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.

Detection of concurrent infection of dairy cattle with Blastocystis, Cryptosporidium, Giardia, and Enterocytozoon by molecular and microscopic methods.

Of fecal specimens examined from 47 dairy cattle ranging in age from neonates to multiparous cows, 9, 10, 24, and 17 were positive for Blastocystis spp., Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi, respectively, as determined by PCR. Eight 3- to 5-month-old cattle were concurrently infected with three or four of these parasites. This is the first report to identify multiple concurrent infections with these four potentially zoonotic protist pathogens in cattle. None of the cattle exhibited signs of illness or effects of infection on growth and are regarded as healthy carriers. A commercially available immunofluorescence (IFA) microscopic test confirmed six of seven available PCR-positive Blastocystis specimens and identified one IFA-positive cow that was PCR negative.

Associations between vaccinations against protozoal and viral infections and Salmonella in broiler flocks.

Reducing the burden of Salmonella in broiler flocks presents a challenge for public health. Worldwide, grow-out broilers are routinely vaccinated to prevent or lessen clinical manifestation of other infections. In this exploratory analysis we tested if details of a routine vaccination programme delivered to conventional grow-out broilers were associated with the burden of Salmonella in the flock as it progressed through its production cycle. None of the flocks studied were vaccinated against Salmonella or received a competitive exclusion product. The flocks were reared on conventional grow-out farms in southeastern USA, and sampled in a prospective field observational study. We observed significant associations between the content and design of a grow-out vaccination programme targeting other infections and the probability of detecting Salmonella in the broiler flock at different time points throughout the production cycle. To the best of the authors' knowledge, this is the first field report of such associations.

Disseminated neoplasia in flat oysters Ostrea edulis from Galicia (NW Spain): occurrence, ultrastructural aspects and relationship with bonamiosis.

Disseminated neoplasia (DN) was one of the most important pathological conditions found in cultured flat oysters (Ostrea edulis) from different geographical origins grow in Galicia (NW Spain), during a two years selective breeding programme to produce oysters less susceptible to bonamiosis. Histological characteristics observed in oysters affected by DN included intense infiltration of connective tissue of various organs (gills, stomach, digestive gland and gonad) by large undifferentiated cells, with a large nucleus and a high nucleus-to-cytoplasm ratio. The main ultrastructural features were predominance of euchromatin over heterochromatin that was arrayed in small clumps in the nucleus, prominent granular nucleolus, swollen mitochondria with few cristae and high number of free ribosomes in the cytoplasm. A seasonal pattern of DN prevalence was detected, with higher values in spring-summer, but there were no significant differences between geographic origins or families within these origins. However, the intensity of the disease was significantly different between origins; oysters originating outside of Galicia (particularly those originating from Ireland) were more susceptible to develop advanced DN. DN (8%) and bonamiosis (4.9%) were found concurrently in oysters. The nature and significance of this association warrants more investigation to determine its importance, if any.

Systemic spironucleosis in 2 immunodeficient rhesus macaques (Macaca mulatta).

Spironucleus spp are parasites of fish and terrestrial vertebrates, including mice and turkeys, that rarely cause extraintestinal disease. Two rhesus macaques (Macaca mulatta) were experimentally inoculated with simian immunodeficiency virus mac251. Both progressed to simian acquired immune deficiency syndrome within 1 year of inoculation and developed systemic protozoal infections in addition to common opportunistic infections, including rhesus cytomegalovirus, rhesus lymphocryptovirus, and rhesus adenovirus. In the first case, the protozoa were associated with colitis, multifocal abdominal abscessation, and lymphadenitis. In the second case, they were one of a number of organisms associated with extensive pyogranulomatous pneumonia and colitis. Ultrastructural, molecular, and phylogenetic analysis revealed the causative organism to be a species of Spironucleus closely related to Spironucleus meleagridis of turkeys. This report is the first of extraintestinal infection with Spironucleus sp in higher mammals and expands the list of opportunistic infections found in immunocompromised rhesus macaques.

A case of "misplaced" Tritrichomonas foetus infection in a dog in Northern Italy.

