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Neurolisteriosis, a common disease of small ruminants, is most often caused by Listeria monocytogenes. Here we describe 25 cases of caprine neurolisteriosis diagnosed in our laboratory over a 5-y period and compare our fluorescent antibody test (FAT) results with immunohistochemistry (IHC) and polymerase chain reaction (PCR) testing for diagnostic confirmation. Neurohistologic changes consistent with neurolisteriosis affected the pons in all cases, extending rostrally to the mesencephalon in 6 cases, caudally to the medulla oblongata in 6 cases, and/or dorsally to the cerebellum in 4 cases. Acute inflammatory changes were observed in 17 cases, and included neuroparenchymal microabscesses, neuronal necrosis and neuronophagia, axonal swelling, microgliosis and astrogliosis, and perivascular neutrophils with macrophages, lymphocytes, and plasma cells that occasionally extended to the leptomeninges. Subacute-to-chronic changes (8 cases) consisted of neuroparenchymal and perivascular clusters of macrophages with rare neutrophils, lymphocytes, and plasma cells admixed with glial nodules. Bacterial bacilli were observed within neutrophils or macrophages in H&E-stained tissue sections in 4 cases. Gram stain highlighted gram-positive bacilli in 13 cases. Neurolisteriosis was confirmed by FAT in 2 cases, by IHC in 19 cases, and by PCR in 20 cases.
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Coronaviruses cause infections in humans and diverse species of animals and birds with a global distribution. Bovine coronavirus (BCoV) produces predominantly two forms of disease in cattle: a respiratory form and a gastrointestinal form. All age groups of cattle are affected by the respiratory form of coronavirus, whereas the gastroenteric form causes neonatal diarrhea or calf scours in young cattle and winter dysentery in adult cattle. The tremendous impacts of bovine respiratory disease and the associated losses are well-documented and underscore the importance of this pathogen. Beyond this, studies have demonstrated significant impacts on milk production associated with outbreaks of winter dysentery, with up to a 30% decrease in milk yield. In North America, BCoV was identified for the first time in 1972, and it continues to be a significant economic concern for the cattle industry. A number of conventional and molecular diagnostic assays are available for the detection of BCoV from clinical samples. Conventional assays for BCoV detection include virus isolation, which is challenging from clinical samples, electron microscopy, fluorescent antibody assays, and various immunoassays. Molecular tests are mainly based on nucleic acid detection and predominantly include conventional and real-time polymerase chain reaction (PCR) assays. Isothermal amplification assays and genome sequencing have gained increased interest in recent years for the detection, characterization, and identification of BCoV. It is believed that isothermal amplification assays, such as loop-mediated isothermal amplification and recombinase polymerase amplification, among others, could aid the development of barn-side point-of-care tests for BCoV. The present study reviewed the literature on coronavirus infections in cattle from the last three and a half decades and presents information mainly on the current and advancing diagnostics in addition to epidemiology, clinical presentations, and the impact of the disease on the cattle industry.
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Canine infectious respiratory disease complex (CIRDC) is caused by different viruses and bacteria. Viruses associated with CIRDC include canine adenovirus type 2 (CAV-2), canine distemper virus (CDV), canine influenza virus (CIV), canine herpesvirus type 1 (CHV-1), canine respiratory coronavirus (CRCoV), and canine parainfluenza virus (CPIV). Bacteria associated with CIRDC include Bordetella bronchiseptica, Streptococcus equi subspecies zooepidemicus (S. zooepidemicus), and Mycoplasma spp. The present study examined the prevalence of CIRDC pathogens in specimens received by a Veterinary Diagnostic Laboratory in Georgia, USA., from 2018 to 2022. Out of 459 cases, viral agents were detected in 34% of cases and bacterial agents were detected in 58% of cases. A single pathogen was detected in 31% of cases, while two or more pathogens were identified in 24% of cases. The percentages of viral agents identified were CAV-2 (4%), CDV (3%), CPIV (16%), CRCoV (7%), and CIV (2%). The percentages of bacterial agents were B. bronchiseptica (10%), Mycoplasma canis (24%), Mycoplasma cynos (21%), and S. zooepidemicus (2%). Over the five-year period, the positive cases ranged from 2-4% for CAV-2, 1-7% for CDV, 1-4% for CHV-1, 9-22% for CPIV, 4-13% for CRCoV, and 1-4% for CIV. Overall, the most prevalent pathogens associated with CIRDC were CPIV, M. canis, and M. cynos.
