Some diagnostic, biologic and morphologic characteristics of Francisella tularensis strains isolated from the ticks Ixodes ricinus (L.) in the Prague agglomeration

Four infectious agents were isolated from the ticks Ixodes ricinus (L.) collected in the recreational area and park-forest of Prague. On the basis of cultivation, staining, biochemical, serologic properties, pathogenicity for animals and histological tests they were identified as Francisella tularensis with the following features: they are short, gram-negative rods of approximate dimensions of 0.3 X 0.8 micron, growing in enriched media after 3-4 day incubation at 37 degrees C. They form small circular, at first transparent, later greyish turbid colonies with regular rims. They are little active biochemically. They are susceptible to streptomycin and some broad spectrum antibiotics. They react positively with tularemic serum, but in lower titres than those in which this serum reacts with standard antigen. The microbes are highly pathogenic for mice, guinea-pigs, young rats, in which a massive bacteriemia occurs before death, but they do not kill rabbits. They multiply well in chick embryo, but do not grow in cell or tissue cultures. The most important histologic changes were observed in liver and spleen of mice. No pathologic changes were found in brain, lungs, heart, kidneys. Necroses were found in liver and in their marginal zones the microbes were present. Conspicuous were changes in numerous hepatocytes which became enlarged due to microbial multiplication and finally transformed into "sacs" packed with microbes. Histological and electronoptical examination showed that these are intracellular parasites fringed with a light lytic zone. Discussed is the problem to what extent the properties of the isolated strains are typical of F. tularensis as well as the importance of their detection from the aspect of epidemiology and differential diagnostics.     

Further characterization of dandelion yellow mosaic virus from lettuce and dandelion

A damaging virus isolated in the Netherlands from lettuce was studied and compared with a virus isolated from dandelion orginating from Czechoslovakia. It was found to biologically resemble dandelion yellow mosaic virus incompletely described from dandelion and lettuce in Great Britain (Kassanis, 1944, 1947) and from dandelion in Germany (Hein, 1963). Mechanical transmission was greatly improved by buffer solution and transmission byMyzus persicae seemed to be in the non-persistent manner. Longevity in vitro of the virus hardly exceeded one day. Thermal inactivation was between 60 and 65 °C and the dilution end-point was between 10 000 and 100 000. It was still infectious in leaf material dried and stored over CaCl₂ at 4 °C for 6 1/2 years. The virus was isolated and purified with difficulty and was found to consist of one type of spherical particle of ca 30 nm diameter, with a sedimentation coefficient of 159 S, a buoyant density of 1.42 g.cm^?3 and an A₂₆₀/A₂₈₀ ratio of 1.67. An antiserum was prepared with a titre of 256 in the agar double-diffusion test. The virus could be identified in crude extracts from lettuce andChenopodium amaranticolor by enzyme-linked immunosorbent assay (ELISA), but not by agar double diffusion. It could only be visualized in crude sap in the electron microscope after trapping of virus particles on antiserum-coated grids. The virus cannot yet be assigned to any known virus group. It is of potential economic importance to lettuce because of its occurrence in widely differing regions in Europe, its aggressiveness and virulence on 22 out of 23 lettuce cultivars tested (and on endive) and its pathogenicity toLactuca genotypes which are resistant to lettuce mosaic virus and other important pathogens of lettuce. ‘Laibacher Eis’ was the only cultivar showing some tolerance.     

Effect of dietary protein content on live and carcase performance of heterozygous naked neck and normally feathered broilers

1. An experiment was conducted to determine the effect of different dietary protein contents on the performance of naked neck (Na/na) and normally feathered (na/na) broilers.

2. Chicks from the two genotypes were reared in wire‐floored cages and divided at random into 3 groups. Birds were fed on high protein (HP, 12.99 MJ ME, 238 g crude protein/kg and 12.94 MJ ME, 216 g crude protein/kg from 0 to 3 and 3 to 7 weeks, respectively), medium protein (MP, 12.99 MJ ME, 219 g crude protein/kg and 12.87 MJ ME, 201 g crude protein/kg from 0 to 3 and 3 to 7 weeks), and low protein (LP, 12.94 MJ ME, 205 g crude protein/kg and 12.75 MJ ME, 184 g protein/kg from 0 to 3 and 3 to 7 weeks) diets.

