Abortion In Cattle Research Articles

An estimation of vertical transmission of Neospora caninum in dairy cattle, in Khorasan Razavi Province, Iran

Neospora caninum is an important agent of abortion and reproductive loss in dairy cattle. The objective of this study was to determine the rate of vertical transmission of N.caninum in dairy cattle, KorasanRazavi province, Iran. Two dairy farms with history of Neospora abortion were selected and two hundred -eighty blood samples of dairy cattle in the third trimester of gestation were collected from January 2018 to April 2019. The serum samples of each dairy cattle were examined by an indirect ELISA. At the calving, the colostrum samples of seropositive dams with blood samples of their precolostral calves were collected and again examined by the ELISA method. The seroprevalence of Neospora infection was determined by 16.59% (41/247) in dairy farm 1 and 12.12% (4/33) in dairy farm 2 (P>0.05). In the present study, 100% of precolostral calves born of seropositive dairy cattle had antibodies against Neospora caninum before colostrum ingestion. Based on the seroprevalence of dam and daughter, the vertical transmission was estimated at the highest rate (100%). In conclusion, the results are shown the importance of vertical transmission to remain and contribute to N.caninum infection in two dairy farms.

Indirect ELISAs with sucrose subcellular fractions of Neospora caninum as antigens for diagnosis of neosporosis in cattle

Neosporosis is one of the major causes of abortion in cattle, and it is responsible for significant economic losses in those animals. Thus, this study aimed to evaluate indirect ELISA using subcellular fractions of Neospora caninum obtained via sucrose gradient separation. Eighty-five sera from dairy cattle previously tested using indirect immunofluorescence assay (IFA) were used. Three distinct bands were separated at 1.0 M, 1.4 M, 1.6 M, and the pellet at 1.8 M, which were identified as fractions one (F1), two (F2), three (F3), and four (F4), respectively. These fractions showed parasite membranes in the F1, rhoptry and conoids in the F2, mitochondria in the F3, and tachyzoite ghosts remain in F4. Indirect ELISAs for IgM, and IgG were performed. Additionally, sensitivity, specificity, and kappa values were defined considering the IFA as the gold standard. The highest and lowest specificities were observed for F1 (76 %) and F3 (16 %), respectively. F2 and F4 showed the highest sensitivity (93.3 %), kappa agreement (0.46), and Negative Preventive Value (NPV) (73 %) respectively. It was possible to standardize indirect ELISAs using whole soluble antigen and subcellular fractions of N. caninum, and F2 and F4 showed higher sensitivity (93.3 %), kappa (0.41), and NPV values (75 %) than F1, and F3, which could be used for epidemiology studies such as screening.

Assessment of Risk Factors Associated with and Practices of Cattle Farmers in Kirehe District Rwanda with Respect to Vector-Borne and Zoonotic Pathogens.

Rural Rwandan communities face health challenges for humans and animals, and the topography and climate of the Kirehe District of Rwanda put farmers at high risk for mosquito-borne diseases. Individuals from 92 Rwandan farms were surveyed about farm practices, as well as animal and human health histories between December 2017 and February 2018. Human, animal, and environmental factors were investigated to determine whether there is a pattern of risk for abortion incidence and/or history of malarial disease on the farm. Iterative, complementary logistic regression models were used to determine whether there was an association between variables and abortion history in animals. These factors were then used to investigate association with a reported history of malaria. Of the 92 farms in our study, 82 were family farms and 10 were commercial farms. On average, 88% of the farms had cattle, and 30% of farms had experienced a cattle abortion in the past 2 years. There was no observed statistical significance in the risk factors for history of abortion in cattle and the measured variables. Using One Health as a guiding framework, we sought to determine whether human, animal, and environmental factors were statistically associated with observed disease outcomes. From our study of the practices of the farmers with respect to biosafety and self-protection against disease, we have identified potential sources of risk that could be targeted to enhance education and protection on these farms.

Identification and phylogenetic analysis of bovine herpesvirus 4 isolated from aborted bovine foetuses in Türkiye.

