Preliminary evaluation of four immunological tests for the early diagnosis of Hypoderma tarandi causing hypodermosis in reindeer

294 serum samples from five Norwegian reindeer herds were examined for antibodies against Hypoderma tarandi L. The first and second larval instars of H. tarandi were tested as antigens in immunodiffusion tests, passive haemagglutination and an Enzyme-Linked Immunosorbent Assay (ELISA). The latter technique, using first instar antigens, produced the best results. A significant difference (P less than 0.1%) was observed between the antibody value of naive reindeer bred in France and those from infected Norwegian herds. No correlation was observed between the antibody titre and the number of warbles recovered at necropsy from the 294 Norwegian reindeer.     

Redescription of Besnoitia tarandi (Protozoa: Apicomplexa) from the reindeer (Rangifer tarandus)

Besnoitia tarandi tissue cysts were found in naturally-infected reindeer (Rangifer tarandus) from Finland. Infectivity of its tissue cysts, bradyzoites, and tachyzoites to animals and cell culture was studied. The bradyzoites and tissue cysts were not infectious to out-bred mice, rabbits or gerbils. When fed tissue cysts, neither cats nor dogs excreted oocysts. However, the parasite was lethal to interferon-gamma gene knock out mice irrespective of the route of inoculation. The parasite was grown successfully in African Green Monkey cells from tissues of two reindeer for the first time. Non-dividing, uninucleate tachyzoites from smears from cell cultures were 5.6 x 1.4 microm (4.5-7.4 x 1.0-1.9, n=50) in size. Longitudinally-cut bradyzoites in tissue sections measured 7.4 x 1.3 microm (6.5-7.8 x 1.0-1.6, n=30). Ultrastructurally, tachyzoites and bradyzoites were similar to those in other Besnoitia species, and in particular to parasites described from cattle (Besnoitia besnoiti) and equids (Besnoitia bennetti) in that their bradyzoites lacked enigmatic bodies. Based on comparative analysis of three portions of nuclear ribosomal DNA (the small and large subunits and the first internal transcribed spacer) B. tarandi was found to be more closely related to the other congeners described from ungulates. The parasite was formally redescribed and specimens deposited in the US National Parasite Collection.     

Serological diagnosis of brucellosis caused by Brucella canis

Blood serum samples from 2,328 dogs were tested to detect antibodies against Brucella canis with the agar gel immunodiffusion (AGID) and 2-mercaptoethanol slide agglutination test (ME-SAT) using Brucella ovis as the antigen. All blood serum samples were also evaluated for antibodies against Brucella abortus and Brucella melitensis using the Rose Bengal test. Twentyfive (1.07%) of the sera evaluated were considered positive with AGID test. Only 4 (16%) of these blood serum samples were positive when evaluated with ME-SAT. The 25 AGID positive samples and 25 AGID negative serum samples were also examined by: the complement fixation test (CFT) using B. ovis hot saline extract (HSE) as the antigen, indirect enzyme linked immunosorbent assay (ELISA) and immunoblotting (IB) using B. canis and B. ovis HSE antigens. Two positive canine sera from culture positive dogs and the serum of an experimentally RM6/66 B. canis-infected rabbit were employed as positive controls and one serum from a known uninfected dog as a negative control. ELISA with B. canis antigen gave 9 (18%) positive results (6 AGID-positive and 3 AGID-negative sera). ELISA performed with B. ovis antigen detected 15 (30%) positive samples (10 AGID-positive, 5 AGID-negative and 8 B. canis ELISA positive sera). IB analysis of known positive controls sera employing B. canis antigen detected bands with molecular weights of 94-80, 64-50, 35, 32-30, 28, 23, 20-18, 15-12 kDa. The same sera tested with B. ovis antigen revealed bands of 35, 32-30, 25, 23, 20-18, 15-12 kDa. No bands were observed with the negative control serum and the 50 canine tested sera.     