3. The LP diets resulted in a significantly lower daily body weight gain of males from 0 to 3 weeks. Dietary protein content had no effect on body weight gain from 3 to 7 weeks, body weight at 7 weeks, and the food intake of birds. Carcase composition of birds from both genotypes was unaffected by dietary protein.

4. Naked neck birds had significandy higher body weights at 7 weeks. Yields of carcase and breast of Na/na males were significantly higher than those of na/na males. There were no significant differences between females from the two genotypes as regards carcase yield.

5. It was concluded that the dietary protein requirements of naked neck birds were similar to those for normally feathered birds.     

Effect of progesterone on ovulation in the hypophysectomised hen

1. One hundred and seven laying hens were hypophysec‐tomised to clarify the relationship between hypophysectomy and the effect of progesterone (P4) on ovulation.

2. Hens were hypophysectomised at 6 intervals from 4.5 to 11.0 h before the expected time of ovulation. Ovulation occurred in the hens operated on from 4.5 to 7.3 h, but did not take place in birds operated on from 9.2 to 9.8 h before the expected time of ovulation.

3. When a single dose of P4 (2 mg/hen) was injected iv immediately after the removal of the anterior pituitary from 7.5 to 9.8 h before the expected time of ovulation, ovulation was induced. However, the percentage of hens responding decreased in proportion to the lapse of time between the hypophysectomy and the expected time of ovulation. No ovulation was induced in hens which were hypophysectomized and given P4 10.2 to 11.0 h before the expected time of ovulation.

4. It is suggested that ovulation is induced by P4 alone possibly in the absence of preovulatory gonadotrophins and that P4 acts directly on the ovary to induce follicle rupture.     

Histochemistry of gland cells of Eurytrema pancreaticum cercariae.

The cercariae of Eurytrema pancreaticum (Janson, 1889) possess four types of gland cells - proper cystogenic, penetration, ventral and dorsal gland cells. The secretion of ventral and dorsal gland cells is released into the tegument. The proper cystogenic gland cells are the largest and their contents serve for the formation of the cyst wall of metacercariae in the second intermediate host. The secretion of proper cystogenic gland cells contains besides neutral mucosubstances also acid mucosubstances with both carboxyl- and sulphogroups digestible with beta-glucuronidase. The secretion of penetration gland cells contains neutral mucosubstances and proteins with tyrosine, tryptophan and SS groups. The ventral gland cells contain mostly acid mucosubstances with sulphogroups, which are digested with beta-glucuronidase, and proteins with tyrosine, tryptophan and SH groups. The rudimentary dorsal gland cells contain a small amount of acid mucosubstances. The whole tegument of cercariae and the two main collecting canals of the excretory system exhibit a high alkaline phosphatase activity. The nerve ring and the main nerve truncs contain proteins with SH groups and hydrophilic lipids and exhibit a cholinesterase activity. The suckers contain a larger amount of glycogen.     

Detection of squash mosaic virus in seeds of melon (Cucumis melo) by enzyme-linked immunosorbent assay (ELISA)

