Assessment of the Virulence Spectrum and Its Association with Genetic Diversity in Magnaporthe oryzae Populations from Sub-Saharan Africa.

A collection of 122 isolates of Magnaporthe oryzae, from nine sub-Saharan African countries, was assessed for virulence diversity and genetic relatedness. The virulence spectrum was assessed by pathotype analysis with a panel of 43 rice genotypes consisting of differential lines carrying 24 blast resistance genes (R-genes), contemporary African rice cultivars, and susceptible checks. The virulence spectrum among isolates ranged from 5 to 80%. Five isolates were avirulent to the entire rice panel, while two isolates were virulent to ∼75% of the panel. Overall, cultivar 75-1-127, the Pi9 R-gene donor, was resistant to all isolates (100%), followed by four African rice cultivars (AR105, NERICA 15, 96%; NERICA 4, 91%; and F6-36, 90%). Genetic relatedness of isolates was assessed by single nucleotide polymorphisms derived from genotyping-by-sequencing and by vegetative compatibility tests. Phylogenetic analysis of SNPs of a subset of isolates (n = 78) revealed seven distinct clades that differed in virulence. Principal component analysis showed isolates from East Africa were genetically distinct from those from West Africa. Vegetative compatibility tests of a subset of isolates (n = 65) showed no common groups among countries. This study shows that blast disease could be controlled by pyramiding of Pi9 together with other promising R-genes into rice cultivars that are adapted to East and West African regions.

Assessment of Aflatoxin and Fumonisin Contamination of Maize in Western Kenya.

We conducted a survey of aflatoxin and fumonisin in maize in western Kenya. In a regional survey of aflatoxin conducted in 2009 across three agroecological zones within three administrative regions, milled maize samples were collected from 985 patrons of 26 hammer mills. Aflatoxin contamination was detected in 49% of samples and was above the regulatory (10 ppb) in 15% of the samples overall; 65% of samples from a drought-prone area were over the limit. In a detailed survey in Bungoma County, we investigated aflatoxin and fumonisin contamination in four popular maize varieties at harvest and after 2 and 4 months of storage. We collected whole-grain samples from farmers' storage sheds and milled samples from patrons of local mills. Mean aflatoxin contamination was identical for storage sheds and mills at 2.3 ppb. In all, 41% of the samples from mills had detectable aflatoxin, with 4% over the regulatory limit, whereas 87% had detectable fumonisin, with 50% over the regulatory limit (1 ppm). Mean contamination levels did not change during storage. Maize varieties differed in fumonisin contamination, with the most popular varieties vulnerable to both mycotoxins and weevils, which are potential factors in exacerbating mycotoxin contamination. Mycotoxin surveillance is important not just in areas known previously for aflatoxin contamination and acute poisoning but also is needed in all maize-producing regions.

Extent and drivers of mycotoxin contamination: inferences from a survey of kenyan maize mills.

The prevalence of aflatoxin and fumonisin was investigated in maize intended for immediate human consumption in eastern Kenya at a time in 2010 when an aflatoxin outbreak was recognized. Samples were collected from people who brought their maize for processing at local commercial mills. Sites were selected using a geographical information system overlay of agroecological zones and Kenya's administrative districts. Interviews and collection of maize flour samples was conducted from 1,500 people who processed maize at 143 mills in 10 administrative districts. Mycotoxins were analyzed using enzyme-linked immunosorbent assays for aflatoxin and fumonisin, leading to detection at levels above the respective maximum tolerable limits in 39 and 37% of the samples, respectively. Samples with aflatoxin contamination above the legal limit ranged between 22 and 60% across the districts. A higher occurrence of aflatoxin was associated with smaller maize farms, lower grain yield, and monocropping systems, while a larger magnitude of the toxin was observed in the subhumid agroecological zone, in samples with more broken kernels, and, curiously, less maize ear damage at harvest. Analysis of paired grain samples (visually sorted and unsorted) showed that sorting reduced fumonisin by 65%, from above to below the legal limit of 1,000 ppb. Sorting did not, however, reduce aflatoxin levels. Although the aflatoxin problem is widely acknowledged, the high prevalence of fumonisin has not previously been reported. There is need for surveillance of the two mycotoxins and establishment of intervention strategies to reach vulnerable small-scale farmers.

Clinical, metabolic, and molecular genetic characterization of hereditary methemoglobinemia caused by cytochrome b5 reductase deficiency in 30 dogs.

