The etiology of foul brood]

Five hundred and thirty-six samples of honeycombs were examined in a laboratory in the years 1971-1974. In all the samples clinically determined as the foul brood, B. alvei was isolated as a pure culture, and enterococci, or both microorganisms were isolated in mixed form. Twenty-five strains of the isolated streptococci were analyzed microbiologically and biochemically; on the basis of their culture and biochemical characteristics five strains were designated as Streptococcus faecalis, 14 strains as Streptococcus faecalis var. liquefaciens, five strains as Streptococcus faecium and one strain as Streptococcus durans. After checking the used taxonomic key of the culture and biochemical classification of B. alvei it may be stated that the culture and biochemical characteristics are stable. The strains of B. alvei (very dried strains), which persisted in the dried slant meat-peptone agar, were viable under the laboratory conditions, which proves the high resistance of the spores to the environment.     

东方蜜蜂抗欧洲幼虫腐臭病的实验研究

选择 5群健康的云南东方蜜蜂蜂群 ,每群有 5脾蜂 ,分别引入受欧洲幼虫腐臭病感染的幼虫脾 ,以后每隔2 4h检查蜂群内的幼虫脾 ,统计被东方蜜蜂清理的受感染幼虫数 ,患欧洲幼虫腐臭病的幼虫数 ,以及这些幼虫受感染后被工蜂清除的情况。另外 ,选择 3群群势健康的西方蜜蜂蜂群 ,每群有 5脾蜂 ,分别引入受欧洲幼虫腐臭病感染的幼虫脾做对比实验。研究结果表明 :云南东方蜜蜂有很强的抗欧洲幼虫腐臭病的特性 ,4天后 ,基本上可以清除原引入的西方蜜蜂幼虫脾上被欧洲幼虫腐臭病感染的幼虫。 8天后 ,在东方蜜蜂幼虫脾上也发现有少量的幼虫被欧洲幼虫腐臭病感染 ,但在其后的 5天内 ,所有东方蜜蜂幼虫脾上染病幼虫均被清理 ,5群东方蜜蜂均保持健康状况。而做对比群的西方蜜蜂在 5天后发现其幼虫被欧洲幼虫腐臭病感染 ,此后疫情不断加重 ,并出现“爬蜂病” ,约 30天后实验对比群消亡。     

健康羚牛肠道芽孢杆菌的分离与鉴定

为了研究羚牛肠道微生态环境,试验对健康羚牛新鲜粪便进行了细菌培养和鉴定。结果表明:共分离出3株革兰阳性长杆菌,均具有芽孢;其中2株菌为兼性厌氧菌,1株为需氧菌,都可在pH值为5.7的培养基和7%NaCl培养基中生长,但对糖和盐的利用具有特殊性;通过细菌形态观察和生化试验最终确定这3株菌依次为苛求芽孢杆菌、巨大芽孢杆菌和栗褐芽孢杆菌。     

In vitro pre-exposure of Haemonchus contortus L3 to blood eosinophils reduces their establishment potential in sheep

Different authors have reported that eosinophils are capable of immobilising infective larvae of different species of nematodes in vitro. However, classifying larvae as mobile or immobile is so subjective that it does not always mean all apparently immobile larvae are dead or those that are mobile are capable of surviving further immune responses if administered to their natural hosts. The objective of this experimental study was therefore to substantiate the role of eosinophils in the killing of Haemonchus contortus infective larvae by comparing the infectivity in sheep of larvae that had been incubated with eosinophil-enriched cell suspensions with control larvae. Since it was not possible to isolate pure eosinophils from sheep blood, we were compelled to evaluate the effects of other blood cells contaminating our eosinophil-enriched suspensions. Although eosinophils and neutrophils were the only cells found adherent to H. contortus infective larvae in vitro, induced eosinophils in the presence of immune serum were primarily responsible for the drastic reduction in larval motility compared to the minor effects of neutrophils and mononuclear cells. Corresponding reductions in faecal egg count and worm numbers were observed when the incubated larvae were transferred intra-abomasally to sheep. Interestingly, the proportion of larvae that failed to establish was much higher following incubation with induced eosinophils compared with other cells or with immune serum alone. Although this study did not address the in vivo role of eosinophils in sheep, the results strongly indicate that sheep blood eosinophils have a larval killing potential in vitro, and a larval mobility test alone may not fully explain the level of damage inflicted on the larvae.     

