Evidence for melano-macrophage centres of teleost as evolutionary precursors of germinal centres of higher vertebrates: an immunohistochemical study

The melano-macrophage centres (MMCs) of the haemolymphopoietic organs of teleost fish trap and retain antigens and are closely associated with immunoglobulin-secreting cells. The hypothesis that they are the phylogenetic precursors of the germinal centres of higher vertebrates has been questioned due to their apparent lack of organising cells. In this study the immunoreactivity of MMC cells from spleen and kidney of the teleosts Cyprinus carpio, Odontesthes bonariensis and Solea senegalensis to CNA-42, an antibody usually employed for labelling follicular dendritic cells of higher vertebrates was investigated. Free melano-macrophages and MMCs in the spleens of all three species were labelled by the antibody. This finding adds new evidence to the hypothesis that an evolutionary relationship exists between the MMCs of fish and the germinal centres of many birds and mammals.     

Eimeria procyonis sp. n., an Isospora sp., and a Redescription of E. nuttalli Yakimoff and Matikaschwili, 1932 (Protozoa: Eimeriidae) from the American Raccoon (Procyon lotor)

SYNOPSIS. Thirty-two of 48 raccoons examined were infected with a previously undescribed species of Eimeria which is herein named E. procyonis. Of the 32 infected animals, 10 also harbored E. nuttalli and 1 had Isospora sp. oocysts.
The ellipsoid to ovoid oocysts of E. procyonis measured 23.4 × 18.0 (16–29 × 13–24) μm; its sporocysts measured 12.1 × 9.3 (11.5–15 × 7–10) μm, each containing a slightly flattened substiedal body. The sporocyst residuum consisted of numerous scattered granules each ∼1 μm in diameter. The oocyst wall was double-layered. The outer layer appeared rough and pitted, measuring 1.5 μm, except at the micropyle where it was 1 μm thick.
The oocysts of the Isospora sp. measured 16.8 × 13.7 (16–18.5 × 12.5–15.5) μm. The wall consisted of a single layer ∼0.5 μm thick. The sporocysts measured 11.2 × 9.1 (9.5–11.5 × 8–10) μm, and each contained 4 elongate sporozoites. The oocysts of E. nuttalli measured 17.5 × 13.6 (12-21 × 11-15) μm, with a smooth single-layered wall approximately 0.7 μm thick. The sporocysts measured 12.2 × 7.1 (9-13 × 5.5–11) μm. Each sporocyst had a thin, dark, Stieda body and the sporocyst residuum consisted of many fine granules.     

Experimental Isospora canis and Isospora felis Infection in Mice,Cats, and Dogs*

SYNOPSIS. Two 6-month-old gnotobiotic dogs and 4 five-day-old dogs became infected but did not shed oocysts within 15 days after ingesting the feline coccidian, Isospora felis. Infection of the dogs was evidenced by the shedding of I. felis oocysts by cats consuming extra-intestinal organs of dogs fed I. felis. Likewise, cats became infected with the canine coccidian, Isospora canis, without producing oocysts. Dogs also became infected after ingesting mice previously fed I. canis oocysts. The prcpatent period for I. canis was slightly shorter in dogs fed infected mice (8–9 days) than in those fed oocysts (9–11 days).     

Liver histopathology and accumulation of melano-macrophage centres in Hoplias malabaricus after long-term food deprivation and re-feeding

In the Neotropical traíra Hoplias malabaricus , hepatocyte surface area declined after 30 days of fasting due to reserve utilization. Changes in normal organization of liver were not confirmed until 180 days of fasting. Severe histopathological changes occurred after 240 days. Pigment accumulation in the hepatocytes and increase in number and size (surface area) of melano-macrophage centres (MMC) were also verified during long-term food deprivation. The melano-macrophages were rich in ferric compounds, probably haemosiderin. This suggests that the activity of hepatic macrophages is related to the intense erythrocyte degradation that occurred in traíra following long-term food deprivation. After re-feeding for 30 days, the liver presented a partial restoration, but the large MMC remained. When compared to the respective starved group, the hepatocytes of re-fed fish increased in size, revealing recovering cell activity and storage of some energy reserves.     

