Chicken infectious anemia virus (CAV) is a worldwide-distributed infectious agent that affects commercial poultry. Although this agent was first detected in Argentina in 1994, no further studies on CAV in this country were reported after that. The recent increased occurrence of clinical cases of immunosuppression that could be caused by CAV has prompted this study. Our results confirmed that CAV is still circulating in commercial flocks in Argentina. Phylogenetic analysis focusing on the VP1 nucleotide sequence showed that all Argentinean isolates grouped together in a cluster, sharing a high similarity (>97%) with genotype B reference strains. However, Argentinean isolates were distantly related to other strains commonly used for vaccination in this country, such as Del-Ros and Cux-1. Sequence analysis of predicted VP1 peptides showed that most of the Argentinean isolates have a glutamine residue at positions 139 and 144, suggesting that these isolates might have a reduced spread in cell culture compared with Cux-1. In addition, a particular amino acid substitution at position 290 is present in all studied Argentinean isolates, as well as in several VP1 sequences from Malaysia, Australia, and Japan isolates. Our results indicate that it is possible to typify CAV strains by comparison of VP1 nucleotide sequences alone because the same tree topology was obtained when using the whole genome sequence. The molecular analysis of native strains sheds light into the epidemiology of CAV in Argentinean flocks. In addition, this analysis could be considered in future control strategies focused not only on breeders but on broilers and layer flocks.

Two primary broiler breeder lines, A and B, were examined for their potential to produce nitric oxide (NO) after stimulating splenocytes from 20-day-old embryos with lipopolysaccharide and interferon-γ. Significant differences were found between lines A and B. Overall, line A had a higher response than line B, but line A also had a large degree of variation between individual sire families. Selection for high and low responders within line A resulted in the segregation of high- and low-responder sire families. Offspring from sire families selected for high and low NO responses and from a nonselected control group from line A were challenged with RB-1B Marek's disease (MD) virus to determine whether these differences could be used to select for improved resistance to MD. Virus isolation rates at 6 and 10 days postinfection were not significantly different, but unexpectedly, the MD incidence in the high-responder group was significantly higher than in the other two groups.

Reticuloendotheliosis virus (REV) fragments are a common contaminant in some commercial vaccines such as fowl poxvirus (FPV) and Marek's disease virus. However, only those strains integrating or containing a near-intact REV provirus are more likely to cause problems in the field. We confirm here, by PCR assays and animal experiments, that vaccines against FPV and herpes virus of turkeys were contaminated with full genome sequences of REV. Further, we determined the complete proviral sequence of two REV isolates from contaminated vaccines. Two REV isolates (REV-99 and REV-06) present in the vaccines were both replication competent, and their proviral genome was 8286 nucleotides in length with two identical long terminal repeats (LTR). The complete genome in these two REV isolates shared 99.8% identity to APC-566 and fowl poxvirus REV proviral inserts (FPV-REV). REV-99 and REV-06 LTR showed over 99% identity to chicken syncytial virus (CSV), but an identity of only 75.8 % and 78.0%, respectively, to SNV. Alignments with other available REV gag, pol, and env sequences revealed high similarity at the nucleotide level. The results further indicated that the prototype CSV may be the most-important REV contaminant in the commercial vaccines, and distinct genotypes of REVs may cocirculate in chicken flocks of China at the present time.

Infectious bursal disease virus (IBDV) is an immunosuppressive virus which primarily infects IgM B-cells in the bursa of Fabricius. Flow cytometric analysis was used to phenotype B-cell populations in the bursa and spleen following IBDV infection. In the bursa, two IgM B-cell subpopulations, designated as A and B, were identified based on cell size and granularity. While both subpopulations differentially expressed IgM and Bu-1b surface markers, both groups displayed major histocompatibility complex class II surface antigens at equal levels. Following IBDV challenge of nonvaccinated birds, the B subpopulation was significantly reduced between 7 and 21 days postchallenge compared to either nonchallenged birds or vaccinated–challenged birds. However, the reduction of subpopulation B in the bursa, following IBDV exposure, did not reduce the levels of total serum IgA, IgG, and IgM, nor did it affect IgG and IgA B-cells in the spleen. Phenotypic analysis of the subpopulations identified differential expression of Lewis^x, IgM, Bu-1b, and MUI78 surface antigens between the subpopulations. Overall, these are the first studies to identify two distinct IgM B-cell subpopulations in the chicken bursa, and the first to describe the decrease in the IgM B-cell population relative to IgA and IgG B-cells following IBDV infection.

