West Nile Virus in Farmed Alligators

Seven alligators were submitted to the Tifton Veterinary Diagnostic and Investigational Laboratory for necropsy during two epizootics in the fall of 2001 and 2002. The alligators were raised in temperature-controlled buildings and fed a diet of horsemeat supplemented with vitamins and minerals. Histologic findings in the juvenile alligators were multiorgan necrosis, heterophilic granulomas, and heterophilic perivasculitis and were most indicative of septicemia or bacteremia. Histologic findings in a hatchling alligator were random foci of necrosis in multiple organs and mononuclear perivascular encephalitis, indicative of a viral cause. West Nile virus was isolated from submissions in 2002. Reverse transcription-polymerase chain reaction (RT-PCR) results on all submitted case samples were positive for West Nile virus for one of four cases associated with the 2001 epizootic and three of three cases associated with the 2002 epizootic. RT-PCR analysis was positive for West Nile virus in the horsemeat collected during the 2002 outbreak but negative in the horsemeat collected after the outbreak

Taxonomic and Functional Metagenomic Profile of Sediment From a Commercial Catfish Pond in Mississippi

Metagenomic analyses of microbial communities from aquatic sediments are relatively few, and there are no reported metagenomic studies on sediment from inland ponds used for aquaculture. Catfish ponds in the southeastern U.S. are eutrophic systems. They are fertilized to enhance algae growth and encourage natural food production, and catfish are fed with commercial feed from spring to fall. As result, catfish pond sediment (CPS) contains a very dense, diverse microbial community that has significant effects on the physiochemical parameters of pond dynamics. Here we conducted an in-depth metagenomic analysis of the taxonomic and metabolic capabilities of a catfish pond sediment microbiome from a southeastern U.S. aquaculture farm in Mississippi using Illumina next-generation sequencing. A total of 3.3 Gbp of sequence was obtained, 25,491,518 of which encoded predicted protein features. The pond sediment was dominated by Proteobacteria sequences, followed by Bacteroidetes, Firmicutes, Chloroflexi, and Actinobacteria. Enzyme pathways for methane metabolism/methanogenesis, denitrification, and sulfate reduction appeared nearly complete in the pond sediment metagenome profile. In particular, a large number of Deltaproteobacteria sequences and genes encoding anaerobic functional enzymes were found. This is the first study to characterize a catfish pond sediment microbiome, and it is expected to be useful for characterizing specific changes in microbial flora in response to production practices. It will also provide insight into the taxonomic diversity and metabolic capabilities of microbial communities in aquaculture. Furthermore, comparison with other environments (i.e., river and marine sediments) will reveal habitat-specific characteristics and adaptations caused by differences in nutrients, vegetation, and environmental stresses

Avaliação do emprego de cartões FTA para o transporte do DNA de Pasteurella multocida e pesquisa de genes associados à virulência em cepas isoladas de cólera aviária nos Estados Unidos

Fowl Cholera (FC) is a disease caused by Pasteurella multocida. The severity of this disease is partly caused by virulence factors. Genes encoding fimbriae, capsule, sialidases and proteins for iron metabolism may be related to P. multocida’s ability to infect the host. Besides to examining DNA for the presence of virulence genes, DNA is essential for the diagnostic and FTA cards are an alternative for genetic material transport. The study aims to evaluate the viability of P. multocida DNA transport using the cards and to detect 14 virulence genes in 27 strains isolated from FC cases in the United States by multiplex-PCR. No growth was observed in any of the FTA cards, which was essential to assess the security. Furthermore, DNA detection was possible in 100% of the samples, independent of the storage period (7 to 35 days) and temperature (4°C and 37°C). ptfA, exbd-tonB, hgbA, nanB, oma87, hyaD-hyaC, sodC, hgbB, sodA, nanH and pfhA genes were detected in more than 80% of the samples. FTA cards have proven to be a viable and safe tool for DNA transport of P. multocida. A majority of genes showed a high frequency, which was similar to strains isolated from FC cases.Cólera aviária (CA) é uma doença causada pela bactéria Pasteurella multocida e a severidade dos casos é em parte justificada por fatores de virulência. Genes codificando fímbrias, cápsulas, sialidases, dismutases e proteínas do metabolismo férrico podem ser relacionados à capacidade do agente em infectar o hospedeiro. Além da obtenção do DNA para pesquisa de genes de virulência, o material genético é fundamental para o diagnóstico, e os cartões FTA seriam uma alternativa no transporte de microrganismos. Os objetivos da presente pesquisa foram avaliar a viabilidade do transporte de DNA de P. multocida através dos cartões e detectar 14 genes de virulência em 27 cepas isoladas de CA nos Estados Unidos, por meio de multiplex-PCR. Nenhuma das amostras para análise microbiológica da segurança dos cartões apresentou crescimento. Foi possível a detecção do DNA em 100% das amostras, independentemente do tempo de estocagem (sete a 35 dias) e das temperaturas (4°C e 37°C) avaliadas. Genes ptfA, exbd-tonB, hgbA, nanB, oma87, hyaD-hyaC, sodC, hgbB, sodA, nanH e pfhA foram detectados em mais de 80% das amostras. Os cartões FTA demonstraram ser uma ferramenta viável e segura para o transporte do DNA de P. multocida. A maioria dos genes apresentou uma alta frequência, compatível com isolados de CA

