Combination of herbal components (curcumin, carvacrol, thymol, cinnamaldehyde) in broiler chicken feed: Impacts on response parameters, performance, fatty acid profiles, meat quality and control of coccidia and bacteria.

The objective of this study was to determine whether curcumin and a commercial microencapsulated phytogenic supplement containing thymol, cinnamaldehyde and carvacrol in broiler chicken feed would improve health and meat quality (fatty acid profile), as well as to determine the coccidiostatic and bactericidal potential of the additives. The broiler chickens were divided into five groups: NC - negative control feed; PC - positive control; CU - with 50 mg/kg of curcumin, PHY - 100 mg/kg phytogenic; and PHY + CU, a combination of both additives at 50 mg/kg (curcumin) and 100 mg/kg (phytogenic). We observed significantly higher levels of total proteins associated with increased circulating globulins, as well as lower levels of uric acid, cholesterol and triglycerides in the PHY + CU group than in the NC. There were significantly fewer oocysts in birds supplemented with additives in the NC group on day 21; on day 35, the NC, PHY and PHY + CU groups had significantly lower counts than the PC and CU groups; however, at 44 days, the lowest counts were in PC group. The bacterial counts were significantly lower on day 21 in all groups that received additives than those of the control group; however, at 44 days, the bacterial and Escherichia coli counts in these groups were significantly higher than those of the control. Curcumin with or without phytogenic agent improved meat quality, with increased antioxidant levels and reduction of lipid peroxidation. There were significantly lower total saturated fatty acid levels and significantly greater monounsaturated/polyunsaturated fatty acid levels in broilers that consumed additives individually and in combination. The combination of additives significantly increased the crypt/villus ratio, a marker of improved intestinal health and performance. Additives potentiated their individual effects, suggesting they can replace conventional growth promoters without compromising health, intestinal mucosa or meat quality.

Life cycle of Cystoisospora felis (Coccidia: Apicomplexa) in cats and mice.

Cystoisospora felis is a ubiquitous apicomplexan protozoon of cats. The endogenous development of C. felis was studied in cats after feeding them infected mice. For this, five newborn cats were killed at 24, 48, 72, 96, and 120 h after having been fed mesenteric lymph nodes and spleens of mice that were inoculated with C. felis sporulated sporocysts. Asexual and sexual development occurred in enterocytes throughout the villi of the small intestine. The number of asexual generations was not determined with certainty, but there were different sized merozoites. At 24 h, merogony was seen only in the duodenum and the jejunum. Beginning at 48 h, the entire small intestine was parasitized. At 24 h, meronts contained 1-4 zoites, and at 48 h up to 12 zoites. Beginning with 72 h, the ileum was more heavily parasitized than the jejunum. At 96 and 120 h, meronts contained many zoites in various stages of development; some divided by endodyogeny. The multiplication was asynchronous, thus both immature multinucleated meronts and mature merozoites were seen in the same parasitophorous vacuole. Gametogony occurred between 96 and 120 h, and oocysts were present at 120 h. For the study of the development of C. felis in murine tissues, mice were killed from day 1 to 720 d after having been fed 10(5) sporocysts, and their tissues were examined for the parasites microscopically, and by bioassay in cats. The following conclusions were drawn. (1) Cystoisospora felis most frequently invaded the mesenteric lymph nodes of mice and remained there for at least 23 mo. (2) It also invaded the spleen, liver, brain, lung, and skeletal muscle of mice, but division was not seen based on microscopical examination. (3) This species could not be passed from mouse to mouse.

Developmental stages of Hepatozoon seurati (Laveran and Pettit 1911) comb. nov., a parasite of the corned viper Cerastes cerastes and the mosquito Culex pipiens from Egypt.