An 8-year-old, spayed female Rottweiler dog, under immunosuppressant treatment for protein-losing enteropathy (PLE) and intestinal lymphangiectasia, was presented for anorexia, poor general conditions and episodes of diarrhea. A subcutaneous mass between the caudal abdominal mammary glands was found. A fine-needle aspiration cytology was performed and revealed the presence of inflammatory cells mixed with pear- or round-shaped microorganisms with cytomorphological features of flagellated protozoan trophozoites, belonging to Trichomonadida order. The final diagnosis of Tritrichomonas foetus infection was achieved with molecular analysis (Real-Time PCR) and sequencing. T. foetus has often been reported as a pathogenic agent in cattle and cats, and rarely reported in dogs, where its pathogenetic role is poorly understood. To the author's knowledge, this is the first case of T. foetus infection described in a location different from the gastrointestinal tract in a dog.

Diagnostic studies of abortion in Danish cattle 2015-2017.

BACKGROUND: Abortion is a major source of economic losses in cattle breeding. Abortion occurs due to a wide range of causes, but infections are the most frequently diagnosed. However, establishing an aetiological diagnosis remains challenging due to the large variety of bacteria, protozoa, viruses, and fungi that have been associated with abortion in cattle. Economic restraints limit the range of diagnostic methods available for routine diagnostics, and decomposition of the conceptus or lack of proper fetal and/or maternal samples further restrict the diagnostic success. In this study, we report recent diagnostic findings from bovine abortions in Denmark, a country that has a large dairy sector and is free from most infectious agents causing epizootic abortion in cattle. The aims of the study were: (i) to identify infectious causes of bovine abortion in Denmark, (ii) to categorise the diagnostic findings based on the level of diagnostic certainty, and (iii) to assess the diagnostic rate. Due to economic restraints, only a limited panel of routine diagnostic methods were available. Placentas and/or fetuses from mid- to late-term abortions and stillbirths (n = 162) were submitted to the Danish National Veterinary Institute between January 2015 and June 2017. The aborted materials were examined macroscopically, histologically, and by bacterial culture. Maternal blood samples were tested for bovine viral diarrhoea virus (BVDV) antibodies. RESULTS: The likely aetiology of the abortion was diagnosed in 52 cases, resulting in a diagnostic rate of 33%. The most common cause was protozoal infection (19%) followed by infection with Trueperella pyogenes (3%), Staphylococcus aureus (2%), and non-haemolytic Escherichia coli (2%). Lesions in fetuses with a protozoal infection were consistent with neosporosis. In many cases (38%), inflammatory changes were found in the placenta and/or fetal organs but no specific aetiology was identified. Neither infection with Brucella spp. nor maternal BVDV antibodies were detected. The majority of submitting herds (92%) were each represented by fewer than three abortion cases over the study period. CONCLUSIONS: Protozoal infection, most likely neosporosis, was the most commonly diagnosed cause of abortion and the only one associated with potential epizootic abortion events. Despite using a reduced number of diagnostic methods in comparison to other abortion studies, the diagnostic rate of this study was within the range reported in an earlier Danish study, as well as in recent international studies. The low number of submitted cases per herd and the sparse anamnestic information provided at submission hampered conclusions on the potential epizootic character of the abortion events in question.

A restriction site to differentiate Plasmodium and Haemoproteus infections in birds: on the inefficiency of general primers for detection of mixed infections.

Avian Plasmodium and Haemoproteus parasites are easily detected by DNA analyses of infected samples but only correctly assigned to each genus by sequencing and use of a phylogenetic approach. Here, we present a restriction site to differentiate between both parasite genera avoiding the use of those analyses. Alignments of 820 sequences currently listed in GenBank encoding a particular cytochrome B region of avian Plasmodium and Haemoproteus show a shared restriction site for both genera using the endonuclease Hpy CH4III. An additional restriction site is present in Plasmodium sequences that would initially allow differentiation of both genera by differential migration of digested products on gels. Overall 9 out of 326 sequences containing both potential restriction sites do not fit to the general rule. We used this differentiation of parasite genera based on Hpy CH4III restriction sites to evaluate the efficacy of 2 sets of general primers in detecting mixed infections. To do so, we used samples from hosts infected by parasites of both genera. The use of general primers was only able to detect 25% or less of the mixed infections. Therefore, parasite DNA amplification using general primers to determine the species composition of haemosporidian infections in individual hosts is not recommended. Specific primers for each species and study area should be designed until a new method can efficiently discriminate both parasites.

