Antimicrobial and Synergistic Activity of 2,2′,4-Trihydroxybenzophenone Against Bacterial Pathogens of Poultry

In poultry farming, the spread of bacterial pathogens results in disease outbreaks causing significant economic losses to this industry. Many of these pathogenic bacteria are zoonotic and have a substantial impact on public health. Antimicrobials are essential for the prevention and treatment of these bacterial infections. However, the indiscriminate use of these agents provides favorable conditions for selection, propagation and persistence of bacteria and development of antimicrobial resistance. We developed a new antimicrobial candidate that could be used alone or in synergy with research protocols for therapeutic, prophylactic and growth promoter uses in the poultry industry. The present study aimed at evaluating the antimicrobial activity of the synthetic compound 2,2′,4-trihydroxybenzophenone against pathogenic bacteria that cause important diseases in poultry and public health. We tested the hemolytic effect of this compound, studied its synergistic effect with conventional antimicrobials and analyzed the site of action on the bacteria. The results of our study showed antimicrobial activity of benzophenone against Gram-positive and Gram-negative bacteria with a similar effect in ATCC (American type culture collection) and field isolates. This compound was non-hemolytic. 2,2′,4-trihydroxybenzophenone acted on the bacterial cell wall. We identified the synergistic effect between 2,2′,4-trihydroxybenzophenone and bacitracin, this effect indicate that antimicrobial synergism may be useful for the treatment of necrotic enteritis in poultry. This compound may also be used as a growth promoter by reducing the dose of bacitracin and thus decreasing the pressure of bacterial resistance in poultry which would circumvent the development of cross-resistance in humans

Cattle immunization with two recombinant peptides derived from peptide SBm7462®. Lymph node response and gut histological changes of tick Rhipicephalus (Boophilus) microplus (Canestrini, 1887).

Devido à sua capacidade de transmitir diversos agentes infecciosos, os carrapatos são importantes para a saúde pública e a produção animal. Dentre estes se destaca o carrapato Rhipicephalus microplus, responsável por perdas econômicas nos países das regiões tropicais e subtropicais. Entre as medidas de controle deste ectoparasito, o controle imunológico se tornou uma alternativa viável, que além de não gerar populações de carrapatos resistentes oferece inocuidade aos produtos de origem animal. A vacina sintética SBm7462®, cuja eficácia já foi demonstrada em bovinos, contém três epítopos imunogênicos (4822, 4824 e 4823) derivados da proteína Bm86. Análises genéticas mostraram que o fragmento correspondente ao peptídeo SBm7462® está conservado nas populações do R. microplus de diferentes regiões da América do Sul. Para avaliar os eventos de imunidade celular em bezerros e as alterações histológicas em fêmeas do R. microplus. Previamente foram desenhados e construídos dois genes que se expressaram na levedura Pichia pastoris cepa Km71. O gene rBmseq1 foi desenhado para produzir os epítopos imunogênicos em tandem e o gene rBmseq4 para produzir a sequência sem repetições. Os peptídeos expressos se recuperaram do meio extracelular e se caracterizaram por SDS-PAGE e Western blotting. Esses peptídeos recombinantes foram inoculados três vezes com intervalos de 30 dias em 4 bezerros Bos taurus taurus, na dose de 2 mg mais 1,5 mg de saponina. Em seguida, os linfonodos foram coletados e processados para análise histológica. Os cortes foram submetidos à coloração por Hematoxilina-eosina (H&E) e imunoistoquímica por peroxidase - anti - peroxidase (PAP), demonstrando que foi induzida uma resposta T- dependente, com desenvolvimento da imunidade nos animais imunizados. Além disso, se observaram claras estruturas que conferem afinidade e imunidade nos animais inoculados com o rBmseq4. Aos 21 dias após a última imunização, foi feito o desafio com 4500 larvas de R. microplus por animal. Com o começo da caída das fêmeas aos 21 dias, foram escolhidas, aleatoriamente, teleóginas de cada grupo. As fêmeas do R. microplus, alimentadas em bovinos do grupo rBmseq1 apresentaram conservação da integridade do epitélio intestinal, similar ao grupo controle. No entanto, alças intestinais de teleóginas, alimentadas em bovinos do grupo rBmseq4 mostraram desnudamento da membrana basal e erosão celular. Demonstrou-se que o peptídeo rBmseq4 possui maior capacidade de gerar uma resposta imune com habilidade para induzir reações adversas nos carrapatos R. microplus.Ticks are important to public health and animal production because of their ability to transmit various infectious agents. Among ticks, Rhipicephalus microplus is responsible for economic losses in tropical and subtropical countries. Among the ways to control this ectoparasite, the immune option is viable, which doesn t generate resistant tick populations and offers safety to the animal products. The synthetic vaccine SBm7462® contains three immunogenic epitopes (4822, 4824 and 4823) derived from the Bm86 protein. Genetic analysis showed that the fragement corresponding to the SBm7462® is conserved in the population of R. microplus from different regions on South America. To evaluate the events of cellular immunity in calves and histology changes in female R. microplus. Previously were designed and constructed two genes expressed in Pichia pastoris Km71 strain. The rBmseq1 gene was designed to express immunogenic epitopes in tandem and rBmseq4 gene designed expressed this without repetitions. The recombinant peptides were recovered from the extracellular medium and were characterized by SDS-PAGE and Western blotting. These peptides were inoculated three times at intervals of thirty days in five Bos taurus taurus calves at doses of 2 mg plus 1.5 mg saponin. Afterwards, the lymph nodes were collected and processed histologically. Histological sections were stained by (H&E) and immunohistochemical staining for peroxidase-anti-peroxidase (PAP). It was shown that they developed a T-dependent response with immunity induction in animals immunized. Hence, showed clear structures whit confers immunity and affinity in animals immunized whit rBmseq4. However, 21 days after the last immunization, the challenge was made with R. microplus 4500 larvae per animal. At 21 days with the beginning of fallen females, these were randomly selected from each group. R. microplus females fed on animal s rBmseq1 group showed integrity of the intestinal epithelium, similar to control group. However, guts of ticks fed on animal s rBmseq4 group showed denudation and erosion of basal membrane cell. It demonstrates the peptide rBmseq4 peptide has a great ability to generate immune responses which induce adverse reactions in R. microplus