Larval biology of rhipicephalus (Boophilus) decoloratus (Acarina: Ixodidae) in Free State Province, South Africa

The objective of this study was to determine certain aspects of the biology of Rhipicephalus (Boophilus) decoloratus larvae under laboratory and field conditions. Larvae allowed 48 h to select a vertical questing substrate preferred 90 cm rods in length to those of 60 or 30 cm, while in a separate experiment migration from rods 5 cm or 25 cm in length to rods 45 cm in length continued between 48 h and 72 h after larval release. Hatching of the larval progeny of engorged female ticks exposed to ambient field temperatures during the period June to August, occurred synchronously during the third or fourth week of November. With a single exception, larvae that hatched during November and between April and July survived for 38 days or longer, while those that hatched from December to March survived for 31 days or less. Questing larvae were present on vegetation throughout the year, with most being recovered during January and February. Parasitic larvae were present on cattle from October to May with most being collected during January and February.     

Detection of Babesia bigemina infection in strains of Rhipicephalus (Boophilus) microplus collected from outbreaks in south Texas

The sudden death of several cattle infested experimentally with Rhipicephalus (Boophilus) microplus led to a clinical investigation into the reasons for the unexpected mortality. Microscopic evidence for Babesia bigemina infection was found in blood smears from the affected animals and a PCR assay was designed to detect the presence of B. bigemina and Babesia bovis in all R. microplus strains received and propagated at the laboratory. The assay utilizes a nested PCR approach with the first PCR amplifying a well-conserved segment from the Babesia 18S ribosomal RNA gene followed by a nested PCR with Babesia species-specific primers and annealing temperatures enabling amplification of the 18S ribosomal RNA gene fragment specific to either B. bigemina or B. bovis. DNA from groups of 50 larvae was extracted using a rapid DNA preparation protocol, which consisted of grinding the frozen tick larvae in PCR buffer and boiling the mixture for 5min. The assay sensitivity allowed for the detection of the equivalent of a single infected tick larva. R. microplus eggs were also analyzed, but yolk protein viscosity created inconsistent results with the crush and boil DNA isolation protocol, necessitating the use of a more extensive proteinase K digestion-based DNA purification method. We detected the presence of B. bigemina in all strains of R. microplus currently reared at the laboratory and 4 of 26 strains collected from infestation outbreaks in Texas by the U.S. Cattle Fever Tick Eradication Program.     

Evidence for plasmid-mediated tetracycline resistance in Paenibacillus larvae, the causal agent of American Foulbrood (AFB) disease in honeybees

Paenibacillus larvae is the causal agent of American Foulbrood (AFB) disease, the most virulent bacterial disease of honeybee (Apis mellifera L.) brood. Oxytetracycline is the main antibiotic used for prevention and control of AFB. Using the polymerase chain reaction, isolates were screened for the presence of the tetracycline resistance tet(K) and tet(L) determinants. Four isolates (5%), which correlated with the Tc-resistant phenotypes, were found to carry the tet(K) determinant, whereas none carried the tet(L) determinant. P. larvae cells were also screened for the presence of extrachromosomal DNA and evidence obtained that tetracycline resistance is plasmid-encoded. A few P. larvae isolates were found to be able to transfer the tet(K) determinant to Bacillus subtilis, suggesting that a conjugation mechanism may be involved in the transfer of the tetracycline-resistant phenotype. Minimum inhibitory concentrations to tetracycline were determined for 75 isolates of P. larvae from different geographical origins and found to range between 0.062 and 128 microg tetracyclineml(-1), with MIC(50) and MIC(90) values of 1 and 4, respectively. According to results from P. larvae populations, isolates could be considered as susceptible when their MICs were <4, intermediate for MICs values 4-8 and resistant for MICs > or = 16. To our knowledge, this is the first report of Tc(r)Paenibacillus species carrying a tet(K) gene, and also the first record of P. larvae strains carrying tet(K) determinants and its correlation with the presence of extrachromosomal DNA.     

