NEWS

Research Article

Elvira Ramovic and 9 colleagues reported a pilot study to assess the rate of MAP infection in Irish dairy and beef cattle herds, and much more.  The abstract is below and the comments that follow discuss the dairy survey prevalence data specifically. The article is in the Irish Veterinary Journal and is Open Access.

Abstract

Background: Dairy and beef cattle can be reservoirs of many pathogens, including Salmonella and Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne’s disease (JD). Farm environments may provide potential entry points for the transmission of infectious agents into the food chain. Antibiotics are used to treat a wide variety of infections on farms, and administration of antimicrobial agents to cattle is considered to be a driving factor for antimicrobial resistance (AMR). Control of JD and AMR are priority for animal health initiatives in Ireland. A national JD pilot programme was introduced by Animal Health Ireland in 2014, while the national action plan launched by Department of Health and Department of Agriculture, Food and Marine introduced in 2017 aims to improve the surveillance of AMR. The current investigation was undertaken as a pilot study to determine the proportion of herds positive for MAP, Salmonella species (Salmonella spp), commensal Escherichia coli (E. coli), Extended-spectrum beta-lactamase (ESBL) AmpC β-lactamase and carbapenemase-producing E. coli from 157 environmental faecal samples in Irish farms.

Results: MAP was detected in 10.2% of samples collected; on culture in 4 (4.9%) of the dairy herds and from 1 (1.3%) of the beef/suckler herds, and by PCR in 10 (12.3%) and 6 (7.9%) of these herds respectively. All culture positive herds were also positive by PCR. An additional 11 herds were positive by PCR only. Salmonella was not detected, while commensal E. coli were isolated from 70.7% of the samples (111/157) with 101 of these isolates shown to be fully susceptible to all antimicrobials tested. Of the 27 presumptive ESBL AmpC β-lactamase producing E. coli detected, one isolate was resistant to ten antimicrobials, nine isolates were resistant to nine antimicrobials, and four isolates were resistant to eight antimicrobials. Carbapenemase-producing E. coli were not isolated.

Conclusions: The results highlight the importance of monitoring farm environments for Johne’s disease. This disease is a growing concern for dairy and beef producers in Ireland, and sampling the farm environment may offer a useful means to rapidly screen for the presence of MAP. Non-pathogenic common enteric commensal and multiple-drug-resistant E. coli may contribute to AMR acting as a reservoir and transferring resistance to other species/pathogens in the environment.

Comment: As they say, “the devil is in the details”.

The above graphic contrasts the two similar surveys for MAP on dairy farms, both done using environmental fecal samples.

The Irish survey used culture methods on the first sample finding 4 farms positive of the 78 sampled (5.1%). The second sample was tested using IS900 PCR finding 10 of 78 (12.8%) positive. All 4 culture-positive farms were also PCR-positive. Were more farms detected because PCR is more sensitive, or because a second sample increases the chances of detecting an infected farm?

The US survey only used culture methods to judge the farm’s MAP infection status but collected 6 samples per farm. The US data indicates that: the more you look the more you find. Over 70% of US dairy farms had a least one culture-positive sample. From this Lombard et al. estimated the true herd-level MAP infection ate in US dairy herds to be 91.1%.

Clearly there are more MAP-infected farms in the U.S. than in Ireland, however, it is tricky to estimate just how many would have been detected if only 2 samples were tested, i.e. which 2 of the 6 tested samples would you count? I will leave that for the modelling experts. For the purposes of this news item it is not that important.

However, since this was a pilot study, it seems that a larger study is warranted that: 1) randomly selects farms, 2) uses PCR as the probably more sensitive and definitely more cost-effective diagnostic test, 3) measures the analytical sensitivity of the test based on spiked samples to better understand the accuracy of the measurement tool, and 4) collects more than 2 environmental fecal samples per farm.

