家养观赏地图鱼肺炎克雷伯氏菌、维氏气单胞菌与黏质沙雷氏菌的分离鉴定及耐药性分析

本试验旨在通过细菌分离鉴定,明确家养观赏地图鱼的死因,筛选敏感药物。采用常规方法分离纯化细菌后,进行细菌形态学观察,并通过小白鼠致病性试验、细菌主要生化鉴定、16SrDNA序列测定分析、药敏试验及耐药基因检测等方法对分离的细菌进行鉴定及耐药分析。分离出3株革兰氏阴性短杆菌,根据细菌形态特征及理化特性,结合16SrDNA序列测定与系统发育分析结果,判定其分别为肺炎克雷伯氏菌(Klebsiella pneumoniae)、维氏气单胞菌(Aeromonas veronii)和黏质沙雷氏菌(Serratia marcescens),其中肺炎克雷伯氏菌具有较强致病性。3株细菌均对洛美沙星、氧氟沙星、阿米卡星、卡那霉素敏感,对阿莫西林、氟苯尼考、克林霉素、甲硝唑等具有较强耐药性。结果表明,肺炎克雷伯氏菌、维氏气单胞菌、黏质沙雷氏菌的混合感染是家养观赏地图鱼的死亡原因。     

In vitro antagonistic activities of Lactobacillus spp. against Brachyspira hyodysenteriae and Brachyspira pilosicoli

The sensitivity of Brachyspira hyodysenteriae and Brachyspira pilosicoli, respectively the causative agents of Swine Dysentery and Porcine Intestinal Spirochaetosis to two probiotic Lactobacillus strains, L. rhamnosus CNCM-I-3698 and L. farciminis CNCM-I-3699 was studied through viability, motility and coaggregation assays. The cell-free supernatant of these lactobacilli contains lactic acid, that is stressful for Brachyspira (leading to the formation of spherical bodies), and lethal. It was demonstrated for the first time the in vitro coaggregation properties of two probiotic Lactobacillus strains (active or heat-treated) with two pathogenic strains of Brachyspira, leading to (1) trapping of spirochaetal cells in a physical network as demonstrated by SEM; (2) inhibition of the motility of Brachyspira. Such in vitro studies should encourage in vivo studies in animal model to evaluate the potential of the use of probiotic lactobacilli through a feeding strategy for the prevention of B. hyodysenteriae and B. pilosicoli.     

Phylogentic analysis of Anaplasma ovis strains isolated from sheep and goats using groEL and mps4 genes

Evidence of Anaplasma spp. in goats and sheep in Cyprus has been demonstrated by previous research. Herein, further research was performed for the identification of the exact Anaplasma spp. resulting in the identification of Anaplasma ovis strains in all samples examined. We used a bioinformatics as well as a molecular approach (study of groEl and mps4 genes) in order to verify the validity of the results. All samples depicted the presence of A. ovis regardless of the host (goat or sheep).     

The genome of Brucella melitensis

The genome of Brucella melitensis strain 16M was sequenced and contained 3,294,931 bp distributed over two circular chromosomes. Chromosome I was composed of 2,117,144 bp and chromosome II has 1,177,787 bp. A total of 3198 ORFs were predicted. The origins of replication of the chromosomes are similar to each other and to those of other α-proteobacteria. Housekeeping genes such as those that encode for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis were found on both chromosomes. Genes encoding adhesins, invasins, and hemolysins were also identified.     

Seroprevalence of antibodies to Encephalitozoon cuniculi and Encephalitozoon intestinalis in humans and animals

