Diagnosis of Neospora caninum in bovine fetuses by histology, immunohistochemistry, and nested-PCR

SUMMARYNeospora caninuminfection is a significant cause of abortion in cattle. We investigated the tissue distribution ofN. caninumin aborted bovine fetuses and dam blood samples by a nested PCR assay, and compared the nested PCR with ELISA in the diagnosis ofN. caninuminfection. In total, 26 aborted fetuses and 813 blood samples were collected from 8 dairy herds in Beijing (n=212) and Tianjin (n=601), China. Fifteen fetuses (57·7%) were testedN. caninum-positive by the nested PCR.N. caninumDNA was detected from the brain of 52%, kidneys of 22%, skeletal muscle of 18%, and heart of 4% of the aborted fetuses. The PCR-positive cases (55%, 11/20) were higher than seropositive cows (40%, 8/20) in a subset of 20 fetuses, but the PCR results of blood samples of the 20 cows were all negative. The seroprevalence of the 813 samples was 15·5% (43·4% of samples from Beijing, 5·7% of samples from Tianjin), compared to the PCR-positive blood samples of 0·9%. Our study showed that the nested PCR is a valuable diagnostic tool for the primary diagnosis ofN. caninumin aborted fetuses, while ELISA is the preferred assay for testing blood samples collected from cows. The two assays are complementary in determining whether abortions are associated withN. caninuminfection in cattle.

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DETECÇÃO DE DNA DE NEOSPORA CANINUM EM PARDELA-PRETA (PROCELLARIA AEQUINOCTIALIS) - RELATO DE CASO

Archives of Veterinary Science ◽

10.5380/avs.v15i5.77091 ◽

2020 ◽

Vol 15 (5) ◽

Author(s):

Ana Paula Sato ◽

Tiffany Christiny Emmerich da Silva ◽

Adrien Wilhelm Dilgerc Sanches ◽

Aline Luiza Konell ◽

Flávia Moreira da Fonseca ◽

...

Keyword(s):

Nested Pcr ◽

Neospora Caninum ◽

Procellaria Aequinoctialis

A pardela-preta (Procellaria aequinoctialis) é uma ave procellariiforme, possui distribuição circumpolar ao longo de todo o oceano austral e migra para o hemisfério sul durante o inverno. É considerada uma espécie globalmente vulnerável pela União Mundial para a Conservação da Natureza (IUCN, 2020), devido a mortalidade acidental da espécie na pesca com espinhel e a degradação do seu habitat natural causando rápido declínio populacional. Neospora caninum é um protozoário intracelular formador de cisto que infecta aves e mamíferos. Os canídeos domésticos e silvestres são os hospedeiros definitivos e eliminam os oocistos nas fezes. As aves se infectam pela ingestão de alimentos e água contaminados com oocistos esporulados e por meio da ingestão de cistos em tecidos infectados (DONAHOE et al.,2015). O objetivo do presente estudo foi relatar a presença de DNA de Neospora caninum no coração de uma ave pardela-preta (P. aequinoctialis), fêmea e adulta. Amostras de tecidos (musculatura peitoral, cérebro e coração) foram coletadas para a realização do diagnóstico molecular de N. caninum e acondicionadas e fixadas em formalina 10% tamponada para análise histopatológica. A extração de DNA foi realizada com kit comercial. Para a amplificação de DNA N. caninum foi empregada a reação de cadeia da polimerase nested-PCR com os primers JS4, CT2c, JS4b e CT2b, que amplificam a região ITS1. Foi detectado DNA de N. caninum na amostra do coração. O produto da PCR foi submetido a purificação e sequenciamento. Foi obtida sequência nucleotídica (MW023246) que apresentou identidade de 99% com isolados de N. caninum depositados no Genbank. Na análise histopatológica os tecidos não apresentavam alteração macroscópica e a análise microscópica revelou congestão no miocárdio. O presente relato demonstra que a pardela-preta (P. aequinoctialis) pode ser naturalmente infectada por N. caninum e até o momento é primeira descrição nesta espécie de ave. Existem poucos estudos sobre a ocorrência de N. caninum na fauna marinha e as possíveis fontes de infecção ainda não foram bem elucidadas. Estudos demonstraram que mamíferos marinhos (lontras, morsas e golfinhos) estão expostos ao protozoário indicando uma possível contaminação das águas marinhas por oocistos O achado nesse relato sugere que as aves marinhas podem ser potenciais hospedeiros intermediários de N. caninum, atuando como sentinela e indicador de contaminação do ambiente marinho, além da sua participação no ciclo silvestre do parasito.

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Detection of Neospora caninum DNA in cases of bovine and ovine abortion in the South-West of Scotland

Parasitology ◽

10.1017/s0031182019000301 ◽

2019 ◽

Vol 146 (07) ◽

pp. 979-982 ◽

Cited By ~ 2

Author(s):

P. M. Bartley ◽

S. Guido ◽

C. Mason ◽

H. Stevenson ◽

F. Chianini ◽

...

