Genetic Features of Mycobacterium avium subsp. paratuberculosis Strains Circulating in the West of France Deciphered by Whole-Genome Sequencing.

Paratuberculosis is a chronic infection of the intestine, mainly the ileum, caused by Mycobacterium avium subsp. paratuberculosis in cattle and other ruminants. This enzootic disease is present worldwide and has a negative impact on the dairy cattle industry. For this subspecies, the current genotyping tools do not provide the needed resolution to investigate the genetic diversity of closely related strains. These limitations can be overcome by the application of whole-genome sequencing (WGS), particularly for clonal populations such as M. avium subsp. paratuberculosis. The purpose of the present study was to undertake a WGS analysis with a panel of 200 animal field M. avium subsp. paratuberculosis strains selected based on a previous large-scale longitudinal study of Prim'Holstein and Normande dairy breeds naturally infected with M. avium subsp. paratuberculosis in the West of France. The pangenome analysis revealed that M. avium subsp. paratuberculosis has a closed pangenome. The phylogeny, based on alignment of 2,786 nonhomoplasic single nucleotide polymorphisms (SNPs), showed that the strain population is structured into three clades independently of the cattle breed or geographic distribution. The increased resolution of phylogeny obtained by WGS confirmed the homoplasic nature of the markers variable-number tandem repeat (VNTR) and short sequence repeat (SSR) used for M. avium subsp. paratuberculosis genotyping. These phylogenetic data also revealed independent introductions of the different genotypes in two main waves since at least 2003. WGS applied to this sampling demonstrated the presence of mixed infections in herds and at the individual animal level. Collectively, the phylogeny results inferred with French isolates compared to M. avium subsp. paratuberculosis isolates from around the world suggest introductions of M. avium subsp. paratuberculosis genotypes through the animal trade. Relationships between genetic traits and epidemiological data can now be investigated to better understand transmission dynamics of the disease. IMPORTANCE Mycobacterium avium subsp. paratuberculosis causes Johne's disease in ruminants, which is present worldwide and has significant negative impacts on the dairy cattle industry and animal welfare. Prevention and control of M. avium subsp. paratuberculosis infection are hampered by knowledge gaps in strain virulence, genotype distribution, and transmission dynamics. This work has revealed new insights into M. avium subsp. paratuberculosis strains currently circulating in western France and how they are related to strains circulating globally. We applied whole-genome sequencing (WGS) to obtain comprehensive information on genome evolution and discrimination of closely related strains. This approach revealed the history of M. avium subsp. paratuberculosis infection in France, refined the pangenomic characteristics of M. avium subsp. paratuberculosis, and demonstrated the existence of mixed infection in animals. Finally, this study identified predominant genotypes, which allow a better understanding of disease transmission dynamics. This information will facilitate tracking of this pathogen on farms and across agricultural regions, thus informing transmission pathways and disease control points.

New insights into the genetic resistance to paratuberculosis in Holstein cattle via single-step genomic evaluation.

BACKGROUND: Bovine paratuberculosis, or Johne's disease (JD), is a contagious and incurable disease caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has adverse effects on animal welfare and is very difficult to control, leading to serious economic consequences. An important line of defense to this disease is host genetic resistance to MAP, which, when it will be more fully understood, could be improved through selective breeding. Using a large dataset of Holstein cows (161,253 animals including 56,766 cows with ELISA serological phenotypes and 12,431 animals with genotypes), we applied a single-step single nucleotide polymorphism (SNP) best linear unbiased prediction approach to investigate the genetic determinism underlying resistance to this disease (heritability estimate and identification of relevant genomic regions) and estimated genetic trends, reliability, and relative risk factors associated with genomic predictions. RESULTS: Resistance to JD was moderately heritable (0.14) and 16 genomic regions were detected that accounted for at least 0.05% of the breeding values variance (GV) in resistance to JD, and were located on chromosomes 1, 3, 5, 6, 7, 19, 20, 21, 23, 25, and 27, with the highest percentage of variance explained by regions on chromosomes 23 (0.36% GV), 5 (0.22% GV), 1 (0.14% GV), and 3 (0.13% GV). When estimated for the whole chromosomes, the autosomes with the largest overall contributions were chromosomes 3 (5.3% GV), 10 (4.8%), 23 (4.7%), 1 (3.6%), 7 (3.4%), 5 (2.9%), 12 (2.5%), 11 (2.2%), and 13 (2%). We estimated a slightly favorable genetic trend in resistance to JD over the last two decades, which can be explained by a low positive genetic correlation between resistance to JD and total merit index (+ 0.06). Finally, in a validation population of 907 cows, relatively reliable genomic predictions (reliability = 0.55) were obtained, which allowed the identification of cows at high risk of infection. CONCLUSIONS: This study provides new insights into the genetic determinism of resistance to JD and shows that this trait can be predicted from SNP genotypes. It has led to the implementation of a single-step genomic evaluation that should rapidly become an effective tool for controlling paratuberculosis on French Holstein farms.

