Phospholipase activity of yeasts from wild birds and possible implications for human disease

Over the last decades, reports on yeast infections in humans have increased especially with respect to immunocompromised individuals. Phospholipases are enzymes which may be associated with pathogenic processes caused by opportunistic yeasts. Phospholipase activity (ph.a.) was investigated in 163 isolates of 13 species of yeasts. A total of 133 isolates were obtained through the screening of a total of 768 cloacae of wild birds (Group I: 182 birds of prey; Group II: 165 passeriformes and Group III: 421 other wild migratory birds), while 30 isolates were recovered from the droppings of birds housed in 32 distinct aviaries (Group IV). Phospholipase production was evaluated and quantified at 2 and 5 day pre-incubation (Pr.t) and incubation times (I.t). Isolates from cloacae (48.1%) and excreta (73.3%) produced ph.a. with the highest values registered after 5 days of I.t. Candida albicans, C. tropicalis, C. glabrata, C. lusitaniae, C. pelliculosa, Cryptococcus albidus, C. laurentii, Trichosporon beigelii, and Saccharomyces cerevisiae displayed the highest ph.a. after 2 days of Pr.t while Candida famata, C. guilliermondii and Cryptococcus neoformans after 5 days of Pr.t. Ph.a. was never found in Rhodotorula rubra isolates recovered from the cloacae of wild birds. Isolates (73.3%) from bird droppings showed a higher ph.a. than those from cloacae thus indicating that wild birds not only act as carriers but may also spread phospholipase-producing yeasts in the environment.     

Physical, epidemiological, and molecular evaluation of infection by Cryptosporidium galli in Passeriformes

Due to the scarcity of information related to the epidemiology of Cryptosporidium infection in passerine birds, this study aimed to determine the periodicity of fecal shedding of Cryptosporidium spp. oocysts, after natural infection, and its clinical signs, mortality, and molecular characterization. Four hundred eighty fecal samples were collected from 40 birds, including 372 samples from 31 adult birds and 108 samples from nine young birds (up to 12 months old), housed in five aviaries, monthly from September 2007 to September 2008, with the exception of April. The birds originated from aviaries in which the following species were raised: great-billed seed-finch (Oryzoborus maximiliani), lesser seed-finch (Oryzoborus angolensis), ultramarine grosbeak (Cyanocompsa brissonii), and rusty-collared seedeater (Sporophila collaris). The samples were preserved in 2.5% potassium dichromate at 4°C until processing. The oocysts were purified by centrifugal flotation in Sheather’s solution, followed by genomic DNA extraction and molecular characterization of oocysts using the nested polymerase chain reaction for amplification of fragments of the 18S subunit of rRNA gene. Intermittent shedding of oocysts was observed by positive amplification for Cryptosporidium spp. in 91 (24.5%) samples of adult birds and 14 (13%) of young birds. The sequencing of the amplified fragments enabled the identification of Cryptosporidium galli. Although all the aviaries had birds positive for C. galli, morbidity or mortality was observed in only one aviary and was associated with concomitant infection with Escherichia coli and Isospora sp.     

Isolation of Newcastle disease virus from birds of prey

In the 4 year period 1971-74 11 isolations of Newcastle Disease Virus (NDV) were made from 44 birds of prey that died in captivity. Three species of Falconiformes were involved, including one red-headed falcon (Falco chicquera), 5 European kestrels (F. tinnunculus), and 2 secretary birds (Sagittarius serpentarius), also 2 species of Strigiformes, comprising 2 barn owls (Tyto alba) and one little owl (Athene noctua). All NDV isolates were of the velogenic type.     