Toxoplasma gondii in small ruminants in northeastern areas of Colombia: Seroprevalence and risk factors

Sheep and goats are susceptible to infections with Toxoplasma gondii and could play an important role in the transmission of the zoonotic parasite to human. We conducted a cross sectional study to estimate the seroprevalence and to assess the risk factors for T. gondii seropositivity in small ruminants under traditional husbandry systems. This study was carried out from November 2015 to April 2016 in randomly selected small ruminants (n = 1038) from 48 farms located in Colombia, in the departments of northern Cesar in the north and La Guajira in the south. An indirect ELISA was used to detect IgG antibodies to T. gondii in the animals. A standardized questionnaire was used to obtain information on putative risk factors. We conducted the association analyses by using univariable and multivariate logistic regression and report odds ratios (OR) with 95% confidence interval (C.I). The overall seroprevalence in small ruminants was 23.5% (C.I: 21–26.2%). Sheep showed a higher seroprevalence (25.1% C.I: 22.4–28.6%) than goats (18.4% C.I: 22.4–28.6%). The association analysis recognized as risk factors for T. gondii seropositivity farming pigs in addition to small ruminants (OR = 1.96 C.I: 1.414–2.743), the inexistence of manure heap (OR = 2.254 C.I: 1.480–3.433) and drinking water from locally aqueducts (OR = 1.489 C.I: 1.006–2.204). The results of the study confirmed that exposure to T. gondii is common in sheep and goats in dry Caribbean regions of Colombia

Cattle seroprevalence and risk factors associated with bovine viral diarrhea in the northeastern of Colombia

