Outbreak of Abortions and Infertility in Thoroughbred Mares Associated with Waterborne <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

At a thoroughbred equine breeding farm near Hissar (Haryana), three mares aborted in their seventh month of pregnancy. The vaginal swabs of all aborted mares, and stomach contents, heart blood, liver, spleen and placenta of aborted fetuses yielded pure culture of Aeromonas hydrophila. In addition, A. hydrophila was also isolated from the vaginal swabs of three repeat breeding mares and faecal sample of a diarrheic foal. The source of infection was possibly water supply as all the water samples collected from taps, mother tank and storage tank were found to be positive for A. hydrophila. The antibiogram of all the isolates was similar showing resistance to ampicillin, carbenicillin, gentamicin, kanamycin and amikacin but sensitive to chloramphenicol, ciprofloxacin, cefuroxime, ceftriaxone, cotrimoxazole, cotrimazine, nitrofurantoin, streptomycin and tetracycline. All the 20 sera samples collected from three aborted and three repeat breeding, and eight in-contact mares, a diarrheic foal, three cows and two male buffaloes maintained at the same farm contained antibodies against A. hydrophila with titres ranging from 80 to 640. The water supply was instantly chlorinated using 0.05% sodium hypochlorite for three consecutive days and all the culturally positive mares were treated with intravaginal administration of 1 g ciprofloxacin, while the foal was given nitrofurantoin for three days. After one month, A. hydrophila could not be isolated either from mares or from their environment and antibody titre in all the seropositive animals showed a declining trend. Later, all the aborted and repeat breeding mares were confirmed to be pregnant. Thus, the present study indicated that water-borne A. hydrophila might be associated with equine abortions and infertility, and diarrhea in newborn foals.     

Isolation, Enumeration, and Characterization of Aeromonas from Polluted Waters Encountered in Diving Operations

Counts of total viable, aerobic, heterotrophic bacteria, indicator organisms, and Aeromonas spp. were made at a diver training site on the Anacostia River in Washington, D.C. The numbers of Aeromonas cells in Anacostia River sediment and water increased during periods of elevated water temperature, to maxima of 4 × 10⁵ cells per g of sediment and 300 cells per ml of water. Correspondingly, Aeromonas counts dropped 2 to 4 logs as the water temperature decreased to 0 to 0.5°C. Cultures taken by sterile swabs from the ears and face masks of divers after a 30-min swim in the Anacostia River yielded bacterial types and numbers similar to those found in the river. The nasal passages of the divers apparently did not become contaminated by swimming, possibly because of the protective effect of the face masks used by the divers. Properties associated with virulence in Aeromonas hydrophila and Aeromonas sobria strains isolated from the river, sediment, and divers were investigated. Nearly 40% of the strains of both species collected during the study produced cytotoxic activity for mouse Y-1 adrenal cells, as well as elastase. Enterotoxin activity, as detected by the Y-1 assay, was observed in 3% (1 of 35) of the strains of A. sobria and in 6% (19 of 330) of the A. hydrophila strains. Fluid accumulation in rabbit ileal loops induced by both species of Aeromonas varied greatly among the 17 strains examined. Fluid accumulation of at least 0.4 ml/cm was correlated with positive cytotoxin- or enterotoxin-like response in the Y-1 tissue culture assay.     

Sequence Analysis of Amplified DNA Fragments Containing the Region Encoding the Putative Lipase Substrate-Binding Domain and Genotyping of Aeromonas hydrophila

