Occurrence of Salmonella spp. in the broiler production chain / [Ocorrência de Salmonella spp. na cadeia de produção de carne de frango]

ABSTRACT Brazilian chicken meat is exported to more than 150 countries and consumed by consumer markets that demand high quality and food safety, thus, requiring very strict control of pathogens present in food to guarantee these rigorous safety standards. This study evaluates the reports from the Salmonella spp. Control and Monitoring Program of the Brazilian Ministry of Agriculture, Livestock and Food Supply of seven slaughterhouses inspected by the Federal Inspection Service from the western region of Paraná state, Brazil, from March 2017 to February 2019. The broiler litter swab and carcass analyses revealed a Salmonella spp. positivity ratio of 5.9% (19/319) and 23.5% (75/319), respectively. The concomitant presence of Salmonella spp. in the broiler litter swab and chicken carcasses occurred in 58% of the positive samples. The most frequently isolated serovar in the carcasses was Salmonella Heidelberg (85.3%) followed by Salmonella spp. (10.6%). During slaughter, carcass positivity to Salmonella spp. was significantly different (p=0.047) between the first (19.6%) and the second (29.4%) shifts. The results alert for the possibility of carcass contamination during slaughtering and, therefore, more stringent hygiene measures between shifts must be implemented to mitigate carcass contamination.

RESUMO A carne de frango brasileira é consumida em mais de 150 países, em mercados exigentes com a qualidade e a produção de alimentos seguros, o que justifica o controle de patógenos nesse alimento, a fim de assegurar tais requisitos. O presente estudo analisou dados constantes dos relatórios do Programa de Controle e Monitoramento da Salmonella spp. do Ministério da Agricultura, Pecuária e Abastecimento do Brasil (MAPA), realizado em sete unidades avícolas e frigoríficas da região oeste do estado do Paraná, com Serviço de Inspeção Federal, no período entre março 2017 e fevereiro de 2019. A análise dos dados revelou a presença de Salmonella spp. no suabe de cama de frango em 5,9% dos lotes analisados e em 23,5% das carcaças oriundas desses lotes. A presença concomitante de Salmonella spp. no suabe de cama e nas carcaças de frango do lote ocorreu em 58% das amostras positivas. O sorovar mais frequentemente isolado nas carcaças foi Salmonella Heidelberg (85,3%), seguido de Salmonella spp. (10,6%). Durante o abate, observou-se diferença significativa (P=0,047) na positividade das carcaças para Salmonella spp. entre o primeiro (19,6%) e o segundo turno (29,4%). Os resultados indicam a possibilidade de contaminação das carcaças durante o abate, portanto a adoção de medidas mais rigorosas de higienização entre os turnos deve ser implementada a fim de mitigar a contaminação das carcaças.

Sertolioma difuso em cão criptorquídico / Diffuse sertolioma in a criptorquidic dog

O presente trabalho tem como objetivo relatar um caso de sertolioma associado a criptorquidia em um cão Poodle de 8 anos que apresentava uma massa na região inguinal há aproximadamente 8 meses de evolução e histórico prévio compatível com síndrome de feminilização. Constatou-se considerável aumento de volume no testículo criptorquídico (esquerdo). O animal foi submetido a orquiectomia no testículo criptorquídico e apresentou boa recuperação. O exame histopatológico revelou se tratar de sertolioma difuso, possivelmente com manifestação sistêmica de alopecia simétrica bilateral não pruriginosa associada à ocorrência do tumor. Após a castração, houve progressiva e completa remissão da dermatopatia com posterior cura clínica do paciente.

The present work aimed to report a case of a Sertoli cell tumor associated with cryptorchidism in a Poodle dog of eight years old, that presented a mass in the inguinal region eight months ago. The previous history and evolution time were compatible with testicular feminization syndrome. It was observed a considerable increase in volume of the affected testis (left). The animal was submitted to an orchiectomy of the cryptorchidic testis and presented a good recovery. The histopathological examination revealed a diffuse sertolioma, and possibly the systemic manifestation of non-pruriginous bilateral symmetric alopecia was related to the presence of the tumor.

