Checklist of tests to consider for anemia

! CBC w/ reticulocyte count (Check PCV/TP in house before sendout)

! Chemistry (r/o chronic disease as cause of anemia)

! U/A (if you find azotemia on your chem you will wish you had this )

! In-saline agglutination

! Abdominal Radiographs (It stinks to find the zinc or splenic torsion a week into it)

! Chest Radiographs

! Coomb's

! Infectious disease testing (Microscopy, IFA and PCR)

! ANA

! Heartworm antigen

! Bone marrow (If non-regenerative anemia or concurrent ITP)

PRACTICAL APPROACH TO IHMA Adam Birkenheuer, DVM, PhD, DACVIM (Small Animal Internal Medicine) College of Veterinary Medicine North Carolina State University, Raleigh, NC, USA Signalment: Dogs with IMHA are usually older than 1 year of age. There does not appear to be a significant gender predisposition. Several breeds have a higher incidence of IMHA, such as cocker spaniels, miniature poodles, old English sheepdogs, and Doberman pinschers. Hereditary erythrocyte disease (PK and PFK deficiency osmotic fragility). History: Dogs are usually presented for weakness, lethargy, anorexia, pale mucous membranes, jaundice, or discolored urine. There is a weak association between recent vaccination and IMHA. Assessment of historical tick attachment, drugs (sulfas), toxins (zinc, onions), other concurrent systemic signs may make infectious or neoplastic causes more likely. Some studies suggest a seasonal increase of IMHA cases in the spring and summer. Babesiosis has been associated with a recent dog bite by an American Pit Bull Terrier. Physical Examination: Pallor, weakness, depression, or jaundice. Fever may be present and does not always indicate and infectious etiology. Systolic heart murmur may be present due to decrease viscosity of the blood. Tachycardia and tachypnea may be present due to low oxygen delivery. Animals with concurrent ITP may have petechia or ecchymoses. Ocular exam may reveal uveitis or petechia.

Treament: Once you have diagnosed IMHA, set your client up for the long haul. A treatment commitment of at least one week is important, since that is about how long it takes for the immunosuppressive therapy to be effective. Treatment will continue at least 4-6 months and possibly the rest of the animal's life. The following are tables that describe the way that I treat IMHA. I break them into two categories. These are guidelines only. I realize there is more than one right way to do things, and that each patient requires custom management. Over the years, I have become more inclined to start a second immune suppressive drug at the time of diagnosis. This is mainly due to

the increasing costs of care and to facilitate long-term management. After the initial crisis is over owners frequently want to decrease the glucocorticoids due to their side-effects. Thromboembolic complications are an important cause of morbidity and mortality in dogs with IMHA. I typically recommend thromboprophylaxis with low dose aspirin, which can be given safely with glucocorticoids. In cases that are at higher risk of thromboembolism (i.e. already having thromboembolic complications, intravascular hemolysis, SIRS, hyperbilirubinemic, hypoalbuminemic etc.) I will often add heparin. Some clinicians prefer clopidogrel because it is newer and more expensive than aspirin. We have some exciting research at NCSU evaluating optimal dosing and adjustment using unfractionated heparin.

How I Treat Uncomplicated IMHA Criteria • Patient is eating and drinking • Not tachypneic at rest • Hct is ≥ 15% at presentation • Reticulocytosis is ≥ 60,000 at presentation • No agglutination of RBC's • Not intravascular Drug/Dose • Prednisone 2mg/kg PO divided BID until the PCV is normal • Azathioprine 2mg/kg PO Q24 for at least 2wks (I have more recently started this on every case in hopes I

can wean the pred faster) • GI protectant

• Omeprazole (1-2mg/kg PO BID) • Aspirin 0.5mg/kg PO Q24 or Clopidogrel 2-3mg/kg PO Q24 Expected Response • Stable or rising hematocrit within 7 days Drug Tapering/withdrwal • Prednisone

• Always recheck the PCV before changing drug doses • Once the PCV is normal I will administer the same daily amount once daily for 7-14 days • i.e. dog was receiving 20mg PO BID, I will switch to 40mg PO Q24 • Decrease the current dose by approximately 25% • DO NOT decrease the dose more frequently than every 2-4 wks • DO NOT change dose without rechecking the PCV

• Azathioprine • After 2-4 weeks I will administer the azathioprine every other day • i.e. Dog was receiving 25mg PO Q24, then I administer 25 mg PO EOD

• GI protectant • I usually stop when the prednisone dose is ≤ 1mg/kg/day

If there is no response within 7-10 days then I treat them as complicated IMHA cases

How I Treat Complicated IMHA Criteria • Patient is not eating and drinking • IS tachypneic at rest • Hct is ≤ 15% at presentation • Reticulocytosis is ≤ 60,000 at presentation • Agglutination of RBC's • Intravascular hemolysis present • Fails to respond to prednisone alone after 7 days Drug/Dose • Prednisone 2mg/kg PO divided BID until the PCV is normal • Azathioprine 2mg/kg PO Q24 for at least 2wks • IV fluids as needed (avoid jugular catheterization if possible) • GI protectant

• Omeprazole (1-2mg/kg PO BID) • Aspirin 0.5mg/kg PO Q24 or Clopidogrel 2-3mg/kg PO Q24 • Heparin? We use heparin and are trying to determine best practical way to dose and monitor Expected Response • Stable or rising hematocrit within 14 days Drug Tapering/withdrwal • Prednisone

• See uncomplicated IMHA • Occasionally I will "taper" by giving the prednisone and azathioprine on alternate days

• Azathioprine • After 2-4 weeks I will administer the azathioprine every other day • i.e. Dog was receiving 25mg PO Q24, then I administer 25 mg PO EOD

• IV fluids are discontinued when the patient is eating and drinking well • Heparin is discontinued when IV catheters are removed • GI protectant

• See uncomplicated IMHA If the patient fails to respond, then I "pull out all of the stops" see adjunctive treatments.

Adjunctive treatments: Cyclosporine (Atopica®) 10 mg/kg/day orally. Use of the Atopic® and Neoral® formulation results in more stable serum concentrations. Monitoring drug levels is still recommended if side effects or lack of response are observed. Therapeutic serum concentrations are 200-400ng/ml. Many people use as a first choice treatment for complicated IMHA cases. Mycophenolate mofetil (Cellcept®) 10-20mg/kg PO BID. Very little information in veterinary medicine for use in IMHA/ITP, but experiences are promising. Human gamma globulin 0.5-1.0 gram/kg intravenously over 4-12 hours. May be repeated within 48 Hrs. Leflunomide 2-4 mg/kg/day. Used to be very expensive treatment that has shown some efficacy for the treatment of immune mediated disease. Price seems to fluctuate now. Cyclophosphamide Due to reports of decreased survival, I am NOT using or recommending this drug for IMHA. However I have several colleagues that use it routinely and feel they have good success. Splenectomy Absolute "last resort" for me. I have never performed a splenectomy for IMHA. Some recent studies may show benefit. Make sure that these patients do not have infectious diseases (especially Babesia) before doing this. Plasmapheresis Special facilities and wealthy clients required, with unknown benefits at this time. Supportive Therapy: Intravenous fluid therapy: IV fluid therapy should be avoided if possible since IV catheterization increases the risk of thromboembolism. Blood transfusion: I don't have a "magic" number as to when I will administer a transfusion. I base it on clinical signs and the rate at which the PCV is dropping. Ideally, cross-matched blood will be administered, but at a minimum, donors should be screened for infectious diseases. Antibiotics: Antibiotics are not indicated for idiopathic IMHA, but antimicrobials such as doxycycline or imidocarb diproptionate are administered pending the results of infectious disease testing. Heparin: While I think that all dogs with IMHA should be on ultra-low-dose aspirin or clopidogrel, some (all?) dogs may also benefit from heparinization. It is not easy to identify which dogs will benefit or for how long to treat them. Dogs that are hypercoagulable based on thromboelastography are good candidates for heparin. We typically give 300-900 U/kg/day as a CRI with the goal of increasing their APTT 1.5 times their baseline. Recent studies have demonstrated that using individually adjusted heparin dosing based on antifactor Xa activity was associated with improved survival. Difficult to use in practice. Plavix ®: Plavix should have the same effect as aspirin, but there is less data in veterinary medicine justifying its use over aspirin. The optimal dose has yet to be determined, but 3mg/kg PO Q24 has been shown to reduce platelet aggregation. Some institutions use a loading dose of up to 10mg/kg PO once followed by a maintenance dose of 2-3mg/kg POQ24.