Genetic Variants of Ehrlichia phagocytophila1, Rhode Island and Connecticut

Primers were used to amplify a 561-bp region of the 16S rRNA gene of Ehrlichia phagocytophila from Ixodes scapularis ticks and small mammals collected in Rhode Island and Connecticut. DNA sequences for all 50 E. phagocytophila-positive samples collected from 1996 through 1998 in southwestern Connecticut were identical to the sequence reported for E. phagocytophila DNA from confirmed human cases. In contrast, the sequences from 92 of 123 E. phagocytophila-positive Rhode Island samples collected from 1996 through 1999 included several variants differing by 1-2 nucleotides from that in the agent infecting humans. While 11.9% of 67 E. phagocytophila-positive ticks collected during 1997 in Rhode Island harbored ehrlichiae with sequences identical to that of the human agent, 79.1% had a variant sequence not previously described. The low incidence of human ehrlichiosis in Rhode Island may in part result from interference by these variant ehrlichiae with maintenance and transmission of the true agent of human disease

Plasma Dynamics

Contains reports on seventeen research projects split into two sections.National Science Foundation (Grant ENG77-00340)U. S. Energy Research and Development Administration (Contract E(11-1)-2766)U. S. Energy Research and Development Administration (Contract EY-76-S-02-2766)U. S. Air Force - Office of Scientific Research (Grant AFOSR-77-3143)U. S. Department of Energy (Grant EG-77-G-01-4107

The Rickettsia: an Emerging Group of Pathogens in Fish

Piscirickettsia salmonis is the first of the previously unrecognized rickettsial pathogens of fish to be fully characterized. Since the recognition of P. salmonis in 1989, the impact of rickettsial pathogens in fish has become increasingly apparent. Growing awareness of the emergence of these fastidious intracellular organisms has led to the discovery of rickettsial diseases among diverse species of fish from different geographic locations and aquatic environments. The source, reservoir, and mode of transmission of these agents as well as appropriate methods of disease prevention and control remain to be established

Polymerase chain reaction detection efficiency of the human granulocytic ehrlichiosis agent (Rickettsiaceae: Ehrlichieae) in ticks (Acari: Ixodidae) is dependent on the DNA extraction method

Several methods of extracting DNA from ticks were examined to improve the efficiency of polymerase chain reaction (PCR) detection of the human granulocytic ehrlichiosis (HGE) agent. DNA was extracted from laboratory-reared uninfected and HGE-infected ticks using 3 separate methods. In one treatment, unfed nymphs and engorged larvae of Ixodes scapularis Say, either individually or in pools of 3, were homogenized in 40 μl of 1x PCR buffer and boiled for 30 min. A 2nd group of ticks was extracted using the QiaAmp Tissue kit, a silica column separation method. A 3rd group was extracted with DNA-STAT, a guanidinium thiocynate method. Five microliters of each extract was used for PCR amplification. Pathogen-free tick DNA samples did not amplify a product. Laboratory-infected ticks extracted either with the QiaAmp kit or those homogenized and boiled in PCR buffer amplified product in 37.5% and 87.5% of the samples, respectively. Infected ticks extracted with DNA STAT-60 amplified a product in 100% of samples. No differences were observed in detection efficiency between ticks tested singly or in pools