Lethal infection caused by Tetratrichomonas gallinarum in black swans (Cygnus atratus).

BACKGROUND: Tetratrichomonas gallinarum is parasitic protozoa with a wide host range. However, its lethal infection is rare reported. CASE PRESENTATION: Here, we described the first lethal cases of T. gallinarum infection in black swans in China. Five black swans died within a week in succession without obvious symptoms except mild diarrhea. At necropsy, severe lesions were observed in caeca with thickened caecal walls and hemorrhages in the mucosa. A large number of moving trophozoites were found in the contents of the cecum by microscopic examination. The livers were enlarged with multiple bleeding spots on the surface. Histopathology of the livers showed mononuclear cell infiltration and moderate hyperplasia of fibrous tissue. The histopathology of the cecum showed that the villi of the cecum were edematous. Finally, the presence of T. gallinarum was determined by specific PCR andin-situ hybridization assay. Additionally, common pathogens that can cause similar symptoms were excluded. CONCLUSIONS: The death of the black swan was caused by T. gallinarum, suggesting that the parasite might be a new threat to the Cygnus birds.

Spotlight on Histomonosis (blackhead disease): a re-emerging disease in turkeys and chickens.

Histomonosis, or blackhead disease, is a well-known disease in turkeys that can cause high mortality, but outbreaks with lower losses are also observed. The disease is less fatal in chickens but is economically important due to reduced performance and its co-appearance with colibacillosis. The lack of specific prophylactic and therapeutic interventions has led to a re-emergence of the disease in recent years, mainly in turkeys, free-range layers and chicken parent stock.

Molecular and pathological investigations of Plasmodium parasites infecting striped forest whiptail lizards (Kentropyx calcarata) in Brazil.

The genus Plasmodium (Plasmodiidae) ranks among the most widespread intracellular protozoan parasites affecting a wide range of mammals, birds, and reptiles. Little information is available about lizard malaria parasites in South America, and the pathological features of the resulting parasitoses remain unknown or poorly understood. To partially fill in these gaps, we conducted blood smear analysis, molecular detection, and phylogenetic and pathological investigations in lizards inhabiting an Atlantic Forest fragment in Paraiba, Brazil. From 104 striped forest whiptails (Kentropyx calcarata) screened for the presence of haemosporidian parasites, 67 (64.4%) were positive. Four of five Amazon lava lizards (Strobilurus torquatus) we collected from this same area were also positive. A total of 27 forest whiptails were infected with a new genetic lineage of Plasmodium kentropyxi and other Plasmodium lineages were also detected. Histopathological analysis in infected forest whiptails revealed systemic intraerythrocytic Plasmodium stages, mainly gametocytes, in the liver, lung, and heart. Also, the liver of infected lizards had mild to moderate levels of Kupffer cell and melanomacrophage hypertrophy/hyperplasia with sinusoid leukocytosis. Overall, our findings suggest that an endemic Plasmodium species causes histological alterations that are not related to major pathological processes in striped forest whiptails.

Preliminary study of Cytauxzoon felis infection in outdoor cats in Mashhad, Iran.

The objectives of the current study were to assess the preliminary status of Cytauxzoon felis (C. felis) infection among outdoor cats in Mashhad, Iran and also to compare clinicopathological findings between C. felis infected and non-infected cats. Blood samples were collected from 100 outdoor domestic cats between April and September in 2019. Infection with C. felis was determined using microscopic observation of giemsa-stained blood smears and molecular analysis. The piroplasms was microscopically detected in 5 (5%) of the blood smears with low parasitemia. The presence of C. felis was confirmed in one positive microscopy sample by PCR. The molecular assay revealed that 19 cats (19%) were infected with C. felis. Hematological and some serum biochemical factors were evaluated in both of the infected and non-infected cats. There was no association between C. felis infection and age, gender, and laboratory findings except for hematocrit (Hct) and concentration of total protein and globulin. Clinical signs such as fever, dehydration, lethargy, and icterus were observed only in 15.78% (3/19) of the infected cats, while 84.22% (16/19) were asymptomatic. Laboratory findings such as non-regenerative anemia, thrombocytopenia, neutrophilic leukocytosis hyperproteinemia, hypoalbuminemia, and hyperbilirubinemia were detected in the clinically infected cats. This study revealed the relatively high frequency of C. felis infection in outdoor domestic cats in Mashahd, Iran. The predominance of asymptomatic infection likely indicates that these cats may be infected with low-virulent strains of C. felis.