Turkey coronavirus (TCoV) is a causative agent associated with poult enteritis and mortality syndrome (PEMS) in turkeys worldwide. The disease is an acute, highly contagious enteric disease that is characterized by depression, anorexia, diarrhea, and high mortality in commercial turkey flocks. The presence of TCoV in 12 intestinal-content samples, from turkey flocks aged between 10 and 104 days and exhibiting severe enteritis, was monitored during the period of 2004 to 2006. TCoV detection was accomplished by a reverse transcriptase-polymerase chain reaction (RT-PCR) through amplification of the 3′ UTR region, followed by amplification of genes 3 and 5. Molecular characterization of the viruses was done through amplification of genes 3 and 5 and showed evidence of genetic similarity between them, although they differed from sequences of other TCoVs described in the literature. In relation to gene 3, samples showed a greater relationship with chicken infectious bronchitis virus (IBV), while gene 5 showed greater identity with pheasant coronavirus (PhCoV). Our results suggest that the strategy of amplification of the 3′ UTR region, followed by sequencing of genes 3 and 5, has proven to be an effective means of detecting TCoV in intestinal contents.

Duck enteritis virus (DEV) causes substantial losses on duck farms; however, its molecular biology is poorly understood. Here, an open reading frame of a US3-like gene of DEV was identified from a DEV genomic library. Its existence was confirmed by cloning from DEV-infected duck embryo fibroblasts (DEFs) and DNA sequencing. The US3-like gene was then subcloned into a prokaryotic protein expression vector and expressed as a six-histidine–tagged fusion protein in Escherichia coli. The protein was purified and inoculated into rabbits for antiserum production. A primary antibody specific to the gene was successfully generated and used to detect the US3-like protein in DEV-infected duck cells. In vivo expression of the US3-like protein in DEV-infected DEFs was demonstrated with indirect immunofluorescence assay and regular fluorescence microscopy, whereas uninfected DEFs did not show any specific fluorescent staining. Furthermore, indirect immunofluorescence assay and confocal microscopy were used to study the time course and subcellular localization of the protein expression. The protein was found to be expressed as early as 2 hr postinfection, and its expression was increased by time at 4, 8, 12, and 24 hr postinfection. The protein was found to be localized mostly around the perinuclear area and in the cytosol, and also in the nucleus at later time points. In addition, a US3 protein phylogenetic tree was constructed and showed that the evolutionary relationship of DEV is close to the genus Mardivirus. In short, the DEV US3-like gene and its in vivo protein expression were found for the first time, and DEV classification and the gene's functions were suggested.

The Virginia avirulent strain (VAS) of turkey hemorrhagic enteritis virus (THEV), which is commonly used in live vaccines for commercial turkeys, was studied to determine characteristics of infection. It has been observed that turkeys infected with the VAS maintain protective antibody levels in excess of 20 wk postvaccination. It is theorized that this immune response is modulated by either a persistent or latent infection. A series of studies have been undertaken to determine changes in virus location and serology over time. A trial was also conducted to evaluate the effect of corticosteroid administration on viral recrudescence, and an attempt was made to isolate live virus from tissues of birds 10 wk postinfection (pi). Antibody titers were determined by enzyme-linked immunosorbent assay, and PCR was used to detect viral DNA. Histopathology was performed on formalin-fixed paraffinized tissues. Viral DNA was detected in various tissues through 15 wk pi in the presence of high antibody titers. Viral DNA was detected at 3–5 days pi in the spleens of susceptible turkeys inoculated with tissues collected from infected birds at 10 wk pi. It is unknown whether the viral DNA is associated with live virus or rather is the result of persistent maintenance of the viral genome within lymphoid/macrophage target cells. Future studies will test for viral RNA in order to confirm the presence of replicating THEV. Regardless of the actual status of the THEV DNA detected at 10–15 wk pi, it is clear that THEV does not cause a simple acute infection. The characteristics of THEV infection are identical to the nonlytic persistent infections seen in human adenoviruses, and therefore THEV may serve as a model for the study of virus–cell interactions mediating persistence.