Keyword(s):

Neospora Caninum ◽

Economic Losses ◽

The South ◽

Farming Community ◽

South West ◽

Reproductive Losses ◽

Bovine Fetuses ◽

Cattle Farming ◽

Ovine Fetal ◽

Cattle And Sheep

AbstractNeospora caninum is a commonly diagnosed cause of reproductive losses in farmed ruminants worldwide. This study examined 495 and 308 samples (brain, heart and placenta) which were collected from 455 and 119 aborted cattle and sheep fetuses, respectively. DNA was extracted and a nested Neospora ITS1 PCR was performed on all samples. The results showed that for bovine fetuses 79/449 brain [17.6% (14.2–21.4)], 7/25 heart [28.0% (12.1–49.4)] and 5/21 placenta [23.8% (8.2–47.2)] were PCR positive for the presence of Neospora DNA. Overall 82/455 [18.0% (14.6–21.7)] of the bovine fetuses tested positive for the presence of N. caninum DNA in at least one sample. None (0/308) of the ovine fetal samples tested positive for the presence of Neospora DNA in any of the tissues tested. The results show that N. caninum was associated with fetal losses in cattle (distributed across South-West Scotland), compared to sheep in the same geographical areas where no parasite DNA was found. Neospora is well distributed amongst cattle in South-West Scotland and is the potential cause of serious economic losses to the Scottish cattle farming community; however, it does not appear to be a problem amongst the Scottish sheep flocks.

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A survey of the incidence of Neospora caninum infection in aborted and stillborn bovine fetuses in England and Wales

Veterinary Record ◽

10.1136/vr.136.24.602 ◽

1995 ◽

Vol 136 (24) ◽

pp. 602-606 ◽

Cited By ~ 35

Author(s):

A. Otter ◽

M. Jeffrey ◽

I. Griffiths ◽

J. Dubey

Keyword(s):

Neospora Caninum ◽

Caninum Infection ◽

England And Wales ◽

Bovine Fetuses

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Immune responses in pregnant cattle and bovine fetuses following experimental infection with Neospora caninum

Parasitology Research ◽

10.1007/s004360100442 ◽

2001 ◽

Vol 87 (10) ◽

pp. 817-825 ◽

Cited By ~ 59

Author(s):

Andrianarivo A. ◽

Barr B. ◽

Anderson M. ◽

Rowe J. ◽

Packham A. ◽

...

Keyword(s):

Immune Responses ◽

Experimental Infection ◽

Neospora Caninum ◽

Bovine Fetuses

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Neospora caninum in bovine fetuses of Minas Gerais, Brazil: genetic characteristics of rDNA

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612011000400005 ◽

2011 ◽

Vol 20 (4) ◽

pp. 281-288 ◽

Cited By ~ 8

Author(s):

Domingos Sávio dos Santos ◽

Michele Placedino Andrade ◽

Mary Suzan Varaschin ◽

Antônio Marcos Guimarães ◽

Christian Hirsch

Keyword(s):

Skeletal Muscle ◽

Genetic Differentiation ◽

Phylogenetic Tree ◽

Neospora Caninum ◽

Minas Gerais ◽

Genetic Characteristics ◽

Three Samples ◽

Bovine Fetuses ◽

Host Parasite ◽

The Brain

Neosporosis is an important cause of abortion in cattle and information on their genetics and host parasite relationships are desirable. Neospora caninum samples obtained from 24 bovine fetuses from Minas Gerais, were genetically analyzed in part of the rDNA region, coding for rRNAs. Previously, brain, heart, liver, skeletal muscle, lung, kidney, spleen, thymus, lymph nodes, ovary or testis, uterus and skin of the ear were analyzed by conventional histopathology and immunohistochemistry. Of these, eight had lesions compatible in the brain and heart and two also in skeletal muscle and liver. Three brains showed antigen identification in immunohistochemistry. Brain and heart tissues were subjected to DNA extraction for PCR, whose product of 588 bp of ITS-1 region was sequenced in three samples. We obtained 96% similarity with dozens of sequences N. caninum deposited in GenBank. The phylogenetic tree showed great conservation among isolates of N. caninum in this study and those deposited in GenBank, while well-defined and specific branches were generated against other Apicomplexa. PCR for this region is useful as a diagnosis, with good analytical specificity, but the ITS-1 region is not suitable for genetic differentiation intra species because the sequences obtained were identical to the others analyzed.