Prognosis:

Favorable prognostic criteria • Reticulocytosis • Coomb's test becomes negative • Hematocrit ≥ 15% at presentation • Resolution of RBC agglutination

Unfavorable prognostic criteria • Sustained absence of reticulocytosis • Hematocrit ≤ 15% at presentation • Persistent RBC agglutination • CHAOS ≥3 • ASA classification ≥3 • Increased Bilirubin • Pigmenturia • Increase Urea Nitrogen

How to improve your diagnostic skills: The science behind clinical reasoning and why smart people do dumb things. Adam J. Birkenheuer DVM, PhD, DACVIM North Carolina State University, College of Veterinary Medicine Raleigh, North Carolina

Cognitive errors are reported to be one of the most common sources for diagnostic error in medical practice. The most common type of cognitive error is faulty reasoning. Many of these “reasoning errors” are believed to have provided survival benefits to our ancestors (AKA: It’s not your fault…). Skeptical cavemen did not survive long…and it is probably their fault that we are prone to some cognitive bias.

What do “they” mean when “they” say bias?

•predictable deviations from rationality

•In the context clinical reasoning, it is inherently bad…

•If it’s good it’s intuition

•If it’s bad it’s bias

Prior to doing some studying about clinical reasoning, I had no bias towards the word bias.

Common cognitive errors clinicians commit and how to avoid them:

From Mckenzie: JAVMA, Vol 244, No. 3, February 1, 2014, pp271-76.

Availability:

• Work hard on your “walk around” knowledge base: Beware of academic bulimia

• Skim a chapter or a list BEFORE you go in to the exam room

Anchoring:

• Make sure you get a complete list of concerns from the client that they would like to have addressed during the visit

• Always start with open-ended questioning and let the client tell their story first (make sure you set an agenda with the client including time available so they can try to be concise)

• Use reflective listening to make sure that your interpretation of the clinical picture/problem representation is aligned with the client’s perception

• Use non-verbals and funnel (open to closed questioning) when you are getting the information you “want” but be careful not to stop there!

• WHAT ELSE COULD THIS BE? WHAT DOESN’T FIT or CAN’T BE EXPLAINED?

Overconfidence:

• Perfection is a myth…get over it!!!!

• You will be wrong!

• You will not know the answer to ever question!

• It is better to have great plan than it is to have a great answer!!!

(Overly?) heuristic:

If you are like me then you might need an explanation of what heuristic means…

• Approach to problem solving, learning, or discovery that employs a practical methodology not guaranteed to be optimal or perfect, but sufficient for the immediate goals

• Examples of this method include using a rule of thumb, an educated guess, an intuitive judgment, stereotyping, profiling, or common sense

Tips to avoid bias:

• Work hard to improve your walk-around knowledge base

• Supplement your available knowledge frequently, take advantage of our tendency to remember things we just saw (pre-game)

• Trust (but verify) your gut

• Challenge yourself constantly

• What can’t be explained?

• What pieces of information did I discard/devalue/ignore to arrive at this conclusion

• Always have a plan B! and probably a C, D, E…

Where is that coming from? Managing fever of unknown origin Adam Birkenheuer, DVM, PhD, DACVIM NCSU College of Veterinary Medicine 4700 Hillsborough St. Raleigh, NC 27603

Definition Fever is defined as a higher than normal body temperature (>102.5) due to altered thermoregulatory mechanisms in the hypothalamus. Fever of unknown origin (FUO) is a fever that does not resolve spontaneously and for which no obvious cause is identified. Infectious disease, immune-mediated conditions and neoplasia account for over 75% of FUO cases. With aggressive diagnostics usually only 10% of FUO cases are considered idiopathic. Prior to beginning a diagnostic investigation for FUO, one must rule out non-pyrogenic causes of an elevated body temperature (i.e. heatstroke, over-exertion). Pathophysiology The hypothalamus is responsible for thermoregulation. Fever occurs when the hypothalamic setpoint is reset to higher than normal. Inflammation and bacterial endotoxins increase the hypothalamic set point by causing the release of endogenous pyrogens such as interleukin (IL)-1, IL-6 and tumor necrosis factor alpha. Differential Diagnoses Infectious, neoplastic and immune-mediated diseases account for over 75% of FUO cases. Below is a list of possible causes of FUO. C=Canine, F=Feline. o Infectious

! Localized or Systemic Bacterial Infections (C /F) • Discospondylitis, osteomyelitis, bacterial endocarditis, septic arthritis,

prostatitis, pyelonephritis, septic meningitis, cholangiohepatitis, abscesses, pyothorax, peritonitis, pneumonia, pyometra, catheter site infections

! Specific Bacterial Infections • Leptospirosis (C), Lyme disease (C), brucellosis (C), mycobacterium

(C/ F), bartonellosis (C/ F), hemotrophic Mycoplasma (Haemobartonella) (C /F), tularaemia (C/F), salmonellosis (C/F)

! Viral • Canine distemper virus (C), parvovirus (C/F), FeLV(F), FIV(F), FIP(F)

! Rickettsial • Ehrlichiosis/anaplasmosis (C/F), Rocky Mountain Spotted Fever

(Rickettsia rickettsii)(C) ! Fungal (C/F)

• Histoplasmosis, blastomycosis, coccidiomycosis, cryptococcosis, aspergillosis

! Protozoal • Babesiosis (C), leishmaniasis (C), trypanosomiasis (C),

hepatozoonosis (C), toxoplasmosis (C/F), cytauxzoonosis (F)

o Immune- mediated ! Immune-mediated polyarthritis (C/F), SLE (C/F), rheumatoid arthritis (C),

vasculitis (C), meningitis (C), pemphigus, immune-mediated hemolytic anemia (IMHA) (C/F), immune-mediated thrombocytopenia (IMT) (C/F), transfusion reaction (C/F)

o Neoplastic (C/F) ! Lymphoma, leukemia, multiple myeloma, solid necrotic tumors

o Other (C/F) ! Pancreatitis ! Drug induced (tetracyclines, penicillins, sulfas), toxins, metabolic bone

disorders, hyperthyroidism, tissue necrosis Diagnostic Plan The diagnostic goal is directed towards identifying a specific underlying cause of fever and instituting specific therapy. When a specific organ system is obviously affected, diagnostics should target that system. The following tests should be performed on all FUO cases.

o CBC, chemistry profile, and urinalysis o Urine bacterial culture and sensitivity should be performed in all cases of FUO

even if urine sediment is inactive. Helpful with detection of pyelonephritis or prostatitis.

o FeLV antigen and FIV antibody tests for all felines The following tests should be considered in cases without an obvious source of fever:

o Serial blood cultures: to detect bacteremia associated with discospondylitis, endocarditis or other foci of infection. A negative culture does not rule out bacteremia

o Cytology of enlarged lymphnodes or affected organs – neoplasia or identification of infectious agents

o Specific serologic tests for infectious agents: antibody titers or antigen tests are obtained for evidence of infectious disease. If infectious disease is suspected and initial titers are negative repeat in 2-4 weeks

o Polymerase chain reaction testing for specific infectious agents o Fungal cultures and/or serology o Cytology and Cultures of CSF and/or synovial fluid o Immune-mediated polyarthritis will frequently NOT be associated with detectable

joint swelling, therefore arthrocentesis is indicated in all FUO cases where no underlying cause has been identified

o CT/MRI often indicated before CSF tap to rule out an intracranial mass and decrease risk of herniation

o Tests to support immune-mediated diseases: ANA test if suspected systemic lupus erythematosus (SLE), Coombs test if suspicion of immune mediated hemolytic anemia (IMHA) or thrombocytopenia (ITP), serum protein electrophoresis

o Thoracic radiographs: to evaluate for evidence of neoplasia, effusions or pulmonary infiltrates

o Abdominal radiographs: evaluate for abdominal masses, effusions, free gas

o Spinal and longbone radiographs: examine for evidence of discospondylitis, osteomyelitis, periosteal proliferation

o Nuclear scintigraphy: to find “hotspots” which are often associated with infection o Abdominal ultrasound: rule out pyelonephritis, prostatitis or pyometra as well as

identifying and aspirating any enlarged abdominal organs or masses o Echocardiogram: evaluate for vegatative valvular lesions o Bone marrow aspirates and/or biopsy: If CBC changes are reflective of bone

marrow involvement or neoplasia is suspected o Muscle biopsy – hepatozoonosis o Abdominocentesis – peritonitis and pancreatitis o Transtracheal wash or bronchoalveolar lavage if respiratory involvement

Treatment

The goal in all cases of FUO is to obtain a specific diagnosis and treat accordingly. Therapeutic trials should only be initiated when a specific diagnosis cannot be ascertained. Patients with persistent fevers that are < 105F should be treated symptomatically with IV fluids and mechanical cooling. Anti-pyretic drugs should only be reserved for patients with fevers > 105F and those that have failed to respond to fluids and mechanical cooling, as they can mask the effects of other therapies and can be associated with adverse effects such as GI ulceration, hepatic and or renal toxicity.