Pathology and Case Definition of Severe Perkinsea Infection of Frogs.

Severe Perkinsea infection (SPI) is an emerging disease of frogs responsible for mass mortalities of tadpoles across the United States. It is caused by protozoa belonging to the phylum Perkinsozoa that form a distinct group referred to as the Pathogenic Perkinsea Clade of frogs. In this work, we provide detailed description of gross and histologic lesions from 178 naturally infected tadpoles, including 10 species from 22 mortality events and 6 amphibian health monitoring studies from diverse geographic areas. On external examination, we observed abdominal distension (10, 5.6%), cutaneous erythema and petechia (3, 1.7%), subcutaneous edema (3, 1.7%), and areas of white skin discoloration (3, 1.7%). On macroscopic examination of internal organs, we found hepatomegaly (68, 38.2%), splenomegaly (51, 28.7%), nephromegaly (47, 26.4%), ascites (15, 8.4%), segmental irregular thickening and white discoloration of the intestine (8, 4.5%), pancreatomegaly (4, 2.2%), and pancreatic petechia (1, 0.6%). Histologically, over 60% of the liver (148/165, 89.7%), kidney (113/147, 76.9%), spleen (96/97, 99%), and pancreas (46/68, 67.6%) were invaded by myriad intracellular and extracellular Perkinsea hypnospore-like and trophozoite-like organisms. Numerous other tissues were affected to a lesser extent. Mild histiocytic inflammation with fewer lymphocytes or eosinophils was commonly observed in areas of infection that were not obscured by lympho-granulocytic hematopoietic tissue. In light of these observations, we suggest a logical pathogenesis sequence. Finally, we propose a "case definition" for SPI to promote standardized communication of results and prevent misdiagnosis with epidemiological and pathologically overlapping diseases such as ranavirosis.

Fatal Leucocytozoon Infection in a Captive Grey-headed Parrot (Poicephalus robustus suahelicus).

A necropsy was conducted on a female grey-headed parrot (Poicephalus robustus suahelicus) that died following signs of depression, ruffled feathers, and inappetence. Microscopic examination revealed the presence of hemoprotozoa in the liver. A nested polymerase chain reaction (PCR), targeting the mitochondrial cytochrome b gene of Haemoproteus species, Plasmodium species, and Leucocytozoon species, was performed on frozen tissue samples collected at necropsy. The hemoprotozoa were identified by PCR analysis as Leucocytozoon species. Hemoprotozoa are rarely reported in African parrots, and this is the first report of a Leucocytozooon species infection in a Poicephalus robustus suahelicus.

Stress effects of amyloodiniosis in gilthead sea bream Sparus aurata.

Amyloodiniosis is a parasitological disease caused by one of the most common and important parasitic dinoflagellates in fish, Amyloodinium ocellatum (Brown), that represents a major bottleneck for gilthead seabream semi-intensive aquaculture in Southern Europe. In this experiment, we analyzed some metabolic, osmoregulatory and stress indicators to elucidate some of the physiological responses of gilthead sea bream when exposed to an A. ocellatum outbreak. We observed significant differences between Control and Infection groups in the cortisol, lactate and gill Na+/K+-ATPase (NKA) activity levels but that glucose, osmolarity, pH and total protein did not present such differences. This could indicate that the presence of the parasite induced a stress response, possibly enhancing the metabolization of glucose and subsequently lactate to cope with the higher energy requirements of the organism. There was also a decrease in gill NKA activity possibly due to severe epithelial damage and increased mucus production caused by the parasite A. ocellatum, which could induce anoxia and osmoregulatory impairment in the organism. However, further works must be performed to fully understand the physiological reactions of fish for A. ocellatum outbreaks.

An Outbreak of Histomoniasis in Backyard Sanhuang Chickens.

