Immunomodulatory effect of Lacticaseibacillus casei CB054 supplementation in calves vaccinated against infectious bovine rhinotracheitis.

Probiotics are live microorganisms that, confer health benefits to the host when supplemented in adequate amounts. They can promote immunomodulation by inducing phagocyte activity, leukocyte proliferation, antibody production, and cytokine expression. Lactic acid bacteria (BAL) are important probiotic specimens with properties that can improves ruminant nutrition, productivity and immunity. The aim of the present study was to evaluate the immunomodulatory effect of the supplementation with Lacticaseibacillus casei CB054 in calve vaccinated against bovine infectious rhinotracheitis (IBR). Calve were vaccinated with a commercial IBR vaccine, on day 0 and received a booster dose on day 21. L. casei CB054 was orally administered (4 ×109 UFC) for 35 days, while a non-supplemented control group received Phosphate Buffer Saline (PBS). Stimulation of bovine splenocytes with L. casei CB054 markedly enhanced mRNA transcription levels of cytokines IL2, IL4, IL10 and IL17 genes. Calves supplemented with L. casei CB054 showed significantly higher (p < 0.05) specific anti-BoHV-1 IgG levels, higher serum neutralization, as well as higher mRNA transcription for IL2, IL4, IL10 and IL17 genes in Peripheral Blood Mononuclear Cells (PBMCs) comparing with control calves. Supplemented calve had an average weight gain of ∼14 kg more than non-supplemented during the experimental period. These results suggest that L. casei CB054 supplementation increase immunogenicity of a commercial IBR vaccine in cattle and improve weight gain.

Melaleuca alternifolia formulations in the treatment of experimental pythiosis.

Essential oils (EO) are aromatic compounds from the plant secondary metabolism. Melaleuca alternifolia EO is well known for its medicinal properties and promising use as an antimicrobial agent. Pythiosis is a difficult-to-treat and emerging disease caused by the oomycete Pythium insidiosum. This study evaluated a nanoemulsion formulation of M. alternifolia (NEMA) in topical and intralesional application to treat experimental pythiosis. Dermal toxicity tests were performed on M. alternifolia EO in Wistar rats. Pythiosis was reproduced in rabbits (n = 9) that were divided into groups: group 1 (control), cutaneous lesions with daily topical application of a non-ionizable gel-based formulation and intralesional application of sterile distilled water every 48 h; group 2 (topical formulation), lesions treated daily with topical application of a non-ionizable gel-based formulation containing 5 mg/ml of NEMA; and group 3 (intralesional formulation), lesions treated with NEMA at 5 mg/ml in aqueous solution applied intralesionally/48 h. The animals were treated for 45 days, and the subcutaneous lesion areas were measured every 5 days. M. alternifolia EO showed no dermal toxicity. The lesion areas treated with intralesional NEMA reduced at the end of treatment, differing from groups 1 and 2 (P < 0.05). In the topically treated group, the lesion areas did not differ from the control group, although the number of hyphae significantly reduced (P < 0.05). Under the experimental conditions of this study, the NEMA formulations presented a favorable safety profile. However, further studies are required to evaluate if this safety applies to higher concentrations of NEMA and to validate its use in clinical pythiosis.

Immunomodulatory effect of short-term supplementation with Bacillus toyonensis BCT-7112T and Saccharomyces boulardii CNCM I-745 in sheep vaccinated with Clostridium chauvoei.