Parameters of intestinal inflammation in mice given graded infections of the nematode Trichinella spiralis

Four parameters of the intestinal inflammatory response (numbers of mucosal mast cells (MMC) and Paneth cells, villus:crypt ratios and mitotic figures) were measured in mice exposed to varying doses of infective larvae of Trichinella spiralis.The aim of the experiments was to determine whether generation of these components of inflammation required a threshold level of infection and whether, once triggered, inflammation became pan-mucosal. Near maximal MMC and Paneth cell responses were elicited even with infections as low as 35 larvae; changes in villus:crypt ratios and in mitotic indices also occurred at this level of infection, but were progressively greater with increasing levels of infection. In all infected mice, including those infected with 35 larvae, MMC and Paneth cell responses extended over most of the small intestine. These data are interpreted as showing: (i) that the intestinal mucosa is highly responsive to T. spiralis infection; (ii) that once triggered, components of the inflammatory response are amplified by T cell-dependent mechanisms, becoming pan-mucosal; and (iii) that MMC and Paneth cell responses, which require cell division and differentiation, become maximal at a lower infection threshold than changes in the villus:crypt ratio or in mitotic indices, which directly reflect increased rates of division in crypt cells.     

Experimental cryptosporidiosis in fetal lambs

Fetal lambs were infected in utero with purified sporulated oocysts of Cryptosporidium parvum in order to study pathogenesis and host cellular response to the enteropathogen. Ileal loops (IL) of fetuses, 124-130 days of gestation, were inoculated with 1-4 x 10(6) oocysts usually via cannulae in the abdominal wall of the ewe. Oocysts, both free and phagocytosed, were observed in the IL content as early as day 1 post-inoculation (PI). The percentage of oocysts phagocytosed by the host's polymorphonuclear neutrophils (PMN's) and mononuclear cells remained high up to day 13, the last day of examination. Numerous parasites were observed at days 6, 7, and 12 PI in the microvilli of the ileum with hypercellularity of the lamina propria, which consisted of a mixed infiltration of PMN's, mononuclear cells, including lymphoid cells, and a few eosinophils. Cytolysis and extrusion of epithelial cells, often heavily parasitized by various stages of the parasite, as well as inflammatory cells, were prominent in luminal contents. Germinal centers were prominent in mesenteric lymph nodes draining the infected loops by day 12 PI. Depletion of lymphoid cells was already present in Peyer's patches by day 4 PI.     

True Intermediate Hosts for Eimeria funduli (Apicomplexa) from Estuarine Fishes1

Grass shrimp (Palaemonetes pugio) fed liver containing sporulated oocysts of Eimeria funduli permitted development of sporozoites that became infective to a variety of killifishes. The shrimp's gastric mill mechanically ruptured the oocysts. Sporozoites then excysted through an opening in the sporocyst, and by 12 and 13 h postinfection (p.i.) numerous empty sporocysts and free sporozoites occurred extracellularly in the intestine of the grass shrimp. Even at 5, 7, 8, 11, 46, 79, and 83 days p.i., and presumably for many months, numerous sporozoites still occurred free in the alimentary tract or between intestinal cells. The coccidium did not infect killifish at either 2 or 4 days p.i., but did at 5 days; after release from the sporocyst, it became more elongate with a distinct nucleus and two relatively large refractile bodies. Infections of E. funduli resulted in about one half of the fish that were fed either entire hepatopancreas or tips of hepatopancreas from experimentally infected shrimp. Feeding either the entire alimentary tract proximal to the first abdominal segment or any portion of that section from experimentally infected shrimp produced infections in nearly all tested fish. Feeding portions of the cephalothorax without any attached hepatopancreas or alimentary tract failed to produce an infection. Feeding killifish with wild grass shrimp from an enzootic area produced infections in only a fourth of the fish sample; however, feeding experimentally infected wild, laboratory-reared, and juvenile grass shrimp produced infections in nearly all fish. Palaemonid shrimps other than P. pugio also can serve as intermediate hosts for E. funduli, and these shrimps include Palaemonetes vulgaris, P. paludosus, P. kadiakensis, and Macrobrachium ohione. In contrast, a penaeid shrimp, mysidacean, amphipod, and crab fed liver with sporulated oocysts did not produce infections when fed to killifish.     