Rumen fluke infections (Paramphistomidae) in diarrhoeal cattle in western France and association with production parameters.

This study was conducted to assess the impact of rumen flukes (RFs) (Paramphistomatidae) on various production parameters of cattle in Normandy. Faecal and blood samples were taken between 1 January 2010 and 31 December 2019 as part of the routine diagnostic activity for diarrhoea in weaned cattle, including a quantitative parasitological analysis coupled with a search for Johne's disease (paratuberculosis). Information on slaughter and carcass weight, inter-farm movements and mortality was obtained from the French national registration database (BDNI). The study was conducted at two levels using adapted categorical RF variables: 1) at the cattle level (>12 months), an estimation of presence of adult parasites using egg count in faecal samples (negative vs. positive) and 2) at the herd level, an estimation of 6-24 months of the dairy heifers exposure to larval forms on the basis of the proportion of dairy cattle shedding eggs in the herd (three classes). At the cattle level, the outcome variables were carcass weight (model 1), case-control status for mortality within 30 days of diagnosis (model 2) and case-control status for slaughter within 365 days of diagnosis (model 3). At the herd level, the outcome variable was the mortality ratio for dairy heifers aged 6-24 months (Model 4). Other important covariates were used to improve model fit. Multivariate analyses were performed using a linear mixed model (model 1), generalised estimating equations (GEE) (models 2 and 3) and a multinomial logistic model (model 4). Overall, 1291 out of 4315 cattle (29.9%) were found to excrete RF eggs and 19.6% of the positives had an excretion score of >200 eggs/g. The prevalence increased steadily with age class and was higher in beef cattle than dairy cattle (42.7% vs. 26.9%) in the years 2015-2019 compared to 2010-2014 (33.3% vs. 26.5%) and in November-February (33.2%) compared to March-June (28.9%) and July-October (27.3%). Rumen fluke variables were not found to be explanatory factors of outcome variables at both animal and herd levels. In contrast, significant negative associations were observed between outcome variables and other health covariates, such as Johne's disease, GI nematode, bovine viral diarrhoea and coccidia statuses. In conclusion, RFs are prevalent in cattle reared in Normandy but this does not result in significant production losses. Therefore, the value to farmers of oxyclozanide treatment at an effective dose for paramphistomosis after simple identification of RF eggs in the faeces seems limited.

Draft Genome Sequences of 142 Mycobacterium avium subsp. paratuberculosis Strains Isolated from Naturally Infected Dairy Cattle.

Mycobacterium avium subsp. paratuberculosis is the etiological agent of Johne's disease in ruminants. Here, we report the annotated draft genome sequences of 142 M. avium subsp. paratuberculosis strains that were isolated from dairy cattle in France between 2014 and 2018. The genomes of these strains were sequenced using Illumina technology.

Identification of the ABCC4, IER3, and CBFA2T2 candidate genes for resistance to paratuberculosis from sequence-based GWAS in Holstein and Normande dairy cattle.

