Presumptive heterophil extracellular traps recognized cytologically in nine reptile patients with inflammatory conditions.

BACKGROUND: Neutrophil extracellular traps (NETs) represent a novel cellular mechanism of antimicrobial defense activity. Intravascular neutrophils produce extracellular web-like structures composed of chromatin, histones, and cytoplasmic granule proteins to attack and kill microbes. They may impact both pathogen and host; NETs correlate strongly with disseminated intravascular coagulation and mortality in critically ill humans. The mechanism was first discovered in human neutrophils in 2004. Presumptive heterophil extracellular traps (HETs) in a non-avian reptile species were first described in blood films of a gopher tortoise with systemic inflammation. OBJECTIVE: While prior reports are limited to blood film review and in vitro studies, this descriptive case series highlights the cytological identification of presumptive HETs in nine reptile patients. METHODS: Subjects included six gopher tortoises, one blood python (Python curtus), one Burmese python (P. bivittatus), and one desert king snake (Lampropeltis getula splendida). All six gopher tortoises (Gopherus polyphemus) had upper respiratory disease with bacterial etiology (including Helicobacter sp. and/or Mycoplasma sp.), and snakes had upper respiratory tract infection confirmed with serpentovirus (n = 2) or bacterial dermatitis (n = 1). RESULTS: Cytology samples with identified HETs included tissue imprints (n = 4), nasal discharge (n = 3), an oral swab (n = 1), and a fine needle aspirate of a skin lesion (n = 1). The identification of specific bacterial (n = 6) and/or viral pathogens (n = 2) was notable. CLINICAL RELEVANCE: To the authors' knowledge, this is the first report of presumptive HETs recognized in reptile cytology specimens, suggesting an active cellular process in vivo in response to systemic inflammation in non-avian reptiles, and contributing to further understanding of extracellular traps in these species.

Retrospective evaluation of the efficacy of isolating bacteria from synovial fluid in dogs with suspected septic arthritis.

OBJECTIVE: To evaluate the efficacy of synovial fluid culture in obtaining the causative organism from dogs with suspected septic arthritis. METHODS: In this retrospective evaluation, synovial fluid cytology and microbiology submissions from dogs with suspected septic arthritis from March 2007 to August 2011 were reviewed. Synovial fluid cytology consistent with joint sepsis was identified. Cultures of synovial fluid from dogs with clinical histories and abnormalities consistent with septic arthritis were used to evaluate the efficacy of bacterial isolation. RESULTS: In total, 36 dogs met the inclusion criteria. Initial aerobic cultures of joint fluid yielded bacterial growth in 44% of these dogs. All anaerobic cultures were negative. In 19% of the dogs with positive cultures, antibiotics had been administered prior to arthrocentesis compared with 10% of dogs with negative cultures. There was no association between culture efficacy and the administration of antimicrobial treatment prior to synovial fluid culture or recent surgery involving the affected joint (P=0.637 and P=0.106, respectively). CONCLUSION: Culture of synovial fluid from dogs with suspected septic arthritis has a low yield, necessitating a more effective means of identifying bacteria from suspected septic joints in dogs.

Comparison of synovial fluid culture and 16S rRNA PCR in dogs with suspected septic arthritis.

OBJECTIVE: To prospectively compare the sensitivity and specificity of 16S rRNA PCR with culture for identifying the causative organism in synovial fluid obtained from dogs with suspected septic arthritis. METHODS: Synovial fluid cytology, PCR analysis and aerobic, anaerobic and Mycoplasma culture of samples from the affected joints of 18 dogs presenting with suspected septic arthritis were performed. Synovial fluid samples from the corresponding contralateral joints of 7 dogs were also analysed as negative controls. RESULTS: There was no significant difference between the sensitivity of bacterial detection via culture (63.2%) versus PCR (73.7%) of synovial fluid (P=0.728) or between culture and combined PCR and culture (89.5%) of synovial fluid (P=0.124). The specificity of PCR (42.9%) was significantly lower than culture specificity (100%) (P=0.07). CONCLUSION: Although 16S PCR may hold potential as an ancillary diagnostic test for identifying the causative organism in dogs with septic arthritis, our study failed to demonstrate improved accuracy compared with traditional synovial fluid culture.

Massive Muscular Infection by a Sarcocystis Species in a South American Rattlesnake (Crotalus durissus terrificus).