The presence of antibodies against Encephalitozoon cuniculi (E. cuniculi) and Encephalitozoon intestinalis (E. intestinalis) was examined in 215 samples from humans and in 488 samples from five different species of domestic and companion animals in Slovakia. The 215 human samples and samples from 90 swine, 123 non-infected cattle (cattle), 24 cattle infected with bovine leukosis virus (BLV-positive cattle), 140 sheep and 111 dogs were examined by the enzyme-linked immunosorbent assay (ELISA). Samples with serum titres 1:200 or higher were considered as positive. Specific anti-E. cuniculi antibodies were found in humans (0.9%), swine (52%), cattle (2%), sheep (9%) and dogs (15%) except for the BLV-positive cattle at the titre of 1:200. The titre of 1:400 was detected only in humans (0.5%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:200 was confirmed in humans (6%), swine (51%), cattle (11%), BLV-positive cattle (13%) and dogs (6%) but not in sheep. The anti-E. intestinalis antibodies reached the 1:400 in humans (1%), swine (4%) and BLV-positive cattle (17%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:600 was observed only in one swine (1%). Significant differences were observed in animals at titres 1:200 and 1:400 (chi-squared test: p < 0.0001) for both pathogens and in humans only for E. cuniculi at the titre of 1:400 (chi-squared test: p < 0.0075).     

Anaplasma marginale infection in a Japanese Black cow 13 years after eradication of Rhipicephalus (Boophilus) microplus in Okinawa, Japan

In October 2007, a 15-year-old Japanese Black cow on Ishigaki Island, Okinawa, Japan, was diagnosed with Anaplasma marginale infection based on clinical symptoms, blood examination, smear observation, 16S rRNA and groEL gene sequence analysis, and the result of a CF test. The cow was introduced into the farm from mainland Japan as a calf in 1993, one year before the eradication of Rhipicephalus (Boophilus) microplus, the main vector of A. marginale in Okinawa Prefecture. It is possible that the cow was first infected with A. marginale as a calf in Ishigaki Island and had been persistently infected since then. This is the first reported clinical case of A. marginale infection of cattle since the eradication of R. microplus in Okinawa Prefecture. Additional analysis of major surface protein 1α amino acid sequences revealed that the A. marginale Okinawa strain presented four new repeat forms which were not seen in other strains. This indicates that the Okinawa strain may be a unique geographical variant of A. marginale.     

Mycoplasma synoviae invades non-phagocytic chicken cells in vitro

Mycoplasma synoviae and Mycoplasma gallisepticum are major poultry pathogens, but their strains differ significantly in invasiveness and pathogenicity. Recent studies have demonstrated that M. gallisepticum invades chicken erythrocytes (CER) and chicken embryonic fibroblasts. The aim of this study was to determine whether M. synoviae also invades chicken cells. Using the gentamicin invasion assay, relative invasion frequency (RIF) of four M. synoviae strains was determined for CER, chicken embryonic cell line (CEC-32) and/or primary chicken chondrocytes (CCH). All tested strains of M. synoviae were capable of invading chicken cells within 24 h after infection. The type strain WVU 1853 showed significantly higher invasiveness in CER (RIF 7.5 ± 1.5%) and CEC-32 (RIF 7.0 ± 0.3%) than field strain ULB 02/T6 and M. gallisepticum strain R_low. Surprisingly, WVU 1853, which is capable of causing synovitis and arthritis in chickens, was less invasive for CCH with a RIF (1.2 ± 0.3%) similar to that of R_low (1.1 ± 0.1%). This is the first study documenting the invasiveness of M. synoviae strains for non-phagocytic chicken cells.     

Deletion of sodCI and spvBC in Salmonella enterica serovar Enteritidis reduced its virulence to the natural virulence of serovars Agona, Hadar and Infantis for mice but not for chickens early after infection

Salmonella enterica serovars Typhimurium, Enteritidis, Dublin, Choleraesuis or Gallinarum can colonise liver and spleen in particular hosts while infections with serovars Infantis, Agona, Hadar, etc. are usually limited to gastrointestinal tract. Reasons for this behavior are unknown, although it has been shown that sodCI and spv genes exhibit a strict distribution between more and less virulent serovars and they influence Salmonella virulence. However to what extent the presence or absence of these genes is associated with the increased virulence of serovars which possess them has never been addressed experimentally. In this study we therefore first confirmed the exclusive association of spvB and sodCI genes with the former group of serovars. In the next step we removed these two genes from S. Enteritidis genome and compared the virulence of such a mutant with the virulence of S. Infantis, S. Agona and S. Hadar for chickens and highly sensitive Balb/C mice. Single strain infection showed that the deletion of these two genes from S. Enteritidis resulted in the reduction of its virulence for mice but not for chickens. Mixed infection further confirmed these observations and indicated that in mice but not in chickens the virulence of sodCI and spv mutant was reduced to the natural virulence of serovars Infantis, Agona and Hadar. Although sodCI and spv genes do not influence S. Enteritidis virulence for chickens directly, they may be of an indirect effect through the increased persistence of S. Enteritidis in mice and increased probability of the reintroduction of S. Enteritidis into poultry flocks.     