• Antibiotic trials: o Broad spectrum antibiotic therapy may be initiated after all culture

specimens have been collected. Therapy should be based on the agents most likely present and their known antibiotic sensitivity

• Glucocorticoid trials o Used when immune-mediated disease is suspected or confirmed o Should only be used when infectious disease has been ruled out o A dramatic response (fever reduction) should be seen within 24-48 hours

Drug therapy trials without a definitive diagnosis may interfere with future diagnosis and may exacerbate an undiagnosed condition that may be life threatening.

References available upon request

VECTOR-BORNE DISEASE “CLIFF NOTES” Adam Birkenheuer, DVM, PhD, DACVIM

Vector Borne Disease Diagnostic Laboratory NCSU College of Veterinary Medicine

4700 Hillsborough St. Raleigh, NC 27603

Background: Infectious diseases are recognized with increased frequency in both general and referral practices. While many practitioners are diagnosing patients with classic signs and presentations, those with atypical presentations are frequently missed. A missed diagnosis often results in therapies that can lead to persistent illness or in some cases may actually worsen the outcome. Many factors are contributing the emergence of infectious diseases and include climate changes, urbanization, vector epidemiology, alternative forms of disease transmission, ease of animal transport and advanced diagnostic techniques. Veterinarians need to be vigilant for infectious diseases, especially in cases that don’t respond to treatment and those in which things “just don’t add up.”

Babesia: Canine babesiosis is an emerging infectious disease in North America and is typically caused by

either Babesia gibsoni or Babesia canis vogeli. Babesia gibsoni is most commonly detected in American Pit Bull Terriers. Babesia canis is most commonly diagnosed in greyhounds. In addition to tick-transmission, infection via blood transfusions, dog-fights and perinatal routes have been documented. There are at least 9 Babesia sp. that have been identified in dogs (B. canis vogeli, B. canis canis, B. canis rossi, B. gibsoni, B. conradae, B. annae, Babesia sp. Coco, as well as several un-named Babesia sp.)

Signs: Babesiosis can be acute or chronic in nature. There are variations in the clinical presentation depending on which species of piroplasm, age and breed of the host and presence of concurrent disease. The most common hematological effects are thrombocytopenia and anemia. Despite the fact that many practitioners associate babesiosis with anemia, most studies have demonstrated that thrombocytopenia is the most common hematological abnormality in dogs with babesiosis. Thrombocytopenia is suspected to be immune mediated (ITP). The thrombocytopenia can be severe (< 50,000 plt/ul), but evidence of bleeding is rare. Some cases have had ITP without anemia. The anemia is primarily due to immune-mediated destruction and is often (> 85%) Coombs positive. The degree of anemia is variable and can be severe (PCV < 10%), but some infected dogs have normal hematocrits. No matter what the hematocrit is, some degree of RBC regeneration is usually detected. The effects on the leukon are variable and inconsistent. Some cases have a profound leukocytosis with a left shift that often accompanies a strong regenerative response. Some cases have had transient leukopenia. The most common abnormalities detected on a biochemical profile include mild increases in liver enzymes and hyperglobulinemia. Other clinical signs that may be seen include fever, lymphadenopathy, splenomegaly, pigmenturia, and jaundice. A common misconception exists that all cases of babesiosis exhibit intravascular hemolysis. For B. gibsoni, the most commonly diagnosed form of Babesia in the US, this seems to be a rare finding. Renal failure and proteinuria have been associated with B. annae (T. annae) in Spain and more recently with B. gibsoni in the USA. Hypoglycemia is identified in 20% of dogs with B. canis rossi infections. Dogs that have undergone splenectomy or that are receiving chemotherapy appear to be at increased risk of disease with a large Babesia sp. (Coco).

Diagnosis: Microscopy or PCR can easily rule babesiosis in, but it is very difficult to rule out babesiosis completely. At this point in time I recommend that microscopy, PCR and serology are all considered to maximize your chances of identifying the infection. The organisms stain well with a modified Wright’s stain (i.e. Diff-quik® or Leukostat®). Evaluation of capillary blood (ear or toenail) may improve parasite recovery. There is variable seroreactivity, so serology against both B. canis and B. gibsoni and is warranted. Convalescent (3-4wks) titers may be helpful in cases with acute onset of illness and low or negative acute titers. Seroreactivity of > 1:64 is suspicious for exposure in most labs. PCR tests should be able to identify and differentiate all common canine Babesia spp. PCR is the most accurate way to identify which species of Babesia is present. In one study, a single PCR test identified 85% of B. gibsoni and 2 consecutive PCR tests identified 100%.

Treatment: Currently imidocarb dipropionate (Imizol® 6.6mg/kg IM, repeat in 2 wk.) is the only approved treatment for canine babesiosis in the U.S. Atovaquone 13.5mg/kgPO TID (with a fatty meal) and azithromycin 10mg/kg PO Q24 in combination for 10 days has been shown to reduce or eliminate B. gibsoni (Asian) parasitemias as determined by PCR. A combination therapy of doxycycline, clindamycin and metronidazole has shown some promise in a few studies.

Follow-up: Babesia canis is likely to be cured by imidocarb. Babesia gibsoni may be cured by atovaquone and azithromycin combination or doxycycline, clindamycin and metronidazole therapy. Other treatments may result

in a clinical remission with persistent parasitemia. These dogs are at risk for recrudescence, and may act as a reservoir. We recommend two consecutive blood smear evaluations AND PCR 60 and 90 days post treatment. Serology is unlikely to be helpful for short-term follow-up, since antibody titers may persist for months following treatment. Feline babesiosis has not been reported in the U.S. However, cats can be infected by Cytauxzoon felis (see cytauxzoonosis)

Ehrlichiosis/Anaplasmosis: Canine ehrlichiosis/anaplasmosis is caused by several different species of

Ehrlichia/Anaplasma. Those species reported in the US include E. canis, E. ewingii, E. chafeensis, A. phagocytophilum (E. equi), and A. platys (E. platys). Ehrlichiosis can either be acute or chronic. Most cases are recognized during the chronic stage.

Signs: The hematological effects of ehrlichiosis/anaplasmosis can be variable, but the most common abnormality detected is thrombocytopenia. Despite the fact that many clinicians suspect ehrlichiosis/anaplasmosis in dogs with hemolytic anemia, a non-regenerative anemia is more commonly identified. Ehrlichiosis is only occasionally associated with a secondary IMHA. Ehrlichiosis can also cause pancytopenia. The effects on the leukon are variable. Both leukocytosis and leukopenia have been reported. Lymphocytosis can be seen. The accompanying clinical signs are often vague including; fever, lethargy, anorexia, weight loss, and vomiting. Bleeding tendencies such as epistaxis, petechia, or ecchymosis may also be present. Hyperglobulinemia (polyclonal much more commonly than monoclonal), hypoalbuminemia, lymphadenopathy, proteinuria, polyarthritis (common with E. ewingii), and/or uveitis may be present. The lack of thrombocytopenia does not rule out ehrlichiosis. Anaplasma platys only appears to cause thrombocytopenia, and not systemic illness. Anaplasma phagocytophilum is associated with acute febrile illness in dogs and thrombocytopenia is a common finding. Co-infection with A. phagocytophilum and Borrelia burgdorferi was associated with worse illness in on study.

