Multiomics and bioinformatics identify differentially expressed effectors in the brain of Toxoplasma gondii infected masked palm civet.

Introduction: The masked palm civet (Paguma larvata) serves as a reservoir in transmitting pathogens, such as Toxoplasma gondii, to humans. However, the pathogenesis of T. gondii infection in masked palm civets has not been explored. We studied the molecular changes in the brain tissue of masked palm civets chronically infected with T. gondii ME49. Methods: The differentially expressed proteins in the brain tissue were investigated using iTRAQ and bioinformatics. Results: A total of 268 differential proteins were identified, of which 111 were upregulated and 157 were downregulated. KEGG analysis identified pathways including PI3K-Akt signaling pathway, proteoglycans in cancer, carbon metabolism, T-cell receptor signaling pathway. Combing transcriptomic and proteomics data, we identified 24 genes that were differentially expressed on both mRNA and protein levels. The top four upregulated proteins were REEP3, REEP4, TEP1, and EEPD1, which was confirmed by western blot and immunohistochemistry. KEGG analysis of these 24 genes identified signaling cascades that were associated with small cell lung cancer, breast cancer, Toll-like receptor signaling pathway, Wnt signaling pathways among others. To understand the mechanism of the observed alteration, we conducted immune infiltration analysis using TIMER databases which identified immune cells that are associated with the upregulation of these proteins. Protein network analysis identified 44 proteins that were in close relation to all four proteins. These proteins were significantly enriched in immunoregulation and cancer pathways including PI3K-Akt signaling pathway, Notch signaling pathway, chemokine signaling pathway, cell cycle, breast cancer, and prostate cancer. Bioinformatics utilizing two cancer databases (TCGA and GEPIA) revealed that the four genes were upregulated in many cancer types including glioblastoma (GBM). In addition, higher expression of REEP3 and EEPD1 was associated with better prognosis, while higher expression of REEP4 and TEP1 was associated with poor prognosis in GBM patients. Discussion: We identified the differentially expressed genes in the brain of T. gondii infected masked palm civets. These genes were associated with various cellular signaling pathways including those that are immune- and cancer-related.

Immune responses following neonatal vaccination with conserved F4 fragment of VtaA proteins from virulent Glaesserella parasuis adjuvanted with CAF®01 or CDA.

Glaesserella parasuis is a Gram-negative bacterium that colonizes the upper airways of swine, capable of causing a systemic infection called Glässer's disease. This disease is more frequent in young post-weaning piglets. Current treatments against G. parasuis infection are based on the use of antimicrobials or inactivated vaccines, which promote limited cross-protection against different serovars. For this reason, there is an interest in developing novel subunit vaccines with the capacity to confer effective protection against different virulent strains. Herein, we characterize the immunogenicity and the potential benefits of neonatal immunization with two different vaccine formulations based on the F4 polypeptide, a conserved immunogenic protein fragment from the virulence-associated trimeric autotransporters of virulent G. parasuis strains. With this purpose, we immunized two groups of piglets with F4 combined with cationic adjuvant CAF®01 or cyclic dinucleotide CDA. Piglets immunized with a commercial bacterin and non-immunized animals served as control groups. The vaccinated piglets received two doses of vaccine, at 14 days old and 21 days later. The immune response induced against the F4 polypeptide varied depending on the adjuvant used. Piglets vaccinated with the F4+CDA vaccine developed specific anti-F4 IgGs, biased towards the induction of IgG1 responses, whereas no anti-F4 IgGs were de novo induced after immunization with the CAF®01 vaccine. Piglets immunized with both formulations displayed balanced memory T-cell responses, evidenced upon in vitro re-stimulation of peripheral blood mononuclear cells with F4. Interestingly, pigs immunized with F4+CAF®01 controlled more efficiently a natural nasal colonization by a virulent serovar 4 G. parasuis that spontaneously occurred during the experimental procedure. According to the results, the immunogenicity and the protection afforded by F4 depend on the adjuvant used. F4 may represent a candidate to consider for a Glässer's disease vaccine and could contribute to a better understanding of the mechanisms involved in protection against virulent G. parasuis colonization.

Insight into the current Toxoplasma gondii DNA vaccine: a review article.