Variation in early developmental stages in two populations of an intertidal crab, Neohelice (Chasmagnathus) granulata

Duration of embryonic development, egg size, larval size at hatching, and starvation tolerance of the first zoeal stage were studied in an intertidal crab from the southwestern Atlantic, Neohelice (formerly Chasmagnathus) granulata. These reproductive traits were quantified comparing (a) two populations living in ecologically contrasting coastal habitats in Argentina, a brackish lagoon, Mar Chiquita, MC vs. an open marine habitat near San Antonio, Patagonia, SA, (b) beginning vs. end of the reproductive season, and (c) two temperatures during egg development (18 vs. 27°C). Eggs in an early stage of embryonic development were in both populations larger at the beginning than at the end of the season, and were consistently larger in the SA population. These size differences persisted through larval hatching, independent of the temperature during embryogenesis. At 18°C, eggs produced at the beginning of the season developed in both populations more rapidly than those from the end of the reproductive season, while the opposite trend was observed at 27°C. The stage duration of the zoea I was in both populations shorter at the beginning as compared to the end of the season. The nutritional flexibility of the zoea I stage was compared using as indices the point-of-reserve-saturation (PRS₅₀) and the point-of-no-return (PNR₅₀). The PRS₅₀ was consistently lower in larvae from SA than in those from MC. In the MC population, this index was lower at the beginning than at the end of the season, while no significant seasonal difference was observed in larvae from SA. The PNR₅₀ varied between temperatures of embryonic development and populations, showing also significant interactions between all three factors. The PRS₅₀ was on average lower, and the PNR₅₀ was higher, than values previously reported for N. granulata, suggesting a stronger nutritional flexibility in the larvae used in the present study. Our results indicate significant intraspecific variability among separate populations, seasonal variation, and carry-over effects of environmental conditions prevailing during the embryonic phase, all of which may affect the performance of the larval phase.     

Protective devices of early developmental stages in Pyrrhalta viburni (Coleoptera,Chrysomelidae)

Summary Larvae and eggs of the leaf beetle Pyrrhalta viburni were investigated for protective devices against predators. The eggs are covered with faeces, which appeared to have no feeding deterrent activity against the ant Myrmica sabuleti. Chemical analyses of the material covering the eggs by gas chromatography-mass spectrometry (GC-MS) showed the triterpenes -amyrin and -amyrin as main components. Both compounds are also present in the hostplant Viburnum lantana. GC-MS analyses of eggs and larvae showed that 1,8-dihydroxy-3-methylanthraquinone (=chrysophanol) was present in both developmental stages. In the larvae, 1,8-dihydroxyanthraquinone (=chrysazin) and 1,8,9-trihydroxyanthracene (dithranol) were also detected. Neither hydroxylated anthraquinones nor dithranol were found in bark and leaf extracts of the hostplant. After assessing the total amounts of these compounds in a single larva, their ecological significance was studied in feeding bioassays with M. sabuleti. Both P. viburni larvae and equivalent amounts of anthraquinones and dithranol deterred feeding by the ants. The role of anthraquinones and triterpenes in P. viburni is discussed.     

Mating behavior and thermoregulation of the reindeer warble fly,Hypoderma tarandi L. (Diptera: Oestridae)

Hypoderma (=Oedemagena) tarandi L. (Diptera: Oestridae) is characterized by a mating strategy in which both sexes meet and mate at two types of distinct topographical landmarks. In the expansive, treeless vidda (= tundra-like) biome, mating places are unique, rocky areas located along rivers and streams or in rocky areas of drying river and stream beds. In wooded valleys below the vidda, flies mated at certain topographical areas along dirt road tracks/paths. Thermoregulatory activities of males occupying perches at mating places included selection of substratum at perch site, orientation of body to sun's rays, crouching, stilting, and flights into upper cooler air. On warm sunny days males perched for just 1–2 min before flying up into cooler air to promote cooling. Laboratory and field studies revealed that flies could not metabolically cool down when held at 25–38°C. Time spent at mating places depended on temperature, duration of sunshine, and wind velocity. Males were very aggressive in pursuing allHypoderma-sized objects that passed by them or that landed near them, but they did not defend specific perch sites. Males either pursued and caught females in flight, or they hopped onto females that landed near them. During 5 years, 74 males and 14 females were seen at mating places. Dissection of six females caught at mating places revealed them to be recently eclosed flies full of fat body and with all eggs intact; two not paired with males were non-inseminated. Three experimentally paired females remainedin copulo for 10, 13, and 19.5 min.     