For detection of MAP-infected dairy farms with a low infection prevalence, e.g. 0 to 12%, as in the Irish situation, Lombard et al. estimated the herd-level sensitivity of a single environmental fecal culture at 33.3% and two environmental fecal cultures at 44.4% (see Table 1 in Lombard, 2013). If Lombard et al. are correct, the Irish survey may have under-estimated the true MAP infection status of its dairy farms.

BUT to be fair - it was only a pilot study! (and nobody asked my opinion). :-)

Research Article

Cristobal Verdugo, Maria Francisca Valdes, and Miguel Salgado from the Instituto de Medicina Preventiva Veterinaria, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile described herd level risk factors for Mycobacterium avium subsp. paratuberculosis (MAP) infection and clinical incidence in dairy herds in Chile. Their publication will appear in Preventive Veterinary Medicine, volume 176, March 2020 and is available online now (6 pages with 56 references). Unfortunately, it is not Open Access.

Holstein with clinical Johne's disease photographed in Chile by M.T. Collins.

Abstract

The study objective was to identify risk factors associated to: i) the infection by Mycobacterium avium subsp. paratuberculosis (MAP), and ii) paratuberculosis clinical incidence in Chilean dairy herds. A random sample of forty herds with previous history of MAP infection was selected. At herd level, all lactating cows were tested using a commercial ELISA kit. On the sampling date, a questionnaire gathering information on herd demographics, husbandry practices, and biosecurity measures was applied. Additionally, the farm manager/owner was surveyed regarding the number of paratuberculosis compatible clinical cases (CCC) in the last 12 months. Two Bayesian generalized linear mixed effect models were used to evaluate the association between the questionnaire data, and the proportion of truly infected animals (model 1) or the number of CCC (model 2).

A total of 4963 animals were sampled with an average apparent prevalence of 6.3 % (95 % confidence interval (4.0–8.0%). All sampled herds presented seropositive animals. Forty eight percent of the herds did not observe any CCC in the last year. Although, among those herd that did report CCC, a median of two cases per year was estimated. Model outputs showed that the proportion of truly infected animals and CCC reporting rates are associated to management practices. Specifically, positive associations were observed for feeding of calves exclusively with milk replacer, and the distance between the milking parlor and the calves’ barn. Additionally, CCC reporting rates were higher in farms that recently purchased animals, and where the distance between the milking parlor and the calves’ barn was less than 30 m.

Author’s Conclusions : “…the present research was able to find herd level factors associated to an increase in the risk of infection and the chances of reporting CCC. The reported factors put an emphasis on the need of introducing or improving basic biosecurity measures, especially in relation to the hygiene of calves, limiting the contact of young animals with manure from adult cows. The investment in such measures would not only be beneficial for MAP control but would improve the general health of the herd, possibly reducing the use of veterinary drugs.

Comment: The association of MAP infections with feeding calves milk replacer should be interpreted with caution. While milk replacer has been shown to harbor viable MAP (Grant 2017), it is also possible that preparation of the milk replacer on farms allowed fecal contamination which carried MAP into the re-hydrated product before feeding.

Research Article

K. Fechner and colleagues from the Department of Animal Sciences, Faculty of Agricultural Sciences, University of Göttingen reported about on-farm HTST (high-temp. short-time) pasteurizer's ability to kill MAP in the December 2019 issue of the Journal of Dairy Science. [not Open Access]

[caption id="attachment_3192" align="aligncenter" width="640"] HTST pasteurizer on a farm in Wisconsin[/caption]

Abstract

Feeding pasteurized milk to suckling calves is a popular practice used increasingly on dairy farms. Waste milk is frequently fed to calves because of its high nutritional value and economic benefits compared to milk replacement products. However, one of the disadvantages of feeding waste milk is the potential for exposure to a high number of bacterial contaminants, which may lead to serious illnesses or infections in calves. One of these contaminants is Mycobacterium avium ssp. paratuberculosis (MAP), the causative agent of Johne's disease (paratuberculosis). The transmission and distribution of paratuberculosis in dairy herds occurs mostly through the feeding newborn calves with contaminated colostrum or milk, because this age group is believed to be most susceptible to infection. To reduce the risk of transmission of pathogens, on-farm pasteurization of milk has become increasingly popular.