Effect of Tetramicra brevifilum (Microspora) infection on respiratory-burst responses of turbot (Scophthalmus maximus L.) phagocytes

In vitro assays were performed to investigate microsporidian-induced intracellular and extracellular production of reactive oxygen species (ROS) by peritoneal-exudate adherent (PEA) cells from turbot. ROS production was quantified using the fluorescent reagents OxyBURST Green H2HFF BSA (extracellular) and OxyBURST Green H2DCFDA succinimidyl ester (intracellular). Five days before assay, the cells had been elicited in vivo by intraperitoneal injection of sodium thioglycollate or spores of Tetramicra brevifilum. Elicitation with spores led to a marked increase in the proportion of neutrophils among PEA cells. PEA cells from normal turbot showed considerable extracellular and intracellular ROS production in response to microsporidian spores. By contrast, PEA cells from microsporidian-infected turbot showed considerably reduced extracellular and intracellular ROS production in response to microsporidian spores. Extracellular ROS production was affected by the addition of infected turbot serum to the assay medium, regardless of whether the PEA cells had been obtained from normal or infected fish. The presence of microsporidian-infected turbot serum significantly reduced intracellular ROS production by PEA cells elicited with microsporidian spores. These results suggest that (a) microsporidian spores partially suppress the repiratory-burst response of turbot phagocytes; and (b) infected turbot serum contains substances capable of modulating the respiratory-burst response of turbot phagocytes to microsporidian spores.     

Microsporogenesis and tapetal development in fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.)

Summary The development of sporogenous and tapetal cells in the anthers of male-fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.) plants was studied using light and transmission electron microscopy. In general, male-sterile anthers showed a much greater variability in developmental pattern than male-fertile anthers. The earliest deviation from normal anther development was observed to occur in sterile anthers at meiotic early prophase: there was a degeneration or irregular proliferation of the tapetal cells. Other early aberrant events were the occurrence of numerous small vesicles in the microspore mother cells (MMC) and a disorganized chromatin condensation. Deviations that occurred in sterile anthers at later developmental stages included: (1) less distinct inner structures in the mitochondria of both MMC and tapetal cells from middle prophase onwards. (2) dilated ER and nuclear membranes at MMC prophase, in some cases associated with the formation of protein bodies. (3) breakdown of cell walls in MMCs and tapetal cells at late meiotic prophase. (4) no massive increase in tapetal ER at the tetrad stage. (5) a general dissolution of membranes, first in the MMC, then in the tapetum. (6) abortion of microspores and the occurrence of a plasmodial tapetum in anthers reaching the microspore stage. (7) no distinct degeneration of tapetal cells after microspore formation. Thus, it seems that the factors that lead to abortive microsporogenesis are structurally expressed at widely different times during anther development. Aberrant patterns are not restricted to the tetrad stage but occur at early prophase.     

Response of splenic melanomacrophage centers of Oreochromis niloticus (Linnaeus, 1758) to inflammatory stimuli by BCG and foreign bodies

The study objective was to make histological, histochemical and morphometric evaluations on the splenic Melanomacrophage centers (MMCs) of tilapias, Oreochromis niloticus (Linnaeus, 1758), that were subjected to chronic inflammation stimuli by implantation (IMP) of a glass coverslip in the subcutaneous tissue and through inoculation of the bacillus Calmette‐Guerin (BCG). Randomly distributed in four groups were 150 tilapias: IMP (n = 45); IMP+BCG (n = 45); BCG (n = 45); and control (n = 15). Nine fish per treatment and three control fish were sampled on days 3, 7, 14, 21 and 33. The results demonstrated that increased numbers and areas of these structures were related to the type of stimulus, and that these were greater for the specific response. The principal pigment component identified was hemosiderin. Results suggest that the intensity of the MMC response in O. niloticus depended on the type of inflammatory stimulus used, and that it was greater in fish inoculated with BCG, which induced a granulomatous inflammation when compared to the foreign body inflammatory response induced by the glass coverslips.     

