Intracellular infections in dogs and cats:
spirochetes, persisters and the integrative approach

Borrelia, Ehrlichia, Leishmania, Leptospira and related pathogens share one property: they escape the immune system via intracellular hiding, endothelial invasion or morphological metamorphosis. Why standard antibiotic treatment is inadequate, what makes spirochetes so special, what ozone therapy adds and how a phased integrative approach works better.

By Stefan Veenstra DVM

What are intracellular pathogens?

Most bacteria are extracellular: they multiply outside cells, in tissue or blood, which antibiotics and immune cells can reach relatively well. Intracellular pathogens have a fundamentally different strategy: they actively invade the cells of the immune system and use them as a shelter and source of nutrition. Macrophages, monocytes and dendritic cells, the very cells built to destroy invaders, become their dwelling.

From that position, they undermine the immune system in two ways. First, they escape immune recognition: they modify their surface proteins, inhibit phagocytosis, and prevent the host cell from initiating apoptosis. Second, they actively undermine the immune response: Leishmania suppresses the NF-kB-mediated pro-inflammatory response via epigenetic modifications, Ehrlichia blocks the fusion of the phagosome with the lysosome, and Borrelia varies its surface antigens so rapidly that the immune system cannot keep up with the recognition.

The result is a chronically activated but ineffective immune system: it fights, but does not hit the enemy. Owners see an animal that does not fully recover: varying joint complaints, recurring fever, persistent fatigue, or a dog that tests positive but is getting worse clinically.

Spirochetes: the most complex group

Within the intracellular pathogens, spirochetes form a special category. Borrelia burgdorferi, the causative agent of Lyme disease, and related species such as Borrelia afzelii and Borrelia garinii are spirochetes: corkscrew-shaped bacteria with unique motility and an exceptional ability to escape treatment.

Morphological metamorphosis

Spirochetes are not static bacteria. Under pressure from the immune system or antibiotics, Borrelia species undergo active morphological changes that guarantee their survival. The three main forms are the spiral, actively motile form that is sensitive to antibiotics; the round bodies or cyst form in which the spirochete encapsulates itself in a protective membrane structure and is metabolically inactive; and the biofilm form in which several bacteria together form a protective matrix that largely excludes antibiotics.

Borrelia persisters (dormant borrellia bacteria) are cells with low metabolic activity that can exist for a long time without replication. They can reversibly return to the actively motile form once conditions are more favorable. Persisters are present in significant numbers in biofilms, which provides the explanation for the antibiotic tolerance of biofilms.

Antigenic variation as an escape strategy

Borrelia has one of the most advanced systems for antigenic variation known in bacteriology: the VlsE system (Variable major protein-like sequence, Expressed). Through constant recombination of surface proteins, Borrelia produces an almost inexhaustible variety of surface patterns. The immune system produces antibodies, but as soon as they are recognized, a new variant appears. This explains why the infection can persist even with a well-functioning immune system.

Intracellular seclusion and erythrocytes

In addition to extracellular residence in joints and connective tissue, Borrelia can also survive intracellularly in fibroblasts, endothelial cells and neuroglia. From that position, the bacterium is protected against antibiotics that do not penetrate the cell wall, if at all. This is a key explanation for therapy resistance in chronic Lyme.

The relationship with erythrocytes is nuanced and species-specific. Borrelia burgdorferi (Lyme) does not actively invade red blood cells but resides primarily extracellularly in connective tissue. The relapsing fever Borrelia species, including Borrelia miyamotoi that also occurs in Dutch Ixodes ticks, have a different mechanism: they bind to erythrocyte membranes and can become completely covered by red blood cells. This forms an extra layer of immune evasion in which the erythrocyte-covered spirochetes avoid contact with phagocytic cells and B cells and thus slow down the production of antibodies. In mouse models, motile B. miyamotoi spirochetes have been observed in infected erythrocytes. This makes blood transfusion a theoretical transmission route and has diagnostic implications: standard Lyme serology does not recognize B. miyamotoi, which requires separate PCR or specific serology.

Leptospira: the Dutch spirochete

Leptospira is an underestimated but growing threat in the Netherlands, directly linked to the rat population. Brown rats (Rattus norvegicus) are chronically asymptomatic carriers that store the spirochetes in their proximal renal tubules and excrete them in urine for a long time. In urban environments with high rat densities — and the Netherlands has one of the densest rat populations in Europe — the risk to dogs is considerable. Infection occurs through contact with contaminated water or wet soil, through skin wounds or mucous membranes. Dogs that walk outside, swim in ditches or visit puddles are particularly vulnerable.

Mechanistically, Leptospira differs from Borrelia. Leptospira is not primarily an intracellular pathogen in the classical sense, but enters the circulation and organs via endothelial and epithelial cells. Pathogenic leptospires activate increased vascular permeability and a violent inflammatory response mediated by IL-1β and TNF-α via TLR4 and NF-kB. The preferred target organs are kidney and liver: leptospires colonize the renal tubules and cause tubulointerstitial nephritis and acute renal insufficiency. Liver damage leads to icterus. In severe cases, pulmonary hemorrhage occurs due to endothelial damage in the pulmonary vessels.

Clinically, the veterinarian sees an acutely ill animal with fever, vomiting, icterus, oliguria or anuria and sometimes bleeding tendency. In less severe or subclinical leptospirosis, the symptoms are vague: varying anorexia, mild renal value disorders, muscle weakness. It is precisely this subclinical form that is regularly missed and can lead to chronic renal insufficiency.

Vaccination provides partial protection: the available leptospirosis vaccines cover the most common serovars (Icterohaemorrhagiae, Canicola, Australis, Grippotyphosa) but not all circulating strains in the Netherlands. For dogs in high-risk areas (urban environments with lots of water and rat populations, farms, nature reserves), annual vaccination is standard but not an absolute guarantee. If leptospirosis is suspected, PCR on urine is the most sensitive early test; serology via MAT only gives reliable titers after two to four weeks.

Treatment consists of doxycycline as the first choice for elimination of carrier status, combined with intensive supportive care for kidney and liver. In case of severe involvement, intravenous fluid therapy and sometimes dialysis is necessary. The NGD Care Intracellular Microbe Protocol is relevant in leptospirosis in the recovery phase after acute treatment: recovery of renal tubules via L-glutamine, intestinal barrier repair after antibiotic treatments, liver support via glutathione and mitochondrial repair via CoQ10 and Longevity Support.

Why standard antibiotic treatment falls short

Doxycycline is the most commonly prescribed antibiotic for tick-borne diseases in dogs. In acute infections, it is effective and well-founded. In chronic or persistent infections, there are fundamental limitations.

Doxycycline: mechanism of action and indications

Doxycycline is a tetracycline antibiotic that inhibits bacterial protein production by binding to the 30S ribosomal subunit. It has a broad spectrum and works against Ehrlichia, Anaplasma, Borrelia and Rickettsia. An additional advantage: doxycycline has significant anti-inflammatory properties via inhibition of TNF-α, IL-1β and IL-6, which is therapeutically relevant in infections where inflammation determines a large part of the clinical damage. In case of acute Ehrlichia infection, a 28-day course is the standard; in Lyme 21 to 28 days.

Deficiencies in chronic and persistent infections

The fundamental problem of doxycycline in chronic infections is twofold. First, it is primarily effective against actively dividing bacteria. Borrelia persisters in cyst form or biofilm, with minimal metabolic activity, hardly respond to doxycycline. The MBIC for biofilm-associated Borrelia is 64 times higher than the MIC for free-floating spirochetes. In practice, this means that standard doses for biofilm infections are pharmacologically insufficient.

Second, doxycycline reaches intracellular reservoirs in fibroblasts and endothelial cells to a limited extent. The intracellular concentration depends on active transport, which in some cell types is insufficient for bactericidal concentrations.

Side effects of long-term use

Side effects play a real role in the treatment times required for chronic infections.

Other antibiotics for intracellular infections

In Leishmania, allopurinol and meglumine antimoniate (Glucantime) or miltefosine are used. Both have significant toxicity: antimoniates are nephrotoxic and hepatotoxic when used long-term and require injection; Miltefosine is oral but has gastrointestinal side effects and is teratogenic. Allopurinol also has many side effects: intestines, liver and kidneys. For Ehrlichia and Anaplasma, doxycycline is the first choice; Rifampicin is an alternative for resistance but has its own toxicity profile. In complex cases, combinations of doxycycline with cefuroxime or azithromycin are used in Borrelia to target both active and persistent forms, but the evidence for combination therapy in veterinary patients is limited.

Ozone therapy: mechanism of action and added value

Ozone therapy is one of the most promising supplements in the treatment of chronic intracellular infections. The principle of action is paradoxical: ozone is a strong oxidant that, in controlled doses, activates the body’s endogenous antioxidant capacity and at the same time has an immediate antimicrobial effect.

Mechanism

The therapeutic action of ozone therapy is based on the controlled and moderate oxidative stress produced by reactions of O3 with biological components. The calculated and transient oxidative stress induces several second messengers in intracellular signaling pathways. This is called the paradoxical action of ozone: it acts as an oxidizing molecule but at the same time can increase the antioxidant properties of the areas affected by the disease.

In practical terms, ozone works on three levels. First, directly antimicrobial: ozone oxidizes the membrane lipids and proteins of microorganisms, preventing them from surviving. This is also true for intracellular forms when ozone is administered systemically via autohemotherapy. Second, immunomodulating: ozone activates macrophages and dendritic cells, increases interferon production, and stimulates NK cell activity. It restores the very immune functions that suppress intracellular pathogens. Third, mitochondrial: ozone stimulates mitochondrial ATP production and enhances cellular oxygen utilization, which is relevant in the energetic depletion that chronic infections cause.

Evidence in Leishmania

Cabral et al. treated Leishmania-infected mice with ozone therapy in a variety of delivery forms. All treatment groups showed significant reductions in lesions, particularly the combination of meglumine antimoniate and topical ozone. Ozone treatment also showed better wound healing and immunomodulatory activity.

Forms of administration in veterinary practice

Two main routes are used in veterinary practice. Major autohemotherapy is the most effective systemic route: blood is taken, treated outside the body with ozone and infused back. This brings activated immune cells and ozone products directly into circulation. Rectal insufflation is the most accessible route for practice and home treatment: ozone gas is delivered via the rectal route, absorbs through the colon mucosa and reaches systemic circulation. This is also the route that is most practical for long-term chronic use, without additional liver burden.

In the NGD Care Intracellular Microbe Protocol, ozone therapy is recommended as an optional addition in phase 2, Usually we use a high dose rectal insufflation as a basic therapy where we treat 2 x a week for 5 weeks. There is also the possibility of a large autohemotherapy through the integrative specialist veterinarian. The combination of ozone therapy with the supplement protocol enhances the antimicrobial activity on multiple pathways at once.

The integrative approach: the NGD Care protocol in three phases

The three phases are strictly ordered. Moving to stage 2 too early increases the risk of a Herxheimer reaction. Each phase mechanistically builds on the previous one.

Herxheimer reaction: what to expect

The Jarisch-Herxheimer reaction is a temporary but sometimes significant deterioration that occurs with massive cell death of pathogens. Released bacterial toxins, endotoxins and cell debris activate the immune system acutely. In spirochete infections, this is a well-known and well-documented phenomenon: classically described in the treatment of syphilis and later also in Lyme treatment.

Clinically, owners see: sudden fever, worsening of joint complaints, extreme fatigue, sometimes vomiting or diarrhea, in severe cases neurological symptoms. The reaction typically occurs in the first days after the start of phase 2, when transitioning from phase 1 to phase 2, and when dose increases. It is paradoxically good news: it proves that an effective response to the pathogens takes place. But it requires careful guidance.

Timeline: What to expect

This article is educational in nature and does not replace a veterinary consultation. The Intracellular Microbe Protocol is the heaviest protocol in the NGD Care offering and always requires veterinary supervision. Never adjust the protocol independently without consulting a veterinarian.