Diagnosis: Serology is helpful in the diagnosis of ehrlichiosis/anaplasmosis. Acute and convalescent (3-4wks) titers should be performed if the clinical signs are acute and initial titers are low or negative. A four-fold change is consistent with ehrlichiosis/anaplasmosis. If the signs are chronic (> 4wks.), then a single high titer is consistent with infection. In-house tests (SNAP®) are not designed for diagnostic use, but a positive test in conjunction with appropriate clinical findings is supportive of a diagnosis of ehrlichiosis/anaplasmosis. The current in-house assays fail to differentiate antibodies against E. ewingii and E. canis and cannot differentiate antibodies against A. phagocytophilum from those against A. platys. Occasionally a morula can be identified in a white blood cell or platelet on a blood smear, but the parasitemia is often very low so the sensitivity of microscopy is poor. The low levels of circulating organisms can also hamper PCR. A positive PCR test is useful to rule-in the presence of infection and identify which species is present. Accurate species identification is important for client education regarding zoonoses as well as expected response to treatment. A negative PCR test does not rule out the possibility of ehrlichiosis. Therefore the resolution of clinical signs in response to therapy remains an important “test.”

Treatment: Doxycycline (10mg/kg/day for 4 wk.) is considered the treatment of choice. Other drugs that are reported to be effective include; tetracycline, oxytetracycline, minocycline, and chloramphenicol. Imidocarb dipropionate does not appear to be an effective treatment.

Follow-up: Resolution of clinical signs after therapy is probably the most important follow-up and further specific diagnostics for ehrlichiosis/anaplasmosis are not indicated. Serology is generally a poor way to assess recovery as antibody titers may persist for months. If the animal is seroreactive, but does not respond to therapy, the PCR should be performed since some species, such as E. chafeensis, may not respond as well to therapy. If the PCR is negative, then an alternative diagnosis should be considered.

Zoonosis: Although humans can be infected with some of the same species of Ehrlichia as dogs, dogs do not appear to transmit infections directly to humans. Since dogs and humans are often exposed to the same population of ticks, dogs can act as a sentinel for ehrlichiosis/anaplasmosis.

Feline ehrlichiosis: The clinical signs appear similar to what is seen in dogs, and it should be considered when more common causes of disease are not apparent.

Bartonellosis: Bartonella vinsonii and B. henselae appear to be the primary causes of bartonellosis in dogs. Signs: The full spectrum of canine disease caused by Bartonella species has yet to be elucidated. Has been

shown to be a cause of endocarditis, and has been associated with granulomatous inflammation and hepatic disease in dogs. Although polyarthritis has only been confirmed in a handful of cases of canine bartonellosis, lameness and stiffness were the most common presenting signs for dogs with confirmed Bartonella endocarditis suggesting that it may be more common finding. Anemia and thrombocytopenia have been detected in nearly half of the dogs diagnosed with Bartonella vinsonii. The pathogenicity of Bartonella infection in cats is unclear, and it has not consistently been found to have any specific or characteristic hematological or biochemical effects. Some

studies have detected an association between stomatitis and lymphadenopathy in cats co-infected with Bartonella and FIV, while other studies have not detected associations between Bartonella and clinical diseases in cats.

Diagnosis: Serology is suggestive of exposure or infection. PCR is also available and positive test results are indicative of current infection. PCR tests should be able to identify and differentiate which species of Bartonella are present. A combination of culture (BAPGM) and PCR appears to be an excellent method for the detection of Bartonella spp. For unknown reasons, there are frequently discordant results between serological and molecular/bacteriologic assays, so a combination of these techniques is recommended.

Treatment: The optimal treatment for bartonellosis is unknown. Currently patients are being treated with a combination of a tetracycline and a fluoroquinolone for a minimum of 6 weeks and sometimes 6 months. Azithromycin (5-10mg/kg PO Q24 for 5 days then every other day for 45 days) has also been used in combination with a tetracycline or fluoroquinolone but resistance to azithromycin can been seen rapidly in vitro. Some cases have had additional clinical responses when rifampin, enrofloxacin or doxycycline were used in combination with the azithromycin.

Follow-up: Resolution of clinical signs and culture. Since the full spectrum of disease is unknown and a large percentage of normal animals can test positive for Bartonella cautious interpretation of test results is warranted and consideration of alternative diagnoses when patients signs fail to resolve with treatment. Zoonosis: Humans, especially immune compromised people, have been infected with Bartonella so client education is warranted.

Rocky Mountain Spotted Fever (RMSF): RMSF caused by Rickettsia rickettsii is an acute systemic

disease of dogs and humans. RMSF is generally seasonal (Apr.-Sept.) correlating with the Dermacentor sp. lifecycle.

Signs: Thrombocytopenia is the most common hematological abnormality (>85%). The degree of thrombocytopenia ranges from moderate (≈ 75,000plt/ul) to severe (< 5,000 plt/ul). The major mechanism is consumption secondary to vasculitis, but there is some evidence for immune mediated destruction. Leukocytosis is the second most common hematological finding. The degree of leukocytosis can be severe (> 50,000WBC/ul), and tends to increase along with the duration of the disease. RMSF is not known to commonly cause immune mediated hemolytic anemia, but the clinical picture can be confusing due to the presence of concurrent anemia and hyperbilirubinemia. The anemia associated with RMSF is often mild (PCV 25-30%). The hematological effects are rarely seen without accompanying clinical signs, such as fever, lethargy, anorexia, pain, petechia, jaundice and neurologic signs. Common biochemical abnormalities identified in dogs with RMSF included hypoalbuminemia, hyponatremia and hyperbilirubminemia.

Diagnosis: Serology is very helpful in the diagnosis of RMSF. If the signs are acute, then paired acute and convalescent (2-4 weeks after the acute) titers must be submitted. A four-fold change is diagnostic for an active infection. If the patient is sick > 10-14 days, then a single high titer (> 1:1024) is consistent with an active infection. Positive Immunofluorescence of skin biopsies or positive nested PCR results also indicates active infection. Response to therapy (doxycycline, tetracycline, enrofloxacin, or chloramphenicol) is suggestive, but not diagnostic.

Treatment: Doxycycline (5mg/kg BID or 10mg/kg Q24), chloramphenicol (15-30mg/kg TID), and enrofloxacin (5mg/kg BID) for 2 weeks are effective treatments. Resistance has not been reported, so if signs persist after treatment an alternative diagnosis should be considered.

Follow-up: An accurate diagnosis is important, since the dog can serve as a sentinel for human infections, so a convalescent titer is indicated even if the animal has responded to treatment. Resistant RMSF has not been reported. RMSF has not been reported in cats. Zoonosis: Casual contact should not pose a major risk, but direct exposure is a potential. Also, common vector transmission is possible (sentinel).

Borrelia burgdorferi: Lyme disease is not endemic throughout the US and is most common in the

Northeastern part of the country. Lameness is the most commonly reported sign. In endemic areas up to 80% of all dogs have been exposed to Lyme.

Signs: Fever and lameness are the most common signs of Lyme disease. Canine Lyme disease caused by the spirochete, Borrelia burgdorferi, and usually does not usually have any hematological or biochemical effects (except possibly “lyme-associated nephritis). “Lyme-associated nephritis” is a form of glomerulonephritis that seems to occur in 1-2% of dogs with Lyme disease, but the cause and effect association with lyme disease remains tenuous. The majority of patients with lyme-associated nephritis have been young retrievers and the prognosis is poor.

Diagnosis: Serology is helpful if there is no history of vaccination. The C6 peptide-based antibody tests (SNAP®) are useful for discriminating vaccinal and natural exposures. With treatment anti-C6 antibody concentration decrease, but the clinical relevance of this decrease is not completely clear. Western immunoblotting

or possibly PCR (of tissues NOT blood) may be helpful in diagnosing exposure to or infection with Borrelia. Unfortunately since 95% of dogs exposed to Borrelia do not develop clinical disease associations between infection/exposure and disease are difficult.