Sertolioma difuso em cão criptorquídico / Diffuse sertolioma in a criptorquidic dog

O presente trabalho tem como objetivo relatar um caso de sertolioma associado a criptorquidia em um cão Poodle de 8 anos que apresentava uma massa na região inguinal há aproximadamente 8 meses de evolução e histórico prévio compatível com síndrome de feminilização. Constatou-se considerável aumento de volume no testículo criptorquídico (esquerdo). O animal foi submetido a orquiectomia no testículo criptorquídico e apresentou boa recuperação. O exame histopatológico revelou se tratar de sertolioma difuso, possivelmente com manifestação sistêmica de alopecia simétrica bilateral não pruriginosa associada à ocorrência do tumor. Após a castração, houve progressiva e completa remissão da dermatopatia com posterior cura clínica do paciente.(AU)

The present work aimed to report a case of a Sertoli cell tumor associated with cryptorchidism in a Poodle dog of eight years old, that presented a mass in the inguinal region eight months ago. The previous history and evolution time were compatible with testicular feminization syndrome. It was observed a considerable increase in volume of the affected testis (left). The animal was submitted to an orchiectomy of the cryptorchidic testis and presented a good recovery. The histopathological examination revealed a diffuse sertolioma, and possibly the systemic manifestation of non-pruriginous bilateral symmetric alopecia was related to the presence of the tumor.(AU)

SERTOLIOMA DIFUSO EM CÃO CRIPTORQUÍDICO

O presente trabalho tem como objetivo relatar um caso de sertolioma associado a criptorquidia em um cão Poodle de 8 anos que apresentava uma massa na região inguinal há aproximadamente 8 meses de evolução e histórico prévio compatível com síndrome de feminilização. Constatou-se considerável aumento de volume no testículo criptorquídico (esquerdo). O animal foi submetido a orquiectomia no testículo criptorquídico e apresentou boa recuperação. O exame histopatológico revelou se tratar de sertolioma difuso, possivelmente com manifestação sistêmica de alopecia simétrica bilateral não pruriginosa associada à ocorrência do tumor. Após a castração, houve progressiva e completa remissão da dermatopatia com posterior cura clínica do paciente.

Effect of orthodontic force associated with cigarette smoke inhalation in healthy and diseased periodontium. A histometric and immunohistochemistry analysis in rats.

BACKGROUND: The aim of the present study is to evaluate the effect of orthodontic forces in healthy or diseased periodontium of rats submitted/not submitted to cigarette smoke inhalation. MATERIAL AND METHODS: Fifty-six male Wistar rats were allocated into two groups of conditions: smoking and non-smoking. Each group was divided into the following subgroups: control (C), orthodontic tooth movement (OTM), ligature-induced periodontitis (P) and P+OTM (POTM), with n = 14 each. Periodontitis was induced in the lower first molar by cotton ligature, and a 4 mm closed stainless steel spring was used for orthodontic movement. Animals were exposed to the smoke of 10 cigarettes for 8 minutes, 3 times a day for 60 days before P induction and OTM. Evaluation parameters were macroscopic analysis of dental movement, bone loss and bone density. In addition, the receptor activator of nuclear factor-kappaB (RANK) immunostaining and RANK ligand/osteoprotegerin ratio in the furcation region were assessed. RESULTS: There was no statistically significant difference between groups, ie, smoking and non-smoking conditions (P = .338). Bone loss intragroup analysis between the P and POTM groups was not significant in smoking (P = 1) and non-smoking (P = .5) conditions; both were different from OTM and C in each condition. Regarding bone density, POTM and P were significant to C (P < .05). The POTM group was significant to the P and C (P = .001) regarding dental movement. The RANK ligand/osteoprotegerin ratio in the non-smoking condition was higher in P and POTM compared to C and OTM and to P and POTM in the smoking condition. RANK immunostaining was significant in the smoking condition for the P and POTM groups (P < .05). CONCLUSION: Within the limitations of the present study, it was concluded that cigarette smoke inhalation had no influence on the evaluated groups, even with the presence of low levels of nicotine, carbon monoxide and tar. The POTM groups did not present greater bone loss compared to P groups, thus periodontal disease is essential for bone loss.

