Eryngium foetidum reported as a new host of Meloidogyne enterolobii in the state of Pará, in the eastern Amazon region, Brazil.

Amazon chicory (Eryngium foetidum L. [Apiaceae]), also known as culantro, is native to Tropical America and the West Indies. It belongs to the unconventional food plants (UFPs) group, and in addition to be consumed as a spice herb, it possesses a wide range of ethnomedicinal uses (Paul et al. 2011). In 2019, in the eastern Amazon region of Brazil, state of Pará, producers of E. foetidum in the municipality of Castanhal (01°15'363" S 047°10'232" W) reported the occurrence of underdeveloped plants with leaf yellowing and a large number of galls in the root system, which are typical symptoms of root-knotting nematode. Soil and root samples were collected and sent to the Nematology Laboratory (LabNema) located at the Faculty of Agrarian and Veterinary Sciences, UNESP, Jaboticabal, São Paulo, Brazil. A total of 46 second-stage juveniles (J2s) were extracted per 100 cm3 of soil, and a total of 460 eggs and J2s Meloidogyne spp. were found per gram of root. Morphological and molecular techniques were used to identify the species. The analysis of the perineal patter of ten females revealed thin striations in an oval shape with a high and semi-trapezoidal dorsal arch. No striations were observed in the perivulvar region. The labial region of the ten males analyzed exhibited a non-prominent labial disc, fused and slightly recessed submedian lips, with no apparent annulations. The morphological characteristics observed in the adults were consistent with those originally described for Meloidogyne enterolobii (Yang; Eisenback, 1983), confirming the species purity of the recovered population. Three individual nematodes had their 18S rDNA region sequenced (Holterman et al. 2006) which showed an average identity of 99.7% with other sequences of M. enterolobii available in the GenBank database. A Bayesian phylogenetic tree was constructed, providing insights into the specific relationship of M. enterolobii recovered from E. foetidum with other related nematodes. Each of the three sequenced nematodes represented a unique haplotype, resulting in their separation into distinct clades. Moreover, the obtained sequences presented polymorphisms that differed from the M. enterolobii sequences already available in the database, highlighting the genetic diversity of this species in relation to its original host (Silva et al. 2021). The species M. enterolobii was also confirmed using species-specific primers for M. incognita, M. javanica, and M. enterolobii (Zijlstra et al. 2000; Tigano et al. 2010). To confirm the pathogenicity of M. enterolobii on E. foetidum, a modified Koch Postulate was conducted. Six seedlings of E. foetidum were transplanted individually to 10-liter pots containing autoclaved soil. Each pot was then inoculated with 5 mL of a suspension containing 3,000 eggs and J2s from the original population of M. enterolobii obtained from E. foetidum. After 90 days, the inoculated plants exhibited root galls with a plentiful egg mass, in contrast to the healthy non-inoculated plants. The average number of M. enterolobii nematodes recovered from the roots of the inoculated plants was 42,040 eggs and J2s, resulting in a reproduction factor (RF) of 14.0. The importance of reporting the occurrence of M. enterolobii in E. foetidum is due to the fact that this plant species is cultivated in a crop rotation system with other vegetables such as lettuce and coriander, which are also hosts of M. enterolobii. Consequently, different crop rotation strategies and control alternatives need to be considered in areas where E. foetidum is grown. This is the first report of E. foetidum serving as a host for the root-knot nematode M. enterolobii worldwide.

First Report of Root-Knot Nematode Meloidogyne enterolobii Infecting Sweetpotato in the State of Rio Grande do Norte, Brazil.