In this study, we analyzed the efficacy of a new commercial high-temperature, short-time pasteurizer (73.5°C for 20 to 25 s) in terms of MAP inactivation under experimental on-farm conditions. The pasteurizer uses a newly developed steam-heating technique, allowing for the pasteurization of the transition milk without clumping. In 3 independent trials, we spiked fresh raw milk samples to a level of 10⁷ or 10⁴ viable MAP cells/mL before pasteurization. We examined the thermal inactivation and viability of MAP using culture and a D29 bacteriophage-based assay. To verify the identity and number of MAP cells, we also performed PCR assays.

Pasteurization of the inoculated milk (10⁷ and 10⁴ MAP cells/mL) resulted in a remarkable reduction in viable MAP cells. The mean inactivation rate of MAP ranged from 0.82 to 2.65 log₁₀ plaque-forming units/mL, depending on the initial MAP amount inoculated and the addition of conservative agents to the pasteurized milk. Nevertheless, approximately 10³ MAP cells/mL remained viable and could be transferred to calves after high-temperature, short-time pasteurization of milk.

[caption id="attachment_3193" align="aligncenter" width="640"] Milk: an effective way to transmit Johne's disease.[/caption]

Comment: Yet again, research has shown that HTST pasteurization [minimum: 161F (72C) x 15 sec] fails to reliably kill 100% of MAP bacteria. And, this study used a slightly higher temperature and longer time [73.5°C for 20 to 25 s]. Achieving 100% kill of MAP by pasteurization is primarily a function of temperature AND time AND the number of MAP in the raw milk.

Recent research by P. Steuer and colleagues in Chile reported finding up to 10⁶ MAP/mL raw milk on Chilean dairy farms with ELISA-positive animals (see P. Steur et al., Is the transmission of Mycobacterium avium subspecies paratuberculosis (MAP) infection through milk intended to feed calves an overlooked item in paratuberculosis control programs?, Tropical Animal Health and Production (vol 52 issue 1, pp 89-94, January 2020).

Many explanations for the heat-resistance of MAP have been suggested, e.g. formation of clusters of bacterial cells (up to 100) hindering heat penetration, location inside host cells (protected), and perhaps most importantly the ability to form tenacious spore-like structures. For more on spore formation see Lamont et al. PLOS ONE, January 24, 2012.)

Research Article

S. Patterson and colleagues in the UK reported on the impact of a dairy cow’s Johne’s ELISA status on the likelihood her calf will also be MAP-infected (ELISA-positive). Their study is reported in the journal Preventive Veterinary Medicine (in press, corrected proof, available online – not Open Access).

Abstract

Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic condition of dairy cattle, and is endemic in the UK. Lack of understanding of the relative importance of different transmission routes reduces the impact of control scheme recommendations.

The long incubation period for Johne’s disease makes evaluation of control schemes difficult, and so this long-term cohort study offers a rare and valuable insight into the disease epidemiology. A longitudinal study was carried out following a cohort of 440 UK dairy cows in 6 herds recruited in 2012-2013. Individuals entering the milking herd were routinely monitored for the presence of MAP using quarterly milk ELISA testing. Using a Cox proportional-hazards regression model the relationship between time until first detection of infection and dam MAP status was investigated. We then compared the magnitude of the effect of dam status with that of other risk factors in order to understand its relative importance.

Dam status was found to be the only observed factor that was significantly associated with time to an individual testing MAP-positive (p = 0.012). When compared to negative dams, we found a marginally significant effect of having a positive dam at time of calving, that increased the hazard of an individual testing positive by a factor of 2.6 (95% confidence interval: 0.89–7.79, p = 0.081). Further positive associations were found with dams becoming positive after the birth of the subject; a dam seroconverting within 12 months post parturition being associated with a 3.6 fold increase in hazard (95% confidence interval: 1.32–9.77, p = 0.013), and dams seroconverting more than a year after calving increased the hazard by a factor of 2.8 (95% confidence interval: 1.39–5.76, p = 0.004).