Prevention: Several safe and effective vaccines are available against B. burgdorferi. Outer surface protein A (OSPA) appears to be a critical component in commercially available vaccines. Vaccination seems prudent in endemic regions and has been associated with preventable fractions of 92-95% in fully vaccinated dogs.

Treatment: Tetracyclines or Clavamox are considered the treatments of choice. Follow-up: Resolution of clinical signs is probably the best follow-up. There is no evidence to suggest that

treatment of naturally infected asymptomatic dogs will prevent clinical disease. Zoonosis: NA

Cats appear to be relative resistant to the development of Lyme disease

Leishmaniasis: Leishmaniasis is an uncommon canine disease in the US. The vector in the US has not been identified. Leishmaniasis typically presents as a chronic disease. To date the majority of cases in the US have been Foxhounds.

Signs: The signs are often non-specific and include weight loss, lethargy and anorexia. Skin lesions are common. Non-regenerative anemia and mild thrombocytopenia. Hypoalbuminemia, hyperglobulinemia, proteinuria and azotemia have been noted.

Diagnosis: Organisms may be seen in macrophages in tissue or blood. There is an IFA test available, but antibodies are not always detectable in infected dogs. If the signs are acute, then acute and convalescent titers should be performed. A four-fold change is suggestive of active infection. It can take some dogs years to develop a positive titer. If the signs are chronic then a single titer can be performed. If the titer is > 64 or the clinical signs are highly suspicious, then a PCR should be performed. It has been demonstrated that PCR can accurately identify over 85% of dogs with leishmaniasis. PCR of lymphnode or bone marrow is slightly more sensitive than peripheral blood alone. Leishmania may also be cultured from infected tissues.

Treatment: There is currently no treatment that is known to clear the infection. Antimony compounds are only available through the Center for Disease Control. Chronic treatment with allopurinol and antimony compounds can induce remission, but dogs remain infected.

Follow-up: Monitor clinical signs, PCR, and serology. Do not expect a cure. Zoonosis: Casual contact should not pose a major risk, but direct exposure is a potential. Also, common

vector transmission is possible (sentinel). Feline leishmaniasis does not appear to be endemic to the US at this time.

Cytauxzoonosis: Cyauxzoonosis is an emerging infectious disease of cats in North America caused by the protozoal parasite Cytauxzoon felis. It is transmitted via the tick vectors Dermacentor variabilis and Amblyomma americanum. Cats typically present acutely and historically the mortality rate is very high (over 90%). Over 90% of the cases are diagnosed between April and September. Outdoor cats are at higher risk for infection and there appear to be hyperendemic areas of C. felis transmission. Bobcats appear to be the reservoir host and only rarely develop severe disease. Without treatment most cats have died within 5-7 days of the onset of clinical signs. The majority of clinical signs are due to obstruction of small vessels with schizont-laden macrophages which results in ischemia and thrombosis.

Signs: The most common signs are lethargy, depression and fever. Pancytopenia is the classic hematologic finding for cytauxzoonosis, but there may only be reductions of one or two cell lines in affected cats. Thrombocytopenia and leucopenia appear to be the most common hematological abnormalities. Hemolytic anemia appears to be most prominent in days 7-14 after presentation. Hyperbilirubinemia and increased ALT/ALP concentrations (often not proportional to the degree of hyperbilirubminemia) are common biochemical findings. Physical examination typically reveals fever, and hepato-splenomegaly. Cats are often dyspneic, moribund, hypothermic and neurologic in the end stages of disease.

Diagnosis: Cytologic diagnosis is the most common and rapid means of diagnosing cytauxzoonosis. The earliest stage of infection is the multiplication of schizonts in macrophages. These infected macrophages can be identified in tissue aspirates (particularly the liver, lung, lymphnode or spleen) or on the feathered edge of peripheral blood smears. These infected macrophages are frequently mistaken for platelet clumps and can measure over 100 microns in diameter. In endemic areas tissue aspirates may be warranted in highly suspicious cases if organisms are not seen on a blood smear. The parasite may be identified in red blood cells on wright-giemsa stained blood smears

as the classic signet ring. There are no commercially available serologic tests. PCR is now available, is sensitive, specific and can be performed rapidly to aid in the diagnosis or confirmation of cytauxzoonosis. Treatment: Supportive care with IV fluids and anti-coagulants are the standard of care for the treatment of cytauxzoonosis. Heparin is my anti-coagulant of choice (100-300 U/kg SQ TID). Atovaquone (15mg/kg PO TID with a fatty meal) and azithromycin (10mg/kg PO Q24) combination therapy is the anti-protozoal treatment of choice at this time. Survival rates in a multi-center trial were >60% compared to a 25% survival with imidocarb dipropionate. Survival rates at NC State have approached 85%. Diminazene aceturate has also shown promise for treatment but is not easily available or FDA approved. Other anti-biotics are frequently administered to cats with cytauxzoonosis presumably to prevent secondary infections as many cats are neutropenic, although some antibiotics (doxycycline and clindamycin) do have anti-protozoal activity.

Follow-up: If cats survive more than 7 days, the prognosis for long-term survival is excellent. Since there appears to be hyperendemic areas client education regarding their other cats and tick prevention are warranted. Prospective testing of cats in the same household as infected cats has identified carrier cats. Dogs and humans do not appear to be susceptible to infection with C. felis.

Haemobartonellosis (Haemoplasmas AKA Hemotropic Mycoplasmas): Mycoplasma spp. are distinct from Bartonella spp. Hemotropic Mycoplasma spp. (AKA Hemobartonella spp.) are one of the most commonly diagnosed infectious cause of IMHA in cats. There are several species that can infect cats. The majority of clinical information exists regarding Mycoplasma haemofelis and candidatus M. haemominutum. Mycoplasma haemofelis is the larger of these two species and is considered to be the more pathogenic of the two. They are presumed to be transmitted via the cat flea, Ctenocephalides felis, although recent transmission studies have failed to demonstrate this. Unlike M. haemofelis, M. haemominutum is not considered to be highly virulent and is not usually associated with clinical disease unless there is concurrent retroviral infection. Canine haemobartonellosis is caused by M. haemocanis, and has been experimentally transmitted by ticks. At least two other species, M. turicensis and M. haematoparvum, have been identified. Clinical disease is more common in cats. One group has recently made an association between canine haemoplasmosis and babesiosis suggesting a similar route of transmission.

Signs: Anemia is the most common hematological sign in cats affected by M. haemofelis. Many cases may not show any signs at all including anemia. When present, other accompanying clinical signs are vague and may include depression, anorexia, weight loss, pale mm, icterus, and splenomegally. Infection in dogs is most often silent, unless the dog is splenectomized or immunosuppressed in some other way. M. haemomintum appears to be minimally pathogenic and in most studies is NOT associated with anemia. The pathogenic potential of M. turicensis and M. haematoparvum are not as well known.

Diagnosis: These organisms have never been cultured in vitro and there are no commercially available serologic tests. The diagnostic modalities available include microscopy and molecular techniques such as the polymerase chain reaction (PCR). Epicellular organisms may be seen on peripheral blood smears however microscopy is generally considered to have poor sensitivity since the number of organisms present in circulation can wax and wane. It is important that PCR-based tests are able to differentiate between the M. haemofelis and M. haemominutum; a positive result with the latter species appears to be less clinically significant.

Treatment: Doxycyline (5mg/kg PO BID for 3 wk.) is considered the treatment of choice, as an alternative fluoroquinolones (Marbofloxacin) can be used.

Follow-up: If there is complete resolution of signs, then serial rechecks of peripheral blood smears may be adequate. Almost all studies have demonstrated that treatment with antibiotics will reduce parasitemias, but not clear the infections.

Zoonosis: NA

Hepatozoonosis: Chronic systemic disease of dogs caused by Hepatozoon canis or Hepatozoon americanum. Transmission of Hepatozoon occurs via the ingestion of an infected tick (Amblyomma maculatum) or prey species. H. americanum is the species most commonly identified in the US and has been diagnosed most frequently in the SE.

Signs: Leukocytosis (20,000-200,000 WBC’s/ul) is common in dogs infected with H. americanum. Some studies have shown and eosinophilia. A mild non-regenerative anemia is often present. The platelet counts have been variable, with some cases displaying a thrombocytosis and others (w/ concurrent E. canis) having thrombocytopenia. Dogs infected with H. americanum are almost always systemically ill. They display signs such as fever, malaise, anorexia, emaciation, stiffness, ocular discharge and pain. Periosteal reaction on longbones are commonly identified.