Transgenic lines of Arabidopsis thaliana producing recombinant σC protein were developed. The S1 gene encoding σC protein of an avian reovirus (ARV) was amplified by reverse-transcription PCR (RT-PCR). The amplified product was cloned into a plant-expression vector, pE1857, with a strong promoter for expression. The resulting construct with the BAR gene cassette for bialaphos selection was designated rpE-σC and was introduced into Agrobacterium tumefaciens by electroporation. Agrobacterium containing the rpE-σC constructs that transform A. thaliana and transgenic plants were selected using bialaphos selection. The presence of S1 transcript in plants and their relative level of expression were determined by real time RT-PCR. Western blot analysis further confirmed the presence of σC protein in the plants. Transgenic lines with high levels of σC protein were selected for immunization experiments using specific-pathogen free chickens. Recombinant σC protein produced in plants induced a variety of immunoglobulin G (IgG) antibody responses in chickens. Recombinant protein administered either subcutaneously or orally in birds showed significant protection against challenge. Results suggested that the recombinant σC protein produced in plants has the potential for large-scale vaccination against ARV in commercial poultry.

The efficacy of killed vaccine of Avibacterium paragallinarum with mineral oil adjuvant and aluminum hydroxide gel adjuvant was tested for antibody titers and protection. The autogenous vaccines at a concentration of 10¹⁰ colony-forming units (CFU)/ml were administered to 5-wk-old male layers by subcutaneous injection in the neck twice at a 3-wk interval. Each chicken was challenged with 10⁸ CFU/ml in 400 µl of an homologous isolate of A. paragallinarum serotype A, IR1, at 4 wk after the second vaccination via the nasal route. Sera were collected and the antibodies were tested by the hemagglutination inhibition test. The results revealed that the autogenous mineral oil adjuvant vaccine provided the antibody titer significantly faster than the other groups (P < 0.05). The average antibody titers of the group vaccinated with autogenous mineral oil adjuvant vaccine were higher than those of the group vaccinated with autogenous aluminum hydroxide gel adjuvant vaccine. The protective ability of vaccines was assessed by infraorbital sinus swab after 5 days postchallenge. The autogenous vaccines prepared with mineral oil adjuvant and aluminum hydroxide gel adjuvant protected all the chickens after challenge. No bacteria were isolated from the infraorbital sinuses of chickens in either autogenous vaccine group with either high or low antibody titers. The commercial vaccines prepared from mineral oil or aluminum hydroxide gel adjuvant revealed some protection. This is in contrast to the unvaccinated control group, in which facial edema and serous nasal discharge was found, and bacteria could be isolated from all chickens in the group.

Marek's disease virus (MDV), which is the causative agent of Marek's disease (MD), is shed by infected chickens and transmitted to other chickens through the respiratory route. Experimental reproduction of MD has been commonly done either by intra-abdominal inoculation of cell-associated MDV or by exposure to MDV-infected ‘seeder’ chickens. The former method does not mimic the natural route of MDV infection, whereas the latter method suffers from lack of uniformity in the timing and amount of virus transmission from seeder chickens to susceptible birds. The aim of the present study was to establish an infection model of MDV that mimics the natural route of infection. Here we report that when chickens were exposed for 20 min to aerosols (particle size 1.91 µm) of cell-free MDV suspensions containing 1280 plaque-forming units/ml, which were generated using a nebulizer, pathological and clinical signs of MD were observed in 95%–100% of the aerosol-exposed chickens by 21 days post-infection (dpi). Chickens that were exposed to aerosols and sampled at 1, 2, 3, 10, and 21 dpi showed MDV replication as early as 1 dpi in lungs as well as in other tissues such as spleen and bursa of Fabricius. This infection model will facilitate the studies directed to elucidate MDV-host interaction at the site of virus entry.

