Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis in domestic ruminants and New World Camelids (NWC). Hepatitis E virus (HEV) is an important public health concern worldwide. The virus has been identified in several species, some of them serving as a reservoir for zoonotic HEV strains. Husbandry and breeding of llamas and alpacas have increased in Austria in recent years. Therefore, the aim of the present study was to evaluate the prevalence of MAP and HEV in NWC in Austria. Altogether 445 animals, originating from 78 farms were enrolled in the study. Of the animals sampled, 184 (41.35%) were llamas and 261 (58.65%) were alpacas. 443 blood samples for MAP-ELISA and 399 faecal samples for quantitative PCR (qPCR) and culture for MAP as well as for HEV detection by RT-qPCR have been collected. All of the 399 animals tested for shedding of MAP were negative by faecal solid culture. Using qPCR, 15 (3.8%) of the animals were MAP positive and 384 (96.2%) negative. Out of the 443 serum samples examined for specific antibodies against MAP by ELISA, 6 (1.4%) were positive, 1 (0.2%) was questionable and 436 (98.4%) samples were negative. All faecal samples were tested negative for HEV.
- Mycobacterium avium
- Hepatitis E
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Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease (JD) or paratuberculosis, an infectious bacterial disease characterised by granulomatous enteritis, diarrhoea, loss of bodyweight and death (Fecteau and others 2013). The primary hosts are domestic ruminants such as cattle, sheep and goats, with an estimated prevalence of 30–50 per cent affected cattle herds in countries with a significant dairy industry (Barkema and others 2010). In 2004, a seroprevalence of 19.05 per cent MAP-positive cattle herds was determined in Austria (Baumgartner and others 2005). In a recent survey, 7.6 per cent of cattle farms were detected MAP-positive by boot swab samples in the Austrian province of Tyrol (Khol and others 2015).
Ruminants infected with MAP regularly shed the bacterium with faeces leading to a faecal-oral transmission, which is the primary route of disease transmission. Additionally, the bacterium also is shed and transmitted with milk and colostrum of infected animals. To diagnose infections with MAP direct detection methods, namely bacteriological culture and PCR, as well as indirect methods to detect specific antibodies (Ab) with ELISA being the most important test, can be applied. Unfortunately, the sensitivity of these tests is low in animals with subclinical JD, hampering the early diagnose of MAP infections (Collins 1996; Nielsen and Toft 2008).
Besides other domestic and wild ruminants, there are individual case reports of JD in llamas (Lama glama) and alpacas (Vicugna pacos) (Belknap and others 1994, Fecteau and others 2009, Salgado and others 2009). As in small ruminants, not all New World Camelids (NWC) infected with MAP show symptoms of JD such as weight loss and diarrhoea (Fecteau and others 2009). In a survey in NWC in North America, 6 per cent of the herds tested were found positive for JD in faecal PCR (Fecteau and others 2013) and an investigation in wild guanacos in Tierra del Fuego Island (Argentina) revealed a MAP herd prevalence of 4.2 per cent and also 21 of 501 (4.2 per cent) animals were faecal culture-positive for MAP (Salgado and others 2009). All MAP strains isolated in this study corresponded to the cattle-type (C-type) strain of MAP (Salgado and others 2009). In another study, 15 out of 85 (17.6 per cent) alpacas were found MAP-faecal positive by culture in the Chilean Altiplano (Salgado and others 2016). As in other species, due to the lack of diagnostic tests with an appropriate sensitivity, the detection of MAP is difficult in camelids (Fecteau and others 2013). Although ELISA-Kits have been adapted for the use in NWC, the sensitivity of these tests of 67.0 per cent is still rather low (Kramsky and others 2000).
Husbandry and breeding of llamas and alpacas have increased in Europe in recent years. Although there is no legal obligation for the registration, the population of NWC in Austria has been estimated to be approximately 4000–6000 individuals (Trah and Wittek 2013). The increasing import of llamas and alpacas from different European and non-European countries as well as the frequent transfer of animals between herds can easily lead to a spread of diseases within the population, including JD (Miller and others 2000). In case of JD, this risk is enhanced by undetected shedders spreading MAP with their faeces, combined with the chronic nature and long subclinical phase of the infection and the high tenacity of MAP in the environment, representing significant challenges for prevention and control (Miller and others 2000, Fecteau and others 2013). The authors hypothesised that NWC may pose a risk for transmission of MAP to cattle and other domestic animals as they are often kept in the same areas.
The hepatitis E virus (HEV) causes hepatitis E in humans and is an important public health concern in many parts of the world (Yugo and Meng 2013). HEV is considered epidemic in many developing countries (Teo 2009), whereas cases of hepatitis E are sporadic in high-income countries (WHO 2016). Notably, the majority of these sporadic cases are zoonotic with HEV originating from domestic pigs, wild boar or cervids serving as a reservoir of HEV (WHO 2016). The virus has been identified in other animal species, including camel (Rasche and others 2016). Additionally, specific Ab against HEV have been reported in cattle, sheep and goats (Meng 2013, Sanford and others 2013). HEV infections in animals usually are subclinical with mild lesions in the liver, but infected animals can shed large amounts of the virus with their faeces posing a possible risk of human infections (Yugo and Meng 2013). As per our knowledge, no reports about HEV or specific Ab in NWC are available.
The aim of the present study was to evaluate the prevalence of MAP in llamas and alpacas in Austria to determine the risk for the spread of JD both in the NWC and to other domestic and wild ruminants. Furthermore, the role of NWC as possible carriers of HEV should be examined.
Materials and methods
Study population and sample collection
The estimate actual total population of NWC in Austria is approximately 4000–6000 individuals, unequally distributed throughout the country (Trah and Wittek 2013). The total number of individuals, as well as the number of animals kept in each of the nine Austrian federal states is unknown and was therefore estimated from size and geological conditions, as some federal states are situated in the Alps and therefore, fewer animals are kept there. As a sample size of 10 per cent of the total population is desirable from the epidemiological and statistical aspects, the study was designed to sample some 10 per cent of the estimated population. This results is an intended sample size of 400–600 NWC, including samples from all Austrian federal states.
Regardless of the actual herd size, a maximum of seven animals was sampled from any herd. Animals to be sampled were selected randomly by drawing of numbers and had to be older than one year of age to be enrolled in the study. Individuals with any clinical illness or treatment at the time of sampling were excluded from the study. After a clinical examination according to Baumgartner and others (2005), blood samples from the jugular vein, using Vacuette tubes with Z Serum Clot Activator (Greiner bio-one, Kremsmünster, Austria) and faecal samples from the ampulla recti were obtained in selected animals. Serum was harvested from the blood samples after centrifugation at approximately 5000 g for 10 minutes immediately after sample collection. Serum and faecal samples were transported cooled and stored at a temperature of −83°C until further processing.
This study was approved by the institutional ethics committee of the University of Veterinary Medicine Vienna and the national authority in accordance with the Austrian Law on Animal Experiments, Tierversuchsgesetz—TVG, GZ 68.205/0171-II/3b/2013. It was conducted in accordance with the guidelines for Good Scientific Practice and in compliance with the relevant national legislation.
Detection of specific MAP Ab by ELISA
ELISA for the detection of specific Ab against MAP was performed at the University Clinic for Ruminants at the University of Veterinary Medicine Vienna. All sera were tested for specific Ab against MAP using the ID Screen M avium Indirect ELISA (IDvet, Montpellier, France). This multispecies ELISA is designed for the detection of anti-M avium Ab in pigs and ruminants. The same antigen as in the ID Screen Paratuberculosis Indirect ELISA (IDvet) for the detection of MAP in cattle is used in this kit but a different conjugate showing a high binding affinity to camelids is included. The specificity reported by the company of the test is 100 per cent with an estimated sensitivity of 50–60 per cent.
The tests were carried out according to the manufacturers’ instructions. A sample to positive ratio of 50 per cent ore above was considered positive, a ratio above 40 per cent and below 50 per cent as doubtful and samples with a sample to positive ratio below 40 per cent were considered negative.
Detection of MAP by bacteriological culture and PCR
Detection of MAP by bacteriological culture and quantitative PCR (qPCR) was performed at the OIE Reference Laboratory for Paratuberculosis at the Veterinary Research Institute Brno. Culture of MAP was carried out according to Whipple and others (1991) and modified by Pavlik and others (2000) using Herrold's egg yolk medium. Approximately 1 g of faeces from each sample was decontaminated using 0.75 per cent hexadecylpyridinium chloride (cetylpyridinium chloride, No. 102340 Merck) for 72 hours. After decontamination, 200 μl of the samples were transferred to each of three culture media, two with and one without mycobactine J and incubated at 37°C for a total time of 3 months. The cultures were checked for bacterial growth weekly, positive results were confirmed with conventional PCR of colonies as described by Moravkova and others (2008).
Faecal samples were tested for the presence of MAP by qPCR detecting the IS900 and F57 genes according to Slana and others (2008). The DNA from faecal samples was isolated by using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) with slightly modified isolation protocol. Both qPCR duplex assays include internal amplification controls (IAC) and plasmid gradients for accurate quantification (Slana and others 2008).
Detection of HEV by RT-qPCR
Nucleic acid from faeces was isolated using QIAamp Viral RNA kit (Qiagen). The detection of HEV RNA was performed by triplex real-time RT-PCR using two sets of oligonucleotides targeting two different loci of the HEV genome and one set of oligonucleotides specific for IAC as described previously (Vasickova and others 2012).
For statistical analysis, the prevalence and CIs were calculated using Microsoft Excel V.14.0 software package (Microsoft Cooperation, Redmond, Washington, USA). To show the pathogen occurrence in the different federal states, a descriptive statistic was applied.
Altogether 445 animals, originating from 78 farms were enrolled in the study. Of the animals sampled, 184 (41.35 per cent) were llamas and 261 (58.65 per cent) alpacas, respectively. On 9 (11.54 per cent) of the visited farms, the NWC were kept together with cattle or small ruminants and on 40 (51.3 per cent) farms NWC had been imported from abroad in the past. The distribution and species of the sampled animals is presented in Table 1. Blood samples could be collected in 443 animals but faecal samples could not be obtained in every individual and not enough material both for culture and PCR was available in some other animals, resulting in 399 faecal samples (157 llamas, 242 alpacas), both for PCR and culture.
All of the 399 animals tested for shedding of MAP were negative by faecal solid culture (see Table 1). Using qPCR, 15 (3.8 per cent) of the animals were MAP positive and 384 (96.2 per cent) negative, both in the IS900 and F57 protocol (see Table 1). Eleven of the qPCR-positive animals were llamas and four alpacas, respectively, leading to a MAP prevalence of 7.2 per cent in llamas and 1.6 per cent in alpacas. Out of the 443 serum samples examined for specific Ab against MAP by ELISA, 6 (1.4 per cent) were positive, 1 (0.2 per cent) was questionable and 436 (98.4 per cent) samples were negative (see Table 1). Two of the positive animals were llamas and four alpacas, therefore 1.1 per cent of the examined llamas and 1.5 per cent of the alpacas were MAP-seropositive. The comparison of the results, age and other characteristics of all animals found positive throughout the study are presented in Table 2. Based on these results, a MAP prevalence in NWC of 3.8 per cent with a 95 per cent CI between 1.9 per cent and 5.6 per cent could be calculated based on the examination of faecal samples by qPCR. Detection of specific Ab by ELISA revealed a seroprevalence of 1.4 per cent with a 95 per cent CI between 0.3 per cent and 2.4 per cent in NWC in Austria. On the herd level, a prevalence of 14.1 per cent (11 positive herds) with a 95 per cent CI between 6.4 per cent and 21.8 per cent based on faecal results and a seroprevalence of 6.4 per cent (5 positive herds) with a 95 per cent CI between 1.0 per cent and 11.83 per cent could be calculated.
All 399 faecal samples tested for presence of HEV RNA were negative, the virus could not be detected by qPCR.
The present study revealed a MAP prevalence in NWC of 3.8 per cent faecal-PCR and 1.4 per cent seropositive animals. While faecal detection tended to occur more frequently in llamas than in alpacas, the later showed a non-significant higher number of positive ELISA results. On the herd level, the detected prevalence was 14.1 per cent using faecal-PCR and 6.4 per cent as diagnosed by ELISA. When compared with results from other areas, the Austrian single animal MAP prevalence of NWC is comparable to 4.2 per cent faecal positive animals in Argentina (Salgado and others 2009), but well below the reported 17.6 per cent positive animals in Chile (Salgado and others 2016). The Austrian herd prevalence however is higher than the 6 per cent found in North America (Fecteau and others 2013) and the herd prevalence of 4.2 per cent in Argentina (Salgado and others 2009). No data about the MAP seroprevalence in NWC are available for other countries or regions, but the seroprevalence found in Austrian NWC on the herd level is considerably below the 19.05 per cent MAP-positive cattle herds reported previously (Baumgartner and others 2005).
Because of impaired diagnostic methods for the early diagnoses of MAP infection, the true prevalence might actually be higher than indicated by this study (Nielsen and Toft 2008). On the other hand, passive shedding without infection, as known to occur in cattle, might lead to an overestimation of the prevalence (Pradhan and others 2011). The frequency of passive faecal shedding of MAP in affected cattle herd is discussed controversially and more recent results indicate a lower incidence of passive shedding than previously reported (Sweeney and others 1992; Pradhan and others 2011). Furthermore, passive shedding is believed to occur after contamination of feed with faces from animals shedding high amounts of MAP within the same herd (Fecteau and Whitlock 2010). As only three of the positive camelids in our study (animal number 28,111 and 204) had contact with other ruminant livestock, contamination of the feed and environment with MAP, leading to passive shedding probably occurred by infected herd mates. No data concerning passive shedding of MAP by NWC are available and as no necropsy or follow-up investigations were performed in the present study, possible passive MAP shedding by some of the positive animals remain doubtful.
Both overestimation and underestimation could be especially important in NWC as little is known about the epidemiology of JD in these species including the shedding patters of MAP with faeces and the development of specific Ab. Furthermore, diagnostic tests are not sufficiently adapted and evaluated for NWC and have therefore been interpreted with caution.
The mismatch between faecal and serum results seen in the present study also is known in cattle, because the two tests detect different subsets of animals, in different stages of infection (Nielsen 2008). Furthermore, due to its poor sensitivity, ELISA is used as a screening tool on the herd level, rather than for single animals and results therefore have been interpreted with caution (Nielsen and Toft 2008). The results presented here indicate that the qPCR is more sensitive in NWC for the detection of MAP in faeces than solid culture. No difference between the results of the qPCR targeting IS900 and F57 was seen in the presented study. Sensitivity and specificity, both of faecal culture and PCR, are objects of ongoing discussions with no clear advantages of one single method (Bölske and Herthnek 2010). Fecteau and others (2013) also reported no positive MAP result of faecal solid culture in PCR-positive alpaca. Possible causes for this discrepancy between the two test methods might be the freezing of faecal samples before testing hampering the culture or, that NWC harbour a MAP strain which is difficult to cultivate overall (Fecteau and others 2013). Although the latter would be in contrast to the finding, that alpacas most often are infected by the cattle strain of MAP (Cousins and others 2000, Kennedy and Allworth 2000). Unfortunately, due to the study design of our survey it was not possible to determine the true MAP status of the animals included by tissue samples, repeated testing or necropsy. Nevertheless, the results indicate that the MAP prevalence in Austrian NWC assumable lies below 5 per cent at the single animal and around 10 per cent at the herd level.
Of the 22 PCR-positive or ELISA-positive animals, 4 were two years old and 13 had an age of five years or older (see Table 2), indicating an increase of MAP shedding and the production of specific Ab with age. The same increase with age can be seen in cattle and other domestic ruminants, due to the chronic nature of JD with a late onset of MAP shedding and production of specific Ab (Fecteau and Whitlock 2010). Again, no epidemiological data concerning MAP shedding and Ab production in NWC are available and warrant further investigation. Six of the positive animals were imported from abroad, four from Germany, one from Chile and one from the USA. Although based on a few results only, the fact that 27.3 per cent of the positive animals were imported from abroad, compared with an overall percentage of 50 per cent of farms to which animals were imported included in the study, indicate no major impact of animal purchase from abroad to the distribution of MAP among Austrian NWC.
Four of the MAP-positive animals were housed in close contact with sheep, goat or cattle, leading to the risk of interspecies transmission of the bacterium. No genotyping of the MAP strains has been performed in our study, but it has been shown that alpaca frequently are infected with the C1 cattle subtype of MAP (Cousins and others 2000, Kennedy and Allworth 2000). Clinical JD in domestic ruminants is a notifiable disease in Austria (Khol and others 2007), but NWC are not affected by this regulation. Nevertheless, as NWC are getting more popular and the number of animals is constantly increasing, there are rising opportunities for contact and disease transmission to ruminant livestock. NWC shedding MAP with their faeces might pose a reservoir for the bacterium which has to be considered as a part of control programmes for JD. This is especially true as NWC regular shed the bacterium without clinical signs of JD (Fecteau and others 2013), as also seen in our study.
No HEV was found in faecal samples of llamas and alpacas by RT-qPCR in our study. This lack of NWC shading the virus with their faeces may indicate no involvement of these animals in the transmission of HEV. Unfortunately, serum samples were not tested for specific Ab in the present study. Therefore, the question if NWC in Austria have contact with the virus, resulting in a seroconversion or not, remains unanswered.
From the results of the presented study it can be concluded that, although not a major pathogen, MAP is distributed among the population of NWC in Austria to a certain extent and might pose a possible risk for infection of other domestic and wild ruminants. Therefore, testing of NWC for MAP should be considered in case of clinical symptoms of JD and in the course of control programmes in other ruminant livestock.
The results also indicate that NWC in Austria are not infected by HEV and therefore might not present a risk for transmission of hepatitis E to humans.
Provenance: Not commissioned; externally peer reviewed
Funding This study was supported by the Ministry of Agriculture and Forestry of the Austrian government, the Ministry of Health of the Austrian government and by the Ministry of Agriculture of the Czech Republic.
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