<Emphasis Type="Italic">Cryptosporidium</Emphasis> spp. parasitize exotic birds that are commercialized in markets,commercial aviaries,and pet shops

The purpose of this study was to genetically characterize and phylogenetically analyze the Cryptosporidium spp. isolated from exotic birds commercialized in popular markets, commercial aviaries, and pet shops located in Rio de Janeiro, Brazil. Fecal samples from individually housed birds were collected and subjected to centrifuge–flotation technique using saturated sugar solution. DNA was isolated from Cryptosporidium positive samples, and 18S subunit rDNA was amplified and processed using nested-polymerase chain reaction (PCR). To identify the protozoan species, the PCR amplicons were used for restriction fragment length polymorphism and sequencing analyses. Of the 103 analyzed fecal samples, seven (6.8%) were positive for Cryptosporidium oocysts. Sequencing and further phylogenetic analyses allowed us to identify the following species: Cryptosporidium parvum in Bengalese finch (Lonchura striata domestica) and avian genotype III in Java sparrow (Padda oryzivora) and cockatiel (Nymphicus hollandicus). The sequences of the Cryptosporidium spp. isolated from canaries (Serinus canarius) were not identifiable within the groups of known species, but they presented a higher genetic similarity with C. parvum. This is the first report in Brazil showing that C. parvum parasitizes Bengalese finches and that avian genotype III parasitizes Java sparrows.     

Feral pigeons as carriers of Cryptococcus laurentii, Cryptococcus uniguttulatus and Debaryomyces hansenii.

We collected fresh droppings and cloaca samples from feral pigeons Columba livia in the southern Swedish city of Malm?, and isolated the following fungi: Debaryomyces hansenii var. hansenii, Cryptococcus laurentii and Cryptococcus uniguttulatus. The first two species are known to be pathogenic to humans. No strains of Cryptococcus neoformans var. neoformans were found. Our results indicate that feral pigeons can be carriers of medically significant fungi other than Cryptococcus neoformans var. neoformans.     

Occurrence of Leucocytozoon and Haemoproteus (Apicomplexa, Haemosporina) in Falconiformes and Strigiformes of Italy

Blood smears from Falconiformes (91 birds of 10 species) and Strigiformes (23 birds of 5 species) captured in Italy, were examined for haematozoa. Leucocytozoon were found in Falco tinnuculus, Buteo buteo, Circus cyaneus, Circus pygargus, Accipiter nisus from Falconiformes and in Strix aluco, from Strigiformes. Haemoproteus were found in Falco tinnuculus and Strix aluco; this latter species harbored mixed infections Leucocytozoon-Haemoproteus. Prevalences were 20.80% in Falconiformes and 21.74% in Strigiformes.     

Isolation of Cryptococcus neoformans var. neoformans from pigeon droppings collected throughout Turkey.

The six hundred and thirty-four samples of pigeon droppings were collected throughout Turkey, from 54 of 80 provinces. Cryptococcus neoformans was isolated from 29 (4.6%, overall) of 634 samples and 29 isolates were from 18 provinces. Interestingly, 16 (88.9%) of these provinces occur on the three different coastlines of Turkey, therefore the ecological role of a humid climate was speculated. Almost all isolates [28/29] were recovered from samples collected from roofs (n=14) and dovecotes (n=14). All isolates were found to be C. neoformans var. neoformans.     

Cryptococcus neoformans isolated from human dwellings in Rio de Janeiro, Brazil: an analysis of the domestic environment of AIDS patients with and without cryptococcosis.

One hundred and fifty-four human dwellings in the metropolitan area of Rio de Janeiro, Brazil were studied. A total of 824 samples of indoor dust, outdoor soil and avian droppings were collected. Cryptococcus neoformans var. neoformans was isolated from 20 (13%) dwellings, comprising five (15.6%) of 32 dwellings of patients with AIDS-associated cryptococcosis; four (8.9%) of 45 dwellings of patients with AIDS but without cryptococcosis; and 11 (14.3%) of 77 dwellings of apparently healthy individuals (P>0.05). The principal factor associated with domiciliar contamination by C. neoformans var. neoformans was the presence of avians in the domestic environment or nearby the home. Cryptococcosis was more frequent among AIDS patients residing in dwellings from which C. neoformans var. neoformans was isolated than among AIDS patients from whose domestic environment the fungus was not demonstrated by the methods used (odds ratio (OR)=2.05). These findings suggest that the distribution of C. neoformans var. neoformans in Rio de Janeiro is not restricted to the classically known biotopes as well as reinforce the possibility of exogenous infection in opportunistic cryptococcosis, including exogenous infection acquired in the domestic environment.     

