Draft Genome Sequence of Aeromonas hydrophila Strain Ae25, Isolated from a Septicemic Moribund Koi Carp (Cyprinus carpio) in Sri Lanka.

Motile aeromonad septicemia caused by mesophilic strains of Aeromonas hydrophila is a widespread problem in cultured freshwater fish. We announce here the draft genome sequence of the multidrug-resistant A. hydrophila strain Ae25, isolated from a koi carp (Cyprinus carpio) with motile aeromonad septicemia that was collected from an ornamental fish-breeding farm in Sri Lanka.

Characterization and antimicrobial susceptibility of motile aeromonads isolated from freshwater ornamental fish showing signs of septicaemia.

A total of 74 phenotypically identified presumptive motile Aeromonas isolates recovered from septicaemic freshwater ornamental fish in Sri Lanka were genetically characterized by sequencing of rpoD and gyrB genes. rpoD/gyrB phylogeny confirmed only 53 isolates as Aeromonas, among which A. veronii was the predominant species (79.2%), followed by A. hydrophila (7.5%), A. caviae (5.7%), A. jandaei (1.9%), A. dhakensis (3.8%) and A. entero pelogenes (1.9%). The aeromonads confirmed by sequencing were further subjected to 16S rDNA PCR-RFLP which substantiated sequencing results for 83% of isolates. Fingerprinting of A. enteropelogenes (n = 42) using ERIC-PCR revealed no dominant clones, and the majority were genetically distinct. All isolates were screened by PCR for 7 virulence determinant genes (aer, act, ast, alt, fla, ser, exu) and 2 integrase encoding genes (intI1, intI2). Each isolate contained ≥3 of the virulence genes tested for, with a heterogeneous distribution. Of the isolates, 77% harboured the intI1 gene, while none had intI2. In vitro antimicrobial susceptibility testing showed highest resistances towards tetracycline (58.5%) and erythromycin (54.7%). Our results indicate the diverse range of aeromonads that could potentially be associated with motile aeromonad septicaemia in ornamental fish. This is the first isolation of A. dhakensis from a septicaemic ornamental fish since its original description from the same host.

Occurrence of nontuberculous mycobacteria in aquatic sources of Sri Lanka.

Nontuberculous mycobacteria (NTM) have been reported to cause opportunistic infections with increasing frequency, especially in immunocompromised patients. Water plays a major role in the epidemiology of nontuberculous mycobacterial infection in humans, as it is one of the natural sources for transmission of this group of organisms. The current study focused on determining the occurrence of NTM in different aquatic sources of Sri Lanka by using phenotypic tests and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the rpoß gene. Of 290 water samples, 45 (15%) were positive for NTM on culture. The percentage of mycobacteria identified at species level by phenotypic tests and PCR-RFLP analysis were 44% (20/45) and 73% (33/45), respectively. The frequency of isolation of mycobacteria from aquarium water, surface water, ground water and chlorinated water were 29% (20/70), 26% (20/76), 5% (4/76) and 1% (1/68), respectively. Eleven different NTM species were identified by PCR-RFLP analysis. M. fortuitum type I was the most frequently isolated species from all the four water sources. The current study suggests that water is an environmental source harboring NTM, a potential public health hazard especially for those with immunodeficiency.

Infectivity of Cryptosporidium parvum isolated from asymptomatic adult goats to mice and goat kids.

An experimental study was carried out in neonatal goat kids to examine the infectivity of Cryptosporidium oocysts, pattern of oocyst shedding and morphological changes in the intestine during the infection. Cryptosporidium oocysts isolated from adult asymptomatic goats, and identified as C. parvum by polymerase chain reaction (PCR) were used in this study. Of three 4-day-old goat kids, two were orally infected with C. parvum oocysts (10(5) oocysts in 10 ml PBS/kid). One goat kid given 10 ml PBS only by the oral route served as a control. Cryptosporidium oocysts were detected in the faeces of one infected kid on day 3 post-inoculation (pi) whereas in the other 6 days pi. The faecal oocyst counts gradually increased and the peak counts in the two kids were 2 x 10(6)g(-1) (on day 12 pi) and 3.2 x 10(6)g(-1) (on day 14 pi). The increase in faecal oocyst output coincided with diarrhoea in an infected kid from days 10-17 pi. Although the oocyst excretion declined gradually after the peak, both infected kids excreted oocysts until euthanized on days 20 and 22 pi. Light and scanning electron microscopic investigations of the ileum revealed the endogenous stages on the brush border of the enterocytes, infiltration of neutrophils and mononuclear cells into the lamina propria, atrophy, stunting and fusion of villi. For purposes of comparison, goat Cryptosporidium oocysts were inoculated orally (10(3) oocysts/mouse) to eight, 1-week-old mice. All experimental mice excreted oocysts from day 3 pi, and four infected mice continued to excrete oocysts up to day 42 pi. The experimental infection described in goat kids resembled the natural disease in terms of oocyst excretion, clinical signs and intestinal pathology. The ability of oocysts excreted by asymptomatic goats, to infect goat kids and mice is likely to have a major impact on the epidemiology of cryptosporidiosis in livestock and man.

