Molecular characterization of carnivore protoparvovirus strains circulating in cats in Turkey / Caracterização molecular de cepas de protoparvovírus carnívoros circulantes em gatos na Turquia

Cats are susceptible to feline panleukopenia virus (FPV) and canine parvovirus type 2 (CPV-2). Therefore, coinfection and superinfection with multiple parvovirus strains may occur, resulting in high heterogeneity and recombination. Considering the importance of cats as a potential source of genetic diversity for parvoviruses, we investigated the frequency of parvovirus infection in cats using their blood and fecal samples and performed molecular characterization of parvovirus strains circulating in cat populations. Accordingly, the fecal and blood samples of 60 cats with gastroenteritis symptoms were collected from Turkey's Burdur, Isparta, and Izmit provinces. Of these 15 fecal samples tested as parvovirus-positive by PCR, 14 were confirmed to have been infected with true FPV strains by sequencing analysis. Through the phylogeny analysis, those were located in the FPV cluster, closely related to CPV-2, and one was discriminated in the CPV-2b cluster. Additionally, sequence analysis of the VP2 gene of CPV and FPV revealed that the FPV strains detected in Turkey and the vaccine strains were highly related to each other, with a nucleotide identity of 97.7- 100%. Furthermore, 13 variable positions were detected in VP2 of the field and reference FPV strains. Three synonymous mutations were determined in the VP2 gene. Some amino acid mutations in the VP2 protein-affected sites were considered responsible for the virus's biological and antigenic properties. The partial sequence analysis of the VP2 gene revealed that four FPV strains detected in Turkey have a single nucleotide change from T to G at the amino acid position 384 between the nucleotides 3939-3941, which was reported for the first time. Therefore, these four isolates formed a different branch in the phylogenetic tree. The results suggest that both FPV and CPV-2b strains are circulating in domestic cats in Turkey and cats should be considered as potential sources of new parvovirus variants for cats, dogs and other animals.

Os gatos são suscetíveis ao vírus da panleucopenia felina (FPV) e ao parvovírus canino tipo 2 (CPV-2). Portanto, coinfecção e superinfecção com múltiplas cepas de parvovírus podem ocorrer, resultando em alta heterogeneidade e recombinação. Considerando a importância dos gatos como uma fonte potencial de diversidade genética para parvovírus, investigamos a frequência da infecção por parvovírus em gatos usando suas amostras de sangue e fezes e realizamos a caracterização molecular de cepas de parvovírus circulantes nas populações de gatos. Amostras fecais e de sangue de 60 gatos com sinais de gastroenterite foram coletadas nas províncias de Burdur, Isparta e Izmit, na Turquia. Destas, 15 amostras fecais testaram positivas para parvovírus por PCR e 14 foram confirmadas como infectadas com cepas verdadeiras de FPV por análise de sequenciamento. Através da análise filogenética, aqueles foram localizados no agrupamento FPV que está intimamente relacionado com o CPV-2, e um foi discriminado no agrupamento CPV-2b. Além disso, a análise da sequência do gene VP2 de CPV e FPV revelou que as cepas de FPV detectadas na Turquia e as cepas vacinais eram altamente relacionadas entre si, com uma identidade de nucleotídeos de 97,7-100%. Além disso, 13 posições variáveis foram detectadas em VP2 das cepas de campo e FPV de referência. Três mutações sinônimas foram determinadas no gene VP2. Algumas mutações de aminoácidos nos locais afetados pela proteína VP2 foram consideradas responsáveis pelas propriedades biológicas e antigênicas do vírus. A análise da sequência parcial do gene VP2 revelou que quatro cepas de FPV detectadas na Turquia têm uma única mudança de nucleotídeo de T para G na posição do aminoácido 384 entre os nucleotídeos 3939-3941, o que foi relatado pela primeira vez. Portanto, esses quatro isolados formaram um ramo diferente na árvore filogenética. Os resultados sugerem que ambas as cepas FPV e CPV-2b estão circulando em gatos domésticos na Turquia e os gatos devem ser considerados como fontes potenciais de novas variantes de parvovírus para gatos, cães e outros animais.

The sequence analysis of M2 gene for identification of amantadine resistance in avian influenza virus (H9N2 subtype), detected from broiler chickens with respiratory syndrome during 2016-2018, in Isfahan-Iran

Amantadine and rimantadine are used for prevention and treatment of influenza A virus (IAV) infection. The rates of resistant IAVs have been increasing globally. However, amino acid substitutions in the M2 transmembrane channel lead to amantadine resistance. The residues of 26, 27, 30, 31 or 34 are marker of amantadine resistance in IAVs. In this study, 15 pooled tracheal samples collected from 15 chicken farms with severe respiratory sign and mortality in 2016-2018. After identification of influenza A and H9 subtype, the 1027 bp fragment of M gene was sequenced for molecular evaluation of amantadine resistance in AIV strains. Results showed 12 out of 15 pooled samples were positive for IAV and H9 subtype. Based on M2 gene analysis, 8 out of 12 (66.66%) were resistance to amantadine. Four out of 8 (50%) showed S31N substitution (serine to asparagine) and four out of 8 (50%) have V27A substitution (valine to alanine). There was no dual amantadine resistance mutation in any specimens. In conclusion, the emergence of amantadine resistance variants of AIV in Iran, can raise concerns about controlling of the seasonal and the future pandemic influenza. Therefore, greater caution is needed in the use of adamantanes.(AU)

Estudo in vitro da eficácia do óleo essencial de cravo-da-índia (Syzygium aromaticum, Myrtaceae) sobre carrapato Rhipicephalus sanguineus

Resumen: Rhipicephalus sanguineus se ha convertido en una plaga urbana, con informes de infestaciones en hogares y poblaciones resistentes a los acaricidas, lo que genera preocupación tanto a los médicos veterinarios como a la salud pública. Con el objetivo de alternativas más sostenibles para el control ambiental de R. sanguineus, el objetivo de este estudio fue evaluar el efecto in vitro del aceite esencial de clavo, Syzygium aromaticum, sobre las larvas de Rhipicephalus sanguineus. Este aceite es rico en eugenol, un compuesto aromático ampliamente explorado por la industria alimentaria y farmacéutica, y que a pesar de su excelente acción acaricida, existen pocos estudios de su acción sobre R. sanguineus. Se utilizaron larvas con 15 días de vida para realizar las pruebas, y se probaron concentraciones de 0.5%, 1% y 2% del aceite esencial. La sensibilidad larvaria se probó mediante pruebas de inmersión larvaria (TIL) con adaptaciones. Se encontró una eficacia larvicida superior al 99% sobre R. sanguineus con concentraciones superiores al 0,5% de aceite de Syzygium aromaticum diluido en solución hidroalcohólica. Demostrando efecto prometedor acaricida del aceite esencial de clavo para el control de larvas de R. sanguineus.