Developmental stages of Hepatozoon seurati (Laveran and Pettit 1911) comb. nov. are described from the tissues of the corned viper Cerastes cerastes, and from the vector Culex pipiens. The parasite described in the present study is firstly recorded as Haemogregarina seurati (Laveran and Pettit 1911) in the same host. After demonstration of the sporogonous development in the mosquito vector (C. pipiens) which showed all characteristics of the genus Hepatozoon (large oocysts containing many sporocysts producing numerous sporozoites), the parasite should be transferred into the genus Hepatozoon. The infected erythrocytes measured 20 ± 0.95 × 7.3 ± 0.85 µm; while uninfected cells measured 13.3 ± 1.04 × 7.5 ± 0.16 µm. Hypertrophy and faintly stained cytoplasm are mostly occurred in infected erythrocytes. Blood stages of the parasite were found exclusively in the erythrocytes in two forms: (1) small trophozoites (10.0 ± 0.52 × 3.0 ± 0.4 µm) and (2) long (mature) sausage-shaped (16.5 ± 1.5 × 3.5 ± 0.4 µm). Merogony occurred in the endothelial cells of the blood capillaries of lung, liver, and spleen. Mature meronts was 27.6 ± 0.7 × 17.5 ± 0.5 µm in diameter and contained 20-35 merozoites (averaged in 26). These merozoites measured 16.5 ± 1.5 × 3.5 ± 0.4 µm. Syzygy and gamogony occurred in the mosquito myxocoel till the 5th day post-infection (p.i.) while sporogony took place after 15 days p.i. On the third day p.i., a large spherical macrogamete of 29.0 ± 0.8 × 20.5 ± 0.6 µm containing a distinct nucleus in association with a single microgamete were observed. The microgamete was pyriform measured 8 ± 02 µm in length. It had a prominent nucleus and a long flagellum of at least 20.4 ± 1.3 µm in length. Fertilization occurred on the 3rd to the 4th days p.i. and the formed zygote developed into an oocyst in which repeated mitotic divisions with centripetal invaginations occurred producing sporoblasts. After sporulation, each sporoblast termed as sporocyst, and contained 18 banana-shaped sporozoites measured 14.0 ± 1.6 × 3.2 ± 0.6 µm. Experimental transmission was successful by intraperitoneal inoculation of the infective stages (sporozoites) to uninfected vipers and led to the appearance of blood stages after 5-6 weeks.

Description of gamontogonic and sporogonic stages of Hepatozoon spp. (Apicomplexa, Hepatozoidae) from Caudisoma durissa terrifica (Serpentes, Viperidae).

Three specimens of Caudisoma durissa terrifica infected with Hepatozoon spp. were studied. One was parasitized by one type of gamont and the other two were each infected by two morphologically different gamonts. Utilizing morphology and morphometry analysis, we concluded that three types of gamonts were very similar and may represent the same Hepatozoon species, but at least three different Hepatozoon species were infecting the C. durissa terrifica snakes in this study. Some of this species caused erythrocyte modifications. The sporogonic development of Hepatozoon sp. was observed from 12 h to the 20th day after female Culex quinquefasciatus blood meals.

Effect of diatomaceous earth on parasite load, egg production, and egg quality of free-range organic laying hens.

The effectiveness of diatomaceous earth (DE) as a treatment against parasites and to increase feed efficiency and egg production of organically raised free-range layer hens was evaluated in 2 breeds of commercial egg layers [Bovan Brown (BB) and Lowmann Brown (LB)] that differ in their resistance to internal parasitic infections. Half the hens of each breed were fed diets supplemented with DE (2%). Their internal parasite loads were assessed by biweekly fecal egg counts (FEC) and by postmortem examination of the gastrointestinal tract. Supplementing DE in diets of LB hens, the more parasite-resistant breed, did not significantly affect their FEC and adult parasite load. However, BB hens treated with dietary DE had significantly lower Capillaria FEC, slightly lower Eimeria FEC, fewer birds infected with Heterakis, and significantly lower Heterakis worm burden than control BB hens. Both BB and LB hens fed the diet containing DE were significantly heavier, laid more eggs, and consumed more feed than hens fed the control diet, but feed efficiency did not differ between the 2 dietary treatments. Additionally, BB hens consuming the DE diet laid larger eggs containing more albumen and yolk than hens consuming the control diet. In a subsequent experiment, the effectiveness of DE to treat a Northern fowl mite (Ornithonyssus sylviarum) infestation was tested. Relative to controls, both breeds of hens that were dusted with DE had reduced number of mites. The results of this study indicate the DE has the potential to be an effective treatment to help control parasites and improve production of organically raised, free-range layer hens.

Haemogregarine blood parasites in the lizards Podarcis bocagei (Seoane) and P. carbonelli (Pérez-Mellado) (Sauria: Lacertidae) from NW Portugal.

