Molecular and immunological characterisation of Theileria parva stocks which are components of the 'Muguga cocktail' used for vaccination against East Coast fever in cattle

The 'Muguga cocktail' which is composed of three Theileria parva stocks Muguga, Kiambu 5 and Serengeti-transformed has been used extensively for live vaccination against East Coast fever in cattle in eastern, central and southern Africa. Herein we describe the molecular characterisation of the T. parva vaccine stocks using three techniques, an indirect fluorescent antibody test with a panel of anti-schizont monoclonal antibodies (MAb), Southern blotting with four T. parva repetitive DNA probes and polymerase chain reaction (PCR)-based assays detecting polymorphism within four single copy loci encoding antigen genes. The Muguga and Serengeti-transformed stocks exhibited no obvious differences in their reactivity with the panel of MAbs, whereas Kiambu 5 differed with several MAbs. Kiambu 5 DNA was very distinct from the Muguga and Serengeti-transformed isolates in the hybridisation pattern with all four nucleic acid probes, whereas Muguga and Serengeti-transformed isolates exhibited minor differences and could not be discriminated with one of the probes. PCR amplification in combination with restriction fragment length polymorphism analysis indicated that Kiambu 5 was also markedly divergent from the Muguga and Serengeti-transformed stocks within two of the four antigen coding genes. The T. parva Serengeti-transformed stock did not contain a 130 base pair insert within the p67 sporozoite antigen gene, which has been observed previously in most T. parva parasites isolated from buffalo, and could not be discriminated from T. parva Muguga at any of the four single copy loci. Collectively the data indicate that two of the cocktail components T. parva Serengeti-transformed and Muguga are genetically closely related, while the third component Kiambu 5 is quite distinct. Based on the findings, there may be a need to include only one of the T. parva Muguga and Serengeti-transformed components in the immunising cocktail. The study demonstrates the value of molecular characterisation data for monitoring of live vaccines.     

Variations among unbred heifers in the activities of polymorphonuclear leucocytes from the mammary gland and blood

The phenotypic characteristics are described for the activity of polymorphonuclear leucocytes NMN) obtained by either lavage of the cavity system of juvenile mammary glands stimulated with a synthetic muramyl dipeptide analogue or isolation from the peripheral blood. Attention was paid to the variability of characteristics and its sources, and to correlations among them. The following characteristics were investigated in 27 clinically healthy, unbred Bohemian Red Pied x Holstein heifers: migration activity in situ, number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and unstimulated and zymosan-stimulated luminol-dependent chemiluminescence. Considerable individual variation was found in the characteristics. Significant differences between blood PMN and PMN from lavages after influx induction were found for bactericidal activity (P < 0.05) and chemiluminescence (P < 0.01). A significant correlation between blood PMN and mammary gland PMN was found only for the number of phagocytosing cells (r = 0.329; P < 0.01). Highly significant positive correlations (P < 0.01) were demonstrated between the number of phagocytosing PMN [a], phagocytotic index [b], and bactericidal activity [c] in both blood PMN (r(ab) = 0.602; r(ac) = 0.565; r(bc) = 0.529) and mammary gland PMN (r(ab) = 0.730, r(ac) = 0.618, r(bc) = 0.589). No significant correlation was demonstrated for non-stimulated (NS), zymosan-stimulated (ZS), or opsonized zymosan-stimulated (OZS) chemiluminescence with any of the other characteristics of phagocytotic activity, in either blood PMN or mammary gland PMN (P > 0.05). The animal was a highly significant source of variability for all the phagocytotic activity characteristics (P < 0.01). Udder quarter was a non-significant source of variability for all the characteristics of phagocytotic activity except for NS chemiluminescence (P < 0.05) and ZS or OZS chemiluminescence (P < 0.01). However, udder quarter was a non-significant source of variability of chemiluminescence indices ZS/NS and OZS/NS (P > 0.05). It has been demonstrated that in situ migration activity, the number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and chemiluminescence indices of PMN collected from juvenile mammary glands of unbred heifers after influx induction can be regarded as candidate early markers of resistance to mammary infections.     

Ocular fundus images with confocal scanning laser ophthalmoscopy in the dog, monkey and minipig

Confocal scanning laser ophthalmoscopy (CSLO) is a new technique that enables ocular fundus image recording and retinal dynamic angiography to be performed. The ocular fundus image is acquired sequentially, point by point, and is reconstructed on a video monitor at the rate of 25 images per second. The feasibility of performing both ocular fundus image recordings and retinal angiography image recordings were tested on two dogs, two monkeys and two minipigs using a 40° field I + Tech CSLO. Fundus area of each dog, monkey and minipig were examined without any additional optical devices. The ocular fundus and angiography images were recorded, stabilized and analyzed under the same conditions. For each species, all images were easily recorded without any additional optical device in a lighted room and the morphology of the retinal images generated was similar to those obtained with a camera or angiography of higher resolution. Capillary phase or venous times are presented. Image recording at 25 frames/second enabled more retinal dynamics to be demonstrated than with use of regular angiography. This technique is noninvasive and easy to perform if the eye is fixed and eyelids maintained open. It also allows exploration of retinal microvascularization and could be utilized for clinical, pharmacologic and toxicologic investigations as well.     