INTRODUCTION: Toxoplasma gondii (T.gondii) is a widespread protozoan with significant economic losses and public health importance. But so far, the protective effect of reported DNA-based vaccines fluctuates widely, and no study has demonstrated complete protection. AREAS COVERED: This review provides an inclusive summary of T. gondii DNA vaccine antigens, adjuvants, and some other parameters. A total of 140 articles from 2000 to 2021 were collected from five databases. By contrasting the outcomes of acute and chronic challenges, we aimed to investigate and identify viable immunological strategies for optimum protection. Furthermore, we evaluated and discussed the impact of several parameters on challenge outcomes in the hopes of developing some recommendations to assist better future horizontal comparisons among research. EXPERT OPINION: In the coming five years of research, the exploration of vaccine cocktails combining invasion antigens and metabolic antigens with genetic adjuvants or novel DNA delivery methods may offer us desirable protection against this multiple stage of life parasite. In addition to finding a better immune strategy, developing better in silico prediction methods, solving problems posed by variables in practical applications, and gaining a more profound knowledge of T.gondii-host molecular interaction is also crucial towards a successful vaccine.

SARS-CoV-2: An Updated Review Highlighting Its Evolution and Treatments.

Since the SARS-CoV-2 outbreak, pharmaceutical companies and researchers worldwide have worked hard to develop vaccines and drugs to end the SARS-CoV-2 pandemic. The potential pathogen responsible for Coronavirus Disease 2019 (COVID-19), SARS-CoV-2, belongs to a novel lineage of beta coronaviruses in the subgenus arbovirus. Antiviral drugs, convalescent plasma, monoclonal antibodies, and vaccines are effective treatments for SARS-CoV-2 and are beneficial in preventing infection. Numerous studies have already been conducted using the genome sequence of SARS-CoV-2 in comparison with that of other SARS-like viruses, and numerous treatments/prevention measures are currently undergoing or have already undergone clinical trials. We summarize these studies in depth in the hopes of highlighting some key details that will help us to better understand the viral origin, epidemiology, and treatments of the virus.

Seroprevalence and risk factors of tropical theileriosis in smallholder asymptomatic large ruminants in Egypt.

Knowledge of the prevalence and epidemiological determinants of tropical theileriosis in large ruminants, particularly in the asymptomatic carrier, is crucial for designing and implementing effective host-specific control measures. This study aimed to estimate the seroprevalence of tropical theileriosis in asymptomatic cattle and water buffaloes and identify the potential risk factors of theileriosis in large ruminants raised under smallholder-production system in Egypt. A cross-sectional study was conducted in five districts of the Sharkia governorate from March 2019 to February 2020. In total, 350 serum samples were collected from cattle and water buffaloes under smallholder-production system and tested for Theileria annulata antibodies using the indirect antibody fluorescence test (IFAT). Data on species, host characteristics, presence of ticks, season, and districts were collected at sampling using a questionnaire. A multivariable mixed-effects logistic regression model was built to determine the potential risk factors associated with T. annulate seropositivity of the animals. The overall apparent seroprevalence of T. annulata in 350 tested animals was 70%. In the univariable analyses, cattle compared to buffaloes, younger animals compared to older ones, animals with ticks on their bodies, and warmer seasons were all associated with a higher likelihood of seropositive results in the study population while sex of the animals was not associated with seropositivity. The final multivariable model showed that animals with ticks on their bodies had 3.5× higher odds of seropositivity than those with no ticks (P < 0.001), and warmer seasons were associated with the higher odds of infection compared to winter (P = 0.003). The high seroprevalence of tropical theileriosis in the study region indicates that the disease is endemic among smallholders of large ruminants. The identified risk factors of T. annulata-seropositivity in asymptomatic carrier animals provides evidence-based guidance for adopting effective intervention measures.

Four Chemotherapeutic Compounds That Limit Blood-Brain-Barrier Invasion by Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii, an intracellular protozoan parasite, exists in the host brain as cysts, which can result in Toxoplasmic Encephalitis (TE) and neurological diseases. However, few studies have been conducted on TE, particularly on how to prevent it. Previous proteomics studies have showed that the expression of C3 in rat brains was up-regulated after T. gondii infection. METHODS: In this study, we used T. gondii to infect mice and bEnd 3 cells to confirm the relation between T. gondii and the expression of C3. BEnd3 cells membrane proteins which directly interacted with C3a were screened by pull down. Finally, animal behavior experiments were conducted to compare the differences in the inhibitory ability of TE by four chemotherapeutic compounds (SB290157, CVF, NSC23766, and Anxa1). RESULTS: All chemotherapeutic compounds in this study can inhibit TE and cognitive behavior in the host. However, Anxa 1 is the most suitable material to inhibit mice TE. CONCLUSION: T. gondii infection promotes TE by promoting host C3 production. Anxa1 was selected as the most appropriate material to prevent TE among four chemotherapeutic compounds closely related to C3.