Histomonas meleagridis is a facultative anaerobic parasite, which can cause a common poultry disease known as histomoniasis. The species and age of the birds impacts on the susceptibility, with turkey being the most susceptible species. Chickens are less susceptible to H. meleagridis than turkeys and usually serve as reservoir hosts. Here, the diagnosis of an outbreak of histomoniasis in backyard Sanhuang chickens is described. The primary diagnosis was made based on clinical symptoms, general changes at necropsy, histopathology, and the isolation and cultivation of parasites. The pathogen was further confirmed by cloning, PCR identification, and animal inoculation tests. A strain of H. meleagri- dis, named HM-JSYZ-C, with a higher pathogenicity level in chickens was obtained. The study lays a foundation for further investigations into H. meleagridis and histomoniasis in chickens.

CLINICAL EFFECT OF HEMOPARASITE INFECTIONS IN SNOWY OWLS ( BUBO SCANDIACUS).

Vector-borne hemoparasites are commonly found in avian species. Plasmodium spp., the causative agent of avian malaria, are intraerythrocytic parasites that can cause signs ranging from subclinical infection to severe acute disease. In raptor species, most hemoparasites are associated with subclinical infection and are generally not treated when seen on blood evaluation. This case series reviews five cases of hemoparasite infection in snowy owls ( Bubo scandiacus). These animals were infected with a variety of hemoparasites, including Plasmodium, Haemoproteus, and Leukocytozoon spp. Death of one of these birds due to hemoparasite burden led to a change in the monitoring for and treatment of subclinical hemoparasitic infections in this species. Three subsequently infected snowy owls have been treated with primaquine and chloroquine. The birds that were treated survived infection, and parasite burdens in peripheral blood diminished. Postulated reasons for increased morbidity and mortality associated with hemoparasitic infections in captive snowy owls, as opposed to other raptor species, include stress, concurrent disease, novel pathogen exposure, and elevated environmental temperatures.

Pathogenesis of mixed infection by Spironucleus sp. and Citrobacter freundii in freshwater angelfish Pterophyllum scalare.

The present study was carried out to identify and describe the pathology of the freshwater angelfish Pterophyllum scalare during chronic mortality in an in-door aquaculture system. Scraping of the integument and gills and the collection of intestinal contents to search for external and internal parasites were performed. Kidneys were collected aseptically for the microbiological analysis and the isolates were subjected to antibiotics to test for susceptibility. Subsequently, necropsy for macroscopic assessment and collection of internal organs for histopathology were performed. The fish exhibited lethargy, lip tumor, hemorrhage and liver granuloma. No ectoparasites were diagnosed. Endoparasites of the genus Spironucleus were found in large numbers in the intestine of the affected fish. In the microbiological analysis, Citrobacter freundii was isolated from the kidney and identified by colony PCR. This bacterium showed susceptibility to three of the eight antibiotics evaluated: ciprofloxacin, cefoxitin and tetracycline. For the pathological analysis, liver and spleen granulomas were present. In the intestinal tissue, a large and unusual amount of mast cells and their free granules were described and discussed in detail. The present study showed that mast cells play an important role during the chronic infection of freshwater angelfish.

Mechanisms of mortality in Culicoides biting midges due to Haemoproteus infection.

We examined the effects of Haemoproteus infection on the survival and pathology caused in the biting midges. Forty-six females of Culicoides impunctatus were exposed experimentally by allowing them to feed on a naturally infected red-backed shrike infected with Haemoproteus lanii (lineage hRB1, gametocytaemia 5·2%). Seventeen females were fed on an uninfected bird (controls). Dead insects were collected, counted and used for dissection, histological examination and polymerase chain reaction-based testing. Parasites were present in all experimentally infected biting midges, but absent from control insects. Survivorship differed significantly between the control and infected groups. Twelve hours post-exposure (PE), 45 (98%) experimentally infected midges were dead, but all control midges remained alive, and many of them survived until 7 day PE. The migrating ookinetes of H. lanii overfilled midgut, markedly damaged the midgut wall, entered the haemocoel and overfilled the abdomen and thorax of exposed biting midges. Massive infection by migrating ookinetes led to damage of abdomen and thorax of biting midges. The parasites often present in large clumps in the haemocoel in abdomen and thorax, leading to the interruption of the haemolymph circulation. These are the main reasons for rapid death of biting midges after feeding on high-intensity infections of Haemoproteus parasites.