In Iberian and Canarian lizards, haemogregarines have been recorded infecting erythrocytes, but most of the records correspond to mature gametocytes. We analysed blood smears from 75 specimens of Podarcis bocagei (Seoane) and 33 specimens of P. carbonelli (Pérez-Mellado) from localities of north-western Portugal. We found haemogregarines in 74.7% of P. bocagei and 69.7% of P. carbonelli. Our observations show characteristics of the haemogregarines other than the morphology of the mature gametocytes. In histological sections of the liver of four hosts latent cysts with sporozoites and meronts with merozoites were detected. Both traits have been described as typical of the genera Hepatozoon Miller, 1908 and Hemolivia Petit, Landau, Baccam & Lainson, 1990. We suggest that not only P. bocagei and P. carbonelli from Portugal but other species of Iberian and Canarian lacertids might also be infected by species belonging to one or both genera.

Complete development of Cryptosporidium in cell culture.

Protozoan parasites of the genus Cryptosporidium cause a short-term, flu-like, gastrointestinal illness in immunocompetent persons and severe, persistent, life-threatening diarrhea in immunodeficient individuals. No effective therapy is available for the treatment of cryptosporidiosis in the immunodeficient host. Complete development (from sporozoite to sporulated oocyst) of a human isolate of Cryptosporidium was achieved in cultured human fetal lung cells and primary chicken kidney and porcine kidney cells. The growth of this newly recognized zoonotic agent in cell culture now provides a means of studying its behavior, development, and metabolism, and a mechanism for evaluation of potentially useful therapeutic agents.

Hammondia hammondi: A new coccidium of cats producing cysts in muscle of other mammals.

Predominant muscle parasitism, and an obligatory two-host cycle (cat-mouse-cat), distinguishes an otherwise similar organism from Toxoplasma. The presence of multiplicative stages in the cat gut separate it from Sarcocystis. Antibody that cross reacts with Toxoplasma antigen is developed in mice and other experimental intermediary hosts, but not in cats, the final host. Recognition of the two-host cycle is essential for the experimental isolation and transmission of the parasite, and for prevention of the infection.

The evolution of the knowledge of cat and dog coccidia.

Before the discovery of Toxoplasma gondii as a coccidium of the cat in 1970, cat and dog coccidia were classified in the genus Isospora and considered of little clinical or zoonotic significance. Since 1970, several new (Hammondia sp., Neospora sp.) and previously described species, including Sarcocystis, Besnoitia, and Cryptosporidium have been found as coccidians of cats and dogs with clinical and zoonotic significance. In the present paper I review salient features of the evolution of cat and dog coccidia.

Effect of pine bark (Pinus radiata) extracts on sporulation of coccidian oocysts.

A series of experiments have been undertaken to determine the effect of water extracts from pine bark (Pinus radiata) on the inhibition of the sporulation of oocysts of three species of avian coccidia. Tubes containing coccidian oocysts isolated from droppings of coccidia-infected chickens were randomly assigned to 0, 250, 500 and 1000 microg/ml pine bark extract (PBE). The tubes were incubated at 25-29 degrees C for 48 h depending on the species of Eimeria. Sporulation inhibition bioassay was used to evaluate the activity of PBE on the sporulation of coccidian oocysts. The oocysts were gently aerated with an air pump away from sun light. The results show for the first time that water-soluble extracts from pine bark containing 35% condensed tannins have anticoccidial activity as evidenced by their ability to decrease significantly the sporulation of the oocysts of three species of Eimeria, namely Eimeria tenella (Railliet et Lucet, 1891), E. maxima Tyzzer, 1929 and E. acervulina Tyzzer, 1929, under laboratory conditions. Incubation of unsporulated oocysts of these parasites in water containing 500 microg PBE per ml resulted in inhibition of sporulation of these oocysts by about 28-84% relative to the oocysts in the control incubations. In addition, up to 12% of E. maxima oocysts exposed to 500-1000 microg pine bark/ml were containing abnormal sporocysts in terms of size, number and shape.

Hepatozoon ursi n. sp. (Apicomplexa: Hepatozoidae) in Japanese black bear (Ursus thibetanus japonicus).