Genotype-phenotype correlations of humans and dogs with hereditary methemoglobinemia are not yet well characterized. We determined total hemoglobin and methemoglobin (MetHb) concentrations, cytochrome b5 reductase (CYB5R) enzyme activities, genotypes, and clinical signs in 30 dogs with persistent cyanosis without cardiopulmonary disease. Erythrocytic CYB5R enzyme activities were low in all dogs assayed. Owner-reported quality of life ranged from subclinical to occasional exertional syncope. Two previously reported and two novel CYB5R3 missense variants were identified among the methemoglobinemic cohort and were predicted to impair enzyme function. Two variants were recurrent: a homozygous Ile194Leu substitution was found in Pomeranians and other small dogs, and a homozygous Arg219Pro change occurred predominately in pit bull terriers. The other two variants were Thr202Ala and Gly76Ser substitutions in single dogs. Of the two common CYB5R3 genotypes, Arg219Pro was associated with a more severe metabolic phenotype. We conclude that CYB5R3 deficiency is the predominate cause of canine hereditary methemoglobinemia. Although this finding is unlikely to alter the clinical approach to hereditary methemoglobinemia in dogs, it demonstrates the possibility of how genotype-phenotype cohort analysis might facilitate precision medicine in the future in veterinary medicine.

Serum Iron Analytes in Healthy and Diseased Florida Manatees (Trichechus manatus latirostris).

Serum iron concentration is usually decreased in true iron deficiency and with inflammatory disease in man and domestic animals. Serum total iron binding capacity (TIBC) may be increased in true iron deficiency and decreased with inflammatory disease. This prospective study was designed to measure serum iron analytes in healthy free-ranging and housed Florida manatees (Trichechus manatus latirostris) of both sexes and various ages and to evaluate the effects of diseases common to manatees on these analytes. Blood samples were collected without anticoagulant from 137 healthy free-ranging manatees, 90 healthy housed manatees and 74 free-ranging diseased manatees, and serum was prepared by centrifugation. Serum iron concentration and unsaturated iron binding capacity were measured colourimetrically, and TIBC and percent transferrin saturation with iron were calculated. Serum amyloid A (SAA) was measured to assist in the health assessment of manatees and provide evidence of inflammation in diseased manatees. Based on the serum iron analytes, iron availability was lower in immature manatees compared with adults, and it was lower in housed manatees compared with free-ranging manatees. In contrast to other mammals studied, serum iron concentration was elevated rather than depressed in late pregnancy. Serum iron concentrations and transferrin saturation with iron percentages were significantly lower, and SAA concentrations were significantly higher, in diseased (ill and injured) manatees compared with healthy manatees. Serum iron concentration and transferrin saturation with iron values were negatively correlated with SAA concentrations, and manatees with the highest SAA concentrations had lower serum TIBC values. These findings indicate that inflammation is the major factor responsible for alterations in iron analytes in diseased manatees. Consequently, hypoferraemia may be used as supportive evidence of inflammatory disease in manatees (unless haemorrhage is also present). A decision threshold of ≤13.8 µmol/l was determined for hypoferraemia using receiver operating curve analysis. Based on studies in man and domestic animals, iron therapy is unnecessary for manatees with hypoferraemia associated with inflammation and has the potential for causing tissue damage and increased susceptibility to bacterial infections.

Primary leiomyosarcoma of bone.

A primary tumor of bone, the light microscopic features of which were suggestive of malignant fibrous histiocytoma, proved to be a primary leiomyosarcoma upon electron microscopic examination. Ultrastructurally the tumor cells were smooth muscle cells having all the characteristic features, such as cytoplasmic filaments, cytoplasmic and sarcolemmal dense bodies, and pinocytotic vesicles, with a basal lamina surrounding the cells. This example emphasizes the importance of electron microscopy in diagnostic pathology. This is the second ultrastructural report of a primary leiomyosarcoma of bone.

Metabolic and work capacity of skeletal muscle of PFK-deficient dogs studied in situ.

Mechanical and metabolic relationships of muscle lacking phosphofructokinase (PFKD) activity were compared with muscle having normal phosphofructokinase (NORM) activity by using the gastrocnemius-plantaris muscle group with isolated circulation in situ. Muscle contractile properties were similar in both groups. Initial power output (W) during repetitive tetanic (200 ms, 50 impulses/s) isotonic contractions was similar in both groups; however, W declined significantly more (30-80%) in PFKD than in NORM muscle over time, with a constant O2 uptake (VO2)/W. Despite similar O2 and substrate delivery, PFKD muscle had a lower VO2 (42-55%), less glucose uptake, similar free fatty acid uptake, and lactic acid uptake rather than output, during contractions. Muscle venous H+ concentration, strong ion difference, and PCO2 increased during contractions, the magnitude of change being smaller in PFKD muscle. Elevating arterial lactate concentration before contractions in PFKD muscle resulted in significant improvements in W and VO2 without altering the acid-base exchange at the muscle. Increasing O2 delivery by increasing arterial O2 concentration in PFKD dogs did not improve W or VO2. We conclude that, despite no inherent mechanical or contractile differences, PFKD muscle has a severely limited oxidative capacity and exaggerated fatigue and blood flow responses to contractions due to limited substrate metabolism resulting from the inability to utilize glycogen and/or glucose.

