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Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis and causes a persistent infection that can leave cattle with no symptoms. Many countries have been able to successfully eradicate BLV through improved detection and management methods. However, with the increasing novel molecular detection methods there have been few efforts to standardize these results at global scale. This study aimed to determine the interlaboratory accuracy and agreement of 11 molecular tests in detecting BLV. Each qPCR/ddPCR method varied by target gene, primer design, DNA input and chemistries. DNA samples were extracted from blood of BLV-seropositive cattle and lyophilized to grant a better preservation during shipping to all participants around the globe. Twenty nine out of 44 samples were correctly identified by the 11 labs and all methods exhibited a diagnostic sensitivity between 74 and 100%. Agreement amongst different assays was linked to BLV copy numbers present in samples and the characteristics of each assay (i.e., BLV target sequence). Finally, the mean correlation value for all assays was within the range of strong correlation. This study highlights the importance of continuous need for standardization and harmonization amongst assays and the different participants. The results underscore the need of an international calibrator to estimate the efficiency (standard curve) of the different assays and improve quantitation accuracy. Additionally, this will inform future participants about the variability associated with emerging chemistries, methods, and technologies used to study BLV. Altogether, by improving tests performance worldwide it will positively aid in the eradication efforts.
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Leucose Enzoótica Bovina , Vírus da Leucemia Bovina , Provírus , Vírus da Leucemia Bovina/isolamento & purificação , Vírus da Leucemia Bovina/genética , Animais , Bovinos , Leucose Enzoótica Bovina/diagnóstico , Leucose Enzoótica Bovina/virologia , Leucose Enzoótica Bovina/sangue , Provírus/genética , Provírus/isolamento & purificação , Reação em Cadeia da Polimerase/veterinária , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase em Tempo Real/métodos , DNA Viral/sangueRESUMO
The practice of feeding raw meat-based diets to dogs has grown in popularity worldwide in recent years. However, there are public health risks in handling and feeding raw meat-based dog diets (RMDDs) to dogs since there are no pathogen reduction steps to reduce the microbial load, which may include antimicrobial-resistant pathogenic bacteria. A total of 100 RMDDs from 63 suppliers were sampled, and selective media were used to isolate bacteria from the diets. Bacterial identification, antimicrobial susceptibility testing, and whole-genome sequencing (WGS) were conducted to identify antimicrobial resistance (AMR). The primary meat sources for RMDDs included in this study were poultry (37%) and beef (24%). Frozen-dry was the main method of product production (68%). In total, 52 true and opportunistic pathogens, including Enterobacterales (mainly Escherichia coli, Enterobacter cloacae) and Enterococcus faecium, were obtained from 30 RMDDs. Resistance was identified to 19 of 28 antimicrobials tested, including amoxicillin/clavulanic acid (23/52, 44%), ampicillin (19/52, 37%), cephalexin (16/52, 31%), tetracycline (7/52, 13%), marbofloxacin (7/52, 13%), and cefazolin (6/52, 12%). All 19 bacterial isolates submitted for WGS harbored at least one type of AMR gene. The identified AMR genes were found to mediate resistance to aminoglycoside (gentamicin, streptomycin, amikacin/kanamycin, gentamicin/kanamycin/tobramycin), macrolide, beta-lactam (carbapenem, cephalosporin), tetracycline, fosfomycin, quinolone, phenicol/quinolone, and sulfonamide. In conclusion, the results of this study suggest that feeding and handling RMDDs may pose a significant public health risk due to the presence of antimicrobial-resistant pathogens, and further research and intervention may be necessary to minimize these risks.
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Enterococcus faecium , Quinolonas , Bovinos , Cães , Animais , Enterobacter cloacae , Enterococcus faecium/genética , Escherichia coli , Aminoglicosídeos/farmacologia , Kentucky , Antibacterianos/farmacologia , Carne/microbiologia , Tetraciclina , Salmonella , Resistência beta-Lactâmica , Canamicina , Gentamicinas , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana/genéticaRESUMO
Reverse transcription fluorescence resonance energy transfer-polymerase chain reaction (FRET-PCRs) were designed against the two most common mutations in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (A23403G in the spike protein; C14408T in the RNA-dependent RNA polymerase). Based on high-resolution melting curve analysis, the reverse transcription (RT) FRET-PCRs identified the mutations in american type culture collection control viruses, and feline and human clinical samples. All major makes of PCR machines can perform melting curve analysis and thus further specifically designed FRET-PCRs could enable active surveillance for mutations and variants in countries where genome sequencing is not readily available.