Destruction of Anoplocephala perfoliata Eggs by the Nematophagous Fungus Pochonia chlamydosporia

The in vitro effect of an isolate of the nematophagous fungus Pochonia chlamydosporia (VC1) on the eggs of Anoplocephala perfoliata was evaluated. The eggs were morphologically analyzed for their integrity using light microscopy (10× objectives), plated on 9.0-cm diameter petri dishes containing 2% WA culture medium with and without fungal isolate (control), grown for 10 days, and 10 replicates were prepared per group. In all, 1000 eggs of A perfoliata were plated on petri dishes containing 2% water agar culture medium with (VC1) and without the fungal isolate (control). After 3, 5, 7, and 10 days, approximately 100 eggs were removed from each plate and classified on the basis of the following parameters: without alteration; type 1, lytic effect without morphological damage to eggshell; type 2, lytic effect with morphological alteration of embryo and eggshell; and type 3, lytic effect with morphological alteration of embryo and eggshell, in addition to hyphal penetration and internal egg colonization and destruction. The P chlamydosporia fungus demonstrated ovicidal activity (P < .05) on the eggs of A perfoliata in the studied intervals presenting type 3 effects of 35%, 42.5%, 53.83%, and 71.17% for the intervals 3, 5, 7, and 10 days, respectively. P chlamydosporia is a potential biological control agent for the eggs of A perfoliata.     

Effect of Staphylococcus aureus and Streptococcus uberis on apoptosis of bovine mammary gland lymphocytes

The aim of this study was to determine whether lymphocyte apoptosis is modulated by infections caused by Staphylococcus aureus and Streptococcus uberis. Samples of cell populations were obtained by lavage of the mammary glands at 4 intervals (24, 48, 72 and 168 h) following infection. The percentage of apoptotic lymphocytes peaked at 168 h after challenge with S. aureus or S. uberis. Subsequent experiments focused on in vitro cultivation of mammary gland lymphocytes with S. aureus and S. uberis. These experiments showed a lower percentage of apoptotic lymphocytes following 3 h of cultivating cells with bacteria than after cultivation without bacteria. The results demonstrate that during both experimental infection of bovine mammary glands with S. aureus or S. uberis and during in vitro cultivation of lymphocytes with S. aureus or S. uberis, apoptosis of lymphocytes is delayed.     

Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in sheep from Mossoró, Rio Grande do Norte, Brazil

Toxoplasma gondii is an obligate intracellular parasite with a variety of hosts, responsible for reproductive problems and economic losses in sheep flocks. Neospora caninum was recently identified and its clinical presentation in sheep is similar to that of toxoplasmosis, which can cause repeated abortions, though less frequently in this species. In order to confirm the prevalence of these agents in the city of Mossoró, Rio Grande do Norte, Brazil, 409 serum samples from adult sheep (364 females and 45 males) were tested by the indirect immunofluorescence antibody test, using cut-off point at a dilution of 1:64 and 1:50 for T. gondii and N. caninum, respectively. From the 35 properties examined, 23 (65.7%) had at least one seropositive animal for T. gondii and six (17.1%) for N. caninum. The prevalence of seropositive animals for T. gondii was 20.7% and for N. caninum 1.8%. There was no association between the presence of the agent’s antibody and gender, reports of reproductive problems and presence of dogs and/or cats in the properties. T. gondii is well distributed and N. caninum has low prevalence in sheep and in the properties of the studied region.     