Bovine herpesvirus 4 (BoHV-4) is responsible for reproductive disorders and abortions in cattle. The aim of this study was to investigate the potential role of BoHV-4 in abortions in dairy cows when the possible aetiological cause could not be determined in Türkiye. The study also provided the molecular characterization of BoHV-4 strains based on the gB gene. In this study, foetuses (n = 188) and serum (n = 50) were retrospectively used for PCR and indirect ELISA assays, respectively. In addition, foetuses with BoHV-4 were tested for the isolation of the BoHV-4 strain in MDBK cell culture. The BoHV-4 gB gene was detected in the brain, liver, lung, kidney, and spleen tissues of three foetuses out of 188 aborted foetuses. The BoHV-4 strain was isolated from the kidney of one foetus in MDBK cell culture. The three aborted foetuses were from different cows in the same herd in 2021. On this dairy farm, the BoHV-4 seroprevalence rate in cattle with a history of abortion was 96% (48/50). Phylogenetic analysis revealed that the detected strains belong to genotype II. The present study shows that the impact of BoHV-4 on abortions in cattle should not be ignored. It can therefore be informative to focus on BoHV-4 in routine laboratory diagnostics for abortions without a proven aetiology or in cattle herds that have previously been infected with BoHV-4.

Seroprevalence and risk factors for Q fever and Rift Valley fever in pastoralists and their livestock in Afar, Ethiopia: A One Health approach.

Coxiella burnetii, the causative agent of Q fever, and Rift Valley fever virus are two under-researched zoonotic pathogens in Ethiopia. Potential outbreaks of these diseases, in light of the high dependency of nomadic pastoralists on their livestock, poses a risk to both human and animal health in addition to risking the pastoralists livelihoods. Our study aimed to determine the seroprevalence and associated risk factors for Q fever and Rift Valley fever in pastoral communities in the Afar region of north-eastern Ethiopia. This cross-sectional study screened pastoralists (n = 323) and their livestock (n = 1377) for IgG antibodies to Coxiella burnetii and Rift Valley fever virus. A seroprevalence for Q fever of 25.0% (95%CI 18.6-32.6) was found in pastoralists and 34.3% (95%CI 27.9-41.3) in livestock overall; with 51.9% in goats (95%CI 44.9-58.8), 39.9% in sheep (95%CI 24.6-51.2), 16.3% in camels (95%CI 10.4-24.6) and 8.8% in cattle (95%CI 5.0-15.0). For Rift Valley fever the seroprevalence in pastoralists was 6.1% (95%CI 3.3-11.0) and 3.9% (95%CI 2.6-5.7) in livestock overall; cattle had the highest seroprevalence (8.3%, 95%CI 3.3-19.2), followed by goats (2.7%; 95%CI 1.4-5.1), sheep (2.5%; 95%CI 1.0-5.9) and camels (1.8%; 95%CI 0.4-6.9). Human Q fever seropositivity was found to be associated with goat abortions (OR = 2.11, 95%CI 1.18-3.78, p = 0.011), while Rift Valley fever seropositivity in livestock was found to be associated with cattle abortions (OR = 2.52, 95%CI 1.05-6.08, p = 0.039). This study provides evidence for a notable exposure to both Q fever and Rift Valley fever in pastoralists and livestock in Afar. The outbreak potential of these pathogens warrants ongoing integrated human and animal surveillance requiring close collaboration of the human and animal health sectors with community representatives following a One Health approach.

