Penicillium and Talaromyces spp. emerging pathogens in dogs since 1990s.

Penicillium and Talaromyces spp. are environmental saprophytic molds rarely encountered as infectious agents in humans and animals. This article summarizes the clinical features, treatment, and outcomes of proven infections caused by Penicillium or Talaromyces in four dogs in France. Two dogs had disseminated infections, while the other two had a localized form. All dogs had positive histopathological results showing the presence of hyaline septate hyphae and a positive fungal culture with typical Penicillium conidiophores. Talaromyces georgiensis (n = 1), Penicillium labradorum (n = 2), and Penicillium from section Ramosa series Raistrickiorum (n = 1), were identified based on Internal Transcribed Spacer (ITS) Sanger sequencing. The dogs were initially treated with ketoconazole or itraconazole. Second-line treatment was initiated in three dogs, but after several relapses, the prognosis remained poor. Since the 1990s, 18 cases of Penicillium or Talaromyces infections in dogs have been described worldwide. This series of four reports brings new cases to those already reported in the literature, which are probably underestimated in the world.

Penicillium and Talaromyces spp. are molds found in the environment that rarely cause infections in humans and animals. This article summarizes the clinical features and treatment of proven infections caused by Penicillium or Talaromyces species in four dogs in France.

Nematode dermatitis due to Angiostrongylus vasorum infection in a dog.

BACKGROUND: Angiostrongylus vasorum is a nematode that primarily infects Canidae. The adult parasites are found in the pulmonary arterial circulation and the right side of the heart. The most common clinical sign is respiratory dysfunction. Bleeding, neurological, ocular, cardiovascular and gastrointestinal disorders are also reported. Skin lesions are very unusual. HYPOTHESIS/OBJECTIVES: This report describes a nematode dermatitis due to A. vasorum infection. To the best of the authors' knowledge, this is the first case of a dog infected with this parasite that initially presented with skin lesions only. ANIMAL: A 3-year-old female Weimaraner dog presented with a crusted papular dermatitis on the bridge of the nose and on the pinnae, and an erythematous pododermatitis with erosions and perionyxis of one digit of 1 week's duration. Two weeks later the dog developed respiratory distress. METHODS AND RESULTS: Skin scrapings and fungal culture were negative for parasites and dermatophytes. Histopathological examination showed dermal granulomas and pyogranulomas with eosinophils centred around parasitic elements compatible with nematode larvae. Angiostrongylus vasorum DNA was demonstrated in skin biopsies. Chest radiographs were compatible with verminous pneumonia and a Baermann test revealed A. vasorum larvae. The dog was treated orally with fenbendazole, with rapid improvement and complete cure after 3 months. CONCLUSIONS AND CLINICAL IMPORTANCE: Angiostrongylus vasorum should be considered in dogs presented with skin lesions and respiratory signs. Skin biopsy, chest radiographs and Baermann test should be included in the diagnostic investigation.

[Zoonoses (synthetic tables): animal reservoirs of pathogens and way of transmission to man]. / Les zoonoses (tableaux synthétiques: animaux réservoirs de pathogènes et modes de transmission).

The knowledge of animals which act as reservoirs for human pathogenic agents is essential for the understanding of the epidemiology of zoonotic diseases and for the application of relevant methods of control. In the present article, synthetic tables present the main zoonoses due to viruses, bacteria, protozoa, helminths, arthropods and fungi. A list of the main pathogenic agents, the animal reservoirs and the way of transmission to man is detailed in each table.

First case of peritoneal cysticercosis in a non-human primate host (Macaca tonkeana) due to Taenia martis.

BACKGROUND: Infections with larval stages (metacestodes) of a variety of taeniid species have been described in primates, including humans, with partial to severe clinical consequences. Taenia martis is a tapeworm of mustelids, and martens are mainly their definitive hosts in Central Europe. In the rodent intermediate host cysticerci develop in the pleural and peritoneal cavities. The present report describes a case of T. martis peritoneal cysticercosis in a Tonkean macaque. FINDINGS: An abdominal mass was detected in a 3-year-old male Tonkean macaque (Macaca tonkeana) born and raised in a primate colony in France. Examination of the mass after laparotomy showed numerous vesicles identified as cysticerci of T. martis, based on the morphology of scolex and hooks, with confirmation by PCR amplification and sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes. Exeresis of the lesion was not possible and praziquantel (5.7 mg/kg) was given twice at an interval of 3 days. The abdominal mass was greatly diminished upon examination 2 months later and no signs of recurrence were noticed during the following 4 years. CONCLUSIONS: This is the first report of T. martis cysticercosis in a monkey. This record and the recent first description of an ocular T. martis cysticercosis in a human show the susceptibility of primates to T. martis and its zoonotic potential. This taeniid species must be considered in the differential diagnosis of cysticercosis in primates.