Seeking a niche: putative contributions of the hfq and bacA gene products to the successful adaptation of the brucellae to their intracellular home

Long-term residence of the brucellae in the phagosomal compartment of host macrophages is essential to their ability to produce disease in both natural and experimental hosts. Correspondingly, the Brucella spp. appear to be well adapted to resist the multiple environmental stresses they encounter in their intracellular home. This brief review will focus on the contributions of the hfq and bacA gene products to this adaptation. Studies with Brucella hfq mutants suggest that stationary phase physiology is critical for successful long-term residence in host macrophages. Analysis of Brucella bacA mutants, on the other hand, reveal very striking parallels between the strategies employed by the rhizobia to establish and maintain protracted intracellular residence in their plant host and those used by the brucellae during their long-term survival in the phagosomal compartment of host macrophages.     

First isolation and identification of Elizabethkingia meningoseptica from cultured tiger frog, Rana tigerina rugulosa

Elizabethkingia meningoseptica has been recognised as an occasional but serious opportunistic bacterial pathogen to human beings. Recently, it was frequently isolated from tiger frog, Rana tigerina rugulosa, with cataract disease, which is the most common disease of unknown aetiology of frogs in Hainan, China. The purpose of this study was to identify and characterise the bacterial strains isolated from the recent outbreaks of cataract disease in farmed tiger frog in Hainan, China, and to evaluate their pathogenicity to the frog and their sensitivity to 20 chemotherapeutic agents.The 16S rRNA gene sequences of strains W0701 (1478 bp), W0702 (1477 bp) and W0703 (1478 bp) showed 98.6–98.7% similarity with the sequence of E. meningoseptica type strain (ATCC 13253) and 99.9–100% similarity with that of E. meningoseptica NTU 870424-IL. Six strains (W0701–W0706) were selected to represent 24 isolates retrieved from six moribund frogs. The morphological, physiological and biochemical characteristics of the six representative isolates were consistent with those of E. meningoseptica strains. The organisms were only susceptible to vancomycin and moderately susceptible to cefoperazone among the 20 investigated chemotherapeutic agents. Virulence test with strain W0702 was conducted and pathogenicity (by intramuscular injection) was demonstrated in the tiger frog. In conclusion, 24 isolates obtained from frogs with cataract disease were the E. meningoseptica strains highly pathogenic to tiger frog, and this is the first report of E. meningoseptica as a pathogen for tiger frog.     

The first report of Gyrodactylus salaris Malmberg, 1957 (Platyhelminthes, Monogenea) on Italian cultured stocks of rainbow trout (Oncorhynchus mykiss Walbaum)

The monogenean Gyrodactylus salaris Malmberg, 1957 is considered one of the most important parasites of wild salmonids in the European Community due to the heavy ecological and economical damage it has inflicted on Atlantic salmon (Salmo salar) parr populations. Rainbow trout (Oncorhynchus mykiss) is susceptible to G. salaris and can act as a suitable carrier host and, consequently, its trade in EU territory is restricted in relation to the status of “recognized free” zones. Despite the economic importance of rainbow trout farming in Italy, information on the Italian gyrodactylid fauna is lacking and prior to this study, G. salaris had not been officially reported. During a routine health examination of farmed rainbow trout stock throughout Central and Northern Italy in 2004–2005, five fish farms were found to be infected with G. salaris alongside three other gyrodactylids. Morphological and molecular characterisation confirmed the presence of G. salaris, Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel et Vigneulle, 1999 and Gyrodactylus derjavinoides Malmberg, Collins, Cunningham et Jalali, 2007, while Gyrodactylus truttae Gläser, 1974 was identified by morphological analysis only. The findings from this study extend the distribution of G. salaris within Europe and highlight the importance of the rainbow trout trade in its dissemination.     