The bacterium Clostridium chauvoei is the causative agent of blackleg in livestock, and vaccination is the most effective means of prevention. The aim of this study was to assess the effect of short-term supplementation with Bacillus toyonensis and Saccharomyces boulardii on the immune response to a C. chauvoei vaccine in sheep. Sheep were vaccinated subcutaneously on day 0 and received a booster dose on day 21, with 2 mL of a commercial vaccine formulated with inactivated C. chauvoei bacterin adsorbed on aluminum hydroxide. Probiotics were orally administered B. toyonensis (3 × 108 cfu) and S. boulardii (3 × 108 cfu) over five days prior to the first and second doses of the vaccine. Sheep supplemented with B. toyonensis and S. boulardii showed significantly higher specific IgG, IgG1, and IgG2 titers (P<0.05), with approximately 24- and 14-fold increases in total IgG levels, respectively, than the nonsupplemented group. Peripheral blood mononuclear cells from the supplemented group had increased mRNA transcription levels of the IFN-γ, IL2, and Bcl6 genes. These results demonstrate an adjuvant effect of short-term supplementation with B. toyonensis and S. boulardii on the immune response against the C. chauvoei vaccine in sheep.

Recombinant bovine IL17A acts as an adjuvant for bovine herpesvirus vaccine.

The Bovine herpes virus type 5 glycoprotein D (gD) is essential for viral penetration into host permissive cells. The Herpes virus gD glycoprotein has been used for bovine immunization, being efficient in reduction of viral replication, shedding and clinical signs, however sterilizing immunity is still not achieved. Recombinant subunit vaccines are, in general, poorly immunogenic requiring additional adjuvant components. Interleukin 17A (IL17A) is a pro-inflammatory cytokine produced by T helper 17 cells that mediate mucosal immunity. IL17 production during vaccine-induced immunity is a requirement for mucosal protection to several agents. In this study, we investigated the potential of a recombinant IL17A to act as an adjuvant for a recombinant BoHV-5 glycoprotein D vaccine in cattle. Three cattle groups were divided as: group 1) rgD5 + alumen + rIL-17A; 2) rgD5 + alumen; and 3) PBS + alumen. The cattle (3 per group) received two doses of their respective vaccines at an interval of 21 days. The group that received rIL17 in its vaccine formulation at the 7th day after the prime immunization had significant higher levels of specific rgD-IgG than the alumen group. Addition of rIL17 also led to a significant fold increase in specific anti-rgD IgG and neutralizing antibodies to the virus, respectively, when compared with the alumen group. Cells stimulated with rIL17A responded with IL17 transcription, as well IL2, IL4, IL10, IL15, Bcl6 and CXCR5. Our findings suggest that the rIL17A has adjuvant potential for use in vaccines against BoHV-5 as well as potentially other pathogens of cattle.

Biogenic silver nanoparticles in the treatment of experimental pythiosis Bio-AgNP in pythiosis therapy.

Pythiosis is a rapidly progressing disease that can be lethal to affected individuals due to resistance to available therapeutic protocols. The disease affects mammals, with the largest number of reports in horses and humans. The present study investigated the activity of biogenic silver nanoparticles (bioAgNP) in the treatment of experimental pythiosis. The disease was reproduced in nine female 90-day-old New Zealand rabbits. Animals were divided into three groups: group1 (control, n = 3) daily and topically treated with a nonionized gel-based formulation and 1 ml of sterile distilled water intralesion administered every 48 hours; group 2 (n = 3), daily and topically treated with gel-based formulation containing 1 µg/ml bio-AgNP; group 3 (n = 3), treated with 1 ml bio-AgNP in 1 µg/ml aqueous solution intralesion administered every 48 hours. Animals were treated for 45 days, and the area of subcutaneous lesions was measured every 5 days. Results showed that groups 2 and 3 differed from control group (P < .05) in the lesion area, as well as the amount of hyphae within the lesions. It was observed that lesions of treated animals (groups 2 and 3) did not differ from each other, showing that the application route did not influence the regression of lesions. However, it was observed that one animal from group 2 reached clinical cure at 35 days of treatment. This research is pioneer in the application of nanocomposites for the treatment of experimental pythiosis and showed that bio-AgNP can be powerful allies of integrative medicine and can be included in pythiosis therapeutic protocols.

In vitro anti-Pythium insidiosum activity of biogenic silver nanoparticles.