Incidence and recurrence of bovine abortion in dairy cattle from Costa Rica

The reproductive efficiency and milk yield of cows are crucial factors in a dairy farm's profitability. However, abortions can have a negative impact on these factors. While the morbidity of abortion has been estimated in many countries, information on the burden on dairy cattle in tropical conditions is limited, and Costa Rica is a good example. This study aims to assess the incidence and recurrence of bovine abortion in dairy cattle from Costa Rica. The study analysed the morbidity of abortion in Costa Rican dairy herds between 2010 and 2022. The incidence rate (IR) and the recurrence rate (ReR) were calculated per 100 cow-months at risk using data from the Veterinary Automated Management and Production Control Programme (VAMPP). The dataset comprised 1032,457 lactations from 330,265 cows in 1134 specialized dairy herds. Abortions were classified either as early foetal mortality (EFM) or late foetal mortality (LFM). Rates were estimated based on cow breed, lactation number, and ecological zone to which the farm belongs. The IR of general abortion, EFM, and LFM cases were 0.98, 0.41, and 0.57 per 100 cow-months at risk, respectively. No statistically significant differences were found in the IR between cow breed, lactation number, and ecological zone, nor for the trend of abortions over calving years. The first ReR (for cows that had one previous abortion during the lactation) was 0.95, and the second ReR (for cows that had two previous abortions during the lactation) was 1.41 per 100 cow-months at risk. These results suggest that bovine abortions are an important ongoing problem in dairy farms in Costa Rica with potentially detrimental effects on the reproductive and productive performance of cows and may be representative of other specialized tropical dairy systems in Latin America.

Identification of the polymorphism of the <i>APAF1</i> gene and its association with economically useful traits of Holstein cattle

A decrease in the reproductive ability of cows leads to losses in dairy farming, as dairy production decreases due to the lack of a lactation period in cows. Haplotype HH1 is responsible for a mutation in the protein apoptotic protease-activating factor 1 (APAF1), as a result of which its structure is shortened, resulting in a decrease in fertility, an increase in the number of embryonic mortality and abortions in Holstein dairy cattle. To study the polymorphism of the APAF1 gene (SNP p.Q579X [c.1741C>T]) and its associations with economically traits, we used data and DNA from 254 cows of Holstein population of indigenous selection of Breeding Farm Integrated Agricultural Production Center of the Republic of Tatarstan. As a result, 2 alleles (Q and X) and 2 genotypes (QQ and QX) were identified in the APAF1 – BstC8 I gene locus in cattle. It was established that the dominant population is represented by carriers of the homozygous QQ genotype of the APAF1 gene - 98.0 %, and the frequency of the Q allele reached 0.990. The percentage of QX-type animals is minimal - 2.0 % of the total number of cows subjected to the study, as a result of which the occurrence of the X allele was only 0.010. An assessment of the influence of APAF1 gene polymorphism on economically significant traits showed that, depending on the genotype, such reproduction traits as the age of the first fertile insemination, the duration of the dry period and the Doha fertility index vary statistically significantly. Analysis of dairy productivity traits that cows with the QQ genotype are significantly superior to individuals with the QX genotype in all compared positions, with the exception of the content of the mass fraction of fat. Through selection using genetic marking, damage to livestock farms can be reduced by reducing the frequency of carriers of lethal alleles in the parent generation. This research was supported by FASO Russia project, registration number 122011800138-7.

In Vitro Virucidal Activity of Different Essential Oils against Bovine Viral Diarrhea Virus Used as Surrogate of Human Hepatitis C Virus.

The hepatitis C virus (HCV) is a major hepatotropic virus that affects humans with increased risk of developing hepatocellular carcinoma. The bovine viral diarrhea virus (BVDV) causes abortion, calf mortality and poor reproductive performance in cattle. Due the difficulties of in vitro cultivation for HCV, BVDV has been used as surrogate for in vitro assessment of the efficacy of antivirals. Essential oils (EOs) display antiviral and virucidal activity on several viral pathogens. In this study, the virucidal activity of five EOs, Salvia officinalis L. EO (SEO), Melissa officinalis L. EO (MEO), Citrus lemon EO (LEO), Rosmarinus officinalis L. EO (REO) and Thymus vulgaris L. EO (TEO) against BVDV was evaluated in vitro at different concentrations for several time contacts. MEO and LEO were able to considerably inactivate BVDV with a time- and dose-dependent fashion. MEO and LEO at the highest concentrations decreased viral titer by 2.00 and 2.25 log10 TCID50/50 μL at 8 h contact time, respectively. SEO, REO and TEO displayed mild virucidal activity at the highest concentrations for 8 h contact times. In this study, the virucidal efficacies of MEO and LEO against BVDV were observed regardless of compound concentration and contact time. Further studies are needed to confirm the potential use of MEO and LEO as surface disinfectants.