Unveiling of brain transcriptome of masked palm civet (Paguma larvata) with chronic infection of Toxoplasma gondii.

BACKGROUND: The aim of this study was to gain an understanding of the transcriptomic changes that occur in a wild species when infected with Toxoplasma gondii. The masked palm civet, an artifically domesticated animal, was used as the model of a wild species. Transcriptome analysis was used to study alterations in gene expression in the domesticated masked palm civet after chronic infection with T. gondii. METHODS: Masked palm civets were infected with 105 T. gondii cysts and their brain tissue collected after 4 months of infection. RNA sequencing (RNA-Seq) was used to gain insight into the spectrum of genes that were differentially expressed due to infection. Quantitative reverse-transcription PCR (qRT-PCR) was also used to validate the level of expression of a set of differentially expressed genes (DEGs) obtained by sequencing. RESULTS: DEGs were screened from the sequencing results and analyzed. A total of 2808 DEGs were detected, of which 860 were upregulated and 1948 were downregulated. RNA-Seq results were confirmed by qRT-PCR. DEGs were mainly enriched in cellular process and metabolic process based on gene ontology enrichment analysis. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that transcriptional changes in the brain of infected masked palm civets evolved over the course of infection and that DEGs were mainly enriched in the signal transduction, immune system processes, transport and catabolic pathways. Finally, 10 essential driving genes were identified from the immune signaling pathway. CONCLUSIONS: This study revealed novel host genes which may provide target genes for the development of new therapeutics and detection methods for T. gondii infection in wild animals.

Bayesian Estimation of Diagnostic Accuracy of Three Diagnostic Tests for Bovine Tuberculosis in Egyptian Dairy Cattle Using Latent Class Models.

The aim of the present study was to calculate the sensitivity (Se) and specificity (Sp) of the single cervical tuberculin test (SCT), rapid lateral flow test (RLFT), and real-time polymerase chain reaction (RT-PCR) for the diagnosis of Mycobacterium bovis (M. bovis) infection in Egyptian dairy cattle herds within a Bayesian framework. The true M. bovis infection within-herd prevalence was assessed as a secondary objective. Data on the test results of SCT, RLFT, and RT-PCR for the detection of M. bovis were available from 245 cows in eleven herds in six major governorates in Egypt. A Bayesian latent class model was built for the estimation of the characteristics of the three tests. Our findings showed that Se of SCT (0.93 (95% Posterior credible interval (PCI): 0.89-0.93)) was higher than that of RT-PCR (0.83 (95% PCI: 0.28-0.93)) but was similar to the Se of RLFT (0.93 (95% PCI: 0.31-0.99)). On the contrary, SCT showed the lowest Sp estimate (0.60 (95% PCI: 0.59-0.65)), whereas Sp estimates of RT-PCR (0.99 (95% PCI: 0.95-1.00)) and RLFT (0.99 (95% PCI: 0.95-1.00)) were comparable. The true prevalence of M. bovis ranged between 0.07 and 0.71. In conclusion, overall, RT-PCR and RLFT registered superior performance to SCT, making them good candidates for routine use in the Egyptian bovine tuberculosis control program.

Characterization of Foot and Mouth Disease Virus Serotype SAT-2 in Swamp Water Buffaloes (Bubalus bubalis) under the Egyptian Smallholder Production System.