Tachyzoite-induced life cycle of Toxoplasma gondii in cats

The tachyzoite-induced cycle of Toxoplasma gondii was studied in 46 cats. Tachyzoites of the M-7741 or Me-49 strain of T. gondii were administered orally to cats by pouring into the mouth or by stomach tube, or by intraintestinal inoculation. Ten weaned cats that had been inoculated with tachyzoites directly in the intestine were killed 1, 3, 6, 9, 12, 15, 18, or 25 days later, and their tissues were studied histologically and bioassayed in mice. Toxoplasma gondii was demonstrable in the blood of 8 cats and in other tissues of all these 10. Four out of five 1- to 8-day-old cats fed tachyzoites by stomach tube became infected with T. gondii, and 1 became ill because of toxoplasmosis. All 19 weaned cats fed tachyzoites (poured into the mouth) became infected, and 6 died of acute toxoplasmosis 9-15 days after being fed T. gondii. Six out of 12 weaned cats fed tachyzoites by stomach tube became infected but were asymptomatic. Overall, 12 out of 26 cats observed for 19 days or more shed oocysts with a prepatent period (pp) of 19 days or more, with the sole exception of 1 cat that shed oocysts with a pp of 5 days. Enteroepithelial stages of T. gondii were not found in any cat before oocysts were shed. Cats shed up to 360 million oocysts in a day, and oocysts were shed for 4-6 days.     

Non-glial phagocytes within the degenerating optic nerve of the newt (Triturus viridescens).

Two non-glial phagocytes were found to participate along with ependymoglial cells in Wallerian degeneration of the severed optic nerve of the newt (Triturus viridescens). The first type of non-glial cell (polymorphonuclear phagocyte) was positively identified as a neutrophil and participates in the early stages of degeneration. Cells of this type make a brief appearance, reaching a peak by the second postoperative day (2 p.o.d.), and quickly diminish until few can be found by 4 p.o.d. Neutrophils invade the degenerating optic nerve from surrounding connective tissue spaces, most likely, through channels which penetrate the nerve parenchyma. The second type of non-glial cell is an invading mononuclear phagocyte which exhibits characteristics of microglial cells reported in other vertebrate species. Such cells appear in the nerve much later than the neutrophils and towards the end of Wallerian degeneration (6-10 p.o.d.). Their mode of entry and exit appears to be the same as that reported for neutrophils. The neutrophils and microglial-like, mononuclear phagocytes may serve to supplement the histolytic action of the ependymoglial cells, picking up scattered fragments of degenerating myelin and axons.     

Sporulation and Survival of Toxoplasma gondii Oocysts in Seawater

ABSTRACT. We have been collaborating since 1992 in studies on southern sea otters ( Enhdyra lutris nereis ) as part of a program to define factors, which may be responsible for limiting the growth of the southern sea otter population. We previously demonstrated Toxoplasma gondii in sea otiers. We postulated that cat feces containing oocysts could be entering the marine environment through storm run-off or through municipal sewage since cat feces are often disposed down toilets by cat owners. The present study examined the sporulation of T. gondii oocysts in seawater and the survival of sporulated oocysts in seawater. Unsporulated oocysts were placed in 1.5 ppt artificial seawater, 32 ppt artificial seawater or 2% sulfuric acid (positive control) at 24 C in an incubator. Samples were examined daily for 3 days and development monitored by counting 100 oocysts from each sample. From 75 to 80% of the oocysts were sporulated by 3 days post-inoculation under all treatment conditions. Groups of 2 mice were fed 10,000 oocysts each from each of the 3 treatment groups. All inoculated mice developed toxoplasmosis indicating that oocysts were capable of sporulating in seawater. Survival of sporulated oocysts was examined by placing sporulated T. gondii oocysts in 15 ppt seawater at room temperature 22–24 C (RT) or in a refrigerator kept at 4 C. Mice fed oocysts that had been stored at 4C or RT for 6 months became infected. These results indicate that T. gondii oocysts can sporulate and remain viable in seawater for several months.     