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Laboratory diagnoses of animal diseases has advanced tremendously in recent decades with the advent of cutting-edge technologies such as real-time polymerase chain reaction, next generation sequencing (NGS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and others However, most of these technologies need sophisticated equipment, laboratory space and highly skilled workforce. Therefore, there is an increasing market demand for point-of-care testing (POCT) in animal health and disease diagnostics. A wide variety of assays based on antibodies, antigens, nucleic acid, and nanopore sequencing are currently available. Each one of these tests have their own advantages and disadvantages. However, a number of research and developmental activities are underway in both academia and industry to improve the existing tests and develop newer and better tests in terms of sensitivity, specificity, turnaround time and affordability. In both companion and food animal disease diagnostics, POCT has an increasing role to play, especially in resource-limited settings. It plays a critical role in improving animal health and wellbeing in rural communities in low- and middle-income countries. At the same time, ensuring high standard of quality through proper validation, quality assurance and regulation of these assays are very important for accurate diagnosis, surveillance, control and management of animal diseases. This review addresses the different types of POCTs currently available for companion and food animal disease diagnostics, tests in the pipeline and their advantages and disadvantages.
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Identification of unknown pathogens in pigs displaying enteric illness is difficult due to the large diversity of bacterial and viral species found within faecal samples. Current methods often require bacterial or viral isolation, or testing only a limited number of known species using quantitative PCR analysis. Herein, faeces from two 25-day-old piglets with diarrhoea from Texas, USA, were analysed by metagenomic next-generation sequencing to rapidly identify possible pathogens. Our analysis included a bioinformatics pipeline of rapid short-read classification and de novo genome assembly which resulted in the identification of a porcine enterovirus G (EV-G), a complete genome with substantial nucleotide differences (>30â%) among current sequences, and a novel non-structural protein similar in sequence to the Torovirus papain-like cysteine protease (PLpro). This discovery led to the identification and circulation of an EV-G with a novel PLpro in the USA that has not been previously reported.
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Cisteína Proteases/genética , Diarreia/veterinária , Infecções por Enterovirus/veterinária , Enterovirus Suínos/classificação , Enterovirus Suínos/enzimologia , Fezes/virologia , Doenças dos Suínos/virologia , Animais , Análise por Conglomerados , Biologia Computacional , Diarreia/virologia , Infecções por Enterovirus/virologia , Enterovirus Suínos/genética , Enterovirus Suínos/isolamento & purificação , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , Análise de Sequência de DNA , Suínos , TexasRESUMO
OBJECTIVE To determine titers of serum antibodies against 3 genotypes of bovine parainfluenza 3 virus (BPI3V) in unvaccinated ungulates in Alabama. ANIMALS 62 cattle, goats, and New World camelids from 5 distinct herds and 21 captured white-tailed deer. PROCEDURES Serum samples were obtained from all animals for determination of anti-BPI3V antibody titers, which were measured by virus neutralization assays that used indicator (reference) viruses from each of the 3 BPI3V genotypes (BPI3V-A, BPI3V-B, and BPI3V-C). The reference strains were recent clinical isolates from US cattle. Each sample was assayed in triplicate for each genotype. Animals with a mean antibody titer ≤ 2 for a particular genotype were considered seronegative for that genotype. RESULTS Animals seropositive for antibodies against BPI3V were identified in 2 of 3 groups of cattle and the group of New World camelids. The geometric mean antibody titer against BPI3V-B was significantly greater than that for BPI3V-A and BPI3V-C in all 3 groups. All goats, captive white-tailed deer, and cattle in the third cattle group were seronegative for all 3 genotypes of the virus. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that BPI3V-A may no longer be the predominant genotype circulating among ungulates in Alabama. This may be clinically relevant because BPI3V is frequently involved in the pathogenesis of bovine respiratory disease complex, current vaccines contain antigens against BPI3V-A only, and the extent of cross-protection among antibodies against the various BPI3V genotypes is unknown.