BACKGROUND: Bovine paratuberculosis is a contagious disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), with adverse effects on animal welfare and serious economic consequences. Published results on host genetic resistance to MAP are inconsistent, mainly because of difficulties in characterizing the infection status of cows. The objectives of this study were to identify quantitative trait loci (QTL) for resistance to MAP in Holstein and Normande cows with an accurately defined status for MAP. RESULTS: From MAP-infected herds, cows without clinical signs of disease were subjected to at least four repeated serum ELISA and fecal PCR tests over time to determine both infected and non-infected statuses. Clinical cases were confirmed using PCR. Only cows that had concordant results for all tests were included in further analyses. Positive and control cows were matched within herd according to their birth date to ensure a same level of exposure to MAP. Cows with accurate phenotypes, i.e. unaffected (control) or affected (clinical or non-clinical cases), were genotyped with the Illumina BovineSNP50 BeadChip. Genotypes were imputed to whole-genome sequences using the 1000 Bull Genomes reference population (run6). A genome-wide association study (GWAS) of MAP status of 1644 Holstein and 649 Normande cows, using either two (controls versus cases) or three classes of phenotype (controls, non-clinical and clinical cases), revealed three regions, on Bos taurus (BTA) chromosomes 12, 13, and 23, presenting significant effects in Holstein cows, while only one of those was identified in Normande cows (BTA23). The most significant effect was found on BTA13, in a short 8.5-kb region. Conditional analyses revealed that only one causal variant may be responsible for the effects observed on each chromosome with the ABCC4 (BTA12), CBFA2T2 (BTA13), and IER3 (BTA23) genes as good functional candidates. CONCLUSIONS: A sequence-based GWAS on cows for which resistance to MAP was accurately defined, was able to identify candidate variants located in genes that were functionally related to resistance to MAP; these explained up to 28% of the genetic variance of the trait. These results are very encouraging for efforts towards implementation of a breeding strategy aimed at improving resistance to paratuberculosis in Holstein cows.

Longitudinal study of Mycobacterium avium ssp. paratuberculosis fecal shedding patterns and concurrent serological patterns in naturally infected dairy cattle.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a disease that affects ruminants worldwide. Despite global interest in the control of this disease, gaps exist in our knowledge of fecal shedding patterns and concurrent serological patterns. This longitudinal study in dairy cattle herds with high MAP seroprevalence in France aimed at accurately describing fecal shedding patterns over 1 year; relating those shedding patterns to individual animal characteristics (age, breed, parity); and exploring the association between fecal shedding patterns and serological patterns. To describe temporal fecal shedding patterns and continuity of shedding, along with the standard quantitative PCR (qPCR) threshold cycle we used a cutoff value that related to low or nonculturable fecal shedding. We also defined a threshold cycle indicative of shedding in high quantities to describe infection progression patterns. Twenty-one herds completed the study, and 782 cows were tested 4 times each. We obtained 4 sets of paired fecal qPCR and serum ELISA results from 757 cows. Although we targeted highly likely infectious animals, we found a large diversity of shedding patterns, as well as high variability between herds in the proportion of animals showing a given pattern. The fecal qPCR results of almost 20% of the final study sample were positioned at least once in the range that indicated low or nonculturable fecal shedding (between the adjusted and the standard cutoff value). Although these animals would typically be classified as non-shedders, they could be important to infection dynamics on the farm. Animals that shed at least twice consecutively and animals that shed in high quantities rarely reverted to negativity. Repeated fecal qPCR can be used to detect temporal fecal shedding traits, and the decision to cull an animal could practically be based on temporal, semiquantitative results. Overall, we found a mismatch between fecal shedding and ELISA seropositivity (637 animals were ELISA-negative 4 times, but only 13% of those animals were qPCR-negative 4 times). We found that having more than 2 ELISA-positive samples was strongly related to persistent and continuous shedding. We suggest that although serological testing is much less sensitive than qPCR, it can also be used, particularly over the course of multiple testing events, to identify animals that are most likely to contribute to the contamination of the farm environment.

Sensitivities of a bulk-tank milk ELISA and composite fecal qPCR to detect various seroprevalence levels of paratuberculosis in cattle herds in Normandy, France.

This study evaluated an ELISA on bulk tank milk (BTM) samples and a qPCR on a single composite fecal sample to detect paratuberculosis seropositive cattle dairy herds. Individual serum (n = 15 372), BTM and composite fecal samples were obtained from 192 herds. The within-herd apparent seroprevalence was categorized and compared with BTM ELISA and fecal qPCR results. The BTM ELISA had poor overall sensitivity (16%) to detect seropositive herds but higher sensitivity (53%) in the higher apparent seroprevalence group of > 9%. Using an optimized cut-off point (5.0% S/P), sensitivities overall and in the high apparent seroprevalence group were 53% and 88%, respectively. The BTM ELISA gave 5% positive results in seronegative herds and 25% using the optimized cut-off. Fecal qPCR had 72% sensitivity to detect seropositive herds and 88% in the higher apparent seroprevalence group, but gave 25% positive results in fully seronegative herds. The combination of BTM ELISA and composite fecal qPCR improved the sensitivity to detect seropositive herds.