Assessment of Aspergillus fumigatus burden in lungs of intratracheally-challenged turkeys (Meleagris gallopavo) by quantitative PCR, galactomannan enzyme immunoassay, and quantitative culture.

Aspergillus fumigatus remains a major respiratory pathogen in birds and treatment is still difficult. We challenged different groups of few-day-old turkeys via intratracheal aerosolisation with increasing concentrations (10(5) up to 10(8)) of conidia using a MicroSprayer(®) device. The fungal burden was assessed by real-time PCR, galactomannan dosage, CFU counting and histopathological evaluation in order to provide a comparison of these results within each inoculum groups. Significant mortality, occurring in the first 96h after inoculation, was only observed at the highest inoculum dose. Culture counts, GM index and qPCR results on the one hand and inoculum size on the other hand appeared to be clearly correlated. The mean fungal burden detected by qPCR was 1.3log10 units higher than the mean values obtained by CFU measurement. The new model and the markers will be used to evaluate the efficacy of antifungal treatments that could be used in poultry farms.

Mutations in the Cyp51A gene and susceptibility to itraconazole in Aspergillus fumigatus isolated from avian farms in France and China.

Azole resistance in the fungal pathogen Aspergillus fumigatus is an emerging problem and may develop during azole therapy in humans and animals or exposure to azole fungicides in the environment. To assess the potential risk of azole-resistance emergence in avian farms where azole compounds are used for the control of avian mycoses, we conducted a drug susceptibility study including A. fumigatus isolates from birds and avian farms in France and Southern China. A total number of 175 isolates were analyzed: 57 isolates were collected in France in avian farms where chemoprophylaxis with parconazole was performed; 51 isolates were collected in southern China in avian farms where no chemoprophylaxis was performed; and 67 additional isolates came from the collection of a mycology laboratory. No resistant isolate was detected, and the distribution of minimum inhibitory concentrations was similar for isolates collected in farms with or without azole chemoprophylaxis. For 61 randomly selected isolates, the full coding sequence of the Cyp51A gene was determined to detect mutations. Nine amino acid alterations were found in the target enzyme, 3 of which were new.

Assessment of Aspergillus fumigatus pathogenicity in aerosol-challenged chickens (Gallus gallus) belonging to two lineages.

Infection due to the mold Aspergillus fumigatus remains a common and life-threatening infection in many animals, especially birds. Animal models are still required to better understand the physiopathology of infection and evaluate diagnostic tools and treatment procedures. The aim of the present study was to assess the pathogenicity of A. fumigatus in two lineages of chicken (Gallus gallus): SPF White Leghorn PA12 layers and conventional JA657 broilers. Four-day-old birds were experimentally infected in an inhalation chamber in order to reproduce a "natural" contamination and to obtain a large repartition of conidia into the respiratory tract. Half of the chicks were injected subcutaneously with dexamethasone for 4 days before the infective challenge. At days 0 and 7, the effects of chicken lineage and immunosuppressive treatment on pulmonary fungal burden were analyzed using two linear mixed models. The pathogenicity of A. fumigatus varied according to the lineage: no clinical signs and no mortality were observed in layer chickens whereas more than 50% of mortality occurred in broilers. The effect of immunosuppressive treatment was also demonstrated, notably on animals weight but also on mortality.

Generalized dermatitis associated with Malassezia overgrowth in cats: A report of six cases in France.

We recently observed six cases of generalized dermatitis associated with Malassezia overgrowth in cats presented to the Veterinary College of Alfort, France. Elevated numbers of yeasts were observed in lesional skin by cytology and culture. Skin lesions occurred on the face, ventral neck, abdomen and ear canals and were characterized by some degree of alopecia, erythema and crusting. In most cases, pruritus was intense. The species M. pachydermatis was systematically isolated.

Simple and highly discriminatory VNTR-based multiplex PCR for tracing sources of Aspergillus flavus isolates.