The sweetpotato (Ipomoea batatas L., Convolvulaceae family) originated in Latin America and is currently cultivated worldwide. The storage roots, rich in calories, have made this crop one of the main caloric sources for low-income populations, especially in developing countries. Brazil annually produces about 805,000 tons, with the Northeast region responsible for 34% of this production (Albuquerque et al. 2020). In October 2019, sweetpotato plants cv. Campina, from a field in the region of Touros, state of Rio Grande do Norte (RN), Brazil (5°12'31"S 35°34'42"W), presented deformed storage roots, with galls, typical of root-knot nematodes. The roots were sent to the Nematology Laboratory (LabNema) where 14,032 eggs and 3,312 second-stage juveniles (J2s) of Meloidogyne sp., in 10 g of roots, were recovered. The species of adults was identified through morphological, biochemical, and phylogenetic analysis. The perineal region of females (n = 10) presented an oval shape, with a high and semi-trapezoidal dorsal arch and streak-free perivulval region. The labial region of males (n=10) presented high and rounded head cap, labial region slightly set off from the body, without annulations. The morphological characters were compatible with the original description of Meloidogyne enterolobii (Yang and Eisenback 1983). The phenotype of esterase isoenzymes showed two major bands (VS1-S1) also characteristic of M. enterolobii (Esbenshade and Triantaphyllou 1985). Sequences of 18S rDNA (~1200bp) of individual females (Holterman et al. 2006) obtained from sweetpotatoes before (SPme1 and 2) and after inoculation (SPme3 and 6), and from guava, used as M. enterolobii species control, were submitted to Bayesian analysis. The sequences presented genetic diversity among them resulting from seven SNPs (Single Nucleotide Polymorphism) and 99.4 to 99.9% identity with M. enterolobii sequences deposited in the NCBI GenBank (accession numbers MW209034-MW209039). The pathogenicity test was carried out under greenhouse conditions, in which 3,000 eggs and J2s from the original population isolated of M. enterolobii were inoculated in sweetpotato seedlings cv. Campina (n = 6). After three months, the roots presented galls and deformations typical of root-knot nematodes, while non-inoculated plants did not present any symptoms. An average of 15,900 eggs and J2s of M. enterolobii (RF = 5.3) were recovered from the roots, proving that sweetpotatoes were a host of this species. Meloidogyne enterolobii is known to cause great damage to sweetpotato (Ye et al. 2020). In Brazil, Meloidogyne nematode had been reported once, isolated from a sweetpotato field in the Ceara state and the species suggested by the authors according to esterase electrophoresis was M. enterolobii. Nonetheless, the authors did not present taxonomic, isoenzyme phenotypes and molecular species identification integratively, nor included pathogenicity tests (Silva et al. 2016). Therefore, it is the first time that M. enterolobii, with reliable identification by different methods, including sequencing, was detected in commercial sweetpotato fields in the RN state and in Brazil. The local farmers reported that this nematode deforms the storage roots which make them useless for commercialization, resulting in minimal losses of 50% of production in the infested areas. Furthermore, as sweetpotatoes are vegetatively propagated, the spread of this nematode through planting material is favored. Considering the importance of this crop in Brazil, this report is essential for control measures of this pathogen to be taken in order to avoid its spread to other regions.

First Report of Meloidogyne incognita Infecting Stachys byzantina in Brazil.

Stachys byzantina C. Koch (Lamiaceae alt. Labiatae), commonly known as lamb's ear, is an important medicinal plant with anti-inflammatory, antitumor, anticancer, antispasmodic, sedative and diuretic properties (Asnaashari et al. 2010). This plant is widely consumed in Europe and Asia as aromatic teas. In Brazil, it is an unconventional food plant, nonetheless, its medicinal properties have been recognized as well as its production. In May 2019, in a Sao Paulo State municipality, Jaboticabal, (21°14'38.7"S 48°17'10.6"W), S. byzantina plants presented reduced growth and chlorotic leaves associated with root galls. In the phytopathological clinic, 7,983 eggs and juveniles of Meloidogyne sp. were counted in 10 g of the plant roots. In 100 cm³ of soil surrounding the plant, 532 second-stage Meloidogyne sp. juveniles (J2) were found. Morphological, enzymatic and molecular identification of the nematode species found were performed (Fig. S1). For morphological analysis, perineal pattern of females (n = 10) and labial region of males (n = 10) were analyzed. In the perineal region of females, a high and trapezoidal dorsal arch with thick striations was observed, whereas the males presented the trapezoidal labial region with the prominent labial disc in relation to the sub-median lips and transverse streaks in the head region, typical characteristics of M. incognita (Kofoid and White, 1919) Chitwood 1949. (Netscher and Taylor 1974; Eisenback and Hirschmann 1981). The esterase enzyme profile, obtained individually from 8 females, was compatible with phenotype I1 [Rm (x100) = 46.25], also associated with M. incognita (Esbenshade and Triantaphyllou 1985). Molecular analysis was realized (n = 3) by applying the primers Finc/Rinc (Zijlstra et al. 2000) in the DNA of individual females, which resulted in the amplification of an amplicon of 1200 bp specific for M. incognita. Pathogenicity testing was conducted in a greenhouse by inoculation of 5,000 eggs and juveniles from the original population into S. byzantina seedlings (n = 4). After 90 days, the inoculated plants, unlike the non-inoculated ones, exhibited symptoms similar to those initially observed in the field. The nematodes were extracted from the roots of the inoculated plants, quantified, and the identity of M. incognita was confirmed. The average reproductive factor obtained was 136.6, confirming the pathogenicity of M. incognita to S. byzantina. Thus, this is the first report of M. incognita associated with S. byzantina in Brazil and in the world. Lamb's ear is a horticultural plant, and its high reproductive factor to M. incognita can also result in damage to the subsequent crops. In addition, Lamb's ear is propagated vegetatively and this favors the spread of nematodes to other areas. This new report is important in order to alert producers to realize the proper management of this nematode in S. byzantina.