Pythium insidiosum belongs to the phylum Oomycota. It is capable of infecting mammals causing a serious condition called pythiosis, which affects mainly horses in Brazil and humans in Thailand. The objective of the present study was to verify the in vitro anti-P. insidiosum activity of a biogenic silver nanoparticle (bio-AgNP) formulation. The in vitro assays were evaluated on P. insidiosum isolates (n = 38) following the M38-A2 protocol. Damage to the P. insidiosum hyphae ultrastructure was verified by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Bio-AgNP inhibition concentrations on P. insidiosum isolates ranged from 0.06 to 0.47 µg/ml. It was observed through SEM that P. insidiosum hyphae treated showed surface roughness, as well as cell walls with multiple retraction areas, loss of continuity, and rupture in some areas. The TEM of treated hyphae did not differentiate organelle structures; also, the cellular wall was rarefied, showing wrinkled and partly ruptured borders. The bio-AgNP evaluated has excellent in vitro anti-P. insidiosum activity. However, further studies on its in vivo action are necessary as so to determine the possibility of its use in the treatment of the disease in affected hosts.

Paracoccidioides spp. in Soil from the Pampa Biome in Southern Brazil.

High rates of paracoccidioidomycosis disease and infection have been recently reported in southern Brazil. However, the knowledgment about the ecoepidemiology of Paracoccidioides spp. in the region is limited, therefore, this study aimed to evaluate the presence of Paracoccidioides spp. DNA in soil samples from a rural area in Southern Rio Grande do Sul, Brazil. Thirty pools of soil samples from Bagé, RS (31º19'53"S 54º06'25"W) were submitted to physicochemical analysis, and to fungal DNA extraction by Norgen Biotek® Kit (Thorold, Canada), followed by Nested PCR technique with ITS4 and ITS5 as external primers, and PBITS-E and PBITS-T as internal primers. DNA amplification products of about 424 bp compatible with Paracoccidioides spp. were detected in eight (26.7%) of the 30 pools of samples, being three were sequenced and identified as P. brasiliensis. Positive soils were characterized by high levels of humidity, organic matter, basic saturation, and pH. This study shows for the first time the presence of Paracoccidioides spp. DNA at soils from the Brazilian Pampa Biome, proving that people living in those areas are exposed to the main agent of paracoccidioidomycosis.

Risk factors and infection due to Cryptosporidium spp. in dogs and cats in southern Rio Grande do Sul.

This study investigated the frequency of oocysts of Cryptosporidium spp. in feces from dogs and cats in five municipalities in the southern region of the state of Rio Grande do Sul. The risk factors associated with infection were also investigated. Feces samples from 110 dogs and 18 cats were stained using the auramine method. At the time of feces sampling, a questionnaire with semi-open-ended questions was applied to the animal guardians and all data obtained underwent statistical analysis. The real frequency of oocysts of Cryptosporidium spp. was 24.63% (27 dogs and two cats). Only four samples of dog feces were diarrheic and no presence of oocysts was observed in any of them. Variables that represented risk factors for infection were: homemade food, untreated water, circulation of animals on grassy terrain and living in the same environment as other animals (cattle). The results made it possible to inferring that within the population studied, the frequency of parasitism due to Cryptosporidium spp. in dogs was relevant and emphasize the asymptomatic nature of this infection. The adopting control measures are highlighted, particularly in relation to variables that represent risk factors for this infection.