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Teste Sorológico para COVID-19/métodos , Reação em Cadeia da Polimerase , RNA Polimerase Dependente de RNA , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Animais , COVID-19/diagnóstico , COVID-19/virologia , Gatos , RNA-Polimerase RNA-Dependente de Coronavírus/análise , RNA-Polimerase RNA-Dependente de Coronavírus/imunologia , Humanos , Mutação , RNA Viral/genética , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/análise , Glicoproteína da Espícula de Coronavírus/imunologia , TemperaturaRESUMO
OBJECTIVE: This nationwide study aimed to investigate risk factors associated with FIP and determine optimal sample submission strategies for its diagnosis. METHODS: A total of 14,035 clinical samples from cats across the US were analyzed by means of reverse transcriptase quantitative PCR to detect replicating feline coronavirus (FCoV). χ2 and logistic regression analyses were conducted to assess the association between FCoV detection rates and risk factors such as age, gender, breed, and types of submitted samples. RESULTS: Higher FCoV detection rates were observed in younger cats, particularly those aged 0 to 1 year, and in male cats. Purebred cats, notably British Shorthairs [OR: 2.81; P < .001], showed a higher incidence of FCoV infection than other cats. Peritoneal fluid (OR, 7.51; P < .001) exhibited higher FCoV detection rates than other samples, while lower rates were seen in blood samples (OR, 0.08; P < .001) than in other samples. High FCoV detection rates were found in urine, kidney, and lymph node samples. CONCLUSIONS: The study identified significant risk factors associated with FIP. Optimal sample submission strategies, particularly emphasizing the use of peritoneal fluid, kidney, and lymph node, were identified to improve FIP detection rates. Urine yielded a relatively high frequency of infection and viral loads compared with most other samples. CLINICAL RELEVANCE: Understanding the risk factors and optimizing sample selection for FIP diagnosis can aid in the early detection and management of the disease, ultimately improving outcomes for affected cats. These findings contribute valuable insights to FIP epidemiology and underscore the importance of continued research to enhance diagnostic strategies and disease management approaches.
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Chlamydia trachomatis (Ct) infections are the most common sexually transmitted infection (STI). Despite effective antibiotics for Ct, undetected infections or delayed treatment can lead to infertility, ectopic pregnancies, and chronic pelvic pain. Besides humans, chlamydia poses similar health challenges in animals such as C. suis (Cs) in pigs. Based on the similarities between humans and pigs, as well as their chlamydia species, we use pigs as a large biomedical animal model for chlamydia research. In this study, we used the pig model to develop a vaccine candidate against Ct. The vaccine candidate consists of TriAdj-adjuvanted chlamydial-protease-like activity factor (CPAF) protein. We tested two weekly administration options-twice intranasal (IN) followed by twice intramuscular (IM) and twice IM followed by twice IN. We assessed the humoral immune response in both serum using CPAF-specific IgG (including antibody avidity determination) and also in cervical and rectal swabs using CPAF-specific IgG and IgA ELISAs. The systemic T-cell response was analyzed following in vitro CPAF restimulation via IFN-γ and IL-17 ELISpots, as well as intracellular cytokine staining flow cytometry. Our data demonstrate that while the IN/IM vaccination mainly led to non-significant systemic immune responses, the vaccine candidate is highly immunogenic if administered IM/IN. This vaccination strategy induced high serum anti-CPAF IgG levels with strong avidity, as well as high IgA and IgG levels in vaginal and rectal swabs and in uterine horn flushes. In addition, this vaccination strategy prompted a pronounced cellular immune response. Besides inducing IL-17 production, the vaccine candidate induced a strong IFN-γ response with CD4 T cells. In IM/IN-vaccinated pigs, these cells also significantly downregulated their CCR7 expression, a sign of differentiation into peripheral-tissue-homing effector/memory cells. Conclusively, this study demonstrates the strong immunogenicity of the IM/IN-administered TriAdj-adjuvanted Ct CPAF vaccine candidate. Future studies will test the vaccine efficacy of this promising Ct vaccine candidate. In addition, this project demonstrates the suitability of the Cs pre-exposed outbred pig model for Ct vaccine development. Thereby, we aim to open the bottleneck of large animal models to facilitate the progression of Ct vaccine candidates into clinical trials.
