Improvement of the enrichment used in the EMJH medium (Ellinghausen-McCullough-Johnson-Harris) for the cultivation of Leptospira spp.

Changes were made to the original formulation of the EMJH medium (Ellinghausen-McCullough-Johnson-Harris) enrichment and some aspects such as growth time of Leptospira and utilization in the microscopic agglutination test (MAT) were evaluated and compared to the original enrichment and to a commercially available enrichment (DIFCO™). Leptospira samples (24 antigens) that make up our panel of antigens used in MAT were used, among them, reference and autochthonous strains isolated in Brazil. The samples were grown individually in the EMJH medium under the three previously mentioned conditions (adapted enrichment, original enrichment and commercial enrichment). In addition, 89 blood serums from domestic and wild animals were analyzed by MAT using the antigens grown in these media. All samples tested grew efficiently with the adapted enrichment, and the MAT results were satisfactory. Therefore, other laboratories could also benefit from the use of this adapted enrichment when culturing the Leptospira strains applied in their MAT panels.

Molecular and serological characterization of pathogenic Leptospira spp. isolated from symptomatic dogs in a highly endemic area, Brazil.

BACKGROUND: Leptospirosis is an endemic zoonosis in Brazil, with a great impact on human and animal health. Although dogs are frequently infected by pathogenic Leptospira, the current epidemiological understanding of canine leptospirosis is mainly based on serological tests that predict the infecting serogroup/serovar. Thus, the present study aimed at identifying the causative agent for severe cases of canine leptospirosis in a highly endemic area through the isolation and characterization of the isolated strains. RESULTS: Urine, serum and blood samples were collected from 31 dogs with suspected acute leptospirosis treated at the Veterinary Hospital Service of Santo Amaro University between 2018 and 2019. Acute infection was confirmed in 17 dogs (54.8%) by the associated use of Polymerase Chain Reaction (PCR), Microscopic Agglutination (MAT) and bacteriological culture. Eleven dogs (35.5%) had titers ≥800, with the most frequent serogroups being Autumnalis and Icterohaemorrhagiae (n = 4 each) and Canicola (n = 2). Leptospires were recovered from four dogs, and Multilocus Sequence Analysis (MLSA) revealed infection caused by L. interrogans, which were further characterized as serogroups Canicola (n = 1) and Icterohaemorrhagiae (n = 3). CONCLUSION: The identity of the isolates and serological pattern of MAT suggest that dogs are highly exposed to the serogroup Icterohaemorrhagiae and Canicola, also indicating possible circulation of serogroups not yet isolated in Brazil, notably serogroup Autumnalis. Our findings also reinforce the usefulness of using multiple diagnostic approaches to confirm acute canine leptospirosis.

Temporal and geographical comparison of bulk tank milk and water microbiota composition in Brazilian dairy farms.

This study investigated the relationship between the bacterial communities of bulk tank milk (BTM) and water used in Brazilian dairy farms. We also explored the association between BTM microbiota and its quality parameters (i.e., somatic cell count [SCC] and total bacteria count [TBC]). Water and BTM samples were collected twice for high throughput metataxonomic characterization. Milk samples were collected for SCCs, TBCs, and antimicrobial residue analysis. Water samples were submitted for physico-chemical and microbiological analyses. Overall, the BTM bacterial community was not influenced by the water microbiota. Furthermore, a higher number of Bacillus spp. and a tendency toward a higher number of Lactococcus spp. was associated with a higher TBC, and consequently could be used as an indicator of milk quality. A higher number of Streptococcus spp. and a tendency toward a higher number of Staphylococcus spp. were associated with a higher SCC. Apart from a variation on the content of the Bacillus taxa, no effect of sampling time on BTM bacterial community was observed. Finally, a negative correlation between the number of different species (richness) on BTM and SCC was found.

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.

Leptospira spp., rotavirus, norovirus, and hepatitis E virus surveillance in a wild invasive golden-headed lion tamarin (Leontopithecus chrysomelas; Kuhl, 1820) population from an urban park in Niterói, Rio de Janeiro, Brazil.

