CHINESE EDITION IS WRITTEN BY DR. WANG, SHIH-HAO
/ ENGLISH EDITION IS TRANSLATED AND EDITED BY DR. LIN, WEN-YANG (WESLEY)
The canine parvovirus (CPV) is a common, acute, high morbidity and high morality virus that mainly infect canine population. This virus possess highly survival rate for 5 weeks in the natural environment. It is highly contagious and easily transmitting among canine population by the fecal-oral route through contacting contaminated feces. CPV usually attack digestive system. Sometimes it may induce myocarditis among canine and cause sudden death. All ages, sexes and breeds of dogs could be susceptible to CPV, especially puppies. Clinical sighs of infected dogs may include fever, lethargy, continuous vomiting, continuous diarrhea, stinky viscous diarrhea with blood, dehydration and abdominal pain etc. Canine show signs of the disease would usually die within 3 to 5 days. There are no specific drugs for curing CPV until now. Supportive care such as consuming water-electrolyte fluid is the only present solution to maintain physiological function and relieve symptoms. The infected canine should have medical care as soon as possible; otherwise, more severe conditions like acute dehydration, hypovolemic shock, bacterial infections and death will occur. Infection prevention measures include environmental disinfection and routine vaccines.
The canine parvovirus (CPV) is an ssDNA virus, which belongs to the species carnivore protoparvovirus 1 within the genus protoparvovirus in the family parvovirus (parvoviridae). CPV is 98% identical to feline panleukopenia virus (FPLV) with variant in six coding nucleotide of structural proteins VP2: 3025, 3065, 3094, 3753, 4477, 4498 that makes CPV-2 infect canine host instead of replicating in cats. Two types of canine parvovirus were discovered – canine minute virus (CPV1) and CPV2, both can attack canine population and canidae family such as raccoons, wolves and foxes. Canine parvovirus may be susceptible to cats without pathogenic, and it is an inapparent infection. CPV2 could stably survive in feces for 5 months with ideal condition. Furthermore, CPV-2a, CPV-2b and CPV-2c type viruses have been isolated and sequenced from animals. Other than targeting on canine, large cats are susceptible to CPV-2a, CPV-2b. CPV-2c type viruses have high prevalence on infecting leopard cats.
Figure 1. Model of CPV evolution showing VP2 amino acid differences between each virus and indicating the virus host ranges. (Karla M. Stucker, Virus Evolution In A Novel Host: Studies Of Host Adaptation By Canine Parvovirus, Published in 2010)
In 1978, a novel infectious canine disease was firstly occurring in the east coast of America. Within 12 months, scientists identified CPV-2 as the aetiological key of severe symptoms among canine. Due to characters of highly contagious and potential environmental resistance, CPV-2 spread swiftly over entire USA, European countries, Australia and Asia. In 1978, canine parvovirus also invade among canine in Taiwan. Therefore, CPV caused large scale of canine death at the early stage of pandemic. By the establishment and development of CPV vaccine, global wide spreading of CPV has been rarely happen today. However, canine parvovirus still widely exists in domestic dogs and wild canidae. It became one of the canine endemic disease.
Incubation period of CPV-2 lasts 4 to 5 days. The virus mostly attacks rapidly dividing cells especially lymphopoietic tissues, the bone marrow, crypt epithelia of the jejunum, ileum and (in young dogs under 4 weeks old) myocardial cells. Rottweilers, black Labrador Retrievers, Doberman Pinschers, and American Pit Bull Terriers are more susceptible than other species; once they are infected, would suffer severer conditions.
Besides, CPV-2 take the major place to affect canine and wild canids. After entering into hosts’ body, CPV-2 firstly replicates in oropharynx lymphoid tissues, mesenteric lymph nodes and thymus gland, then spreading to other lymph nodes, lung, liver, kidney and rapidly dividing tissues (e.g. bone marrow, intestinal epithelial cell and myocardial cell) by the blood stream. 4 to 5 days after, clinical sighs like diarrhea, vomiting, lymphopenia, anorexia, depression, dehydration, hypothermia, thrombocytopenia and neutropenia would appear. Severe dehydration and hypovolemic shock may happen due to lose large amount of fluid and protein by vomiting and diarrhea.
Fecal-oral route is the main transmission pathway of CPV-2. Large amount of virus would be detected in feces of infected canine within 1 to 2 weeks of acute phase. An infected pregnant canine could transmit virus to fetus through placenta. Fomites include contaminated shoes, cages, food bowls and other utensils could serve as CPV transmitting objects also.
There are four clinical forms according to distinct signs and lesions: enteric, myocardial, systemic infection and inapparent Infection.