A larval development test for the detection of anthelmintic resistance in nematodes of sheep

First stage larvae of a number of species of parasitic nematodes of sheep have been shown to develop to third stage larvae in the presence of a defined medium consisting of Earle's balanced salt solution and yeast extract. A larval development test, based on this culture technique, was used as a screen for detecting the presence of anthelmintic resistance in nematodes of sheep. It was found to be sensitive and simple to use and also appeared capable of detecting resistance to any of the main anthelmintic groups. Available anthelmintic sensitive and resistant strains of Haemonchus contortus and Ostertagia circumcincta showed differences in development when incubated in the presence of either thiabendazole, levamisole and ivermectin. These differences were expressed as the minimum inhibitory concentration required to prevent larval development over the incubation period.     

Diagnosis of Bacillus larvae --the causative agent of American foulbrood]

The authoress studied the growth of Bacillus larvae on different nutrient media and its ability of decomposing hydrogen peroxide and reducing nitrates. There are also instructions for rapid cultivation and biochemical diagnosis of Bacillus larvae. It can be performed in any microbiological laboratory, the culture medium can be prepared from available commercially produced preparations, and B. larvae can be detected in suspected material even out of the season, if the method of selective cultivation and repeated pasteurizing is used. Catalase test is proposed to be added to the examination; the result of this test was negative in all 96 strains studied. Photographic documentation of different developmental stages of B. larvae can serve as an aid in microbiological examination.     

A Selective-Differential Medium for Detection of Streptococcus Agalactiae

A practical culture medium which allows direct plating of milk samples for detection and differentiation of Streptococcus agalactiae within 48 hours is described. Most other micro-organisms likely to be present in these samples are inhibited. Although some strains of Staphylococcus species and ofStreptococcus faecalis are able to grow, they may be differentiated on the basis of reaction in the medium surrounding the colonies.     

Effectiveness of tilmicosin against Paenibacillus larvae, the causal agent of American Foulbrood disease of honeybees

American Foulbrood (AFB) of honeybees (Apis mellifera L.), caused by the Gram-positive bacterium Paenibacillus larvae is one of the most serious diseases affecting the larval and pupal stages of honeybees (A. mellifera L.). The aim of the present work was to asses the response of 23 strains of P. larvae from diverse geographical origins to tilmicosin, a macrolide antibiotic developed for exclusive use in veterinary medicine, by means of the minimal inhibitory concentration (MIC) and the agar diffusion test (ADT). All the strains tested were highly susceptible to tilmicosin with MIC values ranging between 0.0625 and 0.5 microg ml(-1), and with MIC(50) and MIC(90) values of 0.250 microg ml(-1). The ADT tests results for 23 P. larvae strains tested showed that all were susceptible to tilmicosin with inhibition zones around 15 microg tilmicosin disks ranging between 21 and 50mm in diameter. Oral acute toxicity of tilmicosin was evaluated and the LD(50) values obtained demonstrated that it was virtually non-toxic for adult bees and also resulted non-toxic for larvae when compared with the normal brood mortality. Dosage of 1000 mg a.i. of tilmicosin applied in a 55 g candy resulted in a total suppression of AFB clinical signs in honeybee colonies 60 days after initial treatment. To our knowledge, this is the first report of the effectiveness of tilmicosin against P. larvae both in vitro and in vivo.     

Infection of Melissococcus plutonius clonal complex 12 strain in European honeybee larvae is essentially confined to the digestive tract

Melissococcus plutonius is an important pathogen that causes European foulbrood (EFB) in honeybee larvae. Recently, we discovered a group of M. plutonius strains that are phenotypically and genetically distinct from other strains. These strains belong to clonal complex (CC) 12, as determined by multilocus sequence typing analysis, and show atypical cultural and biochemical characteristics in vitro compared with strains of other CCs tested. Although EFB is considered to be a purely intestinal infection according to early studies, it is unknown whether the recently found CC12 strains cause EFB by the same pathomechanism. In this study, to obtain a better understanding of EFB, we infected European honeybee (Apis mellifera) larvae per os with a well-characterized CC12 strain, DAT561, and analyzed the larvae histopathologically. Ingested DAT561 was mainly localized in the midgut lumen surrounded by the peritrophic matrix (PM) in the larvae. In badly affected larvae, the PM and midgut epithelial cells degenerated, and some bacterial cells were detected outside of the midgut. However, they did not proliferate in the deep tissues actively. By immunohistochemical analysis, the PM was stained with anti-M. plutonius serum in most of the DAT561-infected larvae. In some larvae, luminal surfaces of the PM were more strongly stained than the inside. These results suggest that infection of CC12 strain in honeybee larvae is essentially confined to the intestine. Moreover, our results imply the presence of M. plutonius-derived substances diffusing into the larval tissues in the course of infection.     