Massive numbers of sarcocysts of a previously undescribed species of Sarcocystis were observed in the skeletal muscles throughout the body of an adult, female South American rattlesnake (Crotalus durissus terrificus). Examination of tissue sections by light microscopy demonstrated that sarcocysts were present in 20 to 40% of muscle fibers from 5 sampled locations. Sarcocysts were not present in cardiac muscle, smooth muscle, or other organs. Sarcocysts were 0.05-0.15 mm wide, had variable length depending on the viewed orientation and size of the muscle fiber, and had a sarcocyst wall less than 1-µm thick. Sarcocysts were subdivided by septa and had central degeneration in older sarcocysts. Host induced secondary encapsulation or an inflammatory response was not present. By transmission electron microscopy (TEM), the sarcocyst wall was Type I, with a parasitophorous membrane of approximately 100 nanometers in width arranged in an undulating pattern and intermittently folded inward in a branching pattern. The sarcocysts contained metrocytes in different stages of development and mature bradyzoites. The nucleic acid sequence from a section of the 18S small subunit rRNA gene was most closely related to S. mucosa that uses marsupials as intermediate hosts and has an unknown definitive host. This is apparently the third report of muscular Sarcocystis infection in snakes and is the first to describe the ultrastructure of the sarcocysts and use sequencing methods to aid in identification.

Marine mammal zoonoses: a review of disease manifestations.

Marine mammals evoke strong public affection as well as considerable scientific interest. However, the resultant close contact with marine wildlife poses human health risks, including traumatic injury and zoonotic disease transmission. The majority of zoonotic marine mammal diseases result in localized skin infections in man that resolve spontaneously or with appropriate medical therapy. However, other marine mammal zoonoses, if left untreated, induce life-threatening systemic diseases that could pose public health risks. As the number of zoonotic diseases rises, the diagnosis of and treatment for these emerging pathogens pose special challenges requiring the expertise of physicians, veterinarians and wildlife biologists. Here, we provide a comprehensive review of the bacterial, viral and fungal marine mammal zoonotic diseases that we hope will be utilized by public health professionals, physicians, veterinarians and wildlife biologists to better understand, diagnose and prevent marine mammal zoonotic diseases.

Treatment of tarsal joint deformities with hinged transarticular external fixators in three young birds.

Pelvic limb deformities are common in many avian species. Three young birds, including a six-week-old Cockatoo and two three-month-old goslings, were presented with tarsal joint deformities. They were treated with an experimental prototype of a hinged linear external fixator, placed in a transarticular fashion, in order to maintain joint function during treatment. All birds had close to normal leg function at six to ten weeks postoperatively. These results suggest that the hinged external fixator may be a viable treatment option for tarsal joint deformities in young birds.

Massive visceral pentastomiasis caused by Porocephalus crotali in a dog.

The testes of a 5-year-old, male, crossbred Schnauzer dog were the indicator organs for detection of massive pentastomiasis. Necropsy revealed numerous additional encysted parasites within the mesenteric lymph nodes, omentum, liver, sub-serosa of the small and large intestines, mesentery, and lungs. The nymphs had a pseudosegmented body, containing large eosinophilic glands and a chitinous cuticle with characteristic pores. Their hook configuration was consistent with that of Porocephalus. A pentastomid-specific 18S rRNA polymerase chain reaction (PCR) was designed and used to amplify template for sequencing. The sequence of the PCR product was 99.7% homologous with the reference sequence for P. crotali. This pentastomid parasite has been reported in North American snakes of genera Crotalus and Agkistrodon. Mammals are intermediate hosts, and snakes are the definitive hosts. Porocephalus crotali has been reported in dogs only once, and molecular methods have not been used previously to identify the species in clinical pentastomiasis.

Intranuclear coccidiosis in tortoises: nine cases.

Chelonian intranuclear coccidiosis has been reported once, in two radiated tortoises (Geochelone radiata), and is apparently rare. We describe intranuclear coccidiosis diagnosed histologically in two radiated tortoises, three Travancore tortoises (Indotestudo forstenii), two leopard tortoises (Geochelone pardalis), one bowsprit tortoise (Chersina angulata), and one impressed tortoise (Manouria impressa). Infection was systemic and involved alimentary, urogenital, respiratory, lymphoid, endocrine, and integumentary systems. Trophozoites, meronts, merozoites, macrogametocytes, microgametocytes, and nonsporulated oocysts were seen histologically or by electron microscopy. Intracytoplasmic and extracellular stages of parasite development also were identified histologically. Sequencing of a coccidial 18S rRNA consensus polymerase chain reaction (PCR) product revealed a novel sequence that provided phylogenetic information and may be useful for further diagnostic test design. Intranuclear coccidiosis was associated with variable degrees of inflammation in all cases, was considered the cause of death in six tortoises, and was a substantial contributing factor to the cause of death in two tortoises.

Mycoplasmosis in captive crows and robins from Minnesota.

Mycoplasma sturni is a recently described organism previously associated with conjunctivitis in European starlings (Sturnus vulgaris), northern mockingbirds (Mimus polyglottos) and blue jays (Cyanocitta cristata). Herein we describe the isolation of M. sturni from an American crow (Corvus brachyrhynchos) presenting with conjunctivitis. A nested-PCR was designed for identification of M. sturni in clinical specimens and the sensitivity of the reaction was found to be 10 colony-changing units. The organism was found in asymptomatic American crows caged with a nestmate of the crow with conjunctivitis. Mycoplasma sturni also was found in asymptomatic American robins (Turdus migratorius) and in a European starling (Sturnus vulgaris) housed at the same facility as the crows. Heterogenity of M. sturni isolates from different host species was found by random amplified polymorphic DNA (RAPD) analyses. Heterogeneity also was found among M. sturni isolates recovered from American crows. We suggest that M. sturni can successfully infect American crows and American robins with or without the presence of clinical disease. Furthermore, we demonstrate that nested-PCR is an effective method for the detection of M. sturni and that substantial genetic heterogeneity exists among natural isolates of this bacterial pathogen.