Functional characterization of three novel dense granule proteins in Neospora caninum using the CRISPR-Cas9 system

Neospora caninum is an obligate intracellular parasite that infects a wide range of mammalian species, and particularly causes abortions in cattle and nervous system dysfunction in dogs. Dense granule proteins (GRAs) are thought to play an important role in the mediation of host-parasite interactions and facilitating parasitism. However, a large number of potential GRAs remain uncharacterized, and the functions of most of the identified GRAs have not been elucidated. Previously, we screened a large number GRAs including NcGRA27 and NcGRA61 using the proximity-dependent biotin identification (BioID) technique. Here, we identified a novel GRA protein NcGRA85 and used C-terminal endogenous gene tagging to determine its localization at the parasitophorous vacuole (PV) in the tachyzoite. We successfully disrupted three gra genes (NcGRA27, NcGRA61 and NcGRA85) of N. caninum NC1 strain using CRISPR-Cas9-mediated homologous recombination and phenotyped the single knockout strain. The NcGRA61 and NcGRA85 genes were not essential for parasite replication and growth in vitro and for virulence during infection of mice, as observed by replication assays, plaque assays and in vitro virulence assays in mice. Deletion of the NcGRA27 gene in the NC1 strain reduced the in vitro replication and growth of the parasite, as well as the pathogenicity of the NC1 strain in mice. In summary, our findings provide a basis for in-depth studies of N. caninum pathogenesis and demonstrate the importance of NcGRA27 in parasite growth and virulence, most likely a new virulence factor of N. caninum.

Simultaneous detection of bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) using recombinase polymerase amplification

Bovine viral diarrhea virus (BVDV) is considered to be the most common agent of severe diarrhea in cattle worldwide, causing fever, diarrhea, ulcers, and abortion. Bovine herpesvirus 1 (BoHV-1) is also a major bovine respiratory disease agent that spreads worldwide and causes extensive damage to the livestock industry. Recombinase polymerase amplification (RPA) is a novel nucleic acid amplification method with the advantages of high efficiency, rapidity and sensitivity, which has been widely used in the diagnosis of infectious diseases. A dual RPA assay was developed for the simultaneous detection of BVDV and BoHV-1. The assay was completed at a constant temperature of 37 °C for 30 min. It was highly sensitive and had no cross-reactivity with other common bovine viruses. The detection rate of BVDV RPA in clinical samples (36.67%) was higher than that of PCR (33.33%), the detection rate of BoHV-1 RPA and PCR were equal. Therefore, the established dual RPA assay for BVDV and BoHV-1 could be a potential candidate for use as an immediate diagnostic.

Laboratory investigation of causes of bovine abortion and stillbirth in the Republic of Korea, 2014-2020.

Bovine abortion is a critical problem in the cattle industry. Identifying causes of abortion is key to establishing appropriate herd management and prevention strategies. We used pathology examinations, detection of etiologic agents, and serology to determine the cause of bovine abortions in Korea. We analyzed 360 abortion and stillbirth cases submitted to the Animal and Plant Quarantine Agency from December 2014 to January 2020. The putative cause of abortion was identified in 140 of 360 (38.9%) cases; 124 of the 140 (88.6%) cases were attributed to infections. The most common etiologic agents detected were bovine viral diarrhea virus (65 of 360; 18.1%), Coxiella burnetii (19 of 360; 5.3%), Leptospira spp. (13 of 360; 3.6%), Listeria monocytogenes (9 of 360; 2.5%), and Neospora caninum (8 of 360; 2.2%). Minor abortifacient pathogens included Brucella abortus (2 of 360; 0.6%), bovine alphaherpesvirus 1 (2 of 360; 0.6%), Akabane virus (2 of 360, 0.6%), and bovine ephemeral fever virus (1 of 360; 0.3%). Non-infectious conditions included congenital anomalies (7 of 360; 1.9%), goiter (7 of 360; 1.9%), and vitamin A deficiency (2 of 360; 0.6%). Our diagnostic rate in cases with placenta submitted (42 of 86; 48.8%) was significantly higher than in cases without placenta (98 of 274; 35.8%), which highlights the value of submitting placentas. Our results confirm the status of the large variety of causative agents associated with abortions in cattle in Korea.

