SARS-CoV-2 infection impacts carbon metabolism and depends on glutamine for replication in Syrian hamster astrocytes.

COVID-19 causes more than million deaths worldwide. Although much is understood about the immunopathogenesis of the lung disease, a lot remains to be known on the neurological impact of COVID-19. Here, we evaluated immunometabolic changes using astrocytes in vitro and dissected brain areas of SARS-CoV-2 infected Syrian hamsters. We show that SARS-CoV-2 alters proteins of carbon metabolism, glycolysis, and synaptic transmission, many of which are altered in neurological diseases. Real-time respirometry evidenced hyperactivation of glycolysis, further confirmed by metabolomics, with intense consumption of glucose, pyruvate, glutamine, and alpha ketoglutarate. Consistent with glutamine reduction, the blockade of glutaminolysis impaired viral replication and inflammatory response in vitro. SARS-CoV-2 was detected in vivo in hippocampus, cortex, and olfactory bulb of intranasally infected animals. Our data evidence an imbalance in important metabolic molecules and neurotransmitters in infected astrocytes. We suggest this may correlate with the neurological impairment observed during COVID-19, as memory loss, confusion, and cognitive impairment.

Anti-SARS-CoV-2 equine F (Ab')2 immunoglobulin as a possible therapy for COVID-19.

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab')2 immunoglobulin using a newly developed SARS-CoV-2 viral antigen that was purified and inactivated by radiation. Cell-based and preclinical assays showed that the F(ab')2 immunoglobulin successfully neutralizes the virus, is safe in animal models, and reduces the severity of the disease in a hamster model of SARS-CoV-2 infection and disease.

Sperm viability, serological, molecular, and modified seminal plasma agglutination tests in the diagnosis of Leptospira in the semen and serum of bovine bulls.

The present study investigated the serum microscopic agglutination test (MAT) among 203 bovine bulls with reproduction by natural means, without apparent signs of orchitis or inflammation of accessory reproductive glands. Simultaneously, the semen of all bulls was subjected to sperm viability analysis and PCR based on the 16S rRNA gene. PCR-positive results of semen samples were confirmed by sequencing. A modified seminal plasma agglutination (MSPA) test, replacing the blood serum of all bulls in the MAT with seminal plasma was performed as well. Eight (8/203 = 3.9%) semen samples from bulls were considered nonviable (necrospermia and azoospermia) without relation to the PCR diagnosis. No agglutinin titers were identified in MSPA test. A high frequency (132/203 = 65%) of leptospiral agglutinin titers was identified in the MAT, particularly for the Sejroe serogroup (Hardjo CTG, 100/203 = 49.3%; Wolffi 74/203 = 36.4%; Guaricura 72/203 = 35.5%; and Hardjoprajitno 56/203 = 27.6%). Three (3/203 = 1.5%) semen samples of bulls were positive in the PCR, but these results were not confirmed by sequencing. The high frequency of serovars from the Sejroe serogroup typically adapted to bovines indicates the need for measures for the prophylaxis/control of the pathogen on the sampled farms. Discrepancies among the MAT, sperm viability, and molecular detection of leptospires in semen highlight the need for a combination of methods to diagnose leptospirosis in bovine bulls. To our knowledge, modified seminal plasma agglutination is described for the first time here to investigate anti-Leptospira antibodies produced locally in the genital tract in the diagnosis of bovine leptospirosis among bulls that reproduce by natural means.

Leptospira interrogans serogroup Pomona strains isolated from river buffaloes.

At present, little is known regarding the prevalence of buffalo leptospirosis worldwide, especially with respect to which Leptospira strains may infect this animal species. Furthermore, most investigations into this disease in buffaloes have only been performed with serological studies. In Brazil, particularly in the Amazon, buffalo production is growing and is just as important as cattle production, although few studies have been performed on buffalo compared to cattle. Thus, the aim of this study was to isolate and characterise Leptospira strains from river buffaloes raised in the Brazilian Amazon region. We collected 109 kidney samples from slaughtered buffaloes raised in the Amazon Delta region of Brazil. The samples were analysed by bacteriological culture for the isolation of leptospires, and the obtained isolates were serologically and molecularly characterised by microscopic agglutination test (MAT), DNA sequencing and multiple locus variable-number tandem repeat analysis (MLVA). Five isolates were obtained, and in serogrouping analyses, these isolates were only reactive for the Pomona serogroup, with an observed titre of 25,600. The DNA sequencing results revealed that all the isolates belonged to the species Leptospira interrogans, and the MLVA results showed that the VNTR loci 4, 7 and 10 profile of all the isolates was 4-1-10. In this study, we observed that Pomona serogroup strains circulate in buffaloes in the Amazon, showing that in Brazil, buffaloes can be affected by Leptospira strains other than the Sejroe group, which are adapted to cattle.