Abstract: Rhipicephalus sanguineus has become an urban plague, with reports of infestations on residences and resistant populations to acaricides, bringing concerns both to veterinarians and public health. Looking for alternatives more sustainable for environmental control of R. sanguineus, the aim of this study was to evaluate the in vitro effect of the clove essential oil, Syzygium aromaticum, on Rhipicephalus sanguineus larvae. This oil is abundant in eugenol, an aromatic compound widely explored by the pharmaceutical and food industry, despite its strong acaricidal activity there are not many studies on its impact on R. sanguineus. In the present study, it was used 15 old day larvae, which were tested the 0,5%, 1% and 2% concentrations of the essential oil. The sensibility of the larvae was tested by the larvae immersion tests (LIT) with adaptations. It was noted the larvicidal efficacy above 99% on R. sanguineus in concentrations above 0,5% of Syzygium aromaticum oil diluted in hydroalcoholic solution. Demonstrating promising acaricidal effect of clove essential oil on tick R. sanguineus.

Resumo: Rhipicephalus sanguineus vem se tornando uma praga urbana, com relatos de infestações em residências e populações resistentes a acaricidas, trazendo preocupações tanto a clínicos veterinários como também em saúde pública. Visando alternativas mais sustentáveis para o controle ambiental de R. sanguineus, o objetivo deste estudo foi avaliar o efeito in vitro do óleo essencial de cravo-da-índia, Syzygium aromaticum, sobre larvas de Rhipicephalus sanguineus. Esse óleo é rico em eugenol, composto aromático amplamente explorado pela indústria alimentícia e farmacêutica, e que apesar da sua excelente ação acaricida, existem poucos estudos de sua ação sobre R. sanguineus. Para realizar os testes foram utilizadas larvas com 15 dias de vida, sendo testadas as concentrações de 0,5%, 1% e 2% do óleo essencial. A sensibilidade das larvas foi testada pelos testes de imersão larval (TIL) com adaptações. Foi encontrada eficácia larvicida acima de 99% sobre R. sanguineus com concentrações acima de 0,5% de óleo de Syzygium aromaticum diluído em solução hidroalcóolica. Demonstrando promissor efeito acaricida do óleo essencial de cravo-da-índia sobre larvas de R. sanguineus.

Estudo in vitro da eficácia do óleo essencial de cravo-da-índia (Syzygium aromaticum, Myrtaceae) sobre fase larval de Rhipicephalus sanguineus / In vitro effect of clove essential oil (Syzygium aromaticum, Myrtaceae) on the larval phase of Rhipicephalus sanguineus / Efecto in vitro del aceite esencial de clavo de olor (Syzygium aromaticum, Myrtaceae) sobre la fase larval de Rhipicephalus sanguineus

Rhipicephalus sanguineus vem se tornando uma praga urbana, com relatos de infestações em residências e populações resistentes a acaricidas, trazendo preocupações tanto a clínicos veterinários como também em saúde pública. Visando alternativas mais sustentáveis para o controle ambiental de R. sanguineus, o objetivo deste estudo foi avaliar o efeito in vitro do óleo essencial de cravo-da-índia, Syzygium aromaticum, sobre larvas de Rhipicephalus sanguineus. Para realizar os testes foram utilizadas larvas com 15 dias de vida, sendo testadas as concentrações de 0,5%, 1% e 2% do óleo essencial. A sensibilidade das larvas foi analisada pelos testes de imersão larval (TIL) com adaptações. Foi encontrada eficácia larvicida acima de 99% sobre R. sanguineus com concentrações acima de 0,5% de óleo de S. aromaticum diluído em solução hidroalcóolica. Demonstrando promissor efeito acaricida do óleo essencial de cravo-da-índia sobre larvas de R. sanguineus.(AU)

Rhipicephalus sanguineus has become an urban plague, with reports of infestations on residences and resistant populations to acaricides, bringing concerns both to veterinarians and public health. Looking for alternatives more sustainable for environmental control of R. sanguineus, the aim of this study was to evaluate the in vitro effect of the clove essential oil, Syzygium aromaticum, on Rhipicephalus sanguineus larvae. In the present study, it was used 15 old day larvae, which were tested concentrations of 0,5%, 1% and 2% of the essential oil. The sensibility of larvae was tested by the larvae immersion tests (LIT) with adaptations. It was noted the larvicidal efficacy above 99% on R. sanguineus in concentrations above 0,5% of Syzygium aromaticum oil diluted in hydroalcoholic solution. Demonstrating promising acaricidal effect of clove essential oil on tick R. sanguineus.(AU)

Rhipicephalus sanguineus se ha convertido en una plaga urbana, con informes de infestaciones en hogares y poblaciones resistentes a los acaricidas, lo que genera preocupación tanto a los médicos veterinarios como a la salud pública. Con el objetivo de alternativas más sostenibles para el control ambiental de R. sanguineus, el objetivo de este estudio fue evaluar el efecto in vitro del aceite esencial de clavo, Syzygium aromaticum, sobre las larvas de Rhipicephalus sanguineus. Se utilizaron larvas con 15 días de vida para realizar las pruebas, y se probaron concentraciones de 0.5%, 1% y 2% del aceite esencial. La sensibilidad larvaria se probó mediante pruebas de inmersión larvaria (TIL) con adaptaciones. Se encontró una eficacia larvicida superior al 99% sobre R. sanguineus con concentraciones superiores al 0,5% de aceite de Syzygium aromaticum diluido en solución hidroalcohólica. Demostrando efecto prometedor acaricida del aceite esencial de clavo para el control de larvas de R. sanguineus.(AU)

Agents of pneumonia in slaughtered pigs in southern Brazil / Agentes patogênicos de pneumonia em suínos abatidos no sul do Brasil