Risk factors and infection due to Cryptosporidium spp. in dogs and cats in southern Rio Grande do Sul / Fatores de risco e infecção por Cryptosporidium spp. em cães e gatos no sul do Rio Grande do Sul

Abstract This study investigated the frequency of oocysts of Cryptosporidium spp. in feces from dogs and cats in five municipalities in the southern region of the state of Rio Grande do Sul. The risk factors associated with infection were also investigated. Feces samples from 110 dogs and 18 cats were stained using the auramine method. At the time of feces sampling, a questionnaire with semi-open-ended questions was applied to the animal guardians and all data obtained underwent statistical analysis. The real frequency of oocysts of Cryptosporidium spp. was 24.63% (27 dogs and two cats). Only four samples of dog feces were diarrheic and no presence of oocysts was observed in any of them. Variables that represented risk factors for infection were: homemade food, untreated water, circulation of animals on grassy terrain and living in the same environment as other animals (cattle). The results made it possible to inferring that within the population studied, the frequency of parasitism due to Cryptosporidium spp. in dogs was relevant and emphasize the asymptomatic nature of this infection. The adopting control measures are highlighted, particularly in relation to variables that represent risk factors for this infection.

Resumo Este estudo verificou a frequência de oocistos de Cryptosporidium spp. em fezes de cães e gatos em cinco municípios da região sul do Rio Grande do Sul e fatores de risco associados à infecção. Amostras de fezes de 110 cães e 18 gatos foram coradas pelo método de auramina. No momento da coleta de fezes aplicou-se um questionário aos tutores dos animais com questões semiabertas e os dados foram submetidos à análise estatística. A frequência verdadeira de oocistos de Cryptosporidium spp. foi de 24,63% (27 cães e 2 gatos). Apenas quatro amostras de fezes caninas eram diarreicas e todas sem oocistos. As variáveis que representaram fatores de risco para a infecção foram: alimentos de preparo caseiro, água não tratada, circulação dos animais em terreno gramíneo e convivência com outros animais, principalmente bovinos. Os resultados sugerem que a frequência de cães parasitados por Cryptosporidium spp. é relevante, reforçando o caráter assintomático da infeção. Destaca-se a importância da adoção de medidas de controle, particularmente das variáveis que representaram fatores de risco à infecção.

Influence of temperature on in vitro zoosporogenesis of Pythium insidiosum.

This study verified the influence of different temperatures on P. insidiosum in vitro zoosporogenesis. P. insidiosum isolates (n = 26) were submitted to zoosporogenesis and incubated at 5°C, 15°C, 20°C and 37°C (1st stage). Grass fragments were evaluated under optical microscopy at 4, 8, and 24 hours of incubation. Afterward, all isolates were incubated at 37°C and assessed at the same periods of time (2nd stage). The development of hyphae, presence of vesicles, zoosporangia and zoospores were checked. Only the presence of short hyphae was observed at 5°C. At 15°C, the hyphae were either under development or elongated and two isolates produced zoospores. When the isolates were submitted to 20°C for 4 hours, the presence of long and mycelial hyphae, vesicles, zoosporangia and zoospores was observed, which also happened at the other periods evaluated. In the second stage, the isolates which were initially at 5°C and 15°C evidenced long developing hyphae with the presence of vesicles, zoosporangia, and zoospores within 4 hours of incubation, and these characteristics were kept at the other evaluated periods. The isolates kept at 37°C showed evident zoosporogenesis in the first 4 hours of evaluation. It was concluded that temperatures of 20°C and 37°C support P. insidiosum zoosporogenesis process. On the other hand, 5°C and 15°C temperatures do not kill the microorganism.

Development of a weight-estimation model to use in pregnant criollo-type mares / Modelo para estimativa de peso em éguas gestantes mestiças crioulas