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Anticorpos Antivirais/sangue , Vírus da Parainfluenza 3 Bovina/isolamento & purificação , Infecções por Respirovirus/veterinária , Alabama , Animais , Camelídeos Americanos , Bovinos , Cervos , Genótipo , Cabras , Vírus da Parainfluenza 3 Bovina/genética , Vírus da Parainfluenza 3 Bovina/imunologia , Infecções por Respirovirus/sangue , Infecções por Respirovirus/virologiaRESUMO
The objective of the present study was to compare the pathogenicity of early and recent isolates of avian metapneumovirus subtype-C (aMPV-C) in turkeys. Two-week-old turkeys were inoculated with early and recent isolates of aMPV-C. Clinical signs were monitored. Tissues were examined for viral ribonucleic acid (RNA), lesions, and viral antigen by reverse transcription-polymerase chain reaction (RT-PCR), histopathology and immunohistochemistry, respectively. Birds infected with the recent isolate had higher clinical sign scores than those infected with the early isolate. Only the recent isolate produced a multifocal loss of cilia in the nasal turbinate of infected birds. Immunohistochemistry revealed intense staining of aMPV antigen in turbinate and trachea of birds infected with the recent isolate. The findings indicate that the recent isolate produced more severe clinical signs and lesions in turkeys compared to the early isolate. The recent isolate could be ideal for the development of a challenge model for aMPV infection in turkeys.
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Antígenos Virais/análise , Metapneumovirus/patogenicidade , Infecções por Paramyxoviridae/veterinária , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , RNA Viral/análise , Animais , Feminino , Imuno-Histoquímica/veterinária , Infecções por Paramyxoviridae/imunologia , Infecções por Paramyxoviridae/patologia , Infecções por Paramyxoviridae/virologia , Doenças das Aves Domésticas/imunologia , RNA Viral/genética , Distribuição Aleatória , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , PerusRESUMO
The length of the published glycoprotein (G) gene sequences of avian metapneumovirus subtype-C (aMPV-C) isolated from domestic turkeys and wild birds in the United States (1996-2003) remains controversial. To explore the G gene size variation in aMPV-C by the year of isolation and cell culture passage levels, we examined 21 turkey isolates of aMPV-C at different cell culture passages. The early domestic turkey isolates of aMPV-C (aMPV/CO/1996, aMPV/MN/1a-b, and 2a-b/97) had a G gene of 1,798 nucleotides (nt) that coded for a predicted protein of 585 amino acids (aa) and showed >97% nt similarity with that of aMPV-C isolated from Canada geese. This large G gene got truncated upon serial passages in Vero cell cultures by deletion of 1,015 nt near the end of the open reading frame. The recent domestic turkey isolates of aMPV-C lacked the large G gene but instead had a small G gene of 783 nt, irrespective of cell culture passage levels. In some cultures, both large and small genes were detected, indicating the existence of a mixed population of the virus. Apparently, serial passage of aMPV-C in cell cultures and natural passage in turkeys in the field led to truncation of the G gene, which may be a mechanism of virus evolution for survival in a new host or environment.
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Metapneumovirus/genética , Metapneumovirus/isolamento & purificação , Infecções por Paramyxoviridae/veterinária , Doenças das Aves Domésticas/virologia , Deleção de Sequência , Proteínas do Envelope Viral/genética , Animais , Sequência de Bases , Chlorocebus aethiops , Dados de Sequência Molecular , Infecções por Paramyxoviridae/virologia , Inoculações Seriadas , Turquia , Estados Unidos , Células VeroRESUMO
Ornithobacterium rhinotracheale (ORT) is an infectious respiratory pathogen of chickens, turkeys, and wild birds. There are 18 serotypes of ORT reported worldwide. In this study, enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction and random amplified polymorphic DNA assay with Universal M13 primer-based fingerprinting techniques were investigated for their ability to differentiate ORT isolates. The authors examined 50 field isolates and 8 reference strains of ORT for their genetic differences. The fingerprint patterns were compared with serotyping results of ORT by the agar gel precipitation test. M13 fingerprinting revealed different patterns for 6 reference serotypes of ORT that were tested, namely, C, D, E, I, J, and K. Ornithobacterium rhinotracheale reference serotypes A and F yielded indistinguishable fingerprints with M13 fingerprinting. The ERIC 1R technique discerned only 5 of the 8 reference serotypes of ORT. Distinct fingerprints were also found within the ORT serotypes with both techniques. From 58 isolates of ORT that were fingerprinted belonging to 8 ORT serotypes, 10 different fingerprints were obtained with M13 fingerprinting and 6 different fingerprints were obtained with ERIC 1R fingerprinting. M13 fingerprinting technique was found to be more discriminative in differentiating ORT isolates than the ERIC 1R fingerprinting technique. These results suggest that fingerprinting techniques may be a more discerning tool for characterizing ORT isolates than the serological test using the agar gel precipitation test. This fingerprinting technique could potentially be a valuable tool in identifying an isolate from a clinical outbreak of ORT infection for development of an autogenous vaccine.
