed on the caruncle conjunctival surface with undulating movements that increased with light intensity. In total, eight worms were collected and morphologically identified as T. callipaeda (seven mature females and one male). PCR amplification of a 689 sequence of partial cytochrome c oxidase subunit 1 target gene confirmed the nematodes were T. callipaeda, haplotype 1. The dog was treated with a single subcutaneous injection of ivermectin combined with additional topical application of ophthalmic fusidic acid drops and oral milbemycin oxime. One week after treatment, no worms were detected and the ocular clinical signs resolved. The most recent reports of canine thelaziosis in the Iberian Peninsula should alert local health authorities to the zoonotic potential of infestation with T. callipaeda, which should be included in the differential diagnosis of conjunctivitis in dogs and humans.     

Simultaneous Infection of Pigs and People with Triple‐Reassortant Swine Influenza Virus H1N1 at a U.S. County Fair

Influenza‐like illness was noted in people and pigs in attendance at an Ohio county fair in August 2007. The morbidity rate in swine approached 100% within 1–2 days of initial clinical signs being recognized, and approximately two dozen people developed influenza‐like illness. Triple‐reassortant swine H1N1 influenza viruses were identified in both pigs and people at the fair. The identified viruses (A/Sw/OH/511445/2007, A/Ohio/01/2007, and A/Ohio/02/2007) were similar to H1N1 swine influenza viruses currently found in the U.S. swine population. This case illustrates the possibility of transmission of swine influenza in settings where there is close human/swine interaction.