A high prevalence of pneumonic lesions has been reported to affect slaughtered pigs in southern Brazil. In order to identify which microorganisms have been causing those lesions, 30 pig lungs presenting pneumonic gross lesions were collected from five different slaughterhouses, totaling 150 lungs. Samples for bacterial isolation, molecular, histopathologic and immunohistochemistry (IHC) evaluation were taken from each lung. The pneumonic lesion scoring ranged from 1.53 to 2.83. The most frequent histopathological lesions found was the concomitant Influenza A virus (IAV) and Mycoplasma hyopneumoniae infection, corresponding to 55.3% (83/150), and Pasteurella multocida type A was isolated in 54.2% (45/83) of these cases. In 102 samples (68%), there was histopathologic suggestion of involvement of more than one infectious agent. M. hyopneumoniae was the most frequent agent associated with pneumonic lesions, being present in 92.1% (94/102) of the lungs with coinfections, followed by IAV in 89.2% (91/102). Besides the coinfections, IAV lesions were observed also in six samples without another pathogenic microorganism detected. A total of 46 samples with acute and subacute IAV suspected lesions in histopathological examination were assessed for IHC and real time RT-PCR for IAV. A total of 35% (16/46) of them were positive by IHC and 13% (6/46) by real time RT-PCR. Regarding M. hyopneumoniae, 79.3% (119/150) of samples were positive by qPCR and 84.9% (101/119) of them also presented M. hyopneumoniae suspected lesions in the histopathological examination. The results of this study suggest the importance of IAV in respiratory diseases in finishing pigs, even though this virus is more frequently reported in the nursery phase. In addition, our results emphasize the importance of lung coinfections in finishing pigs.(AU)

Lesões sugestivas de pneumonia são frequentemente encontradas em altas prevalências em suínos abatidos no sul do Brasil. Para identificar quais microrganismos causam essas lesões, foram coletados 30 pulmões de suínos com lesão macroscópica sugestiva de pneumonia em cinco frigoríficos diferentes, totalizando 150 pulmões. Amostras para isolamento bacteriano, avaliação molecular, histopatológica e imuno-histoquímica (IHC) foram coletadas de cada pulmão. O escore de lesão pulmonar variou entre 1,53 a 2,83. O achado histopatológico mais observado foi a lesão sugestiva de infecção concomitante pelo vírus Influenza A (IAV) e Mycoplasma (M.) hyopneumoniae, correspondendo a 55,3% (83/150), e em 54,2% (45/83) desses casos Pasteurella (P.) multocida tipo A foi isolado. Em 102 amostras (68%), houve lesão histopatológica sugestiva do envolvimento de mais de um agente infeccioso. M. hyopneumoniae foi o microrganismo mais frequente associado a lesões de pneumonia, estando presente em 92,1% (94/102) dos pulmões com coinfecções, seguido de IAV, que foi encontrado em 89,2% (91/102). Além das coinfecções, lesões de IAV foram observadas em mais seis amostras que não aparentavam envolvimento de outro agente infeccioso. Um total de 46 amostras com suspeita de lesão aguda e subaguda de IAV no exame histopatológico foram avaliadas para IHC e RT-PCR em tempo real para IAV e 35% (16/46) delas foram positivas por IHC e 13% (6/46) foram positivas por RT-PCR em tempo real. Com relação a M. hyopneumoniae, 79,3% (119/150) das amostras foram positivas por qPCR e 84,9% (101/119) delas também apresentaram lesões suspeitas de M. hyopneumoniae no exame histopatológico. Os resultados deste trabalho sugerem a importância do IAV como agente causador de pneumonias em suínos de terminação, embora esse vírus seja mais frequentemente relatado na fase de creche. Além disso, os achados deste trabalho demonstram a presença frequente de coinfecções pulmonares em suínos de terminação.(AU)

Agents of pneumonia in slaughtered pigs in southern Brazil / Agentes patogênicos de pneumonia em suínos abatidos no sul do Brasil

A high prevalence of pneumonic lesions has been reported to affect slaughtered pigs in southern Brazil. In order to identify which microorganisms have been causing those lesions, 30 pig lungs presenting pneumonic gross lesions were collected from five different slaughterhouses, totaling 150 lungs. Samples for bacterial isolation, molecular, histopathologic and immunohistochemistry (IHC) evaluation were taken from each lung. The pneumonic lesion scoring ranged from 1.53 to 2.83. The most frequent histopathological lesions found was the concomitant Influenza A virus (IAV) and Mycoplasma hyopneumoniae infection, corresponding to 55.3% (83/150), and Pasteurella multocida type A was isolated in 54.2% (45/83) of these cases. In 102 samples (68%), there was histopathologic suggestion of involvement of more than one infectious agent. M. hyopneumoniae was the most frequent agent associated with pneumonic lesions, being present in 92.1% (94/102) of the lungs with coinfections, followed by IAV in 89.2% (91/102). Besides the coinfections, IAV lesions were observed also in six samples without another pathogenic microorganism detected. A total of 46 samples with acute and subacute IAV suspected lesions in histopathological examination were assessed for IHC and real time RT-PCR for IAV. A total of 35% (16/46) of them were positive by IHC and 13% (6/46) by real time RT-PCR. Regarding M. hyopneumoniae, 79.3% (119/150) of samples were positive by qPCR and 84.9% (101/119) of them also presented M. hyopneumoniae suspected lesions in the histopathological examination. The results of this study suggest the importance of IAV in respiratory diseases in finishing pigs, even though this virus is more frequently reported in the nursery phase. In addition, our results emphasize the importance of lung coinfections in finishing pigs.(AU)