Vaccine effectiveness and use of collar impregnated with insecticide for reducing incidence of Leishmania infection in dogs in an endemic region for visceral leishmaniasis, in Brazil.

Although a national programme for control of visceral leishmaniosis (VL) is being run in Brazil, the disease continues to spread. This programme is essentially based on culling infected dogs from endemic regions. Thus, there is an urgent need to develop other control measures against VL to deter its advance. Here, a subunit vaccine, a recombinant vaccine, an insecticide-impregnated collar and the associations between these measures were evaluated for reducing the incidence of Leishmania infection in dogs. This was through a cohort study conducted in an endemic region of Brazil, considering the incidence and time of total exposure over a period of 1 year. The incidence of VL was estimated by means of serological and molecular diagnostic tests, 180 and 360 days after the application of the control measures. The estimates of the effectiveness (EF) were not significant in any cohort. The EF of the subunit vaccine, the recombinant vaccine and the collar were 26.4%, 32.8% and 57.7% and the upper limit of the 95% confidence interval for EF were 63.7%, 67.9% and 82.5%, respectively. In conclusion, under the conditions of this study, none of the immunogens for VL control was sufficiently effective to protect dogs against infection. On the other hand, use of collars impregnated with insecticide seems to constitute a method with better prognosis, corroborating other studies in this field.

Dog skin parasite load, TLR-2, IL-10 and TNF-α expression and infectiousness.

Visceral leishmaniosis is a zoonotic disease that is transmitted by Lutzomyia longipalpis sandflies. Dogs are the main peri-urban reservoir of the disease, and progression of canine leishmaniosis is dependent on the type of immune response elaborated against the parasite. Type 1 immunity is characterized by effective cellular response, with production of pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-α). In contrast, Type 2 immunity is predominantly humoral, associated with progression of the disease and mediated by anti-inflammatory cytokines such as interleukin 10 (IL-10). Although seemly important in the dynamics of leishmaniosis, other gene products such as toll-like receptor 2 (TRL-2) and inducible nitric oxide synthase (iNOS) exert unclear roles in the determination of the type of immune response. Given that the dog skin serves as a micro-environment for the multiplication of Leishmania spp., we investigated the parasite load and the expression of TLR-2, iNOS, IL-10 and TNF-α in the skin of 29 infected and 8 control dogs. We found that increased parasite load leads to upregulation of TLR-2, IL-10 and TNF-α, indicating that abundance of these transcripts is associated with infection. We also performed a xenodiagnosis to demonstrate that increased parasitism is a risk factor for infectiousness to sandflies.

Serological and molecular diagnostic tests for canine visceral leishmaniasis in Brazilian endemic area: one out of five seronegative dogs are infected.

Euthanasia of infected dogs is one of the measures adopted in Brazil to control visceral leishmaniasis (VL) in endemic areas. To detect infected dogs, animals are screened with the rapid test DPP® Visceral Canine Leishmaniasis for detection of antibodies against K26/K39 fusion antigens of amastigotes (DPP). DPP-positives are confirmed with an immunoenzymatic assay probing soluble antigens of promastigotes (ELISA), while DPP-negatives are considered free of infection. Here, 975 dogs from an endemic region were surveyed by using DPP, ELISA and real-time PCR (qPCR) for the diagnosis of VL. When DPP-negative dogs were tested by qPCR applied in blood and lymph node aspirates, 174/887 (19·6%) were positive in at least one sample. In a second sampling using 115 cases, the DPP-negative dogs were tested by qPCR in blood, lymph node and conjunctival swab samples, and 36/79 (45·6%) were positive in at least one sample. Low-to-moderate pairwise agreement was observed between all possible pair of tests. In conclusion, the official diagnosis of VL in dogs in Brazilian endemic areas failed to accuse an expressive number of infected animals and the impact of the low accuracy of serological tests in the success of euthanasia-based measure for VL control need to be assessed.

