Glanders Diagnosis in an Asymptomatic Mare from Brazil: Insights from Serology, Microbiological Culture, Mass Spectrometry, and Genome Sequencing.

ABSTRACT - This manuscript elucidates the occurrence of glanders in an asymptomatic mare from Brazil presenting positive Burkholderia mallei antibody titers. The diagnosis was established through a multi-pronged approach encompassing microbiological culture, mass spectrometry, and genome sequencing. The outbreak occurred in 2019 in Tatuí, São Paulo, Brazil, and the infected mare, despite displaying no clinical symptoms, had multiple miliary lesions in the liver, as well as intense catarrhal discharge in the trachea. Samples were collected from various organs and subjected to bacterial isolation, molecular detection, and identification. The strain was identified as B. mallei using PCR and confirmed by MALDI-TOF mass spectrometry. Whole-genome sequencing revealed a genome size of 5.51 Mb with a GC content of 65.8%, 5871 genes (including 4 rRNA and 53 tRNA genes), and 5583 coding DNA sequences (CDSs). Additionally, 227 predicted pseudogenes were detected. In silico analysis of different genomic loci that allow for differentiation with Burkholderia pseudomallei confirmed the identity of the isolate as B. mallei, in addition to the characteristic genome size. The BAC 86/19 strain was identified as lineage 3, sublineage 2, which includes other strains from Brazil, India, and Iran. The genome sequencing of this strain provides valuable information that can be used to better understand the pathogen and its epidemiology, as well as to develop diagnostic tools for glanders

Situação da resistência de helmintos gastrintestinais de ovinos aos principais grupos de anti-helmínticos no estado de São Paulo.

O controle da verminose por vermífugos é cada vez menos eficiente em função da resistência que os nematóides têm desenvolvido a anti-helmínticos. O objetivo do trabalho foi verificar a situação atual da eficácia anti-helmíntica em propriedades situadas em várias regiões do Estado de São Paulo. O trabalho foi feito em 19 propriedades, situadas nas seguintes regiões (municípios): Noroeste (Valentim Gentil, Votuporanga), Extremo Oeste (Araçatuba, Gabriel Monteiro, Guararapes, Itapura), Centro Norte (Monte Aprazível, Nova Granada), Centro Leste (São Carlos), Centro Oeste (Piratininga), Centro Sul (Engenheiro Coelho, Nova Odessa, São Pedro), Sudoeste (Itapetininga), Região Leste (Amparo, Serra Negra) e Vale do Paraíba (Guaratinguetá, Pindamonhangaba). As colheitas foram realizadas no período de novembro de 2008 a junho de 2009, assumindo um protocolo estabelecido para a implantação dos experimentos pelos responsáveis em cada região. Em cada propriedade, cerca de 60 ovinos semelhantes em número, sexo e categoria, com OPG superior a 200, foram alocados, ao acaso, em 5 tratamentos anti-helmínticos, a base de quatro grupos químicos: a) lactonas macrocíclicas: ivermectina 1% (IVM, 1mL/50kg), e moxidectina 1% (MOX, 1mL/50kg); b) benzimidazol: sulfóxido de albendazol (ABZ, 1mL/30kg); c) imiditiazol: cloridrato de levamisol (LEV, 1mL/10kg); d) salicilanilidas e substitutos fenólicos: closantel sódico 10% (CLO, 1mL/10kg), e um tratamento controle, não medicado. Em uma propriedade somente testou-se o ABZ, e em outra, ABZ e MOX. Entre 10 e 16 dias após a vermifugação, procedeu-se à colheita das fezes dos animais de todos os tratamentos para efetuar o teste de redução de contagem de ovos nas fazes (calculado pelo programa RESO 2.0 modificado), e coproculturas. A eficácia média geral nos diferentes grupos químicos foram: a) IVE, 35% (0 ? 77%)e MOX, 47% (0 - 99%); b) ABZ, 26% (0 ? 83%); c) LEV, 73% (0 ? 100%); d) CLO, 53% (2 ? 95%). Para a eficácia de redução de Haemonchus: a) IVE, 34% (0 ? 88%), MOX, 46% (0 ? 98%); b) ABZ, 27% (0 ? 85%); c) LEV, 73% (0 ? 100%); d) CLO, 68% (0 ? 99%). Com relação à redução de Trichostrongylus: a) IVE, 39% (0 ? 100%) e MOX, 54% (0 ? 100%); b) ABZ, 52% (0 ? 100%); c) LEV, 68% (0 ? 100%); d) CLO, 34% (0 ? 100%). Trichostrongylus e especialmente Haemonchus foram os nematóides mais encontrados após a vermifugação, em todos os tratamentos. Constatou-se que ABZ foi o que apresentou maior número (18, 95%) de propriedades com resistência (eficácia inferior a 80%); apenas uma propriedade apresentou quatro produtos com eficácia superior a 81%, as outras apresentaram resistência a pelo menos dois grupos; LEV apresentou eficácia superior a 80% em 10 (59%) propriedades. Concluiu-se que a resistência múltipla está presente em várias regiões do Estado de São Paulo, e ações conjuntas no manejo geral dos animais devem ser adotadas, visando mitigar ou reverter esse fenômeno