Necrotic Enteritis Caused by Clostridium perfringens in Blue and Gold Macaws (Ara ararauna).

Clostridium perfringens types A and C, which are gram-positive, anaerobic, spore-forming bacteria, can cause necrotic enteritis in birds. Although Clostridium perfringens is considered a commensal organism in the avian intestinal tract, in association with severe stress, other infectious agents, or immunosuppressive conditions, it can cause disease outbreaks. This report describes a disease occurrence of necrotic enteritis caused by C perfringens in macaws (Ara ararauna). Two adult male blue and gold macaws maintained in a zoo exhibit were presented for postmortem examinations after histories of sudden death. Based on the gross examinations and microscopic evaluation of submitted tissue from both birds, the cause of death was determined to be necrotic enteritis. Microbiologic assays followed by polymerase chain reaction analyses identified the isolated strains as C perfringens type A, indicated by only being positive for the cpa gene that encodes the α-toxin. The birds were maintained in an exhibit in which patrons can interact with the animals within their environment. Thus, organisms, such as this pathogen, may present a danger for other birds because visitors could disperse the bacterium to other parts of the zoo.

Management of the American cockroach's oothecae: The potential of entomopathogenic fungi control.

The Periplaneta americana species is an annoyance to man, causing allergies and damage to clothes and documents. It has the ability to spread pathogens and requires control measures. Control with natural enemies is less aggressive and can currently be applied with less risk than other techniques, such as chemical control, which is the main method used worldwide to control its post-embryonic stages. The potential microbial control of nymphs and adults of this pest has been shown, but little is known about its oothecae. There are isolates of fungal species that can be used to achieve this aim, but they may have innate differences in their virulence and ability to spread. This study aimed to identify fungal isolates JAB 68 and IBCB 35 through genetic sequencing of the ITS1-5.8S-ITS2 region, analyze their ability to synthesize chitinase, and investigate and compare their aggressiveness against P. americana oothecae and their influence on nymph eclosion. Fungal suspensions were inoculated into minimal medium containing glucose (control) as the sole carbon source and 1% colloidal chitin to determine the chitinolytic activity on the 4th, 7th and 10th days and sporulation on the 10th day. To obtain mortality, extrusion and the compiled number of hatched nymphs, oothecae were sprayed with suspensions of the isolates as follows: T1 - no application; T2 - aqueous solution of Tween 80® 0.1% (vehicle suspension for treatments T3 to T8); T3 - 2 × 109 conidia/mL of the JAB 68 isolate; T4 - 2 × 108 con./mL of the JAB 68 isolate; T5 - 2 × 107 con./mL of the JAB 68 isolate; T6 - 2 × 109 con./mL of the IBCB 35 isolate; T7 - 2 × 108 con./mL of the IBCB 35 isolate; T8 - 2 × 107 con./mL of the IBCB 35 isolate. The JAB 68 and IBCB 35 isolates were identified as belonging to the species Metarhizium anisopliae and Beauveria bassiana, respectively. Chitinolytic activity and extrusion were good parameters for evaluating the fungi's action on oothecal control. The most aggressive entomopathogen was M. anisopliae isolate JAB 68, with shorter time for fungus extrusion at a concentration of 2 × 107 con./mL. B. bassiana reduced the number of hatched nymphs at a concentration of 2 × 108 con./mL. Both fungi are capable of infecting and killing P. americana's oothecae and reducing the number of nymphs hatched.

Diversity of Mammomonogamus (Nematoda: Syngamidae) in large African herbivores.

Four species of Mammomonogamus are known from large African herbivores. A recent study demonstrated that a single Mammomonogamus species was shared by both western lowland gorillas (Gorilla gorilla gorilla) and African forest elephants (Loxodonta cyclotis) in Central African Republic, suggesting lower species diversity than previously described in literature. We examined more than 500 fecal samples collected from sympatric African forest elephants, western lowland gorillas, and African forest buffaloes (Syncerus caffer nanus) at four study sites across Central Africa and examined them by coproscopic methods to detect Mammomonogamus eggs, which were found at three of the study sites. Subsequently, sequences of 18S rDNA, 28S rDNA, and cox1 amplified from individual eggs were analyzed. Phylogenetic analyses of both nuclear and mitochondrial DNA revealed two clades: one formed by sequences originating from Gabonese buffaloes and the other comprising gorillas and elephants. The gorilla-elephant clade was further differentiated depending on the locality. We show the existence of at least two distinct species of Mammomonogamus, M. loxodontis in elephants and gorillas and M. nasicola in buffaloes. The available information on Mammomonogamus in African herbivores is reviewed.