The world currently faces severe biodiversity losses caused by anthropogenic activities such as deforestation, pollution, the introduction of exotic species, habitat fragmentation, and climate changes. Disease ecology in altered environments is still poorly understood. The golden-headed lion tamarin (GHLT, Leontopithecus chrysomelas) is an endangered species that became invasive in an urban park in Niterói, Rio de Janeiro, Brazil. The initially few invasive GHLT individuals became hundreds, adapted to living in proximity to humans and domestic animals. These GHLTs were captured as part of a conservation project; some animals were translocated to Bahia and some were kept in captivity. This study tested 593 GHLT for Leptospira serology; 100 and 95 GHLT for polymerase chain reaction (PCR) toLeptospira and hepatitis E virus genotype 3 (HEV-3), respectively, and 101 familiar groups for PCR to viruses (rotavirus A, norovirus GI and GII, and HEV-3). One animal had antibodies for Leptospira serovar Shermani and another for serovar Hebdomadis. One saprophyticLeptospira was found by the 16S PCR and sequencing. Viruses were not detected in samples tested. Findings suggest that the epidemiological importance of such pathogens in this GHLT population is either low or nonexistent. These data are important to understand the local disease ecology, as well as monitoring a translocation project, and to contribute data for species conservation.

Time for change? A systematic review with meta-analysis of leptospires infecting dogs to assess vaccine compatibility in Brazil.

Dogs are thought to be highly exposed to environmental pathogenic leptospires, possibly acting as potential sources of infection for zoonotic transmission. Vaccination stands as the cornerstone strategy to prevent disease and urinary shedding in dogs, yet the success of vaccination is highly dependent on the correspondence of leptospires circulating locally with those used in vaccine compositions. To provide evidence for vaccine compatibility, and to assess whether there are regional differences on serogroup distribution, we conducted a systematic review with meta-analysis on serological data, characterization of leptospiral isolates and risk factors for seropositivity in dogs from Brazil. Studies reporting canine leptospirosis within the Brazilian territory were eligible for inclusion, and methodology was validated by PROSPERO under registration CRD42020204187. Six electronic databases were searched, and data regarding population, methods, and outcomes were extracted. Sixty-one studies were included to access serogroup distribution and risk factors, with a pooled positivity rate of 19.7% in dog population. Serological evidence indicates that Canicola, Icterohaemorrhagiae and Autumnalis are the most frequently found serogroups. Twenty-eight records were included to access leptospiral strains isolated in Brazil, with n = 56 strains characterized as serogroups Canicola, n = 37 as Icterohaemorrhagiae, n = 2 as Pomona, and n = 1 strain as Australis and Sejroe each. Risk factor analysis revealed that stray dogs, puppies or elderly dogs, male dogs and dogs kept by tutors with poor social and economic conditions are at high risk for infection. The present study revealed overall good compatibility of leptospiral strains circulating locally with those used in vaccines against canine leptospirosis in Brazil. The circulation of serovars Pomona and Grippotyphosa has not been consistently demonstrated, and the inclusion of these serovars in local vaccines cannot be supported by our results. The results also provided serological evidence for the circulation of Serogroup Autumnalis among the studied populations.

Isolation of an atypical Leptospira strain assigned to the Sejroe serogroup from a water buffalo in Brazil.

The isolation of leptospires from buffaloes worldwide is still limited to a few strains. Thus, the aim of this study was to describe the first Leptospira isolate from buffalo urine, assigned to the Sejroe serogroup, which does not belong to the Wolffi subgroup, traditionally isolated in Brazil. A total of 244 urine samples of water buffaloes (Bubalus bubalis) raised in the Brazilian Amazon were subjected to bacteriological culturing and polymerase chain reaction (PCR) for the detection of leptospires. The obtained isolate was characterized by serogrouping using polyclonal antibodies, partial DNA sequencing, Hardjo-Bovis-specific PCR, multiple-locus variable-number tandem repeat analysis (MLVA/VNTR) and experimental infection in hamsters. PCR was performed on the urine samples; 11/244 were positive (4.5 %) for Leptospira, and only one isolate was recovered (0.4 %). Regarding characterization, the isolate was assigned to the Sejroe serogroup with high titers (12,800) for the Saxkoebing and Sejroe serovar antisera. The isolate was negative for Hardjo-Bovis-specific PCR, and the species Leptospira borgpetersenii was identified by DNA sequencing. The MLVA results showed that the VNTR profile of the isolate was 1-2-5, compatible with that of serovars Sejroe/Istrica. In the experimental infection in hamsters, the animals did not develop clinical signs, and no macroscopic lesions were observed on the organs at necropsy; however, the strain was detected in the kidneys, uterus, and testicles of the animals. The isolate described herein highlights infection by Sejroe strains that may be overlooked in buffaloes and that may be different from those normally isolated and used in serological studies.