DNA fragments were amplified by PCR from all tested strains of Aeromonas hydrophila, A. caviae, and A. sobria with primers designed based on sequence alignment of all lipase, phospholipase C, and phospholipase A1 genes and the cytotonic enterotoxin gene, all of which have been reported to have the consensus region of the putative lipase substrate-binding domain. All strains showed lipase activity, and all amplified DNA fragments contained a nucleotide sequence corresponding to the substrate-binding domain. Thirty-five distinct nucleotide sequence patterns and 15 distinct deduced amino acid sequence patterns were found in the amplified DNA fragments from 59 A. hydrophila strains. The deduced amino acid sequences of the amplified DNA fragments from A. caviae and A. sobria strains had distinctive amino acids, suggesting a species-specific sequence in each organism. Furthermore, the amino acid sequence patterns appear to differ between clinical and environmental isolates among A. hydrophila strains. Some strains whose nucleotide sequences were identical to one another in the amplified region showed an identical DNA fingerprinting pattern by repetitive extragenic palindromic sequence-PCR genotyping. These results suggest that A. hydrophila, and also A. caviae and A. sobria strains, have a gene encoding a protein with lipase activity. Homologs of the gene appear to be widely distributed in Aeromonas strains, probably associating with the evolutionary genetic difference between clinical and environmental isolates of A. hydrophila. Additionally, the distinctive nucleotide sequences of the genes could be attributed to the genotype of each strain, suggesting that their analysis may be helpful in elucidating the genetic heterogeneity of Aeromonas.     

Isolation and Partial Characterization of Two Aeromonas hydrophila Bacteriophages

Two Aeromonas hydrophila bacteriophages, Aeh1 and Aeh2, were isolated from sewage. Both phages showed binal symmetry. The dimensions of A. hydrophila phages Aeh1 and Aeh2 differed from those of the other Aeromonas phages. Also, phage Aeh2 was the largest Aeromonas phage studied to date. Phage Aeh1 formed small, clear plaques, and phage Aeh2 formed turbid plaques with clear centers. Both phages were sensitive to chloroform treatment, being totally inactivated after treatment for 1 h at 60°C at pH 3 and 11. However, the infectivity of Aeh1 phage stocks increased by approximately fivefold after they were treated at pH 10 for 1 h at 22°C. Phages Aeh1 and Aeh2 were serologically unrelated and had latent periods of 39 and 52 min, respectively. The average burst sizes of phages Aeh1 and Aeh2 were 17 and 92 PFU per cell, respectively. Phage Aeh1 infected 13 of 22 A. hydrophila strains tested, whereas phage Aeh2 infected only its original host. Phage Aeh1 infected some A. hydrophila strains only at or below 37°C. Neither phage infected the two A. (Plesiomonas) shigelloides strains used in this study.     

Natural Infection of Pregnant Cows with Schmallenberg Virus – A Follow-Up Study

Schmallenberg virus (SBV), an orthobunyavirus discovered in European livestock in late 2011 for the first time, causes premature or stillbirth and severe fetal malformation when cows and ewes are infected during pregnancy. Therefore, cattle of two holdings in the initially most affected area in Germany were closely monitored to describe the consequence for fetuses and newborn calves. Seventy-one calves whose mothers were naturally infected during the first five months of pregnancy were clinically, virologically, and serologically examined. One calve showed typical malformation, another one, born without visible abnormalities, was dead. Two cows aborted during the studied period; spleen and brain samples or meconium swabs were tested by real-time PCR, in none of the fetuses SBV-specific RNA was detectable and the tested fetal sera were negative in a commercially available antibody ELISA. In contrast, in nine clinically healthy calves high SBV-antibody titers were measurable before colostrum intake, and in meconium swabs of six of these animals viral RNA was present as well. The mothers of all nine seropositive calves were presumably infected between days 47 and 162 of gestation, which is within the critical timeframe for fetal infection suggested for SBV and related viruses.     

Epizootic abortion related to infections by Chlamydophila abortus and Chlamydophila pecorum in water buffalo (Bubalus bubalis)

Water buffalo (Bubalus bubalis) are affected by high rates of embryonic mortality and abortion related to infectious diseases and non-infectious factors. A number of viral and bacterial infections have been associated with reproductive failure, but there is limited information on the role of chlamydial infections.

In order to investigate the presence and the role of Chlamydiaceae in water buffalo a retrospective study was performed in a herd with a history of reproductive failure. During an 11-month period, the pregnant heifers suffered an abortion rate of 36.8% between the 3rd and 7th month of pregnancy. Antibodies to Chlamydiaceae were detected in 57% of the aborted cows, and in 0% of the overtly healthy cows used as control. By a nested-PCR assay, three of 14 vaginal swabs from aborted animals tested positive for Chlamydophila agents and, additionally, three out of seven aborted fetuses tested positive for Chlamydophila spp., with two being co-infections by Cp. abortus and Cp. pecorum and one being characterised as Cp. abortus. Sequence analysis of the amplicons confirmed the results of the nested-PCR. The presence of anti-Chlamydiaceae antibodies in more than half of the aborting animals (P < 0.002) and the detection of Chlamydophila agents in several fetal organs and in the vaginal swabs are consistent with the history of abortions observed in the herd and suggest an abortifacient role by Chlamydophila spp. in water buffalo (B. Bubalis) herds.     