Epidemiology, Virulence and Antimicrobial Resistance of Escherichia coli Isolated from Small Brazilian Farms Producers of Raw Milk Fresh Cheese.

This study aimed to identify contamination sources in raw milk and cheese on small farms in Brazil by isolating Escherichia coli at various stages of milk production and cheese manufacturing. The study targeted EAEC, EIEC, ETEC, EPEC, STEC, and ExPEC pathotypes, characterizing isolates for the presence of virulence genes, phylogroups, antimicrobial susceptibility, and phylogenetic relationships using PFGE and MLST. The presence of antimicrobial resistance genes and serogroups was also determined. Three categories of E. coli were identified: pathogenic, commensal, and ceftriaxone-resistant (ESBL) strains. Pathogenic EPEC, STEC, and ExPEC isolates were detected in milk and cheese samples. Most isolates belonged to phylogroups A and B1 and were resistant to antimicrobials such as nalidixic acid, ampicillin, kanamycin, streptomycin, sulfisoxazole, and tetracycline. Genetic analysis revealed that E. coli with identical virulence genes were present at different stages within the same farm. The most frequently identified serogroup was O18, and MLST identified ST131 associated with pathogenic isolates. The study concluded that E. coli was present at multiple points in milk collection and cheese production, with significant phylogroups and high antimicrobial resistance. These findings highlight the public health risk posed by contamination in raw milk and fresh cheese, emphasizing the need to adopt hygienic practices to control these microorganisms.

Epidemiology and Integrative Taxonomy of Helminths of Invasive Wild Boars, Brazil.

Wild boars (Sus scrofa) are a significant invasive species in Brazil. We evaluated the helminth diversity of 96 wild boars in São Paulo state. Helminth infection descriptors were calculated, the species were identified and their 18S, 28S rDNA and internal transcribed spacer (ITS) regions were amplified for phylogenetic analyses. Ascarops strongylina, Strongyloides ransomi, Globocephalus urosubulatus, Oesophagostomum dentatum, Trichuris suis, Metastrongylus salmi, Metastrongylus pudendotecus, Ascaris suum and Stephanurus dentatus and Macracanthorhynchus hirudinaceus were identified. Globocephalus urosubulatus had the highest prevalence and mean abundance, and most animals had mixed infections with three parasite species. There was no association between parasite intensity and prevalence and host sex and body condition index (p > 0.05). Novel DNA sequences were obtained from G. urosubulatus, A. strongylina, and S. dentatus. This is the first study on the helmint diversity of non-captive wild boars in Brazil, and the first report of the occurrence of M. hirudinaceus, G. urosubulatus and S. dentatus in Brazilian wild boars. Non-captive wild boars of São Paulo State did not act as capture hosts for native helminth species but maintained their typical parasites, common to domestic pigs. They may act as parasite dispersers for low-tech subsistence pig farming and for native Tayassuidae.

Integrative taxonomy of Metastrongylus spp. in wild boars from Brazil.

BACKGROUND: Wild boars (Sus scrofa) may cause substantial damage to crops and can spread zoonotic parasites to domestic animals, posing a risk to health and animal production. Metastrongylus spp. can negatively affect the wild boar population, increasing piglet mortality. In addition to that, studies with Metastrongylus genetic characterization are still scarce in Brazil. The present study aims to characterize Metastrongylus spp. from wild boars hunted in the states of São Paulo, Paraná, and Rio Grande do Sul, Brazil, using traditional morphological description and DNA sequences in an integrative taxonomic approach. METHODS: After nematode collection from 58 wild boars, the parasites were morphologically identified and genetically characterized by the amplification of 18S ribosomal DNA (rDNA), 28S rDNA, internal transcribed spacer (ITS) region, and cox-1 mitochondrial DNA (mtDNA). Descriptors of infection were determined and Pearson's Chi-square test was applied to compare the prevalence of infections among the identified parasite species, host age group (juveniles and adults), and sex. The Mann-Whitney U test was performed to compare the mean intensity between the age groups and sex. RESULTS: Metastrongylus salmi, Metastrongylus apri, and Metastrongylus pudendotectus were identified in 77.6% (45/58) of the necropsied wild boars. Metastrongylus salmi was the most prevalent and abundant species (70.7%, 11.1), followed by M. pudendotectus (18.9%, 4.3) and M. apri (17.2%, 2.2). Metastrongylus pudendotectus showed the highest mean intensity and range (25.2, 1-93), followed by M. salmi (15.7, 1-58) and M. apri (12.6, 3-27). We found a significantly higher prevalence of Metastrongylus spp. and M. salmi in adult wild boars, probably associated with a more prolonged time of exposure to intermediate host species. The phylogenetic analysis revealed that ITS2 region and cox-1 mtDNA are the most suitable genetic markers for Metastrongylus species characterization. Genetic variability between M. apri and M. salmi isolates was verified. CONCLUSIONS: We expand the knowledge about the Metastrongylus community in the non-captive wild boar population from Brazil as well as the importance of this exotic species in the maintenance of Metastrongylus spp. in its areas of occurrence. The novel genetic sequences obtained may help further studies to understand the genetic diversity in other nematode populations from Brazil and other countries.