A. Enteric form :
It is known that CPV-2 caused enteritis symptoms. This form infect host with low virus titers (around 100 TCID50). Symptoms in initial stage are sopor, loss of appetite, acute diarrhea, vomiting, dehydration, slight elevated body temperature, frailty and acting like in extreme pain. Severity of illness vary according to the age of canine, healthy condition, infectious dose of the virus, and other pathogens in intestine and so on. Typical signs of CPV induced enteritis and its course include loss of appetite, sopor, fever (39.5℃-41.5℃) within 48 hours follow vomiting. 6 to 24 hour after vomiting follow watery stool in yellow or white color, mucus stool or bloody stool with stench in severe cases. Due to consistent diarrhea and vomiting, dogs suffer worsen dehydrated condition. Common clinical pathologic examination consist assessing dehydrated condition and significant decreasing of white blood cell of dogs (400 to 3000 /μL).
B. Myocardial form:
This form only appear in puppies around 3 to 12 weeks of age. Major cases show pups’ age under 8 weeks. Mortality rate is extremely high with myocardial form (almost up to 100%). Clinical signs include irregular breathing, cardiac arrhythmia. Collapse, hard breathing may happen to acute cases follow death within 30 minutes. Most cases would die within 2 days. The subacute form would also die from hypoplastic heart syndrome within 60 days. Nevertheless female adult canine acquire antibodies against myocardial form by vaccination or infection, puppies may prevent from infection by receiving maternally derived antibodies. Thus, myocardial form is less common by now.
C. Systemic infection form:
Paper reported that a 2-week’s old puppy infected and died from CPV-2 invasion. Autopsy method revealed hemorrhagic necrosis over several visceral organs.
D. Inapparent Infection form:
CPV-2 replicate in infected canines and spread out by feces. However, dogs express no clinical signs. The infected canines’ age over one years old or dogs received inactivated vaccine against CPV-2 would show inapparent symptoms.
There two main diagnostic methods of CPV-2 – clinical diagnosis and laboratory test. Clinical signs include lethargy, depression, anorexia, vomiting, smelly/bloody diarrhea, dehydration and fever. Common laboratory tests which were performed include HA (Haemagglutination) , Electron Microscopy (EM), biochemical tests, virus isolation, Enzyme Linked Immunosorbent Assay (ELISA), Latex Agglutination Test (LAT), Fluorescent Antibody Test (FAT), CIE test, Virus neutralization test, traditional PCR, real time PCR, loop-mediated isothermal amplification (LAMP), nucleic acid hybridization or dot blot, in situ hybridization, nucleic acid sequencing etc.
Figure 2. Diagnosis for CPV-2 infection (Minakshi Prasad et al., An Insight into Biomarkers for Canine Parvovirus Diagnosis: A Minireview, 2017, volume 7, issue 1, page 9)
I. Clinical diagnosis
A. Prevalent age: CPV-2 easily infect young puppies around 6 weeks to 6 months of age.
B. Suspicious signs: acute sudden vomiting, diarrhea, apparent stench bloody diarrhea, low body weight.
C. Cardiokymograph examination (CKG): CKG could detect abnormal wave pattern if ill canines suffer myocardial damage or chronic myocarditis by CPV-2 infection.
II. Laboratory diagnosis
A. Hematological methods:
CPV-2 usually cause canine’s leukopenia. White blood cells (WBC) would decrease to 500- 2000 WBC/μl, even lesser amount in severe cases. For enteritis symptoms, lymphopenia would be more significant diagnostic indicator than leukopenia. If white blood cell index recover, this indicate infected canine had passed crisis stage and restored gradually. Besides, packed cell volume (PCV) is variant from the condition of dehydration or intestinal bleeding. Bloody diarrhea would likely induce mild dehydration and cause elevated PC. Whereas, gastrointestinal hemorrhage may decrease PCV degree especially after fluid infusion.
B. Biochemical test: biochemical tests usually show several conditions like electrolyte imbalance (hypopotassemia), prerenal azoturia, increasing bilirubin high serum CRP, high serum TNF, low serum cholesterol and low serum albumin.
C.Endocrine analysis: CPV-2 infection would cause low serum thyroxin and high serum cortisol.
D. Virus identification:
This is the most specific diagnostic method for detecting CPV-2, which include electron microscopy, haemagglutination (HA) assay, Isolation of CPV-2, ELISA, PCR and real-time PCR etc. However, it require infected samples with timeliness and perform virus test from 3 to 12 days shortly after CPV-2 infection.
D-1. Electron Microscopy
CPV-2 could be easily detected by negative staining and use of electron microscopy during acute phase. IEM could also be used to identify CPV or FPV.
D-2. Immunochromatography Test (ICT)
Commercialized IC test is an easy and rapid tool for clinical examination, which detects all three CPV-2 variants (CPV-2a, -2b, and -2c).