Ultrastructure of erythrocytes parasitized by Haemobartonella felis.

Experimental Haemobartonella felis infections were studied in 3 mature, intact cats by examining peripheral blood, lung, and spleen by electron microscopy. Coccoid, rod, or ring forms of the organism were found on or close to the erythrocytic membrane, and adjacent parasitized erythrocytes often were attached. Intracytoplasmic crystalloid inclusions occupying most of erythrocytic cytoplasm were seen in the 3 infected cats. The cat with the highest parasitemia had inclusions in about 10% of the erythrocytes. Less than 0.01% of the erythrocytes of a control cat contained inclusions. Parasitized erythrocytes, with and without inclusions, were seen in capillaries of the lung and spleen of infected cats. Macrophages in the lung and spleen of infected cats contained parasitized erythrocytes, either with or without inclusions. Some macrophages contained erythrocyte-free organisms in phagocytic vacuoles.

Haematology of foals up to one year old.

Packed cell volume, haemoglobin concentration, erythrocyte counts, erythrocyte indices, serum iron, iron binding capacities, total and differential leucocyte counts, platelet counts, total plasma protein, fibrinogen, haptoglobin and icterus index values were determined at 14 different ages in eight Thoroughbred and 14 Quarterhorse foals during the first year of life. Absolute neutrophil numbers in blood decreased and lymphocyte numbers increased during the first months. Absolute eosinophil numbers tended to increase until three months old. Haemoglobin concentration and packed cell volume decreased significantly during the first two weeks and generally stayed in the lower portion of adult horse normal ranges during the remainder of the first year. Mean cell volume decreased to minimum values at approximately four months and then gradually increased. Serum iron was high at birth and decreased rapidly to a minimum at three days old. Total iron-binding capacity increased to a maximum at one month. Serum haptoglobin was generally within the adult normal range at birth. A moderate, but significant, decrease occurred at one week old. Fibrinogen concentration increased to a maximum at five months. Icterus index values decreased rapidly during the first two weeks of life. Minor changes occurred in other parameters measured. Possible causes for the various changes in haematological parameters are discussed.

Hematologic changes associated with serum and hepatic iron alterations in dogs with congenital portosystemic vascular anomalies.

Serum and hepatic iron determinations and hematologic parameters were measured in 10 dogs with congenital portosystemic vascular anomalies. Anemia, hypoferremia, and microcytosis were present in 70%, 70%, and 60% of the dogs, respectively. An increase in hepatic iron content was observed in all dogs. These results suggest a relationship between altered hepatic blood flow and abnormal iron metabolism in dogs with congenital portosystemic vascular anomalies.

Mammalian erythrocyte glutathione reductase: kinetic constants and saturation with cofactor.

Glutathione reductase (GR) was studied in erythrocytes of horses, cats, dogs, and man. Glutathione reductase activity was measured in hemolysates with and without preincubation of hemolysates with flavinadenine dinucleotide. The percentage saturation of GR apoenzyme with cofactor (flavin-adenine dinucleotide) was lower in cats and dogs than in horses or man. The greatest amount of inactive apoenzyme was in feline erythrocytes. Total GR activity listed in order by species is cat greater than man greater than dog greater than horse. Kinetic constants for oxidized glutathione and reduced nicotinamide-adenine dinucleotide phosphate were determined in each species. Although kinetic constant (reduced nicotinamide-adenine dinucleotide phosphate) values for GR were similar, considerable species variation was observed in the kinetic constant (oxidized glutathione) for GR. The kinetic constant (oxidized glutathione) for equine GR was approximately 3 times that for human GR, with intermediate values determined for feline and canine GR.

Serologic diagnosis of infectious cyclic thrombocytopenia in dogs using an indirect fluorescent antibody test.