Spontaneous mutations are a common characteristic of the foot and mouth disease virus (FMDV), leading to wide antigenic variations resulting in the emergence of new topotypes and lineages of FMDV, which contributes to occasional vaccination failures. The objectives of the present study were to genetically characterize FMDV isolated from water buffaloes and study the biochemical and histopathological indicators of infected animals. Fifty-four water buffaloes of both sexes and different ages suffered from acute symptoms of FMD were clinically examined and randomly selected for inclusion in this study. Oral desquamated epithelial and oropharyngeal fluid samples have been tested for FMDV by reverse transcriptase PCR (RT-PCR). Tissue and serum samples were also collected from the diseased buffaloes and subjected to histopathological and biochemical analysis. Our findings showed that all examined samples were confirmed to be positive to FMDV serotype SAT-2 and were adjusted to be responsible for the recent disease outbreak in this study. Phylogenetic analysis revealed that the circulating viruses were of the SAT-2 serotype, closely related to the lineage of lib12, topotype VII, with 98.9% identity. The new lineage of SAT-2 showed a high virulence resulting in the deaths of water buffaloes due to heart failure, confirmed by high serum levels of inflammatory and cardiac markers, including haptoglobin, ceruloplasmin, cardiac troponin I and creatine phosphokinase-MB, indicating an unfavorable FMD-infection prognosis. In conclusion, we document the presence of new incursions circulating in water buffalo populations in Egypt in early 2019, explaining the high morbidity rate of FMD outbreak in early 2019. Furthermore, the newly identified serotype SAT-2 lib12 lineage, topotype VII, showed an aggressive pattern in water buffaloes of the smallholder production system.

Sensitivity and specificity of piroplasm indirect fluorescent antibody test and PCR for Theileria annulata infection in clinically asymptomatic large ruminants using Bayesian latent class analysis.

There is limited information about the accuracy of molecular and serological diagnostic assays for tropical theileriosis in asymptomatic carrier large ruminants. This study has estimated the sensitivity (Se) and specificity (Sp) of PCR and an indirect fluorescent antibody test (IFAT) in the diagnosis of tropical theileriosis in cattle and buffaloes via a Bayesian latent class analysis (BLCA) framework. Blood samples were collected from 70 cattle and water buffaloes (Bubalus bubalis) raised under a smallholder production system in different Egyptian localities. T. annulata infection status was detected by PCR, and IFAT and the test results were subjected to BLCA without assuming the existence of a reference test. Our findings showed that the performance of PCR was superior to that of IFAT. PCR showed a higher Se [0.83 (95% PCI: 0.63-0.98)] in comparison to IFAT [0.72 (95% PCI: 0.68-0.75)]. Similarly, PCR showed a higher Sp [0.95 (95% PCI: 0.77-1.00)] than IFAT [0.82 (95% PCI: 0.80-0.84)]. Se and Sp of the two tests did not differ by species implying that the diagnostics' performance for T. annulata infection in bovines is the same regardless of the species under consideration. In conclusion, PCR outperforms IFAT in the detection of T. annulata infection and can thus be applied to routine control of tropical theileriosis in endemic situations where cattle and buffaloes are kept under traditional smallholder production systems.

Chromosome-Scale Genome of Masked Palm Civet (Paguma larvata) Shows Genomic Signatures of Its Biological Characteristics and Evolution.

The masked palm civet (Paguma larvata) is a small carnivore with distinct biological characteristics, that likes an omnivorous diet and also serves as a vector of pathogens. Although this species is not an endangered animal, its population is reportedly declining. Since the severe acute respiratory syndrome (SARS) epidemic in 2003, the public has been particularly concerned about this species. Here, we present the first genome of the P. larvata, comprising 22 chromosomes assembled using single-tube long fragment read (stLFR) and Hi-C technologies. The genome length is 2.41 Gb with a scaffold N50 of 105.6 Mb. We identified the 107.13 Mb X chromosome and one 1.34 Mb Y-linked scaffold and validated them by resequencing 45 P. larvata individuals. We predicted 18,340 protein-coding genes, among which 18,333 genes were functionally annotated. Interestingly, several biological pathways related to immune defenses were found to be significantly expanded. Also, more than 40% of the enriched pathways on the positively selected genes (PSGs) were identified to be closely related to immunity and survival. These enriched gene families were inferred to be essential for the P. larvata for defense against the pathogens. However, we did not find a direct genomic basis for its adaptation to omnivorous diet despite multiple attempts of comparative genomic analysis. In addition, we evaluated the susceptibility of the P. larvata to the SARS-CoV-2 by screening the RNA expression of the ACE2 and TMPRSS2/TMPRSS4 genes in 16 organs. Finally, we explored the genome-wide heterozygosity and compared it with other animals to evaluate the population status of this species. Taken together, this chromosome-scale genome of the P. larvata provides a necessary resource and insights for understanding the genetic basis of its biological characteristics, evolution, and disease transmission control.

Transcriptome Analysis of Testes and Uterus: Reproductive Dysfunction Induced by Toxoplasma gondii in Mice.