Gray meat in the Atlantic sea scallop, Placopecten magellanicus, and the identification of a known pathogenic scallop apicomplexan.

Atlantic sea scallop (Placopecten magellanicus) meats are normally firm and creamy white. However, scallops with small, darkened and stringy adductor muscle (gray meat) episodically occur along the Eastern Seaboard, most recently in the rotational management areas of Georges Bank after extended fishing closures. These gray meat scallops are associated with reduced harvestable biomass and mass mortality events. We tested age, nutritional stress and disease as causative agents for this condition. Adult scallops of different shell heights (SH) ranging from (90-145mm) were collected from Georges Bank and analyzed for meat quality and the presence of pathogens using biochemical, histopathological and molecular methods. Gray meat occurrence was weakly correlated with shell height only explaining 8.49% of the variance in a generalized additive model (GAMS). Gray meat weights were lower than white meat (p<0.001) and there was a dramatic reduction in protein content (p<0.05) in gray meat scallops associated with extensive myodegeneration. Amino acid profiles confirmed the breakdown of muscle tissue with an increase in free hydroxyproline in gray meat scallops. Infection by an apicomplexan parasite was detected in the muscle tissue of all gray meat scallops tested. An intermediate pathology stage (brown meat) was also identified. As the parasitic infection increased, meat quality decreased. Numerous developmental stages of the parasite were present in various organs of the scallops. This apicomplexan has an identical SSU rDNA sequence to a novel parasite occurring in the Iceland scallop during a recent mass mortality event. The range of this parasite in Atlantic sea scallops and the effect of abiotic/biotic stressors on pathogenicity are currently unknown. Results from this study link an apicomplexan species, known to be highly pathogenic in scallops, to gray meat occurrence with a potentially high impact on the fishery.

Protozoal-related mortalities in endangered Hawaiian monk seals Neomonachus schauinslandi.

Protozoal infections have been widely documented in marine mammals and may cause morbidity and mortality at levels that result in population level effects. The presence and potential impact on the recovery of endangered Hawaiian monk seals Neomonachus schauinslandi by protozoal pathogens was first identified in the carcass of a stranded adult male with disseminated toxoplasmosis and a captive monk seal with hepatitis. We report 7 additional cases and 2 suspect cases of protozoal-related mortality in Hawaiian monk seals between 2001 and 2015, including the first record of vertical transmission in this species. This study establishes case definitions for classification of protozoal infections in Hawaiian monk seals. Histopathology and immunohistochemistry were the primary diagnostic modalities used to define cases, given that these analyses establish a direct link between disease and pathogen presence. Findings were supported by serology and molecular data when available. Toxoplasma gondii was the predominant apicomplexan parasite identified and was associated with 100% of mortalities (n = 8) and 50% of suspect cases (n = 2). Incidental identification of sarcocysts in the skeletal muscle without tissue inflammation occurred in 4 seals, including one co-infected with T. gondii. In 2015, 2 cases of toxoplasmosis were identified ante-mortem and shared similar clinical findings, including hematological abnormalities and histopathology. Protozoal-related mortalities, specifically due to toxoplasmosis, are emerging as a threat to the recovery of this endangered pinniped and other native Hawaiian taxa. By establishing case definitions, this study provides a foundation for measuring the impact of these diseases on Hawaiian monk seals.

Histological location of myxosporean plasmodia in fish tissue with Luna's method.

In the present study, a staining technique, Luna's method, has been used for detection and tissue location of myxosporean spores/plasmodia in histological sections of gills and liver. The Luna's staining was performed on the sections of the tissue fixed in Bouin's, embedded in paraffin wax. The mature plasmodia were stained in bright red color and pansporoblasts and gill tissue stained in blue. This staining helped to detect and locate even a single myxospore of less than 15 µm in size located within the tissues of the host.

Protozoal nodular dermatitis and panniculitis in a Rottweiler puppy caused by Caryospora bigenetica.