Aspergillus flavus is second only to A. fumigatus in causing invasive aspergillosis and it is the major agent responsible for fungal sinusitis, keratitis and endophthalmitis in many countries in the Middle East, Africa and Southeast Asia. Despite the growing challenge due to A. flavus, data on the molecular epidemiology of this fungus remain scarce. The objective of the present study was to develop a new typing method based on the detection of VNTR (Variable number tandem repeat) markers. Eight VNTR markers located on 6 different chromosomes (1, 2, 3, 5, 7 and 8) of A. flavus were selected, combined by pairs for multiplex amplifications and tested on 30 unrelated isolates and six reference strains. The Simpson index for individual markers ranged from 0.398 to 0.818. A combined loci index calculated with all the markers yielded an index of 0.998. The MLVA (Multiple Locus VNTR Analysis) technique proved to be specific and reproducible. In a second time, a total of 55 isolates from Chinese avian farms and from a Tunisian hospital have been evaluated. One major cluster of genotypes could be defined by using the graphing algorithm termed Minimum Spanning Tree. This cluster comprised most of the isolates collected in an avian farm in southern China. The MLVA technique should be considered as an excellent and cost-effective typing method that could be used in many laboratories without the need for sophisticated equipment.

Aspergillus fumigatus in Poultry.

Aspergillus fumigatus remains a major respiratory pathogen in birds. In poultry, infection by A. fumigatus may induce significant economic losses particularly in turkey production. A. fumigatus develops and sporulates easily in poor quality bedding or contaminated feedstuffs in indoor farm environments. Inadequate ventilation and dusty conditions increase the risk of bird exposure to aerosolized spores. Acute cases are seen in young animals following inhalation of spores, causing high morbidity and mortality. The chronic form affects older birds and looks more sporadic. The respiratory tract is the primary site of A. fumigatus development leading to severe respiratory distress and associated granulomatous airsacculitis and pneumonia. Treatments for infected poultry are nonexistent; therefore, prevention is the only way to protect poultry. Development of avian models of aspergillosis may improve our understanding of its pathogenesis, which remains poorly understood.

Relative efficiencies of two air sampling methods and three culture conditions for the assessment of airborne culturable fungi in a poultry farmhouse in France.

Fungal elements represent a significant part of the biological contaminants that could be detected in the air of animal facilities. The aim of this study was to assess the relative efficiencies of two air sampling methods and three culture conditions for the quantification of airborne culturable fungi in a poultry farmhouse in France. Air samples were collected every week throughout a 15-week period. Two devices were simultaneously used-a rotative cup air sampler (CIP 10-M, Arelco, France) and an air sampler based on filtration (AirPort MD8, Sartorius, Germany). Culture of airborne viable fungi was performed on malt extract agar (ME) and dichloran glycerol-18 (DG18) at 25 or 37°C. CIP 10-M and AirPort MD8 were shown to display comparable performances but significant differences were observed between culture conditions for Aspergillus spp. (p<0.01), Scopulariopsis spp. (p=0.02) and unidentified molds (p<0.01).

Ocular thelaziosis in dogs, France.

During 2005-2008, veterinary practitioners reported ocular infection by Thelazia spp. nematodes in 115 dogs and 2 cats in southwestern France. Most cases were detected in Dordogne, particularly in 3 counties with numerous strawberry farms, which may favor development of the fruit fly vector. Animal thelaziosis may lead to emergence of human cases.

Multiple-locus variable-number tandem repeat analysis for molecular typing of Aspergillus fumigatus.

BACKGROUND: Multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) is a prominent subtyping method to resolve closely related microbial isolates to provide information for establishing genetic patterns among isolates and to investigate disease outbreaks. The usefulness of MLVA was recently demonstrated for the avian major pathogen Chlamydophila psittaci. In the present study, we developed a similar method for another pathogen of birds: the filamentous fungus Aspergillus fumigatus. RESULTS: We selected 10 VNTR markers located on 4 different chromosomes (1, 5, 6 and 8) of A. fumigatus. These markers were tested with 57 unrelated isolates from different hosts or their environment (53 isolates from avian species in France, China or Morocco, 3 isolates from humans collected at CHU Henri Mondor hospital in France and the reference strain CBS 144.89). The Simpson index for individual markers ranged from 0.5771 to 0.8530. A combined loci index calculated with all the markers yielded an index of 0.9994. In a second step, the panel of 10 markers was used in different epidemiological situations and tested on 277 isolates, including 62 isolates from birds in Guangxi province in China, 95 isolates collected in two duck farms in France and 120 environmental isolates from a turkey hatchery in France. A database was created with the results of the present study http://minisatellites.u-psud.fr/MLVAnet/. Three major clusters of isolates were defined by using the graphing algorithm termed Minimum Spanning Tree (MST). The first cluster comprised most of the avian isolates collected in the two duck farms in France, the second cluster comprised most of the avian isolates collected in poultry farms in China and the third one comprised most of the isolates collected in the turkey hatchery in France. CONCLUSIONS: MLVA displayed excellent discriminatory power. The method showed a good reproducibility. MST analysis revealed an interesting clustering with a clear separation between isolates according to their geographic origin rather than their respective hosts.

Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms.

BACKGROUND: Aspergillus fumigatus, a saprophytic mould, is responsible for life-threatening, invasive pulmonary diseases in immunocompromised hosts. The role of the airway epithelium involves a complex interaction with the inhaled pathogen. Antimicrobial peptides with direct antifungal and chemotactic activities may boost antifungal immune response. RESULTS: The inducible expression of defensins by human bronchial epithelial 16HBE cells and A549 pneumocyte cells exposed to A. fumigatus was investigated. Using RT-PCR and real time PCR, we showed an activation of hBD2 and hBD9 defensin genes: the expression was higher in cells exposed to swollen conidia (SC), compared to resting conidia (RC) or hyphal fragments (HF). The kinetics of defensin expression was different for each one, evoking a putative distinct function for each investigated defensin. The decrease of defensin expression in the presence of heat-inactivated serum indicated a possible link between defensins and the proteins of the host complement system. The presence of defensin peptide hBD2 was revealed using immunofluorescence that showed a punctual cytoplasmic and perinuclear staining. Quantification of the cells stained with anti hBD2 antibody demonstrated that SC induced a greater number of cells that synthesized hBD2, compared to RC or HF. Labelling of the cells with anti-hBD-2 antibody showed a positive immunofluorescence signal around RC or SC in contrast to HF. This suggests co-localisation of hBD2 and digested conidia. The HBD2 level was highest in the supernatants of cells exposed to SC, as was determined by sandwich ELISA. Experiments using neutralising anti-interleukine-1beta antibody reflect the autocrine mechanism of defensin expression induced by SC. Investigation of defensin expression at transcriptional and post-transcriptional levels demonstrated the requirement of transcription as well as new protein synthesis during A. fumigatus defensin induction. Finally, induced defensin expression in primary culture of human respiratory cells exposed to A. fumigatus points to the biological significance of described phenomena. CONCLUSION: Our findings provide evidence that respiratory epithelium might play an important role in the immune response during Aspergillus infection. Understanding the mechanisms of regulation of defensin expression may thus lead to new approaches that could enhance expression of antimicrobial peptides for potential therapeutic use during aspergillosis treatment.

Dermatophytoses in animals.

Dermatophytoses are one of the most frequent skin diseases of pets and livestock. Contagiousness among animal communities, high cost of treatment, difficulty of control measures, and the public health consequences of animal ringworm explain their great importance. A wide variety of dermatophytes have been isolated from animals, but a few zoophilic species are responsible for the majority of the cases, viz. Microsporum canis, Trichophyton mentagrophytes, Trichophyton equinum and Trichophyton verrucosum, as also the geophilic species Microsporum gypseum. According to the host and the fungal species involved, the typical aspect of dermatophytic lesions may be modified. As a consequence, an accurate clinical examination, a good differential diagnosis and laboratory analyses are required for a correct identification. Few antifungal agents are available and licenced for use in veterinary practice, and the use of systemic drugs is limited in livestock due to the problems of residues in products intended for human consumption. The high resistance of the dermatophyte arthroconidia in the environment, the multiplicity of host species, and the confinement of animals in breedings are cause of an enzootic situation in many cases. Prevention is difficult, but research development on the immune response to dermatophytes and the use of vaccination, especially in cattle, have brought some interesting results.

Evaluation of fungal aerosols using Temporal Temperature Gradient Electrophoresis (TTGE) and comparison with culture.

Information obtained from fungal air samples can assist in the assessment of health hazards and can be useful in proactive indoor air quality monitoring. The objective of the present study was to evaluate the PCR-TTGE technique for the analysis of fungal diversity in the air. Eleven air samples were collected in five different sites using the bioimpactor CIP 10-M (Arelco). After a 2 hours sampling period, the collection liquid was recovered for subsequent cultivation and PCR-TTGE. A set of three fungi-specific primers (Fungcont 1, Fungcont 2+GC and Fungcont 3) was designed for the partial amplification of the 18S rRNA gene. The amplification was obtained in a single reaction tube by a semi-nested PCR. For identification, the TTGE bands were extracted and sequenced. PCR-TTGE allowed the clear separation of amplicons corresponding to distinct fungal species (both Ascomycota and Basidiomycota) that may be encountered in air. The number of fungal taxa detected after culture was systematically higher than the number of taxa found using PCR-TTGE. However, few fungal species were detected by PCR-TTGE and not by cultivation, suggesting that the combination of these approaches may provide a better analysis of fungal diversity in air samples than either method alone.