An indirect fluorescent antibody test was used for detection of serum antibodies to the platelet-specific rickettsial organism that is the causative agent of infectious cyclic thrombocytopenia (ICT) in dogs. The test converted from negative to positive in 7 of 7 experimentally inoculated dogs. One of 2 attempts to recover the rickettsial agent of ICT from naturally occurring seropositive dogs, by blood inoculation of experimental dogs, was successful. Seemingly, the test did not detect antibodies to Ehrlichia canis, nor did a similar test, using E canis antigen slides, detect antibodies to the rickettsial agent of ICT. The rickettsial agent of ICT has been classified tentatively as E platys. When applied to sera from a group of healthy random-source dogs, the test revealed a relatively low (5%) occurrence of positive reactions. A higher occurrence of positive reactions (35%) was noticed in sera from a group of thrombocytopenic dogs from the University of Florida. A majority of these positive sera were also positive for antibodies to E canis. The highest occurrence of positive reactions was found (greater than 50%) in E canis-positive sera from dogs at the University of Florida, as well as from dogs from 9 other states.

Serum ferritin, serum iron, and erythrocyte values in foals.

Twenty-one healthy Thoroughbred and Quarter Horse foals were studied from birth until 1 year of age. Foals had access to an iron-supplemented creep feed before weaning and were fed an iron-supplemented concentrate as part of their diet after weaning at 4 months of age. Initial blood samples were taken before foals were allowed to nurse. Serum iron concentration, total iron-binding capacity, and PCV decreased during the foal's first 24 hours of life. Serum iron concentration decreased rapidly from 446 +/- 16 micrograms/dl (mean +/- SE) at birth to 105 +/- 11 micrograms/dl at 3 days of age. Serum ferritin concentration increased from a mean of 85 +/- 8 ng/ml at birth to 159 +/- 11 ng/ml at 1 day of age. Thereafter, ferritin concentration decreased gradually to a minimum of 61 +/- 6 ng/ml at 3 weeks of age, and then at 6 months increased to values similar to those from reference adult horses. The ferritin concentration in colostrum at birth was 354 +/- 42 ng/ml, compared with 25 +/- 2 ng/ml in milk 1 day later. The decrease and then increase in serum ferritin concentration occurred concomitantly with opposite changes in serum total iron-binding capacity. The mean PCV decreased gradually to a minimum at 3 months of age. This decrease was associated with an increasing number of microcytes, as determined with a cell-size distribution analyzer.

Ultrastructure of the intraerythrocytic stage of Cytauxzoon felis.

The erythrocytes of 2 cats experimentally infected with Cytauxzoon felis were examined by light and electron microscopy. In stained blood smears, parasitized erythrocytes usually contained a single, roundish organism, but occasionally up to 4 were present in a cell. Chains of these roundish organisms also were seen. Elongated parasites, sometimes with ear-like projections, were present in a few erythrocytes. By electron microscopy, the parasite contained a poorly defined nucleus, rough endoplasmic reticulum, ribosomes, nonplicated mitochondria, food vacuoles, and a cytostome on its limiting membrane. Usually, the parasite was oval, but budding forms also were evident. Crystalloid inclusions were present in parasitized and nonparasitized erythrocytes.

Ultrastructure of amastigotes of Leishmania donovani in the bone marrow of a dog.

Visceral leishmaniasis was diagnosed in a dog which had been in Spain for 4 years before being brought to Florida. A bone marrow aspirate from the dog was examined by electron microscopy. Phagocytized amastigotes by macrophages had an electron-dense plasma membrane and contained ribosomes, rough endoplasmic reticulum, a well-developed Golgi apparatus, an intracellular flagellum, lipid, a kinetoplast, a row of microtubules immediately beneath the cell membrane, and a nucleus with marginated chromatin. Mean diameter and microtubule number of the dog isolate were similar to those reported for Leishmania donovani amastigotes of human origin. Plasma cells were congregated in the vicinity of parasitized macrophages.

Ultrastructure of schizonts in the liver of cats with experimentally induced cytauxzoonosis.

Schizonts in the liver of 2 cats with cytauxzoonosis were studied by both light and electron microscopies. By light microscopy, the cytoplasm of macrophages in the sinusoids and small vascular channels contained schizonts with cytomeres or both cytomeres and mature merozoites. By electron microscopy, it was determined that schizogony occurred in 4 stages. The earliest stage was the presence of a multilobed structure containing finely granular protoplasm in the cytoplasm of the macrophage. The 2nd stage was an increase in height and number of the lobulations on the surface of the schizont. The 3rd stage involved the development of cytomeres and the appearance of a polar ring and rhoptries in everted sacculations on the cytomere membrane. Nuclei and mitochondria were incorporated into the sacculations before the release of mature merozoites into the host cell cytoplasm. In the last stage of schizogony, following massive merozoite formation and reduction in size of the schizont, residual nuclei divided by multiple fission. Each nuclear division became incorporated into a developing merozoite having preformed rhoptries, mitochondria, and a polar ring.

Familial nonspherocytic hemolytic anemia in poodles.