Bovine viral diarrhea (BVD) is one of the most common and economically significant viral infections in cattle. Different risk factors have been associated with viral exposure and transmission, generating endemic regions with different biotype circulations. To find the BVD status in the northeastern region of Colombia, this study aimed to determine the seroprevalence and risk factors associated with the disease in non-vaccinated farms. For this purpose, a two-time point sampling strategy was developed, obtaining serum from 1157 animals housed in 25 farms distributed in 46 townships, thus for antibody detection against nonstructural protein 3 (NS3, p80) of bovine viral diarrhea virus (BVDV) using an indirect ELISA test. For the first time in Colombia, the presence of serological persistent cattle was evaluated in 1047 animals 3 weeks later of first sampling. The information on a standardized questionnaire with closed and dichotomic answers was used to calculate the putative risk factors. The association analysis by univariate and multivariate logistic regression reported odds ratios (OR) with a 95% of confidence interval (C.I). The overall seroprevalence was 65% (95% CI 62.5–72.9%), with variations according to age ranges. The prevalence of persistent serological positive corresponded to 37.7% (95% CI 34.2–41.5). The risk factors found by multivariate analyses were the lease of pastures (OR = 2.071 CI 1.485–3.690), the use of the same needle (OR = 2.249 CI 1.354–3.736), the molasses supplementation (OR = 2.742 CI 1.156–5.807), and the native Creole breed (OR = 1.895 CI 1.416–2.804). The results of this study confirmed the endemism and higher common exposure to BVDV, as well as the presence of serological persistent cattle in Valledupar, Colombia.Incluye referencias bibliográfica

Genetic diversity and population structure of Rhipicephalus sanguineus sensu lato across diferent regions of Colombia

Background: There has been a long-standing debate over the taxonomic status of Rhipicephalus sanguineus sensu lato. Different studies worldwide have reported the occurrence of different well-defined lineages, in addition to Rhipicephalus sanguineus sensu stricto. To date, there are very few studies examining the diverse aspects of this tick in Colombia. We assessed the population structure and genetic diversity of R. sanguineus s.l. in eight departmental regions across Colombia. Methods: A total of 170 ticks were collected from dogs in different departments of Colombia. All specimens were morphologically compatible with R. sanguineus s.l. and subjected to genetic analysis. DNA sequences were obtained for the 12S rDNA, cytochrome oxidase I (COI) and internal transcribed spacer 2 (ITS2) markers. A concatenated set of all mitochondrial markers was also constructed. Next, maximum likelihood phylogenetic trees were constructed using the sequences generated herein and sequences available in GenBank. Finally, we assessed different summary statistics and analysed population structure and divergence with Fst and Dxy and demographic changes with Tajima's D and Fu and Li’s statistical tests. Results: Analysis of the 12S rDNA and COI revealed that all R. sanguineus s.l. specimens collected across different regions of Colombia clustered within the tropical lineage. Micro-geographical analyses showed that the tick population from Amazonas formed a distinct cluster separated from the other sequences, with moderate Fst and Dxy values. However, no signs of a robust population structure were found within the country. The results of Fu’s FS tests, together with the haplotype networks and diversity values, signal a possible population expansion of this tick species in Colombia. Conclusions: Evidence provided herein supports the tropical lineage as the main circulating lineage in Colombia, exhibiting a general lack of genetic structure except for the Amazonas regionIncluye referencias bibliográfica

Seroprevalence and risk factors for Leptospira spp. in small ruminants of semi-arid zone in northeastern Colombia

Leptospirosis in small ruminants is an economically important disease that causes important losses by pathologies of the reproductive tract. The present study aimed to estimate the seroprevalence and risk factors associated with Leptospira spp. in small ruminants under traditional husbandry systems. A cross-sectional epidemiological study was carried out in a sample of 1039 animals which included 793 sheep and 245 goats from 49 farms of 10 municipalities of northeastern areas of Colombia. The MAT test using a panel of 13 laboratory serovars was performed to diagnose the Leptospira spp. antibodies in the serum samples of animals. A structured questionnaire was flled in each farm to obtain information on putative risk factors, and its association with Leptospira spp. infection was detected by univariate and multivariate logistic regression. The general seroprevalence showed a rate of 13.9%, the 79.6% of farms showed at least one positive animal, the 14.2% and 0.4% of positive animals showed titers≥1:100 and 1:200, respectively. The seroprevalence in sheep and goats showed a rate of 13.1% and 16.2%, respectively. The most common serovars in positive animals were Autumnalis (3.6%), Tarassovi (3.4%), Bratislava (2.9%), Mini (2.9%), Bataviae (1.4%), Canicola (0.3%), and Grippotyphosa (0.3%). The univariate and multivariate analysis showed as risk factors the absence of housing system (OR=3.412 CI: 1.347–8.152), the absence of quarantine measures to new animals (OR=1.371 CI: 0.943–1.978), and the protective factor identifed was the use of Toggenburg breed (OR=0.571 CI: 0.249–1.074). This exploratory study showed the Leptospira spp. positive small ruminants located in the northeastern areas of Colombia with titer associated with chronic and subclinical infection.Incluye referencias bibliográfica