Nonselectivity of Rimler-Shotts Medium for Aeromonas hydrophila in Estuarine Environments

Thirty presumptive Aeromonas hydrophila isolates were collected from estuarine sources using Rimler-Shotts media. Many of the isolates, especially the strains from a low-salinity site, were also identified as A. hydrophila using the API 20E system and two differential techniques devised for the identification of A. hydrophila. All of the isolates, however, had deoxyribonucleic acid base compositions with guanine-plus-cytosine ratios between 40 and 49 mol%, which excludes these strains from the genus Aeromonas.     

Results of a sensibility test on bacteria in developing countries

Humans, animals and drinking water were examined for pathogenic germs in Sudan, Colombia and the Cap Verde Island. Samples taken from humans mainly consisted of wound,--throat,-- and vaginal swabs plus stools. From animals we took feces resp. anal swabs. All isolated bacterial strains were examined for their reaction to various chemotherapeutics. Gentamicine showed the most comprehensive effects. Co-trimoxazole was second best.     

Toxoplasma gondii and Neospora caninum infections in goat abortions from Argentina

The aims of this study were to identify the occurrence of Toxoplasma gondii and Neospora caninum abortions in goats from Argentina by serological, macroscopical and microscopical examination and bioassay, and to characterize the obtained isolates by molecular techniques. For this purpose, 25 caprine fetal fluids, 18 caprine fetal brains and 10 caprine placentas from 8 dairy/meat goat farms from Argentina were analyzed. Gestational age of the aborted fetuses was determined in 18 cases. Protozoal infections were detected by at least one of the applied diagnostic techniques in 44% (11/25) of examined fetuses; specifically, 24% (6/25) were positive to T. gondii, 8% (2/25) were positive to N. caninum and 12% (3/25) were positive to both parasites. In this study IFAT titers were similarly distributed in younger and older fetuses. Macroscopical and microscopical examination of one placenta revealed chalky nodules in the fetal cotyledons and normal intercotyledonary areas, as well as necrosis and calcification of mesenchymal cells in villi. Tachyzoites were observed in peritoneal wash from 2 mice inoculated with brain and a pool of brain and placenta of two fetuses. Cell culture growth of tachyzoites was achieved from one inoculated mouse, and confirmed as T. gondii by PCR. The T. gondii isolate was identified as atypical or non-canonical by nested-PCR-RFLP. This is the first study that investigated the involvement of N. caninum and T. gondii in cases of goat abortion in Argentina.     

Behavior of an Aeromonas hydrophila aroA Live Vaccine in Water Microcosms

Genetically modified auxotrophic mutants of different fish pathogens have been used as live vaccines in laboratory experiments, but the behavior of the strains after release into aquatic ecosystems has not been characterized. We previously constructed and characterized an aroA mutant of Aeromonas hydrophila and studied the protection afforded by this mutant as a live vaccine in rainbow trout. In this work, we describe the survival of this strain in aquatic microcosms prepared from fish water tanks. The aroA mutant disappeared rapidly in nonfiltered, nonautoclaved fish tank water, declining below detection levels after 15 days, suggesting an inhibitory effect of the autochthonous microflora of the water. When the aroA strain was used to inoculate sterilized water, its culturability was lower than that of wild-type strain A. hydrophila AG2; after long periods of incubation, aroA cells were able to enter a viable but nonculturable state. Entry into this nonculturable state was accompanied by changes in the cell morphology from rods to spheres, but the cells appeared to remain potentially viable, as assessed by the preservation of cell membrane integrity. Supplementation of the culture medium with sodium pyruvate favored the culturability and resuscitation of the two A. hydrophila strains at low temperatures (6 and 16°C). These results contribute to a better understanding of the behavior of the aroA strain in natural environments and suggest that the inactivation of the aroA gene may be beneficial for the safety of this live vaccine for aquacultures.     

