Causes of fetal death in the Flemish cattle herd in Brazil.

Background and Aim: Flemish cattle in Brazil are on the brink of extinction and are found only in one herd in Lages, Santa Catarina State. This study aimed to uncover the reasons for the recurring abortions in the Flemish cattle herd. Materials and Methods: Seventeen Flemish fetuses underwent postmortem examinations, with samples collected for histopathology and microbiology culture tests, polymerase chain reaction (PCR) test for Neospora caninum, and reverse transcription-PCR (RT-PCR) test for bovine viral diarrhea virus (BVDV) from 2015 to 2020. Results: Of the 17 fetuses, N. caninum was the most common diagnosis and was found in 88% (15/17). One fetus (5.8%) had a coinfection with N. caninum and Citrobacter amalonaticus, leading to fibrinonecrotic pericarditis. All fetuses tested negative for BVDV by RT-PCR. Of the 107 dams tested by indirect immunofluorescence assay, 26 (25.2%) were anti-N. caninum seropositive, with 17 (65.4%) aborting and 5 (19.2%) having estrus repetition. Reverse transcription-PCR results showed that 9 (8.4%) of the serum samples collected from dams tested positive, which tested follow-up test 3 months later, indicating a BVDV transient infection. The factors that contributed to neosporosis included dogs' access to pastures and improper disposal of fetal remains, which made it easier for dogs to consume them. Conclusion: This study warns the occurrence of N. caninum as a cause of reproductive disorders that can lead to abortion in the studied Flemish cattle herd.

Experimental infection with Neospora caninum in Texel ewes at different stages of gestation.

In this study Texel sheep, at different stages of pregnancy, were experimentally infected with Neospora caninum. Eleven ewes, seronegative for N. caninum and Toxoplasma gondii, were inoculated 30 days before breeding (Group A), or at 65, 100, and 120 days of gestation (Groups B, C, and D). The group E (control) was inoculated with PBS. Blood samples were collected at -2, 2, 5, and 7 days post-infection (dpi), and weekly up to 42 dpi, for hematology, parasitemia (PCR), and serology (RIFI) assessments. Blood and tissue samples were collected from the lambs for molecular and histological analyses. All animals in Groups B, C, and D were seroconverted, whilst those in groups A and E remained seronegative. Parasitic DNA was detected in the blood of two ewes (groups B and D) and a lamb (group D), and in the brain of a lamb (group B). The parasitemia-positive ewe in group B delivered a weak and seropositive lamb, and had parasitic DNA in its placenta. These results confirm the vertical transmission of N. caninum in ewes inoculated at the beginning and end of pregnancy. The absence of abortions and other clinical signs suggest that Texel sheep may potentially have resistance to N. caninum.

Neospora caninum-associated abortions in cattle from Southern Brazil: Anatomopathological and molecular characterization.

This study aimed to evaluate the N. caninum associated abortions in cattle in the state of Santa Catarina, in the southern Brazil. Aborted bovine fetuses were necropsied, submitting organ samples for histopathological evaluation. Brain fragments were analyzed by polymerase chain reaction (PCR). The diagnosis of abortion due to N. caninum was established through histopathology and molecular analysis in 53.84% (28/52) of the cases, with PCR detection in 71.42% (20/28). The histopathological evaluation showed lesions in 75% of the cases, characterized by mononuclear necrotizing encephalitis, mononuclear myocarditis, mononuclear myositis, mixed placentitis, and mononuclear pneumonia. Neospora caninum was the primary etiological agent associated with causes of abortion in cattle in the present study.

Prevalence and risk factors for Dirofilaria immitis in dogs from Laguna, Santa Catarina, Brazil.

Dirofilaria immitis is a nematode found worldwide and able to infect several species, such as dogs, cats and, under rare circumstances, humans. In Brazil, most of the infections are related to coastal areas. The present study aimed at determining Dirofilaria immitis prevalence and evaluating the possible risk factors for infection in dogs, in Laguna, Santa Catarina, Brazil. Blood samples from 238 dogs were collected and a questionnaire was applied to the owners in order to obtain general information about the animals (age, gender, breed, coat color, home location (region), the use of dewormer/ectoparasiticide drugs, habits and behavior, roaming, insect control and the owner's knowledge about the disease). Samples were processed by blood smear techniques, modified Knott's test, immunochromatographic assay and PCR. Data were statistically analyzed by χ2 and OR. 4.62% was the overall prevalence of canine dirofilariasis and the suburb of Bananal, in Laguna, showed the highest prevalence (18.8%) when comparing with other regions (p < 0.05). However, there was no other significant factor related to the prevalence of D. immitis infection. Thus, the research have confirmed the presence of D. immitis in Laguna region, with a high prevalence in Bananal suburb, a dense forested area.