Filarids (Spirurida: Onchocercidae) in wild carnivores and domestic dogs from the Brazilian Atlantic forest.

Onchocercidae nematodes are heteroxenous parasites with worldwide distribution, and some of the species associated to animals may present zoonotic potential. Climatic changes and anthropic influences on the environment may result in vectors' proliferation, facilitating the spillover to humans and/or non-typical animal hosts. The Iguaçu National Park (PARNA Iguaçu), one of the most important Brazilian natural remanescents of Atlantic rainforest, is strongly affected by human activities such as tourism and agriculture. The complexity of this area is especially characterized by the close nexus between the rich wildlife, humans, and domestic animals, especially domestic dogs. Based on this, this research aimed to diagnose the Onchocercidae nematodes in wild carnivores and domestic dogs in the PARNA Iguaçu and the surrounding areas. For this, we collected 162 samples of seven species of wild carnivores and 225 samples of domestic dogs. The presence of microfilariae in the blood samples was diagnosed by the modified Knott's test and molecular screening, and the specific identification was based on sequencing of the myoHC and hsp70 genes. Microfilariae were detected only in ring-tailed coatis, in which we found five species: Mansonella sp. 1, Mansonela sp. 2, Onchocercidade gen. sp. 1, Onchocercidade gen. sp. 2, and Dirofilaria immitis. The morphological analysis supported the molecular findings. The domestic dogs were parasitized by Acanthocheilonema reconditum, representing a new locality record for this species. Phylogenetic analysis showed high genetic similarity among the four undetermined species and Mansonella spp., Brugia spp., and Wuchereria bancrofti. The presence of D. immitis in ring-tailed coatis may be result of spillover from dogs, even though the parasite was not diagnosed in the sampled dogs. The presence of several undetermined Onchocercidae species indicates the necessity of continuous investigations on wild and domestic animals from Neotropical area, especially considering the growing anthropic influence on forest remnants.

Toxoplasma gondii infection in wild boars (Sus scrofa) from the State of São Paulo, Brazil: Serology, molecular characterization, and hunter's perception on toxoplasmosis.

The consumption of wild boar meat, common in many countries, became popular in Brazil after the hunting of these animals was authorized in 2013. The meat of these animals is often consumed by hunters and their social groups, and their offal is occasionally used as supplemental food in the diet of hunting dogs. Given the high frequency of foodborne diseases related to wild boar meat consumption in other countries, including toxoplasmosis, knowledge on these diseases is essential for risk assessment and elaboration of education campaigns for the exposed public. Thus, this study aimed diagnosing, isolating, and genotyping Toxoplasma gondii in hunted wild boars. For that, we obtained samples of serum and tissues (brain, tongue, diaphragm, and heart) from 26 wild boar hunted in three areas in São Paulo State, Brazil, based on convenience sampling strategy. The serum samples were submitted to the indirect immunofluorescence reaction test (IFAT) test while the tissue samples (n = 22) were used to perform a bioassay in mice to isolate the parasite. The isolated samples were genetically characterized by PCR-RFLP with SAG1, 5' and 3' SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico markers. Questionnaires were also formulated and applied to wildlife hunters to assess knowledge about toxoplasmosis. The seroprevalence of T. gondii was 76.9% (20/26), with titers ranging from 16 to 1024. Viable parasites accounted for 4.5% (1/22) of the samples. The ToxoDB #6 genotype of TgJava1 alone was detected. Most interviewed hunters, 84.2% (16/19) consume game meat and a few of them (15.7%; 3/19) prefer undercooked meat. Also, 15.7% (3/19) of the hunters reported supplementing their hunting dogs' diet with wild boar meat and/or offal. As antibodies to T. gondii were detected in 76.9% (20/26) of the studied wild boars, we concluded that infection by T. gondii is frequent in wild boars used for human and animal consumption in the studied areas. Although genotype #6 is commonly found in Brazil in domestic animals, wild animals, and humans, causing everything from mild clinical symptoms to death, this study found, for the first time, the detection of this genotype in wild boars. These results also reaffirm the importance of these animals as a possible source of T. gondii infection for humans and domestic animals.

Purpureocillium lilacinum and Metarhizium marquandii as plant growth-promoting fungi.