Enzyme-linked immunosorbent assay for detection of Aeromonas hydrophila serogroup O:19

An enzyme-linked immunosorbent assay has been developed for the detection of Aeromonas hydrophila serogroup O:19 isolated from epizootics in eels. The enzyme-linked immunosorbent assay specificity was confirmed after testing A. hydrophila O:19 and non-O:19 strains from different origins, as well as other Aeromonas species and other fish pathogens such as Vibrio vulnificus biotype 2, V. furnisii, V. damsela, Yersinia ruckerii and Edwardsiella tarda. The detection limits for A. hydrophila O:19 cells were around 10⁴–10⁵ cells/well. Artificially infected eels were analyzed and the immunodetection was confirmed by cultural methods. With this methodology A. hydrophila O:19 was successfully detected in infected eels and water samples. We described two subgroups within the serogroup O:19 (Guinée and Jansen system), one of them presents a 50 kDa outer membrane protein as a strong thermostable antigen which is not present in the other group.     

The effect of maternal nutrition level during the periconception period on fetal muscle development and plasma hormone concentrations in sheep

The effect of maternal nutrition level during the periconception period on the muscle development of fetus and maternal–fetal plasma hormone concentrations in sheep were examined. Estrus was synchronized in 55 Karayaka ewes and were either fed ad libitum (well-fed, WF, n=23) or 0.5×maintenance (under-fed, UF, n=32) 6 days before and 7 days after mating. Non-pregnant ewes (WF, n=13; UF, n=24) and ewes carrying twins (WF, n=1) and female (WF, n=1; UF, n=3) fetuses were removed from the experiment. The singleton male fetuses from well-fed (n=8) and under-fed (n=5) ewes were collected on day 90 of gestation and placental characteristics, fetal BWs and dimensions, fetal organs and muscles weights were recorded. Maternal (on day 7 after mating) and fetal (on day 90 of pregnancy) blood samples were collected to analyze plasma hormone concentrations. Placental characteristics, BW and dimensions, organs and muscles weights of fetuses were not affected by maternal feed intake during the periconception period. Maternal nutrition level did not affect fiber numbers and the muscle cross-sectional area of the fetal longissimus dorsi (LD), semitendinosus (ST) muscles, but the cross-sectional area of the secondary fibers in the fetal LD and ST muscles from the UF ewes were higher than those from the WF ewes (P<0.05). Also, the ratio of secondary to primary fibers in the ST muscle were tended to be lower in the fetuses from the UF ewes (P=0.07). Maternal nutrition level during the periconception period did not cause any significant changes in fetal plasma insulin and maternal and fetal plasma IGF-I, cortisol, progesterone, free T3 and T4 concentrations. However, maternal cortisol concentrations were lower while insulin concentrations were higher in the WF ewes than those in the UF ewes (P<0.05). These results indicate that the reduced maternal feed intake during the periconception period may alter muscle fiber diameter without affecting fiber types, fetal weights and organ developments and plasma hormone concentrations in the fetus.     

Pathogenic Aeromonas hydrophila Serogroup O:14 and O:81 Strains with an S Layer

Five autoagglutinating Aeromonas hydrophila isolates recovered from eels and humans were assigned to serogroups O:14 and O:81 of the Sakazaki and Shimada (National Institutes of Health) scheme. They had the following properties in common: positive precipitation after boiling, moderate surface hydrophobicity (salt-aggregation-test value around 1.2), pathogenicity for fish and mice (50% lethal dose, 10^4.61 to 10^7.11), lipopolysaccharides that contained O-polysaccharide chains of homogeneous chain length, and an external S layer peripheral to the cell wall observed by electron microscopy. A strong cross-reactivity was detected by immunoblotting between the homogeneous O-polysaccharide fraction of O:14 and O:81 strains but not between them and the lipopolysaccharide of A. hydrophila TF7 (O:11 reference strain). Outer membrane fractions of these strains contained a predominant 53- to 54-kDa protein which was glycine extractable under low-pH (pH 2.8) conditions and was identified as the surface array protein. The S-layer proteins of the O:14 and O:81 A. hydrophila strains seemed to be primarily different from those previously purified from strains A. hydrophila TF7 and Aeromonas salmonicida A450 on the basis of colony hybridizations with both the structural genes vapA and ahsA. This is the first report of the presence of an S layer in mesophilic Aeromonas strains not belonging to serogroup O:11.     