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Authors are often faced with the decision of whether to maximize traditional impact metrics or minimize costs when choosing where to publish the results of their research. Many subscription-based journals now offer the option of paying an article processing charge (APC) to make their work open. Though such "hybrid" journals make research more accessible to readers, their APCs often come with high price tags and can exclude authors who lack the capacity to pay to make their research accessible. Here, we tested if paying to publish open access in a subscription-based journal benefited authors by conferring more citations relative to closed access articles. We identified 146,415 articles published in 152 hybrid journals in the field of biology from 2013-2018 to compare the number of citations between various types of open access and closed access articles. In a simple generalized linear model analysis of our full dataset, we found that publishing open access in hybrid journals that offer the option confers an average citation advantage to authors of 17.8 citations compared to closed access articles in similar journals. After taking into account the number of authors, Journal Citation Reports 2020 Quartile, year of publication, and Web of Science category, we still found that open access generated significantly more citations than closed access (p < 0.0001). However, results were complex, with exact differences in citation rates among access types impacted by these other variables. This citation advantage based on access type was even similar when comparing open and closed access articles published in the same issue of a journal (p < 0.0001). However, by examining articles where the authors paid an article processing charge, we found that cost itself was not predictive of citation rates (p = 0.14). Based on our findings of access type and other model parameters, we suggest that, in the case of the 152 journals we analyzed, paying for open access does confer a citation advantage. For authors with limited budgets, we recommend pursuing open access alternatives that do not require paying a fee as they still yielded more citations than closed access. For authors who are considering where to submit their next article, we offer additional suggestions on how to balance exposure via citations with publishing costs.
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Complexos Atriais Prematuros , Publicação de Acesso Aberto , Humanos , Salários e Benefícios , Benchmarking , BiologiaRESUMO
BACKGROUND: Rotavirus is the leading cause of severe diarrhea in infants and young children worldwide including Bangladesh. Unlike what was seen in high-income countries, the licensed rotavirus vaccines did not show high efficacy in Bangladeshi trials. We assessed rotavirus prevalence and genotypes in Bangladesh over six-year period to provide baseline information on the rotavirus burden and changing profile in the country. METHODS: This study was conducted from June 2006 to May 2012 in Matlab, Bangladesh. Group A rotaviruses were detected in stools collected from diarrhea patients by ELISA and genotyped using multiplex reverse transcription PCR followed by nucleotide sequencing. RESULTS: Of the 9678 stool samples, 20.3% were positive for rotavirus. The most predominant genotype was G1P[8] (22.4%), followed by G9P[8] (20.8%), G2P[4] (16.9%) and G12P[8] (10.4%). Mixed infections were detected in 14.2% of the samples. Emergence of an unusual strain, G9P[4] was documented during 2011-12. Several amino acid mismatches in the antigenic epitopes of VP7 and VP4 between Bangladeshi and the vaccine strains were identified. CONCLUSIONS: Our study provides important information on rotavirus genotypes that should be considered for the selection and introduction of rotavirus vaccines in Bangladesh.
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Infecções por Rotavirus/virologia , Rotavirus/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Antígenos Virais/imunologia , Bangladesh/epidemiologia , Diarreia/epidemiologia , Diarreia/virologia , Fezes/virologia , Genótipo , Humanos , Dados de Sequência Molecular , Estudos Prospectivos , Rotavirus/imunologia , Rotavirus/isolamento & purificação , Infecções por Rotavirus/epidemiologia , Vacinas contra Rotavirus/administração & dosagem , Vacinas contra Rotavirus/imunologiaRESUMO
Feline infectious peritonitis (FIP), caused by feline coronavirus (FcoV), is considered one of the most enigmatic diseases in cats. Developing effective drugs for FIP is crucial due to its global prevalence and severity. In this study, six antiviral drugs were tested for their cytotoxicity, cell viability, and antiviral efficacies in Crandell-Reese feline kidney cells. A cytotoxicity assay demonstrated that these drugs were safe to be used with essentially no cytotoxicity with concentrations as high as 250 µM for ruxolitinib; 125 µM for GS441524; 63 µM for teriflunomide, molnupiravir, and nirmatrelvir; and 16 µM for ritonavir. GS441524 and nirmatrelvir exhibited the least detrimental effects on the CRFK cells, with 50% cytotoxic concentration (CC50) values of 260.0 µM and 279.1 µM, respectively, while ritonavir showed high toxicity (CC50 = 39.9 µM). In the dose-response analysis, GS441524, nirmatrelvir, and molnupiravir demonstrated promising results with selectivity index values of 165.54, 113.67, and 29.27, respectively, against FIPV. Our study suggests that nirmatrelvir and molnupiravir hold potential for FIPV treatment and could serve as alternatives to GS441524. Continued research and development of antiviral drugs are essential to ensure the well-being of companion animals and improve our preparedness for future outbreaks of coronaviruses affecting animals and humans alike.
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Theileria orientalis Ikeda genotype, a parasite causing a disease in cattle that leads to significant economic challenges in Asia, New Zealand, and Australia, has been identified in seven U.S. States since 2017. Two previously validated PCR tests for Theileria followed by DNA sequencing were performed to test blood samples collected from 219 cattle in Alabama, USA, during the period of 2022-2023. Bidirectional Sanger sequencing revealed that the MPSP gene sequences (639-660 bp) from two cattle in Lee and Mobile Counties of Alabama exhibited a 100% match with those of recognized T. orientalis Ikeda strains, and showed similarities ranging from 76% to 88% with ten other T. orientalis genotypes. A high copy number of T. orientalis Ikeda was detected in the blood of infected cattle (ALP-1: 1.7 × 105 and 1.3 × 106/mL whole blood, six months apart; ALP-2: 7.1 × 106/mL whole blood). Although the confirmed competent vector for T. orientalis Ikeda, Haemaphysalis longicornis tick, has not yet been identified in Alabama, the persistent nature of T. orientalis Ikeda infection and the detection of a high pathogen burden in seemingly healthy cattle in this study suggest that other tick species, as well as shared needles and dehorning procedures, could facilitate pathogen transmission within the herd. Continued investigations are necessary for the surveillance of T. orientalis Ikeda and Haemaphysalis longicornis ticks in Alabama and other U.S. states, along with assessing the pathogenicity of T. orientalis Ikeda infections in cattle.
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Salmonella and Campylobacter are common foodborne pathogens in chickens, but their persistence mechanisms within flocks are not fully understood. In this study, 4 groups of SPF Leghorn chickens (n = 50) were orally inoculated with 108Salmonella Enteritidis and 108Campylobacter jejuni, housed in BSL-2 rooms inside containers with autoclaved bedding and beetles (n = 200). Phase I (wk 1-3): the infected chickens remained in the containers and were then euthanized while beetles and litter remained in the container (group A), beetles were removed and litter remained in the container (group B), beetles remained and litter was removed (group C), and beetles and litter were removed (group D). Phase II (wk 5-7): autoclaved bedding was added to containers in groups C and D, and new SPF chickens (n = 50) were introduced and kept. Phase III (wk 8-20): all chickens were euthanized, and the litter and/or beetles remained in the containers for 17 wk. The prevalence of Salmonella Enteritidis and Campylobacter was significantly higher when detected by PCR compared to culture. In phase II, when infected chickens were removed and new chickens were introduced, 1 fecal sample in group B and 3 litter samples in groups B and C were found positive for Salmonella Enteritidis, and Campylobacter was still detected in groups A, B, and C litter samples, but not in beetles. In phase III, when all chickens were removed, Salmonella Enteritidis was identified in beetle samples from group A and the litter samples of all tested groups A, B, and C, and C. jejuni was positive in litter samples from groups A and B but not in the beetle. Sixty-nine days after removing infected chickens, culturable Salmonella was still found in beetles. Salmonella and Campylobacter were detectable in litter up to 127 d after removing infected chickens. This study highlights the transmission of Salmonella and Campylobacter via beetles and litter to new flocks in successive rearing cycles. Intensive control programs should target insect exclusion and implement strict poultry litter management or litter changes between flocks.
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BACKGROUND: Heartworms, Dirofilaria immitis, are known to be widespread in dogs and cats in the USA, but there have been no country-wide prevalence studies performed to date. There have also been no large-scale studies to determine whether the closely related species, Dirofilaria repens, occurs in the USA. METHODS: To provide this large-scale data, we examined whole blood samples (n = 2334) submitted from around the USA to the Molecular Diagnostic Laboratory at Auburn University between 2016 and 2022. Quantitative PCRs for D. immitis (targeting 16S rRNA) and D. repens (targeting cytochrome c oxidase subunit 1 gene) were performed to determine the presence of Dirofilaria DNA. DNA sequencing was performed to confirm the results. RESULTS: Dirofilaria immitis DNA was found in 6.3% (68/1080) of the dogs from 17/39 states, and 0.3% (4/1254) of the cats from 4/42 states. None of the dogs or cats were positive for D. repens. The average 16S rRNA copy number of D. immitis in the dogs was 1,809,604 in 200 µl whole blood, while only a single copy was found in each of the four D. immitis-positive cats. The prevalence of D. immitis in dogs of different ages, sexes, and breeds did not differ significantly, but the prevalence in Southern states (7.5%, 60/803) was significantly higher than in the Western (1.7%, 1/58), Midwest (3.3%, 4/120), and Northeastern states (3.1%, 3/98) (P < 0.05). Dogs positive for D. immitis were identified in each study year (2016: 4.2%, 2/48; 2017: 9.8%, 4/41; 2018: 5.1%, 8/156; 2019: 4.9%, 15/306; 2020: 9.8%, 26/265; 2021: 4.9%, 13/264). Interestingly, dogs infected with Hepatozoon spp. (11.8%, 37/313) were significantly more likely to also be positive for D. immitis than dogs without evidence of Hepatozoon infection (3.9%, 30/760) (P < 0.0001). CONCLUSIONS: To our knowledge, this is the first nationwide molecular survey of Dirofilaria spp. in dogs and cats in the USA, and the largest molecular survey of canine and feline dirofilariosis worldwide. Further studies are warranted to combine PCR with standard heartworm diagnostics to better understand the prevalence of Dirofilaria spp. and aid in determining the risks posed to dogs and cats in the USA.
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Doenças do Gato , Dirofilaria immitis , Dirofilaria repens , Dirofilariose , Doenças do Cão , Animais , Doenças do Gato/diagnóstico , Doenças do Gato/epidemiologia , Gatos , Dirofilaria immitis/genética , Dirofilaria repens/genética , Dirofilariose/diagnóstico , Dirofilariose/epidemiologia , Doenças do Cão/diagnóstico , Doenças do Cão/epidemiologia , Cães , Complexo IV da Cadeia de Transporte de Elétrons/genética , Animais de Estimação , Prevalência , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase em Tempo Real , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: Accurate identification of mosquito species is essential for the development and optimization of strategies to control mosquitoes and mosquito-borne diseases. Problems with the morphological identification of mosquito species have led to the use of molecular identification techniques, in particular the Folmer cytochrome c oxidase subunit I (COI) PCR system (FCOS), originally designed to identify a range of other invertebrates. METHODS: As there can be difficulties identifying mosquitoes using FCOS, we re-evaluated the FCOS primers and developed a new COI-based SYBR PCR (the Auburn COI system-AUCOS) to improve the molecular identification of mosquitoes. Sequence data in GenBank for 33 species from 10 genera of mosquitoes were used to develop our AUCOS primers. Two molecular assays (AUCOS, FCOS) and morphological identification were carried out on mosquitoes collected from the field in Auburn, Alabama (USA) and on Saint Kitts. RESULTS: With a convenience sample of individual mosquitoes comprising 19 species from six genera in Saint Kitts (n = 77) and Auburn (n = 48), our AUCOS provided higher-quality sequence data than FCOS. It also proved more sensitive than FCOS, successfully amplifying 67.5% (85/126) as opposed to 16.7% (21/126) of the samples. The species determined by morphology, or genus with damaged samples, matched that as determined by AUCOS for 84.9% (62/73) of the samples. Morphological classification was confirmed by FCOS with 81.0% (17/21) of samples producing utilizable sequences. While both FCOS and AUCOS correctly identified all the Aedes, Anopheles, Deinocerites, and Uranotaenia species in the study, identification of Culex species was less successful with both methods: 50.0% (3/6) by FCOS and 35.7% (5/14) by AUCOS. CONCLUSIONS: The AUCOS DNA barcoding system for mosquito species described in this study is superior to the existing FCOS for the identification of mosquito species. As AUCOS and FCOS amplify the same variable region of the COI, the large amount of existing data on GenBank can be used to identify mosquito species with sequences produced by either PCR.
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Aedes , Anopheles , Culex , Animais , Complexo IV da Cadeia de Transporte de Elétrons/genética , Primers do DNA/genéticaRESUMO
Knowledge of SARS-CoV-2 variants is essential for formulating effective control policies. Currently, variants are only identified in relatively small percentages of cases as the required genome sequencing is expensive, time-consuming, and not always available. In countries with facilities to sequence the SARS-CoV-2, the Delta variant currently predominates. Elsewhere, the prevalence of the Delta variant is unclear. To avoid the need for sequencing, we investigated a RT-FRET-PCR that could detect all SARS-CoV-2 strains and simultaneously identify the Delta variant. The established Delta RT-FRET-PCR was performed on reference SARS-CoV-2 strains, and human nasal swab samples positive for the Delta and non-Delta strains. The Delta RT-FRET-PCR established in this study detected as few as ten copies of the DNA target and 100 copies of RNA target per reaction. Melting points of products obtained with SARS-CoV-2 Delta variants (around 56.1°C) were consistently higher than products obtained with non-Delta strains (around 52.5°C). The Delta RT-FRET-PCR can be used to diagnose COVID-19 patients and simultaneously identify if they are infected with the Delta variant. The Delta RT-FRET-PCR can be performed with all major thermocycler brands meaning data on Delta variant can now be readily generated in diagnostic laboratories worldwide.
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COVID-19/virologia , Transferência Ressonante de Energia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , Alelos , Substituição de Aminoácidos , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Mutação , RNA Viral , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/classificação , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
To provide more complete data on SARS-CoV-2 infections in dogs and cats in the U.S., we conducted a serosurvey on convenience serum samples from dogs (n=1336) and cats (n=956) collected from 48 states of the USA in 2020. An ELISA targeting the antibody against nucleocapsid identified eleven positive and two doubtful samples in cats, and five positive and five doubtful samples in dogs. A surrogate neutralization assay detecting antibodies blocking the attachment of the spike protein to ACE2 was positive with three of the ELISA positive and doubtful samples, and one of 463 randomly selected ELISA negative samples. These four positive samples were confirmed by SARS-CoV-2 virus neutralization testing. All were from cats, in New York, Florida, and New Jersey (n=2). The serosurvey results, one of the largest yet completed on dogs and cats globally, support the OIE and CDC positions that currently there is no evidence that pets play a role in the spread of SARS CoV-2 in humans.
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Anticorpos Antivirais/imunologia , COVID-19/veterinária , Doenças do Gato/epidemiologia , Doenças do Gato/imunologia , Doenças do Cão/epidemiologia , Doenças do Cão/imunologia , SARS-CoV-2/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Doenças do Gato/virologia , Gatos , Doenças do Cão/virologia , Cães , Ensaio de Imunoadsorção Enzimática , Humanos , Testes de Neutralização , Vigilância em Saúde Pública , Estudos Soroepidemiológicos , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: The main vector and reservoir host of Rickettsia felis, an emerging human pathogen causing flea-borne spotted fever, is the cat flea Ctenocephalides felis. While cats have not been found to be infected with the organism, significant percentages of dogs from Australia and Africa are infected, indicating that they may be important mammalian reservoirs. The objective of this study was to determine the presence of R. felis DNA in the blood of domestic dogs and cats in the USA. METHODS: Three previously validated PCR assays for R. felis and DNA sequencing were performed on blood samples obtained from clinically ill domestic cats and dogs from 45 states (2008-2020) in the USA. The blood samples had been submitted for the diagnosis of various tick-borne diseases in dogs and feline infectious peritonitis virus, feline immunodeficiency virus, and Bartonella spp. in cats. Phylogenetic comparisons were performed on the gltA nucleotide sequences obtained in the study and those reported for R. felis and R. felis-like organisms. RESULTS: Low copy numbers of R. felis DNA (around 100 copies/ml whole blood) were found in four cats (4/752, 0.53%) and three dogs (3/777, 0.39%). The very low levels of infection in clinically ill animals is consistent with R. felis being an unlikely cause of disease in naturally infected dogs and cats. The low copy numbers we found emphasize the requirement for very sensitive PCRs in prevalence studies. CONCLUSIONS: The low prevalence of naturally infected PCR-positive cats is further evidence that cats are unlikely to be important reservoirs of R. felis. Similarly, the low prevalence in dogs suggests they are not important reservoirs in the USA. Investigations should continue into the role other mammalian species may be playing in the epidemiology of R. felis infections.
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Animais Domésticos/microbiologia , Doenças do Gato/microbiologia , DNA Bacteriano/sangue , Doenças do Cão/microbiologia , Infecções por Rickettsia/veterinária , Rickettsia felis/genética , Animais , Animais Domésticos/sangue , Doenças do Gato/epidemiologia , Gatos , Estudos Transversais , Ctenocephalides/microbiologia , Doenças do Cão/epidemiologia , Cães , Infestações por Pulgas , Filogenia , Infecções por Rickettsia/sangue , Infecções por Rickettsia/epidemiologia , Rickettsia felis/classificação , Análise de Sequência de DNA , Estados UnidosRESUMO
Leptospirosis is a widespread zoonosis and has been recognized as a re-emerging infectious disease in humans and a variety of wild and domestic animal species. In order to understand the prevalence and diversity of Leptospira spp. in feral pig populations of Alabama, we trapped 315 feral pigs in Bullock County east-central Alabama, and collected 97 environmental samples from riparian areas in Bullock County and Macon County east-central Alabama. Two previously published PCRs followed by DNA sequencing and BLASTn were performed to identify pathogenic Leptospira species in the kidney of feral pigs (3.2%, 10/315) as well as environmental samples collected from the habitats of feral pigs (2.1%, 2/97), but not in the whole blood samples (n = 276) or spleen (n = 51). An ELISA determined that 44.2% of serum samples (122/276) were antibody-positive for Leptospira. The identification of two pathogenic Leptospira species from environmental samples and the high sero-positivity in feral pigs suggests potential pathogen shedding from feral pigs to environments, and to humans and domestic animals. In order to better understand the risk to human health associated with feral swine presence, further studies are warranted to explore the interrelationship between Leptospira spp. shedding in the urine of feral pigs and bacterial culture to explore pathogenicity. Multi-locus sequencing typing (MLST) and microscopic agglutination tests (MAT) should be performed in future studies to make a definite determination of pathogenic Leptospira in feral pigs in Alabama.
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Chlamydia suis is an important, highly prevalent, and diverse obligate intracellular pathogen infecting pigs. In order to investigate the prevalence and diversity of C. suis in the U.S., 276 whole blood samples from feral swine were collected as well as 109 fecal swabs and 60 whole blood samples from domestic pigs. C. suis-specific peptide ELISA identified anti-C. suis antibodies in 13.0% of the blood of feral swine (26/276) and 80.0% of the domestic pigs (48/60). FRET-qPCR and DNA sequencing found C. suis DNA in 99.1% of the fecal swabs (108/109) and 21.7% of the whole blood (13/60) of the domestic pigs, but not in any of the assayed blood samples (0/267) in feral swine. Phylogenetic comparison of partial C. suis ompA gene sequences and C. suis-specific multilocus sequencing typing (MLST) revealed significant genetic diversity of the C. suis identified in this study. Highly genetically diverse C. suis strains are prevalent in domestic pigs in the USA. As crowding strongly enhances the frequency and intensity of highly prevalent Chlamydia infections in animals, less population density in feral swine than in domestic pigs may explain the significantly lower C. suis prevalence in feral swine. A future study is warranted to obtain C. suis DNA from feral swine to perform genetic diversity of C. suis between commercial and feral pigs.