Feline Infectious Peritonitis (FIP)

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1 Feline Infectious Peritonitis (FIP) Introduction Feline Infectious Peritonitis (FIP) is a generally fatal disease of domestic and some wild Felidae (cougars, lions, cheetahs, jaguars, bobcats, and lynx) caused by a mutant variant of feline coronavirus and has worldwide distribution. It can affect cats of any age, although it is principally found in young and old animals and has a higher incidence in purebreds. Feline infectious peritonitis is characterized by three distinct clinical forms namely; the effusive or wet FIP with voluminous quantities of straw-colored fluid in the peritoneal and pleural cavities; noneffusive or dry form which presents with chronic, granulomatous lesions in a wide variety of organs and a mixed form. Sample Guide *Important note: tests on effusion fluids have far greater diagnostic value than tests performed on blood. Therefore, if fluid can be collected this should be submitted to the laboratory in preference to blood. FIP Body Fluid / Serum analysis Total Protein increased total protein in cases of FIP are due to increased globulins. Effusions: > 35g/L with globulins > 50% Cerebrospinal fluid: normal < 25mg/dL while in FIP mg/dl Serum: raised total protein > 60g/L up to 120g/L with low albumin. Comparison of total serum protein, γ-globulin (electrophoresis) and albumin: globulin ratio reveals that albumin: globulin ratio has a statistically significantly better diagnostic value than total serum protein and γ-globulin. Serum albumin: globulin ratio of < 0.8 has a 92% positive predictive value for FIP. 169

2 Use of electrophoresis for quantification of γ-globulin levels and to distinguish polyclonal from monoclonal hypergammaglobulinaemia to differentiate FIP and other chronic infections from multiple myeloma or other plasma cell tumours, is considered of limited value. Cell counts in effusions cell counts are normally quite low (< 1000 nucleated cells per ml), while in CSF the range can be cells per ml although many cats with FIP have normal CSF. Cytology examination of centrifuged or cytospin samples provide the best material. Cellular component predominantly neutrophils, lymphocytes and macrophages and best observed in effusion fluids from the body cavities, but may also be seen in CSF. Rivalta s test on effusions the Rivalta s test has a positive predictive value of 86% and negative predictive value of 97% for FIP and should be performed on all effusion fluids. FIP Antibody testing Antibodies are measured by ELISA or fluorescent antibody test (FAT). Effusion fluid the presence of antibodies in effusion fluids has a high positive predictive value (90%) and high negative predictive value (79%) and measurement of antibodies in effusions is more useful than in serum. Serum should be tested at various serum dilutions with the following guidelines Negative at 1:25 = negative Positive at 1:100 1:400 = low positive Positive at 1:1600 = medium positive Positive at 1:3200 = high positive *Please note: the presence of serum antibodies does not indicate FIP and the absence of serum antibodies does not exclude FIP. Cerebrospinal fluid as with serum the presence of CSF antibodies does not indicate FIP and the absence of CSF antibodies does not exclude FIP. 170

3 FIP Antigen tests Polymerase Chain Reaction (PCR) PCR on blood samples should be submitted in EDTA False negative results are a problem in that this is an RNA virus and RNA is extremely labile making sample collection and transport to the laboratory all the more critical. The target sequence of the assay may not identify all variants of the FIP mutants. False positive results are possible as PCR cannot distinguish between virulent and avirulent feline coronavirus strains, nor can PCR differentiate FIP from canine coronavirus (CCV) or transmissible gastroenteritis virus of pigs (TGEV). Felines have been experimentally infected with both CCV and TGEV. PCR on effusions body fluids should be submitted in a sterile container (syringe, urine container or plane blood tube without gel). Initial studies indicate that this is a useful diagnostic procedure. PCR on CSF is not recommended as a diagnostic test because if the blood brain barrier is compromised even cats without FIP can produce positive results. PCR on faeces - is considered a sensitive and reliable method for determining shedding of feline coronavirus (FCV). As there is tremendous variability in the amount of FCV shed, PCR should be performed daily over 4-5 days to accurately determine if a particular cat is shedding. *Please note: Samples MUST be fresh and kept frozen while transporting to the laboratory and the PCR performed as quickly as possible. 171