Morphological and genetic features of a new Hepatozoon species, Hepatozoon ursi n. sp., in Japanese black bear (Ursus thibetanus japonicus) were studied. Schizogonic developmental stages were observed in the lungs of Japanese black bears. The schizonts were sub-spherical in shape and 45.7+/-4.6 x 42.7+/-4.5 microm in size. Each mature schizont contained approximately 80-130 merozoites and 0-5 residual bodies. The merozoites were 7.0+/-0.7 x 1.8+/-0.3 microm in size. Intraleukocytic gametocytes were slightly curved, cigar-like in shape and had a beak-like protrusion at one end. The size of the gametocytes was 10.9+/-0.3 x 3.3+/-0.2 microm. The analyses of the18S rRNA gene sequences supported the hypothesis that H. ursi n. sp. is different from other Hepatozoon species. Mature Hepatozoon oocysts were detected in two species of ticks (Haemaphysalis japonica and Haemaphysalis flava) collected on the bears infected with H. ursi n. sp. Two measured oocysts were 263.2 x 234.0 microm and 331.8 x 231.7 microm, respectively. The oocysts contained approximately 40 and 50 sporocysts, respectively. The sporocysts were sub-spherical in shape and 31.2+/-2.5 x 27.0+/-2.9 microm in size. Each sporocyst contained at least 8-16 sporozoites, with the sporozoites being 12.2+/-1.4 x 3.5+/-0.5 microm in size. H. ursi n. sp. is the first Hepatozoon species recorded from the family Ursidae.

Klossiella quimrensis (Apicomplexa: Klossiellidae) causes renal coccidiosis in western barred bandicoots Perameles bougainville (Marsupialia: Peramelidae) in Western Australia.

Previous studies have described a range of Klossiella species parasitic in marsupial hosts. Klossiella quimrensis is the etiologic agent of renal coccidiosis in the peramelid marsupial hosts Isoodon obesulus and Perameles gunnii in Eastern Australia, but there is no previous report of klossiellosis in Western Australian peramelids. This study describes klossiellosis diagnosed by histology of renal tissue sections collected during necropsy of 20 Perameles bougainville between 2000 and 2005. Sporonts, sporoblasts, and macrogametes were identified within parasitophorous vacuoles of epithelial cells located near the renal corticomedullary junction. The prevalence of renal coccidiosis in P. bougainville diagnosed by renal histology is estimated at 30%. Only a single unsporulated sporocyst was detected by examination of cystocentesis-collected urine, indicating that microscopic evaluation of urine samples is an insensitive diagnostic test for detection of K. quimrensis in P. bougainville. This infection in P. bougainville is indirectly associated with mild multifocal interstitial lymphohistiocytic nephritis and is likely to be only minimally pathogenic in otherwise healthy individuals. Our study also extends the host and geographic range of K. quimrensis to include P. bougainville and Western Australia.

A new species of Hepatozoon (Apicomplexa: Adeleorina) from Python regius (Serpentes: Pythonidae) and its experimental transmission by a mosquito vector.

Hepatozoon ayorgbor n. sp. is described from specimens of Python regius imported from Ghana. Gametocytes were found in the peripheral blood of 43 of 55 snakes examined. Localization of gametocytes was mainly inside the erythrocytes; free gametocytes were found in 15 (34.9%) positive specimens. Infections of laboratory-reared Culex quinquefasciatus feeding on infected snakes, as well as experimental infection of juvenile Python regius by ingestion of infected mosquitoes, were performed to complete the life cycle. Similarly, transmission to different snake species (Boa constrictor and Lamprophis fuliginosus) and lizards (Lepidodactylus lugubris) was performed to assess the host specificity. Isolates were compared with Hepatozoon species from sub-Saharan reptiles and described as a new species based on the morphology, phylogenetic analysis, and a complete life cycle.

Genomic and Proteomic Evidence for the Presence of a Peroxisome in the Apicomplexan Parasite Toxoplasma gondii and Other Coccidia.