Nonspherocytic hemolytic anemia, characterized by marked reticulocytosis, hepatosplenomegaly, hemosiderosis of reticuloendothelial organs and bone marrow myelofibrosis, and osteosclerosis, was diagnosed in 5 related Poodles. The unremitting anemia was clinically evident by 1 year of age, and was fatal as early as 3 years of age. Despite intense diagnostic endeavors including RBC fragility studies, RBC enzyme assays, and hemoglobin electrophoresis, the cause of this nonspherocytic hemolytic anemia remains to be determined.

Hematologic effects of cytauxzoonosis in Florida panthers and Texas cougars in Florida.

Cytauxzoon felis is a long-recognized hemoparasite of free-ranging Florida panthers (Puma concolor coryi), but its prevalence and effect on the population has not been assessed. Red blood cell indices and white blood cell counts were compared between infected and noninfected Florida panthers and Texas cougars (Puma concolor stanleyana) from 1983-1997 in Florida (USA). The prevalence of cytauxzoonosis for both populations was 39% (11/28) for Texas cougars, 35% for Florida panthers (22/63) and 36% overall. Thirteen hematologic parameters were compared between C. felis positive and negative panthers and cougars. Florida panthers had significantly lower mean cell hemoglobin count (MCHC) and higher white blood cell (WBC), neutrophil, monocyte and eosinophil counts (P < or = 0.05) than Texas cougars. Infected Florida panthers had significantly lower mean cell hemoglobin (MCH) and monocyte counts and higher neutrophil and eosinophil counts than infected Texas cougars. Although statistically significant differences were measured for hematologic parameters in C. felis positive panthers and cougars, biologically significant differences were not likely because values were generally within expected reference ranges for healthy animals. Cytauxzoonosis does not appear to have a negative effect on the hematologic parameters of chronically infected panthers and cougars. Potential transient changes during initial infection were not evaluated.

Hemolysis caused by phosphofructokinase deficiency in English springer spaniels: seven cases (1983-1986).

Seven English Springer Spaniels (6 adult males and 1 female) with chronic hemolysis and sporadic intravascular hemolytic crises were determined to have a deficiency in erythrocyte phosphofructokinase (PFK) activity, a key regulatory enzyme of anaerobic glycolysis. Intermittent severe pigmenturia concomitant with weakness, lethargy, and anorexia were the major clinical signs and commonly were related to exercise or other stressful situations that caused panting or barking (hyperventilation). Pale or icteric mucous membranes, fever, mild hepatosplenomegaly, and muscle wasting sometimes were evident. Results of routine laboratory testing indicated a persistent marked bilirubinuria and reticulocytosis with normal PCV, to severe anemia and intermittent hemoglobinuria and hyperkalemia. Erythrocyte PFK activities were severely reduced to 8% to 22% of values for control dogs. The block of glycolysis at the PFK step caused a markedly diminished erythrocyte 2,3-diphosphoglycerate content, resulting in an increased hemoglobin-oxygen affinity and compensatory accelerated erythrocyte production. Phosphofructokinase-deficient erythrocytes had increased alkaline fragility in vitro and in vivo. Hemolytic crises were induced in vivo by hyperventilation that caused transient, mild alkalemia. Studies of family members of a PFK-deficient dog suggested an autosomal recessive mode of inheritance. Carrier dogs with half-normal erythrocyte PFK activities appeared clinically normal.

Phenazopyridine toxicosis in the cat.

Severe illness developed after the oral administration of several drugs, including large doses of phenazopyridine (100 mg TID for 4 days) to a cat with dysuria and hematuria. Hemolysis and icterus were evident in blood serum and plasma after day 4 of drug administration, and many hemolyzed red blood cell "ghosts" containing Heinz bodies were observed on a stained blood smear. The cat became anemic and died within 48 hours after the last dose was administered. In an attempt to confirm a cause-and-effect relationship between drug administration and disease, 100 mg of phenazopyridine was given TID (65 mg/kg/day) for 3 days to a clinically normal cat. Nearly 50% of the hemoglobin was oxidized to methemoglobin during the course of phenazopyridine administration. Lower dosages of phenazopyridine (10 and 20 mg/kg/day) for longer periods of administration to 2 other clinically normal cats did not result in illness or anemia; however, the number and size of Heinz bodies and blood methemoglobin content were increased. Evidence of hepatic injury was observed in the clinically affected cat and in 2 of the experimental cats. The relationship between hepatic injury and toxic signs was not determined. Combination products recommeneded for treatment of cystitis in man often contain phenazopyridine. Such products should be avoided in cats unless a safe, effective dosage for phenazopyridine can be established.