4 Direct Fluorescent Antibody Test Immunofluorescent staining of intracellular FCV antigen in macrophages is a highly specific with a positive predictive value of 100%, therefore if there is positive staining this predicts 100% that the cat has FIP. Unfortunately, the negative predictive value is low (57%) making the incidence of false negatives high. Histopathology and Immunohistochemistry (IHC) Both procedures are performed on tissues fixed in 10% buffered formalin. Vasculitis and pyogranulomatous inflammation are the hallmark histopathological features of the disease. IHC staining enables demonstration of viral antigen within histological lesions, thereby confirming the diagnosis. Further reading 1. Hartmann Feline Infectious Peritonitis. Veterinary Clinics: Small Animal. 35: Hartmann et al Comparison of different tests to diagnose feline infectious peritonitis. Journal of Veterinary Internal Medicine. 17: Gaskell & Dawson FIP related disease. In: Textbook of Veterinary Internal Medicine 5 th edn. Ettinger & Feldman eds. p

5 Feline Leukemia Virus (FeLV) Introduction Feline Leukaemia Virus (FeLV) is classified into 4 types namely FeLV-A, FeLV-B, FeLV-C and FeLV-T based on their env gene sequence. FeLV transmission occurs via oronasal spread of virus in saliva from infected cats (mutual grooming, sharing of water / food bowls), with clinical disease being more common in multi-cat households. Most cats exposed to FeLV (60%) develop protective immunity and are able to rapidly clear their infection (antigen negative), while the minority (30%) become persistently infected (repeatedly antigen positive) developing FeLVrelated disease syndromes within 3 years. The remaining 10% develop a viraemia followed by a systemic immune response and clearance of the virus after a more protracted period (initially antigen positive then revert to antigen negative). FeLV viral antigen testing Antigen ELISA, Immunochromatography Sample Guide These common commercial assays detect free FeLV p27 capsid protein in plasma (EDTA) or serum. There is variability in sensitivity and specificity of these assays and therefore all positive results should be confirmed by either immunofluorescence or PCR or repeat antigen assay in 4-12 weeks. Please note: FeLV antibodies (maternal / natural infection / vaccination) do not interfere with these antigen assays. Kittens may be tested from birth. A positive test would indicate viraemia but this may be transient and therefore the cat should be isolated and the test repeated in weeks. A positive FeLV p27 assay does not always correlate with viraemia. Up to 10% of positive p27 cases could be the consequence of early emerging infection or recovery. Any discordant results should warrant re-test in 4-12 weeks. 173

6 Latently infected cats (sequestered bone marrow infection) test repeatedly antigen negative. Virus isolation from bone marrow remains the only definitive way to detect latently infected animals. Polymerase Chain Reaction (PCR) This is a specific and sensitive procedure which detects FeLV proviral DNA in blood (EDTA), bone marrow (EDTA) or tissues (lymphoid tissues, visceral organs). All samples are packed using the prescribed Triple Packaging System as per regulations SABS 0229:1996 and transported at 4ºC to the laboratory. (See Reference Ranges Section 3). Please note: Probably all cats exposed to FeLV become provirus positive and remain positive even after recovery. Therefore, PCR does not confidently distinguish between viraemic and nonviraemic animals. Direct Fluorescent Antibody Test Immunofluorescence detects the presence of FeLV p27 in circulating leukocytes in a fixed blood smear or smears made from blood (EDTA) or bone marrow (EDTA). This procedure is frequently used as a confirmatory assay in cases positive on antigen ELISA / immunochromatography. Further Reading 1. Carmichael et al Feline Leukemia Virus associated Myelopathy in Cats Veterinary Pathology. 39: Dunham et al Retroviral infections of small animals. Veterinary Clinics: Small Animal. 38: Hartmann et al Quality of different in clinic test systems for feline immunodeficiency virus and feline leukemia virus infection. Journal of Feline Medicine and Surgery. 9(6): Kerr M Veterinary Laboratory Medicine 2 nd edn. Blackwell Science, Oxford