Lesões sugestivas de pneumonia são frequentemente encontradas em altas prevalências em suínos abatidos no sul do Brasil. Para identificar quais microrganismos causam essas lesões, foram coletados 30 pulmões de suínos com lesão macroscópica sugestiva de pneumonia em cinco frigoríficos diferentes, totalizando 150 pulmões. Amostras para isolamento bacteriano, avaliação molecular, histopatológica e imuno-histoquímica (IHC) foram coletadas de cada pulmão. O escore de lesão pulmonar variou entre 1,53 a 2,83. O achado histopatológico mais observado foi a lesão sugestiva de infecção concomitante pelo vírus Influenza A (IAV) e Mycoplasma (M.) hyopneumoniae, correspondendo a 55,3% (83/150), e em 54,2% (45/83) desses casos Pasteurella (P.) multocida tipo A foi isolado. Em 102 amostras (68%), houve lesão histopatológica sugestiva do envolvimento de mais de um agente infeccioso. M. hyopneumoniae foi o microrganismo mais frequente associado a lesões de pneumonia, estando presente em 92,1% (94/102) dos pulmões com coinfecções, seguido de IAV, que foi encontrado em 89,2% (91/102). Além das coinfecções, lesões de IAV foram observadas em mais seis amostras que não aparentavam envolvimento de outro agente infeccioso. Um total de 46 amostras com suspeita de lesão aguda e subaguda de IAV no exame histopatológico foram avaliadas para IHC e RT-PCR em tempo real para IAV e 35% (16/46) delas foram positivas por IHC e 13% (6/46) foram positivas por RT-PCR em tempo real. Com relação a M. hyopneumoniae, 79,3% (119/150) das amostras foram positivas por qPCR e 84,9% (101/119) delas também apresentaram lesões suspeitas de M. hyopneumoniae no exame histopatológico. Os resultados deste trabalho sugerem a importância do IAV como agente causador de pneumonias em suínos de terminação, embora esse vírus seja mais frequentemente relatado na fase de creche. Além disso, os achados deste trabalho demonstram a presença frequente de coinfecções pulmonares em suínos de terminação.(AU)

Active Surveillance and Risk Assessment of Avian Influenza Virus Subtype H9 from Non-Vaccinated Commercial Broilers of Pakistan

A cross-sectional study was conducted to investigate seroprevalence and virus prevalence of the H9 subtype of avian influenza virus in non-vaccinated broiler farms of dense poultry-populated districts, Lahore and Sheikhupura of Punjab-Pakistan. A convenient sampling method was adopted for collection of blood (n=500) and oropharyngeal swab (n=500) samples from 25 broiler farms of each district for hemagglutination inhibition assay and RT-PCR test, respectively. Proportional estimates were calculated using R software and overall seroprevalence of H9 was estimated at 36.3% (95% CI 33.3-39), with no significant difference (p>0.05) between Lahore (37.2 %, 95% CI=31.2-39.59) and Sheikhupura (35.4%, 95% CI= 29.64-39.76). RT-PCR identified 2% (4/200) pool level viral prevalence. None of the farms from Lahore districts were RT-PCR positive for H9. Simple logistic regression followed by multivariable analysis, identified the presence of foot bath/dipping area at the entrance (OR=0.7, 95% CI=0.52-0.93) and availability of rubber shoes for visitors (OR=0.36, 95% CI 0.26-0.48) as protective factors. History of respiratory signs (OR=1.51, 95%=CI 1.12-2.04), history of sudden death in past flocks (OR=3.26, 95% CI=2.41-4.41), and birds previously infected with avian influenza virus (OR=1.33, 95% CI=1-1.76) were significant risk factors. Negligence in preventive measures at farms level was associated with the spread of H9 infection between the farms. To control future outbreaks, biosecurity and continuous monitoring of non-vaccinated flocks are suggested.(AU)

Active Surveillance and Risk Assessment of Avian Influenza Virus Subtype H9 from Non-Vaccinated Commercial Broilers of Pakistan

A cross-sectional study was conducted to investigate seroprevalence and virus prevalence of the H9 subtype of avian influenza virus in non-vaccinated broiler farms of dense poultry-populated districts, Lahore and Sheikhupura of Punjab-Pakistan. A convenient sampling method was adopted for collection of blood (n=500) and oropharyngeal swab (n=500) samples from 25 broiler farms of each district for hemagglutination inhibition assay and RT-PCR test, respectively. Proportional estimates were calculated using R software and overall seroprevalence of H9 was estimated at 36.3% (95% CI 33.3-39), with no significant difference (p>0.05) between Lahore (37.2 %, 95% CI=31.2-39.59) and Sheikhupura (35.4%, 95% CI= 29.64-39.76). RT-PCR identified 2% (4/200) pool level viral prevalence. None of the farms from Lahore districts were RT-PCR positive for H9. Simple logistic regression followed by multivariable analysis, identified the presence of foot bath/dipping area at the entrance (OR=0.7, 95% CI=0.52-0.93) and availability of rubber shoes for visitors (OR=0.36, 95% CI 0.26-0.48) as protective factors. History of respiratory signs (OR=1.51, 95%=CI 1.12-2.04), history of sudden death in past flocks (OR=3.26, 95% CI=2.41-4.41), and birds previously infected with avian influenza virus (OR=1.33, 95% CI=1-1.76) were significant risk factors. Negligence in preventive measures at farms level was associated with the spread of H9 infection between the farms. To control future outbreaks, biosecurity and continuous monitoring of non-vaccinated flocks are suggested.

Histopatological effects of bleaching and disease on the coral Siderastrea stellata from coastal reefs of Brazil

ABSTRACT Histological analysis of bleached samples of Siderastrea stellata Verrill, 1868, with white plague disease and with alteration in the color pattern, revealed drastic tissue and cellular disturbances, such as lysis of the external epithelium, hyperplasia of gastrodermis, apoptosis of epithelial cells and zooxanthellae, and degradation of mesenteric filaments and reproductive cells. Fungal hyphae, diatoms, and cyanobacteria were present in diseased samples and colonies with alteration in the color pattern. Furthermore, bleached and diseased samples showed significant reductions in the number of zooxanthellae per 100 µm2 of gastrodermis and significant reductions in the diameters and volumes of stage V oocytes. We found that bleaching events, diseases, and colonies with alteration in the color pattern promoted degradation of reproductive cells, resulting in the consequent interruption of the reproduction of the coral S. stellata, which is one of the most common Brazilian coral species. The implications of these indirect effects of bleaching, disease, and changes in coral color patterns in the population dynamics of Brazilian reefs are discussed.