Inactivated Flagellin-Containing Vaccine Efficacy against Ovine Enzootic Abortion.

Chlamydia abortus is the etiological agent of abortion and fetal loss in sheep, goats and bovine cattle in many countries. Even though commercially available vaccines can reduce the incidence in sheep, the development of new, safe, and effective vaccines remains high on the agenda. In this study, an evaluation was made of the efficacy of a vaccine candidate, an inactivated antigen based on the extract of outer membrane proteins of a C. abortus strain known as Chlamydia VNITIBP-21, in combination with recombinant flagellin as an adjuvant. Pregnant sheep (n = 43) were divided into three groups: an experimental vaccinated group, a control infected group and a control non-infected group. The sheep were vaccinated twice, with an interval of 3 weeks, then infected with the homologous virulent strain of Chlamydia abortus on pregnancy day 75. The vaccine candidate reduced C. abortus shedding in vaginal swabs considerably, in comparison with the control group. In addition, ewes in the experimental group experienced no abortions, while those in the control group experienced instances of abortion, as well as births of weak and nonviable lambs. The findings show that the vaccine candidate proved itself to be promising in combatting the agent of ovine abortion and fetal loss.

Molecular detection of bacterial zoonotic abortive agents from ruminants in Turkey.

Abortions in cattle and sheep are one of the major causes of economic losses worldwide. Brucella spp. are the most common infectious agent associated with these abortions. However, abortions caused by bacteria such as Listeria spp., Leptospira spp., Campylobacter spp. and Mycoplasma spp. are usually overlooked due to their sporadic nature and their status as non-priority abortion agents. In our study, we investigated the bacteria associated with abortion cases in cattle and sheep using PCR. For this purpose, we collected vaginal swab samples (n: 110) of aborted cattle and sheep, as well as stomach content samples (n: 69) of aborted calves and lambs from various cities in Turkey. The samples were analysed by bacteria-specific PCR to detect Campylobacter fetus, Leptospira spp., Listeria spp., Mycoplasma spp., and Yersinia spp. PCR analyses revealed that the investigated bacterial agents were present in 18.85% and 19.3% of the cattle and sheep samples, respectively, with an overall percentage of 18.99%. While the overall positivity rate for C. fetus, Leptospira spp., and Mycoplasma spp. was 2.79%, 10.06%, and 4.47%, respectively, the positivity rate for co-infection with Leptospira spp. and C. fetus was 1.68%. All samples were found to be negative for Yersinia spp. and Listeria spp. The high C. fetus positivity rate detected in sheep and in the stomach contents was statistically significant (p < 0.05). However, the difference in positivity rates between the cities, hosts, co-infections and causative agents was statistically insignificant (p > 0.05). This study provides preliminary data on the significant involvement of C. fetus, Leptospira spp. and Mycoplasma spp. in cattle and sheep abortions in Turkey indicating that they should not be overlooked in diagnosis. In addition, further research is needed to investigate the zoonotic potential of these pathogens for public health in Turkey.