7 Feline Immunodeficiency Virus (FIV) Introduction Feline immunodeficiency virus exists as at least 5 subtypes (clades) A-E based on their env gene sequence. Divergence between the different clades can be as much as 30% and different clades predominate in different geographical locations. Following virus entry it is integrated into the host genome of lymphoid and myelomonocytic cells resulting in persistent infection. Transient disease coincides with the initial period of viraemia; thereafter animals remain healthy. During the asymptomatic phase, which may last many years and in many cases for the entire life of the cat, there is a gradual decline in CD4+ lymphocytes with waning of the antiviral immune response that eventually allows the plasma viral load to increase. This increased viral load in the latter stages of disease is associated with opportunistic infections. However, the final disease outcome is variable and not necessarily life threatening. Sample Guide Feline Immunodeficiency Virus serum antibody testing ELISA / Immunofluorescence / Western blot These assays demonstrate the presence of antibodies against the virus. A serum sample, collected in a red or yellow stopper tube, is required. Following collection, allow samples to stand for minutes at room temperature to form a clot, after which they should ideally be centrifuged and the serum poured off. Serum or un-spun clotted blood should then be transported at 4 C to the laboratory. Most commercial kits available use ELISA technology. To avoid false positive results all positive results obtained from a commercial kit should be confirmed using another test method (western blot / PCR / immunofluorescence). 175

8 Please note: Antibodies usually take 8-12 weeks to reach detectable levels following initial infection. Therefore FIV antibody negative cats which have been exposed to FIV positive cats or any cat with an inconclusive result should be re-tested in 8-12 weeks to confirm their seronegative status. Kittens born to FIV positive queens often test positive due to maternal antibody transfer via the colostrum. Therefore, any kittens which test seropositive at <6 months of age should be re-tested after they are 8-12 months of age to determine their FIV status. These various serological assays are unable to distinguish antibodies associated with natural infection from those associated with vaccination. Feline Immunodeficiency Virus viral antigen testing Polymerase Chain reaction (PCR) These assays are designed to detect FIV viral or proviral DNA in EDTA blood or tissue (lymph nodes, spleen and bone marrow). Poor sensitivity and specificity are a problem. False negative results may occur if viral loads are lower than the threshold of detection of the assay. Primers have not been designed to detect all 5 FIV variants. Virus isolation Virus isolation used to detect circulating virus in heparin blood or lymphoid tissue (lymph node, spleen, bone marrow). This procedure is rarely employed as the procedure is time consuming and requires an adequately equipped laboratory with considerable expertise. Further Reading 1. Dunham et al Retroviral infections of small animals. Veterinary Clinics: Small Animal Practice 38:

9 Feline Respiratory Disease Complex Introduction The major role-players in feline infectious respiratory disease include most commonly Feline Herpesvirus-1 (FHV-1) and Feline Calicivirus (FCV). Bordetella bronchiseptica is a primary respiratory pathogen in the cat and considered a potential contributor to the feline respiratory disease complex. Chlamydophila felis is an infrequent cause of upper airway disease in the cat, more commonly being associated with conjunctivitis. Mycoplasma spp, feline reovirus and cowpox virus have been implicated in rare instances. Although in general FHV-1 causes more severe disease than FCV, there are some more virulent pneumotropic strains of FCV which induce severe acute to subacute interstitial pneumonia due to their tropism for type I pneumocytes, plus the recently described virulent haemorrhagic strain, which causes alopecia, cutaneous ulcers, subcutaneous oedema and high mortality in affected cats. Polymerase Chain Reaction (PCR) panel Sample Guide The PCR panel includes Feline Herpesvirus-1 (FHV-1), Feline Calicivirus (FCV), Chlamydophila felis and Mycoplasma spp. Specimens suitable for PCR are nasal flushes, tracheal washes, bronchoalveolar lavages, nasal swabs, conjunctival swabs and EDTA blood samples. In the case of FHV-1, running concurrent PCR on EDTA blood and nasal swabs enables identification of carrier animals and shedders. Samples are packed using the prescribed Triple Packaging System as per regulations SABS 0229:1996. (See Reference Ranges Section 3). Specimens are stored and transported at 4 C to the laboratory. 177

10 Microbiology Nasal, oropharyngeal and conjunctival swabs are the preferred samples for microbiological culture. Culture can still be performed on nasal flushes, tracheal washes or bronchoalveolar lavages. Both charcoal and non-charcoal swabs should be collected for culture. All samples are packed using the prescribed Triple Packaging System as per regulations SABS 0229:1996 and transported at 4 C to the laboratory. (See Reference Ranges Section 3). Histopathology and Immunohistochemistry (IHC) Histopathology in conjunction with IHC staining for FHV-1 and FCV on formalin-fixed post mortal tissues enables demonstration of viral antigen within histological lesions. These IHC stains have high diagnostic specificity for FHV-1 and FCV. Serology has little clinical application in feline respiratory disease complex investigation. Further Reading 1. Green C E Infectious Diseases of the dog and cat. 3 rd edn. Saunders-Elsevier, St Louis. 2. Pesavento P A, MacLachlan N J et al Pathologic, immunohistochemical and electron microscopic findings in naturally occurring virulent systemic feline calicivirus infection in cats. Veterinary Pathology 41:

11 Feline Parvovirus / Feline Panleukopaenia Introduction Feline panleukopaenia is a common infectious viral disease affecting the domestic cat, wild felidae, mink and ferret. Target organs for this parvovirus are organs with rapidly dividing cells; the crypt epithelium of the intestine, lymphoid tissue, bone marrow and central nervous tissue in neonates being sites of predilection of this viral agent. The disease is characterized by a marked decrease in circulating white blood cells (panleukopaenia) and destruction of intestinal mucosa leading to enteritis. Infection of pregnant queens, from the middle-third of gestation to immediately post-natally, results in cerebella hypoplasia and the development of the so-called "feline ataxia syndrome". This syndrome develops most commonly when queens are infected or vaccinated during pregnancy (later stages) but can still develop in kittens infected up to 9 days after birth. Feline Panleukopaenia Antigen Detection Polymerase Chain Reaction (PCR) Sample Guide The PCR is an extremely useful diagnostic tool in the case of feline panleukopaenia in that it can detect localized tissue infections with low virus load. Preferred samples include intestine (swabs or tissue), mesenteric lymph node, lung, kidney and brain. The image above-right is from a young kitten with feline ataxia syndrome. Note the severe cerebella atrophy. Feline panleukopaenia virus is detectable by PCR in these cases, but restricted to cerebella tissue. Samples are packed using the prescribed Triple Packaging System as per regulations SABS 0229:1996. (See Reference Ranges Section 3). Specimens are stored and transported at 4 C to the laboratory. 179

12 Histopathology / Immunohistochemistry (IHC) The use of IHC staining in conjunction with histopathology enables demonstration of feline panleukopaenia viral antigen within histological lesions. Target tissues include intestine (especially areas with Payer s patches), lymph node, spleen, thymus, lung, kidney and brain (in cases of feline ataxia syndrome). All tissues are fixed in 10% buffered formalin. Virus Isolation (VI) Neonatal infected kittens lung, kidney, brain, urine and faeces. In CNS infection virus is often restricted to the Purkinje cells of the cerebellum. Post-natal infections intestine, mesenteric lymph node, faeces, lung and kidney. Samples are packed using the prescribed Triple Packaging System as per regulations SABS 0229:1996. (See Reference Ranges Section 3). Specimens are stored and transported at 4 C to the laboratory. 180

13 Faecal antigen capture ELISA Application of the canine parvovirus faecal antigen capture ELISA assays has had success in identifying infected kittens. These assays have been successfully applied to faeces and homogenized post mortal tissues. Feline parvovirus (FPV) is only detectable in faeces for hours post infection and therefore in many cats with clinical symptoms, FPV is no longer detectable by faecal antigen capture ELISA. Samples are packed using the prescribed Triple Packaging System as per regulations SABS 0229:1996. (See Reference Ranges Section 3). Specimens are stored and transported at 4 C to the laboratory. Serology (virus neutralisation / complement fixation / haemagglutination-inhibition / ELISA) has been applied in research facilities, but is rarely used in the diagnostic situation. Further Reading 1. Green C E Infectious Diseases of the dog and cat. 3 rd edn. Saunders-Elsevier, St Louis. 2. Resibois et al Naturally occurring parvovirus-associated feline hypogranular cerebellar hypoplasia a comparison to experimentally induced lesions using immunohistology. Veterinary Pathology. 44:

14 Feline Haemotrophic Mycoplasmosis Introduction Mycoplasma species (formerly Haemobartonella) have now been proven with molecular sequencing to be gram negative, non-acid fast epicellular mycoplasmas. Two species have been identified in cats namely Mycoplasma haemofelis and Mycoplasma haemominutun. M. haemofelis is considered the more common species and causes more severe clinical illness. The mode of transmission of these organisms has not been fully elucidated but biting flies, bloodsucking arthropods and even cat bites are possible routes of infection. The various phases of the disease have been classified into pre-parasitaemic, acute phase, recovery and carrier phases. The pre-parasitaemic phase lasts 1-3 weeks and the cat is usually asymptomatic and parasites are not seen in blood smears. The acute phase which may last a month or longer, represents the time from the first to the last parasitaemic waves. These waves are short-lived, varying from 1-5 days and correspond to high parasitaemias which induce sharp decreases in haematocrit and pyrexic spikes. Often these parasitaemias clear in a very short space of time (as little as two hours has been recorded), which is believed to be due to sequestration of parasitised erythrocytes in the spleen and synchronized detachment of the organisms; they are followed by rising haematocrit and temperatures returning to normal. These parasitaemic waves repeat in a cyclical fashion causing relapsing / recurrent illness. The severity of the anaemia varies from cat to cat and the exact reasons for this have not been identified. Some cats can die from precipitous declines in haematocrit if left untreated. Blood smear examination Sample Guide The anaemia is typically regenerative with anisocytosis, polychromasia and reticulocytosis. There are parasitaemic spikes and therefore failure to demonstrate organisms in blood smears cannot exclude infection (false negatives). There is convincing evidence of an immune-mediated haemolytic component and many of these cats are Coomb s test-positive, even in between the parasitaemic spikes. FeLV-positive cats are more likely to have M. haemofelis infections; the severity of the anaemia being worse in co-infected cats than in those with just one of the diseases. Screening of all M. haemofelis blood smear positive cats for FeLV is indicated. 182

15 M. haemofelis Antigen Detection Polymerase Chain Reaction Molecular diagnostic techniques by means of PCR for 16s ribosomal-rna are the most sensitive and reliable method to confirm a diagnosis. Blood collected into EDTA is the preferred sample. New cats introduced to breeding colonies or multi-cat households should be screened with this PCR. Anaemic cats with regenerative anaemia and positive Coomb s should be tested with this PCR. M. haemofelis PCR positive cats should be screened for FeLV. FeLV-positive cats should be tested for M. haemofelis if a regenerative anaemia is present. All samples are packed using the prescribed Triple Packaging System as per regulations SABS 0229:1996 and transported at 4 C to the laboratory. (See Reference Ranges Section 3). Further Reading 1. Harvey JW Haemotrophic Mycoplasmosis (Haemobartonellosis). In: Infectious Diseases of the Dog and Cat. Greene CE, ed. 3 rd Ed. Saunders, Philadelphia Messick JB New perspectives about Hemotropic mycoplasmas (formerly, Haemobartonella and Eperythrozoon species) infections in dogs and cats. Veterinary Clinics Small Animal Practice. 33: , 3. Hagiwara MK Anemia in cats: is it Mycoplasma? Proceedings of the34th World Small Animal Veterinary Congress. Brazil. 183

16 Feline Babesiosis Introduction Feline babesiosis is a highly significant clinical disease among domestic cats, which seems to occur almost exclusively in South Africa, with rare reports from France, Germany, Thailand, India, and Zimbabwe. Disease incidence is highest along the southern Cape coast, with more sporadic occurrence along the east coast (KZN) and minimal disease reported from the west coast beyond Cape Town. An isolated focus of disease has also been identified in Mpumalanga. Feline babesiosis of domestic cats is caused by the intra-erythrocytic haemoprotozoan parasite currently classified as Babesia felis, which is part of the group of small Babesia parasites. A morphologically similar parasite also occurs in wild felids and gene sequencing reveals that the isolates from caracals are very similar to B. felis, while that from lions is distinct and described as a separate species, Babesia leo. B. felis is highly pathogenic while the pathogenicity of B. leo in domestic cats has not been fully investigated. The vector is thought to be a tick species but has not yet been identified. Typical clinical signs of the disease are chronic and include anorexia and listlessness/depression, anaemia and weight loss. Other, less common clinical signs include icterus, weakness, vomiting, pica and respiratory signs. Macrocytic, hypochromic, regenerative anaemia is the most consistent haematological finding and in-saline agglutination is positive in a number of cases indicating secondary immune-mediated haemolytic anaemia in these patients. Raised ALT and total bilirubin with normal ALP levels represent the most common clinical chemistry findings. Blood Smear Examination Sample Guide Demonstration of the parasites in blood smears in a cat with consistent clinical signs warrants a presumptive diagnosis of feline babesiosis. There are occasions where parasitaemias appear very low and the possibility of coinfections with immunosuppressive viral pathogens (FeLV / FIV) or Mycoplasma haemofelis must be considered. The organisms are very small often occurring as single intraerythrocytic inclusions but paired forms and Maltese-crosses are occasionally seen. Caution must be exercised not to confuse Howell-Jolly bodies for parasites. 184