BACKGROUND: Especially on commodities crops like soybean, maize, cotton, coffee and others, high yields are reached mainly by the intensive use of pesticides and fertilizers. The biological management of crops is a relatively recent concept, and its application has increased expectations about a more sustainable agriculture. The use of fungi as plant bioinoculants has proven to be a useful alternative in this process, and research is deepening on genera and species with some already known potential. In this context, the present study focused on the analysis of the plant growth promotion potential of Purpureocillium lilacinum, Purpureocillium lavendulum and Metarhizium marquandii aiming its use as bioinoculants in maize, bean and soybean. METHODS: Purpureocillium spp. and M. marquandii strains were isolated from soil samples. They were screened for their ability to solubilize phosphorus (P) and produce indoleacetic acid (IAA) and the most promising strains were tested at greenhouse in maize, bean and soybean plants. Growth promotion parameters including plant height, dry mass and contents of P and nitrogen (N) in the plants and in the rhizospheric soil were assessed. RESULTS: Thirty strains were recovered and characterized as Purpureocillium lilacinum (25), Purpureocillium lavendulum (4) and Metarhizium marquandii (1). From the trial for P solubilization and IAA production, seven strains were selected and inoculated in maize, bean and soybean plants. These strains were able to modify in a different way the evaluated parameters involving plant growth in each crop, and some strains distinctly increased the availability of P and N, for the last, an uncommon occurrence involving these fungi. Moreover, the expected changes identified at the in vitro analysis were not necessarily found in planta. In addition, this study is the first to evaluate the effect of the isolated inoculation of these fungi on the growth promotion of maize, bean and soybean plants.

Occurrence of Methicillin-Resistant Staphylococcus spp. on Brazilian Dairy Farms that Produce Unpasteurized Cheese.

Methicillin-resistant Staphylococcus spp. (MRS) have been identified in several foods, including dairy products. Studies are needed about their occurrence and genetic diversity in the dairy production chain in order to gain a better understanding of their epidemiology and control. This study therefore focuses on isolating and characterizing MRS strains detected in milk used in the production of Brazilian artisanal unpasteurized cheeses. To this end, samples were collected from bovine feces, the hands of milkmen, milking buckets, sieves, unpasteurized milk, whey, water, artisanal unpasteurized cheeses, cheese processing surfaces, cheese handlers, cheese trays, cheese molds, and skimmers at five dairy farms located in the state of São Paulo, Brazil. Colonies suggestive of Staphylococcus spp. were subjected to multiplex PCR to confirm the presence of Staphylococcus aureus and to detect the mecA gene. Sixteen isolates containing mecA gene were detected in samples from unpasteurized cheese and from cheese handlers. None of these isolates were positive to enterotoxin genes. These 16 isolates were subjected to antimicrobial susceptibility tests, which revealed they were resistant to oxacillin, penicillin, and cefepime. Using gene sequencing, the MRS isolates were identified as S. haemolyticus, S. hominis, and S. epidermidis. Furthermore, isolates from cheese handlers' hands and artisanal unpasteurized cheese presented high genetic similarity by random amplified polymorphic DNA (RAPD-PCR) analysis, which indicates cross contamination during cheese production. Thus, we found that people directly involved in milking and cheese processing activities at small dairy farms are a potential source of contamination of MRS strains in unpasteurized milk and cheese, representing a risk to public health.

HoBi-like pestivirus infection in an outbreak of bovine respiratory disease.

HoBi-like is an emerging pestivirus of the family Flaviviridae detected in cattle herds and biological products of bovine origin in many parts of the world, causing disease similar to that observed in bovine viral diarrhea virus (BVDV) infections. In this study we reported the detection of HoBi-like pestivirus in an outbreak of respiratory disease in calves from Brazil, seropositive for viruses of the bovine respiratory disease complex (BRDC). Thus, serum samples and nasal swabs were collected from calves up to one year old, presenting or not clinical signs of respiratory disease. Serum samples were submitted to virus neutralization test (VNT) for BVDV-1, BVDV-2, bovine herpesvirus-1 (BoHV-1), bovine respiratory syncytial virus (BRSV) and bovine parainfluenza-3 (BPIV-3). These samples were also tested for the presence of pestiviruses (BVDV-1, BVDV-2 and HoBi-like) and BoHV-1 by RT-PCR and PCR, respectively. Nasal swabs were analyzed by RT-PCR for pestiviruses, BRSV and BPIV-3. VNT results showed high serological prevalence and a wide range of antibodies titers, for all viruses studied, in calves of different age groups. The RT-PCR amplified the 5'UTR and E2 regions of pestiviruses of four calves, from both nasal swabs and serum samples, which sequencing identified the HoBi-like pestivirus. This is the first detection of HoBi-like in nasal secretions of calves in an outbreak of respiratory disease in Brazil, along with the serological detection of other respiratory viruses. We concluded that HoBi-like pestivirus should be considered as part of the BRDC, as a differential diagnosis, to take correct measures of control and prophylaxis.