These results suggest that cows may be transmitting MAP to their offspring at an earlier stage than had previously been thought, and so raise important questions about how this transmission may be occurring. The results of the study may have important practical implications for the management on-farm of the offspring of MAP-positive animals, with the potential to vastly reduce the time required to eliminate this chronic disease.

Comment: For faster control of Johne’s disease in dairy herds it is advisable to tell herd owners not to retain as herd replacements any heifer born to a cow that tests positive for Johne’s disease. This study supports that recommendation (using ELISA as the Johne’s disease test) and extends it to include calves born to cows that test positive AFTER calving. Not culling calves born to test-positive dams will result in more MAP-infected heifers entering the herd as replacements and then infecting more calves thereby slowing progress of a control program. That said, in my experience it is difficult enough to get herd owners to act on positive test results on cows, much less act on the probabilities that their calves are also infected. Hopefully, this report and others like it will help to strengthen culling recommendations to include calves born to ELISA-positive dams resulting in more successful control of Johne’s disease in dairy herds.

Today's news: Another report of probable MAP “spillover” from domestic animals to wildlife.  Monica Cunha from the National Institute for Agrarian and Veterinary Research, Oeiras, Portugal and colleagues report detection of MAP by PCR in 10% red fox (Vulpes vulpes), 6% Egyptian mongoose (Herpestes ichneumon) as well as stone marten (Martes foina) and common genet (Genetta genetta) – a first time report of MAP in genet.  This Open Access publication appeared January 21 in Nature.com,  Scientific Reports. [14 pages with 88 references]

Common genet photo from Wikipedia.

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease or paratuberculosis, a chronic infection affecting domestic ruminants worldwide. Despite sporadic reports of MAP occurrence in non-ruminants, information on the risk factors predisposing for infection is still scarce and evidence of transmission paths linking the livestock-wildlife-environment interfaces also remains lacking. In this study, we predicted that environmental, host-related, land use and human driven disturbance factors would modulate carnivore exposure to MAP. To test these hypotheses, we performed a retrospective survey, based on microbiological and molecular methods, in mainland Portugal including five sympatric species from the Herpestidae, Canidae, Viverridae, and Mustelidae families (n = 202) and examined 16 variables as putative predictors of MAP occurrence. Molecular evidence of MAP using IS900 as proxy was demonstrated in 7.43% (95%CI: 4.55–11.9) of surveyed carnivores, the highest proportions being registered for red fox (Vulpes vulpes) (10%; 95%CI: 4.0–23) and Egyptian mongoose (Herpestes ichneumon) (6.0%; 95%CI: 3.2–11). We demonstrate that important species of the Mediterranean carnivore guild, such as stone marten (Martes foina) and common genet (Genetta genetta), may also be exposed to MAP, being this the first time that occurrence in genet is reported. The high proportion of DNA-positive specimens, concurrent with the apparent lack of gastro-enteric lesions and molecular confirmation of IS900 in feces, argue for the presence of subclinical carriers that occasionally shed bacteria, potentially aiding as source of infection to susceptible species and possibly contributing for environmental contamination. Achievement of MAP isolation would prove beyond any doubt that MAP is present in this wildlife population. Ecological modelling results suggested that the probability of MAP infection using IS900 as proxy in mongoose is positively associated with higher altitude and temperature stability, as well as with lower annual rainfall. Density of livestock farms was found not to be a significant predictor, which may indicate that the livestock-wildlife interface is probably not important as an infection route for mongoose.

Egyptian mongoose photo from Wikipedia.

Concluding remarks excerpt: Suspicion for the ecological spread of MAP reinforces the need for increased surveillance and action. Quantitative information on the prevalence and concentration of MAP in putative contaminated sources of exposure is required.