Newcastle disease (ND) is a major threat to poultry, but the outbreak of the disease is well controlled by the vaccination. Recently, in ovo administration technology has been realized as a safe, efficacious, and convenient method for chicken vaccination. However, no in ovo administration has been applied for ND or other live vaccines that are highly pathogenic against chicken embryos. We found that an attenuated Newcastle disease virus (NDV) was applicable for an in ovo vaccination by adsorbing the virus to aluminum hydroxide (AH). Pathogenicity to chicken embryos of the AH-adsorbed NDV could be decreased compared with the administration of the virus alone. Namely, in ovo administration of the AH-adsorbed attenuated NDV resulted in improved hatchability and survival rate and better antibody responses of protection-level immunity compared with the administration of NDV alone. However, further improvements in hatchability and survival rate are necessary for practical application. From these results, in ovo vaccination with the AH-adsorbed attenuated NDV was revealed to be safe and immunogenic to chicken embryos. The use of AH-adsorbed attenuated live viruses might be applicable for in ovo vaccinations against not only ND but also other avian infectious diseases.

The messenger-RNA (mRNA) expression of selected cytokines and chemokines in primary chicken oviduct epithelial cells (COEC) was determined following in vitro infections with wild-type or type three secretion system (T3SS)–mutant Salmonella enterica serovar Enteritidis (SE) strains. All SE strains examined in this study elicited the expression of proinflammatory immune mediators including inducible nitric oxide synthase (iNOS), CXCLi1 (K60), CXCLi2 (IL-8), CCLi3 (K203), and CCLi4 (MIP-1β). SE also triggered the expression of an anti-inflammatory cytokine, IL-10, but repressed TGF-β3 transcription. Both T3SS-1 (sipA and sipB) and T3SS-2 (pipB and ssaV) mutants showed reduced capacity, compared to the wild-type SE, to stimulate iNOS mRNA expression in COEC. T3SS-1 (sipA and sipB) mutants were significantly impaired in their ability to induce the expression of CXCLi1 and CXCLi2. T3SS-2 mutants displayed a wild-type phenotype in terms of modulating the expression of chemokines and cytokines in COEC. The expression of iNOS, but not CXC chemokines, correlated with the number of intracellular bacteria in COEC. Genetic complementation of the sipA mutation restored a wild-type phenotype. Thus, SE induction of CXCLi1 and CXCLi2 was sipA-dependent. These results provide enhanced insights into the complex interplay between local host innate immune system and bacterial virulence factors.

The sensitivity of field isolates of turkey coccidia from the United States to the anticoccidial drugs amprolium, clopidol, diclazuril, and monensin was investigated. Clopidol and diclazuril were the most effective, followed by monensin and amprolium. Thirty-one isolates were classified as resistant to amprolium, 23 resistant to monensin, 10 resistant to diclazuril, and 6 resistant to clopidol. Six isolates were partially resistant to monensin, 10 partially resistant to clopidol, and 11 partially resistant to diclazuril. Four isolates were sensitive to monensin, 12 sensitive to diclazuril, and 17 sensitive to clopidol.

This study identified and assessed secreted proteins of Clostridium perfringens additional to those previously described for their ability to protect broiler chickens against necrotic enteritis (NE). Secreted proteins of virulent and avirulent C. perfringens were electrophoretically separated and reacted with serum of chickens immune to NE. Three immunoreactive protein bands unique to the virulent C. perfringens were identified by mass spectrometry as the toxin C. perfringens large cytotoxin (TpeL), endo-beta-N-acetylglucosaminidase (Naglu), and phosphoglyceromutase (Pgm). The genes encoding Naglu and Pgm proteins were cloned, and their gene products were purified as histidine-tagged recombinant proteins from Escherichia coli and used in immunizing chickens. Immunized and nonimmunized control broiler chickens were then challenged with two different strains (CP1, CP4) of C. perfringens and assessed for the development of NE. Of the two immunogens, Pgm immunization showed significant protection of broiler chickens against experimental NE, although protection reduced as challenge severity increased. However, birds immunized with Naglu were protected from challenge only with strain CP4. Birds immunized with these proteins had antigen-specific antibodies when tested in an enzyme-linked immunosorbent assay. In conclusion, this study demonstrated the partial efficacy of additional secreted proteins in immunity of broiler chickens to NE. The study also showed that there may be differences in the protective ability of immunogens depending on the infecting C. perfringens strain.