Antagonistic Activity of Cellular Components of Potential Probiotic Bacteria, Isolated from the Gut of Labeo rohita, Against Aeromonas hydrophila

The objective of this study was to characterise the antagonistic activity of cellular components of potential probiotic bacteria isolated from the gut of healthy rohu (Labeo rohita), a tropical freshwater fish, against the fish pathogen, Aeromonas hydrophila. Three potential probiotic strains (referred to as R1, R2, and R5) were screened using a well diffusion, and their antagonistic activity against A. hydrophila was determined. Biochemical tests and 16S rRNA gene analysis confirmed that R1, R2, and R5 were Lactobacillus plantarum VSG3, Pseudomonas aeruginosa VSG2, and Bacillus subtilis VSG1, respectively. Four different fractions of cellular components (i.e. the whole-cell product, heat-killed whole-cell product [HKWCP], intracellular product [ICP], and extracellular product) of these selected strains were effective in an in vitro sensitivity test against 6 A. hydrophila strains. Among the cellular components, the ICP of R1, HKWCP of R2, and ICP of R5 exhibited the strongest antagonistic activities, as evidenced by their inhibition zones. The antimicrobial compounds from these selected cellular components were partially purified by thin-layer and high-performance liquid chromatography, and their properties were analysed. The ranges of pH stability of the purified compounds were wide (3.0–10.0), and compounds were thermally stable up to 90 °C. Considering these results, isolated probiotic strains may find potential applications in the prevention and treatment of aquatic aeromonosis.     

Low Seroprevalence of Brucellosis in Humans and Small Ruminants in the Gambia

BackgroundBrucellosis is a worldwide zoonosis with significant impact on rural livelihoods and a potentially underestimated contributor to febrile illnesses. The aim of this study was to estimate the seroprevalence of brucellosis in humans and small ruminants in The Gambia.MethodsThe study was carried out in rural and urban areas. In 12 rural villages in Kiang West district, sera were collected from humans (n = 599) and small ruminants (n = 623) from the same compounds. From lactating small ruminants, milk samples and vaginal swabs were obtained. At the urban study sites, sera were collected from small ruminants (n = 500) from slaughterhouses and livestock markets. Information on possible risk factors for seropositivity was collected through questionnaires. Sera were screened for antibodies against Brucella spp. with the Rose Bengal Test, ELISA and Micro Agglutination Test (human sera only). PCR was performed on 10 percent of the milk samples and vaginal swabs from small ruminants.ResultsOne human and 14 sheep sera were positive by the Rose Bengal Test. The rest were negative in all serological tests used. The PCR results were all negative.ConclusionsThe results suggest that brucellosis is currently not a generalized problem in humans or small ruminants in The Gambia.     

Distribution and antibacterial drug resistance ofAeromonas spp. from fresh and brackish waters in Southern Turkey

The frequency of antibiotic resistance was compared inAeromonas spp. isolated from fresh and brackish water in Southern Turkey. A total of 97Aeromonas spp. strains were isolated from four zones (three from fresh and one from brackish water). Most of the strains isolated from all samples wereAeromonas hydrophila (79.4%), while the amount ofAeromonas sobria andAeromons caviae, were rather lower in the samples examined (17.5% and 3.1% respectively). A high proportion of isolates from all water sources showed resistance to cephalotin (86.6%) and trimethoprim-sulphamethoxazole (69%). On the other hand, a low proportion of bacteria showed resistance to tetracycline (14.4%), chloramphenicol (11.3%), gentamicin (7.2%) and nitrofurantoin (6.8%). Only one strain showing resistance to amikacin was found. Multiple Antibiotic Resistance Index (MARI) to at least two antibiotics was highest in brackish water (zone 4), followed by fresh water (zone 3). MARI values ranging from 0.2 to 0.8 for the bacteria isolated from brackish water. This study suggest that, multiple antibiotic resistantAeromonas spp., especiallyA. hydrophila, can be easily recovered from fresh and brackish water sources in Turkey and these sources may play as a reservoirs responsible for disease pathogen aeromonads.     