Apicomplexans are successful parasites responsible for severe human diseases including malaria, toxoplasmosis, and cryptosporidiosis. For many years, it has been discussed whether these parasites are in possession of peroxisomes, highly variable eukaryotic organelles usually involved in fatty acid degradation and cellular detoxification. Conflicting experimental data has been published. With the age of genomics, ever more high quality apicomplexan genomes have become available, that now allow a new assessment of the dispute. Here, we provide bioinformatic evidence for the presence of peroxisomes in Toxoplasma gondii and other coccidians. For these organisms, we have identified a complete set of peroxins, probably responsible for peroxisome biogenesis, division, and protein import. Moreover, via a global screening for peroxisomal targeting signals, we were able to show that a complete set of fatty acid ß-oxidation enzymes is equipped with either PTS1 or PTS2 sequences, most likely mediating transport of these factors to putative peroxisomes in all investigated Coccidia. Our results further imply a life cycle stage-specific presence of peroxisomes in T. gondii and suggest several independent losses of peroxisomes during the evolution of apicomplexan parasites.

The flea, Megabothris abantis: an invertebrate host of Hepatozoon sp. and a likely definitive host in hepatozoon infections of the montane vole, Microtus montanus.

In searching for an invertebrate host for Hepatozoon sp. infecting the montane vole (Microtus montanus), we collected fleas, ticks, and mites from live-trapped voles and searched squash preparations for Hepatozoon oocysts. From 1989 through 1996, we identified six species of fleas in Grand Teton National Park: Megabothris abantis, Megabothris asio megacolpus, Aetheca wagneri, Peromyscopsylla selenis, Peromyscopsylla. hesperomys, and Hystrichopsylla dippiei dippiei. We found Hepatozoon oocysts only in M. abantis; we found no oocysts in mites or ticks. We conclude that M. abantis is an invertebrate host of Hepatozoon sp. and is likely to be the definitive host for theHepatozoon spp. of M. montanus.

Diagnosis of Hepatozoon spp. in Amblyomma ovale and its experimental transmission in domestic dogs in Brazil.

Transmission of Hepatozoon spp. to dogs was investigated using four species of ixodid ticks: Rhipicephalus sanguineus, Amblyomma aureolatum, Amblyomma ovale and Amblyomma cajennense. We collected completely or partially engorged adult ticks of these species from dogs that were naturally infested and positive for Hepatozoon spp. We selected some of these ixodids and inoculated them orally in four negative dogs. The other ticks were dissected and examined for oocysts. Of all dogs inoculated orally with R. sanguineus, A. aureolatum, A. cajennense and A. ovale, only the animal that received the macerate of A. ovale was positive; evidence (gametocytes in peripheral blood) of infection was found 63 days after inoculation. Among all dissected ticks, we found only two oocysts; these were similar to those of Hepatozoon canis, and both were recovered from a single A. ovale specimen. We inoculated sporozoites recovered from the oocysts intraperitoneally into a Hepatozoon spp. negative dog, and circulating gametocytes were detected 84 days later. Our study demonstrated that A. ovale can be a vector of Hepatozoon spp. in Brazil.

Intestinal coccidiosis in bluegill, Lepomis macrochirus.

Developmental stages of a coccidial parasite were observed in young-of-year bluegill (Lepomis macrochirus) from an impoundment lake in Norfolk County, Virginia. The fish were anorexic and lethargic. Necropsy examination revealed emaciated bluegill with little or no abdominal fat and no food in the stomach or intestines. Coccidia were present in the posterior intestine in moderately large numbers. Few sporulated oocysts were present, and identification to genus was not possible. Epithelial cell destruction, sloughing of the intestinal mucosa, and hemorrhage were associated with the developing coccidial parasites. Coccidia were not observed within other organ systems. No pathogenic bacteria were isolated from the fish tissues. Our findings indicate that intestinal coccidiosis may pose a significant health problem in young bluegill.

A primary culture of haemocytes isolated from Gryllus bimaculatus (Orthoptera, Gryllidae) and their interactions with two intracellular parasites--Paranosema grylli (Microsporidia) and Adelina grylli (Coccidia).