Mycoplasma gallisepticum (MG) is an economically significant pathogen of poultry species. Among the table egg sector of the poultry industry, live attenuated strains of MG are commonly used to limit production losses associated with MG-induced disease. These vaccines, however, may be problematic to broiler- and turkey-related industries because of associated virulence; therefore, an understanding of the transmissibility of the live MG vaccines is of particular importance. In the present study, a broiler model addresses the effect of vaccine application route and dosage on the transmission of the MG vaccine FVAX-MG® to commingled unvaccinated subjects for 7 wk postvaccination. Vaccinations occurred at 2 wk of age via eyedrop or spray application at 1× (4 × 10⁶ colony-forming units [cfu]), 10⁻³× (4 × 10³ cfu), or 10⁻⁶× (4 cfu) of the manufacturer's recommended dosage, and subsequent transmission to unvaccinated subjects was measured. The serologic response to MG antigen and the presence of MG DNA indicated FVAX-MG transmission only within the 1× FVAX-MG eyedrop treatment. Among no other treatment was transmission of FVAX-MG detected. The results of the present study demonstrate that the dosage and vaccination route may have direct implications on subsequent transmission of FVAX-MG.

Similar to West Nile virus (WNV), Japanese encephalitis virus (JEV) has a history of intercontinental spread, and birds are important for the maintenance and transmission of both of these closely related viruses. We examined viremic and serologic responses of blackbirds (Agelaius phoeniceus), with and without immunity to WNV, following experimental inoculation with two strains of JEV. Japanese encephalitis (JE) viremia was detected in only one of 16 (6.3%) WNV-immune birds, while all 16 nonimmune birds had detectable JE viremia. Two weeks after JEV inoculation, all birds without pre-existing WNV immunity had clearly distinguishable anti-JEV antibodies, while in all birds with pre-existing WNV immunity, antibodies to WNV and JEV were either indistinguishable or the anti-WNV antibody titers were significantly higher. As WNV is endemic throughout much of North America, WNV immunity among birds may dampen transmission while complicating the serologic diagnosis of JEV, should this pathogen be introduced to North America.

Three infectious bronchitis virus (IBV) strains, isolated from suspected Tunisian broiler flocks, were characterized as variant viruses using genotyping and serotyping techniques. They were compared with commonly used vaccine strains, including 793/B, D274, and Massachusetts types. Reverse transcription-PCR-restriction fragment length polymorphism, nucleotide sequencing, and GenBank BLAST database analyses of the hypervariable region of the S1 subunit of the virus spike gene showed that the three isolates, designated TN20/00, TN200/01, and TN335/01, share from 64% to 82% homologies between each other but are very different from the H120 strain, the only infectious bronchitis vaccine used in Tunisia. In addition, they showed from 57% to 78% similarities with the European genotypes, including D274 and 793/B. Phylogenetic data allowed classification of the three Tunisian isolates as new genotypes placed inside the same genetic group as the CR88121 and D274 genotypes but very distant from the Massachusetts genotype. Cross-virus neutralization tests confirmed the genotyping results and showed that both TN200/01 and TN335/01 isolates are serologically related, whereas the TN20/00 is closer to TN335/01 than to TN200/01. Moreover, all three Tunisian isolates are closely related to the European variant serotypes, including the CR88121 and the D274 strains, but none is serologically related to the H120 vaccine strain. These data demonstrated, for the first time in Tunisia, the cocirculation of IBV variant serotypes along with the Massachusetts type, causing severe clinical diseases and high economic losses to the poultry industry.