RESUMO Análises histológicas de amostras de Siderastrea stellata Verrill, 1868 branqueadas, doentes e com alteração no padrão de cloração, revelaram drásticas perturbações teciduais e celulares, como lise do epitélio externo, hiperplasia da mesogleia, apoptose das células epiteliais e das zooxantelas, e degradação dos filamentos mesentéricos e das células reprodutivas. Hifas fúngicas, diatomáceas e cianobactérias estavam presentes em amostras doentes e em tecidos de colônias com alteração no padrão de coloração. Além disso, amostras branqueadas e doentes apresentavam reduções significativas no número de zooxantelas por 100 µm2 da gastrodermis e, reduções significativas nos diâmetros e volumes dos oócitos de estágio V. Constatamos que eventos de branqueamento, doenças e colônias com alteração no padrão de cloração, promoveram degradação das células reprodutivas, resultando na consequente interrupção da reprodução do coral S. stellata, que é uma das espécies de corais brasileiro mais comuns. As implicações desses efeitos indiretos do branqueamento, doenças e das mudanças nos padrões de coloração dos corais na dinâmica populacional dos recifes brasileiros são discutidas.

Molecular diagnosis of avian viruses in grassland passerines and captive yellow cardinals Gubernatrix cristata in Brazil / Diagnóstico molecular de vírus aviários em passeriformes campestres e cardeais-amarelos Gubernatrix cristata cativos no Brasil

Avian influenza viruses (AIVs), Newcastle disease virus (NDV), West Nile virus (WNV), adenovirus (AV) and herpesvirus (HV) play an important role in the health of human and animal populations. However, knowledge of the prevalence of these viruses in wild birds is restricted to some groups (e.g. shorebirds) or regions worldwide. Information on grassland birds of South America, which is essential for their conservation, is scarce. The objectives of the present study were to evaluate occurrences of AIV, NDV, WNV, AV and HV for the first time in a bird community of a unique protected area in southern Brazil, which is home for the critically endangered yellow cardinal (Gubernatrix cristata), and captive yellow cardinals from fauna maintainers of the Brazilian Captive Program of the Yellow Cardinal. Passerine species of wild life were caught, identified and samples (swabs) were collected from the oropharynx and cloaca of 64 passerines of 26 species (including 3 yellow cardinals) and 30 yellow cardinals of captive, for molecular diagnosis. The samples were subjected to RNA and DNA extraction and the real-time polymerase chain reaction (RT-PCR) for AIV, NDV and WNV and nested PCR for AV and HV. One yellow cardinal of captive presented a positive result for AV, this result is important for planning, managing natural attributes and making decisions in relation to integrated conservation of threatened species. This is the first report of AV in yellow cardinal and epidemiological investigation of viruses in wild passerines of the Pampa biome, in Rio Grande do Sul, Brazil.(AU)

Os vírus da gripe aviária (VGA), vírus da doença de Newcastle (VDN), vírus do Nilo Ocidental (VNO), adenovírus (AV) e herpesvírus (HV) desempenham um papel importante na saúde das populações humana e animal. No entanto, o conhecimento da prevalência desses vírus em aves selvagens é restrito a alguns grupos (por exemplo, aves limícolas) ou regiões em todo o mundo. As informações sobre as aves campestres da América do Sul, essenciais para a sua conservação, são escassas. Os objetivos do presente estudo foram avaliar a ocorrência de VGA, VDN, VNO, AV e HV pela primeira vez em uma comunidade de aves de uma área única protegida no Sul do Brasil, que abriga o cardeal-amarelo (Gubernatrix cristata) criticamente ameaçado de extinção e em cardeais-amarelos de cativeiro dos mantenedores de fauna do Programa Brasileiro de Cativeiro do Cardeal-amarelo. Espécies de passeriformes silvestres foram capturadas, identificadas e amostras (swabs) foram coletadas da orofaringe e cloaca de 64 passeriformes de 26 espécies (incluindo 3 cardeais-amarelos) e 30 cardeais-amarelos de cativeiro, para diagnóstico molecular. As amostras foram submetidas à extração de RNA e DNA e à reação em cadeia da polimerase em tempo real (RT-PCR) para VGA, VDN e VNO e nested PCR para AV e HV. Um cardeal-amarelo de cativeiro apresentou resultado positivo para AV, este resultado é importante para o planejamento, manejo dos atributos naturais e tomada de decisões em relação à conservação integrada de espécies ameaçadas. Este é o primeiro relato de AV em cardeal-amarelo e de investigação epidemiológica de vírus em passeriformes silvestres do bioma Pampa, no Rio Grande do Sul, Brasil.(AU)

Molecular diagnosis of avian viruses in grassland passerines and captive yellow cardinals Gubernatrix cristata in Brazil / Diagnóstico molecular de vírus aviários em passeriformes campestres e cardeais-amarelos Gubernatrix cristata cativos no Brasil

Avian influenza viruses (AIVs), Newcastle disease virus (NDV), West Nile virus (WNV), adenovirus (AV) and herpesvirus (HV) play an important role in the health of human and animal populations. However, knowledge of the prevalence of these viruses in wild birds is restricted to some groups (e.g. shorebirds) or regions worldwide. Information on grassland birds of South America, which is essential for their conservation, is scarce. The objectives of the present study were to evaluate occurrences of AIV, NDV, WNV, AV and HV for the first time in a bird community of a unique protected area in southern Brazil, which is home for the critically endangered yellow cardinal (Gubernatrix cristata), and captive yellow cardinals from fauna maintainers of the Brazilian Captive Program of the Yellow Cardinal. Passerine species of wild life were caught, identified and samples (swabs) were collected from the oropharynx and cloaca of 64 passerines of 26 species (including 3 yellow cardinals) and 30 yellow cardinals of captive, for molecular diagnosis. The samples were subjected to RNA and DNA extraction and the real-time polymerase chain reaction (RT-PCR) for AIV, NDV and WNV and nested PCR for AV and HV. One yellow cardinal of captive presented a positive result for AV, this result is important for planning, managing natural attributes and making decisions in relation to integrated conservation of threatened species. This is the first report of AV in yellow cardinal and epidemiological investigation of viruses in wild passerines of the Pampa biome, in Rio Grande do Sul, Brazil.(AU)