Correlations between peripheral parasite load and common clinical and laboratory alterations in dogs with visceral leishmaniasis.

Intensity of peripheral parasite infection has an important role in the transmission of Leishmania spp. from one host to another. As parasite load quantification is still an expensive procedure to be used routinely in epidemiological surveillance, the use of surrogate predictors may be an important asset in the identification of dogs with high transmitting ability. The present study examined whether common clinical and laboratory alterations can serve as predictors of peripheral parasitism in dogs naturally infected with Leishmania spp. Thirty-seven dogs were examined in order to establish correlations between parasite load (PL) in multiple peripheral tissues and common clinical and laboratory findings in canine visceral leishmaniasis (CVL). Quantitative polymerase chain reaction was employed to determine PL in conjunctival swabs, ear skin, peripheral blood and buffy coat. Additionally, a series of hematological, biochemical and oxidative stress markers were quantified. Correlations between net peripheral infection and severity of clinical alterations and variation in laboratory parameters were assessed through a new analytical approach, namely Compressed Parasite Load Data (CPLD), which uses dimension reduction techniques from multivariate statistics to summarize PL across tissues into a single variable. The analysis revealed that elevation in PL is positively correlated with severity of clinical sings commonly observed in CVL, such as skin lesions, ophthalmic alterations, onycogriphosis, popliteal lymphadenomegaly and low body mass. Furthermore, increase in PL was found to be followed by intensification of non-regenerative anemia, neutrophilia, eosinopenia, hepatic injury and oxidative imbalance. These results suggest that routinely used clinical and laboratory exams can be predictive of intensity of peripheral parasite infection, which has an important implication in the identification of dogs with high transmitting ability.

Detection of cross infections by Leishmania spp. and Trypanosoma spp. in dogs using indirect immunoenzyme assay, indirect fluorescent antibody test and polymerase chain reaction.

The aim of this study was to detect cross infections by Leishmania spp. and Trypanosoma spp. using enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody test (IFAT) and polymerase chain reaction (PCR). Thus, 408 blood samples were collected from dogs domiciled in Araçatuba Municipality, São Paulo State, Brazil; the dogs were of both sexes, of several breeds and aged 6 months. For Leishmania spp., 14.95% (61 out of 408) of dogs were reactive using IFAT. Positivity was 20.10% (82 out of 408) using ELISA and 29.66% (121 out of 408) using PCR, with significant differences for the sex and age of these animals (p < 0.05). For Trypanosoma spp., antibody occurrence using ELISA was 10.54% (43 out of 408), while PCR indicated 2.45% (10 out of 408) positive dogs. Using IFAT, 10.29% (42 out of 408) of animals were considered positive and only sex showed a significant difference (p < 0.05). In this study, 10.54% (43 out of 408) of animals were seropositive according to ELISA for Trypanosoma spp., of which 79.07% (34 out of 43) showed positive results in the molecular diagnosis for Leishmania spp., while of the 10.29% (42 out of 408) positive dogs according to IFAT, 95.24 % (40 out of 42) had confirmed infection by this parasite. The obtained results demonstrate evidence of cross infections by both protozoa in the animals analysed in this study.

Targeted nucleotide exchange in bovine myostatin gene.