Aspergillus fumigatus conidia inhibit tumour necrosis factor- or staurosporine-induced apoptosis in epithelial cells.

A major innate immune response to inhaled conidia of the opportunistic pathogen Aspergillus fumigatus (Af) is the synthesis of pro-inflammatory cytokines, which include tumour necrosis factor (TNF)-alpha, a known inducer of apoptosis. Modulation of host cell apoptosis has been reported to be one of the mechanisms whereby pathogens overcome host cell defences. Our study was designed to investigate whether or not Af conidia could modulate apoptosis induced by TNF-alpha or staurosporine (STS). Exposure of epithelial cells treated by these inducers and exposed to Af conidia decreased the number of apoptotic cells detected by Annexin V staining, analysis of nuclear morphology, terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end-labelling reaction and immunoblotting. Inhibition of apoptosis by Af conidia was seen in cells of the A549 pneumocyte II line, human tracheal epithelial 16HBE and primary human respiratory cells. Inhibition of apoptosis by Af conidia was also observed when apoptosis was induced by co-cultivating A549 cells with activated human alveolar macrophages. Unlike Af conidia, conidia of Cladosporium cladosporioides as well as latex beads or killed Af conidia have no inhibitory effect on TNF-alpha or STS-induced apoptosis. For TNF-induced apoptosis, the observed anti-apoptotic effect of Af conidia was found to be associated with a significant reduction of caspase-3.

Frequency, body distribution, and population size of Malassezia species in healthy dogs and in dogs with localized cutaneous lesions.

Malassezia species are commensal organisms of human and animal skin that occasionally act as opportunistic pathogens. The lipid-dependent species are associated with human skin disorders, whereas the non-lipid-dependent species (Malassezia pachydermatis) is considered as an opportunistic secondary pathogen affecting the canine skin surface and ear canal. This study evaluated the relationship between Malassezia yeasts, their population size, and the occurrence of skin lesions from healthy and skin-diseased dogs. The efficiency of cytological examination and fungal culture for Malassezia detection was also evaluated. From March 2002 to July 2003, 33 healthy dogs and 54 dogs with pruritic localized skin diseases were examined; skin swabs (1218) were collected from 7 anatomical sites for culture and cytological examination. Malassezia prevalence according to anatomical site and the agreement between cytological results and fungal cultures were statistically analyzed. Differences in mean colony forming unit counts between positive healthy and diseased dogs were evaluated using the Bonferroni test for post hoc pair-wise comparisons. In healthy dogs, Malassezia yeasts were most frequently isolated in the perianal and perioral areas. The frequency of isolation and population size of Malassezia species were higher in dogs with localized dermatitis, especially in affected areas, indicating a role for Malassezia in the occurrence of skin lesions. Malassezia pachydermatis was the species most commonly cultured from the skin and external ear canal of healthy and diseased dogs; isolation of lipid-dependent yeasts from healthy dogs was less frequent. Using fungal culture as the gold standard, cytological examination showed good relative specificity (95%) but very low relative sensitivity (30%).

Molecular and serological evidence of Pneumocystis circulation in a social organization of healthy macaques (Macaca fascicularis).

Simian populations represent valuable models for understanding the epidemiology of human pneumocystosis. The present study aims to describe the circulation of Pneumocystis organisms within a social organization of healthy crab-eating macaques (Macaca fascicularis) living in a natural setting in France. Animals were followed for up to 2 years. Deep nasal swab and blood samples were collected monthly from each animal under general anaesthesia. Environmental air was sampled for a 1 week period every month in the park where the macaques dwelt. Pneumocystis DNA was detected by nested-PCR of mitochondrial large subunit rRNA (mtLSU) gene in nasal swab and air samples. Anti-Pneumocystis IgG antibodies were detected in serum samples by indirect immuno-fluorescence assay. Pneumocystis DNA was detected in 168 of 500 swab samples examined (33.6 %). The number of macaques with detectable Pneumocystis DNA was highly variable from one month to another. Positive detection of Pneumocystis DNA was not related to the detection of serum anti-Pneumocystis antibody. During the second year of the study, Pneumocystis DNA was amplified more frequently from unweaned macaques than from adults or subadults. The mtLSU sequence showed marked polymorphism with eight Pneumocystis sequence types representing two distinct groups. On the whole, a constant and intensive circulation of Pneumocystis organisms within the community was observed. However, the implication of the various members of the colony was probably different and several levels of colonization by Pneumocystis may occur in immunocompetent macaques.