Toxoplasma gondii (T. gondii) infection in female mammals during pregnancy can result in poor pregnancy. Similarly, it can result in male reproductive disorders in male mammals. Although the testes and uterus have very different biological makeup, they are still both attacked by T. gondii resulting in reproductive dysfunctions. We hypothesized that there are significant common genes in the testes and uterus that interact with T. gondii. Finding out and studying these genes is vital to understand the infection mechanism of T. gondii and the induced disease pathogenesis. To achieve this goal, we built a mice model of acute infection with T. gondii and the testes and uterus of the mice were sequenced by RNA-Seq. A total of 291 and 679 significantly differently expressed genes (DEGs) were obtained from the testes and the uterus, respectively. In the Gene Ontology (GO) analysis, part of the DEGs in the testes and uterus were related to 35 GO functions. When compared with the KEGG database, seven pathways affecting both the testes and uterus during the course of T. gondii infection were identified. In addition, Toxoplasmosis can significantly affect the expression of Nlrp5 and Insc leading to negative outcomes in the host. On the other hand, the host regulates Gbp7, Gbp2b, and Ifit3 to defend against T. gondii infection.

Accuracy of PCR, mycobacterial culture and interferon-γ assays for detection of Mycobacterium bovis in blood and milk samples from Egyptian dairy cows using Bayesian modelling.

The number of bovine tuberculosis (bTB) infected dairy herds in Egypt is growing and this calls for accurate and reliable diagnostic methods at cow level for cost-effective bTB eradication as culling of the whole herd is not economically sustainable. The present study aimed to estimate the sensitivity (Se) and specificity (Sp) of PCR, mycobacterial culture and interferon-γ (IFN-γ) assays for Mycobacterium bovis (M. bovis) detection in blood and milk samples from dairy cows in Egyptian dairy herds within a Bayesian framework. As a secondary objective, the distribution of true within-herd prevalence of M. bovis infection was estimated. Blood and milk samples were collected from 245 Holstein dairy cows in 11 Egyptian dairy herds and subjected to PCR, mycobacterial culture and IFN-γ testing. With respect to the detection of M. bovis in blood, IFN-γ recorded higher Se [0.97 (95% Posterior Credible Interval (PCI): 0.87-1.00)] than PCR [0.68 (95% PCI: 0.53-0.95)] and culture [0.22 (95% PCI: 0.13-0.37)]. However, Sp estimates of PCR [0.98 (95% PCI: 0.95-1.00)], culture [0.99 (95% PCI: 0.98-1.00)] and IFN-γ [0.97 (95% PCI: 0.88-1.00)] were comparable. As for milk samples, Se estimate of PCR [0.29 (95% PCI: 0.01-0.60)] was higher than that of culture [0.08 (95% PCI: 0.001-0.23)]. However, the Sp estimates of both tests were statistically similar. The estimated true within-herd prevalences of M. bovis varied across the tested bovine subpopulations and ranged between 0.06 and 0.66. In conclusion, IFN-γ registered a similar overall performance to PCR but was superior to mycobacterial culture. With its good accuracy and wide applicability, IFN-γ lends itself to use in the Egyptian bTB eradication program.

A Transcriptome Analysis: Various Reasons of Adverse Pregnancy Outcomes Caused by Acute Toxoplasma gondii Infection.

BACKGROUND: Toxoplasma gondii (T. gondii) is an obligate intracellular parasite, which can affect the pregnancy outcomes in infected females by damaging the uterus, and the intrauterine environment as well as and the hypothalamus resulting in hormonal imbalance. However, the molecular mechanisms underlying the parasite-induced poor pregnancy outcomes and the key genes regulating these mechanisms remain unclear. Therefore, this study aimed to analyze the gene expression in the mouse's uterus following experimentally-induced acute infection with T. gondii RH strain. Three groups of female mice were intraperitoneally injected with tachyzoites as follow; 3 days before pregnancy (FBD6), after pregnancy (FAD6), and after implantation (FID8) as the experimental groups. Another corresponding three groups served as control, were injected with normal saline at the same time. Transcriptome analysis of the total RNA extracted from both infected and non-infected mouse uterus samples was performed using RNA sequencing (RNA-Seq). RESULTS: The three experimental groups (FBD6, FAD6, and FID8) had a total of 4,561, 2,345, and 2,997 differentially expressed genes (DEGs) compared to the controls. The significantly upregulated and downregulated DEGs were 2,571 and 1,990 genes in FBD6, 1,042 and 1,303 genes in FAD6 and 1,162 and 1,835 genes in FID8 group, respectively. The analysis of GO annotation, and KEGG pathway showed that DEGs were mainly involved in anatomical structure development, transport, cell differentiation, embryo development, hormone biosynthetic process, signal transduction, immune system process, phagosome, pathways in cancer, and cytokine-cytokine receptor interaction pathways. CONCLUSION: T. gondii infection can induce global transcriptomic changes in the uterus that may cause pregnancy hypertension, destruct the intrauterine environment, and hinder the normal development of placenta and embryo. Our results may help to understand the molecular mechanisms of the acute T. gondii infection, which could promote the development of new therapeutics or prophylactics for toxoplasmosis in pregnancy.