Cryptococcosis outbreak in psittacine birds in Brazil.

An outbreak of cryptococcosis occurred in a breeding aviary in S?o Paulo, Brazil. Seven psittacine birds (of species Charmosyna papou, Lorius lory, Trichoglossus goldiei, Psittacula krameri and Psittacus erithacus) died of disseminated cryptococcosis. Incoordination, progressive paralysis and difficulty in flying were seen in five birds, whereas superficial lesions coincident with respiratory alterations were seen in two birds. Encapsulated yeasts suggestive of Cryptococcus sp. were seen in faecal smears stained with India ink in two cases. Histological examination of the birds showed cryptococcal cells in various tissues, including the beak, choana, sinus, lungs, air sacs, heart, liver, spleen, kidneys, intestines and central nervous system. High titres of cryptococcal antigen were observed in the serum of an affected bird. In this case, titres increased during treatment and the bird eventually died. Yeasts were isolated from the nasal mass, faeces and liver of one bird. Cryptococcus neoformans var. gattii serovar B was identified based on biochemical, physiological and serological tests. These strains were resistant (minimum inhibitory concentration 64 microg/ml) to fluconazole. This is the first report of C. neoformans var. gattii occurring in psittacine birds in Brazil.     

Comparison of the effectiveness of rHVT-H5, inactivated H5 and rHVT-H5 with inactivated H5 prime/boost vaccination regimes in commercial broiler chickens carrying MDAs against HPAI H5N1 clade 2.2.1 virus

Vaccination is the main tool implemented in Egypt since 2007 to control H5N1 avian influenza. The present study aimed at comparing the effectiveness of three avian influenza vaccination regimes in commercial broiler chickens carrying high levels of maternally derived antibodies (MDAs). Day-old chicks were divided into four experimental groups. Group I received only the rHVT-H5 vaccine (recombinant turkey herpesvirus (HVT) which carries a H5 clade 2.2 insert) administered at D1. Group II received only the KV-H5 (an oil emulsion killed vaccine prepared from reassortant HPAI virus (A/duck/Anhui/1/06)) vaccine (inactivated reverse genetic H5N1 clade 2.3.4 virus) administered at D8. Group III received rHVT-H5 and KV-H5 as prime/boost. Group IV served as unvaccinated control. Weekly serological monitoring was conducted using the haemagglutination inhibition test. Two challenge experiments were conducted at D28 and D35 using HPAI H5N1 clade 2.2.1 virus. Birds were monitored daily 14 days post-challenge for morbidity and mortality, and oropharyngeal swabs were collected for virological monitoring. Initially, day-old chicks had high mean MDA titres (9?+?0.9 log₂). The MDA half-life was >7 and <7 days, respectively, for unvaccinated and vaccinated birds. Group III showed the highest post-vaccination humoral immune response and seroconversion rate. The highest protection rate against morbidity (80–90%) and mortality (90–90%) was obtained in Group III after challenge at D28 and D35, respectively, as compared to Group I (70–70%) and (80–90%) and Group II (0–0%) and (30–30%). Groups I and III had lower number of shedder birds. The vaccination regime with prime/boost conferred the highest and earliest protection, and can hence be recommended for the broiler production sector in endemic and high HPAI H5N1 challenge areas.     

Choanotaenia spp. infestation of Australian finches (Estrildidae)

Mortality of 2 to 5% was reported in young adult finches (Emblema, Poephila, Neochmia and Chloebia spp.) from aviaries in Victoria, Australia. Affected birds were depressed and had severe diarrhoea for 2 to 4 days before death. Lesions found on gross postmortem examination were marked distension of the duodenum by 20 to 40 cestodes (Choanotaenia spp.) and fluid-filled distal intestines. Histological examination of the duodenum revealed enteritis. The heavy cestode infection was considered the cause of the diarrhoea and deaths. Oral treatment of remaining finches in two aviaries with praziquantel was associated with cessation of mortality. Specific identification of the cestodes was not possible due to the condition of the specimens and the confused taxonomy of some Australian species of the genus. Control of the internal parasites, and of intermediate hosts in the environment, is important in captive finch management.     