Correct diagnosis of early zoeal stages of Athanas nitescens (Leach, 1814) (Decapoda, Caridea, Alpheidae) using laboratory-raised larvae

The morphology of the first two larval stages of Athanas nitescens(Leach, 1814), reared under laboratory conditions, is redescribed.The present data are compared with previous works, since a clarificationof the morphological characters of the first two larval stagesof A. nitescens is needed, in order to avoid misidentificationof these stages in the future.     

Ultrasonography in early pregnancy diagnosis and measurements of fetal size in reindeer (Rangifer tarandus tarandus)

Transrectal or transabdominal examinations of 13 pluriparous reindeer (Rangifer tarandus tarandus) by ultrasonography from the start of mating until week 20 of gestation were conducted to find out when pregnancy could first be detected and to describe fetal development in early pregnancy. The examinations (n=35 per animal) were performed with a 5 MHz linear transducer from 7th October until 1st January and with a 3 MHz sector transducer from that time until 24th February. Time of pregnancy diagnosis by ultrasonography, the first fetal heartbeat and measurements of crown-rump length, chest width and chest depth were recorded during the examinations. Pregnancy was diagnosed by transrectal ultrasonography between the weeks 3 and 7 of gestation. The accuracy of the pregnancy diagnosis, defined as the proportion of females correctly detected to be pregnant, was 15% at week 3, 46% at week 4, 77% at week 5, and 92% at week 6 of gestation. Fetal heartbeat was first detected between the weeks 5 and 8 of gestation. The first measurements of crown-rump length were made on week 3 of gestation, of chest width on week 4 and of chest depth on week 5 of gestation. Chest width and depth were detectable until the end of the study at week 20 of gestation. Transrectal ultrasonography is an efficient tool in early pregnancy diagnosis of reindeer. The fetal growth curves obtained by ultrasonography resembled those obtained in previous morphological studies.     

Determination of the migratory route of botfly larvae, Cuterebra grisea (Diptera: Cuterebridae) in deermice

HUnter D. M. and Webster J. M. 1973. Determination of the migratory route of botfly larvae, Cuterebra grisea (Diptera: Cuterebridae) in deermice. International Journal for Parasitology3:311–316. Cuterebra grisea larvae introduced into deermice (Peromyscus manicutatus) occurred in the nasal passages (nose or eye entry) or were associated with the esophagus and trachea (mouth entry) in the first three days after entry. During this period, some deermice exhibited a violent wheezing reaction. From day 4 to 6 after entry, larvae migrated to the final development site in the inguinal region of the deermouse. Migrating larvae were found on the front and top of the head, on the back and at the base of the tail. The similarity of this migratory path for C. grisea with the final development sites of other Cuterebra species is discussed.     

Factors affecting size, longevity and fecundity in the reindeer oestrid flies Hypoderma tarandi (L.) and Cephenemyia trompe (Modeer)

1. Laboratory reared reindeer oestrid flies Hypoderma tarandi and Cephenemyia trompe (Diptera: Oestridae) were weighed to determine progressive weight loss and death weights at treatments with various temperature and humidity conditions.
2. Four individual measurements of size were taken: larval weight, wet weight of newly eclosed flies, wing length, and weight of flies after dehydration and fat extraction. In H. tarandi, males were bigger than females (except for wing length), whereas the reverse was true for C. trompe .
3. Size variation was not significantly related to conditions (temperature, humidity, duration) during the pupal stage, but individual reindeer produced flies (both species) of different mean sizes. These size differences were not correlated with larval burden (= number of larvae per individual host), but are hypothesized to be connected to unknown host quality factors.
4. Longevity of flies kept in vials and subjected to various temperature and humidity conditions revealed that C. trompe lived significantly longer than H. tarandi (range: 4–44 and 1.2–27 days, respectively) at 5–33 °C. Male H. tarandi survived longer than females; female C. trompe survived longer than males. Longevity was not significantly correlated to any of the size measures.
5. Most flies had a large portion of their fat reserves left at death.
6. In H. tarandi , mean number of eggs was 609 ± SD 73 (range 354–772, n = 119). Egg number was slightly dependent on larval size, but not on wet weight of newly eclosed flies or wing length. In C. trompe , mean number of eggs was 960 ± SD 208 (range 493–1349, n = 31).
7. The possible adaptive value of large size in oestrids is questioned. Benefits of flexibility in size in oestrids are hypothesized.     