Diagnosis: Organisms are occasionally seen in leukocytes on peripheral blood smears. Infected dogs commonly have a periosteal reaction on multiple long bones. A serum antibody test is available, but H. americanum might not seroreact. A PCR test on whole blood appears sensitive and specific. Organism recovery is high in muscle biopsies, some still consider this the test of choice.

Treatment and Follow-up: Currently, there is no treatment that is effective in eliminating the infection. Some treatments can induce a remission (trimethoprim sulfa/clindamycin/pyramethamie combination therapy followed by decoquinate), and the improvement of clinical signs, but relapses are common after cessation of therapy.

Zoonosis: NA Hepatozoonosis has rarely been reported in cats, most of which have been co-infected with a feline retrovirus. GENERAL INFORMATION ABOUT DIAGNOSTICS:

INTRODUCTION There is NO standardization for molecular testing between different laboratories other than commercially available kits. This means that the results between different labs usually cannot be compared directly. When there are discrepancies between the results from different labs, it is often impossible to tell which lab was "right." You MUST remember that no tests are 100% sensitive and/or specific. The results of ALL tests must be interpreted in light of the patient.

THE USE OF SEROLOGICAL AND MOLECULAR DIAGNOSTICS IN PARALELL INCREASES THE CHANCES OF DETECTING INFECTION WITH OR EXPOSURE TO VECTOR BORNE PATHOGENS!

I.E. “DON’T ASK…IF I CAN ONLY RUN ONE TEST…?

IFA (INDIRECT FLUORESCENT ANTIBODY, IMMUNOFLUORESCENCE ASSAY)

What is it? This is one of the most commonly used techniques used in the aid of diagnosing infectious diseases. This is the methodology that is used to generate titers. Although it is the most common, it is also usually the most difficult to interpret.

What does it test for? IFA is usually used to test for the presence of specific antibodies in a biological fluid. Serum, CSF, and aqueous humor are the most common samples. In other cases (i.e., FeLV bone marrow IFA) you are testing for the presence of antigen in a biological sample. In order to perform an IFA test the operator must have the antigen (usually a whole organism) fixed to a glass slide or a source of specific antibodies.

How do I interpret the results? Sensitivity? Specificity? Predictive value? IFA results are usually reported as titers (1:20, 1:40, 1:80 etc.) The titer reported is the final dilution in which the operator was able to detect the presence of fluorescence on the slide. The use of “cutoff titers” is usually not recommended unless the lab has established the sensitivity and specificity of a given test. The presence of antibodies is only circumstantial evidence for infection. It doesn't even always guarantee exposure to a particular organism (i.e. false positive results due to cross reactivity). A four-fold change (NOT four dilution) in antibody titer between acute and convalescent samples is considered diagnostic for most infections (i.e., 1:20 to 1:80, 1:640 to 1:180). Each dilution represents a 2-fold change. Acute and convalescent titers should always be run at the same lab, and ideally should be run simultaneously. There is no standardization between labs in regards to antigens, reagents, dilutions and interpretation. The sensitivity of most IFA titers have NOT been established. The specificity of most IFA titers have NOT been established. The predictive value of most IFA titers have NOT been established. If you are doing an IFA looking for antigen (i.e., FeLV bone marrow) the specificity and predictive value are very high. For chronic conditions, a single test is usually adequate to rule-in or rule-out an organism as a potential cause. For acute conditions (less than 2 weeks) paired titers should be run.

ELISA (ENZYME LINKED Immunosorbent ASSAY)

What is it? It is another commonly used test modality for several infectious disease. It is the technology that is used in most of the "SNAP®" type test kits.

What does it test for? ELISAs can be used to detect antibodies or antigens. Not all blue dots are created equally. Almost all of the in house " SNAP®" tests are ELISA based assays.

How do I interpret the results? Sensitivity? Specificity? Predictive value? Due to their commercial nature, the majority of snap tests are standardized and the sensitivity and specificity have been determined compared to a gold standard test. Unfortunately the reported results do not always correlate with the result in the real world where infection rates and levels of antigen or antibody may be higher or lower. A positive antigen test is consistent with infection (i.e. heartworm ag). A positive antibody test is only indicative of exposure to the organism. May have false negative tests in the acute phase of infection due to lack of specific antibodies. Not readily quantifiable so rising/falling titers cannot be detected.

WESTERN BLOT

What is it? This is a test that is usually run in a reference laboratory. It is often run as a confirmatory test. In order to run a Western blot, the lab must be able to grow the pathogen in large quantities.

What does it test for? Western blot is still just an antibody test. It does however test for the presence of antibodies against specific pathogen proteins rather than whole organism.

How do I interpret the results? Sensitivity? Specificity? Predictive value? Researchers have determined that the presence of antibodies against particular pathogen proteins are more specific for infection than antibodies detected in a whole pathogen prep IFA. Generally believed to be more sensitive than IFA, but this is not always the case. The predictive value is generally considered to be very high. May have false negative tests in the acute phase of infection due to lack of specific antibodies. Not quantitative so rising/falling titers cannot be detected.

PCR (POLYMERASE CHAIN REACTION)

What is it? Amplification of a specific (hopefully) piece of DNA in test tube.

What does it test for? The presence of a specific piece of pathogen DNA in a small portion of your biological sample.

How do I interpret the results? Sensitivity? Specificity? Predictive value? If a PCR test is positive, then it is very likely that that animal has that particular infection (If the appropriate controls are run). The only way I believe that you should have pathogen DNA in your blood is if you are actively infected. If a PCR test is negative, it only means that the pathogen DNA was not detected in that test tube. It does not mean that the pathogen is not present in that patient. Most PCR tests only evaluate 1-10 microliters of blood equivalent (That is 0.001-0.01 cc of whole blood equivalent). The negative predictive value of a PCR test is only known if the test has been evaluated in a population of patients in which the true prevalence of disease is known. Unfortunately this is

not the case for ANY commercially available PCR test that I know of. All PCR tests are not created equally so careful questioning and evaluation should be performed about quality control and sensitivity and specificity if available rather than "price shopping" when it comes to choosing a diagnostic lab. Unlike antibody titers PCR can detect infections during any phase of infection as long as the appropriate tissue is tested. However, some organisms have lower numbers of circulating in the bloodstream during chronic infection and false negatives can occur.

RT-PCR (REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION)

What is it? A specific (hopefully) fragment of RNA in a test tube.

What does it test for? The presence of a specific piece of RNA in a small portion of your biological sample. RT-PCR is most often used to detect RNA viruses. It has however been proposed that RT-PCR might me more sensitive that standard PCR for diagnosing other infectious diseases due to higher numbers of RNA transcripts compared to gene copy numbers. RT-PCR is also used to detect the presence of specific mRNA transcripts for gene expression.

How do I interpret the results? Sensitivity? Specificity? Predictive value? The same basic principles for PCR apply for the diagnosis of pathogens. Gene expression can be determined as a +/- if that gene is not expressed constitutively. In order to determine up or down regulation, Quantitative competitive RT-PCR must be used and compared to a standard.

REAL-TIME PCR “SOMETIMES KNOWN AS THE OTHER RT-PCR”

What is it? Amplification of a specific fragment DNA or RNA in a test tube where the results are measured real-time rather than at an endpoint as they are with standard PCR or RT-PCR.

What does it test for? The presence of specific (hopefully) DNA or RNA in a test tube. Real-time PCR also has the ability to quantify the amount of template in a sample.

How do I interpret the results? Sensitivity? Specificity? Predictive value? The interpretation of real-time PCR for the diagnosis of infectious diseases is essentially the same as standard PCR. Although there is a big push to do real-time PCR, there is little or no data to show that it is clearly superior to standard PCR. In the future the number of organisms present in the bloodstream may be used as prognostic indicators. One of the major utilities of real-time PCR in human medicine is the quantification of HIV transcripts as an assessment of response to anti-viral treatment. PCR Real-time PCR has a major advantage over standard PCR and RT-PCR when it comes to evaluating gene expression. It allows an investigator to determine if animal have up or down regulation of particular genes in given disease states.

