Paratuberculosis: A Hidden Threat to Dairy Animals

Paratuberculosis: A Hidden Threat to Dairy Animals

Anand Mohan¹, Neelam Kushwaha^2*, Sudhakar P. Awandkar³

¹ Assistant Professor, Department of Veterinary Epidemiology & Preventive Medicine.

²Hospital Registrar, Teaching Veterinary Clinical Complex.

³Assistant Professor and Sectional Head, Department of Veterinary Microbiology.

College of Veterinary and Animal Sciences, Udgir, Dist. Latur, Maharashtra, Pin – 413517 (Maharashtra)

^*Corresponding Author

Abstract

Paratuberculosis or Johne’s disease is a bacterial disease of domestic and wild ruminants. It is caused by Mycobacterium avium subspecies paratuberculosis. The disease, most often, causes intestinal granulomatous inflammation of intestine. The disease has worldwide occurrence with significant economic losses to cattle, sheep, and goat husbandry. Its presence also influencing the livestock trade worldwide. In spite of exhaustive research on diagnosis, control and prevention since its discovery, it is still a challenge to the veterinary profession. Slow disease progression, mild clinical manifestation in adult animals, prolong course, difficulty in disease diagnosis, lack of good diagnostic, inaccurate diagnosis in early stage of disease, lack of treatment available, difficult and prolonged control strategies, legally restriction of positive cattle makes JD as a hidden threat to dairy animals.

Key words: Mycobacterium avium subspecies paratuberculosis, Johne’s disease, Paratuberculosis, Dairy animals.

          Paratuberculosis or Johne’s disease (JD) is caused by a facultative aerobic bacteria Mycobacterium avium subspecies paratuberculosis (Map). It is a slow-growing, non-motile, acid-fast and weakly gram-positive bacilli of 0.5-1.5 µm in length. Map is extremely robust, reported to survive up to 250 days in water, feces and on pastures. Based on host affinity, Map has been grouped as different strains (Table 1). The difference in host affinity among the different strains is related to geographical location and its genetic makeup.

Table 1: Strains of Mycobacterium avium subspecies paratuberculosis (Map)

Host and transmission

Cattle, Sheep and Goats are the principal host of Map but infection in other domestic and wild animals e.g., Horses, Mules, Hogs, Chicken, Monkeys, Camel, Dear, Wild boar, Mouflons, Chamois, Moose, Antelopes, Feral cats, Foxes, Stoats and Wild rabbits, Weasel, Crow, Jacksaw, Wood mouse, Badgers, Mice, Guinea pig, Rats, Hamsters, Human being and Invertebrates have been confirmed. Wild ruminants, most often, act as reservoir for domestic animals.

Disease is transmitted when pasture is shared among domestic and wild animals. Infection is usually introduced to dairy herds through the introduction of infected but clinically normal cattle. Infected animals may not show clinical signs for many years because of its long incubation period, and will often test negative on serologic and/or fecal culture. Infected dairy calves usually start shedding the bacteria in feces as early as at 2 years of age and develop clinical symptoms around 4 years of age. Excretion of Map in feces is the main source of environmental contamination. Infection is commonly acquired early in life via the fecal-oral route by ingestion of contaminated colostrum, milk, water, feed, pasture, soil, feces etc. Young and particularly newborn animals are more susceptible than older animals. A new born calf has an open gut, which allow infection to penetrate the mucosa barrier. On the other hand, genetic resistance to intracellular pathogens and the presence of a functional rumen could reduce susceptibility to JD in older animals. Apart from faeco-oral route of transmission, other routes of transmission are also possible. The calf may also get infection from the mother in utero. Less important routes include sperm from infected bulls and via embryo transfer if donor is infected. However, the chance of transmission of infection by these routes is extremely low.

Disease progression and clinical signs

After ingestion of Map from contaminated food material/milk, bacilli transfer across the mucosal layer occurs via M-cells (Trans-cellular pathway or Para-cellular-trans-cellular pathway) and enterocytes. Once bacilli cross the barrier, it may be phagocytized by subepithelial macrophages, intraepithelial macrophages and/or dendritic cells. Infection might be cleared by intracellular killing within these macrophages. Not all bacilli are killed by this mechanism. The remaining bacteria multiply within macrophage, resulting in the initiation of specific immune responses. At this point, bacteria may be processed and presented to immune cells or remained intact within phagocyte cells. Map has complex mechanism to remain protected within the phagocytic cell. In general, mycobacteria are relatively resistant to the bactericidal action of phagocytic cells. Mycobacteria circumvent macrophage antigen processing by inhibiting phago-lysosomal fusion via the secretion of protein tyrosine phosphatase and acidification. Mycobacteria also suppressed the inflammatory and immune response; it has also antiapoptotic, anti-destructive responses and antimicrobial responses. All these mechanisms of Map are helpful in survival within host mononuclear phagocytes. Resistance to reactive oxygen intermediates (linked to catalase and peroxidase activities, Alkyl hydroperoxidase reductase activity) and low amount of nitric oxide, etc. increase the chances of its survivable.

Clinical symptoms appear only in cows after a number of years, often after the first or second calving. The clinical signs are only seen, if there is enhanced intracellular multiplication of organisms, which intern due to alterations in the hormonal milieu and accumulation of macrophages and dendrite cells. Cattle rarely demonstrate a sign of illness before 2 years of age, as incubation period of disease is long. Animals exposed later in life (after weaning) are less likely to develop the disease. According to the severity of the clinical signs, immune response and potential to detect the infection, the disease progresses is described by four stages viz; preclinical, subclinical, and clinical and advanced clinical infection.

Preclinical infection

Infection in this stage is silent, as infected animals do not show clinical signs of disease. Prolong subclinical stage (2-4 years) is the result of complex interaction of pathogen and innate immune response of host. There is differential expression of set of genes and follow different molecular pathway in paucibacillary, multibacillary and resilient group. Proinflammatory cytokine genes were upregulated in subclinical stages. Animals are asymptomatic and shed low numbers of the organism which are undetectable. In some cases, mycobacterium-specific CMI (Cell-mediated Immunity) such as INF-γ, lympho-proliferation and delayed-type hypersensitivity (DTH) reaction might be detectable.

Subclinical excretory phase

In this phase, no visible clinical signs of JD, but animals may shed low to moderate number of Map in feces. Bacilli are also demonstrated in colostrum or milk, however it most often in later stages of the disease. On the basis of fecal bacterial shedding, subclinical animals are classified into, 1) low (<10 CFU/g); 2) moderate (10-50 CFU/g) and 3) high (>50 CFU/g) fecal shedders. Animal develop strong mycobacterium-specific CMI response while antibodies titre is generally low. CMI response can be detected by assays, such as lymphocyte proliferation to a T-cell-dependent mitogen and DTH or skin tests. A negligible humoral immune response during subclinical infection significantly reduces the usefulness of diagnostic tests measuring antibody titre.

Clinical and advance clinical stages

In contrast to the first and second stages, animal in clinical stage develop progressive disease, characterized by strong humoral immune response and a weak CMI response. Clinical signs in this stage are: intermittent or persistent diarrhea, gradual weight loss, reduced milk production and decreased fertility. Occasionally, in later stage, edema in the submandibular jaw area, which disappears when thirst increase as a result of fluid loss from diarrhea. Animals does not have fever and continue to demonstrate a normal appetite. Homogeneous watery feces without offensive odor, blood, epithelial debris, and mucus are present. In deer, however, the most common clinical signs are sudden death and extreme weight loss. Diarrhea, which is the first clinical sign in disease in cattle, may not occur in sheep or deer. But in some cases, occurrence of diarrhea has been reported in dear. Clinically infected cows shed 10⁶ – 10⁸ CFU/g of feces, that can easily be detected in fecal culture or spread the infection to new calves, the infectious dose is 10³ CFU/animal. Advance clinical stage is somewhat alike clinical stage with enhanced symptoms along with advance stage of cachexia.

Treatments

Mycobacterial infections are extremely difficult to treat. Generally, antimicrobial therapy of livestock is impractical for several reasons; 1) the drugs are expensive and need to be administered over extended periods and when the therapy is discontinued, the disease will progress, 2) the uses of antibiotics don’t result in a complete cure, possibly due to the inaccessibility of drug due to high lipid content and complexity of mycobacteria cell wall. Furthermore, evaluation of antimicrobial susceptibility is not easy as bacterial growth is very slow.

Most common regime tried for JD was isoniazid in combination with rifabutin and/or ethambutol, followed by a daily dose of isoniazid for life long. Daily isoniazid @ 20 mg/kg alone or in combination with rifampin @ 20 mg/kg for the duration of the animal’s life can forestall progression of Map infections but does not cure them. This application was advocated only for animals of high genetic merit or for the purpose of harvesting of germplasm like semen and embryos.

Macrolide drugs (clarithromycin and azithromycin) have the greatest in vitro efficacy. On the other hand, most first-line anti-tuberculosis (ethambutol & isoniazid) or anti-leprosy drugs (dapsone and clofazimine) are not effective against Map, while rifampicin family of drugs being the exception. Combination of amikacin, clarithromycin, and rifabutin may be the efficacious therapy for the treatment of Map. Isoniazid alone was ineffective but triple-drug combination of isoniazid-amikacin-clarithromycin was the most effective, therefore same may be applied for JD.

Attention has also been given to an ionophore antibiotic, monensin, as a possible therapeutic in adult cattle or chemoprophylactic in calves. Monensin poorly absorbed from the gastrointestinal tract. Drug is used as a feed additive to enhance growth rate and milk production efficiency in cattle. Monensin inhibits in vitro growth of Map but found that Map strains vary widely in monensin susceptibility. Provision of monensin to naturally infected adult cattle has been associated with modest improvements in histopathology scores, decline in fecal shedding rate, or reduced odds of testing positive on a milk-ELISA. Monensin used as a chemoprophylactic in calf milk replacer in dairy calves resulted in reduced tissue colonization and Map fecal shedding.

Diagnosis

Diagnosis of JD is essential as treatment is not promising and complete. Early diagnosis of disease is of prime importance as it helps in its control and prevention. Following are the most common methods for diagnosis of JD in domestic animals.

i) Ziehl–Neelsen (ZN) staining- Ziehl–Neelsen (ZN) staining of feces is routinely used to detect the mycobacteria. A positive diagnosis of JD is based on the presence of clumps (three or more micro-organisms) of acid-fast bacilli. Most often ZN staining on fecal samples as primary test at field level because it is a quick, simple and cost- effective test. Direct ZN staining of fecal samples is difficult to interpret as it stains other acid-fast organism such as Nocardia, Cornybacterium, Cryptosporidium also and will not differentiate between different mycobacterial species.

ii) Intradermal johnin test– It is a delayed type of hypersensitivity test, widely used for screening of JD at field level. It is mostly used as a single intradermal test. In this, 0.1 ml Johnin or avian purified protein derivatives (0.5 mg/ml or 25000 U/ml) is inoculated intradermally on caudal fold or in middle of neck. An increase in the skin thickness over 2 mm or 3mm after 72 hours after inoculation is interpreted as positive. Purified protein derivatives (PPD) used in test is a complex, undefined Map-secreted proteins. This test is prone to false-positive results due to cross-reactivity with similar proteins present in other mycobacteria.

iii) Interferon Gamma Assay- This test is based on specific production of cytokine IFN-γ by T lymphocytes after stimulation with PPD. The IFN-γ assay is superior to humoral antibody tests in the detection of subclinical infection. The advantage of the IFN-γ test is the significant secretion of IFN-γ during the early stages of JD and may thus be an attractive tool to detect cattle and sheep in the subclinical stage. However, it has several disadvantages: i) the possible cross-reactions, ii) the need to process the sample quickly since cells must be alive, iii) its high cost and iv) its low sensitivity. For all these reasons, this test is not widely used, although it can be used in control programs in order to reduce transmission to adult animals and to identify infected animals before they develop the disease.

iv) Culture- Culturing of Map is most accepted method of diagnosis of JD. Culture can be done from feces, colostrum, milk or intestinal mucosal scrapings. Culture of Map from feces is a widely used diagnostic test and is considered the reference assay. By this method at least 4-6 week are required to confirm the infection. If the sample found negative for growth of Map after 4- 6 week then, the culture may be continued for more than 7 months or more before declaring it negative. Culture is not used commonly for diagnosis of JD because of long incubation period, requirement of special culture media and possibility of contamination of culture.

v) Serological tests- Various serological tests like complement fixation tests (CFT), agar gel immunodiffusion (AGID) test and enzyme-linked immunosorbent assay (ELISA) have been developed to detect antibodies against Mapin animal sera. Among these, ELISA is most commonly used test. Diagnostic tests measuring antibody response lack sensitivity in early infections and hence their use have been restricted to the diagnostic confirmation of suspected clinical cases. ELISA is most often applied with diagnostic certainty in animals which shows clinical signs. In other words, animals after 2^ndor 3^rd delivery or older than two years develop humoral response which is also correlated with fecal and/or milk excretion of bacilli and hence give good sensitivity to ELISA. It can be applied on milk, because there is a moderate correlation between milk and serum antibodies. Milk antibodies concentration not only depends on the levels of serum antibodies, but also on genetic milk production level, days in lactation, number of calving etc. Based on these parameters, ELISA carried out in milk can detect fewer positive animals that that carried out in serum. Therefore, it is recommended that at least two ELISA determinations are carried out at different times of lactation to establish not only the level of antibodies but also the stability of the result.

vi) Polymerase Chain Reaction (PCR)– It is applied for specific detection of DNA sequences, for which samples can be taken from colostrum, milk, feces, and tissues from the ileocecal valve, ileum, or jejunum, or jejuna or ileocecal lymph nodes. The IS900based PCR has been found to be a rapid and sensitive method of detection of Mapin culture, feces, blood and tissues. PCR on fecal sample is less sensitive than fecal culture but offered great advantage in terms of rapidity of results without the need of viable bacteria in the sample.

Effect of JD on animal production

JD causes persistent diarrhea, progressive weight loss, debilitation and eventually death. Persistent diarrhea is most common clinical finding of disease. Continuous attention, care and treatment of clinically affected animals increase the financial burden on animal owner to a large extent (Table 2). The situation is aggravated when diagnosis is not confirmatory and animal owner continue to lose the money on term of treatment and care of animals.

Table 2: Direct and indirect losses from JD

Decreased in milk production in milch animals are the direct losses in dairy sector. Further it has also been documented that the shedding of bacilli in feces, milk or semen increase the chances of transmission in herd mate before its identification as the diagnostics in the early stage is not proper and specific. Furthermore, if adult animals are infected the clinical manifestation is not severe and hence its identification most often is not tried as losses are limited. However, small aggregated losses in production in large number of animals are actually huge. In this situation a through diagnostic survey is required to confirm the actual cause of production loss. On the other hand, diagnostic expanses increase the actual economic losses in dairy farm. Control of disease is difficult, bacilli is resistant to most disinfectant solution used routinely. Further the shedding of bacilli is intermittent, in this situation actual number of animals infected and its identification is not accurate. Therefore, once a herd declared infected with JD the local eradication of bacilli from farm, required great effort, further to keep the farm uninfected is another challenge. Keeping these points in mind, once animals found infected with JD, it should immediately slaughtered if law permit, otherwise separate the infected animals from another animals and human habitat. This is an attempt to restrict the transmission of bacilli among the susceptible hosts.  All these activities required the financial burden on animal owner in which there is no anticipated monetary gain.

It has also been documented that if a farm detected a single positive case of JD, the 20-24 animals in different stage of infection is present in farm. Segregation these cases which are silent, are not possible and the natural cycle of infection is eventually going on in the farm without noticed. Situation may become worse when the breeding bull is infected, the spread of infection is very fast and anticipated losses are manifold in coming future. In unrestricted transmission of JD in herd the maximum positivity is not more than 40% and herd propagate with disease with significant production losses. Today dairy will not sustain these losses as our resources and animal number is limited. In this situation the control and prevention of this disease is the only solution.

Conclusions

JD is called a hidden treat to dairy industry as it is slow, progressive disease of animals and treatment is not rewarding as well as it is not recommended. The silent transmission and perpetuation of bacilli in farm is progressive and eventual. Control and prevention of JD is laborious and expensive. The expanses in the animals due to this disease are significant in term of direct as well as indirect losses. the expense on control and prevention activities are added burden to the farmers.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8749836/

https://www.pashudhanpraharee.com/paratuberculosis-johnes-disease-in-cattle/

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