Muscovy duck reovirus (MDRV) is an important poultry pathogen that causes high morbidity and mortality in ducklings. The mechanisms by which viruses kill susceptible cells, and ultimately produce diseases, in Muscovy duck remain poorly understood. In this study, we focused on the biologic functions of the MDRV p10.8 protein in vitro. The p10.8 protein is a small protein of MDRV that is encoded by the first open reading frame of the S4 segment. In our study, the p10.8-encoding gene was individually cloned and expressed in bacterial and eukaryotic cells. The p10.8 protein had no potential transmembrane domain; it shared no sequence similarity to other known fusion-associated small transmembrane proteins encoded by the avian reovirus, Nelson Bay virus or baboon reovirus; and it did not show any syncytium formation activity. The p10.8 protein induced apoptosis when expressed by itself in transfected primary Muscovy duck embryonic fibroblasts or in Vero E6 cells. Four assays were used to analyze the apoptosis induced by p10.8: DNA ladder formation; terminal deoxynucleotidyl transferase dUTP nick end labeling; enzyme-linked immunosorbent assay detection of cytoplasmic histone-associated DNA fragments; and nuclear staining with propidium iodide. Two deletion products, p10.8δ1 (1–63aa; amino acid position) and p10.8δ2 (64–96aa), were constructed. Deletion analysis suggests that p10.8δ1 (1–63aa) is important in mediating p10.8-induced apoptosis because its deletion abolishes induction of apoptosis.

The aim of this investigation was to determine the presence of the opportunistic pathogen Clostridium perfringens by PCR and DNA sequencing, without previous cultivation. This methodology was then used to investigate how C. perfringens was affected by different preventive measures, such as ionophores and feed additives, for necrotic enteritis in broilers chickens. DNA was extracted from the intestinal content or intestinal tissue by DNA extraction kits. Detection limits for 16S rRNA, alpha-toxin, and cpb2 PCR gene targets were approximately 1 × 10³, 5 × 10⁴, and 1 × 10⁶ cells per g of intestinal content or tissue, respectively, as determined with samples spiked with C. perfringens. The method was evaluated with samples from single conventional broilers or from pools of six birds of experimentally reared broilers. Conventional chickens, raised with salinomycin in their feed, showed reduced numbers of C. perfringens-positive samples (P < 0.05) for all three PCR tests. With respect to cpb2, a tendency to detect more samples as positive for C. perfringens was observed with increasing age. The addition of sodium butyrate and lactic acid in the feed for experimental birds had a minor effect (P < 0.10) on positive samples, as detected with the 16S rRNA PCR. For experimental birds fed whole wheat, only three out of six pools of six birds allowed detection of C. perfringens by the 16S rRNA PCR, compared to five for the untreated controls or the Avilamycin- or prebiotic-treated birds. All 16S rRNA partial gene sequences obtained were identical and were 99.5% similar to the rrnB gene of the type strain of C. perfringens. Two types of the partial cpb2 gene sequence were detected with a similarity of 93%. One type was translated into protein, whereas a stop codon was found in the other type. Both types were located in the “atypical” phylogenetic group of the cpb2 gene sequences. The PCR test, based on extraction of DNA from intestinal content, provided rapid screening of poultry for C. perfringens without the need to have access to facilities in order to immediately cultivate and identify bacteria at the location of sampling. Further work is suggested to determine the relationship between the degree of necrotic enteritis, the actual level of C. perfringens in the animal, and the detection achieved by PCR.

This retrospective study concerned 41 infectious bursal disease virus (IBDV) isolates obtained from Brazilian broiler and layers flocks by reverse transcription-polymerase chain reaction. Twenty-five of them were identified as very virulent (vv) by restriction enzyme analysis and by further nucleotide and phylogenetic analysis. All of them had the typical amino acid residues, and all clustered in a phylogenetic tree with the vvIBDV strains. Four amino acid substitutions, at positions D213N, G254D, S317R, and D323E, were common to 3 vv isolates, Br/03/DB, Br/03/CK, and Br/04/CR, and differed from other vv isolates and strains. These isolates came from the same locale, but were collected in different years, indicating that the vvIBDVs circulating on Brazilian farms are undergoing slight but continuous exchanges.

Growing concerns about avian influenza, and its effect on agriculture and human health, have highlighted the need to understand the role of wildlife in maintaining and spreading the virus. We surveyed the wildlife inhabiting a poultry farm with recent H3N6 and H4N6 avian influenza virus exposure in Pennsylvania, U.S.A. One raccoon (Procyon lotor) tested positive for H4N6 antibodies. This is the first recorded incident of avian influenza exposure in a wild raccoon. We suggest that raccoons may play a role in the transmission of avian influenza viruses and in compromising biosecurity efforts at poultry operations.