Congenital tremor in piglets: Is bovine viral diarrhea virus an etiological cause?

Congenital tremor in pigs involves several etiologies, including pestivirus, which may cause neurological injuries in different animal species. To evaluate whether bovine viral diarrhea virus (BVDV), an important pestivirus, is one of the etiological agents of congenital tremor in swine, gilts and the fetuses were challenged at 45 days of gestation with BVDV-2. Four pregnant gilts were inoculated oronasally, four gilts underwent fetal intrauterine inoculation, and two gilts constituted the control group. Antibody titers were determined by virus neutralization (VN), and viral RNA was detected by RT-PCR. Blood samples were collected from all gilts and piglets born to obtain whole blood and serum for analysis. One third of the neonates were euthanized at three days old, and samples of the encephalon, brain stem and spinal cord were collected for anatomopathological evaluation and viral RNA detection. The piglets that remained alive were clinically evaluated every day, and blood sampling was performed regularly for 35 days. The piglets from gilts in both inoculation treatment groups showed no clinical neurological signs and were born with no viral RNA in their blood and organs. Piglets born from oronasally inoculated gilts did not present antibodies against BVDV-2 at birth, although they were acquired by passive maternal transfer. In contrast, intrauterine-inoculated piglets were born with high antibody titers (80 to 640) against the agent, which remained high until the end of the experimental period. Microscopically, no noticeable changes were observed. Macroscopically, 29.5% of the total piglets euthanized, from both inoculation groups, were born with a low cerebellar:brain ratio. Nevertheless, some piglets had a high cerebellar:brain ratio, indicating the need for standardizing this value. Thus, it was concluded that BVDV is not an etiological agent for congenital swine tremor.

Molecular characterization and potential sources of aqueous humor bacterial contamination during phacoemulsification with intraocular lens implantation in dogs.

Bacterial contamination of the anterior chamber during cataract surgery is one of the main responsible for endophthalmitis postoperative. Phacoemulsification is a less invasive technique for cataract treatment, although it does not exclude the possibility of contamination. In this study, bacterial contaminants of aqueous humor collected pre- and post-phacoemulsification with intraocular lens implantation (IOL) of twenty dogs were identified. As the conjunctival microbiota constitute a significant source of anterior chamber contamination, bacterial isolates from aqueous humor were genetically compared with those present in the conjunctival surface of the patients. Three dogs presented bacterial growth in both aqueous humor and conjunctival surface samples. Bacterial isolates from these samples were grouped according to their genetic profiles by repetitive-element PCR (rep-PCR) and their representatives were identified by 16S rRNA sequencing. Isolates from conjunctival surface were identified as Enterobacter spp., Staphylococcus spp. and S. aureus; and from aqueous humor samples as Enterobacter spp., Pantoea spp., Streptococcus spp. and Staphylococcus spp., respectively in decreasing order of prevalence. According to the rep-PCR analysis, 16.6% of Enterobacter spp. isolates from conjunctival surface were genetically similar to those from aqueous humor. The rest of isolates encountered in aqueous humor were genetically distinct from those of conjunctival surface. The significant genetic diversity of bacterial isolates found in the aqueous humor samples after surgery denoted the possibility of anterior chamber contamination during phacoemulsification by bacteria not only from conjunctival surface but also from different sources related to surgical environment.

Isolation of Saprolegnia aenigmatica oomycetes and protocol for experimental infection of pacu (Piaractus mesopotamicus) / Isolamento do oomiceto Saprolegnia aenigmatica e protocolo de infecção experimental em pacu (Piaractus mesopotamicus)

Successful disease treatment depends on molecular studies under indoor conditions with experimental infection protocols that facilitate understanding the disease and the drug`s efficacy. The internal transcribed spacer (ITS) region was sequenced from three isolates, which were identified as Saprolegnia aenigmatica. Subsequently, healthy fish were immunosuppressed with dexamethasone (1.2 mg kg-1) and descaled to the skin using a sharp scalpel. These individuals were isolated in individual aquariums maintained at 22°C. Individuals in one group were subcutaneously inoculated with 9,000 zoospores (DDZ treatment), a second group was exposed to oomycetes in water with three colonized baits (DDB), a third group was maintained in water without zoospores (DD), and a control group (C) consisted of healthy animals. After 48 and 96 hours, two animals from each group were euthanized for fungal reisolation. The fish from groups DD and C did not show clinical signs, and no oomycetes were isolated. The animals from the DDZ and DDB groups showed cotton-wool-like masses on the skin, and S. aenigmatica was re-isolated. Thus, for infection using zoospores or baits parasitized by S. aenigmatica, an immunosuppressor (dexamethasone) and a sharp scalpel can be used effectively to establish an experimental infection in P. mesopotamicus.