Canine vector-borne diseases: What tests to run and what to do with the results Adam Birkenheuer, DVM, PhD, DACVIM North Carolina State University College of Veterinary Medicine Vector Borne Disease Diagnostic Laboratory Raleigh, North Carolina Most veterinary practitioners have encountered one or both of these vector-borne disease dilemmas: the lame dog that presents with clinical signs consistent with tick-borne disease, or the asymptomatic dog that comes in for a wellness exam, only to test positive for a tick-borne disease on a screening panel. In the first case, which tests should you run to make a diagnosis? In the second case, how exactly do you interpret those results, and what are your next steps? Finally, is there a single diagnostic test that serves as a “holy grail,” providing the definitive answer you need? Unfortunately, no test is 100% sensitive and/or specific. What’s more, a negative serologic or molecular test result can’t rule out infection. In fact, no test result can be a substitute for your clinical judgment; all results must be evaluated in the context of your patient’s signs and history. In this paper, we’ll start with an overview of the variety of signs and laboratory abnormalities caused by vector-borne diseases (Table 1), serologic and molecular diagnostic tests available and how to interpret them, then examine specific vector-borne diseases and provide you with practical tips on how to get to a diagnosis. Table 1. Common Signs or Laboratory Findings Caused by Selected Vector-Borne Infections Acute

or Chronic Disease

Thrombocytopenia Anemia Immune-Mediated Hemolytic Anemia

Polyarthritis Proteinuria

Ehrlichia spp. A or C Y Y Rare +/- +/- Anaplasma phagocytophilum

A Y Y Rare +/- +/-?

Anaplasma platys A or C Y N N N N Babesia spp. A or C Y Y Y N Rare Rocky Mountain spotted fever

A Y Y N Y Y (but not GN)

Bartonella spp. A or C Y Y Y Y Y Borrelia spp. A or C N N N Y Y Leishmania spp. C Y Y N Y Y Hepatozoon spp. C N Y Y Y (look alike) Y Hemotropic Mycoplasma spp.

A or C N Y Y N N

EHRLICHIOSIS/ANAPLASMOSIS A positive SNAP test merely indicates exposure, but when combined with the appropriate laboratory and clinical findings, the result is supportive of a diagnosis. While the current in-house assays can’t differentiate antibodies against Ehrlichia ewingii from those against Ehrlichia canis or antibodies against Anaplasma phagocytophilum from

those against Anaplasma platys, the distinction may not be critical for treatment, as all organisms respond to doxycycline. However, accurate species identification can be important for client education regarding zoonoses as well as expected response to treatment. If you have a symptomatic dog with a negative SNAP test, the dog may be acutely infected, and there simply aren’t enough circulating antibodies to be detected by serology. IFA testing with acute and convalescent (3 to 4 weeks later) titers may be helpful. A four-fold change is consistent with infection. There may be cross-reactions among different species. Microscopic identification of morulae in circulating neutrophils, monocytes or platelets can confirm the diagnosis, but parasitemia is often very low, so they may be difficult to find on blood smears. A positive PCR test is useful to rule in the presence of infection and identify which species is present. However, low levels of circulating organisms can also hamper PCR, so a negative PCR does not rule out the possibility of infection. Therefore, resolution of clinical signs in response to therapy remains an important “test.” After treatment, serology is generally a poor way to assess recovery, because antibody titers can persist for months. Resolution of clinical signs and laboratory abnormalities is the most logical way to assess response to therapy. However, because studies suggest that Ehrlichia infections may not be cleared with antibiotics, annual screening with a CBC, serum chemistry, and urinalysis is still indicated for the remainder of the pet’s life. Lyme Disease/Borreliosis Although a positive SNAP antibody result typically indicates exposure, one exception is Borrelia burgdorferi, where the amount of exposure required to develop an antibody response, combined with the low immune clearance rate, means that a positive antibody test can mean infection. The C6 peptide-based SNAP antibody tests are also useful for discriminating antibodies to natural exposure from antibodies to vaccination. Other serologic tests, such as IFAs and plate ELISAs performed in reference laboratories, can detect antibodies to Lyme vaccines, resulting in false-positive tests. That’s why a Western blot or C6 peptide-based assays are most helpful for diagnosing exposure to or infection with Borrelia. Because Borrelia is only rarely detected in circulation, PCR on whole blood samples is a poor way to diagnose infection. PCR using tissue (skin or joint) samples can assist in the diagnosis but are often considered more invasive then necessary to confirm infection. Because 95% of dogs exposed to Borrelia do not develop clinical disease, it can be difficult to make meaningful associations between infection/exposure and disease. Treatment of asymptomatic dogs is still controversial. Seropositive dogs should be tested for evidence of proteinuria and serum biochemical abnormalities, as well as for presence other tick-borne disease agents. With treatment, anti-C6 antibodies concentrations decrease, but the clinical relevance of this decrease is not completely clear. Resolution of clinical signs is probably the best follow-up. Similar to E. canis, Borrelia may not be cleared from all animals following antibiotic therapy, so annual screening for active disease with a CBC, serum chemistry, and urinalysis is indicated for the remainder of the pet’s life.

Canine Babesiosis Microscopy or PCR can easily rule babesiosis in, but it is very difficult to rule out the infection completely. The organisms stain well with a modified Wright’s stain. Evaluation of capillary blood (ear or toenail) may improve parasite recovery. However, to maximize your chance of identifying the infection, I recommend that you consider microscopy, serology, and PCR. There is variable seroreactivity, so serology (acute and convalescent IFA titers) against both Babesia canis and Babesia gibsoni is warranted. PCR is the most accurate way to identify which species of Babesia is present. In one study, a single PCR test identified 85% of B. gibsoni and two consecutive PCR tests identified 100% of the species.1 Some treatments may result in a clinical remission with persistent parasitemia. These dogs are at risk for recrudescence and may act as a reservoir. For follow-up, I recommend two consecutive blood smear evaluations and PCR at 60 and 90 days posttreatment. Canine Bartonellosis A combination of culture, serology, and PCR appears to be the best method for the detection of Bartonella spp. For unknown reasons, there are frequently discordant results between these assays, so a combination of these techniques is recommended. Resolution of clinical signs and a negative culture may indicate that the treatment was effective. Because the full spectrum of disease is unknown and a large percentage of normal animals can test positive for Bartonella, cautious interpretation of test results and consideration of alternative diagnoses when a patient’s signs fail to resolve with treatment are warranted. Rocky Mountain Spotted Fever Serology is very helpful in the diagnosis of Rocky Mountain spotted fever. If the signs are acute, then paired acute and convalescent IFA titers should be submitted. A four-fold change is diagnostic for active infection. If the patient is symptomatic for 10 to 14 days or more, a single high titer (≥1:1024) is consistent with active infection. Positive immunofluorescence of skin biopsies or positive nested PCR results also indicates active infection. Response to therapy is suggestive, but not diagnostic.

An accurate diagnosis is important because the dog can serve as a sentinel for human infections, so a convalescent IFA titer is indicated even if the animal has responded to treatment. Leishmaniasis Organisms may be seen in macrophages in tissue or blood. While an IFA test available, antibodies are not always detectable in infected dogs (it can take some dogs years to develop a positive titer). If signs are acute, then acute and convalescent titers should be performed. A four-fold change is suggestive of active infection.

If the signs are chronic, then a single titer can be performed. If the titer is ≥64 or the clinical signs are highly suspicious, then a PCR should be performed. It has been demonstrated that PCR can accurately identify more than 85% of dogs with

leishmaniasis. PCR of lymph node or bone marrow is slightly more sensitive than peripheral blood alone. Leishmania can also be cultured from infected tissue, such as bone marrow, lymph nodes, liver, spleen, and blood (although using blood is not ideal).

