Within-herd transmission of Mycoplasma bovis infections after initial detection in dairy cows.

Mycoplasma bovis outbreaks in cattle, including pathogen spread between age groups, are not well understood. Our objective was to estimate within-herd transmission across adult dairy cows, youngstock, and calves. Results from 3 tests (PCR, ELISA, and culture) per cow and 2 tests (PCR and ELISA) per youngstock and calf were used in an age-stratified susceptible-infected-removed/recovered (SIR) model to estimate within-herd transmission parameters, pathways, and potential effects of farm management practices. A cohort of adult cows, youngstock, and calves on 20 Dutch dairy farms with a clinical outbreak of M. bovis in adult cows were sampled, with collection of blood, conjunctival fluid, and milk from cows, and blood and conjunctival fluid from calves and youngstock, 5 times over a time span of 12 wk. Any individual with at least one positive laboratory test was considered M. bovis-positive. Transmission dynamics were modeled using an age-stratified SIR model featuring 3 age strata. Associations with farm management practices were explored using Fisher's exact tests and Poisson regression. Estimated transmission parameters were highly variable among herds and cattle age groups. Notably, transmission from cows to cows, youngstock, or to calves was associated with R-values ranging from 1.0 to 80 secondarily infected cows per herd, 1.2 to 38 secondarily infected youngstock per herd, and 0.1 to 91 secondarily infected calves per herd, respectively. In case of transmission from youngstock to youngstock, calves or to cows, R-values were 0.7 to 96 secondarily infected youngstock per herd, 1.1 to 76 secondarily infected calves per herd, and 0.1 to 107 secondarily infected cows per herd. For transmission from calves to calves, youngstock or to cows, R-values were 0.5 to 60 secondarily infected calves per herd, 1.1 to 41 secondarily infected youngstock per herd, and 0.1 to 47 secondarily infected cows per herd. Among on-farm transmission pathways, cow-to-youngstock, cow-to-calf, and cow-to-cow were identified as most significant contributors, with calf-to-calf and calf-to-youngstock also having noteworthy roles. Youngstock-to-youngstock was also implicated, albeit to a lesser extent. Whereas the primary focus was a clinical outbreak of M. bovis among adult dairy cows, it was evident that transmission extended to calves and youngstock, contributing to overall spread. Factors influencing transmission and specific transmission pathways were associated with internal biosecurity (separate caretakers for various age groups, number of people involved), external biosecurity (contractors, external employees), as well as indirect transmission routes (number of feed and water stations).

Zoonotic risks of pathogens from sheep and their milk borne transmission.

Sheep were domesticated around 9000 BC in the Middle East, and since then milk from sheep gradually became very popular, not only for drinking but also for making cheeses and other dairy products. Nowadays, these dairy products are also important for people with an allergy to cow milk, and these products are an essential part of the local daily diet in regions of the world that are not suitable for cows and goats. Consumption of raw milk and raw milk products has a zoonotic risk, and with regard to sheep, the main pathogens associated with such dairy products are: Brucella melitensis, Campylobacter spp., Listeria spp., Salmonella spp., Shiga-toxin producing Escherichia coli, Staphylococcus aureus, tick borne encephalitis virus, and Toxoplasma gondii. Especially, young children, elderly people, pregnant women and immunocompromised (YOPI) persons, and those suffering from disease should be aware of the risk of consuming raw milk and raw milk products. This latter risk can be reduced by proper flock health management, prevention of contamination during milking, adequate milk processing, transport, and refrigerated storage. Only processes equaling pasteurization sufficiently reduce zoonotic risks from milk and milk products, but proper cooling is essential and recontamination must be prevented. Therefore, strict hygiene practices throughout the production process and supply chain especially for raw milk and raw dairy products, should be applied. Small scale production systems pose a greater risk compared to industrialized production systems because of a less protocolized and controlled production process. This manuscript describes zoonotic risks of pathogens from sheep and their milk borne transmission. Additionally, routes of contamination, possibilities for multiplication, and prevention measures thereof are described. We summarize some major human outbreaks caused by consumption of sheep milk and products made thereof, and finally discuss their implications.

Molecular characterisation of Mycoplasma bovis isolates from consecutive episodes of respiratory disease on Dutch veal farms.