Proposal to reclassify Paenibacillus larvae subsp. pulvifaciens DSM 3615 (ATCC 49843) as Paenibacillus larvae subsp. larvae. Results of a comparative biochemical and genetic study

The bacterial pathogen Paenibacillus larvae subsp. larvae (P. l. larvae), is the etiological agent of American foulbrood, an extremely contagious and disastrous disease of honeybee brood. In case of American foulbrood the destruction of infected colonies is often considered the only workable control measure. Therefore, the ability to diagnose this disease properly is important to prevent unnecessary economic loss to beekeepers. The development of suitable methods for the early and reliable detection of P. l. larvae is hampered by the fact that the two subspecies of Paenibacillus larvae, P. l. larvae and Paenibacillus larvae subsp. pulvifaciens (P. l. pulvifaciens), seem to be indistinguishable by cultural characteristics as well as by PCR protocols. Here we present an extensive analysis of several P. larvae reference strains. We employed conventional culture techniques, morphological and biochemical identification, PCR-based methods and sequencing of the 16S rDNA. We found indeed that P. l. pulvifaciens strain DSM 3615 is indistinguishable from P. l. larvae (DSM 7030). We did not face any problems to discriminate between P. l. larvae and P. l. pulvifaciens strains DSM 8442 and DSM 8443. Therefore, classification of DSM 3615 as type strain of P. l. pulvifaciens seems not to be justified. We propose to reclassify this strain as P. l. larvae. Former problems in differentiating the two subspecies might have arisen from this misclassification. PCR-based methods as well as appropriate biochemical identification systems provide a reliable means for the discrimination between the two subspecies P. l. larvae and P. l. pulvifaciens.     

Histological studies of the occurrence of organisms shaped like Campylobacter in the abomasum of calves]

The mucosa of the abomasum is a strongly acidic and therefore germ poor, but not entirely germfree environment. Only in isolated areas of the fundic glands zone were straight rods in the foveolas, and even more seldom spiral shaped bacteria could be encountered. They had no relation to the inflammatory infiltrates. On the other hand, Campylobacter-like organisms were more often observed in the pyloric region and, preferably, in a narrow zone following the cutaneous mucosa of the omasum. These organisms appeared to have a different size (1.3 to 2.4 microns length and 0.4 to 0.8 microns width) and they occurred deep inside the glands in an almost pure fashion. Whereas they caused no visible reaction of the tissue in the narrow cardiac zone, their occurrence in the pyloric region was several times connected with neutrophilic-rich infiltration. It ought to be tested, whether Helicobacter are among these Campylobacter-like organisms, which cause a disease leading up to ulcerations in calves similar to gastritis B in humans.     

中意蜂混合群工蜂清理异种蜂卵和幼虫行为的比较

通过比较中蜂和意蜂混合群内工蜂对异种蜂卵和幼虫的清理行为,研究中蜂工蜂和意蜂工蜂的亲属辨识能力。混合蜂群稳定一周后开始第一次实验,再隔一周进行第二次实验。每次实验连续进行5天。结果表明:混合群刚稳定时,中蜂工蜂和意蜂工蜂对异种蜂卵和幼虫均有较强的亲属辨识能力,意蜂工蜂对中蜂幼虫的清理率明显高于中蜂工蜂对意蜂幼虫的清理率。随着混合群稳定3周后,中蜂工蜂和意蜂工蜂对异种蜂的卵和幼虫的清理积极性明显降低。     