Squash mosaic virus (SqMV, comovirus) is seed-transmitted in severalCucurbitaceae. Therefore, the use of virus-free seed is important to prevent establishment of this virus in the Netherlands and to avoid spread to other countries.This study was undertaken to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of SqMV in melon seeds. An antiserum was produced to a serotype 1 isolate from melon. Two ELISA variants were investigated viz. an ELISA variant with simultaneous incubation of sample and enzyme conjugate (ELISA 1) and an ELISA variant with successive incubation of sample and enzyme conjugate (ELISA 2). The sensitivity of ELISA was tested by mixing fluor of ground infected and non-infected seeds in different proportions. SqMV was detected by both ELISA variants at dilutions of 1 160 (1 part of infected flour mixed with 159 parts of non-infected flour) or higher after a substrate incubation period of 4 h. However, ELISA 1 gave relatively higher absorbance values than ELISA 2 for nearly all dilutions. Since ELISA 1 is also faster than ELISA 2, ELISA 1 is advised for routine testing. In these test, using subsamples of 100 melon seeds SqMV is detected reliably. ELISA 1 is now used in the Netherlands for routine-indexing of melon seed lots for SqMV.Samenvatting Het pompoenemozaïekvirus gaat over met het zaad van verscheideneCucurbitaceae. Het gebruik van virusvrij zaad is belangrijk om te voorkomen dat het virus zijn intrede doet in Nederland en zich naar andere landen verspreidt.Een antiserum werd geproduceerd tegen een serotype 1 isolaat van meloen. Met behulp van dit antiserum werd een ELISA ontwikkeld om pompoenemozaïekvirus in zaden van meloen aan te tonen. Twee varianten van ELISA werden vergeleken, namelijk een variant waarbij monster en enzymconjugaat gelijktijdig geïncubeerd werden (ELISA 1) en een variant waarbij monster en enzymconjugaat na elkaar geïncubeerd werden (ELISA 2). De gevoeligheid van de ELISA varianten werd uitgetoetst door meel van zieke zaden in verschillende verhoudingen te mengen met meel van gezonde zaden. Het pompoenemozaïekvirus werd met beide ELISA varianten aangetoond in verdunningen van 1 160 (1 deel meel van zieke zaden gemengd met 159 delen meel van gezonde zaden) of hoger na 4 uur incubatie met substraat. ELISA 1 gaf doorgaans hogere extinctiewaarden dan ELISA 2 voor bijna alle verdunningen. Omdat ELISA 1 ook nog sneller is dan ELISA 2, wordt ELISA 1 aanbevolen voor routinematig gebruik. Wanneer voor routinematig gebruik 100 meloenezaden per submonster getoetst worden, kan het pompoenemozaïek virus betrouwbaar worden aangetoond. In Nederland worden momenteel per zaadpartij 20 submonsters van 100 zaden getoetst.     

Arbovirus isolations from mosquitoes in South Slovakia.

In the years 1973 and 1975 mosquitoes and some other Diptera (Tabanidae, Simuliidae, Hippoboscidae) were tested for virus. 13,924 mosquitoes, 75 horseflies and 60 blackflies were processed in 1973. Five strains of Tahyna virus were isolated from mosquito species Aedes vexans. 3,378 mosquitoes and 12 sheep keds were tested for virus in 1975. Twelve strains of Calovo virus were isolated from Anopheles maculipennis and one strain of Tahyna virus was obtained from Aedes vexans mosquitoes.     

Nerine latent virus: some properties and serological detectability in Nerine bowdenii

Nerine latent virus (NeLV), first found inNerine bowdenii, may occur also in the otherNerine species investigated so far:N. sarniensis, N. flexuosa Alba, andN. Mansellii. Chenopodium amaranticolor, C. quinoa, andGomphrena globosa sometimes reacted with local lesions after mechanical inoculation with NeLV.Nicotiana clevelandii andHippeastrum were symptomless hosts. In this respect NeLV resembled the incompletely describedHippeastrum latent virus (HLV).Serologically NeLV was closely related to HLV and to carnation latent virus (CaLV), but differed from the latter in host plant reaction. A more distant relationship was observed with some other carlaviruses, wheareas NeLV also reacted with an antiserum to potato virus X.Depending on the lot, NeLV could be detected rather reliably with the micro-precipitin test inN. bowdenii Van Roon, but less well in 63. Better results were obtained with the microplate method of enzyme-linked immunosorbent assay (ELISA).The average particle length was 664 nm, the sedimentation coefficient 155 S and the buoyant density 1.298 g/cm³.NeLV can be considered as a member of the carlavirus group. On basis of priority HLV may be considered as NeLV.