Usefulness of the Ranking Technique in the Microscopic Agglutination Test (MAT) to Predict the Most Likely Infecting Serogroup of Leptospira.

The microscopic agglutination test (MAT) used for the serological diagnosis of leptospirosis, as a robust and inexpensive method, is still the reality in many laboratories worldwide. Both the performance and the interpretation of the MAT vary from region to region, making standardization difficult. The prediction of the probable infecting serogroup using this test is indispensable for elucidating the epidemiology of the disease; however, in veterinary medicine, many studies consider any reaction detected with a titer of 100, which may ultimately overestimate some serogroups. Thus, the aim of this study was to evaluate the usefulness of the ranking technique for predicting the probable infecting serogroup identified by the MAT, eliminating cross reactions with other serogroups. Leptospira strains (12 samples) were inoculated in hamsters, and after 30 days, serology was performed by the MAT for these animals to confirm the infecting serogroup. Using the ranking technique, the probable infectious serogroup found with the MAT was the same as that in which the strains of inoculated leptospires belonged; additionally, the technique can be applied in epidemiological studies involving herds.

Immunoprotective Activity Induced by Leptospiral Outer Membrane Proteins in Hamster Model of Acute Leptospirosis.

Leptospirosis is a zoonotic disease of worldwide distribution, affecting both humans and animals. The development of an effective vaccine against leptospirosis has long been pursued but without success. Humans are contaminated after direct contact with the urine of infected animals or indirectly by contaminated water or soil. The vaccines available consist of inactivated whole-bacterial cells, and the active immunoprotective antigen is the lipopolysaccharide moiety, which is also the basis for serovar classification. However, these vaccines are short-lasting, and protection is only against serovars contained in the preparation. The search for prevalent antigens, present in pathogenic species of Leptospira, represents the most cost-effective strategy for prevention of leptospirosis. Thus, the identification of these antigens is a priority. In this study, we examined the immunoprotective effect of eight leptospiral recombinant proteins using hamster as the challenge model. Animals received subcutaneously two doses of vaccine containing 50 µg of each recombinant protein adsorbed on alum adjuvant. Two weeks after the booster, animals were challenged with virulent leptospires and monitored for 21 days. All proteins were able to induce a specific immune response, although significant protective effects on survival rate were observed only for the proteins Lsa14, rLIC13259, and rLIC11711. Of these, only rLIC13259 and rLIC11711 were found to be highly prospective in promoting renal clearance. The sterilizing potential of both proteins will be further investigated to elucidate the immunoprotective mechanisms involved in leptospirosis control. These are the first proteins involved with human complement components with the capacity to protect against virulent challenge and to eliminate the bacteria from the host.

Identification of Pathogenic Leptospira Species in the Urogenital Tract of Water Buffaloes (Bubalus bubalis) From the Amazon River Delta Region, Brazil.

In the current context of deforestation and fire in the Amazon, buffaloes could be a cost-effective and sustainable alternative for cattle production in the region, as they can convert low-quality foods and be raised in floodplain areas. However, little is known about the reproductive diseases that affect these animals; thus, the purpose of this study was to perform the molecular characterization of Leptospira spp. in the urogenital tract of water buffaloes (Bubalus bubalis) raised in the Amazon River Delta region in Brazil. Samples were collected from 114 kidneys, 204 ovaries, and 160 uterine swabs of slaughtered buffaloes in the Macapá microregion of Amapá State (Brazil) and were subjected to PCR to detect bacterial DNA. Positive amplicons were sequenced to identify Leptospira species. Among the total samples, 11/473 were PCR positive (2.3%), including 10 kidney samples and one uterine swab sample. DNA sequencing identified two pathogenic species from the kidney samples: L. interrogans, accounting for 60.0% (6/10) of these samples, and L. borgpetersenii, accounting for 20.0% (2/10), while 20.0% (2/10) were identified only at the genus level. The bacterium in the uterine swab sample was identified as L. interrogans with genetic proximity to strains belonging to the serovar Hardjo. This is the first report of leptospires species identified in buffaloes from the Amazon River Delta region and revealed that these animals may be carriers of different pathogenic Leptospira species, similar to bovines, including showing genital colonization.

Leptospira transport medium (LTM): A practical tool for leptospires isolation.