Excretion of Cryptosporidium oocysts by goats in relation to age and season in the dry zone of Sri Lanka.

Pattern of Cryptosporidium oocyst shedding in relation to age and season was investigated monthly from May 1999 to April 2000 in three groups (24 goats per group) of naturally infected goats (from 1 month of age). The three groups designated 1, 2 and 3 were studied for 12, 6 and 3 months, respectively. An association between Cryptosporidium oocyst counts and age was demonstrated. In Group 1, oocyst excretion in the first, second and fourth months of age were significantly higher than that in 6, 7, 8, 9 and 12 months of age (p<0.01), whereas in Group 2, oocyst excretion in the first month of age was significantly higher than that in 2, 4, 5 and 6 months of age (p<0.01). The 3-month observations made in Group 3 showed high oocyst excretion during 1 and 3 months of age. The mean maximum count for Group 1 was 592 oocyst per gram of feces when the animals were 2 months old, while in Groups 2 and 3, this was observed at 3 months of age and the oocyst counts were 787 and 520, respectively. A close association between the prevalence of the Cryptosporidium infection and age of the animal was also observed (p<0.01). At least one-third of the Group 1 animals were excreting Cryptosporidium oocysts during the first 5 months of age. Goats excreted Cryptosporidium oocysts irrespective of the dry or rainy season. The long periods of Cryptosporidium oocyst shedding by goats may have implications for the epidemiology of the disease in susceptible hosts.

Prevalence of Cryptosporidium infection in goats in selected locations in three agroclimatic zones of Sri Lanka.

The prevalence of Cryptosporidium oocysts in the faeces of 1020 goats in three age categories was examined during 1999 in selected locations of three agroclimatic zones of Sri Lanka. The oocysts were demonstrated using the Sheather's sucrose flotation method followed by staining with the modified Ziehl Neelsen technique. Cryptosporidium oocysts were detected in animals from all agroclimatic zones with the highest prevalence of infection in the dry zone (33.6%) compared with 24.7 and 21.7% in the intermediate zones and wet, respectively (P<0.001). Overall, Cryptosporidium oocyst counts were significantly higher in goats of <6 months and 7-12 months of age groups compared with goats of >12 months of age (P<0.001). Cryptosporidium oocysts were detected in 291/1020 (28.5%) animals, while 194/1020 animals (19%), 84/1020 animals (8.2%) and 13/1020 animals (1.3%) excreted low (1-1000 oocysts per gram of faeces), moderate (1000-5000 oocysts per gram of faeces) and high (>5000 oocysts per gram of faeces) counts, respectively. The mean Cryptosporidium count was 383 oocysts per gram of faeces. The majority of the infected goats were asymptomatic. These animals are likely to play an important role in the epidemiology of cryptosporidiosis in goat kids and humans.

Biased immunoglobulin G1 isotype responses induced in cattle with DNA expressing msp1a of Anaplasma marginale.

Immunization with the native major surface protein 1 (MSP1) (a heterodimer containing disulfide and noncovalently bonded polypeptides designated MSP1a and MSP1b) of the erythrocytic stage of Anaplasma marginale conferred protection against homologous challenge (G. H. Palmer, A. F. Barbet, W. C. Davis, and T. C. McGuire, Science 231:1299-1302, 1986). The MSP1a polypeptide possesses a conserved neutralization-sensitive epitope. In the present study, the immune response to DNA-mediated immunization using msp1a was studied. The plasmid pVCL/MSP1a, which encodes the complete msp1a gene of A. marginale under the control of human cytomegalovirus immediate-early enhancer/promoter and intron A, was constructed. The immune responses elicited by immunization with pVCL/MSP1a into cardiotoxin-induced regenerating muscle were evaluated in mice and cattle. Antibody reactive with native MSP1a was detected in pooled sera of immunized BALB/c mice 3 weeks following primary immunization. Two calves seronegative for A. marginale were immunized four times, at weeks 0, 3, 7, and 13, with pVCL/MSP1a. By 8 weeks, both calves responded to MSP1a with an antibody titer of 1:100, which peaked at 1:1,600 and 1:800 by 16 weeks after the initial immunization. Interestingly, immunoblotting with anti-immunoglobulin G1 (anti-IgG1) and anti-IgG2 specific monoclonal antibodies revealed a restricted IgG1 anti-MSP1a response in both animals. T-lymphocyte lines, established after the fourth immunization, proliferated specifically against A. marginale homogenate and purified MSP1 in a dose-dependent manner. These data provide a basis for an immunization strategy to direct bovine immune responses by using DNA vaccine vectors containing single or multiple genes encoding major surface proteins of A. marginale.