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Outbreak of caprine abortion by Toxoplasma gondii in Midwest Brazil

Pesquisa Veterinária Brasileira ◽

10.1590/s0100-736x2011001100001 ◽

2011 ◽

Vol 31 (11) ◽

pp. 933-937 ◽

Cited By ~ 6

Author(s):

Flávio Henrique Bravim Caldeira ◽

Daniel Guimarães Ubiali ◽

Isabela de Godoy ◽

Valéria Dutra ◽

Daniel Moura de Aguiar ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Brucella Abortus ◽

Nested Pcr ◽

Neospora Caninum ◽

Pcr Assay ◽

Serum Samples ◽

Pathology Laboratory ◽

Mato Grosso ◽

Veterinary Pathology ◽

Gross Lesions

An outbreak of abortion by Toxoplasma gondii in goats on a farm in the Brazilian Midwest is reported. Gross lesions were not observed in seven aborted fetuses submitted to the Veterinary Pathology Laboratory, Federal University of Mato Grosso, for necropsy investigation. The main histologic lesions were mononuclear cell pneumonia and necrotizing encephalitis in varying degrees of intensity. PCR for Brucella abortus and Neospora caninum and aerobic cultures were negative in all cases. Antibody titles against T. gondii varying from 1:1024 to 1:32.768 were detected in serum samples from four aborted goats. Nested-PCR assay for T. gondii were positive in brain samples of all cases submitted. These findings indicate that T. gondii infection should be considered in the diagnosis of abortion in goats in Midwest Brazil.

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Neospora caninum in properties in the west region of Paraná, Brazil: prevalence and risk factors

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612018001 ◽

2018 ◽

Vol 27 (1) ◽

pp. 51-59 ◽

Cited By ~ 6

Author(s):

Alessandra Snak ◽

Felipe Gustavo Garcia ◽

Arielle Aparecida Lara ◽

Hilda Fátima Jesus Pena ◽

Silvia Cristina Osaki

Keyword(s):

Risk Factors ◽

Neospora Caninum ◽

Fluorescent Antibody ◽

Antibody Test ◽

Intermediate Hosts ◽

Serum Samples ◽

West Region ◽

Bovine Fetuses ◽

History Of ◽

Polymerase Chain

Abstract Neospora caninum is a heteroxenous protozoa, whose definitive hosts are canids and intermediate hosts are herbivores, and is of great importance in cattle. The objectives of this study were to determine the prevalence of N. caninum in dairy cattle and dogs, to detect the presence of the protozoa at the molecular level in aborted fetuses, and to identify the risk factors associated with infection in properties in the western region of the state of Paraná. For this study, 600 bovine serum samples from 60 properties, 163 canine serum samples from 52 properties and 17 bovine fetuses from nine properties were collected. Data were collected using an epidemiological questionnaire to verify the risk factors. Serum samples were analyzed using the indirect fluorescent antibody test. Fetal tissues were analyzed using polymerase chain reaction and subsequent DNA sequencing. Of the bovine samples, 23.67% were positive for N. caninum. Among the canine samples, 11.66% were positive for N. caninum. Risk factors in cattle were history of abortion, low milk production, extensive breeding, and Jersey breed (p<0.05). Protozoan DNA was detected in 52.94% of the 17 fetuses and the sequencing presented high similarity with N. caninum.

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Experimental infection with Neospora caninum in pregnant bitches

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612012000300010 ◽

2012 ◽

Vol 21 (3) ◽

pp. 232-236 ◽

Cited By ~ 8

Author(s):

Guacyara Tenorio Cavalcante ◽

Rodrigo Martins Soares ◽

Sandra Mayumi Nishi ◽

Stéfano Carlo Filippo Hagen ◽

Camila Infantoni Vannucchi ◽

...

Keyword(s):

Nested Pcr ◽

Fragment Length Polymorphism ◽

Indirect Fluorescent Antibody Test ◽

Neospora Caninum ◽

Fluorescent Antibody ◽

Antibody Test ◽

Transplacental Transmission ◽

Nested Polymerase Chain Reaction ◽

The Central Nervous System ◽

Polymerase Chain

In this study, transplacental transmission of Neospora caninum in bitches at different stages of pregnancy was evaluated. Three bitches were inoculated in the 3rd week and three in the 6th week of gestation with 10(8) tachyzoites of N. caninum (Nc-1 strain). All the infected bitches and at least one of their offspring presented anti-N. caninum antibodies according to the indirect fluorescent antibody test (IFAT > 400). The pups and their mothers were sacrificed and tissues from the central nervous system (CNS), popliteal lymph nodes, skeletal muscle, brain, lungs, heart and liver were analyzed for the presence of N. caninum using the nested polymerase chain reaction (nested PCR), restriction fragment length polymorphism (RFLP) and immunohistochemistry (IHC). The parasite was found in the pups in lymph node, CNS, heart and liver tissues using nested PCR. There was no difference in perinatal mortality between the offspring from bitches infected in the 3rd week of gestation (60%) and in the 6th week (53.8%).

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