Enzyme Variation of Eimeria arizonensis from Peromyscus truei and P. boylii

ABSTRACT. Cricetid rodents, Peromyscus truei and P. boylii , were inoculated with sporulated oocysts of Eimeria arizonensis collected from wild P. truei maintained in the lab. In P. truei the prepatent period was 4–5 days, the patent period was 9–11 days, and sporulated oocysts were 21.5 × 25.0 (20–23 × 24–26) μm with sporocysts 7.7 × 12.0 (6–8 × 10–13) pm. In P. boylii the prepatent period was 6–7 days, the patent period was 8–9 days, and sporulated oocysts were 20.1 × 23.2 (18–22 × 21–24) pm with sporocysts 6.8 × 10.0 (5–8 × 9–12) pm. Sporulated oocysts from both host species were used in direct side-by-side comparison of isozyme banding patterns using protein electrophoresis. The parasite has polytypic loci for leucine aminopeptidase (LAP), lactate dehydrogenase (LDH), and 6-phosphogluconate dehydrogenase (6-PGD). In oocysts from P. truei , LAP showed one band with fast migration and LDH and 6-PGD each showed two bands, one with fast and one with slow migration. In oocysts from P. boylii , LAP and LDH each had one band with slow migration and 6-PGD had one band with moderate migration. Oocysts of E. arizonensis collected from P. boylii were used to inoculate P. truei. The prepatent and patent periods, structural measurements, and isozyrne banding patterns of the resultant oocysts were the same as those from P. truei when inoculated with oocysts from P. truei.     

Effects of sublethal concentrations of potassium dichromate on the occurrence of splenic melano-macrophage centres in juvenile plaice, Pleuronectes platessa, L.

Plaice were exposed to 0.5 and 2.0 ppm potassium dichromate (K₂Cr₂O₇) concentrations for 27 days. In both water concentrations chromium was accumulated up to a level of 400 ppb in the carcass of the fish. At the same time the number of splenic melano-macrophage centres (MMC) tripled, whereas their average size was reduced to one third. The percentage area of MMC in the splenic tissue remained unchanged. Histological alterations following exposure to K₂Cr₂O₇ included increases both in melanin content of the MMC and in the number of melanin-containing cells within blood vessels; also, the splenic ellipsoids became less clearly demarcated. Splenic melano-macrophages show a decreased ability to aggregate to form large MMC under the influence of K₂Cr₂O₇, which leads to the assumption that the turnover rate of macrophage settlement in, and macrophage elimination from, the spleen is increased.     

Hammondia pardalis sp. n. (Sarcocystidae) from the Ocelot, Felis pardalis, and Experimental Infection of Other Felines

Synopsis.
Hammondia pardals sp. n. (Eimeriorina: Sarcocystidae) from Panama Canal Zone is described as an obligate heteroxenous coccidian, with felids as the final host and laboratory mice as the experimental intermediate host. Ovoid oocysts. measuring 40.8 (36–46) × 28.5 (25–35) m. are shed unsporulated. Oocysts were infective only for the intermediate host. the laboratory mouse, Mus musculus , and the intracellular cysts were infective only for felids. Attempted passage of tissue cysts from mouse to mouse was unsuccessful.
Mice fed 5 × 10⁴ sporulated oocysts were found to harbor small intracellular cysts, 13–16 × 10–15 m, in the mesenteric lymph nodes, lungs, and intestinal submucosa 15 days postinfection. The meronts in these early cysts were stubby and measured 3 × 6 m. The prepatent period in the felids was 5 to 8 days and the patent period 5–13 days. Experimental infections of definitive hosts were successful with 6/6 domestic laboratory-reared kittens, Felis catus ; 5/5 ocelots, F. pardalis ; and 1/1 jaguarundi, F. yagouaroundi. None of the exposed raccoons, Procyon lotor , shed oocysts.     

The fate of Hepatozoon species naturally infecting Florida black racers and watersnakes in potential mosquito and soft tick vectors, and histological evidence of pathogenicity in unnatural host species.

Haemogregarine parasites, derived from the Florida snakes Coluber constrictor and Nerodia fasciata and ingested by Aedes aegypti, completed sporogony within the hemocoeles of nearly all fed mosquitoes in 14-18 days, and produced oocysts typical of Hepatozoon. However, mortalities and morbidity were high in the Culex which had fed on the Coluber. Oocysts were not found in any Ornithodoros turicata (Argasidae) which fed upon either snake host, but many sections of fed ticks had gametocyte-like cells within the gut lumen. Most lizards, Anolis carolinensis and Anolis sagrei, infected per os with oocysts derived from both snake species developed infections. Infections in the lizards were largely confined to hepatic schizonts with few parasites found in erythrocytes. Unlike naturally infected snake hosts, Hepatozoon schizonts in livers of lizards were often either surrounded by an unidentified dark pigment or heavily infiltrated with mononuclear inflammatory cells.     