Leptospira strains isolated from cattle in the Amazon region, Brazil, evidence of a variety of species and serogroups with a high frequency of the Sejroe serogroup.

In Brazil, there have been few leptospires isolated from cattle, especially in the Amazon, implying that the epidemiology of the disease in this region is still largely unclear. In a previous study, 52 Leptospira isolates were obtained from urine of cattle raised in the Brazilian Amazon and, to achieve a greater understanding of Leptospira infection in cattle of this region, the present study aimed to serologically and molecularly characterizes all these isolates. The laboratory assays used were the microscopic agglutination test (MAT) adopting a panel of polyclonal antisera against Leptospira spp. for serogrouping the isolates, DNA sequencing (secY) and multiple locus variable number tandem repeat analysis (MLVA). The isolates belonged to five species: 20/52 were identified as L. borgpetersenii (38.5 %); 18/52 as L. kirschneri (34.6 %); 9/52 as L. santarosai (17.3 %); 3/52 as L. noguchii (5.8 %) and 2/52 as L. interrogans (3.8 %). With serogrouping, nine different serogroups were detected, with a high frequency of the Sejroe serogroup. MLVA showed that all L. borgpetersenii isolates had a profile compatible with serovar Hardjo; moreover, the other isolates demonstrated a diversity of patterns, and some of them may represent strains not yet characterized. In the Brazilian Amazon, the leptospires circulating in cattle revealed the unique aspects of infections in this area which, in addition to a variety of strains, were characterized by a high frequency of the Sejroe serogroup, highlighting the serovar Hardjo, which has not been reported in other regions of Brazil.

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.

Serologic survey for selected infectious diseases in free-ranging Brazilian tapirs (Tapirus terrestris) in the cerrado of central Brazil.

From September 2000 to January 2002, a serologic survey was conducted in a population of free-ranging Brazilian tapirs (Tapirus terrestris) inhabiting Emas National Park and surrounding areas in Goiás state, central Brazil, as part of an ecologic study. Ten tapirs were immobilized with a tiletamine-zolazepam combination, and blood samples were collected. All sera were negative for Leptospira spp., Brucella abortus, and equine infectious anemia; and one of 10 animals was positive for Toxoplasma gondii. This report represents the first serologic survey for selected infectious diseases in a free-ranging population of Brazilians tapirs in central Brazil.

Molecular Typing and Antimicrobial Susceptibility Profile of Staphylococcus aureus Isolates Recovered from Bovine Mastitis and Nasal Samples.

In the present study, we aimed to determine the antimicrobial resistance and molecular typing of Staphylococcus aureus recovered from transient and persistent intramammary infections and nares/muzzles in dairy cows. We investigated the antimicrobial resistance of 189 S. aureus strains using a broad antimicrobial susceptibility profile. Furthermore, 107 S. aureus isolates were strain-typed using staphylococcal protein-A (spa) typing. A large proportion of strains exhibited multidrug resistance to antimicrobials, including resistance to critically important antimicrobials, although no methicillin-resistant S. aureus strains were found. Our study did not strengthen the idea that extramammary niches (i.e., nares/muzzles) are an important source of S. aureus for bovine mastitis. A discrepancy in the antimicrobial resistance between S. aureus strains isolated from nares/muzzles and milk samples was observed. Furthermore, S. aureus isolates from transient and persistent intramammary infections (IMIs) did not differ by spa typing, suggesting that the persistence of bovine IMIs was determined by cow factors. Thus, the high level of multidrug-resistant S. aureus found in the two herds, considered together with the predominance of a well udder-adapted S. aureus strain, may contribute to our knowledge of the history of the high prevalence of mastitis caused by S. aureus, which is of great concern for animal and public health.