O sucesso do tratamento de uma enfermidade depende do estudo de moléculas em condições de laboratório por meio de protocolos de infecção experimental que viabilizam a compreensão da doença e da eficácia dos fármacos. Pela sequência ITS foram identificadas três cepas de Saprolegnia aenigmatica. Dessa forma, pacus sadios foram submetidos à imunossupressão com dexametasona (1,2 mg kg-1), esfoliados com auxílio de bisturi e distribuídos em aquários a 22ºC. Após este procedimento, um grupo de animais foi inoculado com 9.000 zoósporos/peixe subcutâneo (DEZ), a outro foram adicionadas três iscas colonizadas com o oomiceto na água (DEI), um terceiro grupo foi mantido sem contato com o oomiceto (DE) e um quarto grupo, de animais sadios, representaram o controle (C). Após 48 e 96h deste procedimento, foram eutanaziados animais de cada grupo para reisolamento. Os animais do grupo DE e C não apresentaram sinais clínicos e não foi reisolado o oomiceto. Porém, tanto os animais do grupo DEZ quanto como os animais do grupo DEI apresentaram micélio branco na pele e foi reisolado Saprolegnia aenigmatica. Assim, a infecção com zoósporos ou com iscas colonizadas por S. aenigmatica, com o uso de dexametasona e abrasivo epitelial são formas eficazes de infecção experimental em P. mesopotamicus.

Effects of sugarcane juice addition on the population dynamics of Escherichia coli and the presence of Shiga-toxigenic E. coli during the anaerobic codigestion of dairy cattle manure / Efeito da adição de caldo de cana-de-açúcar na dinâmica da população de Escherichia coli e presença de E. coli shigatoxigênicas durante a codigestão anaeróbia de dejetos bovinos leiteiros

ABSTRACT: The objective of this study was to evaluate the effects of the addition of sugarcane juice on the population dynamics of Escherichia coli and the presence of Shiga-toxigenic E. coli (STEC) during the anaerobic codigestion of dairy cattle manure. For the overall analyses at the end of a hydraulic retention time of 90 days, ten two-liter batch-type biodigesters were divided into two treatment groups: biodigester containing manure and water (MW) and the biodigester containing manure, water and sugarcane juice (MSC). For monitoring the population dynamics and presence of microorganisms, pH, and volatile acidity, tests were carried out every ten days, on 36 smaller-scale batch biodigesters made of one-liter plastic bottles (18 for each treatment). The reductions in E. coli population over time were significant in the MW (60 days) and MSC (20 days) biodigesters. Inactivation of STEC occurred in a shorter period (40 days in MW and <10 days in MSC). Significant differences were obtained between the two treatments, with the pH values being lower, the concentrations of volatile acids (VA) being higher, and the inactivation of E. coli and STEC being faster in the biodigester with sugarcane juice added. The amount of sugarcane juice applied (7%) suggests its suitability for the sanitization of dairy cattle manure for use as a biofertilizer, given the high reduction in the E. coli population and inactivation of STEC.

RESUMO: O estudo teve como objetivo avaliar o efeito da adição de caldo de cana-de-açúcar sobre a dinâmica da população de Escherichia coli e presença de E. coli shigatogixênicas (STEC) no processo de codigestão anaeróbia de dejetos de bovinos leiteiros. Foram utilizados dez biodigestores bateladas divididos em dois tratamentos, dejeto sem caldo de cana-de-açúcar (DSC) e dejeto com caldo (DCC), com tempo de retenção hidráulica (TRH) de 90 dias. Para o monitoramento periódico da dinâmica da população E. coli e presença de E. coli shigatoxigenicas, do pH e da acidez volátil, realizados a cada dez dias, foram abastecidos mais 36 biodigestores bateladas, construídos de garrafas de material plástico de um litro, sendo 18 unidades para cada tratamento. A redução das populações de E. coli no decorrer do tempo foi significativa no DSC (60 dias) e no DCC (20 dias). A inativação de E. coli shigatoxigênicas ocorreu em um período mais curto, 40 dias no DSC e menos de 10 dias no DCC. Foram obtidas diferenças significativas entre os tratamentos para os valores de pH, que foram menores, e as concentrações de ácidos voláteis, que foram maiores, com adição de caldo e contribuíram para a inativação mais rápida da E. coli e STEC. A dose de caldo de cana-de-açúcar utilizada (7%) sugere a adequada sanitização do dejeto bovino leiteiro, tendo em vista a alta redução na população de E. coli e a inativação de STEC.