Conference: 18 - 2016    Case: 3     -


Signalment:  

15-day-old, male, Angus x Nellore cross, ox (Bos taurus).


Gross Description:  

In the two necropsied calves (one of each sex), the carcasses were moderated jaundiced, there was splenomegaly and the liver was swollen and had an orange-tan discoloration. The kidneys of one the calves were dark brown and the urine was faint red, although hemoglobinuria was not observed clinically. The grey telencephalic and cerebellar cortices and the basal nuclei had an intense cherry-pink discoloration which contrasted strongly with the white matter. One calf had omphalitis associated with myiasis (Cochliomyia hominivorax).


History:  

On September 2015, in a farm in Midwestern Brazil, eight out of 100, 15-day-old cross bred (Angus x Nelore) calves got sick. Affected calves were unable to follow their mothers and presented with lethargy and ataxia. They were unsuccessfully treated with florfenicol and sodium dipyrone. Five calves died on site; the remaining three sick calves were referred to the Veterinary Teaching Hospital of the Federal University of Mato Grosso do Sul, where they were clinically examined and given supportive therapy. Two of those calves died within 24 hours of the onset of clinical signs and were necropsied. The remaining calf was treated for babesiosis and recovered. Clinical signs in the affected calves included fever, apathy, icterus, stiffness of the neck, and difficulty in keeping a standing position due to incoordination. This latter sign rapidly progressed to sternal decubitus, lateral decubitus, muscle tremors, paddling movements, nystagmus, tachycardia, and tachypnea. One calf had lost of menace reflex and another one had opisthotonus. All calves were parasitized by Rhipicephalus (Boophilus) microplus ticks.


Histopathologic Description:

The most striking lesions are the diffuse and marked capillary engorgement with erythrocytes in the grey matter of the brain. Virtually every erythrocyte within these capillaries contained small paired or single faint basophilic spherical organisms. Perivascular and perineuronal edema are also observed in the grey matter. Mild to minimal mononuclear perivascular cuffings are observed in a few of the slides. There is marked intra-hepatocellular and canalicular cholestasis mainly in centrolobular areas associated with vacuolar hepatocellular degeneration and necrosis. Only minimal hemoglobinuric nephrosis is observed in the kidneys; focal mixed mononuclear cell reaction found in renal interstitium is considered incidental. Changes in the spleen are moderate diffuse congestion. There are no pathological changes in the lungs, heart and gastrointestinal tract.


Morphologic Diagnosis:  

Cerebral cortical congestion, marked, acute, associated with intraerythrocytic organisms with morphology compatible with Babesia bovis.


Lab Results:  

Blood work: Erythrocytes 4.08 p/106µl (reference values 5.5-10.0 p/106µl); hemoglobin 6.2 g/dL (8-15 g/dL); mean corpuscular value 50 reference values 40-60 fL); Fibrinogen: 1,000 mg/dL (reference values 300-700 mg/dL). Serum biochemistry: Aspartate aminotransferase (AST): 297.5 UI/L (reference values 20 a 34 UI/L); g glutamyl transferase (GGT) 37.4 UI/L (reference values 6.1-7.4 UI/L); glucose 5.8 mg/dL (reference values 45 a 75 mg/dL).

In Romanowsky-stained squashes from telencephalic cortex, capillaries appeared clogged with intraerythrocytic small (2 μm-diameter) paired or single spherical basophilic organisms (morphology compatible with Babesia bovis). Similar organisms were also observed within erythrocytes from blood smears.


Contributor Comment:  

The diagnosis of bovine babesiosis caused by Babesia bovis was made based on the pathognomonic gross brain lesions, necropsy and microscopic lesions consistent with the hemolytic crisis, and visualization of the intraerythrocytic parasite in blood imprints, in the capillaries of brain cortical grey matter, in squashes of brain tissue, and HE stained slides.

Babesia was first described by Babés in Romania as a parasite of bovine erythrocytes. Although it is possible for a single Babesia species to infect more than one vertebrate host (e.g., B. microti affects rodent and humans; B. divergens and B. bovis affect cattle and humans), Babesia spp. are typically host specific.2

Bovine babesiosis (aka, piroplasmosis, Texas fever, redwater, and tick fever) is a febrile hemolytic condition caused by one of at least seven species the protozoan organisms Babesia spp. It is characterized by extensive intravascular hemolysis leading to depression, anemia, icterus, hemo-globinuria, and, in the case of B. bovis infections, neurological signs.2