A polymerase chain reaction for the detection of Brucella canis in semen of naturally infected dogs

The objective was to evaluate a PCR assay for the detection of Brucella canis in canine semen, comparing its performance with that of bacterial isolation, serological tests and PCR assay of blood. Fifty-two male dogs were examined clinically to detect reproductive abnormalities and their serum was tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR assays were performed on blood and semen samples. The findings of the semen PCR were compared (Kappa coefficient and McNemar test) to those of blood PCR, culture of blood and semen, RSAT, and 2ME-RSAT. Nucleic acid extracts from semen collected from dogs not infected with B. canis were spiked with decreasing amounts of B. canis RM6/66 DNA and the resulting samples subjected to PCR. In addition, semen samples of non-infected dogs were spiked with decreasing amounts of B. canis CFU and the resulting suspensions were used for DNA extraction and amplification. of the 52 dogs that were examined, the following tests were positive: RSAT, 16 (30.7%); 2ME-RSAT, 5 (9.6%); blood culture, 14 (26.9%); semen culture, 11 (21.1%); blood PCR, 18 (34.6%); semen PCR, 18 (34.6%). The PCR assay detected as few as 3.8 fg of B. canis DNA experimentally diluted in 444.9 ng of canine DNA (extracted from semen samples of noninfected dogs). In addition, the PCR assay amplified B. canis genetic sequences from semen samples containing as little as 1.0 x 10(0) cfu/mL. We concluded that PCR assay of semen was a good candidate as a confirmatory test for the diagnosis of brucellosis in dogs; its diagnostic performance was similar to blood culture or blood PCR. Furthermore, the PCR assay of semen was more sensitive than the 2ME-RSAT or semen culture. Examination of semen by PCR should be included for diagnosis of brucellosis prior to natural mating or AI; in that regard, some dogs that were negative on serological and microbiological examinations as well as blood PCR were positive on PCR of semen. (c) 2007 Elsevier B.V. All rights reserved

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations. (C) 2007 Elsevier B.V. All rights reserved

Resistance to cypermethrin, deltamethrin and chlorpyriphos in populations of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) from small farms of the State of Sao Paulo, Brazil

A field survey of resistance was conducted based on the larval packet test technique with synthetic pyrethroids (cypermethrin and deltamethrin) and organophosphates (chlorpyriphos) in Rhipicephalus (Boophilus) microplus field populations from six different regions of the State of Sao Paulo (Brazil). 82.6% of the populations showed resistance to cypermethrin, 86.36% to deltamethrin and 65.25% to chlorpyriphos, with 50% presenting resistance to both SP and OP acaricide. According to the questionnaires completed by the producers, OP + SP mixtures followed by SP-only formulations were the products most commonly used for controlling the cattle tick in the surveyed areas. The present study showed high occurrence of resistance to SP and OP in the State of Sao Paulo, Brazil and revealed the type of strategy adopted by small dairy farms in this state. This information is fundamental in order to establish the monitoring of resistance on each farm individually, contributing to the rational use of acaricides for the control of R. (B.) microplus. (C) 2011 Elsevier B.V. All rights reserved.FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao Paulo[2006/55934-4]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Multidrug and multispecies resistance in sheep flocks from São Paulo state, Brazil.

The economic importance of sheep production is increasing worldwide simultaneously with the emergence of parasitic resistance. This study aimed to survey the current situation of management practices and parasite resistance in sheep flocks in São Paulo state, Brazil. A questionnaire was given to 35 sheep farmers to obtain information related to flock management practices. Of these flocks, 30 were submitted to the fecal egg count reduction test (FECRT) with at least one of the five following anthelmintics: albendazole, closantel, ivermectin, levamisole, and moxidectin, for comparison against an untreated control group. In the survey, the median number animals per flock was 301, mainly of the Santa Ines breed (in 75.8% of the flocks) and crossbred animals (in 54.5% of the flocks). The predominant farming system was semi-intensive (82.9%), using rotational grazing (80%). Selective treatment was based on FAMACHA grade (47.1%) and in clinical signs (41.2%). The most often applied anthelmintics were macrocyclic lactones (42.9?54.2% in the last three applications). Considering the anthelmintics employed in this study, 10.7% of the farms? flocks were resistant to three, 35.7% to four, and 53.6% to all five anthelmintics. The main helminth genera observed before and after treatments were Haemonchus sp. (75.8%) and Trichostrongylus sp. (19.1%), but all observed genera (Cooperia sp., Oesophagostomum sp., and Strongyloides sp.) were detected by the FECRT. Considering efficacy values less than or equal to 90% in the FECRT as resistant, 100% of flocks were resistant to albendazole and ivermectin, 96.6% to moxidectin, 92.9% to closantel, and 53.6% to levamisole. It is thus possible to conclude that multidrug resistance is widespread in sheep flocks in São Paulo state, Brazil, and this involves all prevalent helminth genera