The isolation of Leptospira is challenging, since the bacteria of this genus are susceptible to adverse environmental conditions and may not remain viable for extend periods in urine samples. This study attempted to develop and evaluate a simple and practical method to isolate leptospires from bovine urine samples. A culture medium for sample transport, named Leptospira Transport Medium (LTM), was described and validated using reference serovars of Leptospira spp. in addition to autochthonous strains isolated in Brazil. We evaluated LTM in the field, by collecting 215 urine samples from slaughtered cattle and immediately seeding them in LTM and Fletcher's medium, used as control. The cultures were sent to a laboratory within 10 days for further processing. Moreover, 16S PCR was also performed on the urine samples directly to detect Leptospira DNA. Using LTM enabled 52 isolates (24.2%) to be obtained in pure culture, and contamination was only observed in 15/215 samples (7.0%). Regarding the samples in Fletcher's medium, 10 (4.6%) isolates were obtained. With 16S PCR performed in the urine samples, 31 samples (14.4%) were determined to be positive. LTM was developed and used in a simple and practical way and can significantly improve the isolation of leptospires from urine samples, as well as being highly useful in remote areas, not only in Brazil but also in other countries where few easily accessible laboratories are available. Furthermore, LTM can be prepared by laboratories and provided to veterinarians and technicians for urine collection in the field.

High discrimination of Mycobacterium bovis isolates in Brazilian herds by spoligotyping.

Bovine tuberculosis is an infectious disease caused by Mycobacterium bovis (M. bovis), that leads to economic losses in infected herds and it is also considered an important zoonosis. The molecular typing methods of M. bovis isolates are fundamental for the bovine tuberculosis surveillance system, and spoligotyping is the standard genotyping technique for this species. Thus, the aim of the present study is to analyze the spatial and cluster distribution of M. bovis strains from several regions of Brazil through molecular typing. Spoligotyping technique was applied on 422 isolates identified as M. bovis, and Ripley's K function was used to perform the spatial and cluster analysis of each identified profile. Forty-three (43) different profiles were identified and spoligotype SB0121 was the most frequent and showed a uniform pattern in the spatial distribution while spoligotypes SB0295, SB1380 and SB1050 formed clusters. In addition, three novel spoligotype profiles (SB2361, SB2362, SB2364) were identified in different herds. In this perspective, it is believed that molecular identification and typing can significantly improve the performance of surveillance systems for bovine tuberculosis in Brazil.

Survey of Leptospira spp. and Brucella abortus in Free-Ranging Armadillos from Pantanal, Brazil.

Out of the 20 recognized species of armadillos in the world, 11 are found in Brazil, and five of them are found in Pantanal, one of the world's largest wetlands. Beef cattle (Bos taurus) farming is the main economic activity in this region, which promotes intense wildlife-livestock contact and increases the likelihood of pathogen exposure, including to agents with zoonotic and economic relevance. Previous studies demonstrated that several wildlife species in Pantanal have been exposed to Brucella abortus and Leptospira spp.; however, little is known regarding the exposure and/or prevalence of zoonotic pathogens in armadillos. We used conventional PCR, the rose Bengal test (RBT), and the microscopic agglutination test (MAT) to investigate the exposure to and infection by Brucella spp. and Leptospira spp. using blood samples from four species of armadillos: nine-banded armadillo (Dasypus novemcinctus, n=2), southern naked-tailed armadillo (Cabassous unicinctus, n=8), yellow armadillo (Euphractus sexcinctus, n=16), and giant armadillo (Priodontes maximus, n=22), captured in Nhecolândia, Pantanal, Brazil. Samples were PCR- and RBT-negative for Brucella spp. infection and exposure. However, MAT revealed a Leptospira spp. seroprevalence of 31% (5/16; 95% confidence interval [CI]=0.11-0.58) in yellow armadillo and 18% (4/22; 95% CI=0.05-0.40) in giant armadillo specimens to serogroups Autumnalis, Cynopteri, and Pomona, with titers ranging from 200 to 1,600. Our results contribute to the understanding of zoonotic pathogens in armadillos in Pantanal and reinforce the importance of wildlife health surveillance in this area.

Development of a pooled antigen for use in the macroscopic slide agglutination test (MSAT) to detect Sejroe serogroup exposure in cattle.