Variations in association of nasal microbiota with virulent and non-virulent strains of Glaesserella (Haemophilus) parasuis in weaning piglets.

Glaesserella (formerly Haemophilus) parasuis causes Glässer's disease, which results in high economic loss in the swine industry. To understand the polymicrobial interactions of G. parasuis and the nasal microbiota, the statistical association patterns of nasal colonizing bacteria with virulent and non-virulent strains of G. parasuis were studied accounting for the farm management practices as potential risk factors for the occurrence of Glässer's disease. The nasal microbiota from 51 weaned-piglets from four farms with Glässer's disease and three farms with no respiratory diseases was previously characterized and included in this study. The presence of virulent and/or non-virulent G. parasuis strains in the nasal cavities was determined in order to establish the potential association with other members of the nasal microbiota. Multivariate logistic and linear regression models were performed among the various members of nasal microbiota and G. parasuis. The multi-site production system and disease presence in the farm were both significantly associated with the presence of G. parasuis virulent strains in the nose of the piglets. Differential bacterial associations were observed with virulent or non-virulent G. parasuis. Chitinophagaceae, Corynebacteriaceae and Corynebacterium were positively associated with the virulent G. parasuis strains, while Enterobacteriaceae, Peptostreptococcaceae, Clostridium XI, and Escherichia/Shigella were negatively associated with virulent G. parasuis. On the other hand, Flavobacteriaceae, Planobacterium, and Phascolarctobacterium were positively associated with the non-virulent G. parasuis strains, while Rikenellaceae, Enterococcaceae, Odoribacter, and Corynebacterium were negatively associated with non-virulent G. parasuis. In conclusion, the nasal microbiota communities showed variations in the association with the G. parasuis strains type.

Comparison of phenotypic and genotypic antimicrobial resistance patterns associated with Staphylococcus aureus mastitis in German and Danish dairy cows.

Staphylococcus aureus is one of the most common pathogens associated with bovine mastitis in Germany and Denmark. Successful therapy is strongly linked to the susceptibility of the pathogen to the administered antimicrobial. An increase in resistant pathogens in human and veterinary medicine has become a concern worldwide and hampers therapy due to reduced susceptibility. In the present study, susceptibility testing was performed for 85 and 93 S. aureus isolates originating from mastitis cases on 12 German and 8 Danish dairy farms, respectively. Phenotypic examination was performed by detection of minimal inhibitory concentration (MIC) values using the broth microdilution method, followed by genotypic investigations of the blaZ and mecA resistance genes via PCR. The tested antimicrobials were the most frequently used ß-lactams in German and Danish dairy farms, including cefquinome, cefoperazone, cephapirin, penicillin, oxacillin, cloxacillin, amoxicillin-clavulanic acid, and cephalexin-kanamycin. Special attention was paid to varying therapy concepts because, in Germany, third- and fourth-generation cephalosporins have been predominantly used in mastitis therapy, whereas in Denmark, restrictive use of penicillin is followed by a general avoidance of cephalosporins. Differences in MIC values were mainly based on determined MIC90 values (MIC at which 90% of isolates are inhibited). In general, Danish S. aureus isolates were inhibited at comparatively lower MIC90 values than S. aureus isolated from German dairy farms for most ß-lactams. No differences were observed regarding cefquinome, because both German and Danish isolates exhibited MIC50 and MIC90 values of 0.5 and 1 µg/mL, respectively. In contrast, the MIC90 for penicillin against German and Danish S. aureus were 0.5 and ≤0.06 µg/mL, respectively. Resistance genes (blaZ, mecA) were only detected in German S. aureus isolates on 3 dairy farms in Germany. A total of 5 isolates tested positive for both blaZ and mecA, whereas 1 isolate carried the blaZ resistance gene only. A direct correlation between frequently used antimicrobials and reduced susceptibility could not be determined based on results of the present study. In addition to further research to determine factors associated with resistance development, we emphasize the urgent need for internationally standardized clinical breakpoints to assess resistance situations more accurately.