In vitro antiviral activity of chestnut and quebracho woods extracts against avian reovirus and metapneumovirus

Field evidences have suggested that a natural extract, containing tannins, could be effective against poultry enteric viral infections. Moreover previous studies have shown that vegetable tannins can have antiviral activity against human viruses. Based on this knowledge three different Chestnut (Castanea spp.) wood extracts and one Quebracho (Schinopsis spp.) wood extract, all containing tannins and currently used in the animal feed industry, were tested for in vitro antiviral activity against avian reovirus (ARV) and avian metapneumovirus (AMPV). The MTT assay was used to evaluate the 50% cytotoxic compounds concentration (CC₅₀) on Vero cells. The antiviral properties were tested before and after the adsorption of the viruses to Vero cells. Antiviral activities were expressed as IC₅₀ (concentration required to inhibit 50% of viral cytopathic effect). CC₅₀s of tested compounds were >200 μg/ml. All compounds had an extracellular antiviral effect against both ARV and AMPV with IC₅₀ values ranging from 25 to 66 μg/ml. Quebracho extract had also evident intracellular anti-ARV activity (IC₅₀ 24 μg/ml). These preliminary results suggest that the examined vegetable extracts might be good candidates in the control of some avian virus infections. Nevertheless further in vivo experiments are required to confirm these findings.     

Effects of Teladorsagia (Ostertagia) circumcincta infection on lambs selected for high fleece weight

The physiological processes leading to the expression of the resilient phenotype, which allow animals to maintain a relatively higher production level during infection, have been investigated in lambs from a closed flock selected for 40 generations for high fleece weight (HFW), but with higher FEC and worm burdens than their unselected control (C) flock run in parallel. After recovery from surgery to implant abomasal cannulae, eight parasite-naïve lambs from each flock were infected intraruminally at 4.5 months-of-age with 50,000 Teladorsagia circumcincta L3. Blood, abomasal fluid and faecal samples were collected daily for measurement of serum gastrin and pepsinogen concentrations, blood eosinophils, abomasal pH and FEC. Four lambs from each flock were euthanased on Day 8 post-infection and the other four on Day 28 post-infection. At necropsy, abomasal contents and tissues were collected for worm counts, abomasal lymph nodes and fundic tissue for cytokine gene expression and fundic tissue for histopathology. Expression of resilience appeared to be age-dependent as there were no significant differences in either FEC or worm burden between lambs from the two flocks, unlike older HFW lambs in a previous study. Abomasal secretion did not differ between flocks. Histopathological changes were typical of parasitism: inflammatory cells, mainly eosinophils and lymphocytes, were numerous in nodular areas and there were fewer TGF-α positive parietal cells, many of which were vacuolated. By Day 28 p.i., globule leucocytes were present. Mucosal thickness was significantly greater on Day 8 than Day 28 p.i. (p = 0.000) and in C than HFW lambs. There were fewer parietal cells on Day 28 than on Day 8 p.i. (p = 0.003) for pooled data. Circulating eosinophil counts increased moderately in both groups, significantly less in the HFW lambs. Fewer tissue and blood eosinophils in the HFW than C group on Day 8 p.i. were consistent with cytokine gene expression patterns, particularly lower IL-5 levels. Worm count decreased by 90% by Day 28 p.i., along with declining tissue eosinophil counts and IL-13 gene expression and increasing IL-10 and IL-4 gene expression. Food intake was depressed less in the HFW lambs, suggesting that maintenance of appetite could be an important aspect of the physiological basis for resilience. Although the resilient phenotype was not apparent at the younger age, lesser effects on food intake, differences in ALN cytokine profiles and lower blood and tissue eosinophil numbers in the HFW lambs may lead to the expression of resilience when older.     