The myostatin gene, known as Growth Differentiation Factor 8 (GDF8), located at chromosome 2 (BTA2) in cattle, is specifically expressed during embryo development and in the adult skeletal muscle. Molecular analysis of this gene reveals the presence of three exons and two introns. Several cattle breeds, such as Piedmontese, Belgian Blue, Blond'Aquitaine, among others, show polymorphisms in this gene, which are directly related to double muscling phenotype. Piedmontese cattle shows a nucleotide transition G --> A (G938A) at exon 3, resulting in the substitution of cysteine to tyrosine, leading to a protein structure change, which determines myostatin inactivation and consequent muscular hypertrophy. The objective of this work was to implant the polymorphism G938A, naturally existent in Piedmontese breed, into in vitro propagated foetal myoblasts, from Nellore cattle. Single strand DNA (ssDNA) oligonucleotides were used to direct the same nucleotidic transition (G938A) to exon 3. Two transfection protocols (cationic lipid solution and electroporation) were tested and, 48 hours after transfection, RNA and DNA were extracted from myoblasts. Reverse transcription and polymerase chain reaction (PCR) were performed, using primers flanking the mutation region. The PCR products were cloned and analyzed by DNA sequencing, and it was possible to detect the nucleotidic CT transition at position 938, in the electroporated myoblasts. The existence of a positive signal in the transfection indicates that ssDNA oligonucleotides can be used to introduce this point mutation in specific functional gene sites.

OCORRÊNCIA DE ANTICORPOS CONTRA Neospora caninum E Toxoplasma gondii E ESTUDO DE FATORES DE RISCO EM CÃES DE ARAÇATUBA - SP.

A ocorrência de anticorpos contra Neospora caninum e Toxoplasma gondii foi determinada através de uma amostra de 108 cães em 63 domicílios e canis da zona urbana do município de Araçatuba, SP, por meio da reação de imunofluorescência indireta (RIFI). Para se verificar possíveis associações entre a presença de anticorpos contra os referidos coccídios e as variáveis raça, sexo, idade, dieta, ambiente, presença de gatos e acesso às ruas, o Teste Qui-Quadrado (ou Teste de Fischer) foi realizado. Também foi realizada uma análise de fatores de risco com as variáveis mais associadas à infecção por T. gondii e N. caninum. Dos 63 locais amostrados, 28 (44,4%) foram soropositivos para T. gondii e 15 (23,8%) para N. caninum. Foi verificada associação entre cães positivos para T. gondii e criados em ambiente de terra ou gramado (p < 0,001), quando comparados aos mantidos em ambiente cimentado. A presença de um dos agentes não apresentou associação com a ocorrência simultânea do outro coccídio (p = 0,84).PALAVRAS-CHAVE: Toxoplasma gondii. Neospora caninum. Cães. Epidemiologia.

Comparative evaluation of an indirect ELISA test for diagnosis of swine cysticercosis employing antigen from Taenia solium and Taenia crassiceps metacestodes.

Taenia solium cysticercosis is still a serious public health problem in several countries where poverty and lack of hygiene favor transmission. Because pigs are the primary intermediate hosts, prevalence of porcine cysticercosis is a reliable indicator of active transmission zones. Serological diagnostic methods are important tools for epidemiological studies since they can be applied to living animals on a large scale. Four antigen preparations (cyst fluid and crude) from T. solium and T. crassiceps metacestodes were compared for swine cysticercosis diagnosis by indirect ELISA (IE). Twenty-eight serum samples from swine naturally and experimentally infected by cysticerci of T. solium and 56 serum samples from swine reared in commercial herds were tested. Best results of overall sensitivity were obtained by the use of cyst fluid and crude antigen of T. crassiceps metacestode (100 and 96.4%, respectively). Using homologous antigen preparations we have observed higher specificity percentage (98.2% for cyst fluid and 96. 4% for crude metacestode T. solium antigen). We concluded that sensitivity is of far more importance than specificity for identification of endemic areas in order to prevent transmission to man. We conclude, therefore, that IE performed with cyst fluid antigen of T. crassiceps metacestode is a better tool for that purpose.

Detection and differentiation of Leptospira spp. serovars in bovine semen by polymerase chain reaction and restriction fragment length polymorphism.