Analysis of the interaction of Aeromonas caviae, A. hydrophila and A. sobria with mucins

Aeromonas species are known to be involved in human gastrointestinal diseases. These organisms colonize the gastrointestinal tract. Aeromonas hydrophila, A. caviae, and A. sobria have been demonstrated microscopically to adhere to animal cell lines that express mucous receptors, but quantitative studies of adherence to mucosal components such as mucin have not been published to date. Purified bovine submaxillary gland, hog gastric mucin, and fish skin mucin were used as a model to study mucin-binding activity among A. caviae, A. hydrophila, and A. sobria strains. Our findings revealed that binding of radiolabeled and enzyme-conjugated mucins to Aeromonas cells varied depending on the labeling procedure. The highest binding was observed when the three mucin preparations were labeled with horseradish peroxidase. Binding of the various horseradish peroxidase-labeled mucins by A. caviae, A. hydrophila, and A. sobria cells is a common property among Aeromonas species isolated from human infections, diseased fish, and from environmental sources. The proportion of Aeromonas strains which bind the various horseradish peroxidase-labeled mucins was significantly higher for A. hydrophila than for A. caviae and A. sobria. Bacterial cell-surface extracts containing active mucin-binding components recognized the horseradish peroxidase-labeled mucins. The molecular masses of the mucin-binding proteins were estimated by SDS-PAGE and Western blot as follows: A. caviae strain A4812 (95 and 44 kDa); A. hydrophila strain 48748 (97, 45, 33 and 22 kDa); and A. sobria strain 48739 (95 and 43 kDa). Mucin interaction with Aeromonas cells was also studied in terms of growth in mucin-rich media. The culture conditions greatly influence the expression of A. hydrophila mucin-binding activity.     

Genomic study of polyhydroxyalkanoates producing Aeromonas hydrophila 4AK4

The complete genome of Gram-negative Aeromonas hydrophila 4AK4 that has been used for industrial production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) was sequenced and annotated. Its chromosome is 4,527,993 bp in size encoding 4,272 genes, including 28 rRNA genes and 104 tRNA genes. Comparative analysis indicated that genome of A. hydrophila 4AK4 was similar to that of the A. hydrophila ATCC 7966^T, an intensively studied aeromonad for its pathogenicity related to its genomic information. Genes possibly coming from other species or even other genus were identified in A. hydrophila 4AK4. A large number of putative virulent genes were predicted. However, a cytotonic enterotoxin (Ast) is absent in A. hydrophila 4AK4, allowing the industrial strain to be different from other A. hydrophila strains, indicating possible reduced virulence of strain 4AK4, which is very important for industrial fermentation. Genes involved in polyhydroxyalkanoate (PHA) metabolism were predicted and analyzed. The resulting genomic information is useful for improved production of PHA via metabolic engineering of A. hydrophila 4AK4.     

Protein fingerprinting profiles in different strains of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> isolated from diseased freshwater fish

Summary Aeromonas hydrophila(Ah) strains isolated from diseased fish in India were studied for protein profiling using the SDS-PAGE protein fingerprinting profile pattern of whole cells of 12 local strains of A. hydrophilaand one reference strain (MTCC 646). Variability among the strains was observed. The average similarity between the 12 strains of A. hydrophila ranged from 0.272 to 0.916. Proteins with molecular mass of 55.6 and 14.67 kDa in Ah1, Ah2 and Ah3, 28.5 and 27.9 kDa in Ah4, Ah5 and Ah6, 21.4 and 19.5 kDa in Ah7, Ah8, Ah9 and 72.9, 91.5 and 71.3 kDa in Ah10, Ah11 and Ah12 were common. The protein polypeptide bands from 19.5 to 86.2 kDa were common in both local strains and reference strain of A. hydrophila. The protein fingerprinting study showed that there is genetic similarity between strains of A. hydrophila and reference strain (MTCC 646). These protein markers may be useful for further strain differentiation in epidemiological study.