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Impressões Digitais de DNA/métodos , Ornithobacterium/genética , Ornithobacterium/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Ornithobacterium/classificação , SorotipagemRESUMO
The objective of this study was to evaluate different preparations of avian metapneumovirus (aMPV) subtype C as vaccine challenge in turkeys. Two aMPV isolates and their respective nasal turbinate homogenates after propagation in turkeys were used in the study. Significantly higher clinical sign scores were recorded in birds inoculated with 20 or 2% turbinate homogenate of recent isolate. Birds in the above groups showed more pronounced histopathological lesions, and a higher percentage of birds showed viral RNA and antigen in tissues. The data demonstrated that nasal turbinate homogenate of recent isolate produced severe clinical signs and lesions in turkeys and could be an ideal candidate for vaccine-challenge studies.
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Metapneumovirus , Infecções por Paramyxoviridae/veterinária , Doenças das Aves Domésticas/virologia , Perus , Animais , Antígenos Virais/análise , Feminino , Histocitoquímica , Pulmão/patologia , Pulmão/virologia , Metapneumovirus/imunologia , Metapneumovirus/isolamento & purificação , Nariz/patologia , Nariz/virologia , Infecções por Paramyxoviridae/imunologia , Infecções por Paramyxoviridae/virologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/patologia , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Traqueia/patologia , Traqueia/virologiaRESUMO
A commercial live attenuated, freeze-dried avian metapneumovirus vaccine, Pneumomune, was assessed for its viability at three different temperatures (5.6 C, 21 C, and 37 C). No significant reduction in virus titer was observed when the vaccine was stored at 5.6 C for a period of 24 hr. However, reductions in virus titer of 1 log10 and 2 log10 were observed after 24 hr at 21 C and 37 C, respectively. Batch-to-batch variation in virus reduction was also observed. The addition of a dye or a vaccine stabilizer to the vaccine preparation did not have any deleterious effect on the survival of vaccine virus.
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Metapneumovirus , Temperatura , Vacinas Atenuadas , Vacinas Virais , Corantes , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Conservantes FarmacêuticosRESUMO
In the last 2 decades, the prevalence of Salmonella enterica serotype Enteritidis (Salmonella Enteritidis) has dramatically increased worldwide, becoming the leading cause of food-borne illnesses and an important public health issue. Many studies have suggested the role of the SEF14 fimbrial protein in the adhesion of Salmonella Enteritidis to the host. In the present study, the sefA gene, which encodes the main subunit of the SEF14 fimbrial protein, was cloned into a temperature-sensitive expression vector and transformed into a nonpathogenic, avirulent strain of Escherichia coli. The recombinant strain was used as a vaccine to elicit specific immune response against the SefA protein of Salmonella Enteritidis in 1-day-old chickens. The recombinant strain was reisolated from the intestines of treated birds for up to 21 days posttreatment, demonstrating its ability to colonize the intestinal tracts of 1-day-old chickens. In addition, immunoglobulin A (IgA) against the SefA protein was detected in intestinal secretions from treated birds at 7 days posttreatment and in bile samples from 14 to 21 days posttreatment by enzyme-linked immunosorbent assay. Nontreated birds did not show any evidence of intestinal colonization by the recombinant strain or anti-SefA IgA response in their bile or intestinal secretions. Preliminary evaluation of the recombinant strain showed a potential use of this strain to elicit protection against Salmonella Enteritidis infection in chickens. Further experiments are needed to study the ability of the recombinant strain to protect birds against Salmonella Enteritidis colonization.
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Galinhas/imunologia , Proteínas de Fímbrias/imunologia , Imunização/veterinária , Doenças das Aves Domésticas/imunologia , Salmonelose Animal/imunologia , Vacinas contra Salmonella/imunologia , Animais , Proteínas de Fímbrias/genética , Doenças das Aves Domésticas/prevenção & controle , Salmonelose Animal/prevenção & controle , Vacinas contra Salmonella/administração & dosagem , Vacinas contra Salmonella/genéticaRESUMO
We report a concurrent increase in the number of isolates of Salmonella enterica serotype Newport and the rate of multidrug resistance in S. Newport isolates from animal and human populations in Minnesota. Antimicrobial susceptibility and pulsed-field gel electrophoresis analysis demonstrated heterogeneity of isolates and showed that 1 pulsed-field gel electrophoresis cluster contained most of the multidrug-resistant isolates with a resistance pattern and most class 1 integron isolates, implying the clonal origin of the isolates.