Behavioural and physiological effects of population density on domesticated Zebra Finches (Taeniopygia guttata) held in aviaries

Zebra Finches (Taeniopygia guttata) are highly social and monogamous birds that display relatively low levels of aggression and coordinate group life mainly by means of vocal communication. In the wild, small groups may congregate to larger flocks of up to 150-350 birds. Little is known, however, about possible effects of population density on development in captivity. Investigating density effects on physiology and behaviour might be helpful in identifying optimal group size, in order to optimise Zebra Finch wellbeing. A direct effect of population density on development and reproduction was found: birds in lower density conditions produced significantly more and larger (body mass, tarsus length) surviving offspring than birds in high density conditions. Furthermore, offspring in low density aviaries produced slightly longer song motifs and more different syllables than their tutors, whereas offspring in high density aviaries produced shorter motifs and a smaller or similar number of different syllables than their tutors. Aggression levels within the populations were low throughout the experiment, but the number of aggressive interactions was significantly higher in high density aviaries. Baseline corticosterone levels did not differ significantly between high- and low density aviaries for either adult or offspring birds. On day 15 post hatching, brood size and baseline corticosterone levels were positively correlated. On days 60 and 100 post hatching this correlation was no longer present. The results of this study prove that population density affects various aspects of Zebra Finch development, with birds living in low population density conditions having an advantage over those living under higher population density conditions.     

Misidentification of clinical yeast isolates by using the updated Vitek Yeast Biochemical Card.

The Vitek Yeast Biochemical Card (YBC) is widely used as a rapid identification (RI) (within 48 h) system for clinical yeast isolates. We compared the RI results obtained by the YBC technique with matched results obtained with the API 20C system. The RI of germ tube-negative yeasts isolated from 222 clinical specimens was performed with the YBC system, and the results were compared with those of standard identifications obtained by using the API 20C system and morphology, with additional biochemical reactions performed as required. Commonly isolated yeasts (Candida albicans [n = 29], Candida tropicalis [n = 40], Torulopsis [Candida] glabrata [n = 28], Candida parapsilosis [n = 12], and Cryptococcus neoformans [n = 14]) were generally well identified (115 of 123 [93%] identified correctly, with only C. albicans, C. tropicalis, and C. neoformans mis- or unidentified more than once). The RI of less commonly isolated yeasts included in the YBC database, however, was less successful (54 of 99 [55%] correct). The YBC card failed to identify 42% (10 of 24) of Candida krusei isolates, 80% (4 of 5) of Candida lambica isolates, 88% (7 of 8) of Trichosporon beigelii isolates, and 83% (10 of 12) of Cryptococcus isolates (non-C. neoformans species). For most identification failures (79%; 42 of 53) there was no identification by the end of 48 h; the other identification failures (21%; 11 of 53) gave definite but incorrect identifications. Of eight rare clinical yeast isolates not included in the Vitek database, six were correctly, not identified, while two (25%) were falsely assigned a definite RI (one Hansenula fabianii isolate was identified as Rhodotorula glutinis, and one Hansenula isolate [non-Hansenula anomala] was identified as Hansenula anomala). While the Vitek YBC rapidly and adequately identifies common yeast isolates, it fails in the RI of more unusual organisms.     

Efficacy of swabbing versus a conventional technique for isolation of Cryptococcus neoformans from decayed wood in tree trunk hollows.