Antimicrobial susceptibility of Staphylococcus intermedius and Staphylococcus schleiferi isolated from dogs

The susceptibility to 23 antimicrobial agents was determined in 114 isolates of Staphylococcus intermedius and eight isolates of Staphylococcus schleiferi of canine origin. Overall, 73% of S. intermedius isolates and 37.5% of S. schleiferi isolates were susceptible to all the 23 antimicrobials tested. The large majority of S. intermedius strains retained susceptibility to antimicrobials currently employed in treatment of pyoderma (cephalosporins, cotrimoxazole and association amoxicillin–clavulanic acid) as well as to those effective against staphylococci (fusidic acid, rifampicin and fluoroquinolones). Resistance in S. intermedius was observed mainly against macrolides, chloramphenicol and lincosamides, while S. schleiferi isolates retained susceptibility to all antimicrobials except three of six fluoroquinolones. Although, our results confirm susceptibility to antimicrobials currently employed in pyoderma treatment, the several different resistance patterns observed for S. intermedius emphasize the importance of antimicrobial susceptibility testing of canine staphylococci to choose the most appropriate treatment of infections and to allow the prudent use of antimicrobial drugs in companion animals.     

Prevalence and molecular characterization of bovine Cryptosporidium in Qazvin province, Iran

The aim of this study was to determine the prevalence, variability with host age, and the genotypes of species of Cryptosporidium in cattle from 15 dairy farms in Qazvin province, Iran. Fecal samples, collected from 272 cattle during May 2006 to December 2007, were characterized microscopically. Oocysts from 51 positive samples were analyzed using PCR assay of 18S SSU rRNA, restriction fragment length polymorphism (RFLP) and sequencing. We identified 72.6% of the positive samples as Cryptosporidium parvum, 17.7% as Cryptosporidium andersoni, 7.8% as Cryptosporidium bovis and 1.9% as a novel genotype of C. parvum possessing a single mutation on MboII restriction. An infection rate of 19.5% of C. parvum among 174 pre-weaned calves was significantly higher than the 3.1% among 98 post-weaned calves (P < 0.0006). This is the first report of C. bovis and the new subgenotype of C. parvum in Iranian cattle.     

Fast detection of Vibrio species potentially pathogenic for mollusc

Vibrio tasmaniensis, Vibrio splendidus and Vibrio neptunius species were distributed worldwide and associated with aquaculture and have been reported as the cause of diseases in aquatic organisms. Polyphasic analyses for bacterial identification are not feasible for routine diagnostic because of the time involved. The aim of this study is to design three PCR primer sets that can assist with fast detection of these species. They were designed from the 16S ribosomal RNA gene, and PCR conditions were found. Each PCR test successfully identified all the tested strains of each target species. The combined specificity of V. tasmaniensis and V. splendidus primer sets offered the best coverage (86%) in terms of separating target organisms from other related species. The primer set of V. tasmaniensis showed a lower sensitivity limit (500 fg of DNA) than the V. splendidus set (1 pg) and both sets gave positive amplification using homogenized tissues from inoculated clams, with 10² and 10⁴ cfu/g of clam, respectively. The primer set of V. neptunius was highly specific, showing only cross-reaction with V. parahaemolyticus species from 44 tested species. Its sensitivity limit was 100 pg of DNA. A small number of biochemical tests were proposed concurrently with the PCR to differentiate the cross-reacting bacteria. The time of detection of the three tested species was reduced and the further affected animals can be diagnosed in a rapid fraction of time. The detection of virulent strains of V. tasmaniensis pointed to the risk of mollusc culture outbreaks.     

Detection of Fusobacterium necrophorum and Dichelobacter nodosus in lame cattle on dairy farms in New Zealand

Lameness in the dairy industry in New Zealand causes a problem in lost production, animal welfare and associated costs. To understand what bacteria may be present on the hooves of lame dairy cattle in this grass-fed system, samples were scraped from lame dairy cows and examined for the presence of Fusobacterium necrophorum (F. necrophorum) and Dichelobacter nodosus (D. nodosus) using the polymerase chain reaction (PCR). The PCR primers were designed to detect the presence of the lktA gene, which encodes a leukotoxin unique to F. necrophorum, and the fimA gene of D. nodosus. A total of 148 hoof scrapings were collected by farm staff over the period September 2005 to May 2006. F. necrophorum was detected in 79/148 of the samples, while D. nodosus was detected in 7/148 of the samples. The frequent finding of F. necrophorum within dairy herds in New Zealand is noteworthy and the occasional finding of D. nodosus on some dairy cattle suggests a possible role in both ovine and bovine hoof pathology.