In Brazil, bovine babesiosis is caused by B. bovis (formerly B. argentina) and/or B. bigemina and is transmitted to cattle by the tick Rhipicephalus (Boophilus) microplus.8,10 The disease is frequently, but not always associated with icterus and hemoglobinuria and the animal may become extremely ill before severe anemia, parasitemia or hemoglobinuria are apparent.5,15

In general, the disease distribution follows that of the vector ticks producing three distinct epidemiological situations. The disease does not occur in areas without the tick vector. In areas of enzootic instability, there is an alternation of warm and cold seasons. The cold period prolongs the free-living stages of the tick, allowing cattle extended periods without vector contact, resulting in a significant drop in antibodies due to the absence of Babesia infection. When the warm period returns, the tick parasite load increases and outbreaks occur. In enzootic areas, weather condition allow the presence of tick on cattle all year round, which confers high level of lasting immunological protection.1

Factors influencing the occurrence of babesiosis outbreak include (1) over infestation by vector ticks resulting in a high inoculum of Babesia: (2) long periods without ticks with resultant loss of immunity, and (3) stress factor and nutritional deficiencies which can induce a drop in immunity and vulnerability to the disease.2 Calves are typically more typically more resistant to infection by Babesia sp. than adult cattle.15  Mortality is lower in enzootic areas due to resistance to infection.

B. bovis is small, pleomorphic apicomplexan parasite, and can occur as single or as paired pear-shaped bodies joined at an obtuse angle within the center of the mature erythrocyte. The round forms measure 1-1.5 µm, and the pear-shaped bodies 1.5 x 2.4 µm in size. Vacuolated signet ring forms are particularly common. Hosts are cattle, buffalo, and deer.14

The incubation period for bovine babesiosis is typically 2-3 weeks1,15 for the natural disease. The natural infection caused by B. bovis tends to present a longer incubation time than that caused by B. bigemina. Cases of extremely short incubation periods (seven days) have been reported for B. bovis.13,14

Infection occurs after the tick vector feeds on the host. After the inoculation of the infectious sporozoites, the parasite penetrates the erythrocytes in the definitive host where they form a parasitophorous vacuole and change to the trophozoite form. Later these trophozoites undergo binary division, usually forming two merozoites. Ticks acquire Babesia infection during feeding on infected animals.2

Affected cattle develop depression, anorexia, paleness of mucous membranes, and fever (40 degrees/C-42 degrees C). Icterus and hemoglobinuria are also common clinical signs, but they can be minimal or absent in cases of peracute or acute disease. The elevation of serum activity of AST and GGT, observed in this case, may be due to the hepatic centrilobular hypoxic necrosis.1 Additionally, this calf had omphalitis, which probably resulted in septicemia (high fibrinogen) which would explain the low blood sugar.4

B. bovis causes the most severe form of babesiosis in cattle in which peripheral circulatory disturbances with sequestration of parasitized erythrocytes in the peripheral circulation are unique features.3,11,12,16 In southern Brazil this form of disease is found in approximately two-thirds of the cases of babesiosis caused by B. bovis and is virtually always fatal.11

Necropsy findings include yellow discoloration (icterus) of mucosae, subcutaneous tissue, muscle fasciae and the intimal surface of arteries. In acute or peracute cases (which include the cases of cerebral babesiosis) icterus can be mild or absent. The serosal membranes of the abdominal viscera have hemoglobin imbibition. The liver is swollen, with rounded edges, and is yellow or tan discolored. The biliary vesicle is usually markedly distended by dark-green inspissated bile. Subepicardial and subendocardial hemorrhages (petechiae and ecchymosis) are virtually always observed. In cases where hemoglobinuria is a prominent sign, the kidneys are diffusely dark red-brown (hemoglobinuric nephrosis) and the urine is dark-red (red water disease). There is always some degree of splenomegaly. In severely enlarged spleens the red pulp prolapses on the cut surface. In cerebral babesiosis, the grey matter of the telencephalic, and cerebellar cortex and that of the basal nuclei has a characteristic cherry pink color. Squashes made from cortical brain, stained with Wright-Giemsa reveal numerous capillaries engorged with parasitized erythrocytes.3,5,6

The encephalic lesion is characteristic of B. bovis infection and does not occur in any other Babesia sp. infection of cattle. However, it can be compared with the brain lesions seen in severe cases of malaria caused by Plasmodium falciparum. Microscopically the brain lesions are characterized by cortical capillaries diffusely engorged with red blood cells, and perivascular and perineuronal edema. In tissue sections of the brain, Giemsa and even HE stains demonstrate the parasites as small paired or single spherical faint basophilic bodies.1,15 Parasitized erythrocytes may also be seen in vessels of virtually all tissues, such as the interstitial capillaries in the kidney, heart, and in skeletal muscle.15 Other changes characteristic of hemolytic anemia are observed such hemoglobinuric nephrosis, centrilobular hepatic necrosis (due to hypoxia) and bile stasis.15