Os vírus da gripe aviária (VGA), vírus da doença de Newcastle (VDN), vírus do Nilo Ocidental (VNO), adenovírus (AV) e herpesvírus (HV) desempenham um papel importante na saúde das populações humana e animal. No entanto, o conhecimento da prevalência desses vírus em aves selvagens é restrito a alguns grupos (por exemplo, aves limícolas) ou regiões em todo o mundo. As informações sobre as aves campestres da América do Sul, essenciais para a sua conservação, são escassas. Os objetivos do presente estudo foram avaliar a ocorrência de VGA, VDN, VNO, AV e HV pela primeira vez em uma comunidade de aves de uma área única protegida no Sul do Brasil, que abriga o cardeal-amarelo (Gubernatrix cristata) criticamente ameaçado de extinção e em cardeais-amarelos de cativeiro dos mantenedores de fauna do Programa Brasileiro de Cativeiro do Cardeal-amarelo. Espécies de passeriformes silvestres foram capturadas, identificadas e amostras (swabs) foram coletadas da orofaringe e cloaca de 64 passeriformes de 26 espécies (incluindo 3 cardeais-amarelos) e 30 cardeais-amarelos de cativeiro, para diagnóstico molecular. As amostras foram submetidas à extração de RNA e DNA e à reação em cadeia da polimerase em tempo real (RT-PCR) para VGA, VDN e VNO e nested PCR para AV e HV. Um cardeal-amarelo de cativeiro apresentou resultado positivo para AV, este resultado é importante para o planejamento, manejo dos atributos naturais e tomada de decisões em relação à conservação integrada de espécies ameaçadas. Este é o primeiro relato de AV em cardeal-amarelo e de investigação epidemiológica de vírus em passeriformes silvestres do bioma Pampa, no Rio Grande do Sul, Brasil.(AU)

Histopatological effects of bleaching and disease on the coral Siderastrea stellata from coastal reefs of Brazil / Efeitos histopatológicos dos eventos de branqueamento e doenças no coral Siderastrea stellata dos recifes costeiros do Brasil

Histological analysis of bleached samples of Siderastrea stellata Verrill, 1868, with white plague disease and with alteration in the color pattern, revealed drastic tissue and cellular disturbances, such as lysis of the external epithelium, hyperplasia of gastrodermis, apoptosis of epithelial cells and zooxanthellae, and degradation of mesenteric filaments and reproductive cells. Fungal hyphae, diatoms, and cyanobacteria were present in diseased samples and colonies with alteration in the color pattern. Furthermore, bleached and diseased samples showed significant reductions in the number of zooxanthellae per 100 µm2 of gastrodermis and significant reductions in the diameters and volumes of stage V oocytes. We found that bleaching events, diseases, and colonies with alteration in the color pattern promoted degradation of reproductive cells, resulting in the consequent interruption of the reproduction of the coral S. stellata, which is one of the most common Brazilian coral species. The implications of these indirect effects of bleaching, disease, and changes in coral color patterns in the population dynamics of Brazilian reefs are discussed.(AU)

Análises histológicas de amostras de Siderastrea stellata Verrill, 1868 branqueadas, doentes e com alteração no padrão de cloração, revelaram drásticas perturbações teciduais e celulares, como lise do epitélio externo, hiperplasia da mesogleia, apoptose das células epiteliais e das zooxantelas, e degradação dos filamentos mesentéricos e das células reprodutivas. Hifas fúngicas, diatomáceas e cianobactérias estavam presentes em amostras doentes e em tecidos de colônias com alteração no padrão de coloração. Além disso, amostras branqueadas e doentes apresentavam reduções significativas no número de zooxantelas por 100 µm2 da gastrodermis e, reduções significativas nos diâmetros e volumes dos oócitos de estágio V. Constatamos que eventos de branqueamento, doenças e colônias com alteração no padrão de cloração, promoveram degradação das células reprodutivas, resultando na consequente interrupção da reprodução do coral S. stellata, que é uma das espécies de corais brasileiro mais comuns. As implicações desses efeitos indiretos do branqueamento, doenças e das mudanças nos padrões de coloração dos corais na dinâmica populacional dos recifes brasileiros são discutidas.(AU)

Histopatological effects of bleaching and disease on the coral Siderastrea stellata from coastal reefs of Brazil / Efeitos histopatológicos dos eventos de branqueamento e doenças no coral Siderastrea stellata dos recifes costeiros do Brasil

Histological analysis of bleached samples of Siderastrea stellata Verrill, 1868, with white plague disease and with alteration in the color pattern, revealed drastic tissue and cellular disturbances, such as lysis of the external epithelium, hyperplasia of gastrodermis, apoptosis of epithelial cells and zooxanthellae, and degradation of mesenteric filaments and reproductive cells. Fungal hyphae, diatoms, and cyanobacteria were present in diseased samples and colonies with alteration in the color pattern. Furthermore, bleached and diseased samples showed significant reductions in the number of zooxanthellae per 100 µm2 of gastrodermis and significant reductions in the diameters and volumes of stage V oocytes. We found that bleaching events, diseases, and colonies with alteration in the color pattern promoted degradation of reproductive cells, resulting in the consequent interruption of the reproduction of the coral S. stellata, which is one of the most common Brazilian coral species. The implications of these indirect effects of bleaching, disease, and changes in coral color patterns in the population dynamics of Brazilian reefs are discussed.

Análises histológicas de amostras de Siderastrea stellata Verrill, 1868 branqueadas, doentes e com alteração no padrão de cloração, revelaram drásticas perturbações teciduais e celulares, como lise do epitélio externo, hiperplasia da mesogleia, apoptose das células epiteliais e das zooxantelas, e degradação dos filamentos mesentéricos e das células reprodutivas. Hifas fúngicas, diatomáceas e cianobactérias estavam presentes em amostras doentes e em tecidos de colônias com alteração no padrão de coloração. Além disso, amostras branqueadas e doentes apresentavam reduções significativas no número de zooxantelas por 100 µm2 da gastrodermis e, reduções significativas nos diâmetros e volumes dos oócitos de estágio V. Constatamos que eventos de branqueamento, doenças e colônias com alteração no padrão de cloração, promoveram degradação das células reprodutivas, resultando na consequente interrupção da reprodução do coral S. stellata, que é uma das espécies de corais brasileiro mais comuns. As implicações desses efeitos indiretos do branqueamento, doenças e das mudanças nos padrões de coloração dos corais na dinâmica populacional dos recifes brasileiros são discutidas.