Experimental infection of tachyzoites of the NC1 strain of Neosporacaninum in female swine.

Neospora caninum is a protozoan that can cause reproductive problems in several animal species. Although N. caninum infection has been reported in swine, the pathogenesis and clinical signs are not fully known in this species. The objective of this work was to evaluate the effect of experimental infection with tachyzoites of the N. caninum strain Nc1 in swine matrices at different stages of gestation. For that purpose, 12 gilts, seronegative for N. caninum and T. gondii, were selected and allocated into four groups of three animals each. Animals in group A were not inoculated (control) and animals in groups B, C, and D were inoculated intravenously with of 2.9 × 107 tachyzoites, 30 days before conception, and at 45 and 90 days of gestation, respectively. Temperature, heart rate, blood, saliva, and vaginal mucus samples from the animals were collected periodically until the time of delivery for the investigation of IgG and IgM antibodies against N. caninum using IFAT and PCR to detect the parasite DNA. All gilts sero-converted from 5 and 7 DPI (days postinoculation) to IgM and IgG, respectively. Two gilts showed hypothermia on the 5th and 7th DPI, and five inoculated animals had leukocytosis on the 7th DPI. It was possible to detect DNA of N. caninum in samples of saliva (33/84), vaginal mucus (17/84), and blood (2/84). Based on serology (IgM) and PCR, three animals in group B showed evidence of reappearance of the infection during pregnancy. It is concluded that N. caninum can cause clinical signs in infected swine females, in addition to indicating saliva as a suitable diagnostic biological material for the detection of N. caninum DNA in this animal species.

Does Neospora caninum cause reproductive problems in pigs?

Neospora caninum is known to cause reproductive disturbances in several animal species, such as cattle, sheep, and goats. However, research on the effects of N. caninum on reproduction in pigs is limited. The objective of this study was to verify the transplacental transmission of N. caninum in pigs during several gestational stages. Twelve healthy Toxoplasma gondii and N. caninum seronegative female pigs were selected and separated into four groups of three animals each. Group A was maintained as a control group. Groups B, C, and D were inoculated intravenously with 2.9 × 107 tachyzoites of the N. caninum strain Nc1, 30 days before conception and at 45 and 90 days of gestation, respectively. Blood samples were collected from females periodically through IFAT for IgG and IgM screening to confirm the infection. At birth, after blood samples were collected from the piglets, they were then euthanized for the collection of the brain, heart, lung, liver, and diaphragm, which were then subjected to PCR. All inoculated gilts seroconverted (IgG) from the seventh day after inoculation. Nine of the 12 females expelled 24 mummified fetuses at the time of delivery, two in group A (eight), two in group B (four), three in group C (nine), and two in group D (three). Of the 24 mummified fetuses, nine were positive for N. caninum (one (25%) fetus of group B, seven (77.8%) of group C, and one (33.3%) of group D). A total of 126 live piglets were born. When the organs of the piglets from the inoculated females were analyzed by PCR for N. caninum, 88 (93.61%) were positive. All gilts inoculated produced at least one positive piglet. This demonstrates that there is transplacental transmission of N. caninum in all phases of gestation, regardless of the time of infection.

Arginine and manganese supplementation on the immune competence of broilers immune stimulated with vaccine against Salmonella Enteritidis.