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Farmacorresistência Bacteriana Múltipla/genética , Salmonelose Animal/microbiologia , Infecções por Salmonella/microbiologia , Salmonella enterica/classificação , Animais , Técnicas de Tipagem Bacteriana , Bovinos , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/microbiologia , Eletroforese em Gel de Campo Pulsado , Humanos , Integrons , Testes de Sensibilidade Microbiana , Minnesota/epidemiologia , Salmonella enterica/isolamento & purificaçãoRESUMO
This study was conducted to reexamine the hypothesis that human metapneumovirus (hMPV) will not infect turkeys. Six groups of 2-week-old turkeys (20 per group) were inoculated oculonasally with 1 of the following: noninfected cell suspension; hMPV genotype A1, A2, B1, or B2; or avian metapneumovirus (aMPV) subtype C. Poults inoculated with hMPV showed nasal discharge days 4-9 postexposure. Specific viral RNA and antigen were detected by reverse-transcription PCR and immunohistochemical evaluation, respectively, in nasal turbinates of birds exposed to hMPV. Nasal turbinates of hMPV-infected turkeys showed inflammatory changes and mucus accumulation. Each of the 4 hMPV genotypes caused a transient infection in turkeys as evidenced by clinical signs, detection of hMPV in turbinates, and histopathologic examination. Detailed investigation of cross-species pathogenicity of hMPV and aMPV and its importance for human and animal health is needed.
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Metapneumovirus/patogenicidade , Infecções por Paramyxoviridae/veterinária , Doenças das Aves Domésticas/virologia , Infecções Respiratórias/veterinária , Perus , Animais , Anticorpos Antivirais/sangue , Linhagem Celular , Efeito Citopatogênico Viral , Ensaio de Imunoadsorção Enzimática/veterinária , Genótipo , Humanos , Imuno-Histoquímica/veterinária , Pulmão/virologia , Metapneumovirus/genética , Infecções por Paramyxoviridae/patologia , Infecções por Paramyxoviridae/virologia , Doenças das Aves Domésticas/patologia , RNA Viral/química , RNA Viral/genética , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Traqueia/virologiaRESUMO
The objectives of the present study were to investigate the pathogenesis of a recent isolate of avian metapneumovirus (aMPV) in turkeys and to evaluate the quantitative distribution of the virus in various tissues during the course of infection. Seventy 2-week-old turkey poults were divided equally into two groups. One group was inoculated with aMPV (MN 19) with a titer of 10(5.5) TCID50 oculonasally. Birds in the second group were maintained as sham-inoculated controls. Birds showed severe clinical signs in the form of copious nasal discharge, swollen sinus, conjunctivitis, and depression from 4 days postinoculation (PI) to 12 days PI. Samples from nasal turbinates, trachea, conjunctiva, Harderian gland, infraorbital sinus, lungs, liver, and spleen were collected at 1, 3, 5, 7, 9, 11, and 14 days PI. Histopathologic lesions such as a multifocal loss of cilia were prominent in nasal turbinate and were seen from 3 to 11 days PI. Immunohistochemistry revealed the presence of aMPV from 3 to 9 days PI in nasal turbinate and trachea. Viral RNA could be detected for 14 days PI from nasal turbinate and for 9 days from trachea. In situ hybridization demonstrated the presence of aMPV from 1 to 11 days PI in nasal turbinates and from 3 to 9 days PI in the trachea. Quantitative real-time polymerase chain reaction data showed the presence of a maximum amount of virus at 3 days PI in nasal turbinate and trachea. Clinically and histopathologically, the new isolate appears to be more virulent compared to the early isolates of aMPV in the United States.