Construction of luciferase-expressing Neospora caninum and drug screening

BackgroundNeospora caninum is an apicomplexan parasite that is particularly responsible for abortions in cattle and neuromuscular disease in dogs. Due to the limited effectiveness of currently available drugs, there is an urgent need for new therapeutic approaches to control neosporosis. Luciferase-based assays are potentially powerful tools in the search for antiprotozoal compounds, permitting the development of faster and more automated assays. The aim of this study was to construct a luciferase-expressing N. caninum and evaluate anti-N. caninum drugs.MethodsLuciferase-expressing N. caninum (Nc1-Luc) was constructed using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9). After testing the luciferase expression and phenotype of the Nc1-Luc strains, the drug sensitivity of Nc1-Luc strains was determined by treating them with known positive or negative drugs and calculating the half-maximal inhibitory concentration (IC50). The selective pan-rapidly accelerated fibrosarcoma (pan-RAF) inhibitor TAK-632 was then evaluated for anti-N. caninum effects using Nc1-Luc by luciferase activity reduction assay and other in vitro and in vivo studies.ResultsThe phenotypes and drug sensitivity of Nc1-Luc strains were consistent with those of the parental strains Nc1, and Nc1-Luc strains can be used to determine the IC50 for anti-N. caninum drugs. Using the Nc1-Luc strains, TAK-632 showed promising activity against N. caninum, with an IC50 of 0.6131 μM and a selectivity index (SI) of 62.53. In vitro studies demonstrated that TAK-632 inhibited the invasion, proliferation, and division of N. caninum tachyzoites. In vivo studies showed that TAK-632 attenuated the virulence of N. caninum in mice and significantly reduced the parasite burden in the brain.ConclusionsIn conclusion, a luciferase-expressing N. caninum strain was successfully constructed, which provides an effective tool for drug screening and related research on N. caninum. In addition, TAK-632 was found to inhibit the growth of N. caninum, which could be considered as a candidate lead compound for new therapeutics for neosporosis.Graphical

Using participatory epidemiology to investigate the causes of cattle abortion in Southwest Ethiopia

Cattle abortion, stemming from both infectious and non-infectious causes, lead to notable financial setbacks in the cattle industry. Between October 2020 and October 2021, an epidemiological investigation took place in Southwest Ethiopia. The objective was to determine the magnitude and seasonal occurrence of the presumed causes of cattle abortion. Information for this research was collected through 30 group discussions, each involving 8–12 participants. Various participatory epidemiological tools, including semi-structured interviews, pairwise ranking, matrix scoring, proportional piling, and seasonal calendars, were employed in the designated areas. By employing the pairwise ranking approach, the relative significance of presumed causes contributing to cattle abortion was established. The identified major presumed causes of cattle abortion, listed in increasing order of importance, were blackleg, foot-and-mouth disease, pasteurellosis, lumpy skin disease, listeriosis, trypanosomosis, Q fever, leptospirosis, and brucellosis. Participants identified brucellosis (6.1%), leptospirosis (6.0%), and Q-fever (5.7%) as the primary presumed causes of abortion, determined through proportional piling. Matrix scoring analysis indicated a robust agreement (W = 0.464–0.989; P < 0.001) among different informant groups regarding both the presumed causes of abortion and the associated clinical signs. Brucellosis and Q-fever were perceived to be more prevalent during the dry season, while leptospirosis, listeriosis, and lumpy skin disease were associated with the wet, hot, and rainy seasons. However, Pasteurellosis, blackleg, and physical/mechanical factors were deemed to be consistently encountered causes of abortion throughout the year. The patterns of seasonal occurrence of suspected abortion causes were widely acknowledged across informant groups (W = 0.977–0.863; P < 0.001). Local practices involving herbal remedies and traditional methods were employed by participants to manage cattle abortion. Moreover, the results underscore the necessity for additional laboratory research to pinpoint the exact causes of abortion in the study areas.

Infectious sporadic bovine abortions: retrospective analysis.

Infectious sporadic abortions in cattle are mainly caused by opportunistic bacteria and fungi usually present in environmental or gastrointestinal and reproductive microbiota of healthy animals. A retrospective analysis was carried out to evaluate the main opportunistic microorganisms involved in bovine abortions recorded at INTA Balcarce (Argentina) from 1997 to 2023, accounting for 2.2% of the total diagnosed etiologies of bovine abortion. The opportunistic agents identified as the cause of abortion in 29 fetuses were bacteria (90%) and fungi (10%). Escherichia coli (n = 8), Trueperella pyogenes (n = 5), and Histophilus somni (n = 4) were the bacterial species most often identified as causing infectious abortions, whereas Aspergillus spp. (n = 3) was implicated in all fungal abortions identified. Pure culture of bacteria or fungus was achieved from abomasal content and/or lung essential. Main microscopic findings were bronchopneumonia, myo- and epicarditis, meningitis, and portal hepatitis. Herein, we highlight the importance of detecting potential infectious bacteria in cultures to improve etiological diagnosis of bovine abortions associated with compatible microscopic findings to confirm the etiology.