ABSTRACT: The aims of this study were: 1) to compare the tape weight and associated weight-estimation formula to evaluate weight gain in pregnant mares, and 2) to develop a mathematical model to estimate the weight of pregnant mares using body measurements. Thirty-four criollo-type mares were evaluated every two weeks during the middle and late pregnancy. The mares were weighed on a livestock scale, and we estimated body weight using tape weights and an associated body-weight estimation formula. Also, heart-girth circumference (heartgirth) and abdominal circumference were measured; the latter at the 12th intercostal space (12th ICS) and 18th rib (18th Rib), to use in a mathematical model to estimate the weight of pregnant mares. Observations were divided into three periods of pregnancy: 5th to 7 h month, 7th to 9 h month, and 9th to 11th month. Mares in late pregnancy showed an increase in actual weight and an increase in 12th ICS and 18th Rib measurements. Tape weight and body-weight estimation formula underestimated the weight of pregnant mares. However, the regression model using heart-girth circumference, 12th ICS, and 18th Rib measurements showed high correlation (r2 = 0.87, P<0.001) with actual weight. Finally, the alternative methods usually used in horses are not accurate to estimate body weight in pregnant mares. In conclusion, the regression model Y=-540.143 + (heartgirth x 3.068) + (12th ICS x 1.278) + (18th Rib x 0.944) can be used to estimate body weight in pregnant mares from the 5th to 11th months of pregnancy.

RESUMO: Este estudo teve como objetivos: 1) comparar a utilização dos métodos alternativos fita de pesagem e peso metabólico em éguas gestantes e; 2) desenvolver um modelo matemático para estimar o peso de éguas gestantes, baseado em medidas corporais. Trinta e quatro éguas foram avaliadas quinzenalmente durante o terço médio e final da gestação. Foi realizada a pesagem em balança comercial; a mensuração com a fita de peso para equinos; o cálculo do peso metabólico; as medidas de perímetro torácico (Ptorac); a mensuração abdominal no décimo segundo espaço intercostal (12EI) e na décima oitava costela (18Cost). As observações foram divididas em três períodos gestacionais: Período de 5 a 7 meses, Período de 7 a 9 meses e Período de 9 a 11 meses. As éguas demonstraram incremento no peso na balança e na mensuração abdominal no 12EI e 18Cost no terço final da gestação. A utilização da fita de pesagem e o cálculo de peso metabólico subestimaram o peso na balança em éguas gestantes. O modelo de regressão utilizando o Ptorac, 12EI e 18Cost apresentou alta correção (r2=0,87; P<0,001) com o peso na balança. Observou-se que os métodos alternativos de pesagem, usualmente utilizados, não apresentaram a mesma eficiência em éguas prenhes. Pode-se concluir que o modelo de regressão Y=-540,143+(Ptorac X 3,068) + (12EI X 1,278) + (18Cost X 0,944) pode ser utilizado para mensurar o peso em éguas gestantes a partir do 5º mês de gestação.

In Vitro Activity of Melaleuca alternifolia (Tea Tree) in Its Free Oil and Nanoemulsion Formulations Against Pythium insidiosum.

Pythium insidiosum is an important aquatic oomycete which can cause pythiosis in both animals and humans. This microorganism shows low susceptibility to antifungal drugs available. This study analyzed the in vitro antimicrobial activity of Melaleuca alternifolia in its free oil (FO) and nanoemulsion (NE) formulations against Brazilian P. insidiosum isolates. The antimicrobial activity evaluation was performed by the broth microdilution method according to CSLI M38-A2 document adapted to phytopharmaceuticals. Twenty-six P. insidiosum isolates were evaluated, and the minimum inhibitory concentration was determined at 100 % growth inhibition. Melaleuca alternifolia essential oil or FO was obtained commercially. The NE containing 1 % M. alternifolia essential oil was prepared by the spontaneous emulsification method. All P. insidiosum isolates evaluated showed minimum inhibitory concentrations (MIC) ranging from 531.5 to 2125 µg/mL for the FO formulation; MIC50 and MIC90 showed values between 1062.5 and 2125 µg/mL, respectively. When the NE formulation was evaluated, MIC values ranged from 132.7 to 2125 µg/mL and both MIC50 and MIC90 corresponded to 1062.5 µg/mL. FO and NE formulations of M. alternifolia showed antimicrobial activity against P. insidiosum. This study demonstrated that M. alternifolia oil can be an additional therapy in pythiosis treatment; however, further studies are needed to evaluate the applicability of the plant essential oils in the treatment of clinical pythiosis.