Follow-up should consist of monitoring clinical signs, PCR, and serology, although no cure should be expected. Hemobartonellosis (Hemoplasmas or Hemotropic Mycoplasmas) The diagnostic modalities available include microscopy and PCR. Epicellular organisms may be seen on peripheral blood smears; however, microscopy is generally considered to have poor sensitivity because the number of organisms present in circulation can fluctuate. No studies have found an association between the presence of hemotropic Mycoplasma spp. in spleen-intact dogs and clinical disease. If dogs don’t improve with treatment, consider an alternative diagnosis. Hepatozoonosis Organisms are occasionally seen in leukocytes on peripheral blood smears. Infected dogs commonly have a periosteal reaction on multiple long bones. A serum antibody test is available, but Hepatozoon americanum might not seroreact. A PCR test on whole blood appears sensitive and specific. Organism recovery is high in muscle biopsies, and some still consider this the test of choice. No treatment is effective in eliminating the infection, and relapses are common after cessation of therapy. Combining Serologic and Molecular Assays: Does It Make Sense? There are times when an accurate diagnosis, including species identification, can be extremely important. A missed diagnosis often results in therapies that can lead to persistent illness or in some cases may actually worsen the outcome. Accurate species identification is important for client education regarding zoonoses as well as expected response to treatment. When an animal doesn’t respond to treatment, there may be coinfection with other organisms. To demonstrate the advantages and limitations of serologic and molecular assays for the diagnosis of canine vector-borne infections, we prospectively tested healthy dogs (n=30) using a panel of serologic and molecular assays for evidence of exposure to, or infection with, 10 genera of organisms that cause canine vector-borne diseases (VBDs).2 Using the same panel, we retrospectively tested three additional groups of sick dogs:

• Dogs that were seronegative and only had serologic testing requested by the attending clinician (n=25)

• Dogs that were seroreactive against at least one organism and only had serologic testing requested by the attending clinician (n=25)

• Dogs that were PCR positive for at least one organism and only had PCR testing requested by the attending clinician (n=24)

Finally, a group of dogs suspected of canine VBDs (n=145) were tested using a panel of serologic and molecular assays for evidence of exposure to, or infection with, 10 genera of organisms.

In these representative populations, a panel combining serologic and molecular assays run in parallel would have resulted in 4% to 60% increased recognition of exposure to, or infection with, canine VBDs. We concluded that the “holy grail” remains elusive and that serologic and PCR assays should be used in parallel to maximize canine VBD diagnosis.

UPDATE ON CYTAUXZOONOSIS: BEWARE THE PROTOZOANS! Adam J. Birkenheuer DVM, PhD, DACVIM NCSU-CVM, Raleigh, NC Cytauxzoonosis History

Cytauxzoonosis is a tick-transmitted protozoan disease of cats caused by Cytauxzoon felis that was first reported in Missouri in 1976. Over the next thirty years the disease was only recognized in the southcentral and southeastern United States. Recently the geographic range of the organism has been recognized to extend east and north. The states in which C. felis has been reported are shown below.

Classification and life-cycle

Based on its DNA sequence and life-cycle, C. felis is closely related to the Theileria spp. Unlike Babesia spp. which have only been found to infect red blood cells, Cytauxzoon and Theileria first enter a white blood cell after tick-transmission to the vertebrate host. Below is a cartoon depicting the proposed life-cycle of C. felis showing Bobcats (Lynx rufus) as the most likely reservoir hosts. Dermacentor variabilis and Amblyomma americanum have been demonstrated to transmit C. felis to domestic cats under laboratory conditions. The disease typically occurs between the months of April and September which correlates with the peak tick activity in most regions. Approximately 13-16 days after being bitten by an infected tick the cat develops non-specific signs of illness, typically characterized by

anorexia, lethargy, pyrexia, icterus, and pallor. The initial infection of macrophages, or schizogenous phase, is responsible for nearly all the pathology associated with cytauxzoonosis. Bobcats do not appear to undergo a profound schizogenous phase of infection and therefore do not evelop severe clinical disease. The erythrocytic phase of the organism alone does not induce severe disease.

Clinical signs and laboratory abnormalities

It is typically characterized by an acute febrile illness that is associated with decreases in one or more cell lines. Physical exam findings are often non-specific. Cats usually have a profound fever, but hypothermia may be identified in moribund cats. Lymphadenopathy and splenomegaly are common but not uniform findings. Cats are typically depressed and vocalization (death yowl) is a common finding in advanced cases. Some cats will have dyspnea as a prominent clinical feature (in our experience this is frequently due to pleural effusion and can be addressed easily with thoracocentesis). Laboratory abnormalities include non-regenerative anemia, leukopenia, thrombocytopenia, hyperbilirubinemia and bilirubinuria, elevations in liver enzymes (often not as severe as would be expected considering the degree of hyperbilirubinemia), hyperglycemia and hypoalbuminemia. In our experience thrombocytopenia and neutropenia are the most common CBC findings. Elevations in aPTT and non-overt DIC seem to be common. Many of the typical laboratory abnormalities become more pronounced as the disease progresses and may be absent or mild if the cat is presented for care early in the course of illness. The course of disease is short, and most cats succumb within 5 days of the onset of clinical signs.

Risk factors

Cytauxzoonosis is most frequently identified in young outdoor cats. They often have a history of tick attachment. There appear to be “hot spots” of infection, and often more than one cat in a household or neighborhood will become infected. In some households where cytauxzoonosis has been diagnosed, the prevalence in the domestic cats may be >30%.

Diagnostic testing

Microscopy: Organisms in red blood cells are most easily identified on 1000X. Schizont infected macrophages can be seen at low power and can look like platelet clumps on the feathered edge of a peripheral blood smear. Fine needle aspiration and cytology of liver, spleen and lymphnodes can facilitate a rapid diagnosis in suspect cases. In house quick stains are usually adequate

Polymerase Chain Reaction: PCR testing can be sensitive and specific.

Treatment

Aggressive supportive care is a mainstay of therapy for cytauxzoonosis. This includes IV fluid therapy and anti-coagulants (unfractionated heparin 100-300U/kg SQ TID or 300-900U/kg IV as a CRI). Corticosteroids do not appear to have a negative effect on outcome but also do not seem to provide a benefit.

Atovaquone and Azithromycin: An atovaquone (15 mg/kg PO TID with a fatty meal) and azithromycin (10mg/kg PO Q24) drug combination is the current treatment of choice for cytauxzoonosis. A feeding tube should be placed at admission to facilitate administration of medications. A randomized clinical trial demonstrated survival rates of 60% for atovaquone and azithromycin compared to 27% for imidocarb dipropionate. Treatment should be initiated within hours of admission and should be started empirically for suspect cases in endemic areas even if a definitive diagnosis is not made easily.

Imidocarb diproprionate: Imidocarb diproprionate (Imizol®, Schering-Plough) 2-4mg/kg IM once. Repeat dose in 2 weeks. Side-effects include pain at injection site and cholinergic effects (SLUD). Pre-treatment with atropine reduces cholinergic effects. Success rate have been reported to range from 0-50%.

Diminazene aceturate: Has been used with some success, but is not FDA approved or widely available in the US.

Follow-up:

Time to respond: The response to therapy is NOT immediate. Most cats begin to improve within 4-7 days.

Recovery: In our experience, cats that survive acute cytauxzoonosis have complete resolution of all clinical signs (following some cats for >4 years). However, they may have persistent infection with the red blood cell form of the parasites. It is unknown whether or not these cats can serve as a reservoir for infection. It is strongly recommended that all cats are kept indoors.

Follow-up testing: Serial blood counts are recommended until PCV, WBC, and platelet count return to within normal range. In our experience, all biochemical abnormalities have returned to normal within 2-3 weeks. Parasites may remain detectable by microscopy and PCR for years.

Adam J. Birkenheuer, DVM, PhD, DACVIMs

Dr. Birkenheuer is a professor of internal medicine and North Carolina State University College

of Veterinary Medicine. He attended veterinary school at the University of Florida. After the

completion of veterinary school he completed a one-year rotating small animal internship at

North Carolina State University and stayed there to complete the Clinician Investigator Program,

a combined PhD and small animal internal medicine residency. He is board certified in small

animal internal medicine by the American College of Veterinary Internal Medicine. He has

worked as an internal medicine specialist at a private referral hospital, an emergency doctor and

a general practitioner prior to starting his current position at NC State. He is a co-director of the

Vector Borne Disease Diagnostic Laboratory at NC State. His research areas include the

epidemiology, diagnosis and treatment of vector-borne diseases of dogs and cats.