Eimeria galateai sp. n. from the Paradise Kingfisher, and Eimeria duncani sp. n. from the Sacred Kingfisher in Papua New Guinea

SYNOPSIS. Eimeria galateai sp. n. from the paradise kingfisher ( Tanysiptera galatea Gray) and Eimeria duncani sp. n. from the sacred kingfisher ( Halcyon sancta Vigors & Horsfield) have been described from Papua New Guinea. Four of 11 paradise kingfishers were infected with E. galateai oocysts, measuring 13 (11–16) × 9 (8–11) μm. The oocysts were ovoid with nipple-like protrusion at one pole. Micropyle and polar granule were absent, while oocyst residuum (5 × 4 μm) was present. Sporocysts, measuring 5 (4–6) × 2 μm, were elongate-ovoid, and had a distinct convex Stieda body; the sporocyst residuum was absent. Two of 9 sacred kingfishers were infected with ovoid-truncated, 22 (19–25) × 16 (12–18) μm oocysts of E. duncani . Polar granule (5 × 2) was present in the oocysts, but there was no micropyle or oocyst residuum. Sporocysts were ovoid, measuring 9 (8–10) × 5 (4–6) μm, with a prominent Stieda body, and granular sporocyst residuum. Eimeria galateai and E. duncani are the first species of this genus to be described from birds of the order Coraciiformes.     

Painful,degenerating intervertebral discs up‐regulate neurite sprouting and CGRP through nociceptive factors

Intervertebral disc degeneration (IVD) can result in chronic low back pain, a common cause of morbidity and disability. Inflammation has been associated with IVD degeneration, however the relationship between inflammatory factors and chronic low back pain remains unclear. Furthermore, increased levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are both associated with inflammation and chronic low back pain, but whether degenerating discs release sufficient concentrations of factors that induce nociceptor plasticity remains unclear. Degenerating IVDs from low back pain patients and healthy, painless IVDs from human organ donors were cultured ex vivo. Inflammatory and nociceptive factors released by IVDs into culture media were quantified by enzyme‐linked immunosorbent assays and protein arrays. The ability of factors released to induce neurite growth and nociceptive neuropeptide production was investigated. Degenerating discs release increased levels of tumour necrosis factor‐α, interleukin‐1β, NGF and BDNF. Factors released by degenerating IVDs increased neurite growth and calcitonin gene‐related peptide expression, both of which were blocked by anti‐NGF treatment. Furthermore, protein arrays found increased levels of 20 inflammatory factors, many of which have nociceptive effects. Our results demonstrate that degenerating and painful human IVDs release increased levels of NGF, inflammatory and nociceptive factors ex vivo that induce neuronal plasticity and may actively diffuse to induce neo‐innervation and pain in vivo.     

Transplacental transmission and abortion in cows administered Neospora caninum oocysts

Neospora caninum infection is a common cause of bovine abortion. One method by which cattle can acquire infection is through ingestion of oocysts; however, this has not yet been proved to cause transplacental infection or abortion. In this study, 19 cows, pregnant between 70 and 176 days, were administered 1500 to 115,000 oocysts through an esophageal tube. Seventeen of the cows became seropositive, indicating acquisition of infection, whereas 8 negative control cows remained seronegative (P < 0.001). Offspring were examined using serology, histology, immunohistochemistry, parasite isolation, and polymerase chain reaction (PCR). Six offspring were infected and 1 of them was aborted. The aborted fetus had typical lesions and positive immunohistochemistry and PCR for N. caninum. All 6 cows with infected offspring had continuously rising antibody titers, whereas 10 of 11 infected cows with uninfected offspring had falling titers after an early apex. The risk of transplacental transmission was increased by later exposure times during gestation and by the dose of oocysts (P < 0.01 for the 2 combined variables). The lowest dose of oocysts, when administered after the 160th day of gestation, caused transplacental infection in 1 of 2 animals. This study demonstrates that infection with N. caninum oocysts can cause transplacental transmission and abortion in cattle.