Hepatozoonosis in a dog with skeletal involvement and meningoencephalomyelitis.

A 15-month-old, female mongrel dog was presented with a 6-week history of inappetence, weight loss, and tetraparesis. Physical examination revealed weakness, poor body condition, mild fever, pale mucous membranes, and diffuse muscle atrophy. The right hind limb was painful and edematous, with large ecchymoses. The femur was irregular on palpation and moderate popliteal lymphadenopathy was evident. Results of a CBC showed severe anemia with mild regeneration, an inflammatory leukogram with 90% of neutrophils parasitized by Hepatozoon sp. gamonts, and moderate thrombocytopenia. A bone marrow aspirate had myeloid hyperplasia and contained a few extracellular Hepatozoon meronts and a few intracellular gamonts within neutrophils. Serum chemistry abnormalities included hypoalbuminemia, hyperglobulinemia, hypoglycemia, hypercalcemia, hyperphosphatemia, and elevated alkaline phosphatase activity. Radiologic findings of the right femur included periosteal bone proliferation and lesions compatible with osteomyelitis. A fine needle aspirate specimen from the bone lesion had neutrophilic inflammation; 36% of the neutrophils contained Hepatozoon gamonts. Results of cerebrospinal fluid analysis included a protein concentration of 37 mg/dL and marked mononuclear pleocytosis (243 cell/microL) with a predominance of lymphocytes. An ELISA was positive for Hepatozoon canis and PCR results with DNA sequencing confirmed infection with this organism. A diagnosis of hepatozoonosis with skeletal involvement and meningoencephalomyelitis was made. The dog recovered almost completely neurologically and had no gamonts in the blood after 60 days of therapy with imidocarb dipropionate and prednisone. This is an unusual case of canine hepatozoonosis involving neurologic signs and a periosteal reaction more typical of H. americanum infection and rarely reported in dogs infected with H. canis.

Tritrichomonas foetus and Mycoplasma felis coinfection in the upper respiratory tract of a cat with chronic purulent nasal discharge.

A 5-year-old indoor male neutered Siamese cat was presented with clinical signs of sneezing and chronic bilateral purulent nasal discharge. Multiple nasal cavity swabs were submitted for bacterial cultures, Mycoplasma felis-DNA qPCR, and cytology. M felisqPCR was positive and cytomorphologic diagnosis was severe, acute, purulent, rhinitis with intralesional protozoal microorganisms consistent with a Trichomonas spp. Nested PCR (nPCR) confirmed the diagnosis of Tritrichomonas foetus. Systemic therapy with doxycycline for M felis and metronidazole for T foetus was started with remission of clinical signs within 2 weeks; however, symptoms relapsed shortly after therapy was discontinued. This study represents the first documented case of T foetus associated with chronic nasal discharge in a cat, which supports the hypothesis that T foetus can live in the nasal cavity. It is also the first reported case of M felis and T foetus coinfection, which indicates that with mycoplasmal feline upper respiratory tract infections, T foetus should be considered as a coinfecting agent.

Malaria and risk of predation: a comparative study of birds.