The efficacy of swabbing versus a conventional sedimentation technique was evaluated for sampling of decayed wood in tree trunk hollows for isolation of Cryptococcus neoformans. Of 52 samples of decayed wood, bark or other plant debris originating from 35 living trees, 42 wood samples yielded C. neoformans. The positive samples included 40 collected from 31 Syzygium cumini trees growing along roadsides in Old Delhi, whereas the remaining two were from inside tree trunk fissures of Ficus religiosa in a New Delhi locality. The number of wood samples found positive by swabbing was 40 (95%) as opposed to 32 (76%) by the conventional technique, and this difference was statistically significant (P < 0.01). Also, the conventional technique showed 24% false-negative results, which was in striking contrast to only 5% by swabbing. Furthermore, swabbing yielded a significantly higher C. neoformans mean colony count than did the conventional technique (P < 0.005), thus highlighting greater efficacy of the former technique. The overall prevalence of C. neoformans in the S. cumini trees investigated was 84% (26/31 trees) which is the highest as yet reported from any tree species in India. Varietal identification and serotyping was done with 33 of the C. neoformans isolates, 31 of which came from 23 tree trunk hollows of S. cumini and two from the tree trunk fissures of F. religiosa. Among the S. cumini isolates, 26 were identified as C. neoformans var. gattii (all serotype B except two untypeable ones) and five as C. neoformans var. neoformans, serotype A (= C. neoformans var. grubii). Both of the F. religiosa isolates belonged to C. n. var. neoformans, serotype A. Being a more efficacious, simple, less time-consuming and less hazardous technique, swabbing is recommended for wider use in order to further elucidate the ecology of C. neoformans.     

New culture medium for the presumptive identificaion of Candida albicans and Cryptococcus neoformans.

A new medium composed of Tween 80, oxgall, caffeic acid, and Davis agar (TOC) that provides for the rapid presumptive identification of Candida albicans and Cryptococcus neoformans is described herein. C. albicans is differentiated from other yeasts by the sequential production of germ tubes and chlamydospores. In a comparison with cormeal agar control plates, there was an increase of chlamydospore-forming strains of C. albicans (97.1% versus 87.2%) and a decrease in the time required for chlamydospore formation (24 h versus 48 h). C. neoformans produced a brown pigment of TOC, which is specific for its identification, thus differentiating it from the other yeasts. A comparison of 24-h pigment production by C. neoformans on TOC with that of birdseed agar showed a dark, coffee brown color in the former cultures and a light brown color in the latter. The change in pigmentation of C. neoformans, as well as morphological changes in C. albicans, can be induced within 3 to 12 h and in not more than 24 h on the TOC medium.     

Effect of thyroid hormone on concentrations of plasma calcitonin in broiler chicks

The purpose of these studies was to determine the effect of thyroidectomy (Tx), and thyroid hormone (T3/T4) treatment on concentrations of plasma CT in chicks. In addition, the turnover of CT in Tx- and T3/T4-treated chicks was estimated using a novel nonradioactive salmon CT preparation. One-week-old broiler chicks (Gallus domesticus) (n = 75) were divided into three groups. Group I was sham-injected daily (i.m. saline), Group II was injected with 50 micrograms/day of T3/T4 while Group III was injected with the goitrogen, methimazole, (150 mg/kg BW per day) for 8 weeks. Chicks (8-9 weeks old) were implanted with catheters in the brachial wing vein and administered ruthenium-labeled salmon CT. Blood samples were collected at 30 s, 1, 2, 4, 8, 20 min, and 3 h after injection. Results showed that concentrations of plasma CT were decreased in T3/T4-injected birds. There was no significant effect of methimazole on circulating concentrations of plasma CT. The half-life of CT was significantly increased (P < 0.05) in both T3/T4-injected (n = 6; 1.34 +/- 0.16 min) and goitrogen-treated birds (n = 2; 5.81 +/- 2.83 min) compared to controls (n = 7; 54 +/- 3 s) The results demonstrate that changes in concentrations of plasma thyroid hormones can significantly affect concentrations of plasma CT.     

Comparison of updated Vitek Yeast Biochemical Card and API 20C yeast identification systems.

