An intracellular nanobody targeting T4SS effector inhibits Ehrlichia infection.

Infection with obligatory intracellular bacteria is difficult to treat, as intracellular targets and delivery methods of therapeutics are not well known. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system (T4SS) effector, is a primary virulence factor for an obligatory intracellular bacterium, Ehrlichia chaffeensis In this study, we developed Etf-1-specific nanobodies (Nbs) by immunizing a llama to determine if intracellular Nbs block Etf-1 functions and Ehrlichia infection. Of 24 distinct anti-Etf-1 Nbs, NbD7 blocked mitochondrial localization of Etf-1-GFP in cotransfected cells. NbD7 and control Nb (NbD3) bound to different regions of Etf-1. Size-exclusion chromatography showed that the NbD7 and Etf-1 complex was more stable than the NbD3 and Etf-1 complex. Intracellular expression of NbD7 inhibited three activities of Etf-1 and E. chaffeensis: up-regulation of mitochondrial manganese superoxide dismutase, reduction of intracellular reactive oxygen species, and inhibition of cellular apoptosis. Consequently, intracellular NbD7 inhibited Ehrlichia infection, whereas NbD3 did not. To safely and effectively deliver Nbs into the host cell cytoplasm, NbD7 was conjugated to cyclized cell-permeable peptide 12 (CPP12-NbD7). CPP12-NbD7 effectively entered mammalian cells and abrogated the blockade of cellular apoptosis caused by E. chaffeensis and inhibited infection by E. chaffeensis in cell culture and in a severe combined-immunodeficiency mouse model. Our results demonstrate the development of an Nb that interferes with T4SS effector functions and intracellular pathogen infection, along with an intracellular delivery method for this Nb. This strategy should overcome current barriers to advance mechanistic research and develop therapies complementary or alternative to the current broad-spectrum antibiotic.

High-Dose Filgrastim Treatment of Nonregenerative Pancytopenia Associated With Chronic Canine Ehrlichiosis.

OBJECTIVE: To report the management and outcome of a dog with canine monocytic ehrlichiosis and nonregenerative pancytopenia, with high doses of filgrastim. CASE DESCRIPTION: An 8-year-old male, mixed-breed dog, weighing 5.6kg, presented with a 1-month history of hyporexia, adynamia, and a weight loss of approximately 1kg. The general condition of the dog was observed to be poor as follows: lethargy, tachycardia, marked pallor of the mucous membranes, petechiae on the abdomen, hepatosplenomegaly, and cervical lymphadenopathy. A complete blood count analysis revealed severe leukopenia, thrombocytopenia, and anemia. A direct immunofluorescence assay using anti-Ehrlichia canis-immunoglobin G (1:400) yielded positive result. The dog was diagnosed with nonregenerative pancytopenia associated with canine monocytic ehrlichiosis. The dog presented poor prognostic signs (neutropenia, thrombocytopenia, and severe anemia). The dog was treated with antibiotics and a short course of high-dose filgrastim (50µg/kg, SC, q 48h for 4 days) to stimulate bone marrow response, prednisone to decrease peripheral platelet destruction, and an iron supplement to compensate for the iron deficiency in the bone deposits. Although temporary side effects associated with filgrastim use, such as bone pain, bleeding, and the worsening of thrombocytopenia, were observed, the treatment improved the clinical course and the cell counts in less than a month. CLINICAL RELEVANCE: The treatment protocol used in this case might be an alternative for treating cases of severe myelosuppression. This treatment plan can substantially change the clinical course of the disease for the better, compared to conventional treatment.

Interstitial pneumonia and pulmonary hypertension associated with suspected ehrlichiosis in a dog.