Predators have been hypothesized to prey on individuals in a poor state of health, although this hypothesis has only rarely been examined. We used extensive data on prey abundance and availability from two long-term studies of the European Sparrowhawk (Accipiter nisus) and the Eurasian Goshawk (Accipiter gentilis) to quantify the relationship between predation risk of different prey species and infection with malaria and other protozoan blood parasites. Using a total of 31 745 prey individuals of 65 species of birds from 1709 nests during 1977-1997 for the Sparrowhawk and a total of 21 818 prey individuals of 76 species of birds from 1480 nests for the Goshawk during 1977-2004, we show that prey species with a high prevalence of blood parasites had higher risks of predation than species with a low prevalence. That was also the case when a number of confounding variables of prey species, such as body mass, breeding sociality, sexual dichromatism, and similarity among species in risk of predation due to common descent, were controlled in comparative analyses of standardized linear contrasts. Prevalence of the genera Haemoproteus, Leucocytozoon, Plasmodium, and Trypanosoma were correlated with each other, and we partitioned out the independent effects of different protozoan genera on predation risk in comparative analyses. Prevalence of Haemoproteus, Leucocytozoon, and Plasmodium accounted for interspecific variation in predation risk for the two raptors. These findings suggest that predation is an important factor affecting parasite-host dynamics because predators tend to prey on hosts that are more likely to be infected, thereby reducing the transmission success of parasites. Furthermore, this study demonstrates that protozoan infections are a common cause of death for hosts mediated by increased risk of predation.

Tritrichomonas foetus infection in cats with diarrhea from densely housed origins.

Tritrichomonas foetus is a protozoan parasite that has been recently identified as a causative agent of chronic diarrhea in domestic cats. Transmission of infection occurs by the fecal-oral route through direct contact among animals. Consequently, feline trichomonosis (FT) is more likely to be present in multi-cat environments. The objective of this work was to study the presence of T. foetus and some associated risk factors in cats from densely housed origins and with a reported history of chronic diarrhea. Animals enrolled in this study were family cats (n=15) acquired from pet shops, shelters or breeding centers and cattery cats belonging to one breeding center (n=28) and two cat shelters (A and B, n=25 each). In the catteries, a follow-up analysis for a period of up to 2 months was also performed to determine the parasite shedding pattern in feces and the incidence of infection. Fecal samples were analyzed using in vitro culture and a PCR technique. T. foetus was detected in a total of 38.7% (36/93) of the cats with chronic diarrhea. Parasite infection was similarly detected in family cats and cattery animals (40% versus 38.4%). In the catteries, the parasite was detected in 50%, 44% and 20% of the animals from the breeding center and shelters A and B, respectively. The follow-up analysis showed that 58.3% of infected cats intermittently shed trophozoites in their feces, with an incidence of 23.1%. Investigation of potential risk factors showed that cats ≤1 year old were more likely to be infected than older cats (57.1% versus 27.3%; P<0.05). No significant differences were found when sex and breed factors were studied. These results confirm the importance of FT as a cause of chronic diarrhea in cats and highlight the relevance of close contact conditions for T. foetus transmission.

The effect of single and concomitant pathogen infections on condition and fecundity of the wild rabbit (Oryctolagus cuniculus).

We assessed the effect of two pathogens (myxoma virus and Eimeria stiedae) and five macroparasites (gastrointestinal helminth species) of the wild rabbit (Oryctolagus cuniculus) upon total host body mass and abdominal fat level. Additionally, we assessed the effects of these organisms on the number of foetuses in adult females during the peak breeding period. Both mass of abdominal fat and total body mass of the rabbit were negatively associated with myxoma virus infection and increasing helminth species richness. Total body mass was also negatively associated with the protozoan parasite E. steidae. No relationship was found between any of the parasites/pathogens and the number of foetuses in adult females, although only relatively small sample sizes were available for this section of the analysis. Increasing host body mass was positively associated with number of foetuses and we propose that mass reduction caused by the pathogen and parasite species could also have the consequence of reducing foetal number.

Blackhead disease in turkeys: direct transmission of Histomonas meleagridis from bird to bird in a laboratory model.