Mycoplasma bovis infections are wide spread in veal calf farms and a major contributor to respiratory disease. M. bovis are genetically diverse. It is unclear how this diversity influences the virulence and epidemiology of infections on veal calf farms over time. Therefore, the aim of this study was to follow the genetic composition of M. bovis isolates on veal farms over time in a fattening round and combine this with presence of disease and presence of other respiratory pathogens. For this, M. bovis isolates were obtained from healthy and diseased calves from ten different farms at different episodes of respiratory disease in the same groups in one fattening round. A new episode of respiratory disease was defined by the practitioner based on clinical diagnosis at least 7 days after end of a previous metaphylactic treatment. These isolates were sequenced using Illumina sequencing and analysed. This resulted in 148 sequenced isolates. The isolates belonged to 9 different clusters and to the known MLST sequence types ST4 (n=9), ST6 (n=2), ST7 (n=1), ST8 (n=1), ST21 (n=32), ST29 (n=30), ST32 (n=1), ST100 (n=36), ST122 (n=17) and ST135 (n=4), and new sequence types ST222 (n=8), ST223 (n=1), ST224 (n=5) and ST225 (n=1). Major sequence types are linked to types, found in other European countries. All farms showed presence of two or more different clusters, however with different distribution patterns. Farms did not show a major shift in type distribution over time. There was a relationship between M. bovis type and region of origin of the calves and the types differed with regards of presence of variable membrane surface lipoprotein (Vsp) genes. Types were not related to disease status of the calves or presence of other major respiratory pathogens. This study underlines the complexity of M. bovis infection on veal calf farms with persistent presence of different types together in both healthy and diseased calves with or without other respiratory pathogens. Prevention of introduction of M. bovis and biosecurity measures combined with optimisation of calf resilience should have priority.

Impact of carbon dioxide concentrations on laboratory sensitivity of Mycoplasma species isolated from dairy cows.

Conventional Mycoplasma spp. diagnostics involve culture, often considered the gold standard in diagnostic test evaluation. However, culture protocols lack empirical derivation and primarily adhere to National Mastitis Council recommendations, tracing back to initial cultivation of Mycoplasma bovis. Despite a wide range of carbon dioxide (CO2) supplementation reported in literature, specific impacts of CO2 on Mycoplasma spp. growth remain unexplored. Our objective was to assess the effect of CO2 concentration on growth detection rates of 24 Mycoplasma spp. isolates from dairy cows. These isolates, mainly M. bovis, were incubated at 37°C in triplicate and three dilution ranges under three CO2 conditions: ambient air or 5% CO2 or 10% CO2. Bacterial growth was evaluated on incubation days 3, 5, 7, and 10. When cultured using ambient air, log10 cfu/mL was lower on days 3, 5, and 7 of incubation compared with isolates incubated in the recommended 5% or 10% CO2, with less variation observed in ambient air compared with 5% or 10% CO2. However, by 10 days of incubation, no differences in the detection of observable growth were noted among isolates incubated in ambient air, 5% CO2, or 10% CO2. Consequently, Mycoplasma spp. isolated from dairy cattle demonstrated growth after the recommended 7-10 days of culture, even in the absence of supplemental CO2. Given the expected concentration of M. bovis in (sub)clinical samples had similar concentrations to those used in our study, with the majority of isolates being M. bovis, we recommend expanding CO2 concentration ranges in M. bovis culture from 10% CO2 to ambient air when incubating for 10 days. However, the turnaround time could be shortened when incubating with supplemental CO2. IMPORTANCE: Current Mycoplasma spp. culture protocols lack empirical derivation concerning carbon dioxide (CO2) supplementation and are primarily based on the initial cultivation of Mycoplasma bovis. This study indicates that the suitable range for CO2 supplementation is broader than what is currently recommended by the National Mastitis Council for culturing within the specified 7-10 days. No differences in bacterial growth detection rates were observed among ambient air, 5% CO2, or 10% CO2 supplementation during the 7- and 10-day incubation intervals. These new insights provide evidence supporting the possibility of culturing Mycoplasma spp. under ambient air conditions in a laboratory setting.

Monitoring and Surveillance of Small Ruminant Health in The Netherlands.