Canine distemper virus in a crab-eating fox (Cerdocyon thous) in Brazil: case report and phylogenetic analyses.

Although canine distemper is enzootic worldwide and has a wide host range, there are no reports of canine distemper virus in crab-eating foxes (Cerdocyon thous) that provide information on virus phylogeny and histopathologic lesions. The objective of this study is report and describe canine distemper in a crab-eating fox (C. thous), with a focus on the phylogeny of the virus strain and the histopathologic lesions in the animal.

Circulating Leptospira species identified in cattle of the Brazilian Amazon.

The present study aimed to detect the most prevalent serogroups and circulating Leptospira species in cows from Brazilian Amazon. Samples of blood serum, urine and kidney of 208 animals were collected at a municipal slaughterhouse in the Baixo Tocantins region of Pará State, Northern Brazil. The tests used were microscopic agglutination test (MAT), bacteriological isolation, polymerase chain reaction (PCR) and DNA sequencing. The frequency of MAT-reactive cows was 46.6% (97/208) with titers ranging from 100 to 3200, being Sejroe serogroup the most prevalent. There was no Leptospira isolation, but the DNA of bacterium was detected in 5.8% (12/208) of the kidney and in 14.9% (31/208) of the urine samples. DNA sequencing was performed directly from PCR products of 30 samples (3 kidneys and 27 urines), with identification of four different species: L. borgpetersenii with 56.7% (17/30), followed by L. kirschneri with 13.3% (4/30), L. interrogans with 6.7% (2/30), L. santarosai with 3.3% (1/30), and 20.0% (6/30) of samples were identified only at the genus level. These results reveal a diversity and peculiarity for bovine leptospirosis in the Amazon region, mainly due to the low frequency of L. santarosai and more surprising, the presence of L. kirschneri, differently of what is observed in other regions of Brazil.

Molecular diversity of Brazilian strains of porcine circovirus type 2 (PCV-2).

Porcine circovirus 2 (PCV-2) is associated with a broad range of syndromes. In this study, eight pig tissue samples from two Brazilian states were analyzed using six PCR primer pairs amplifying a 1705-bp fragment of the PCV-2 genome. The NJ distance-based method was used for the phylogenetic analysis with the eight field strains herein, 15 GenBank sequences and using PCV-1 as an out-group. This yielded two major clusters (A and B) for this viral species, with the Brazilian strains segregating with European and Asian sequences. Nucleotide identity was 99.7 to 100% among the sequences. This information can be used in further studies of pathogenesis related to PCV-2 in Brazil.

Dogs naturally infected by Rangelia vitalii, Babesia canis vogeli, and Ehrlichia canis in São Paulo, Brazil / Cães naturalmente infectados por Rangelia vitalii, Babesia canis vogeli, e Ehrlichia canis em São Paulo, Brasil

Several agents can cause hemoparasitic diseases in dogs, and blood-sucking arthropods transmit these diseases. These agents can cause several clinical manifestations and, in some cases, can kill the host. Because these agents are essential in animal health, this study aims to detect the frequency of Ehrlichia canis, Rickettsia rickettsii, Anaplasma platys, and Rangelia vitalii by real-time PCR and Babesia vogeli in dogs in the southern region of the city of São Paulo, São Paulo. Of the 98 dog samples, 18 (18.4%) tested positive with real-time polymerase chain reaction for at least one studied agent. Of these 18 samples, 17 tested positive for a single agent (11.2% for B. canis vogeli, 1.02% for R. vitalii, and 5.1% for E. canis), and one showed co-infection with B. canis vogeli and R. vitalii. The results demonstrate the presence of hemoparasites in the studied animals, which can influence the quality and life expectancy of these animals. The Rangeliadetection warns small animal clinicians to include it as a differential diagnosis for hemoparasitosis.(AU)