Anti-influenza A virus antibodies in Tayassuidae from commercial rearing farms in Brazil / Detecção de anticorpos contra vírus influenza A em Tayassuidae de criações comerciais no Brasil

Family Tayassuidae in the suborder Suina include two species of peccaries in Brazil: the white-lipped peccary (Tayassu pecari) and the collared peccary (Pecari tajacu). These animals share common pathogens with domestic swine (Sus scrofa); however, their role as potential carrier remains unclear. This study focused on detecting the prevalence of influenza A antibodies in Tayassu pecari and Pecari tajacu from commercial rearing farms from two states in Brazil. A set of 50 blood samples from Pecari tajacu and 55 from Tayassu pecari were analyzed using a commercial indirect ELISA in order to investigate anti influenza A antibodies. Pecari tajacu samples presented 22% (11/50) of seropositivity for the virus. Serological surveillance is an important tool to identify the presence and the spread of the influenza virus in feral pigs.(AU)

A família Tayassuidae pertencente a subordem Suina e compreende duas espécies presentes no Brasil: Queixada (Tayassu pecari) e o Caititu (Pecari tajacu). Ambas as espécies compartilham patógenos com o suíno doméstico (Sus scrofa), entretanto o papel destes animais como carreadores destas infecções permanece indefinido. O presente estudo teve como objetivo detectar a ocorrência de anticorpos contra vírus influenza A em amostras de soro de rebanhos comerciais de queixada e caititu, provenientes de dois estados do Brasil. Um total de 50 amostras de soro de Pecari tajacu e 55 amostras de Tayassu pecari foram testadas por meio de ELISA, sendo que 22% (11/50) das amostras de Pecari tajacu foram soropositivas para o agente. Estudos de vigilância sorológica são importantes para identificar a presença e a disseminação do vírus influenza em suínos selvagens.(AU)

Yersinia pestis detection by loop-mediated isothermal amplification combined with magnetic bead capture of DNA

We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23 CFU for pure culture, whereas 2.3 × 104 or 2.3 × 106 CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3 × 106 CFU, but PCR was negative at the level of 2.3 × 107 CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3 × 103 or 2.3 × 106 CFU, whereas 2.3 × 105 or 2.3 × 107 CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.(AU)

Evaluation of the Viral Interference between Lentogenic Newcastle Disease Virus (Lasota) and Avian Influenza Virus (H9N2) using Real-Time Reverse Transcription Polymerase Chain Reaction in SPF Chicken

Lentogenic Newcastle disease virus (lNDV) such as Lasota strain and low pathogenicity avian influenza such as H9N2 virus are two of the most economically important viruses affecting poultry worldwide, and little attention in recent years has been paid to simultaneous infections in chickens with these two viruses for the reason that co-infection do occur but are not easily detected. In the present study, chickens were inoculated with lNDV (Lasota) and LPAIV (A/chicken/Tehran/ZMT-173/99(H9N2)) simultaneously or sequentially three days apart. Oropharyngeal and cloacal swabs were collected from chickens from 1 to 14 days after inoculation. RRT-PCR for AIV and NDV detection was performed. The rate of viral shedding was measured within 14 days. No clinical symptoms were observed during the experiment however the pattern of virus shed was different with co-infection, thus comparing the results obtained from viral shedding showed that AIV is a much stronger agent than NDV in the occurrence of viral interference. This is due to the fact that in simultaneous inoculation, AIV replication delayed and reduced NDV replication, while replication of Lasota in simultaneous or pre-inoculated inoculation could not significantly disrupt H9N2 virus replication. These findings indicate that the infection with one virus can interfere with the replication of another, modifying the pathogenesis of the viruses. So, infection of the host with both viral agents simultaneously causes higher shedding of LPAIV than lNDV in OP and CL areas. In conclusion, co-infection with LPAVI in chickens did not impact clinical signs but affected the replication dynamics of these viruses.(AU)

Evaluation of the Viral Interference between Lentogenic Newcastle Disease Virus (Lasota) and Avian Influenza Virus (H9N2) using Real-Time Reverse Transcription Polymerase Chain Reaction in SPF Chicken

Lentogenic Newcastle disease virus (lNDV) such as Lasota strain and low pathogenicity avian influenza such as H9N2 virus are two of the most economically important viruses affecting poultry worldwide, and little attention in recent years has been paid to simultaneous infections in chickens with these two viruses for the reason that co-infection do occur but are not easily detected. In the present study, chickens were inoculated with lNDV (Lasota) and LPAIV (A/chicken/Tehran/ZMT-173/99(H9N2)) simultaneously or sequentially three days apart. Oropharyngeal and cloacal swabs were collected from chickens from 1 to 14 days after inoculation. RRT-PCR for AIV and NDV detection was performed. The rate of viral shedding was measured within 14 days. No clinical symptoms were observed during the experiment however the pattern of virus shed was different with co-infection, thus comparing the results obtained from viral shedding showed that AIV is a much stronger agent than NDV in the occurrence of viral interference. This is due to the fact that in simultaneous inoculation, AIV replication delayed and reduced NDV replication, while replication of Lasota in simultaneous or pre-inoculated inoculation could not significantly disrupt H9N2 virus replication. These findings indicate that the infection with one virus can interfere with the replication of another, modifying the pathogenesis of the viruses. So, infection of the host with both viral agents simultaneously causes higher shedding of LPAIV than lNDV in OP and CL areas. In conclusion, co-infection with LPAVI in chickens did not impact clinical signs but affected the replication dynamics of these viruses.