Nemaline Rods in Canine Myopathies: 4 Case Reports and Literature Review

The diagnosis of nemaline rod myopathy (NM) is based on the presence of numerous pathognomonic rods within a fresh frozen muscle biopsy specimen. Three forms of congenital NM have been described in humans, and rods have been found to occur in various other conditions. A similar myopathy was described in 1986 in a family of cats. In this report, we describe a case of congenital NM in a 10-month-old Border Collie, an adult-onset NM in an 11-year-old Schipperke, and 2 acquired myopathies with nemaline rods in adult dogs associated with hypothyroidism and Cushing's syndrome. Common clinical features included exercise intolerance, abnormal electromyography, and the presence of nemaline rods in fresh, frozen, and glutaraldehyde-fixed biopsies from proximal appendicular limb muscles. Staining of cryostat sections of muscle biopsy specimens by the modified Gomori trichrome technique disclosed numerous rod bodies that were localized to type 1 fibers by the histochemical adenosine triphosphatase reaction. Accumulation of rods also was demonstrated by electron microscopy in 2 of the cases with localized enlargement and streaming of Z lines. Documentation of NM in a young Border Collie and the adult-onset form in the Schipperke alerts clinicians to the existence of this disorder in these breeds.     

Presence of Mycobacterium avium subspecies paratuberculosis in suspensions of ovine trichostrongylid larvae produced in faecal cultures artificially contaminated with the bacterium

A reference strain of Mycobacterium avium subspecies paratuberculosis was added to faecal larval cultures of Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis. Samples of the larvae produced were cultured for the presence of the bacterium in modified BACTEC 12B medium, both before and after exposure to gamma irradiation. The water used to wash the larvae off the faecal cultures was also tested for the presence of the bacterium. Positive growth was confirmed as M. avium subspecies paratuberculosis by IS900 polymerase chain reaction and restriction endonuclease analysis of the product. M. avium subspecies paratuberculosis was detected in the unirradiated larval suspensions and wash waters of all three nematode species, and in the irradiated H. contortus larval suspension.     

Symptomatic Black Queen Cell Virus infection of drone brood in Hessian apiaries

The Black Queen Cell Virus (BQCV) can affect brood of the honey bee (Apis mellifera). In general queen cells are endangered showing dark coloured cell walls as typical symptoms. Worker- and dronebrood can be infected by BQCV but normally without clinical symptoms. This paper describes for the first time a symptomatic BQCV-infection of diseased drone brood found on two bee yards in Hessen/Germany in 2001. The drone larvae were seriously damaged and some of them were dead. Samples of the affected brood were tested for BQCV by the PCR detection method. A BQCV specific nucleic acid fragment was found. The PCR product were sequenced and aligned with the relevant GenBank entry. At the nucleic acid level as well as at the deduced protein level the isolate showed a high similarity with the south african isolate noted in GenBank.     

Isolation and characterisation of Bordetella avium and related species and an evaluation of their role in respiratory disease in poultry

A total of 24 Gram negative non fermentative bacteria obtained from poultry were compared with reference strains of Bordetella avium, Alcaligenes faecalis, Bordetella bronchiseptica and a Bordetella avium-like organism. Thirteen isolates were identified as B. avium and 11 were identified as B. avium-like. A commercial microidentification kit (the API2ONE) did not identify the field isolates but did separate them correctly into the 2 groups. A practical identification scheme, suitable for diagnostic laboratories, is proposed for these organisms. The available clinical histories suggest that B. avium is associated with upper respiratory tract disease in turkeys.     

Influence of antibiotics on growth dynamics and movement ability of Salmonella rods

Variety of traits important in diagnostics and epidemiology of pathogenic microorganisms may change due to antibiotics. Movement ability, that is characteristic for every serovar except from Salmonella Gallinarum-Pullorum, is important to salmonellas. In own experiments using semi-fluid MSRV medium, it was found that a decrease in salmonella sensibility to selected antibiotics and chemiotherapeutics due to passage might lead to weakening of its movement ability. Movement ability of all strains (S. Enteritidis, S. Dublin, S. Typhimurium) after passage with amoxycillin, neomycin, colistin and enrofloxacin became weakened as compared to results achieved before passage. The strongest inhibition of movement ability was most often observed in strains after passage on medium with colistin. It seems to be associated with the action mechanism of the antibiotic. Colistin injuries cellular membranes, where flagella (active motoric organ of Salmonella) are anchored. Appearance of drug-resistance as a result of passage at the presence of antibiotics may cause variability of biochemical properties of Salmonella rods and leads to weakening of movement ability of ciliated Salmonella.