TaqMan-quantitative PCR assays applied in Neospora caninum knock-outs generated through CRISPR-Cas9 allow to determine the copy numbers of integrated dihydrofolate reductase-thymidylate synthase drug selectable markers.

As for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in Neospora caninum, an apicomplexan parasite causing abortion in cattle and neuromuscular disease in dogs. A widely used approach for generating knock-out (KO) strains devoid of virulence factors is the integration of a drug selectable marker such as mutated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) into the target gene, thus preventing the synthesis of respective protein and mediating resistance to pyrimethamine. However, CRISPR-Cas9 mutagenesis is not free of off-target effects, which can lead to integration of multiple mdhfr-ts copies into other sites of the genome. To determine the number of integrated mdhfr-ts in N. caninum, a duplex quantitative TaqMan PCR was developed. For this purpose, primers were designed that amplifies a 106 bp fragment from wild-type (WT) parasites corresponding to the single copy wtdhfrs-ts gene, as well as the mutated mdhfrs-ts present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic NC5 gene. Thus, the dhfr-ts to NC5 ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated mdhrf-ts gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the Neospora KO strains NcΔGRA7 and NcΔROP40. For NcΔGRA7, the number of tachyzoites determined by dhfr-ts quantification was twice the number of tachyzoites determined by NC5 quantification, thus indicating that only one mdhfr-ts copy was integrated. The results obtained with the NcΔROP40 strain, however, showed that the number of dhfr-ts copies per genome was substantially higher, indicating that at least three copies of the selectable mdhfr-ts marker were integrated into the genomic DNA during gene editing by CRISPR-Cas9. This duplex TaqMan-qPCR provides a reliable and easy-to-use tool for assessing CRISPR-Cas9 mediated mutagenesis in WT N. caninum strains.

Avaliação do desempenho analítico de um teste imunocromatográfico rápido para o diagnóstico da brucelose bovina baseado em uma proteína recombinante BP26

ABSTRACT Brucellosis is an important bacterial disease of global distribution with zoonotic potencial. Serological tests used in Brazil for diagnosis of bovine brucellosis, including the Rose Bengal test (RBT), 2-mercaptoethanol (2-ME), fluorescent polarization (FPA), and complement fixation (FC), are based on the smooth lipopolysaccharide antigen (S-LPS) of Brucella abortus. The aim of this study was to evaluate a recombinant BP26 protein used as antigen in a rapid lateral flow immunochromatographic assay (rBP26-LFIA) for serological diagnosis of bovine brucellosis. Analytical performance of rBP26-LFIA was evaluated in positive and negative bovine serum samples previously characterized by RBT and 2-ME. Estimates of analytical sensitivity and specificity were 73.91% and 97.14%, respectively. Bovine sera reactive to Neospora, Trypanosoma vivax or Leptospira were used to assess specificity because these diseases are commonly associated with abortion in cattle. In addition to a possible cross-reaction induced by commercial vaccines against Leptospira in serological tests for bovine brucellosis using S-LPS as an antigen. In conclusion, rBP26-LFIA, with its current standardization, had good analytical performance. However, a future evaluation of diagnostic performance by rBP26-LFIA with samples from regions with known prevalence is necessary for its recommendation for use in the Brazilian program for the control and eradication of bovine brucellosis.

Prevalence, Public Health and Socioeconomic Importance of Infectious Diseases Abortion in Cattle in Africa 2007–2022

Prevalence, Public Health and Socioeconomic Importance of Infectious Diseases Abortion in Cattle in Africa 2007–2022

Distribution of Species and Biotypes of Brucella Isolates Obtained from Sheep and Cattle Abortions

Distribution of Species and Biotypes of Brucella Isolates Obtained from Sheep and Cattle Abortions