Molecular detection of Treponema species organisms in foremilk and udder cleft skin of dairy cows with digital dermatitis.

Identification of reservoirs and transmission routes of digital dermatitis (DD)-associated Treponema spp. is considered an effective means for controlling DD infection in dairy cows. The objective of this study is to identify and characterize the potential reservoir niches for DD-associated Treponema spp. from healthy udder cleft skin and foremilk in lactating dairy cows. A large dairy farm was visited weekly from March to July 2015. Clinical investigation revealed that a total of 25 lame cows had DD lesions located at the plantar aspect of the interdigital cleft. A total of 75 samples, three per cow, were collected including deep swabs from DD lesions (n = 25), non-aseptically collected foremilk samples (n = 25) and skin swabs from udder cleft (n = 25). Treponema spp. were identified using nested PCR assays and confirmed by DNA sequencing. Results revealed that Treponema phagedenis (T. phagedenis)-like was the most identified species in the foremilk 40% (10/25), in comparison with DD lesions and udder cleft skin samples with 32% (8/25) and 20% (5/25), respectively. On the other hand, Treponema pedis (T. pedis) was the most identified species in the udder cleft skin 80% (20/25), in comparison with DD lesions and foremilk samples with 68% (17/25) and 60% (15/25), respectively. None of the examined samples were identified by PCR as containing DNA from Treponema medium (T. medium) or Treponema vincentii (T. vincentii)-like. To the best of our knowledge, this is the first report for detection of T. phagedenis-like and T. pedis from healthy skin of udder cleft and foremilk samples. Detection of DD Treponema spp. from udder cleft skin and foremilk samples indicates that these sites could be potential reservoirs for spirochetes involved in DD. Udder cleft skin and foremilk may have a role in transmission routes of DD Treponema in dairy farms.

Transcriptomic analysis of reproductive damage in the epididymis of male Kunming mice induced by chronic infection of Toxoplasma gondii PRU strain.

BACKGROUND: Some researchers have reported that Toxoplasma gondii can cause serious reproductive impairment in male animals. Specifically, T. gondii destroy the quality of sperm in the epididymis, which affects their sexual ability. However, among such studies, none have investigated the male reproductive transcriptome. Therefore, to investigate the relationship between T. gondii and sperm maturation, we infected mice with T. gondii prugniaud (PRU) strain and performed transcriptome sequencing of the epididymis. RESULTS: Compared with the control group, 431 upregulated and 229 downregulated differentially expressed genes (DEGs) were found (P-value < 0.05, false discovery rate (FDR) < 0.05 and |log2 (fold change)| ≥ 1). According to results of a bioinformatics analysis, Gene Ontology (GO) function is divided into three categories: cellular component, molecular function and biological process. Upon performing GO analysis, we found that some DEGs correlated with an integral part of membrane, protein complex, cell surface, ATP binding, immune system process, signal transduction and metabolic process which are responsible for the epididymal injury. DEGs were mapped to 101 unique KEGG pathways. Pathways such as cytokine-cytokine receptor interaction, glycolysis/gluconeogenesis and apoptosis are closely related to sperm quality. Moreover, Tnfsf10 and spata18 can damage the mitochondria in sperm, which decreases sperm motility and morphology. CONCLUSIONS: We sequenced the reproductive system of male mice chronically infected with T. gondii, which provides a new direction for research into male sterility caused by Toxoplasma infection. This work provides valuable information and a comprehensive database for future studies of the interaction between T. gondii infection and the male reproductive system.

Diagnostic performance of direct and indirect methods for assessing failure of transfer of passive immunity in dairy calves using latent class analysis.