Ultrastructurally, capillaries in the brain are dilated and filled with densely packed parasitized erythrocytes. These erythrocytes have scalloped edges with fine strands apparently connecting adjacent red blood, as well as connecting erythrocytes and endothelial cells.5,6 Masses of lysed red blood cells which have undergone lysis but still contain intact parasites are frequently seen in capillaries. Changes in the capillary endothelium of the affected parts in the brain range from swelling of the cytoplasm and nucleus to necrosis. Perivascular and perineuronal spaces are enlarged. In the kidneys, capillaries are not packed as tightly with red blood cells as in the brain, and the parasitemia does not exceed 50% of red blood cells. Other changes are similar to those seen in the brain. Capillaries in the lung are packed with red blood cells, but only a small proportion of the cells are parasitized.5

Although usually reported resistant to babesiosis, calves in enzootic areas can be parasitized by R. microplus on the first day of life.8 Since the protection of neonates calves is conferred by passive immunity through colostral ingestion, a failure in the transfer of passive immunity from the dam can explain very young calves being affected by the disease. A lack of passive immunity caused by the failure of colostral ingestion in the calf of this report could be suspected since omphalitis was also present.

The pathogenesis of the disease caused by B. bovis in cattle is not completely understood, but some mechanisms are proposed.5,15 Acute disease is characterized by a hypotensive shock syndrome with vascular stasis and accumulation of parasitized erythrocytes in the peripheral circulation. It is accompanied by activation of coagulation/complement cascades and the release of vasoactive compounds resulting in vasodilation and circulatory stasis as well as generalized organ damage due to anoxia and toxic products from both parasites and damaged host tissue.

Parasite proteases cause hydrolysis of fibrinogen which results in the accumulation of large quantities of soluble fibrin complexes which are not cross-linked, as well as in altered fibrinogen in the circulation. Thus, the coagulability and viscosity of blood increases but insoluble fibrin is not produced, suggesting that classic disseminated intravascular coagulo-pathy is not a feature of B. bovis infections.5 The cause of cytoadherence of in B. bovis organisms to endothelial cells is also uncertain,5,15 but the Babesia organisms remodel the erythrocyte surface with their erythrocyte surface antigen proteins, which changes the membrane mechanical and adhesive properties.15 In the severe form of malaria caused by Plasmodium falciparum, a disease that in many aspects resemble cerebral babesiosis, the causative organism induce infected red cells to clump together and to stick to endothelial cells lining of small blood vessels (sequestration), which blocks blood flow. Several proteins, including P. falciparum erythrocyte membrane protein 1 (PfEMP1), associated and form knobs on the surface of erythrocytes. PfEMP1 binds to thrombospondin, VCAM-1, ICAM-1, CD36, and E-selectin on the endothelial cells. Erythrocytes sequestration decreases tissue perfusion and leads to ischemia, which is responsible for the manifestations of cerebral malaria, the major cause of death in children with malaria.9 Mild to minimal mononuclear perivascular cuffing can be observed in a few of the slides of this case. This change is of no clinical significance encountered in one-third of symptomless cattle.7


JPC Diagnosis:  

Cerebrum, vessesls: Intraerythrocytic protozoal trophozoites, numerous, Angus x Nellore ox, Bos taurus.


Conference Comment:  

The contributor provides an exhaustive review of Babesia bovis infection in an ox in addition to excellent quality gross photographs of the striking and pathognomonic uniformly cherry-red cerebral grey matter, referred to colloquially as “cerebral flush” or “pink brain”. This disease is one of the leading causes of infectious mortality of cattle in Brazil.2,15 As mentioned by the contributor, this pink discoloration of the brain is secondary to marked congestion of the cerebral microvasculature by infected erythrocytes. In this case, nearly every erythrocyte is infected with at least one round to pyriform trophozoite. Parasitized erythrocytes have a tropism for the cerebral grey matter, kidney, heart, and skeletal muscle, but infected erythrocytes can be found in any tissue. Interestingly, the parasite is only rarely found in about 5% of circulating red blood cells.15

The sequestration of infected erythrocytes within the microvasculature of the cerebrum and other visceral organs leads to occlusion of the vessels and subsequent hypoxic injury. Babesia bovis also releases vasoactive proteases that activate the hypotensive agent, kallikrein, which then activates another potent vasodilator, bradykinin.15 The dilatory effect of these vasoactive proteins shifts blood away from veins and further contributes to apparent anemia. Additionally, infected animals are typically markedly anemic secondary to both intravascular and extravascular hemolysis, described by the contributor.15 The combination of vascular congestion, vasodilation, and hemolysis leads to both metabolic alkalosis and a hemodynamic crisis, often resulting in the death of the animal.

Most conference participants noted congestion and dilation of the cerebral microvasculature in this case; however, some attendees offered a dissenting opinion that the apparent congestion is a result of post-mortem pooling of blood within the brain rather than an antemortem change. The conference moderator agreed with the majority of the participants that the cerebral congestion is part of the pathogenesis of this disease and likely represents a real lesion, rather than an artifact. Participants also astutely noted that while vessels are congested in both the grey and white matter histologically, congestion is only apparent within the grey matter macroscopically.15


References:

1. Barros CSL, Driemeier D, Dutra IS, Lemos RAA. Babesiose cerebral [Cerebral babesiosis]. In: Barros CSL, Driemeier D, Dutra IS, Lemos RAA, eds. Doenças do sistema nervoso de bovinos no Brasil [Diseases of the central nervous system in cattle in tick Brazil]. São Paulo, SP: Coleção Vallée; 2006:87-95.
2. Barros CSL, Fighera RA. Babesiosis. In: Foreign animal diseases. Washington DC, United States: Animal Health Association, 2008:147-157.
3. Callow LL, McGavin MD. Cerebral babesiosis due to Babesia argentina. Aust Vet J. 1963; 39:15-21.
4. Carlson GP. Clinical chemistry tests. In: Smith BP, ed. Large Animal Internal Medicine. 4th ed. St. Louis: Mosby; 2009:393.
5. de Vos AJ, de Waal DT, Jackson LA. Bovine babesiosis. In: Coetzer JAW, Tustin RC, eds. Infectious Diseases of Livestock. 2nd ed., vol. 1. Cape Town: Oxford University; 2004:406-424.
6. Everitt JI, Shadduck JA, Steinkamp C, Claubaugh G. Experimental Babesia bovis infection in Holstein calves. Vet Pathol. 1986;23:556-562.
7. Gavier-Widen D, Wells GAH, Simmons MM, Wilesmith JWW, Ryan J. Histological observations on the brains of symptomless 7-year-old cattle. J Comp Path. 2001; 124:52-59.
8. Kessler RH, Madruga CR, Schenk MAM Ribeiro OC. Babesiose cerebral por Babesia bovis em bezerros no Estado de Mato Grosso do Sul [Cerebral babesiose by Babesia bovis in calves in Mato Grosso do Sul]. Pesq Agropec Bras. 1983;18:931-935.
9. McAdams AJ, Milner, Sharp AH. Parasitic infections. In: Kumar V, Abbas A, Aster J, eds. Robbins & Cotran Pathologic Basis of Disease. 9th ed. Philadelphia: Elsevier Saunders; 2014:389-402.
10. Patarroyo JH, Vargas M.L., Bicudo PL. Description of lesions in cattle in a natural outbreak of Babesia bovis infection in Brazil. Vet Parasitol. 1982;11:301-308.
11. Rodrigues A, Rech RR, Barros RR, Fighera RA, Barros CSL. Babesiose cerebral em bovinos: 20 casos [Cerebral babesiosis in cattle: 20 cases]. Ciência Rural. 2005;35:121-125.
12. Rogers RJ. Observations on the pathology of Babesia argentina infections in cattle. Aust Vet J. 1971; 47:242-247.
13. Santarosa BP, Dantas GTN, Ferreira DOL, et al. Infecção neurológica por Babesia bovis em bovino neonato – relato de caso [Babesia bovis neurological infection in bovine neonates - case report]. Vet. Zootec. 2013; 20:9-14.
14. Taylor MA, Coop RL, Wall RL. Veterinary parasitology. 3rd ed. Oxford: Blackwell; 2007:105-107.
15. Valli VEO, Kiupel M, Bienzle D. Babesiosis. In: Maxie MD, ed. Jubb, Kennedy & Palmer Pathology of Domestic Animals. 6th ed. vol. 3. St. Louis, MO: Elsevier; 2016:118-120.
16. Zlotnic L. Cerebral piroplasmosis in cattle. Vet Rec. 1953; 40:642-643.


Click the slide to view.



3-7. Cerebellum, calf.


3-1. Cadaver, calf.


3-2. Spleen, calf.


3-3. Kidney, calf.


3-4. Cerebrum, calf.


3-5. Cerebrum, calf.


3-6. Cerebrum, calf.



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