Comment:  This report adds to the growing body of literature about MAP spread to wildlife.  It is critical to establish whether the transmission of MAP is a “two-way street”, with MAP going back and forth between domestic animals and wildlife, or instead that wildlife are a “dead-end host”. i.e. transmission only from domestic animals to wildlife ("one-way street"). The answers will likely depend on the type of wildlife, i.e. wild ruminants probably develop permissive infections shedding MAP in ways that can then infect domestic animals, while wild carnivores may be dead-end hosts. Read more about non-ruminants that have been found to be MAP-infected on this website page.

As reported in the Wisconsin State Farmer, September 16, 2019:

Wisconsin's self-proclaimed moniker as "America's Dairyland" is taking on fresh meaning in the 21st century thanks to a growing market for milk from an animal that bleats rather than moos. While the state's traditional dairy cattle industry continues to hemorrhage producers at a record pace, Wisconsin's dairy goat industry is in the midst of a long-term, and accelerating, growth spurt. Indeed, in 2019 Wisconsin can reasonably claim to be America's dairy (goat) land.

Data from the United States Department of Agriculture, which counts livestock across the United States every 5 years, show just how much Wisconsin dominates the nation's dairy goat industry. In 2017, the most recent year the USDA surveyed producers, the size of Wisconsin's dairy goat herd easily topped the nation at more than 83,000-head. California came in a distant second, with some 43,000 dairy goats, while Iowa, Texas and Missouri rounded out the top five.

It's not only the sheer size of Wisconsin's dairy goat herd that stands out: The state also leads the nation in the value of sales from dairy goat operations and is the epicenter of national growth in goat dairy.

Read the rest of this excellent article that contains many useful links here.

Comment:

Rapidly expanding animal industries commonly overlook biosecurity risks feeling compelled to buy animals regardless of health status. Many of these new dairy goat operations have 5,000 to 10,000 goats. Operations only can attain this size by purchase of goats from many different herds. Purchasing animals is the #1 way by which herds, whether of cattle, sheep or goats, become infected with MAP.  This then leads to long-term problems of animal health, animal welfare, animal productivity and animal longevity which all translate to a negative economic impact due to Johne’s disease.

Johne’s disease is common among all goat breeds.

• France found that 55.2% of dairy goat herds had one or more ELISA-positive goats.
• In Cyprus 50% of dairy goat herds were seropositive.
• In Ontario in Canada 83% (estimated true prevalence) of dairy goat herds were MAP-infected.
• In Spain 87.5% of dairy goat herds were seropositive.
• I found no such surveys in U.S. dairy goats but a study of Boer goats in Missouri reported that an estimated 54.7% of Boer goat herds are seropositive for Johne’s disease.

Many owners fail to realize their goats are MAP-infected and therefore do not implement control measures with the result that the infection continues spreading. In this article in the Wisconsin State Farmer, I also observed that links provided in this article to goat associations and experts never once mention biosecurity concerns or the threat of Johne’s disease.  So unfortunate!

Goat owners and veterinarians must be much more aware of Johne’s disease and implement biosecurity protocols and on-farm control measures to mitigate the damage caused by Johne’s disease. When MAP becomes recognized as a food-borne zoonotic pathogen, these large goat herds with face a daunting task to control or eradicate this infection.

The age-old wisdom that “prevention pays” should be heeded. 

A rare and beautiful sight, the Southern Patagonian huemul deer (Hippocamelus bisulcus) is an icon of Patagonia Park and of Chile. A native species, the huemul is Chile’s national animal, found on the country’s coat of arms, alongside the Andean condor. Despite its high level of recognition, the huemul is classified as endangered by the IUCN, with a global population of less than 1,500 individuals. With 10% of these remaining deer residing within Patagonia Park’s boundaries, Conservacion Patagonica has made huemul deer recovery the cornerstone of its wildlife program.