This experiment was conducted to evaluate the combined effects of manganese-amino acid complex and arginine supplementation on the immune competence of broilers. On the day of hatch 640 male Cobb 500 broiler chicks assigned to two study groups (immune stimulate and non-stimulated). A 2 × 2 factorial arrangement of treatments was used with two manganese sources (MnSO4 or manganese-amino acid complex - MnAA) and two digestible Arg:Lys ratios (1.12 or 1.20). The treatments are: IM (80 ppm MnSO4); MnAA (40 ppm MnSO4 + 40 ppm MnAA); IM+Arg: 80 ppm MnSO4+ L-Arg (Arg:DigLys 1.20); MnAA+Arg: 40 ppm MnSO4 + 40 ppm MnAA + L-Arg (Arg:Lys 1.20). For treatments 1 and 2, the digestible Arg:Lys ratio was 1.12, considered normal for corn-soybean meal-based diets. Birds in the immune stimulated group received a dose of Salmonella Enteritidis vaccine. For growth performance and lymphoid organ development, no significant results were observed. Non-stimulated birds fed diets with Arg supplementation had higher percentage of mucosal T helper, T helper and T cytotoxic, compared to the normal Arg:Lys ratio (1.12). In the immune stimulated birds, broilers fed exclusive IM diet had a higher amount of T helper, T cytotoxic, activated T cytotoxic, and APC cells compared to broilers fed MnAA. The inorganic Mn diets, resulted in higher humoral antibody level (increased IgM levels) only when associated with supplementation of L-Arg. However, the use of an associated Mn source, could support high levels of IgM in commercial levels of Arg. No differences were observed to macrophage phagocytic activity analyses.

Hammondia sp. oocysts shed by a Brazilian fox (Lycalopex vetulus) differ from Hammondia heydorni and Hammondia triffittae.

A Brazilian fox (Lycalopex vetulus) was rescued from a highway, and 16 days after maintained in captivity, the fox shed oocysts with sizes compatible with Hammondia sp. and Neospora caninum. DNA extracted from oocysts were initially tested in two PCRs targeting the internal transcribed spacer 1 (ITS-1) of the rDNA of Hammondia heydorni and the Nc-5 gene of N. caninum. A 270-bp product was visualized in the PCR for H. heydorni. No amplification was observed for N. caninum PCR. Since ITS-1-based PCR is not sufficient to differentiate Hammondia species derived from canids, oocyst DNA was examined using multilocus sequence analysis of five genetic fragments [intron 1 of the alpha tubulin gene (intron 1), internal transcribed spaces 1 and 2 (ITS-1 and ITS-2) of the rDNA, 28S rRNA gene (D2/D3 domain), and heat shock protein 70 (Hsp70)]. The Hammondia sp. oocyst from the Brazilian fox, referred here as H-FOXBR isolate, is closely related to H. heydorni and Hammondia triffittae, but differs from these parasites in three genetic markers (alpha tubulin gene, ITS-2, and 28S rRNA). As reported by other research groups, Hammondia spp. excreted by canids are genetically diverse and may encompass additional species besides H. heydorni and H. triffittae. In this study, we confirmed that H-FOXBR has significant genetic differences in comparison to H. heydorni and H. triffittae and may represent a separate species. Further studies are needed to identify the life cycle of this parasite and to characterize the parasite stages in the intermediate and definitive hosts.

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

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.

Neospora caninum in properties in the west region of Paraná, Brazil: prevalence and risk factors / Neospora caninum em propriedades rurais da região Oeste do Paraná, Brasil: prevalência e fatores de risco

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.

Resumo Neospora caninum é um protozoário heteroxeno, cujos hospedeiros definitivos são os canídeos e os hospedeiros intermediários são os herbívoros, apresentando maior importância em bovinos. O objetivo desse trabalho foi determinar a prevalência de N. caninum em bovinos leiteiros e em cães, detectar molecularmente o protozoário em fetos abortados e identificar os fatores de risco associados à infecção em propriedades da região Oeste do estado do Paraná. Para isso, foram coletadas 600 amostras de soro de bovinos provenientes de 60 propriedades, 163 amostras de soro de cães de 52 propriedades e 17 fetos de nove propriedades. Por fim, foram coletados dados em um questionário epidemiológico para verificar os fatores de risco. As amostras de soro foram analisadas por meio da Reação de Imunofluorescência Indireta (RIFI) e os tecidos fetais foram analisados utilizando a PCR e posterior sequenciamento de DNA. Das amostras de bovinos, 23,67% foram positivas para N. caninum. Entre os cães, 11,66% foram positivos. Os fatores de risco nos bovinos foram histórico de aborto, baixa produção de leite, criação extensiva e raça Jersey (p<0,05). Dos 17 fetos analisados, em 52,94% foi possível detectar DNA do protozoário, e o sequenciamento mostrou alta similaridade com N. caninum.