Sensibilités d'un test ELISA sur lait de réservoir et d'une qPCR sur prélèvement composite de fèces pour le dépistage de cheptels bovins à différents niveaux de séroprévalence en paratuberculose en Normandie, France. L'étude a été entreprise pour évaluer les performances diagnostiques d'un test ELISA, effectué sur un échantillon de lait de réservoir (BTM), et d'une qPCR, réalisée sur un échantillon composite de fèces (CF), pour détecter les troupeaux de bovins séropositifs pour la paratuberculose. Les sérums individuels (n = 15 372), les échantillons de BTM et de CF ont été collectés dans 192 troupeaux. La séroprévalence apparente intra-troupeau a été calculée puis catégorisée avant d'être comparée aux résultats de l'ELISA sur BTM et de la qPCR sur CF. Le test ELISA sur BTM a montré une faible sensibilité globale (16 %) mais celle-ci était plus élevée dans les élevages les plus fortement séropositifs > 9 % (53 %). En utilisant un seuil optimisé (E/P 5,0 %), les sensibilités étaient de 53 % et 88 %, respectivement. Le test ELISA sur BTM a donné 5 % de résultats positifs sur des troupeaux entièrement séronégatifs et 25 % en utilisant le seuil optimisé. La qPCR sur CF avait une bonne sensibilité (72 %), en particulier dans les élevages fortement séropositifs (88 %), et a donné 25 % de résultats positifs sur des cheptels entièrement séronégatifs. L'utilisation combinée de BTM ELISA et CF qPCR a permis d'améliorer la sensibilité à dépister des cheptels séropositifs.(Traduit par Docteur Serge Messier).

The association between Ostertagia ostertagi antibodies in bulk tank milk samples and parameters linked to cattle reproduction and mortality.

In Western Europe, gastrointestinal nematodes are widespread in dairy cattle. This study was carried out to evaluate the relationship between optical density ratio (ODR) measured on bulk tank milk with an indirect Ostertagia ostertagi ELISA and reproduction/mortality parameters. Data were collected between 2008 and 2010 from monitoring carried out on 1643 dairy herds (Normandy, Western France). ODR values of 3 samples from each farm taken from November 2008 to 2010 were averaged and then transformed into a categorical variable. Reproductive and mortality data were obtained from 1444 herds using cow records from government databases. Statistical analysis was carried out using ordinary logistic regression (OLR). The outcome variables were the case-control status of a herd for reproductive factors, age at first calving and inter-calving intervals, and mortality ratios of various age classes. The effect of the categorical ODR variable was studied and several potential confounder herd factors were used to improve the model fit. A significant relationship was found between high Ostertagia ODR levels and a late age at the first calving (>34.5 months) (odds ratio (OR)=1.94, p<0.001). No significant relationship was observed with OLR for inter-calving intervals although bivariate analysis showed that herds with high ODR levels had longer inter-calving intervals than herds with low ODR level (first inter-calving interval in herds with low vs. high ODR levels=412 days vs. 422 days, p<0.001; other inter-calving intervals=408 days vs. 413 days, p<0.01). A high ODR level was also associated with high mortality of calves between 0 and 30 days of life (mortality ratio>6%) (OR=1.43, p<0.05) and between 91 and 365 days (ratio>3%) (OR=1.72, p<0.01). No significant relationship was observed with multivariate approach for mortalities in other classes by age, but bivariate analysis showed that herds with high ODR level had higher mortalities than herds with low ODR levels (mortality between 31 and 90 days in herds with low vs. high ODR levels=1.89% vs. 2.91%, p<0.001; mortality after 365 days=1.67% vs. 2.93%, p<0.001). In conclusion, our results confirm the usefulness of ELISA as an indicator for production losses in dairy herds. This inexpensive tool could be advantageous, used to aid farmers and veterinarians to carry out appropriate control measures.

Herd-level risk factors for Cryptosporidium infection in dairy-goat kids in western France.