Paulo Corti and colleagues from Universidad Austral de Chile, Valdivia, Chile describe recovery of MAP from 3 of three fecal samples from huemul deer in Patagonia in The Australian Journal of Veterinary Science (52:33-35, 2020).

Abstract

In a huemul (Hippocamelus bisulcus) population sympatric with cattle, we found evidence of Mycobacterium avium subsp. paratuberculosis (MAP) infection. Three huemul faecal pellet samples and two cow pats were collected and cultured for MAP presence. DNA was then extracted for PCR analysis of all signal-positive cultures. To assess whether MAP isolates obtained from huemul faeces were associated with typical MAP isolated from livestock, positive confirmed culture samples were sub-typed using a combination of five Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat Analysis and one Short Sequence Repeat analysis markers. All faecal samples from both species were MAP positive. One huemul presented a different bacteria profile genotype not described before, suggesting that huemul and cattle in Patagonia could carry a unique MAP strain.

Comment:  The primary reservoir for MAP is in domesticated ruminants such as dairy cattle, beef cattle, sheep and goats.  However, MAP infections in these livestock readily spread to wild ruminants and this first-time report of finding MAP in huemel deer illustrates the concept of infection “spillover”. 

Spillover is the title of an excellent book by David Quamenn which focuses on the spillover of infectious diseases from animals to humans, so called zoonotic infections.  MAP infections clearly spillover to wild ruminants, and non-ruminants, including humans.  This website provides more details about MAP infections of dairy cattle, beef cattle, goats, sheep, deer & elk, bison, water buffalo, wild ruminants, zoo ruminants, and non-ruminants.  A separate page is devoted to the zoonotic potential of MAP.

MAP is a promiscuous, insidious, zoonotic, foodborne pathogen that threatens the economic viability of livestock producers, the health and well-being of wildlife, zoological collections of wild ruminants (many of which are endangered), and humans.  It deserves far more research funding and control program investment than it currently receives.

Research Report

Mathevon and colleagues from Ecole Nationale Vétérinaire de Toulouse, Toulouse Cedex, France reported on optimal sheep sample pooling strategies for serum samples (for ELISA testing) or fecal samples (for qPCR testing). Their report appeared December 26, 2019 in PLoS ONE and is OPEN ACCESS. [18 pages with 47 references]

Abstract

The aim of our study was to evaluate the flock sensitivity and specificity of fecal qPCR and serum ELISA using pooled samples for screening paratuberculosis in French sheep.

Using individual feces with low or high qPCR Ct values from ewes sampled in 14 infected flocks, a total of 555 pools of size 5, 10 and 20 were created by diluting individual materials in negative feces and analysed using a commercial IS900 qPCR kit. The relative performances of pooled serum ELISA analysis were evaluated based on the analysis of 181 different pools of size 5 and 10, composed of individual serum samples of various individual S/P values. Results showed that for pools of size 5, 10 or 20, individual fecal samples with low Ct values were invariably detected. Conversely fecal samples with high Ct values were associated with a lower detection rate in both pools of size 5 (87.0% to 90.0%), 10 (63.0% to 70.7%) and 20 (46.7% to 60.0%). After lowering the decision threshold to 25% and 15% for serum pools of size 5 and 10 respectively, the pooled serum ELISA relative sensitivity ranged between 62.2% and 100.0% depending on the composition of the pools.

Finally, a simulation study was carried out to evaluate the performances of 16 screening strategies at flock level, with varying pool size (5 to 20) and number (5 to 60). The use of pooled serum ELISA led to very false positive detection rate ranging between 37.6% and 91.8% in paratuberculosis free flocks and prevents its further use in that context. For infection prevalence ≤ 5%, the flock sensitivity based on pooled fecal qPCR ranged between 39.0% (5 pools of size 10) and 99.9% (300 sampled individuals, with pools of size 5,10 or20), and was always above 93% when the infection prevalence was greater or equal to 15%. We conclude that pooled-fecal qPCR but not pooled-serum ELISA could be a useful tool to detect sheep flocks infected with paratuberculosis.

Comment: The authors focused their discussion on diagnostic tests useful for screening of sheep flocks for ovine Johne’s disease (OJD) where the primary goal is to detect MAP-infected flocks, i.e. maximize “flock sensitivity” and “flock specificity”, at the least cost. This should not be confused with the use of fecal pooling strategies for qPCR testing in known infected flocks where the goal is to identify individuals in the flock that should be culled or isolated.

In MAP-infected flocks there are two equally important goals: 1) to limit the cost of testing the whole flock, and 2) to limit the cost of testing the individual samples, i.e. to have the fewest number of qPCR-positive pools. This second goal is important because each of the fecal samples comprising a qPCR-positive pool must be individually tested by qPCR in order to identify each MAP-infected sheep adding significant costs, e.g. roughly $30 x 5 = $150 per PCR-positive pool (based on WVDL testing rates).

Using the simulation data from this publication (shown above), for flocks with a MAP infection prevalence of 10%, the simulation data (Fig 4 C & D in the publication as shown above) indicate that using 20 pools of 5 fecals/pool, 93% of flocks would have 7 or fewer positive pools which would then cost an additional $1,050 to test each fecal individually by qPCR (7 pools x 5 fecals/pool x $30/qPCR = $1,050). By contrast, if these same flocks used 10 pools of 10 fecals/pool, then 92% of flocks would have 5 or fewer qPCR-positive pools resulting in a cost of $1,500 to individually test the fecal samples making up those pools (5 pools x 10 fecals/pool x $30 = $1,500).

The cost of testing by pooled PCR at the WVDL is $34/pool so would cost $340 for 10 pools of 10 fecals/pool or $680 for 20 pools of 5 fecals/pool. Thus, while testing 20 pools (5 fecals/pool) would initially cost $340 more, the cost of testing the individual fecals comprising each qPCR-positive pool does not offset the cost of testing fewer pools in this example.

These findings are based on a simulation of random pooling of fecal samples described in the publication. As mentioned in a previous news posting, strategic pooling by animal age can help limit the number of test-positive pools Kalis (2000). This is because animals of similar age experienced similar risks of becoming MAP-infected when young. Thus, age-based pooling (strategic) results in more MAP-infected animals likely to be in the same pool and conversely the fewest number of positive pools for the flock or herd as a whole.

You can read more about a clustering of MAP-infections by age and season in the publication by Zare et al. titled: Evidence of birth seasonality and clustering of Mycobacterium avium subspecies paratuberculosis infection in US dairy herds. Preventive Veterinary Medicine 112: 276– 284, 2013. However, the clustering effect may not be as pronounced for sheep which do not have lambs thought the year.

In closing, this discussion highlights the importance of utility analysis when judging the merits of diagnostic tests for MAP. For more on this topic see Garder et al. Consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis in ruminants. Preventive Veterinary Medicine 101: 18-34, 2011.

Research Report

Brazilian researchers report a small survey of a popular cheese eaten in Brazil for MAP.  Their findings were reported in Arquivo Brasileiro de Medicina Veterinária e Zootecmia (vol.71 no.6 Belo Horizonte Nov./Dec. 2019  Epub Dec 13, 2019).

Abstract

Paratuberculosis is a chronic and incurable disease that affects ruminants and other domestic animals. It is caused by Mycobacterium avium subsp. paratuberculosis (MAP) that may also be involved in some human diseases such as Crohn's disease, type 1 diabetes, sarcoidosis, multiple sclerosis, and Hashimoto's thyroiditis. The objective of this study was to investigate the occurrence of MAP DNA in samples of artisanal coalho cheese purchased in the State of Pernambuco. Forty samples of coalho cheese submitted to the Real Time Polymerase Chain Reaction (qPCR) technique were analyzed for the detection of the MAP region IS900. 11 (27.5%) were positive with a mean of 195.9 MAP colony forming unit (CFU) per gram of each sample, with a minimum of 30.3 CFU/g and a maximum of 324.2 CFU/g. Thus, this type of cheese that is one of the most consumed in this region of Brazil constitutes a source of human exposure to MAP. Further research in this area should be performed to evaluate the viability of the bacteria in this cheese type.