In view of the importance of venereal transmission of bovine leptospirosis, the objective of the present study was to apply the polymerase chain reaction (PCR) to 26 serovars of Leptospira interrogans, L. borgpetersenii, L. santarosai, L. noguchii and L. biflexa, to determine the detection threshold in semen samples and to evaluate the possibility of differentiation among serovars using 19 restriction endonucleases. The results showed that all serovars were amplified and the detection threshold in semen samples of a bull was 100 bacteria/ml. Using endonucleases we could classify the 26 serovars into eight groups. The present results show that PCR is a method of great potential for the detection of Leptospira spp. at bovine artificial insemination centers.

[Presence of larva migrans in sand boxes of public elementary schools, Araçatuba, Brazil]. / Ocorrência de larva migrans na areia de áreas de lazer das escolas municipais de ensino infantil, Araçatuba, SP, Brasil.

There are sandboxes in public elementary school playground areas in Brazil, which can be harmful to children. They are at risk of cutaneous and visceral larva migrans infection caused by Ancylostoma spp. and Toxocara spp., respectively. The study was designed to investigate contamination by Toxocara spp. and/or their eggs and Ancylostoma spp. larvae in sand samples collected from the schools' sandboxes. Five hundred and thirty-five sand samples from 28 public elementary schools were collected during summer and winter and analyzed by both Baerman's method and centrifugal flotation technique. Ancylostoma spp. larvae were found in 35.7% (10/28) schools in summer time and in 46.4% (13/28) schools in the winter time. Eggs of Toxocara spp. could not be recovered from the samples analyzed and eggs from Ancylostoma spp. were seen in 0.56% (3/535) of the samples.

Toxocariasis: serological diagnosis by indirect antibody competition ELISA.

Toxocariasis is caused by infection of man by Toxocara canis and Toxocara cati larvae, the common roundworm of dogs and cats. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. Non specific reactions are observed mainly due to cross-reactivity with Ascaris sp antigens. This investigation aimed at developing and evaluating an indirect antibody competition ELISA (IACE) employing a specific rabbit IgG anti-Toxocara canis excretory-secretory antigens as the competition antibody, in order to improve indirect ELISA specificity performed for toxocariasis diagnosis. For that, the rabbit IgG was previously absorbed by Ascaris suum adult antigens. Sensitivity and specificity of IACE were first evaluated in 28 serum samples of mice experimentally infected with T. canis embryonated eggs. Adopting cut-off value established in this population before infection, sensitivity and specificity were 100% after 20 days post-inoculation. For human population IACE was evaluated using sera from 440 patients with clinical signs of toxocariasis and the cut-off value was established with 60 serum samples from apparently healthy individuals. Using as reference test the indirect ELISA performed by Adolfo Lutz Institute, sensitivity was 60.2%, specificity was 98% and concordance was 77.3%. Repeatability of IACE was evaluated by the inter-reactions variation coefficient (2.4%).

Detection of leptospires in bovine semen by polymerase chain reaction.

OBJECTIVE: In view of the considerable importance of venereal transmission of bovine leptospirosis, the objective of the present study was to compare the polymerase chain reaction (PCR), culture/isolation and serology to detect leptospire infection in bovine semen. DESIGN: Blood for serologic examination and semen for bacterial culture and PCR were collected from 20 bulls at artificial insemination centres in Brazil. Each animal was sampled twice for serology. RESULT: Forty-five percent (9/20) of the serum samples collected showed agglutinin titers to serovar hardjo in the first sample and 25% (5/20) had agglutinin titers to serovar hardjo in the second sample. Eighty percent (16/20) of semen samples were positive by PCR. Leptospires could not be isolated from any of the semen samples examined. CONCLUSION: Polymerase chain reaction can be a method of great potential for the detection of leptospires at artificial insemination centres.

Detection of Leptospira spp. from pure cultures and from experimentally contaminated bovine semen by polymerase chain reaction

Due to the high importance of the venereal transmission of bovine leptospirosis, this study aimed to test the ability of PCR to detect Leptospira interrogans serovar hardjo DNA in experimentally contaminated bovine semen. Employing primers directed to the 16S rRNA gene, 10 bacteria/ml of semen could be detected by PCR. Results achieved in this work show that PCR can have a great potential to detect Leptospira spp. in insemination centers