The updated Vitek Yeast Biochemical Card (YBC) was compared with the API 20C by using 409 germ tube-negative yeasts and Geotrichum spp. that were either clinical or proficiency sample isolates. The API 20C was the reference standard. The 409 isolates represented nine genera and 21 species. Morphology agars were inoculated and interpreted for each isolate. The API 20C identified 406 isolates (99.3%), while the Vitek YBC identified 367 (89.7%). Both systems identified the majority of yeasts after 24 h of incubation--73.4% were identified by the API 20C and 77.4% were identified by the Vitek YBC. The Vitek 24-h reading had some incorrect identifications. These included 14 isolates of Candida tropicalis that were identified as Candida parapsilosis (91 to 97% reliability) and 3 isolates of Candida krusei that were called Blastoschizomyces capitatus (Geotrichum capitatum), Candida rugosa, and Candida zeylanoides. In total, the Vitek YBC misidentified 30 isolates, while the API 20C misidentified 3 isolates. In addition, results for 14 isolates with the Vitek YBC were listed under the category "no identification." Morphology agars were required for identification with 89 isolates (21.9%) when the API 20C was used and with 50 isolates (12.6%) when the Vitek YBC was used. Apart from the price of the Vitek instrument, the API 20C costs $1.28 more per test than the Vitek YBC. Overall, the updated Vitek YBC compares favorably with the API 20C in the identification of common yeasts such as Torulopsis glabrata, C. parapsilosis, and Cryptococcus neoformans. However, problems were encountered with the Vitek system in the identification of C. tropicalis, C. krusei, Trichosporon spp., and some Cryptococcus spp. The routine use of morphology agars with either method is recommended.     

Horizontal transmission of lymphoid leukosis virus. Influence of age, maternal antibodies and degree of contact exposure

Eight groups of 1-day-old or 8-week-old chickens were exposed by contact to lymphoid leukosis virus (LLV) infection. Five groups of about 60 spf chickens were used. Three groups of the same size were progeny from LLV vaccinated hens. Five groups were housed in one chicken house in close contact with a large number of immunologically tolerant chickens (virus "spreaders"). On two occasions infectious LLV was recovered from air/dust samples collected in this house. In the second house a small number of congenitally infected birds generated a mild degree of LLV exposure. It was demonstrated that infection by contact may lead to lymphoma formation and congenital virus transmission. The incidence of virus infection and LL mortality in the groups of birds exposed at 8 weeks of age were significantly lower than in chickens exposed at 1 day of age. In addition, about 100-fold differences in numbers of LLV-associated white blood cells were observed between both age groups. These results indicate that in addition to resistance to tumour formation, resistance to LLV infection develops in the chicken with increasing age. Maternal antibodies, present in three groups exposed at 1 day of age, reduced the rate of infection and the incidence of LL.     

Fatty liver syndrome in captive bustards: Clinical, pathological and epidemiological findings.

Clinical, pathological, and epidemiological findings are presented on fatty liver syndrome mainly in houbara bustards (Chlamydotis undulata macqueenii), but also in some other bustard species. Of 72 houbara bustards, 34 (47%) had fatty liver diagnosed post-mortem. Males and females were equally susceptible, and both adults and juvenile birds were affected. Bustards with fatty liver had significantly greater abdominal fat reserves than unaffected birds. Other predisposing factors included poor husbandry, translocation between aviaries, handling and capture paresis.     

In Vitro Phagocytosis and Intracellular Fate of Variously Encapsulated Strains of Cryptococcus neoformans

Five isolates of Cryptococcus neoformans type A with stable capsular thicknesses were used. Three of the isolates had capsules of medium size, one had a minimal capsule, and the other, a large capsule. Peritoneal exudate cells from Lewis rats were cultured on cover slips in Leighton tubes containing medium 199 and 20% fresh, isologous normal rat serum. Yeast cells were added to the Leighton tube cultures, and, 2 hr later, the extracellular yeasts were rinsed out. Cover slips were removed from some tubes for Wright staining and measurement of both phagocytosis and loss of macrophages. The remaining tubes were reincubated and sampled at 24 or 48 hr. To determine fate of yeast cells after ingestion, washed cover slips were inverted onto agar slide cultures, and specific macrophages were observed in situ for subsequent multiplication of their intracellular yeasts. More than half of the macrophages survived 24 to 48 hr of exposure to different strains of C. neoformans, with small, medium, or large capsules. Phagocytic activity was dependent upon a heat-labile factor in normal rat serum. The number of yeast ingested by macrophages was inversely proportional to the capsular size. Although most of the ingested yeasts were resistant to intracellular killing, the agar culture technique clearly demonstrated that many were unable to multiply, presumably dead. Three of the isolates were more susceptible than the other two, and the fate of these yeasts after engulfment was not correlated with their capsular size.