Immunisation with Neospora caninum subunits rsNcSAG4 and rsNcGRA1 (NcSAG4 and NcGRA1 epitopes construct) in BALB/c mice: the profile of the immune response and controlling the vertical transmission

Neospora caninum is an apicomplexan protozoan that causes neosporosis, which has a high economic impact on cattle herds with no available vaccine. During infection, the secretion of dense granules and the expression of surface antigens play an important role in hosting immunomodulation. However, some epitopes of those antigens are immunogenic, and using these fractions could improve the subunit antigens in vaccine design. This study evaluates the recombinant peptides rsNcGRA1 and rsNcSAG4 derived from NcGRA1 and NcSAG4 native antigens as vaccine candidates produced by a fermentative process in the yeast culture system of Komagataella phaffii strain Km71, confirmed by colony PCR, SDS-PAGE, and western blotting. The assay was conducted in BALB/c mice using the peptides at low (25 μg) and standard (50 μg) dosages in monovalent and combined administrations at three time points with saponin as an adjuvant assessing the immunogenicity by antibodies response and cytokine production. We challenge the females after pregnancy confirmation using 2 × 105 NC-1 tachyzoites previously propagated in Vero cells. We assessed the chronic infection in dams and vertical transmission in the offspring by PCR and histopathology. Mice, especially those immunised with combined peptides and monovalent rsNcGRA1 at a standard dose, controlling the chronic infection in dams with the absence of clinical manifestations, showed an immune response with induction of IgG1, a proper balance between Th1/Th2 cytokines and reduced vertical transmission in the pups. In contrast, dams inoculated with a placebo vaccine showed clinical signs, low-scored brain lesions, augmented chronic infection with 80% positivity, 31% mortality in pups, and 81% vertical transmission. These findings indicate that rsNcGRA1 peptides in monovalent and combined with rsNCSAG4 at standard dose are potential vaccine candidates and improve the protective immune response against neosporosis in miceIncluye referencias bibliográfica

Leishmania infantum ecto-nucleoside triphosphate diphosphohydrolase-2 is an apyrase involved in macrophage infection and expressed in infected dogs

Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum) is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (∼70 kDa and ∼45 kDa) have been found in the L. infantum genome. Here, we studied the ∼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection. We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP  =  UDP> ADP> UTP  =  ATP) in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis. In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in naturally infected dogs. Additionally, the blockade of NTPDases led to lower levels of in vitro adhesion and infection, suggesting that these proteins are possible targets for rational drug design

Immunohistochemistry using anti-rLicNTPDase-2 in the lymph nodes of naturally infected dogs.

<p>(A) Lymph nodes from 48 <i>Leishmania</i>-positive dogs were evaluated by immunohistochemistry (IHC) using anti-rLicNTPDase-2. The results of the IHC are compared with ELISA data of the same samples using a Biomanguinhos Kit. (B) An example of IHC result using polyclonal anti-rLicNTPDase-2. (C) The zoom of section C is from image B.</p

Phylogenetic tree using representatives of the ENTPDases from mammals and Trypanosomatids (<i>Leishmania</i> and <i>T. cruzi</i>).

<p>ENTPDase sequences were aligned by the CLC workbench program and used to construct the phylogenetic tree using the Neighbor Joining method with bootstrap analysis (number in the branches). <i>Mus musculus</i> (Mm); <i>Homo sapiens</i> (Hs). Trypanosomatids have two ENTPDases with exception of <i>T. cruzi</i>, which has only one ENTPDase in databank. Trypanosomatid ENTPDases are more similar to mammalian ENTPDases isoforms 5 and 6 and are grouped at the upper branch of the tree.</p