BACKGROUND: Caryospora bigenetica is an intracellular protozoan parasite in snakes and raptors (primary hosts) and rodents (secondary host). Experimental infection has been documented in mice, pigs and goats; natural infection in dogs is rare. OBJECTIVES: To describe the clinical presentation, histological features, treatment and outcome of a case of protozoal nodular dermatitis and panniculitis in a Rottweiler puppy caused by C. bigenetica. RESULTS: The puppy presented with generalized subcutaneous nodules measuring up to 2 cm in diameter. Histopathology revealed marked suppurative to pyogranulomatous dermatitis and panniculitis with intralesional protozoal organism. PCR and DNA sequencing confirmed infection with C. bigenetica. Treatment with a combination of oral trimethoprim-sulfamethoxazole (TMS), pyrimethamine and high-dose clindamycin (20 mg/kg twice daily) resulted in resolution of lesions in 6 weeks. Discontinuation of the treatment 2 weeks later was followed by a rapid relapse of skin lesions. Clindamycin and TMS were restarted and all lesions resolved within 2 weeks; TMS was discontinued 4 weeks later due to adverse effects. The lesions remained in remission for 2 months while the puppy received clindamycin monotherapy before a second relapse of skin lesions occurred. CONCLUSION AND CLINICAL IMPORTANCE: To the best of the authors' knowledge, this is the first documentation of the treatment and outcome of C. bigenetica cutaneous infection in a dog. Although remission of clinical signs can be achieved with combination therapy of clindamycin and TMS, long-term management is challenging and relapses should be anticipated.

KLOSSIELLA DULCIS N. SP. (APICOMPLEXA: KLOSSIELLIDAE) IN THE KIDNEYS OF PETAURUS BREVICEPS (MARSUPIALIA: PETAURIDAE).

Two cases of renal klossiellosis were diagnosed by histopathology in pet sugar gliders (Petaurus breviceps). In both cases, parasites were associated with tubular dilation and mild interstitial nephritis. Rare schizonts were seen in the proximal convoluted renal tubular epithelium, whereas all other life cycle stages were found within distal convoluted tubule cells or the urinary space of the structures distal to the loop of Henle. Conventional optical and transmission electron microscopies were used to assess the life stages of the parasite. The morphologic characteristics and measurements observed differ from those of previously described species of Klossiella infecting marsupial hosts, and the name Klossiella dulcis n. sp. is hereby proposed. This is the first report of a Klossiella sp. infection in Petaurus breviceps .

Apoptosis and cell proliferation in the mouse model of embryonic death induced by Tritrichomonas foetus infection.

Bovine tritrichomonosis is a sexually transmitted disease caused by the protozoon Tritrichomonas foetus and characterised by embryonic-death and abortion. During pregnancy, the processes of cell proliferation and death play a crucial role for blastocyst implantation and the subsequent maintenance of early pregnancy, and their misbalance may lead to the abortion. In this study, we aimed to investigate whether cell proliferation and death may be altered during tritrichomonosis. For this purpose, we used pregnant BALB/c mice as an alternative experimental animal model that has successfully reproduced the infection. We analysed the immunohistochemical expression of active caspase-3 and proliferating cell nuclear (PCNA) antigens in the endometrium of infected mice. We found an increase in the number of caspase-3 positive cells in infected mice that were not pregnant at the necropsy. Besides, the number of positive proliferating cells increased in the uterine luminal epithelium of infected animals killed at 5-7 days post coitum (dpc). Pregnant infected mice killed at 8-11 dpc showed higher proliferation than control animals. We suggest that the cytopathic effect induced by T. foetus in the uteri of infected mice may induce the apoptosis of the epithelial cells and, as a result, promote a compensatory proliferative response. The information described here will be helpful to further study the pathogenesis of the bovine tritrichomonosis.

Neuropathology of Natural Cytauxzoon felis Infection in Domestic Cats.