BACKGROUND: In dogs with canine monocytic ehrlichiosis (CME), respiratory signs are uncommon and clinical and radiographic signs of interstitial pneumonia are poorly described. However, in human monocytic ehrlichiosis, respiratory signs are common and signs of interstitial pneumonia are well known. Pulmonary hypertension (PH) is classified based on the underlying disease and its treatment is aimed at reducing the clinical signs and, if possible, addressing the primary disease process. PH is often irreversible, but can be reversible if it is secondary to a treatable underlying etiology. CME is currently not generally recognized as one of the possible diseases leading to interstitial pneumonia and secondary PH in dogs. Only one case of PH associated with CME has been reported worldwide. CASE PRESENTATION: A seven-year-old, male intact, mixed breed dog was presented with 2 weeks history of lethargy and dyspnea. The dog previously lived in the Cape Verdean islands. Physical examination showed signs of right-sided congestive heart failure and poor peripheral perfusion. Thoracic radiography showed moderate right-sided cardiomegaly with dilation of the main pulmonary artery and a mild diffuse interstitial lung pattern with peribronchial cuffing. Echocardiography showed severe pulmonary hypertension with an estimated pressure gradient of 136 mm Hg. On arterial blood gas analysis, severe hypoxemia was found and complete blood count revealed moderate regenerative anemia and severe thrombocytopenia. A severe gamma hyperglobulinemia was also documented. Serology for Ehrlichia canis was highly positive. Treatment with oxygen supplementation, a typed packed red blood cell transfusion and medical therapy with doxycycline, pimobendan and sildenafil was initiated and the dog improved clinically. Approximately 2 weeks later, there was complete resolution of all clinical signs and marked improvement of the PH. CONCLUSION: This report illustrates that CME might be associated with significant pulmonary disease and should be considered as a possible differential diagnosis in dogs presenting with dyspnea and secondary pulmonary hypertension, especially in dogs that have been in endemic areas. This is important because CME is a treatable disease and its secondary lung and cardiac manifestations may be completely reversible.

Molecular detection and treatment of tick-borne pathogens in domestic dogs in Khon Kaen, northeastern Thailand.

We determined the prevalence of tick-borne pathogens in domestic dogs using microscopy and polymerase chain reaction (PCR) techniques. A total of 303 EDTA blood samples were collected from domestic dogs in Khon Kaen Province, Thailand, in May 2013. Microscopic observation of Giemsa-stained smears and molecular diagnosis using conventional PCR were performed. Infected dogs were treated with imidocarb dipropionate, a combination of imidocarb dipropionate and doxycycline, or doxycycline alone. Seventy-one (23.4%) out of 303 dogs were positive for DNA of tick-borne pathogens. Of the 303 animals, 13.2% and 1.3% were positive for a single infection with Babesia spp or Ehrlichia canis, respec- tively using microscopy; whereas 19.5% and 3.0% were positive using the PCR technique. Co-infection with Babesia spp and E. canis was observed in 0.7%, and coinfection with Hepatozoon canis and E. canis in 0.3%. Infected dogs were treated with the assigned drugs, and elimination of the pathogens was demonstrated by microscopy and PCR. The results indicated that while both microscopic and PCR diagnostic techniques were useful for tick-borne pathogen detection, PCR was more effective. Imidocarb dipropionate and doxycycline were found to be effective for treatment of babesiosis and ehrlichiosis, respectively. The present study suggests that the PCR technique has high sensitivity and specificity for Babesia and Ehrlichia diagnosis as well as for detection of Babesia spp, E. canis and H. canis DNA in EDTA blood specimens.

Premature parturition, edema, and ascites in an alpaca infected with Anaplasma phagocytophilum.

An 8-year-old alpaca was presented for fever, anorexia, edema, ascites, and premature parturition. She was determined to have Anaplasma phagocytophilum infection based on positive blood polymerase chain reaction (PCR) and positive acute and convalescent serum titers. Antibiotics and supportive therapies were administered and the alpaca made a complete recovery.

Parturition prématurée, œdème et ascite chez un alpaga infecté parAnaplasma phagocytophilum. Un alpaga femelle âgé de 8 ans a été présenté pour une fièvre, de l'anorexie, de l'œdème, de l'ascite et une parturition prématurée. On a déterminé qu'elle avait une infection à Anaplasma phagocytophilum en se fondant sur le résultat positif d'un test d'amplification en chaîne par la polymérase (PCR) effectué sur un échantillon sanguin et des titres sériques aigus et convalescents positifs. Des antibiotiques et des thérapies de soutien ont été administrés et l'alpaga s'est rétabli complètement.(Traduit par Isabelle Vallières).

[Monocytic ehrlichiosis--an emerging zoonotic disease in Israel].

Ehrlichiosis is an emerging zoonotic disease transmitted to man by ticks. Its clinical features include fever, headache, myalgia, nausea and rash. The diagnosis requires a high index of suspicion; the disease has a specific serology, and has never been reported in Israel. We describe a 52-year-old man hospitalized with fever, a diffuse rash, arthralgia and epididymitis. Skin biopsy disclosed necrotizing small vessel disease consistent with periarteritis nodosa. Acute phase serum titer for E. chaffeensis was 1:256. Fever promptly subsided following ciprofloxacin.