Pathogenesis of Alfalfa mosaic virus in Soybean (Glycine max) and Expression of Chimeric Rabies Peptide in Virus-Infected Soybean Plants

ABSTRACT Infection of soybean (Glycine max) plants inoculated with particles of Alfalfa mosaic virus (AlMV) isolate 425 at 12 days after germination was monitored throughout the life cycle of the plant (vegetative growth, flowering, seed formation, and seed maturation) by western blot analysis of tissue samples. At 8 to 10 days after inoculation, the upper uninoculated leaves showed symptoms of virus infection and accumulation of viral coat protein (CP). Virus CP was detectable in leaves, stem, roots, seedpods, and seed coat up to 45 days postinoculation (dpi), but only in the seedpod and seed coat at 65 dpi. No virus accumulation was detected in embryos and cotyledons at any time during infection, and no seed transmission of virus was observed. Soybean plants inoculated with recombinant AlMV passaged from upper uninoculated leaves of infected plants showed accumulation of full-length chimeric AlMV CP containing rabies antigen in systemically infected leaves and seed coat. These results suggest the potential usefulness of plants and plant viruses as vehicles for producing proteins of biomedical importance in a safe and inexpensive manner. Moreover, even the soybean seed coat, treated as waste tissue during conventional processing for oil and other products, may be utilized for the expression of value-added proteins.     

The Role of Ethylene Production in Virulence of Pseudomonas syringae pvs. glycinea and phaseolicola

ABSTRACT The importance of ethylene production for virulence of Pseudomonas syringae pvs. glycinea and phaseolicola was assayed by comparing bacterial multiplication and symptom development in bean and soybean plants inoculated with ethylene-negative (efe) mutants and wild-type strains. The efe mutants of Pseudomonas syringae pv. glycinea were significantly reduced in their ability to grow in planta. However, the degree of reduction was strain-dependent. Population sizes of efe mutant 16/83-E1 that did not produce the phototoxin coronatine were 10- and 15-fold lower than those of the wild-type strain on soybean and on bean, and 16/83-E1 produced very weak symptoms compared with the wild-type strain. The coronatine-producing efe mutant 7a/90-E1 reached fourfold and twofold lower population sizes compared with the wild-type strain on soybean and bean, respectively, and caused disease symptoms typical of the wild-type strain. Experiments with ethylene-insensitive soybeans confirmed these results. The virulence of the wild-type strains was reduced to the same extent in ethylene-insensitive soybean plants as the virulence of the efe mutants in ethylene-susceptible soybeans. In contrast, the virulence of Pseudomonas syringae pv. phaseolicola was not affected by disruption of the efe gene.     

Serological survey for antibodies against selected infectious agents among fallow deer (Dama dama) in central Italy

Sera were collected from 224 fallow deer (Dama dama) in reserves in central Italy. Samples were tested for antibodies against Chlamydia spp., Brucella spp. and Coxiella spp. with the complement fixation (CF) test. Indirect immunofluorescence was used to test for antibodies against Borrelia spp. and agglutination tests were conducted for Leptospira interrogans antibodies. Enzyme-linked immunosorbent assay (ELISA) tests were used to detect antibodies against bovine herpesvirus-1 (BHV-1) and bovine viral diarrhoea virus (BVDV). All samples were negative for antibodies against Brucella spp., L. interrogans, Coxiella spp. and BHV-1. Four samples (1.8%) had antibodies against Chlamydia spp., nine (4%) against Borrelia spp. and 10 (4.5%) against BVDV. These results indicate that fallow deer in central Italy have a low rate of exposure to pathogens typical of domestic livestock.     

Epidemiology of feline leukaemia and feline immunodeficiency virus infections in the Czech Republic

Commercial serological sets were used for the examination of 727 cats kept in larger towns of the Czech Republic. FeLV antigen and antibodies to FIV were demonstrated in 96 (13.2%) and 42 (5.8%) of the animals, respectively. Seven (0.96%) animals were positive for both FeLV and FIV. Most of the FeLV and/or FIV positive patients were intact rambling males aged 1-4 years. Chronic gastrointestinal and respiratory diseases were found in 54.2% and 43.8% of the FeLV-positive patients, respectively. Chronic urinary tract diseases and generalized lymphadenopathy were found in 47.6% and 45.2% of the FIV-positive patients, respectively. The results of this first survey in the Czech Republic have shown prevalence values and clinical patterns similar to those reported formerly from other European countries.     

Aggregation of sow lactobacilli with diarrhoeagenic Escherichia coli

A total of 20 strains of lactobacilli were isolated from the oesophagus and vagina of 20 sows at the time of partus. Aggregation activity was seen between six homofermentative autoaggregative lactobacilli and three strains of pathogenic Escherichia coli with F4, F5 and F6 fimbriae. The highest aggregation activity was observed between vaginal Lactobacillus acidophilus PV 32 or oesophageal OE 2/1 and E. coli with F4 (K88). The presence of aggregation-promoting factor (APF) was confirmed by polymerase chain reaction (PCR) with primers of a specific fragment the apf gene derived from human L. gasseri 4B2 in one oesophageal L. acidophilus strain OE 2/1. We propose that autoaggregative lactobacilli that aggregate with diarrhoeagenic E. coli can express a class of APF proteins that exhibit the function of an aggregation mediator.     

Trichoecius calomysci sp. n. (Acari: Myocoptidae), a new mite species from Iran

A new species of myocoptid mite, Trichoecius calomysci sp. n. (Acari: Myocoptidae), from Calomyscus sp. (Rodentia: Cricetidae) from Iran is described.