Diagnosis and treatment of conjunctivitis in house finches associated with mycoplasmosis in Minnesota.

An ongoing outbreak of Mycoplasma gallisepticum-associated conjunctivitis in house finches (Carpodacus mexicanus) that began in 1994 in the eastern United States has been spreading westward. House finches presenting with the clinical signs of M. gallisepticum-associated conjunctivitis were first seen at the Wildlife Rehabilitation Center of Minnesota (Minnesota, USA) in July of 1996, and 42 cases were admitted from 26 December 1996 to 10 August 1997. A nested PCR was designed for sensitive and specific detection of the presence of the organism. Twelve birds were treated with oral enrofloxacin (15 mg/kg, twice daily for 21 days) and ophthalmic gentamicin (twice daily for 21 days). All treated birds showed resolution of clinical signs. Following treatment, finches were held for up to 6 mo and tested for the presence of M. gallisepticum by culture and nested polymerase chain reaction (PCR). Eight of twelve finches (67%) were positive for M. gallisepticum by nested-PCR and four (33%) were positive by culture. The results suggest that oral enrofloxacin and opthalmic gentamicin are not an effective treatment for the eradication of M. gallisepticum in house finches. Further, the results show that nested PCR is an effective method for detection of M. gallisepticum in house finches and was more sensitive than culture.

Type I external skeletal fixation of radial fractures in microchiropterans.

An adult male big brown bat (Eptesicus fuscus) and an adult female hoary bat (Lasiurus cinereus) were presented with open transverse middiaphyseal left radial fractures. Initial repair was attempted by intramedullary pinning. When the fractures did not heal, intramedullary pins were removed and type I external skeletal fixators were placed. The fractures healed, and the big brown bat regained normal flight but the hoary bat did not.

Molecular epidemiology of Ornithobacterium rhinotracheale.

Ornithobacterium rhinotracheale is a recently described gram-negative rod-shaped bacterium associated with respiratory tract infections in poultry. In order to determine the molecular epidemiology of this bacterium, we characterized 55 O. rhinotracheale isolates from eight countries on four continents by multilocus enzyme electrophoresis (MLEE), repetitive sequence based-PCR (rep-PCR), and 16S rRNA gene sequencing. MLEE discriminated the O. rhinotracheale isolates into six electrophoretic types (ETs), of which only three ETs were recovered from domesticated poultry. The 16S rRNA gene sequence and rep-PCR analyses confirmed the results obtained by MLEE and indicated limited heterogeneity among isolates of O. rhinotracheale recovered from poultry. Taken together, the results of our analysis demonstrate that the majority of O. rhinotracheale isolates recovered from domesticated poultry throughout the world are represented by a small group of closely related clones and suggest that the bacterium was recently introduced to domesticated poultry from wild bird populations.

Overexpression of the D-alanine racemase gene confers resistance to D-cycloserine in Mycobacterium smegmatis.

D-Cycloserine is an effective second-line drug against Mycobacterium avium and Mycobacterium tuberculosis. To analyze the genetic determinants of D-cycloserine resistance in mycobacteria, a library of a resistant Mycobacterium smegmatis mutant was constructed. A resistant clone harboring a recombinant plasmid with a 3.1-kb insert that contained the glutamate decarboxylase (gadA) and D-alanine racemase (alrA) genes was identified. Subcloning experiments demonstrated that alrA was necessary and sufficient to confer a D-cycloserine resistance phenotype. The D-alanine racemase activities of wild-type and recombinant M. smegmatis strains were inhibited by D-cycloserine in a concentration-dependent manner. The D-cycloserine resistance phenotype in the recombinant clone was due to the overexpression of the wild-type alrA gene in a multicopy vector. Analysis of a spontaneous resistant mutant also demonstrated overproduction of wild-type AlrA enzyme. Nucleotide sequence analysis of the overproducing mutant revealed a single transversion (G-->T) at the alrA promoter, which resulted in elevated beta-galactosidase reporter gene expression. Furthermore, transformants of Mycobacterium intracellulare and Mycobacterium bovis BCG carrying the M. smegmatis wild-type alrA gene in a multicopy vector were resistant to D-cycloserine, suggesting that AlrA overproduction is a potential mechanism of D-cycloserine resistance in clinical isolates of M. tuberculosis and other pathogenic mycobacteria. In conclusion, these results show that one of the mechanisms of D-cycloserine resistance in M. smegmatis involves the overexpression of the alrA gene due to a promoter-up mutation.