Accurate diagnosis of failure of transfer of passive immunity (FTPI) in newborn calves is an essential component of dairy farm management plan. Several methods (direct and indirect) are available for diagnosis of FTPI in dairy calves. However, the indirect methods offer an advantage over the direct methods in not requiring an experienced veterinarian, rapid, cost efficient and can be performed under field-setting. The objective of this study was to estimate the diagnostic performance of radial immunodiffusion (RID) assay, transmission infrared (TIR) spectroscopy and digital Brix refractometer for diagnosis of FTPI in dairy calves using latent class models at four cut-off values of digital Brix refractometer. Holstein calves (n = 691) from 40 commercial dairy farms in the four Atlantic Canada provinces were blood-sampled and tested for detection of FTPI. Results showed that the number of calves with FTPI was 253 (36.6%) by RID, 194 (28.1%) by TIR and 204 (29.5%) by Brix refractometer at cut-off value of 8.2%. Estimates of SeRID was higher than SeTIR and SeBrix, at all Brix refractometer cut-offs, but with increase of Brix refractometer cut-off from 8.2 to 8.5%, SeRID and SeTIR were decreased from 96.0% (95% PCI: 88.0-99.0) and 79.0% (95% PCI: 70.0-85.0), to 92.0% (95% PCI: 77.0-99.0) and 74.0% (95% PCI: 61.0-82.0), respectively. SpRID and SpTIR were always higher than SpBrix at all tested cut-offs and were above 92.0%, and 96.0%, respectively. With increasing the cut-off of Brix refractometer from 8.2 to 8.5%, SeBrix estimate has remarkably increased from 79.0% (95% PCI: 70.0-96.0) to 95.0% (95% PCI: 87.0-100.0), respectively. Whilst, SpBrix was decreased from 95.0% (95% PCI: 91.0-98.0) at cut-off 8.2% to 84.0% (95% PCI: 78.0-94.0) at cut-off 8.5%. In conclusion, RID has a higher Se than TIR and Brix, if the latter is used with cut-offs of 8.2% or 8.3%. However, the higher the cut-off, the more comparable sensitivities of RID and digital Brix refractometer. The median estimate of SpTIR was always higher than SpRID and SpBrix at all tested cut-offs. However, the 95% confidence interval estimates of the three tests were overlapping across the tested cut-offs of digital Brix refractometer reflecting the inability to prefer a test over the other based on the Sp estimate.

Association between teat skin colonization and intramammary infection with Staphylococcus aureus and Streptococcus agalactiae in herds with automatic milking systems.

The objective of this study was to investigate the association between teat skin colonization and intramammary infection (IMI) with Staphylococcus aureus or Streptococcus agalactiae at the quarter level in herds with automatic milking systems. Milk and teat skin samples from 1,142 quarters were collected from 300 cows with somatic cell count >200,000 cells/mL from 8 herds positive for Strep. agalactiae. All milk and teat skin samples were cultured on calf blood agar and selective media. A subset of samples from 287 quarters was further analyzed using a PCR assay (Mastit4 PCR; DNA Diagnostic A/S, Risskov, Denmark). Bacterial culture detected Staph. aureus in 93 (8.1%) of the milk samples and 75 (6.6%) of the teat skin samples. Of these, 15 (1.3%) quarters were positive in both the teat skin and milk samples. Streptococcus agalactiae was cultured in 84 (7.4%) of the milk samples and 4 (0.35%) of the teat skin samples. Of these, 3 (0.26%) quarters were positive in both the teat skin and milk samples. The PCR detected Staph. aureus in 29 (10%) of the milk samples and 45 (16%) of the teat skin samples. Of these, 2 (0.7%) quarters were positive in both the teat skin and milk samples. Streptococcus agalactiae was detected in 40 (14%) of the milk samples and 51 (18%) of the teat skin samples. Of these, 16 (5.6%) quarters were positive in both the teat skin and milk samples. Logistic regression was used to investigate the association between teat skin colonization and IMI at the quarter level. Based on bacterial culture results, teat skin colonization with Staph. aureus resulted in 7.8 (95% confidence interval: 2.9; 20.6) times higher odds of Staph. aureus IMI, whereas herd was observed as a major confounder. However, results from the PCR analyses did not support this association. Streptococcus agalactiae was isolated from the teat skin with both PCR and bacterial culture, but the number of positive teat skin samples detected by culture was too low to proceed with further analysis. Based on the PCR results, Strep. agalactiae on teat skin resulted in 3.8 (1.4; 10.1) times higher odds of Strep. agalactiae IMI. Our results suggest that Staph. aureus and Strep. agalactiae on teat skin may be a risk factor for IMI with the same pathogens. Focus on proper teat skin hygiene is therefore recommended also in AMS.