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Metapneumovirus/classificação , Metapneumovirus/patogenicidade , Infecções por Paramyxoviridae/veterinária , Doenças das Aves Domésticas/virologia , Perus/virologia , Animais , Antígenos Virais/isolamento & purificação , Sequência de Bases , DNA Viral/genética , Hibridização In Situ , Metapneumovirus/genética , Metapneumovirus/isolamento & purificação , Minnesota , Infecções por Paramyxoviridae/patologia , Infecções por Paramyxoviridae/virologia , Doenças das Aves Domésticas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , VirulênciaRESUMO
OBJECTIVE: To compare molecular typing methods for the differentiation of Salmonella enterica serovar Enteritidis phage type (PT) 4 isolates that allowed for the determination of their genetic relatedness. SAMPLE POPULATION: 27 Salmonella Enteritidis PT 4 strains isolated in the United States and Europe. PROCEDURE: Several molecular typing methods were performed to assess their ability to genetically differentiate among Salmonella Enteritidis PT 4 isolates. Results of pulse-field gel electrophoresis (PFGE), repetitive polymerase chain reaction (PCR) assay, 16S rRNA gene sequencing, random amplification of polymorphic DNA (RAPD), PCR-restriction fragment length polymorphism of 16S rRNA, and antimicrobial susceptibility were evaluated. RESULTS: Compared with results for other techniques, results for the RAPD typing method with the RAPD1 primer reveal that it was the most discriminatory fingerprinting technique, and it allowed us to cluster Salmonella Enteritidis PT 4 isolates on the basis of their genetic similarity. CONCLUSIONS AND CLINICAL RELEVANCE: This study revealed the value of RAPD with the RAPD1 primer as a tool for epidemiologic investigations of Salmonella Enteritidis PT 4. It can be used in conjunction with PFGE and phage typing to determine the genetic relatedness of Salmonella Enteritidis isolates involved in outbreaks of disease. A reliable and highly discriminatory method for epidemiologic investigations is critical to allow investigators to identify the source of infections and consequently prevent the spread of Salmonella Enteritidis PT 4.
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Técnicas de Tipagem Bacteriana/métodos , Bacteriófago T4/genética , Aves Domésticas/microbiologia , Salmonella enteritidis/classificação , Salmonella enteritidis/genética , Animais , Sequência de Bases , Análise por Conglomerados , Primers do DNA , Farmacorresistência Bacteriana , Eletroforese em Gel de Campo Pulsado , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Salmonella enteritidis/virologia , Análise de Sequência de DNARESUMO
This study reports on the development of a reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific detection of turkey coronavirus (TCoV). Of the several sets of primers tested, 1 set of primers derived from the P gene and 2 sets derived from the N gene of TCoV could amplify the TCoV genome in the infected samples. The RT-PCR was sensitive and specific for TCoV and did not amplify other avian RNA and DNA viruses tested except the infectious bronchitis virus (IBV). To overcome the problem of IBV amplification, a set of separate primers was designed from the spike protein gene of IBV. The RT-PCR under the same conditions as above could effectively differentiate between TCoV and IBV. The closely related bovine coronavirus and transmissible gastroenteritis virus of pigs were differentiated from TCoV using the same RT-PCR with slight modifications. The results of RT-PCR correlated well with the results of the immunofluorescent test for the same samples tested at the Purdue University Animal Disease Laboratory, West Lafayette, Indiana. The nucleotide sequence and projected amino acid sequence comparison of the P gene of different isolates of TCoV from 5 different states in the United States revealed a close association among the different isolates of TCoV.
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Coronavirus do Peru/genética , Enterite Transmissível dos Perus/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Animais , Coronavirus do Peru/patogenicidade , Primers do DNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , PerusRESUMO
The survival of avian pneumovirus (APV) in turkey litter was studied at different temperature (room temperature, [approximately 22-25 C], 8 C, and -12 C) conditions. Built-up turkey litter from a turkey breeder farm known to be free of APV was obtained and was divided into two portions. One portion was sterilized by autoclaving and the other portion was kept nonautoclaved. Both samples were inoculated with a Vero cell-propagated Minnesota isolate of APV subtype C (APV/MN2A) with a titer of 10(5) 50% tissue culture infective dose at 1% level. These samples were then stored at three different temperatures: -12 C, 8 C, and room temperature (20-25 C). The samples were tested for the presence of viral RNA by reverse transcriptase-polymerase chain reaction and for the presence of live virus by virus isolation in Vero cells at the intervals of 1, 2, 3, 7, 14, 30, 60, and 90 days. Our studies revealed the presence of APV RNA even after 90 days in the autoclaved litter samples kept at -12 C and at 8 C. The virus was isolated from the autoclaved litter kept at -12 C up to 60 days. From the nonautoclaved litter, viral RNA was detected up to 60 days and virus was isolated up to 14days. The present study indicated that APV could survive in built-up turkey litter up to 60 days postinoculation at a temperature of-12 C.