17 Polymerase Chain Reaction (PCR) Blood samples collected into EDTA are the preferred samples for PCR. B. felis PCR is a sensitive procedure and usually reserved for cases where a regenerative anaemia is present without any explanation and no parasites can be found on blood smears. Once the presence of babesia DNA is ascertained, the individual species of organisms may be identified by comparing genetic sequences of the 18S ribosomal subunit of the RNA. All samples are packed using the prescribed Triple Packaging System as per regulations SABS 0229:1996 and transported at 4 C to the laboratory. (See Reference Ranges Section 3). Serology for feline babesiosis is not recommended. Further Reading 1. Penzhorn BL, Schoeman T and Jacobson LS Feline Babesiosis in South Africa: a review. Annals of the New York Academy of Sciences. 1026: Schoeman T Clinical and Clinico-pathological changes in feline babesiosis. Post Graduate Thesis, University of Pretoria. 3. Schoeman T, Lobetti RG, Jacobson LS, Penzhorn B Feline babesiosis: signalment, clinical pathology and concurrent infections. South African Veterinary Journal. 72(1):

18 Feline Toxoplasmosis Introduction Toxoplasma gondii is an intracellular coccidian parasite and is one of the most common parasitic diseases of animals and man. Felines are the definitive hosts of Toxoplasma gondii with nonfeline hosts (mammals and birds), as well as cats, serving as intermediate hosts. Cats are most frequently infected by ingestion of tissue cysts in prey species, or less commonly by ingestion of oocysts. Oocysts produced during the sexual reproductive phase are excreted in faeces of the definitive hosts. The major modes of transmission to intermediate hosts on the other hand are ingestion of food or water contaminated with oocysts, eating uncooked meat and congenital infections. The extra-intestinal cycle with intestinal invasion and visceral dissemination of tachyzoites, is clinically the most important phase in the intermediate host. This extra-intestinal cycle also occurs in the cat, usually simultaneously with the entero-epithelial cycle, but rapid development of specific cell-mediated immunity usually prevents any further clinical complications from arising in the feline species. Tachyzoites can multiply in any cell of the body causing the classic multifocal areas of visceral necrosis that characterize acute systemic toxoplasmosis. With a developing immune response in the intermediate host, the tachyzoites undergo transition to bradyzoite filled tissue cysts, which are inert and rarely associated with any clinical disease. Acute systemic toxoplasmosis of felines has been described in neonates, young immunologically immature kittens and immunocompromised cats. Systemic infection during pregnancy can result in placentitis and invasion of the foetus with foetal death. Such congenital transmission occurs when immunologically naïve queens, women or ewes initially become infected. High rates of congenital transmission in mice probably serve to maintain a reservoir of infection in the wild. Toxoplasma Antibody Detection - Serology Sample Guide Indirect Immunofluorescent Antibody (IFA) / Agglutination Assays / ELISA A minimum of 2ml of serum collected into serum tubes with (yellow stopper) or without (red stopper) clot activator. Following collection, allow samples to stand for minutes at room temperature to form a clot, after which they should ideally be centrifuged and the serum poured off. Serum or un-spun clotted blood should then be transported at 4 C to the laboratory. 186

19 Seropositivity is very high in most cat populations. After infection IgM titres initially rise in most infected cats and their detection can be used as an indicator of recent infection and to estimate the risk of oocyst shedding. However, some cats remain persistently IgM-positive (co-infections with FIV appear to play a role here), while some cats never develop detectable IgM titres despite infection. IgG on the other hand can take up to 4-6 weeks to peak and therefore a single positive IgG titre may be well after the date of infection, and critically, after the oocyst-shedding phase. Once IgG or IgA antibodies have been detected, they increase over a relatively short period of 2-3 weeks, and if paired sera are tested during this phase, rising titres (> 4 fold increase), give a strong indication of recent T. gondii infection. However if this short 2-3 week window is missed, paired sera may not show significantly different titres and the only assumption that can be made is that of previous exposure to T. gondii. Failure to detect rising antibody titres cannot exclude a diagnosis of toxoplasmosis. IgG titres can remain at high concentrations for years post-infection, therefore measurement of titres that are considered high does not prove recent or current infection nor give any indication as to stage of oocyst shedding. With IFA assays IgM > 1:64 with concurrent IgG > 1:64 probably indicate active infection. The major drawback of the agglutination tests is that they only detect IgG and therefore may be negative in the acute stages of infection. In cats with encephalitis / uveitis, CSF and aqueous humor is collected for serology and PCR. CSF / aqueous humor antibody titres need to be compared to serum antibody titres as well as CSF / aqueous humor PCR results. Toxoplasma Antigen Detection Polymerase Chain Reaction (PCR) PCR assays have been developed and are highly sensitive. They are based on the DNA amplification of the Toxoplasma gondii-b1 gene. A single organism can be detected directly from a crude cell lysate or as few as 10 parasites in the presence of 100,000 leukocytes. 187