We conducted a cross-sectional study of risk factors for herd-level kid positivity for Cryptosporidium parvum oocysts in dairy-goat farms (Deux-Sèvres, western France). From January to March 2003, faeces from a convenient sample of 879 5- to 30-day-old goat kids from 60 herds were examined microscopically after staining with carbol fuschin. Oocyst shedding was scored semi-quantitatively (0 to 4+) allowing us to obtain a cumulative score per herd. Standardized questionnaires with information about management practices were collected in each farm. We found positive kids in 32 of 60 herds (53.3%) and in 142 animals out of 879 (16.2%). We used logistic regression for two risk-factor model: (1) simple positive (case: herd score > or = 1+, at least one positive kid in the herd, versus control: herd score=0), (2) strongly positive (case: overall herd score > or = 3+ versus control: herd score <3+). Risk factors associated with simple positive herds were period of sampling compared to the peak of births (After versus Before, OR=4.2, 95% CI 1.2, 15.3) and practice of kid grouping by age or weight (Yes versus No, OR=4.4, 95% CI 1.0, 19.1). Risk factors associated with strongly positive herds were period of investigation (February/March versus January, OR=12.7, 95% CI 2.1, 76.6), exposure to graminaceous plants in forage (OR=11.6, 95% CI 1.7, 81.0) and type of ventilation in the goat premises (Vertical versus Wind effect, OR=14.7, 95% CI 2.1, 106.1). No important association was found between kid-management practices and herd positivity. These results suggest a major role of the environment of kids during their first hours of life in the adult-goat premises regarding the transmission of C. parvum infection.

Epidemiology of Trypanosoma vivax infection in cattle in the tse-tse free area of Lake Chad.

A study was conducted in Chad to estimate the prevalence and the incidence of Trypanosoma vivax infection in herds of cattle from the Lake Chad area. The risk factors associated with disease were also identified. A random sample of 933 cattle from 17 herds was initially selected (January 1999, cold dry season). Cattle were identified by ear-tags and sampled in the rainy season (July 1999) and the cold dry season (January 2000). Each animal sampled was treated with diminazene aceturate (3.5mg/kg). Samples were examined for the presence of T. vivax using an antibody (indirect ELISA) and a parasite detection test (buffy-coat technique, BCT). Standardized questionnaires with information about the host and management practices were collected and evaluated for their association with seroprevalence (model 1) and parasitological prevalence (model 2) as indicator of host susceptibility to T. vivax infection. Risk factors were selected using two approaches: ordinary logistic regression (OLR) and generalized estimating equations (GEE) to account for within-herd correlation. The apparent prevalence was 1.6% using BCT and 42.3% with indirect ELISA. The true prevalence in the sample was estimated to (2.0%-8.0%) with two assumptions of BCT sensitivity. Overall, 58.8% (BCT) and 100.0% (ELISA) of the herds had a least one-positive animal. In January-July 1999, apparent monthly incidence was calculated at 0.24% in comparison with 0.76% for August 1999-January 2000. The true monthly incidence was estimated at 0.36%-1.43% for the first period and at 0.94%-3.78% for the second period. Risk factors associated with seroprevalence were age, race, a great number of small ruminants in the herd, and latitude and longitude of pasture area in the rainy season. Risk factors associated with BCT prevalence were duration of seasonal migration and longitude of pasture area in the rainy season. In conclusion, T. vivax is present and widely disseminated in the cattle herds of tse-tse free area of Lake Chad. Farm-level factors, particularly seasonal migration, should be considered as the main risk factors for infection and to host susceptibility to the parasite.

Prevalence of Trypanosoma evansi infection and associated risk factors in camels in eastern Chad.

A cross-sectional study was conducted to estimate the prevalence of Trypanosoma evansi infection (Surra) in herds of camels from the eastern area of Chad. The risk factors associated with disease were also identified. From August 1997 to April 1998, a random sample of 2831 camels from 136 herds was selected. Blood samples were collected and examined for the presence of T. evansi using an antibody (card agglutination test-CATT/T. evansi) and a parasite detection test (buffy-coat technique-BCT). Standardized questionnaires with information about the host and management practices were collected and evaluated for their association with seroprevalence (model 1) and parasitological prevalence (model 2) as indications of host sensitivity. In both models, risk factors were selected using ordinary logistic regression (OLR) and herd effect was evaluated using a generalized estimating equations (GEE) model. The apparent prevalence was 5.3% using BCT and 30.5% with CATT. Real prevalence was estimated at 16.9% +/- 1.4 (alpha = 5%). Overall, 27.9% (BCT) and 94.9% (CATT) of the herds had a least one-positive animal. Real herd prevalence was estimated at 42.6 +/- 8.3% (alpha = 5%). Camels of the large transhumants had the highest prevalence (estimated to 30.3% +/- 2.5; 62.9 +/- 12.0 in herds). Risk factors associated with seroprevalence were age, ethnic group, length of seasonal migration and longitude of pasture area in the dry season. Risk factors associated with BCT prevalence were age, length of seasonal migration, longitude of pasture area in the dry season, latitude of pasture area in the rainy season and season of sampling.