Serological diagnosis of petriellidiosis (Allescheriosis)

Soluble antigens in culture filtrates of three strains of Petriellidium boydii and three strains of Monosporium apiospermum were examined. Antigens were separated from concentrated crude filtrates by anion-exchange chromatography. A single major peak (Antigen 1), constituting a significant proportion of the total recoverable carbohydrate, was the only product isolated from each of four chromatographed filtrates. Depending on the fungus strain, Antigen 1 consisted of 90–96% carbohydrate, 3–4% protein, and 2–4% nucleic acid. Antigen 1 was found to consist of a population of molecules with a heterogeneous molecular size when assayed by gel filtration chromatography; however, isolated fractions of Antigen 1 proved to be immunologically identical when examined by Ouchterlony immunodiffusion. In addition, Antigen 1 from each strain was immunologically identical to similar preparations of Antigen 1 from the other five fungus strains. Chromatography of culture filtrates from two strains of M. apiospermum revealed a second peak (Antigen 2), which was found to consist of 70% carbohydrate, 16% protein, and 4% nucleic acid. Although Antigen 2 contained four times as much protein as Antigen 1, the two preparations were immunologically identical by immunodiffusion tests. Ion-exchange chromatography proved to be a useful procedure for isolating antigens of P. boydii and M. apiospermum from culture filtrates.     

Genes induced during the early developmental stages of the Cane Toad, Bufo (Chaunus) marinus

Metamorphosis, a critical stage in the development of toads and frogs, involves rapid levels of morphological change. In the current study, we have used microarray analysis to identify shifts in gene expression between tadpole and toadlet stages of the cane toad, Bufo (Chaunus) marinus. Here, we report on nine genes that show the greatest induction during metamorphosis; the gut-associated gastrokine and trefoil factor, blood components haemoglobins alpha/beta, apolipoprotein and serum albumin, a nasal gene olfactomedin, a lens gene gamma-crystallin, and a novel gene with low homology to frog harderin. We present both temporal and spatial expression patterns of these genes identified in developing and adult cane toads. This study extends our knowledge of the molecular basis of toad metamorphosis, and not only offers insights to the genes induced during the general remodelling that occurs but also reveals possible targets for control and manipulation of amphibian pest species, for example, the cane toad in Australia.     

Intermediate filament protein expression in early developmental stages of the mouse

Expression patterns of intermediate filament proteins have been studied during early mouse embryo development. For this purpose, pre-implantation embryos at different stages of development after in vitro fertilization were studied using antibodies to cytokeratins, vimentin and lamins, using the indirect immunofluorescence assay. The levels of expression were quantitated and localization of the protein constituents was assessed by means of confocal scanning laser microscopy. Our studies showed that, although the embryos grew in culture, vimentin could not be detected in a filamentous organization. Immunofluorescence for cytokeratins was only positive from the 8-cell stage onwards. In the morula stage an increased level of cytokeratin expression was observed with a transitional staining pattern, combining a filamentous and a diffuse occurrence. In the blastocyst stages profound cytokeratin filaments were seen in trophoblast cells but not in the inner cell mass. When the cytokeratin subtypes were analysed separately, it became apparent that expression levels of cytokeratins 8 and 18 increased gradually up to a filamentous pattern in the blastocyst stage. Cytokeratins 7 and 19, although elevated in the latter stage and showing a filamentous distribution, were not found as prominently as cytokeratins 8 and 18. A-type as well as B-type lamins could be detected in all developmental stages examined, as a faintly reactive nuclear lamina. In blastocysts both lamin types were detected in trophoblast as well as in inner cell mass.     

Errors in the estimation of recruitment of early stages of mesocyclops leuckarti (claus) caused by the diurnal periodicity of egg-production

The seasonal and bathymetrical distribution of Mesocyclops leuckarti (Claus) was investigated during 1969–1975 Development time of egg, naupliar and copepodite stages were found experimentally at three temperatures (15°, 22° and 27°C). The calculations of population dynamics generated some unexplained results.Experimental study of the influence of light-dark conditions and temperature on the process of laying and hatching, together with the field data, helped us to describe an artifact when sampling is done at early morning.Contribution no. 123. Israel Oceanographic & Limnological Research Ltd.