Serological diagnosis of influenza A subtype H1 on family poultry of Belo Horizonte, Minas Gerais and Santa Maria, Rio Grande Do Sul, in Brazil

Serum samples (n=687) from Gallus gallus domesticus were collected for the investigation of antibodies to avian influenza virus (AIV-A) in the family poultry of the surrounding counties of Santa Maria/RS and the metropolitan region of Belo Horizonte/MG, totaling twenty different counties. Additional samples of seventeen (n=17) free-flying ducks (C. moschata pure or hybrid with Anas platyrhynchos) were collected in Belo Horizonte. The chosen tests for the survey were performed as described by the World Organization for Animal Health (OIE), including agar gel immunodiffusion (AGID) for antibodies to AIV-A nucleoprotein (N) and haemagglutination- inhibition (HI) for antibodies to subtype H1. Out of the 704 serum tests performed by AGID, eight (8/704) were revealed positive for antibodies to AIV-A N protein, with six (6/704) retested positive for subtype H1. Two sera tested positive by AGID were shown to be non reactive to the H1 subtype, suggesting specificity to another subtype. A low occurrence of antibodies to influenza A (1.13%) was found, and mostly (75%) specific to subtype H1. This represents an approximately 0,85% overall occurrence for subtype H1 antibodies, with an unknown subtype specific antibodies detected in one free-flying anatid. The low occurrence of antibodies in the family poultry may suggest a low AIV-A activity during the period of study, information which remains to be confirmed by virus detection.(AU)

Serological diagnosis of influenza A subtype H1 on family poultry of Belo Horizonte, Minas Gerais and Santa Maria, Rio Grande Do Sul, in Brazil

Serum samples (n=687) from Gallus gallus domesticus were collected for the investigation of antibodies to avian influenza virus (AIV-A) in the family poultry of the surrounding counties of Santa Maria/RS and the metropolitan region of Belo Horizonte/MG, totaling twenty different counties. Additional samples of seventeen (n=17) free-flying ducks (C. moschata pure or hybrid with Anas platyrhynchos) were collected in Belo Horizonte. The chosen tests for the survey were performed as described by the World Organization for Animal Health (OIE), including agar gel immunodiffusion (AGID) for antibodies to AIV-A nucleoprotein (N) and haemagglutination- inhibition (HI) for antibodies to subtype H1. Out of the 704 serum tests performed by AGID, eight (8/704) were revealed positive for antibodies to AIV-A N protein, with six (6/704) retested positive for subtype H1. Two sera tested positive by AGID were shown to be non reactive to the H1 subtype, suggesting specificity to another subtype. A low occurrence of antibodies to influenza A (1.13%) was found, and mostly (75%) specific to subtype H1. This represents an approximately 0,85% overall occurrence for subtype H1 antibodies, with an unknown subtype specific antibodies detected in one free-flying anatid. The low occurrence of antibodies in the family poultry may suggest a low AIV-A activity during the period of study, information which remains to be confirmed by virus detection.

Infecção pelo vírus influenza A em suínos no sul de Moçambique / Influenza A virus infection in pigs in southern Mozambique

A influenza suína (IS) é uma doença aguda e altamente contagiosa do trato respiratório de suínos, causada pelo vírus influenza A (VIA). A doença provoca perdas econômicas na suinocultura e também, tem importância na saúde pública devido ao seu potencial zoonótico. O objetivo deste trabalho foi relatar a infecção pelo VIA em suínos de Moçambique e realizar a caracterização anatomopatológica e imuno-histoquímica (IHQ) das lesões pulmonares associadas. Pulmões de 457 suínos abatidos foram avaliados e coletados 38 (8,3%) pulmões de suínos com áreas de consolidação pulmonar em um abatedouro na cidade da Matola, no Sul de Moçambique. As áreas de consolidação em cada lobo pulmonar foram classificadas em 4 graus de acordo com a extensão da lesão. Amostras de pulmões com consolidação foram submetidas ao exame histopatológico e IHQ para a detecção de antígenos do VIA, Circovírus suíno tipo 2 (PCV2) e Mycoplasma hyopneumoniae. Os pulmões coletados apresentaram áreas multifocais ou coalescentes de consolidação pulmonar, frequentemente, observadas nos lobos craniais, mediais e acessório. Estas lesões acometiam principalmente um ou três lobos pulmonares e as lesões de grau 1 e 2 foram as mais frequentes. As principais lesões histopatológicas observadas foram bronquiolite necrotizante (23/38), infiltrado de neutrófilos nos alvéolos (24/38), hiperplasia de pneumócitos tipo II (26/38), hiperplasia de tecido linfoide peribronquiolar (28/38), e pneumonia intersticial mononuclear (29/38). No exame de IHQ, antígenos do VIA foram detectados em 84.3% (32/38) dos pulmões com pneumonia, e a nucleoproteína do vírus foi visualizada, no núcleo de células epiteliais de brônquios e bronquíolos e em macrófagos alveolares. Suínos positivos para o VIA na IHQ eram provenientes do distrito de Matutuine (5/32), distrito da Moamba (2/32), distrito de Namaacha (21/32), distrito de Boane (3/32) e Cidade da Matola (1/32). Todas as amostras foram negativas para PCV2 e Mycoplasma hyopneumoniae pelo exame de IHQ. Os resultados demonstram que o VIA está presente e é causa de pneumonia em suínos em Moçambique.(AU)

Swine influenza (SI) is an acute and highly contagious disease of the respiratory tract of pigs caused by influenza A virus (IAV). The disease causes economic losses in swine production and is of great public importance for its zoonotic potential. The aim of the present study was to report IAV infection in pigs from Mozambique and characterize the anatomopathological and immunohistochemical features of associated lung lesions. Lungs from 457 slaughtered pigs were subjected to gross evaluation and 38 (8.3%) lungs with cranioventral consolidation were collected from a slaughterhouse in Matola City, southern Mozambique. Areas of consolidation in each lung lobe were classified in 4 grades according to the lesion extension. Samples with consolidated lung tissue were examined for histopathology and immunohistochemistry (IHC) for the presence of IAV, Porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae antigens. The lungs had multifocal to coalescing areas of consolidation observed most frequently in the cranial, middle, and accessory lobes. The lesions involved mainly one or three pulmonary lobes and grade 1 and 2 lesions were the most frequent. The main histopathological findings were necrotizing bronchiolitis (23/38), alveolar neutrophil infiltration (24/38), type II pneumocytes hyperplasia (26/38), peribronchiolar lymphoid tissue hyperplasia (28/38) and interstitial mononuclear cells infiltrate (29/38). Immunohistochemistry revealed positive staining in 84.3% (32/38) of the samples and IAV nucleoprotein was present in the nucleus of bronchial and bronchiolar epithelial cells and alveolar macrophages. Positive IHC pigs were from Matutuine district (5/32), Moamba district (2/32), Namaacha district (21/32), Boane district (3/32) and Matola city (1/32). All lung samples were immunohistochemically negative for PCV2 and Mycoplasma hyopneumoniae. These results demonstrate that IAV is a cause of pneumonia in pigs in Mozambique.(AU)