In contemporary society and modern livestock farming, a monitoring and surveillance system for animal health has become indispensable. In addition to obligations arising from European regulations regarding monitoring and surveillance of animal diseases, The Netherlands developed a voluntary system for the monitoring and surveillance of small ruminant health. This system aims for (1) early detection of outbreaks of designated animal diseases, (2) early detection of yet unknown disease conditions, and (3) insight into trends and developments. To meet these objectives, a system is in place based on four main surveillance components, namely a consultancy helpdesk, diagnostic services, multiple networks, and an annual data analysis. This paper describes the current system and its ongoing development and gives an impression of nearly twenty years of performance by providing a general overview of key findings and three elaborated examples of notable disease outbreaks. Results indicate that the current system has added value to the detection of various (re)emerging and new diseases. Nevertheless, animal health monitoring and surveillance require a flexible approach that is able to keep pace with changes and developments within the industry. Therefore, monitoring and surveillance systems should be continuously adapted and improved using new techniques and insights.

Dairy Sheep Played a Minor Role in the 2005-2010 Human Q Fever Outbreak in The Netherlands Compared to Dairy Goats.

Q fever is an almost ubiquitous zoonosis caused by Coxiella burnetii. This organism infects several animal species, as well as humans, and domestic ruminants like cattle, sheep and goats are an important animal reservoir of C. burnetii. In 2007, a sudden rise in notified human Q fever cases occurred in The Netherlands, and by the end of 2009, more than 3500 human Q fever patients had been notified. Dairy sheep and dairy goats were suspected to play a causal role in this human Q fever outbreak, and several measures were taken, aiming at a reduction of C. burnetii shedding by infected small ruminants, in order to reduce environmental contamination and thus human exposure. One of the first measures was compulsory notification of more than five percent abortion within thirty days for dairy sheep and dairy goat farms, starting 12 June 2008. After notification, an official farm inspection took place, and laboratory investigations were performed aiming at ruling out or demonstrating a causal role of C. burnetii. These measures were effective, and the number of human Q fever cases decreased; levels are currently the same as they were prior to 2007. The effect of these measures was monitored using a bulk tank milk (BTM) PCR and an antibody ELISA. The percentage PCR positive dairy herds and flocks decreased over time, and dairy sheep flocks tested PCR positive significantly less often and became PCR negative earlier compared to dairy goat herds. Although there was no difference in the percentage of dairy goat and dairy sheep farms with a C. burnetii abortion outbreak, the total number of shedding dairy sheep was much lower than the number of shedding dairy goats. Combined with the fact that Q fever patients lived mainly in the proximity of infected dairy goat farms and that no Q fever patients could be linked directly to dairy sheep farms, although this may have happened in individual cases, we conclude that dairy sheep did not play a major role in the Dutch Q fever outbreak. BTM monitoring using both a PCR and an ELISA is essential to determine a potential C. burnetii risk, not only for The Netherlands but for other countries with small ruminant dairy industries.

An intra-laboratory cultural and real-time PCR method comparison and evaluation for the detection of subclinical paratuberculosis in dairy herds.

Mycobacterium avium subsp. paratuberculosis (MAP) is a vigorous microorganism which causes incurable chronic enteritis, Johne's disease (JD) in cattle. A target of control programmes for JD is to accurately detect MAP-infected cattle early to reduce disease transmission. The present study evaluated the efficacy of two different cultural procedures and a TaqMan real-time PCR assay for detection of subclinical paratuberculosis in dairy herds. Therefore, sixty-one faecal samples were collected from two Dutch dairy herds (n = 40 and n = 21, respectively) which were known to be MAP-ELISA positive. All individual samples were assessed using two different cultural protocols in two different laboratories. The first cultural protocol (first laboratory) included a decontamination step with 0.75% hexadecylpyridinium chloride (HPC) followed by inoculation on Herrold's egg yolk media (HEYM). The second protocol (second laboratory) comprised of a decontamination step using 4% NaOH and malachite green-oxalic acid followed by inoculation on two media, HEYM and in parallel on modified Löwenstein-Jensen media (mLJ). For the TaqMan real-time PCR assay, all faecal samples were tested in two different laboratories using TaqMan® MAP (Johne's) reagents (Life Technologies). The cultural procedures revealed positive reactions in 1.64% of the samples for cultivation protocol 1 and 6.56 and 8.20% of the samples for cultivation protocol 2, respectively. The results of the TaqMan real-time PCR performed in two different laboratories yielded 13.11 and 19.76% positive reaction. The kappa test showed proportional agreement 0.54 between the mLJ media (second laboratory) and TaqMan® real-time PCR method (second laboratory). In conclusion, the TaqMan real-time PCR could be a strongly useful and efficient assay for the detection of subclinical paratuberculosis in dairy cattle leading to an improvement in the efficiency of MAP control strategies.