The American crow (Corvus brachyrhynchos) is known to suffer 100% mortality from infection with the New York 1999 strain of West Nile virus (WNV). Following the initial detection of WNV in North America in 1999, we measured prevalence of WNV-reactive antibodies (“seroprevalence”) in free-ranging American and fish crows (Corvus ossifragus) of central New Jersey after each transmission season through 2005. In 2002, seroprevalence in American crow juveniles increased to 14% from the 5% of the previous year, potentially indicating increased survival in this species. Using the annual seroprevalence measurements and the number of human West Nile neuroinvasive disease cases as a surrogate for WNV transmission intensity, we developed a model to estimate the annual WNV-associated mortality rates among both of these crow species. Our model supports the hypothesis that mortality is changing over time; the WNV-associated mortality rate declined over time by 1.5% for American crow and by 1.1% for fish crow. The probability that the trend in mortality was negative was 90% for the American crow and 60% for the fish crow.

This study appears to represent the first serotyping study of 24 isolates of Avibacterium paragallinarum obtained from different regions of Peru during 1998–2008. All isolates were characterized as β-nicotinamide adenine dinucleotide dependent. According to the Page scheme, modified by Blackall, it was found that eight isolates were classified as serogroup A, seven isolates as serogroup B, and five isolates as serogroup C, while four isolates could not be serotyped. Further serotyping, following the same scheme but using rabbit antiserum raised against Argentinean strains of the three serogroups, allowed allocation of these four unclassified isolates to serogroup B. These results suggest that some of the Peruvian B isolates appear to be similar to the previously described variant B isolates from Argentina. Therefore, inactivated vaccines used in Peru should include the three recognized serogroups (A, B, and C), with the addition of at least one of these variant B isolates. Cross-protection trials are needed to compare the protection conferred by vaccines containing traditional B serovar strains to the protection by experimental vaccines containing variant B serovar isolates from Peru.

The aim of the study was to characterize atherosclerotic changes in African grey parrots (Psittacus erithacus) and Amazon parrots (Amazona spp.) by histochemical and immunohistochemical methods. Samples of the aorta ascendens and trunci brachiocephalici from 62 African grey parrots and 35 Amazon parrots were stained by hematoxylin and eosin and Elastica van Gieson for grading of atherosclerosis in these birds. Four different stages were differentiated. The incidence of atherosclerosis in the examined parrots was 91.9% in African grey parrots and 91.4% in Amazon parrots. To evaluate the pathogenesis in birds, immunohistochemical methods were performed to demonstrate lymphocytes, macrophages, smooth muscle cells, and chondroitin sulfate. According to the missing lymphocytes and macrophages and the absence of invasion and proliferation of smooth muscle cells in each atherosclerotic stage, “response-to-injury hypothesis” seems inapplicable in parrots. Additionally, we found alterations of vitally important organs (heart, lungs) significantly correlated with atherosclerosis of the aorta ascendens.

All fledgling canaries (Serinum canarius) in a small private aviary died from atoxoplasmosis during a single breeding season. The birds were clinically normal when removed from their parents at 2 mo of age, but by 3 mo of age all had died following an illness characterized by progressive lethargy, anorexia, and debilitation. Cachexia, splenomegaly, and pale foci in the liver were evident at necropsy. Microscopically, there was striking infiltration of the intestinal lamina propria by mononuclear cells that contained intracytoplasmic protozoa. Protozoa were also observed in mononuclear cells in splenic and hepatic sinusoids and in vascular or perivascular spaces of other organs, but were much less numerous. Ultrastructural features of infected enteric mononuclear cells were suggestive of lymphocytes, and the majority of parasitized cells in paraffin sections of intestine were positive for CD-79 antigen, consistent with B lymphocytes. CD-3 staining was minimal, suggesting little or no T-cell infection. The following year, after egg-laying was completed, adults were treated with sulfadimethoxine, and no further fledgling losses occurred.