The spread of Histomonas meleagridis infections through groups of turkeys in the absence of the cecal worm vector (Heterakis gallinarum) was studied in a battery cage model. Battery-reared poults were exposed at 2 wk of age by commingling with infected birds into cages that had the floor lined with paper. One treatment received no exposure, whereas other birds were commingled with two, three, or four birds/cage (25%, 37.5%, or 50%) inoculated per cloaca with cultured H. meleagridis (200,000/bird). Inoculated birds died at 7-13 days postinoculation (DPI) showing typical liver and cecal lesions of histomoniasis. By 14 DPI, 87.5% of the directly inoculated birds died or had severe lesions of histomoniasis. Turkeys commingled with two, three, or four infected birds became infected at the rate of 72%, 80%, or 75%, respectively. In another experiment, two birds/cage (25%) were inoculated with Histomonas from culture and allowed to commingle with other birds for 1, 2, 3, or 4 days. Two of 12 (16.7%) birds had minor cecal lesions after contact with inoculated birds for 1 day, but 87.5%-100% became infected if inoculated birds remained in the cage for 2-4 days. Contemporaneous inoculation with cecal coccidia (Eimeria adenoeides) as a predisposing factor in blackhead infections was studied using the model. Turkey poults directly inoculated with Histomonas were allowed to commingle for 5 days with uninoculated birds that had received inoculation with 0, 10(3), or 10(4) sporulated oocysts. The coccidian infection appeared to interfere with transmission of blackhead infection by 7 DPI, as suggested by lessened severity of cecal lesions and a lower percentage of infected birds. These studies confirm that histomoniasis is transmitted readily from directly exposed young turkeys to others in the absence of the cecal worm vector, and that this phenomenon can be reproduced in battery cages as an experimental model.

Spinal curvature of cultured Japanese mackerel Scomber japonicus associated with a brain myxosporean, Myxobolus acanthogobii.

Skeletal deformities were found in the cultured Japanese mackerel Scomber japonicus. External and radiographical observations showed the deformed fish to exhibit a dorso-ventral spinal curvature (kyphosis) without fracture or dislocation of the vertebrae. Numerous myxosporean cysts, ca. 0.3 to 1.0 mm in diameter, formed in the 4th ventricle, the cavity of the optic tectum, the surface of the olfactory lobe and bulb, the optic lobe and the inferior lobe of the brain. Spore morphology and molecular analysis of the small subunit ribosomal RNA gene sequence identified the myxosporean parasite as Myxobolus acanthogobii, a parasite which also causes scoliosis in yellowtail Seriola quinqeradiata. Histopathological observation showed that the myxosporean cysts were encapsulated within the host's collagenous layer although some had disintegrated to disperse mature spores into the cranial cavity. Occasionally, lymphocytic infiltration and local granulomatous inflammation were found to be associated with spore dispersion.

Chronic hepatitis associated with canine leishmaniosis (Leishmania infantum): a clinicopathological study of 26 cases.

Hepatic tissue samples were obtained from 26 dogs humanely destroyed because of naturally occurring leishmaniosis (Leishmania infantum). None of the animals had palpable hepatomegaly or any other physical finding or historical evidence indicative of liver failure. However, serum biochemistry revealed hypoalbuminaemia (6/26), increased alkaline phosphatase (ALP) activity (15/26), and increased concentrations of total bilirubin (2/26) and post-prandial bile acids (4/26). Three main histological patterns were identified. In pattern 1 (3/26), the liver microarchitecture remained unchanged apart from the presence of individual or clustered macrophages in the sinusoids. In pattern 2 (20/26), there was multifocal, mild to moderate, granulomatous to pyogranulomatous infiltration of the hepatic parenchyma, particularly in the portal areas. Pattern 3 (3/26), which was the most severe form, was characterized by marked portal lymphoplasmacytic infiltration with occasional broaching of the limiting plate and extension into the adjacent parenchyma. In this pattern there was also mild portal fibrosis, together with lymphoplasmacytic aggregates within the parenchyma and small clusters of lymphocytes and plasma cells within the sinusoids. All three patterns were associated with hepatocyte vacuolation (15/26 dogs), and haemosiderin accumulation within the hepatocyte cytoplasm. Congestion was present in the liver of five dogs. No correlation was found between histopathological pattern and breed, sex, age, clinical manifestations, serum biochemical profile or parasite load in the hepatic tissue; patterns 1-3 may, however, represent sequential stages of hepatic leishmania infection during the chronic course of the disease.