This study aimed to develop and evaluate a pooled antigen for use in the macroscopic slide agglutination test (MSAT) to detect cattle positive for the Sejroe serogroup. To this end, 193 bovine serum samples from different Pará State regions (Amazonia) were subjected to a reference microscopic agglutination test (MAT) for the serological diagnosis of leptospirosis using 11 serovars representing the Sejroe serogroup: Hardjo-prajitno; Hardjo-bovis; Sejroe; Wolffi; Guaricura (Bov.G.); Guaricura (M4/98); Ricardi; Gorgas; Recreo; Polonica and Medanensis. The three most prevalent serovars in the MAT were selected for the development of a pooled antigen for use in MSAT; subsequently, the 193 serum were assessed with the macroscopic slide agglutination test (MSAT) containing the developed antigen. The Kappa test was used to determine the general agreement between the MAT and MSAT results. As a result, of the 193 serum samples, 155 (80.3%) were reactive, and 38 (19.7%) were non-reactive in the MAT; Hardjo-prajitno, Wolffi and Medanensis were the three most prevalent serovars. Of the 193 serum samples tested in the MSAT using the developed pooled antigen, 114 were reactive (59.0%), and 79 (41.0%) were non-reactive; the Kappa coefficient was 0.52 (CI 95%, 0.40-0.63), indicating moderate agreement between the two tests. The MSAT with the pooled antigen including the most prevalent serovars detected bovines with the Sejroe serogroup exposure, mainly in animals with high titters in the MAT, and could be used to screen herds suspected of acute infection by this serogroup in Pará State.

Genotyping and antimicrobial resistance of Streptococcus uberis isolated from bovine clinical mastitis.

A genotypic characterization of Streptococcus uberis isolated from clinical mastitis (CM) in dairy cows, and the association of Strep. uberis genotypes and antimicrobial susceptibility (AMS) was performed. A total of 89 isolates identified as Strep. uberis from 86 dairy cows with CM in 17 dairy herds of Southeastern Brazil were genotyped using random amplified polymorphic DNA (RAPD) analysis. After genotyping, two clusters (I and II) were created according to RAPD types. A commercial broth microdilution test was used to determine the susceptibility of Strep. uberis isolates to 8 antimicrobials (ampicillin, ceftiofur, cephalothin, erythromycin, penicillin, penicillin+novobiocin, pirlimycin and tetracycline). For each antimicrobial, we determined the minimal inhibitory concentrations that inhibit 50% (MIC50) and 90% (MIC90) of Strep. uberis strains. Differences in AMS among genotypic clusters were evaluated using mixed regression models. Overall, a great polymorphism (56 RAPD-types) was found among Strep. uberis isolates, although a higher genetic similarity (based on the PCR bands features) was observed within herds after genotypic clustering. No differences in AMS were observed among clusters. Strep. uberis isolated from bovine CM were resistant to most antimicrobials, with the exception of cephalothin and penicillin+novobiocin.

Identification of clonal complexes of Mycobacterium bovis in Brazil.

Bovine tuberculosis is a disease that is widely distributed around the world. Its causative agent, Mycobacterium bovis, has characteristics of a microorganism with clonal multiplication in populations with no evidence of genetic exchange between strains, and, consequently, a group of strains can be identified as descending from a common ancestor. The aim of this study was to investigate the clonal complexes of M. bovis isolated from samples of lesions suggestive of bovine tuberculosis collected from slaughterhouses in various states of Brazil between 2006 and 2012. Ninety samples were analyzed, and it was found that 14.4% belonged to the clonal complex European1 and 81.1% to the clonal complex European2, while 4.65% were not identified as any of the four known complexes.

Molecular characterization and antimicrobial susceptibility pattern of Streptococcus agalactiae isolated from clinical mastitis in dairy cattle.

The objectives of this study were to: (a) genotypically characterize Streptococcus agalactiae isolates recovered from clinical mastitis (CM) cases in dairy cows and, (b) determine the association of antimicrobial susceptibility (AMS) and genotypes of Strep. agalactiae clustered according to the genetic similarity. A total of 89 Strep. agalactiae isolates recovered from bovine CM were genotyped using random amplified polymorphic DNA (RAPD) analysis. In addition, the AMS of the isolates was determined using a commercial broth microdilution test composed of 10 antimicrobials (penicillin, ampicillin, oxacillin, cephalothin, ceftiofur, penicillin/novobiocin, erythromycin, pirlimycin, tetracycline, and sulfadimethoxine). Descriptive analysis was used to report the frequency of RAPD-types and genotypic clusters within herd, housing system, season and CM severity scores. The minimal antimicrobial concentrations that inhibited 50% (MIC50) and 90% (MIC90) of the isolates were calculated and survival analysis was completed to verify the differences of AMS among genotypic clusters. Results of RAPD showed a great genotypic diversity of Strep. agalactiae (45 RAPD-types) and three clusters (Ia, Ib and II) were created based on the genetic similarity among genotypes. After clustering, a high genetic similarity was observed within and between herds. Overall, Strep. agalactiae showed high susceptibility to most antimicrobials, except to tetracycline and erythromycin. Differences in the AMS among clusters were observed for ampicillin, ceftiofur, erythromycin, pirlimycin, sulfadimethoxine and tetracycline. In conclusion, Strep. agalactiae is still highly susceptible to most antimicrobials, although differences in susceptibility to certain antimicrobials were observed among genotypic clusters.

Genotyping and rifampicin and isoniazid resistance in Mycobacterium bovis strains isolated from the lymph nodes of slaughtered cattle.

In developing nations, 10-20% of the human cases of tuberculosis are caused by Mycobacterium bovis. However, this percentage may be underestimated because most laboratories in developing countries do not routinely perform mycobacterial cultures, and only a few have the systems in place to identify M. bovis. There are few studies investigating genotypic diversity and drug resistance in M. bovis from animal and/or human infections. The genotypic diversity of M. bovis strains obtained from bovine lymph nodes were investigated by spacer oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive unit-variable-number tandem repeat typing (MIRU-VNTR). The phenotypic resistance to isoniazid and rifampicin and MIC values of the isolates were determined using the resazurin microtiter assay plate method (REMA). The evaluation of the possible genetic basis for such resistance was performed with GenoType MTBDRplus. Sixty-seven isolates were obtained, of which 11 (16%) were MDR-TB, 8 (12%) were isoniazid-resistant, and 2 (3%) were rifampicin-resistant. Mutations associated with drug resistance were not found. Genotyping techniques enabled the grouping of the strains into 12 clusters and 21 isolates with unique profiles. The high frequency of M. bovis reinforces the impact of the pathogen as a major causal agent of bovine tuberculosis in the study area. The resistance of the strains to drugs used for first-line treatment of human tuberculosis raises public health concerns. Further studies are required to elucidate the basis of drug resistance and genotypic diversity in M. bovis.

Mycobacterium bovis in a European bison (Bison bonasus) raises concerns about tuberculosis in Brazilian captive wildlife populations: a case report.

BACKGROUND: Tuberculosis caused by Mycobacterium bovis is an important worldwide zoonosis and has been reported to cause clinical disease in several animal species, including captive wildlife. This report describes a case of M. bovis infection in a European bison from a Brazilian zoo and compiles a number of literature reports that raise concern regarding tuberculosis among captive wildlife in Brazil. CASE PRESENTATION: A 13 year-old captive-born male bison (Bison bonasus) from a Brazilian zoo began presenting weight loss, diarrhea and respiratory symptoms, which inevitably led to his death. At the animal's necropsy, inspection of the thoracic and abdominal cavities revealed multiple enlarged lymph nodes, ranging from 4 to 10 cm, and pulmonary nodules containing caseous masses with firm white materials consistent with mineralization. Histopathology findings showed a significant amount of acid-alcohol resistant bacilli compatible with Mycobacterium spp. Specimens from lymph nodes and lungs were cultured on Petragnani and Stonebrink media, and specific PCR assays of the bacterial isolate identified it as M. bovis. CONCLUSION: The European bison reported herein died from a severe form of disseminated tuberculosis caused by M. bovis. A review of the available literature indicates possible widespread occurrence of clinical disease caused by M. bovis or M. tuberculosis affecting multiple animal species in Brazilian wildlife-related institutions. These likely underestimated numbers raise concern regarding the control of the disease in captive animal populations from Brazil.

Molecular characterization of Leishmania infantum in domestic cats in a region of Brazil endemic for human and canine visceral leishmaniasis.

Leishmaniasis is a "neglected tropical disease" and serious public health issue in Brazil. While dogs are recognized as particularly important reservoirs, recent reports of domestic cats infected with Leishmania sp. in urban areas suggest their participation in the epidemiological chain of the parasite in endemic areas. The aim of this study was to screen domestic cats for Leishmania sp. infection in an area where human and canine visceral leishmaniasis are endemic, followed by the identification of the species circulating in cats. We collected peripheral blood, lymph-node aspirates and bone marrow from 100 adult animals, both male and female, and analyzed the samples using cytological and molecular (PCR) detection techniques. We detected Leishmania in 6% of animals, which were then analyzed by RFLP-PCR to identify the species. Leishmania infantum (synonym: L. chagasi), a species responsible for visceral leishmaniasis in humans and other animals, was identified from all six samples. Amastigotes were observed in the peripheral blood, bone marrow and lymph-node aspirates in 4 of the 6 PCR-positive animals. The presence of infected cats in endemic areas should not be neglected, because it demonstrates the potential role of these animals in the biological cycle of the pathogen.