Cricket haemocytes were derived from either haemolymph or haemopoietic organs (lymph glands) of insects and introduced to a primary culture. Varied isolation protocols, tissue culture vessels, media compositions and cell densities were tested to determine the optimal conditions for in vitro maintenance of haemocytes, and for subsequent light and electron microscopic analysis of monolayers. Freshly prepared Mitsuhashi and Maramorosh (MM;Sigma, Steinheim, Germany) insect medium (420 mOsm), buffered with sodium bicarbonate (pH 7.2) and supplemented with 10 % FCS, was found to be most appropriate for haemocyte maintenance. All tested tissue culture vessels (FLEXiperm units, multiwell plates and Thermanox slides, with the exception of Melineux agar plates), were suitable for cell attachment and haemocyte monolayers formation. Viability of cultured cells was confirmed by LIVE/DEAD Viability/Cytotoxity Kit for Eukaryotic Cells. Free circulating haemocytes were cultivated up to 27 days and then degraded. Infection with the microsporidian Paranosema grylli or the coccidian Adelina grylli caused noticeable swelling of host lymph glands (haemopoietic tissue) and increase in the number of cells comprising the glands. The cells derived from haemopoietic tissue were maintained for maximum 5 days; thereafter multiplication of bacteria normally inhabiting cricket lymph glands destroyed monolayers and killed the cells. Microsporidian and coccidian invasive stages (spores and sporozoites, respectively) were isolated from infected tissues, resuspended in MM medium and added to haemocyte monolayers in ratios 1 zoite per haemocytes or 10 spores per 1 haemocyte. Actively moving zoites contacted and penetrated the cultured cells. Unlike coccidian zoites, microsporidian spores were phagocytized by haemocytes. Application of fluorescent LIVE/DEAD kit allowed to visualize internalized parasites inside host cells as clearly shaped dark areas. The present study has demonstrated that 1) cricket haemocytes from both circulating haemolymph and lymph glands can be short-term cultivated on tissue culture vessel surfaces which made possible their further light and electron microscopic analysis; 2) short-term haemocyte cultures may be employed to study host-parasite interactions, in particular, to follow the initial steps of parasite internalization inside host cell; 3) Fluorescent assay with Viability/Cytotoxity Kit for Eukaryotic Cells (Molecular Probes, Oregon) allows to observe penetration of these parasites into cultured cells.

Food- and waterborne pathogens: you are (infected by) what you eat!

Coccidia are protozoan parasites responsible for disease worldwide. The orally transmissible stages of coccidia make them food- and waterborne threats. The occurrence of multiple, human-infectious coccidia with diverse life cycles suggests that alterations in host range are a frequent occurrence, and can underlie the rapid emergence of pathogens.

Cryptosporidiosis in perspective.

In this review I have examined the vast literature which has accumulated on Cryptosporidium, particularly in the past 3 years, in an attempt to highlight areas in which progress has been made in relation to the organism and the disease, and to indicate areas in which knowledge is still lacking. Since 1982, a global effort by scientists and clinicians has been directed towards determining the nature of the disease in humans and the relative contribution of cryptosporidiosis to gastroenteritis. From published data, the incidence of diarrhoea is 1-5% in most developed countries, and 4-7% in less developed countries, when measured throughout the year and in all age groups. The frequency of cryptosporidiosis is highest in children aged between 6 months and 3 years, and in particular locations (e.g., day-care centres) and at particular times of the year. Although susceptibility to infection is life-long, one suspects that the lower prevalence among older children and adults is due to immunity acquired from frequent exposure. Other important factors contributing to higher prevalence are the season--it is more frequent in a wet, warm climate--association with travel to particular destinations, poor hygiene, intimate contact with certain animals, and congregation of large numbers of young previously unexposed children in day-care centres. The association between cryptosporidiosis and giardiasis presumably results from the existence of a common source of infection. The immune status of the host appears to be a major determinant of whether the infection is self-limiting or persistent. It is clear that both branches of the immune system are required for complete recovery, since T-lymphocyte dysfunction or hypogammaglobulinaemia can both lead to persistent illness. Chronic diarrhoea and malabsorption attributed to cryptosporidiosis also occur in the absence of evidence of immune defect. The importance of respiratory tract infection in humans, other than in the terminal stages of chronic illness, requires investigation. The infection has now been identified in all classes of vertebrates; it has been observed in all domestic animals including pets, and a wide range of wildlife including birds. Cryptosporidiosis seems to cause diarrhoea in young ruminants, less frequently in pets. In birds the parasite has been observed in the gastrointestinal tract, without ill effect, and in the respiratory tract, in which clinical symptoms of variable severity have been described. The mucosal response of the gastrointestinal tract to infection appears to vary among mammals and may be the key to the variable clinical manifestations observed.(ABSTRACT TRUNCATED AT 400 WORDS)