Cytauxzoonosis is a tick-borne disease of felids caused by the protozoan Cytauxzoon felis. This article characterizes the neuropathologic changes in 8 cases of natural C. felis infection in domestic cats with typical clinical signs and pathologic changes in multiple organs. Histologic changes in the brain included intravascular schizont-laden macrophages in leptomeningeal and parenchymal arterioles and venules. Small capillaries throughout the gray and white matter and choroid plexus also appeared occluded. Affected vessels were often surrounded by small to moderate numbers of lymphocytes and plasma cells and multiple areas of hemorrhage. Vasculitis and fibrin thrombi were occasionally present. Parenchymal changes consisted of variable degrees of vacuolation of the gray and white matter, with microgliosis, astrocytosis, astrogliosis, and multiple microhemorrhages. Multiple well-demarcated areas of necrosis observed in 4 cases were characterized by marked vacuolation of the gray or white matter, with necrotic neurons containing shrunken hypereosinophilic cytoplasm and pyknotic nuclei, nuclear debris, swollen axons, and scattered foamy macrophages. The changes in the brains of affected cats were attributed to vascular occlusion and secondary ischemia caused by the protozoal infection.

Aberrant Clinical Appearance and Pathomorphology Noticed During an Outbreak of Histomonosis Indicates a Different Pathogenesis of Histomonas meleagridis Genotype 2.

The present case report describes a remarkable feature of Histomonas meleagridis characterized by aberrant clinical appearance and pathomorphologic lesions, which were mainly confined to the ceca, noticed during a field outbreak of histomonosis. In a flock of meat turkey toms, sudden death was noticed at the end of week 5 in the absence of specific clinical signs. Instead of the well-known sulfur-colored feces, some caseous cores were found in the litter. Mortality up to 17% per week was noticed in the first 2 wk of observation, after which it declined to approximately 1% per week. In the 10th week of life roughly 31% of the birds had died before the remaining birds were killed to preclude further economic losses due to insufficient growth or continuing mortality. Necropsy of affected birds on the farm and during a more detailed investigation of 15 birds prior to the killing of the flock revealed severe lesions in the ceca characterized by thickened cecal walls filled with necrotic and caseous material. Additionally, some ruptured and necrotic ceca were noticed together with localized peritonitis. Despite such severe typhlitis, only one of the sectioned birds showed pathomorphologic changes in the liver. Test tube flotation from collected fecal samples revealed only sporadic occurrence of coccidial oocysts and no nematodes. However, the presence of H. meleagridis was confirmed by PCR and/or immunohistochemistry, with specific antibodies against the parasite in a majority of the investigated ceca and in four liver samples. Remarkably, genetic characterization revealed H. meleagridis genotype 2, about which no detailed investigations have been reported so far. Although PCR detected a concurrent presence of Tetratrichomonas gallinarum, an involvement in the lesions could be excluded based upon histologic investigations. Finally, infection with Escherichia coli and Gallibacterium anatis was demonstrated by bacteriologic smears of internal organs, most likely a secondary infection. Altogether, the results demonstrate an aberrant clinical appearance and pathomorphology in turkeys suffering from histomonosis. Pathomorphologic changes were characterized by severe inflammation of the ceca with minimal liver involvement, indicating a different pathogenesis of H. meleagridis genotype 2.

Pathogenesis of histomonosis in experimentally infected specific-pathogen-free (SPF) layer-type chickens and SPF meat-type chickens.

Several studies have shown differences in the course of histomonosis, the infection with the trichomonad parasite Histomonas meleagridis, in different chicken breeds. In the present study, 10 specific-pathogen-free (SPF) layer-type (LT) chickens and twelve SPF meat-type (MT) chickens were infected intracloacally with 200,000 H. meleagridis trophozoites. One and two weeks postinfection (p.i.), three birds of each group were euthanatized. The remaining birds were euthanatized 3 wk p.i. Infected birds showed severe gross lesions typical for histomonosis in ceca at the first and second week p.i., while livers showed necrotic foci at 2 and 3 wk p.i., but only very rarely at 1 wk p.i. Differences between groups in the severity of lesions were statistically insignificant. In histopathology, LT chickens showed a significantly more-severe necrosis and ablation of the cecal epithelium 1 wk p.i. Parasites without inflammation were also found in most investigated spleens and lungs but only in a few kidneys. Investigation of these organs for histomonal DNA by real-time PCR confirmed these results. In addition, the humoral immune response against histomonal actinin 1 and 3 was measured by an ELISA. The humoral immune response against actinin 1 started sooner and was significantly higher in LT chickens than in MT chickens. In conclusion, the results of the present study suggest that the genetic background of the birds influences the reaction to infection with H. meleagridis.