20 Positive PCR on EDTA blood samples indicates the presence of T. gondii organisms but cannot differentiate acute infections from sub-clinical, chronic encysted infections. False-negative results have been reported in chronically infected humans. Positive PCR s in aqueous humor or CSF in symptomatic cats is more specific for T. gondii infection but not conclusive. Histopathology and Immunohistochemistry (IHC) IHC staining used in conjunction with histopathology enables demonstration of protozoal antigen within histological lesions confirming the diagnosis of toxoplasmosis. The histopathological image above is of a T. gondii IHC stain on the liver of a cat with acute systemic toxoplasmosis, which has been counterstained with haematoxylin. Note the positive brown granular staining of T. gondii tachyzoites within as well as at the periphery of a necrotic focus in the liver. IHC is considered the gold standard diagnostic technique for post mortal tissues. These stains are performed on formalin-fixed paraffin embedded tissue sections. T.gondii antigen is still easily detected by IHC in tissues with advanced autolysis. Faecal examination is not recommended as Toxoplasma oocysts are very small and can easily be missed in faecal floatations. Also naïve cats that are infected only excrete oocyts in the first 1-2 weeks post-infection and during this period are usually clinically normal without any diarrhoea. 188

21 Public health-risk to pregnant women It has been shown that women are more likely to contract T. gondii from eating uncooked meat or ingesting contaminated food or water (soil contaminated with oocysts), than from direct contact with an infected pet. Therefore sound hygiene practices must be recommended to pregnant women to minimize potential exposure to oocysts and these include. Pregnant women do not empty litter trays. Litter trays are not stored in the kitchen; and are cleaned daily. Cats are not fed on kitchen counter-tops. Pregnant women avoid handling raw meat or wear gloves when doing so. Thorough hand cleansing after gardening or handling the cat. Allowing the cat to lick the face must be discouraged. Covering of sandpits to prevent defaecation by infected cats therein. Water from garden sources is boiled completely before consumption. Note: Cats older than 4-5 years of age that have grown up in urban areas, are very likely to already have been exposed to T. gondii and are likely immune with no faecal shedding of oocysts. Serology of cats should form part of the risk assessment but should not be used alone, but interpreted together with all other epidemiological factors. The interpretation of feline serology for estimation of zoonotic risk is as follows: Seronegative (IgM, IgG ) indicates this particular cat is not likely to be currently shedding oocysts, but will most likely shed oocysts if exposed. These cats pose the greatest risk for human infection!! Seropositive (IgM +, IgG ) indicates that this cat has been recently infected and there is a potential risk that it may still be shedding oocysts and so poses a zoonotic risk. Seropositive (IgG +, IgM +/ ) indicates previous infection with a developed immune response and these cats are very unlikely to be shedding oocysts, plus they are less likely to shed oocysts if re-exposed, stressed or immunosuppressed. Such cats are of minimal zoonotic risk. Further Reading 1. AAFP Panel Report American Association of Feline Practitioners Report on Feline Zoonoses. AAFP Guidelines Statements. 2. Dubey J P and Lappin M R Toxoplasmosis and Neosporosis. In: Infectious Diseases of the Dog and Cat. Greene CE, ed. 3 rd Ed. Saunders, Philadelphia Dubey J P Toxoplasmosis in cats and dogs. Proceedings of the 30 th World Small Animal Veterinary Association Congress. 4. Last R D, Suzuki Y et al A case of fatal systemic toxoplasmosis in a cat being treated with cyclosporin A for feline atopy. Veterinary Dermatology 15: