3. Protozoal diseases

Some protozoan species are capable of infecting dogs and can also infect humans, triggering zoonoses. The existence of protozoal coinfection in dogs are already established, one of the combination of Toxoplasma gondii and Neospora caninum sometimes increased by the presence of Leishmania spp. These types of infections exacerbate the clinical state of the animal by co‐ existence of various diseases [5].

The T. gondii causes toxoplasmosis and its definitive hosts are the Felidae. However, its prevalence in dogs is high. Transmission can occur vertically through congenital infection, however, horizontal transmission is the main mean of infection. Dogs are the definitive hosts of N. caninum that causes Neosporidiose disease, which can be transmitted horizontally or vertically, and it has maintained the parasite for generations amongst the canines [84].

#### 3.1. Toxoplasmosis

Toxoplasma gondii, the protozoan that causes a coccidiosis in felines can infect mammals (including humans), birds and reptiles, mainly affecting the central nervous system and occasionally, the reproductive system, muscles and visceral organs [6]. This protozoan is an obligate intracellular parasite and its definitive hosts are the Felidae, which excrete oocysts that house sporozoites, an infectious stage of the parasite. As for the intermediate hosts and therefore not Felidae, which shelter tissue cysts, it was possible to observe two infectious stages of the organism: tachyzoites and bradyzoites [7]. The oocytes containing the sporozoites have a double‐walled, spherical shape and resistance to environmental conditions, Being the parasitic manner that promotes their strength and dissemination. They are developed by a entero‐epitelial cycle inside the felids through sporogony, and are then excreted in their immature form with the faeces of these animals. The tachyzoites Form occurs in the active proliferation stage of T. gondii, or the acute phase of infection Parasitizing preferably macrophages and monocytes but with the potential to infect any nucleated cell. This parasitic stage has an arc shape, is mobile and provides rapid multiplication by endodyogeny. In the chronic phase of infection are found the bradyzoites located in parasitophorous vacuole of a cell whose membrane forms the capsule of the tissue cyst. They have slow multiplication, also endodyogeny, intracisto, where it is protected from the action of the host immune system and drugs, allowing its existence for months and even years. They are poorly resistant to high temperatures [8].

Escherichia coli is the most common bacterium isolated from the canine vagina and is also commonly cultured from the uteri of bitches with metritis and pyometra. E. coli produces an endotoxin that may result in pregnancy loss in the bitch. Streptococcus spp are bacteria that are physically present in the skin and mucosa of dogs and cats. Some microorganisms belonging to this group have been related to the occurrence of neonatal sepsis, abortion and metritis [2, 3]. βH Streptococcus is known cause metritis, pyometra, placentitis and abortion, commonly linked to ascending infection. Streptococcus βH is a major cause of neonatal death. Typically, newborns are infected in the mother's birth canal, through the umbilical cord or, less commonly, from the udders with mastitis. Bitches have high bacterial load in the vaginal canal, which persist throughout pregnancy leading to the infection of the uterus and foetus [2, 3].

Some protozoan species are capable of infecting dogs and can also infect humans, triggering zoonoses. The existence of protozoal coinfection in dogs are already established, one of the combination of Toxoplasma gondii and Neospora caninum sometimes increased by the presence of Leishmania spp. These types of infections exacerbate the clinical state of the animal by co‐

The T. gondii causes toxoplasmosis and its definitive hosts are the Felidae. However, its prevalence in dogs is high. Transmission can occur vertically through congenital infection, however, horizontal transmission is the main mean of infection. Dogs are the definitive hosts of N. caninum that causes Neosporidiose disease, which can be transmitted horizontally or

Toxoplasma gondii, the protozoan that causes a coccidiosis in felines can infect mammals (including humans), birds and reptiles, mainly affecting the central nervous system and occasionally, the reproductive system, muscles and visceral organs [6]. This protozoan is an obligate intracellular parasite and its definitive hosts are the Felidae, which excrete oocysts that house sporozoites, an infectious stage of the parasite. As for the intermediate hosts and therefore not Felidae, which shelter tissue cysts, it was possible to observe two infectious stages of the organism: tachyzoites and bradyzoites [7]. The oocytes containing the sporozoites have a double‐walled, spherical shape and resistance to environmental conditions, Being the parasitic manner that promotes their strength and dissemination. They are developed by a entero‐epitelial cycle inside the felids through sporogony, and are then excreted in their immature form with the faeces of these animals. The tachyzoites Form occurs in the active proliferation stage of T. gondii, or the acute phase of infection Parasitizing preferably macrophages and monocytes but with the potential to infect any nucleated cell. This parasitic stage has an arc shape, is mobile and provides rapid multiplication by endodyogeny. In the chronic phase of infection are found the bradyzoites located in parasitophorous vacuole of a cell whose membrane forms the capsule of the tissue cyst. They have slow multiplication, also endodyogeny,

vertically, and it has maintained the parasite for generations amongst the canines [84].

3. Protozoal diseases

58 Canine Medicine - Recent Topics and Advanced Research

existence of various diseases [5].

3.1. Toxoplasmosis

T. gondii has worldwide distribution and is considered as the most cosmopolitan of all the causative parasite zoonoses. It is an opportunistic organism, independent of the host and has relevance related to animal production and public health by having transmitted through food from infected animals. In transmission between animals, the Felidae assume the lead role as a source of infection [8]. Thus, the transmission can occur by congenital way, via ingestion of infected tissue or contaminated food and water, in addition to transmission by breastfeeding, through transfusion of body fluids or by transplantation of tissues or organs [7].The entero‐ epitelial biological cycle is unique to the definitive hosts in which the oocysts are released in the faeces. Intermediate hosts are infected by ingestion of oocysts and cysts. Oocysts must sporulate in the environment to become infective and once sporulated and ingested by potential hosts, will promote asexual reproduction and infect the animal that ingested it, leading to the formation of tissue cysts. Cysts, in turn, may be ingested by host carnivores, infecting them. Ingestion of raw or undercooked meats are the main sources of this type of transmission. Congenital transmission and placenta are more common in women, sheep, goats and nuts. However, studies have shown that infected female dogs with 56, 40 and 32 days of gestation showed congenital infection that may have led to abortion and thus confirm this relationship.

The evidence of facts relating to such a manifestation of the parasite in dogs is scarce. What is known is that in its life cycle, the T. gondii reaches different host tissues, including male and female reproductive organs of intermediate hosts, which may cause certain adverse effects on the reproductive function. In animals, toxoplasmosis is associated with reproductive failure in males related with some evidence of venereal transmission of T. gondii [9]. Researchers have shown that four bitches inseminated with canine semen samples containing 1 × 106 tachyzoites of T. gondii RH strain, had embryonic resorption in two of them and the protozoan was found in the brain of the four offsprings [10]. The results of this study show, among other things, that dogs with infected semen can cause reproductive problems and, consequently, the vertical transmission to offspring. Additionally, transmission of tachyzoites to the milk intake neonates (via lactogenic) shortly after birth [11] has been reported. The severity and type of clinical signs presented by the Animals infected with T. gondii are variable depending on the degree of tissue injury and location. There is no established reason regarding the different presentations of clinical signs and their absence in dogs. It is assumed that all types of cells are susceptible and some of these differences can be assigned to factors such as age, sex, host species, T. gondii strain, body number and stage of ingested parasites [8]. Specifically in dogs, the occurrence of clinical signals from toxoplasmosis is associated with concomitant infections such as distemper, ehrlichiosis, neosporidiose and leishmaniasis.

Animals infected with T. gondii usually show no clinical symptoms and undergo the primary infection for a latent infection or a chronic stage. In dogs, infection with this parasite is very common, being demonstrated by several studies of serological prevalence. Already the clinical form of infection is rather sparse, usually presenting itself in young animals, usually associated with immunosuppressive factors, infections or to canine distemper virus. Canine symptoms can concentrate in the gastrointestinal tract and respiratory systems and in neuromuscular or be from generalized infection. Therefore, clinical signs usually identified in dogs with toxoplasmosis are ataxia, diarrhoea and respiratory disease and focal necrosis in areas of the lung, liver and brain may also occur, triggering numerous clinical signs [12]. Therefore, toxoplasmosis in dogs and other animals can show symptoms quite similar to many infectious diseases, the Neosporidiose one with the greatest similarity.

The possible interference of Toxoplasmosis in the reproduction of dogs as a whole has been studied in 1970 and found out that toxoplasma infection in dogs at various gestational stages can cause mortality of the puppies from the 4th to 75th postnatal days [13]. Still, abortion and foetal death has been observed, in the middle third and final pregnancy in dogs experimentally infected with T. gondii orally (oocysts) and subcutaneously (tachyzoites) [14]. Later, reproductive problems related to canine toxoplasmosis were again observed, when naturally infected dogs with 30 days of gestation were artificially reinfected. The puppies from infected mothers were positive serologically at birth, but without symptoms, except a neonate who was weakened. With additional tests, the parasite was detected in saliva samples, milk and urine of these animals still being detected positive immunostaining of cysts and / or tachyzoites by immunohistochemistry in 23 organs of experimental animals [15, 16]. Soon, placental transmission and subsequent foetal infection with T. gondii in cats and dogs, have been experimentally observed and their association with foetal death have been found.

Diagnosis of toxoplasmosis is mainly based on clinical suspicion since the symptoms can be similar to other infectious diseases. Therefore, attention should be paid to the epidemiological information and data collected during the interview, making a request for additional tests, a prerequisite. In dogs, the presence of apathy, rhinorrhea, conjunctivitis, pneumonia, fever, convulsions, paralysis, diarrhoea and lymphadenopathy may be clinical signs of T. gondii infection, requiring additional tests to confirm the diseases [6]. The identification of T. gondii presence can be accomplished by in vitro and in vivo isolation, serology, histopathology, immunohistochemistry and PCR. In general, the serological diagnosis is the most used, mainly through modified agglutination test (MAD) and indirect immunofluorescence (IFA), which is the preferred test [17]. Whichever method is used, it is important to collect samples at intervals of about two weeks to determine seroconversion, indicating recent infection [18]. When there is abortion, should be referred to the placenta and the foetus (paying attention to the foetal brain), cooling temperature, as soon as possible to enable the laboratory diagnosis for toxoplasmosis. Submissions will be used for histopathology associated or not with immunohistochemical tissues with compatible lesions [8].

In cases of acute systemic infection in dogs changes can be observed in haematological parameters, such as nonregenerative anaemia, neutrophilic leucocytosis, lymphocytosis, monocytosis and eosinophilia. The biochemical changes are consistent with increased serum activities of alanine aminotransferase (ALT) and alkaline phosphatase may occur when there is liver necrosis. If necrosis is acute, bilirubin levels are likely to be increased. In the event of muscle necrosis, serum creatine kinase activity is also increased [7]. The establishment of the diagnosis can be made through serological tests, but there is no absolute serologic test that can definitely confirm the diagnosis of toxoplasmosis. Furthermore, only the detection of antibody against the parasite

is not sufficient for the establishment of serological diagnosis since previously infected dogs may also exhibit antibodies response. Some tests that may be ordered in addition to the IFT and MAD, such as the reaction of Sabin‐Feldman (SF), the enzyme linked immunosorbent assay (ELISA), complement fixation test (CF) and the reaction of indirect haemagglutination (HI) [8].

be from generalized infection. Therefore, clinical signs usually identified in dogs with toxoplasmosis are ataxia, diarrhoea and respiratory disease and focal necrosis in areas of the lung, liver and brain may also occur, triggering numerous clinical signs [12]. Therefore, toxoplasmosis in dogs and other animals can show symptoms quite similar to many infectious diseases,

The possible interference of Toxoplasmosis in the reproduction of dogs as a whole has been studied in 1970 and found out that toxoplasma infection in dogs at various gestational stages can cause mortality of the puppies from the 4th to 75th postnatal days [13]. Still, abortion and foetal death has been observed, in the middle third and final pregnancy in dogs experimentally infected with T. gondii orally (oocysts) and subcutaneously (tachyzoites) [14]. Later, reproductive problems related to canine toxoplasmosis were again observed, when naturally infected dogs with 30 days of gestation were artificially reinfected. The puppies from infected mothers were positive serologically at birth, but without symptoms, except a neonate who was weakened. With additional tests, the parasite was detected in saliva samples, milk and urine of these animals still being detected positive immunostaining of cysts and / or tachyzoites by immunohistochemistry in 23 organs of experimental animals [15, 16]. Soon, placental transmission and subsequent foetal infection with T. gondii in cats and dogs, have been experimentally observed and

Diagnosis of toxoplasmosis is mainly based on clinical suspicion since the symptoms can be similar to other infectious diseases. Therefore, attention should be paid to the epidemiological information and data collected during the interview, making a request for additional tests, a prerequisite. In dogs, the presence of apathy, rhinorrhea, conjunctivitis, pneumonia, fever, convulsions, paralysis, diarrhoea and lymphadenopathy may be clinical signs of T. gondii infection, requiring additional tests to confirm the diseases [6]. The identification of T. gondii presence can be accomplished by in vitro and in vivo isolation, serology, histopathology, immunohistochemistry and PCR. In general, the serological diagnosis is the most used, mainly through modified agglutination test (MAD) and indirect immunofluorescence (IFA), which is the preferred test [17]. Whichever method is used, it is important to collect samples at intervals of about two weeks to determine seroconversion, indicating recent infection [18]. When there is abortion, should be referred to the placenta and the foetus (paying attention to the foetal brain), cooling temperature, as soon as possible to enable the laboratory diagnosis for toxoplasmosis. Submissions will be used for histopathology associated or not with immunohisto-

In cases of acute systemic infection in dogs changes can be observed in haematological parameters, such as nonregenerative anaemia, neutrophilic leucocytosis, lymphocytosis, monocytosis and eosinophilia. The biochemical changes are consistent with increased serum activities of alanine aminotransferase (ALT) and alkaline phosphatase may occur when there is liver necrosis. If necrosis is acute, bilirubin levels are likely to be increased. In the event of muscle necrosis, serum creatine kinase activity is also increased [7]. The establishment of the diagnosis can be made through serological tests, but there is no absolute serologic test that can definitely confirm the diagnosis of toxoplasmosis. Furthermore, only the detection of antibody against the parasite

the Neosporidiose one with the greatest similarity.

60 Canine Medicine - Recent Topics and Advanced Research

their association with foetal death have been found.

chemical tissues with compatible lesions [8].

The IFA identifies antibodies through specific fluorescent conjugates of IgG and IgM. The presence of IgG is related to a previous exposure, suggesting active and recent infection. The MAD evaluates IgM antibodies indirectly by subtracting antibody titres present in treated and untreated sera [19]. The SF reaction has a high sensitivity and specificity and allows the antibody titration within a few days post‐infection. The enzyme linked immunosorbent assay allows detection of IgM response. In the CF test, different parasite antigens are used and that can be identified as testing positive in case of an early infection. In short, the interpretation of the results: a high titre of IgM with low or zero IgG indicates a disease evolving, otherwise, the high quantitation of IgG and low or zero IgM indicate a chronic state of the disease. Whatever the outcome, a new serology test should be done after 15–21 days due to the possibility of severe immunosuppression until the second post‐infection week, which may lead to false‐ negative results [8]. Identifying the T. gondii in tissue or bodily fluids is possible by polymerase chain reaction that detects the protozoa in biological samples. However, this detection which is based on PCR is still quite limited, as it requires special equipment, which are not available in any clinical laboratory. In addition, the realization of body isolation requires relatively vast and considerable experience time. However, care should be taken that the molecular detection of the parasite does not replace the serological methods in the diagnosis of the disease, making it imperative to associate the two diagnostic methods to identify the presence of infection and the determination of the disease stage [8].

Various infectious and non‐infectious diseases should be evaluated in the differential diagnosis of toxoplasmosis. In dogs, the most relevant are distemper, neosporosis, isosporiasis and strongyloidiasis [8]. In aborted foetuses, macroscopically it is possible to observe necrotic foci white‐greyish punctate located in the lungs and liver, as well as pulmonary congestion and heart pallor. The central nervous system (CNS) is marked with congestion of the brain and cerebellum [7].

Treatment of toxoplasmosis is based on suppression of the replication agent when the disease presents itself in the acute form, and emphasizes the importance of early diagnosis and consistent implementation of measures leading to the reduction in disease transmission [20]. Clindamycin is the drug of choice for the treatment of dogs and cats. The drug administration once started, the clinical signs tend to initiate regression after 24 – 48 hours. In addition to clindamycin, the combined use of pyrimethamine and fast‐acting sulphonamides (e.g., sulfadiazine, sulfamethazine and sulfamerazin) is valid in the treatment of systemic toxoplasmosis infection [7].

Prevention should be taken to avoid ingestion and contact with oocysts and cysts. So for pets that eat meat, the meat should be well cooked. As a complement to prevention, measures should be taken to avoid these same animals hunt and/or eating mechanical hosts such as cockroaches, flies, worms and rodents, potential or intermediate hosts. The prevention becomes even more important because there is no vaccine available for humans or animals [8]. General care in food hygiene, whether in urban or rural areas, considering proper cooking of meat and milk boiling, are the main preventive measures of toxoplasma infection to humans. Personal hygiene and comprehensive hand washing performed after handling raw meat, for example, assists in preventing the disease. Generally, prevention of human toxoplasmosis is based on the maximum avoidance of exposure to susceptible hosts. This disease is not notifiable, except for outbreaks [7].