Comment: It should be noted that MAP numbers (CFU) were actually based 10-fold serially diluted MAP DNA used to obtain the standard curve, in which it was possible to detect from 21 up to 2.12 x 10⁷ MAP cells. In my opinion a more informative titration curve (MAP CFU vs qPCR Ct values) would begin with known CFU/mL MAP suspensions that were then processed by DNA extraction followed by qPCR. Only then can an investigator truly know the net results of both DNA extraction and target IS900) amplification to estimate the CFU of MAP in the original sample.  That said, most molecular biologists opt for titration of extracted DNA as the authors of this study did.

The last line of the abstract is the critical one. The really big question is: Are the detected MAP alive or dead. That said, some investigators postulate that dead MAP may function as a dietary allergen triggering inflammation in the absence of infection. MAP as a food safety threat is a critical knowledge gap urgently requiring research.

Research Report

KST Kanankege and colleagues from the Department of Population Medicine, College of Veterinary Medicine, University of Minnesota reported on use of milk ELISA testing data from a voluntary testing program through DHI testing laboratories as a passive surveillance tool for Johne’s disease in Minnesota. Their report appears in the journal BMC Veterinary Research 15:429, 2019. [Open Access]

Abstract

Background: One of the key steps in the management of chronic diseases in animals including Johne’s disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is the ability to track disease incidence over space and time. JD surveillance in the U.S. dairy cattle is challenging due to lack of regulatory requirements, imperfect diagnostic tests, and associated expenses, including time and labor. An alternative approach is to use voluntary testing programs. Here, data from a voluntary JD testing program, conducted by the Minnesota Dairy Herd Improvement Association, were used to: a) explore whether such a program provides representative information on JD-prevalence in Minnesota dairy herds, b) estimate JD distribution, and, c) identify herd and environmental factors associated with finding JD-positive cows. Milk samples (n = 70,809) collected from 54,652 unique cows from 600 Minnesota dairy herds between November 2014 and April 2017 were tested using a MAP antibody ELISA. Participant representativeness was assessed by comparing the number of JD-tested herds with the number of herds required to estimate the true disease prevalence per county based on official statistics from the National Agricultural Statistical Services. Multivariable logistic regression models, with and without spatial dependence between observations, were then used to investigate the association between herd status to JD (positive/negative), as indicated by milk ELISA results, and available covariates at the herd level.

Results: Within the study population, at least one test-positive cow was found in 414 of 600 (69%) herds. Results indicated that large herds that test frequently and herds located in loamy or silt soils are more likely to have at least one MAP test-positive cow. After adjusting for herd size, testing frequency, and soil type, there was no spatial dependence in JD risk between neighboring dairies within 5 to 20 km. Furthermore, the importance of collecting data on herd management, feed, and biosecurity for insightful interpretations was recognized. The study suggested that, although limited, the voluntary testing database may support monitoring JD status.

Conclusions: Results presented here help elucidate the spatial characteristics of JD in Minnesota and the study may ultimately contribute to the design and implementation of surveillance programs for the disease.

Comments: This excellent article demonstrates the power of utilizing diagnostic testing data for Johne’s disease to explore relationships and environmental factors affecting the prevalence of Johne’s disease. This is data that otherwise would provide no benefit to the dairy industry and simply sit idly on computer hard drives. Kudos to the Minnesota Dairy Herd Improvement Association for their support in data collection! Imagine if all DHI labs and all state veterinary diagnostic labs could/would share data in this same way to provide a comprehensive understanding of the spatial distribution and risk factors affecting Johne’s prevalence in the U.S.: a relatively small investment with potentially big returns.