Persisting learning deficits in rats after exposure to Pfiesteria piscicida.

Pfiesteria piscicida and other toxic Pfiesteria-like dinoflagellates have been implicated as a cause of fish kills in North Carolina estuaries and elsewhere. Accidental laboratory exposure of humans to P. piscicida has been reported to cause a complex syndrome including cognitive impairment. The current project was conducted to experimentally assess the possibility of cognitive effects of P. piscicida exposure in rats. Samples of water from aquaria in which P. piscicida zoospores were killing fish were frozen, a procedure that has been found to induce encystment. Thawed samples were injected into albino Sprague-Dawley rats. A significant learning impairment was documented in rats administered samples of P. piscicida that were recently frozen. Prolonged storage of Pfiesteria samples diminished the effect. No effect was seen in the recall of a previously learned task, but when the rats were called upon to learn a new task, the Pfiesteria-treated animals showed a significant learning deficit. This effect persisted up to at least 10 weeks after a single injection of Pfiesteria. The Pfiesteria-induced learning deficit did not seem to be associated with any obvious debilitation or health impairment of the exposed rats. Deficits in habituation of arousal and rearing behavior were detected using a functional observational battery. No Pfiesteria-induced effects on blood count and white cell differential or in a standard pathological screening of brain, liver, lung, kidney, and spleen tissue were seen at 2 months after exposure. These studies document a persistent learning impairment in rats after exposure to the dinoflagellate P.piscicida in otherwise physically well-appearing rats. This effect may partially model the symptoms of cognitive impairments that humans have shown after Pfiesteria exposure.

Blackhead disease (Histomonas meleagridis) aggravated in broiler chickens by concurrent infection with cecal coccidiosis (Eimeria tenella).

The effect of concurrent cecal coccidiosis infections on severity of Histomonas meleagridis (blackhead disease) in chickens was investigated in a series of experiments. Cecal lesions from H. meleagridis were severe in all inoculated control groups and did not appear to be affected by the introduction of Eimeria tenella infection. However, the severity of liver lesions and number of birds positive for liver lesions of H. meleagridis increased significantly with the presence of E. tenella. The increase was similar when 10(3) or 10(4) oocysts of E. tenella were given and was the same when oocysts were given at the same time as H. meleagridis or 4 days prior. The liver lesions increased directly as doses of H. meleagridis increased from 7.5 x 10(3) cells to 30, 100, or 300 x 10(3) when E. tenella was given along with H. melelagridis but not when H. meleagridis was given alone. Administration of a live coccidiosis vaccine containing very low levels of E. tenella also gave a significant boost to liver lesions but at a much lower level than that observed with larger doses of E. tenella. The positive relationship between infections of cecal coccidiosis and H. meleagridis in chickens suggests that such dual exposure may contribute to increased clinical outbreaks of blackhead disease in chickens under field conditions.

Parasitism by the protozoan Perkinsus atlanticus favours the development of opportunistic infections.

It has been suggested that opportunistic pathogens could contribute to the mortality of Perkinsus atlanticus-infected clams. Examination of Tapes semidecussatus clams from the northern Mediterranean coast of Spain revealed that while 86% of the clams heavily infected with P. atlanticus were co-infected by bacteria and/or viruses, neither non-infected nor lightly P. atlanticus-infected specimens had bacterial or viral infections. The bacteria, which had a Gram-negative cell wall, were always located in the apical pole of gill epithelial cells and enclosed within membranous compartments. Bacteria-containing cells were hypertrophied and showed dysplasia with loss of cilia and microvilli. The viruses shared ultrastructural, morphologic and cytopathic characteristics of a polyomavirus. Viral particles with icosahedral symmetry were found in both the cytoplasm and the nucleus of numerous cell types. Virus-infected cells showed severe alterations, including hypertrophy, reduction of the intracellular compartments and extrusion of the nuclear envelope. Moreover, gill epithelial cells showed disorganization and swelling of the apical region, which affected the ciliary structure. Our findings show that P. atlanticus parasitism favours the development of opportunistic infections which have detrimental effects in this clam population.