As hemoparasitoses em cães podem ser causadas por diversos agentes, sendo essas doenças transmitidas por artrópodes hematófagos. Esses agentes podem causar diversas manifestações clínicas e, em alguns casos, podem matar o hospedeiro. Este estudo teve como objetivo detectar por PCR em tempo real a frequência de Ehrlichia canis, Rickettsia rickettsii, Anaplasma platys, Rangelia vitalii e Babesia canis vogeli em amostras de cães da zona sul da cidade de São Paulo, Brasil. Das 98 amostras de cães, 18 (18,4%) testaram positivo com reação em cadeia da polimerase em tempo real para pelo menos um agente estudado. Destas 18 amostras, 17 testaram positivo para um único agente (11,2% para B. canis vogeli, 1,02% para R. vitalii e 5,1% para E. canis), e uma apresentou coinfecção com B. canis vogeli e R. vitalii. Os resultados demonstram a presença de hemoparasitas nos animais estudados, o que pode influenciar a qualidade e a expectativa de vida desses animais. Além disso, é o primeiro relato da detecção de R. vitalli na zona sul de São Paulo e serve de alerta para os clínicos de pequenos animais incluírem esse agente como diagnóstico diferencial para as hemoparasitoses.(AU)

Septicemia due to Streptococcus dysgalactiae subspecies dysgalactiae in vampire bats (Desmodus rotundus).

Beta-hemolytic Streptococcus dysgalactiae is a well-known pathogen for a wide range of animals and humans. Two subspecies are recognized: (i) equisimilis, associated to disease in horses and humans, and (ii) dysgalactiae mainly isolated from animal illness with only a few humans' cases. This study describes the isolation and characterization of Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) from vampire bats, maintained in captivity for research proposes. Animals presented neurologic, respiratory and gastroenteric symptoms and sudden death. Beta-hemolytic Gram-positive cocci were isolated in blood agar plates and further characterized as Lancefield group C. All isolates were identified as S. dysgalactiae by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and subspecies dysgalactiae was confirmed by 16S rRNA sequencing and phylogenetic analysis. Genotyping through SE-ALFP resulted in three profiles (A1-A3) with one bat being infected by profiles A1 and A3. This is the first report of SDSD causing illness in bats and especially in Desmodus rotundus species.

Author Correction: Septicemia due to Streptococcus dysgalactiae subspecies dysgalactiae in vampire bats (Desmodus rotundus).

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Molecular identification of Giardia duodenalis isolates from humans, dogs, cats and cattle from the state of São Paulo, Brazil, by sequence analysis of fragments of glutamate dehydrogenase (gdh) coding gene.

The nucleotide sequence of glutamate dehydrogenase (gdh) coding genes were obtained from cysts of Giardia duodenalis isolated from feces of naturally infected cats (n=19), dogs (n=27), humans (n=37) and cattle (n=5). The samples were from several municipalities within the state of São Paulo, Brazil and were collected from January 2004 to August 2006. Sequences analysis of the 37 specimens recovered from humans revealed 29 G. duodenalis assemblage AII and 8 G. duodenalis assemblage B. Among samples from cats, 11 were classified into assemblage F and 8 into assemblage AI. Only the host-adapted assemblages C and D were detected in samples from dogs (7 and 20, respectively). Among the samples from cattle, the genotype livestock was found in four samples and the assemblage AI was detected in one sample. The molecular identification of assemblages of G. duodenalis isolates from different hosts reveals that genetic diversity of this protozoon in Brazil is similar to that of Giardias from other parts of the world.

Complete Genome Sequence of a Hobi-Like Virus Isolated from a Nelore Cow with Gastroenteric Disease in the State of São Paulo, Brazil.

The Hobi-like virus presents antigenic and molecular differences in relation to bovine virus diarrhea virus 1 and 2. The description of the complete genome of the Hobi-like virus SV757/15, isolated from a Nelore cow with gastroenteric disease in Brazil, will help in understanding the evolution and diversity of pestiviruses.

Outbreak Control and Clinical, Pathological, and Epidemiological Aspects and Molecular Characterization of a Bovine Herpesvirus Type 5 on a Feedlot Farm in São Paulo State.

This paper describes the control, epidemiological, pathological, and molecular aspects of an outbreak of meningoencephalitis in calves due to bovine herpesvirus 5 at a feedlot with 540 animals in São Paulo State, Brazil. The introduction of new animals and contact between the resident animals and the introduced ones were most likely responsible for virus transmission. Bovine herpesvirus 1 vaccine was used, resulting in the efficacy of the outbreak control, although two bovine herpesvirus 1 positive animals, vaccinated and revaccinated, presented meningoencephalitis, thereby characterizing vaccinal failure.