Figure 3. Immunochromatographic assay for detecting Canine Parvovirus antigen – CPV 2, 2a, 2b & 2c
Figure 4. A rapid reliable commercialized ICT test for detecting canine parvovirus, VETlabs Canine Parvovirus Ag Test
D-3. Haemagglutination (HA) Assay
The HA is an easy and fast test to detect CPV-2’s agglutination effect with Canines’ RBCs at 4 °C. The titer of HA usually located between 128 and 10240. The HA test commonly show nonspecific result when HA titer is under 33, which may be adjusted by adding fluorocarbon or CHCl3 (10% V/V).
D-4. Isolation of CPV-2 Virus
CPV virus can be isolated and performed cell culture in MDCK (Madin-Darby Canine Kidney) or CRFK (Crandell Feline Kidney) from the cases of CPV induced myocarditis and enteritis. A canine cell line (A-72) originated from a canine S/C tumor is especially useful for CPV isolation and growth of CPV. However, low sensitivity, the need of cell culture skill personnel, long incubation period (5-10 days) etc. are listed as disadvantages of viral isolation method.
ELISA test is rapid, inexpensive and easily performed, which based on the antigen–antibody reactions. Recently it reported that monoclonal antibodies could sensitively bind on 1.5 ng of CPV antigen by ELISA test. Furthermore, the double sandwich ELISA is even more rapid, sensitive and suitable test than traditional ELISA. This test has become routine diagnostic method for CPV in canine around the world.
D-6. Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR) technique is a fast, sensitive and accurate diagnosis tool, which has been applied in detecting virus infection. PCR can even identify fewer virus DNA than other tools. Specific designed primers could recognize different types of CPV (CPV2, CPV-2a, CPV-2b and CPV-2c).
D-7. Real Time PCR
According to sequences of VP2 gene, SYBR Green based primers for CPV-2 variants had been designed as forward primer (5’-CAT TGG GCT TAC CAC CAT TT-3’ and reverse primer (5’-CCA ACC TCA GCT GGT CTC AT-3’). The PCR product should locate at 160 base pair (bp). Besides, the TaqMan assay had been developed by utilizing minor groove binder (MGB) probe that can be also applied in single nucleotide polymorphisms of the capsid protein gene between CPV types 2a, 2b and 2c. Real time PCR took several advantages over conventional PCR as without running the agarose gel electrophoresis, monitoring outcome in the computer and quantifying CPV-2 amount of the infected samples.
D-8. Detection of CPV in Fecal Samples Using LAMP
The Loop Mediated Isothermal Amplification of DNA (LAMP) is another molecular tool to identify CPV genomic DNA. LAMP use two outer and two inner primers designed from VP2 gene of CPV genomic DNA. LAMP could reach 100% relative sensitivity and 76.9% of the relative specificity. However, limitation of this method is 0.1 (TCID50)/ml.
D-9. Detection of Canine Parvovirus by In situ Hybridization
CPV-specific DNA probe would target VP-1 and VP-2 mRNA sequences infected tissue sections.
A. Intravenous fluids Infusion:
Administration of polyionic isotonic fluids with potassium and dextrose to relieve symptom of metabolic acidosis. Preventing dehydration by supplying Ringer’s or 0.9 % saline.
B. Restricted diet:
Fasting without taking water for 24 hours. If condition of vomiting relieved, owner could begin to feed ill dog with soft and digestive food like commercial formula and boiled white chicken. Dogs could not have ordinary meal until intestinal function fully recovered.
C. Broad-spectrum antibiotics
Broad-spectrum antibiotics include cephalosporine, gentamicin, ampicillin and amikacin can effectively prevent secondary bacterial infection.
D. Antiemetic drugs
- Chlorpromazine 0.5mg/kg SC, IM, SID-QID or 0.05mg/kg IV TID-QID。
- Metoclopramide tablets 0.2-0.4mg/kg PO, SC, TID or 0.01-0.02mg/kg/h by Continuous intravenous infusion
- Prochlorperazine 0.5mg/kg IM TID-QID or 1mg/kg PO BID
E. Obstipantia (antidiarrheal agent): bismuth subsalicylate or loperamide
A. Using dilute bleach (1:32) to clean and disinfect animal living environment
B. Vaccine injection:
1. 6 to 8 weeks’ old puppies should receive vaccine 3 to 4 weeks a time until arriving 16 to 18 weeks’ old.
2. 16 weeks’ old above or adult canine who had not received vaccine should take CPV vaccine injection and interval of injection should be 3 to 4 weeks.
3. For the convenience of administration of vaccine injection, veterinarian could execute CPV vaccine and canine distemper vaccine at the same time. However, canine’s age under 9 weeks should receive measles plus CPV vaccine. This vaccine should administrate on canine every year.
4. Pregnant canine could receive inactivated vaccine 2 weeks before delivery. This method insure puppies acquire high efficient maternal antibody and prevent CPV infection in the womb or after giving birth.
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